ATCC

PRIMARY CELL CULTURE GUIDE

tips and techniques for culturing primary cells
THE ESSENTIALS OF
LIFE SCIENCE RESEARCH
GLOBALLY DELIVERED
www.atcc.org
Table of Contents
ATCC Primary Cell Solutions...........................................................................1
ATCC Primary Human Endothelial Cell Solutions.............................................5
ATCC Primary Human Smooth Muscle Cell Solutions.......................................9
ATCC Primary Human Epithelial Cell Solutions...............................................12
ATCC Primary Human Fibroblast Solutions.....................................................15
ATCC Primary Human Keratinocyte Solutions................................................19
ATCC Primary Human Melanocyte Solutions..................................................22
ATCC Human Mesenchymal Stem Cell and Dierentiation Solutions..........25
References .......................................................................................................31
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ATCC

PRIMARY CELL SOLUTIONS

Guide to Culturing Human Primary Cells

Primary cells and cell types
Primary cell cultures more closely mimic the physiological state of cells in vivo and generate more
relevant data representing living systems. Primary cultures consist of cells that have been freshly
derived from a living organism and are maintained for growth in vitro. Primary cells can be categorized
according to the genus from which they are isolated, as well as by species or tissue type. Each
mammalian tissue type is derived from the embryonic germ layer consisting of ectoderm, endoderm
and mesoderm, which dierentiate into the many cell types that organize into tertiary structures such
as skin, muscle, internal organs, bone and cartilage, the nervous system, blood and blood vessels
1
. The
cell types most frequently found in primary cell culture are epithelial cells, broblasts, keratinocytes,
melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells.
Basic properties of primary cells
Once adapted to in vitro culture conditions, primary cells undergo a limited, predetermined number of
cell divisions before entering senescence. The number of times a primary cell culture can be passaged
is minimal due to the Hayflick Limit, nutrient requirements and culture conditions, and the expertise
by which they are manipulated and subcultured. In contrast, cell lines that have been immortalized by
viral, hTERT or tumorigenic transformation typically undergo unlimited cell division and have an innite
lifespan. And, unlike tumor cell lines cultured in medium containing 10% to 20% serum, primary cell
cultures are fastidious, requiring optimized growth conditions, including the addition of tissue specic
cytokines and growth factors for propagation in serum-free or low-serum growth media.
Benets of primary cells
Primary cell cultures are commonly used as in vitro tools for pre-clinical and investigative biological
research, such as studies of inter- and intracellular communication, developmental biology, and
elucidation of disease mechanisms, such as cancer, Parkinsons disease, and diabetes. Historically,
investigators have employed immortalized cell lines in research related to tissue function; however, the
use of cell lines containing gross mutations and chromosomal abnormalities provides poor indicators
of normal cell phenotype and progression of early-stage disease. The use of primary cells, maintained
for only short periods of time in vitro, now serves as the best representative of the main functional
component of the tissue (in vivo) from which they are derived.
Isolation of primary cells
The isolation and purication of peripheral blood cells can be easily achieved by dierential
centrifugation or by positive sorting using magnetic beads. On the other hand, the isolation of a pure
population of cells from primary tissue is often dicult to perform, and requires knowledge of how
the cell strata should be teased apart into a suspension containing only one predominant cell type.
Diagram 1 is an illustration of some of the basic steps used to establish a primary cell culture
14
.
Primary cell culture
Growth requirements
Primary cells, except for those derived from peripheral blood, are anchorage-dependent, adherent
cells, meaning they require a surface in order to grow properly in vitro. In most cases, primary cells are
cultured in a flat un-coated plastic vessel, but sometimes a microcarrier, which can greatly increase the
surface area, can be used. A complete cell culture media, composed of a basal medium supplemented
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with appropriate growth factors and cytokines, is required. During establishment of primary cultures, it
may be useful to include an antibiotic in the growth medium to inhibit contamination introduced from
the host tissue. These may include a mixture of gentamicin, penicillin, streptomycin and amphotericin
B. However, long-term use of antibiotics is not advised, since some reagents, such as amphotericin B,
may be toxic to cells over time.
Maintenance
The maintenance phase begins when cells have attached to the surface of the culture dish. Attachment
usually occurs about 24 hours after initiation of the culture. When initiating a culture of cryopreserved
primary cells, it is important to remove the spent media once the cells have attached because DMSO
is harmful to primary cells and may cause a drop in post-thaw viability. When cells have reached a
desired percent of cellular confluence and are actively proliferating, it is time to subculture. It is best
to subculture primary cell cultures before reaching 100% confluence, since post-confluent cells may
undergo dierentiation and exhibit slower proliferation after passaging.
Cellular confluence
Cellular confluence refers to the percentage of the culture vessel inhabited by attached cells. For
example, 100% cellular confluence means the surface area is completely covered by cells, while 50%
confluence means roughly half of the surface is covered. It is an important parameter to track and
assess in primary cell culture because dierent cell types require dierent confluence end points, at
which point they need to be subcultured.
Levels of cellular confluence
Subculture
Anchorage-dependent cells grow in monolayers and need to be subcultured at regular intervals to
maintain exponential growth. Subculturing procedures, including recommended split-ratios and
medium replenishment (feeding) schedules for each ATCC primary cell culture, are provided on the
Tissue Acquisition Dissection Disaggregation Incubation & Growth
Separation &
Purication
Process primary
tissue, removing
fatty and necrotic
cells
Mechanical
or enzymatic
disaggregation.
Enzymes used:
Trypsin
Collagenase II
Elastase
Hyaluronidase
DNase
Incubate dispersed
cells
Change medium 24
hours after initiation
to remove loose
debris & unattached
cells
Further purication
of primary cells
achieved by:
Selective media
Remove cells at
dierent levels of
attachment
Immunomagnetic
beads
Diagram 1. Basic steps used to isolate cells from primary tissue
Human smooth muscle cells between 20%
and 30% confluence
Human melanocyte cells between 50%
and 60% confluence
Human keratinocytes at nearly 100%
confluence (note the formation of
vacuoles and dierentiated cells)
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product information sheet provided with each cell (available online at www.atcc.org). Subcultivation of
monolayers involves the breakage of both inter- and intracellular cell-to-surface bonds. Most adherent
primary cells require the digestion of their protein attachment bonds with a low concentration of a
proteolytic enzyme such as trypsin/EDTA. After the cells have been dissociated and dispersed into a
single-cell suspension, they are counted and diluted to the appropriate concentration and transferred
to fresh culture vessels where they will reattach and divide.
Cell Counting
Hemocytometers are commonly used to estimate cell number and determine cell viability with the aid
of an exclusion dye such as Trypan Blue or Erythrosin B. A hemocytometer is a fairly thick glass slide
with two counting chambers, one on each side. Each counting chamber has a mirrored surface with a 3
3 mm grid consisting of 9 counting squares. The chambers have raised sides that will hold a coverslip
exactly 0.1 mm above the chamber floor. Each of the 9 counting squares holds a volume of 0.0001 mL.
Alternatively, counting cells can also be achieved by using an automated cell counter, such as Vi-CELL.
For a detailed description of cell counting using a hemocytometer, refer to the ATCC Animal Cell
Culture Guide: Tips and Techniques for Continuous Cell Lines available online at www.atcc.org.
Cryopreservation and Recovery
Special attention is needed to cryopreserve and thaw primary cells in order to minimize cell damage
and death during each process. Cryopreservation of human cells is best achieved with the use of a
cryoprotectant, such as DMSO or glycerol. Most primary cell cultures can be cryopreserved in a mixture
of 80% complete growth medium supplemented with 10% FBS and 10% DMSO. The freezing process
should be slow, at a rate of -1C per minute, to minimize the formation of ice crystals within the cells.
Once frozen, cultures are stored in the vapor phase of liquid nitrogen, or below -130C. Additional
information about freezing cells can be found in ATCC Technical Bulletin No. 3: Cryogenic Preservation
of Animal Cells, available online at www.atcc.org.
Thawing cryopreserved cells is a rapid process and is accomplished by immersing frozen cells in a 37C
water bath for about 1 to 2 minutes. Care should be taken not to centrifuge primary cells upon thaw,
since they are extremely sensitive to damage during recovery from cryopreservation. It is best to
plate cells directly upon thaw, and allow cultures to attach for the rst 24 hours before changing the
medium to remove residual DMSO.
Challenges of primary cell isolation and culture
There are several challenges associated with the use of primary cells. One of the greatest hurdles
primary cell culturists face is limited cell accessibility due to issues with donor tissue supply, diculty
with cell isolation/purication, quality assurance and consistency, and contamination risks. Data
comparability is also a serious issue with primary cell use, and arises out of variability among reagents
used and the procedures implemented by individual scientists and laboratories to isolate and culture
primary cells. A comparison between primary cells and continuous cell lines is described in Table 1.
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Table 1. Comparison between primary cells and continuous cell lines
Properties Primary cells Continuous cell lines
Life span & cell
proliferation
Finite; limited to a small number of cell
doublings
Innite when handled properly
Consistency
Variability exists between donors and
preparations
Minimal variability
Genetic Integrity
Retains in vivo tissue genetic makeup
through cell doublings
Subject to genetic drift as cells divide
Biological relevance
More closely mimics the physiology of
cells in vivo
Relevance can drift over time as cells
divide
Ease of use (freeze-thaw
& use)
Requires optimized culture conditions
and careful handling
Well established conditions and robust
protocols exist
Time & expense to use More time and less abundance of cells Less time and more abundance of cells
ATCC Primary Cell Solutions
ATCC has provided a solution to help investigators overcome the high cost and inconsistency found
in routine primary cell culture with the development of ATCC

Primary Cell Solutions

, a standardized
cell culture system that includes high quality cells, media, supplements, reagents and protocols. ATCC

Primary Cell Solutions focuses on providing researchers with superior quality from a trusted source
each lot of ATCC Primary Cell Solutions primary cells is:
Cryopreserved at early passage
ATCC Primary Cell Solutions primary cells are frozen at passages 1 through 3
Early passage material ensures higher viability and optimal plating eciency
Performance tested
ATCC Primary Cell Solutions cells, media, kit supplements, and reagents are tested for optimal
reliability
Growth and morphology are assessed to ensure all components work synergistically
Quality controlled to ensure safety, purity, and functionality
Sterility testing - all cells are tested for bacteria, yeast, fungi, and Mycoplasma
Viral testing - HIV-1, HIV-2, HBV, and HCV tissue screening is performed at isolation
Viability and Growth - viability and growth of each lot of cells is checked before freezing and
after-thawing
Staining - staining for cell-specic marker expression is determined and performed for some
cell types
ATCC Primary Cell Solutions basal media and cell-specic growth kits are designed to support recovery
and proliferation of ATCC Primary Cell Solutions primary cells in vitro. Used as a system, ATCC Primary
Cell Solutions primary cells, basal media and cell-specic growth kits provide all the components
needed to be successful in primary cell research. (Detailed formulations for each growth kit can be
found at www.atcc.org.)
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Endothelial Cells
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ATCC PRIMARY HUMAN ENDOTHELIAL CELL SOLUTIONS
Introduction
Endothelial cells form the endothelium - the thin layer of cells that line
the interior surface of blood vessels forming a smooth anticoagulant
surface that functions as a selective lter to regulate the passage of
gases, fluid, immune cells and various molecules
14
. Human endothelial
cells can be isolated from human umbilical vein, the aorta, the pulmonary
and coronary arteries, and the skin, and serve as useful tools in the study
of angiogenesis, cancer therapy, wound healing, burn therapy, high-
throughput and high-content screening projects, cell signaling studies,
gene expression proling, toxicology screening, tissue engineering and
regeneration.
ATCC Primary Human Endothelial Cells can be cultured in complete growth medium containing either
bovine brain extract (BBE) or vascular endothelial growth factor (VEGF). Use of the Endothelial Cell
Growth Kit-VEGF (ATCC PCS-100-041) will support a faster rate of proliferation, while the Endothelial
Cell Growth Kit-BBE (ATCC PCS-100-040) is recommended if a less dened cell culture medium is
desired.
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Endothelial Cells Growth Kit Options* Basal Medium
Umbilical Vein Endothelial Cells, Normal, Human
(ATCC PCS-100-010)
Choose ONE:
Endothelial Cell Growth Kit-
BBE (ATCC PCS-100-040)
Endothelial Cell Growth Kit-
VEGF (ATCC PCS-100-041)
Vascular Cell Basal Medium
(ATCC PCS-100-030)
Umbilical Vein Endothelial Cells, Normal,
Human, Pooled (ATCC PCS-100-013)
Aortic Endothelial Cells; Normal, Human (ATCC
PCS-100-011)
Coronary Artery Endothelial Cells, Normal,
Human (ATCC PCS-100-020),
Pulmonary Artery Endothelial Cells, Normal,
Human (ATCC PCS-100-022),
Dermal Microvascular Endothelial Cells, Normal,
Human, Neonatal (ATCC PCS-110-010)
For Microvascalar Endothelial
Cells, Choose ONE:
Microvascalar Endothelial Cell
Growth Kit-BBE (ATCC PCS-
110-040)
Microvascalar Endothelial Cell
Growth Kit-VEGF (ATCC PCS-
110-041)
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
*Phenol red and antibiotics may be added if desired, and are listed in the appendix.
Primary Dermal Micovascular
Endothelial Cells, Normal, Human,
Neonatal (ATCC PCS-110-010)
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Endothelial Cells
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Preparation of Complete Growth Media
1. Obtain one growth kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is
necessary to warm the L-glutamine component in a 37C water bath and shake to dissolve any
precipitates prior to adding to the basal medium.
3. Obtain one bottle of Vascular Cell Basal Medium from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium
bottle by spraying them with 70% ethanol.
5. Using aseptic technique, and working in a laminar flow hood or biosafety cabinet, transfer the
volume of each growth kit component (volumes for each growth kit are provided on the product
information sheet; the following table represents the Endothelial Cell Growth Kit-VEGF) to the
bottle of basal medium using a separate sterile pipette for each transfer.
6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored
under these conditions, complete growth media is stable for 30 days.
Endothelial Cell Growth Kit-VEGF (ATCC PCS-100-041)
Component Volume Final Concentration
rh VEGF 0.5 mL 5 ng/mL
rh EGF 0.5 mL 5 ng/mL
rh FGF basic 0.5 mL 5 ng/mL
rh IGF-1 0.5 mL 15 ng/mL
L-glutamine 25.0 mL 10 mM
Heparin sulfate 0.5 mL 0.75 Units/mL
Hydrocortisone hemisuccinate 0.5 mL 1 g/mL
Fetal Bovine Serum 10.0 mL 2%
Ascorbic acid 0.5 mL 50 g/mL
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last
page of the product information sheet for the total number
of viable cells recovered from each lot of ATCC Primary Human
Endothelial Cells.
2. Using the total number of viable cells, determine how much
surface area can be inoculated to achieve an initial seeding
density between 2,500 and 5,000 cells per cm
2
for most Primary
Human Endothelial Cells oered by ATCC; the exception being
Primary Dermal Microvascular Endothelial Cells, which should
be seeded at a density of 5,000 cells per cm
2
.
3. Prepare the desired combination of flasks. Add 5 mL of
complete growth media per 25 cm
2
of surface area. Place
the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-equilibrate to
temperature and pH for 30 minutes prior to adding cells.
Primary Dermal Microvascular Endothelial
Cells dual stained for von Willebrand
factor (green) as a marker for endothelial
cells and smooth muscle -actin (red)
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Endothelial Cells
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4. While the culture flasks equilibrate, remove one vial of ATCC Primary Human Endothelial Cells from
storage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility of
contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately
1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth media [volume = (1 mL x number of flasks to
be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1.0 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C with a 5% CO
2
atmosphere. Incubate for
at least 24 hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
multiple times.
2. 24 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth media per 25 cm
2
of surface area and return the
flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular
confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have
reached approximately 80% confluence, and are actively proliferating (many mitotic gures are
visible), it is time to subculture. Subculture endothelial cells before reaching confluence; post-
confluent primary endothelial cells may exhibit slower proliferation after passaging.
Subculture
1. Passage Primary Human Endothelial Cells when cultures have reached approximately 80%
confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing
Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm complete growth
medium to 37C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual traces of
serum.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away from each other and round up
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Endothelial Cells
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(typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from
several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of Trypsin
Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to
ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the flask to collect any additional cells that might have
been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to
8 mL fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new flasks at a density of 2,500 to 5,000 cells per cm
2
; however, be sure
to seed Primary Dermal Microvascular Endothelial Cells at 5,000 cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
, incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
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Smooth Muscle Cells
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ATCC PRIMARY HUMAN SMOOTH MUSCLE CELL SOLUTIONS
Introduction
Vascular tissue is made up of a diverse population of cell types,
including endothelial cells, smooth muscle cells, pericytes,
broblasts, and other connective tissue cell types. Together
these cell types form tight junctions or connections, which
allow for permeability for both passive and active transport
across the vessel wall. Vascular smooth muscle cells make up
the smooth muscle layer of blood vessels and can be co-cultured
with vascular endothelium
14
. Smooth muscle cells isolated from
human ascending (thoracic) and descending (abdominal) aorta,
coronary artery, and pulmonary artery are useful for studying
vascular diseases such as thrombosis and atherosclerosis.
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Smooth Muscle Cells Growth Kit Options* Basal Medium
Aortic Smooth Muscle Cells, Normal, Human
(ATCC PCS-100-012)
Vascular Smooth Muscle Cell
Growth Kit (ATCC PCS-100-
042)
Vascular Cell Basal Medium
(ATCC PCS-100-030)
Coronary Artery Smooth Muscle Cells, Normal,
Human (ATCC PCS-100-021)
Pulmonary Artery Smooth Muscle Cells, Normal,
Human (ATCC PCS-100-023)
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
*Phenol red and antibiotics may be added if desired, and are listed in the appendix.
Preparation of Complete Growth Media
1. Obtain one growth kit from the freezer; make sure that the
caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding
them to the basal medium. It is necessary to warm the
L-glutamine component in a 37C water bath and shake to
dissolve any precipitates prior to adding to the basal medium.
3. Obtain one bottle of Vascular Cell Basal Medium from cold
storage.
4. Decontaminate the external surfaces of all growth kit
component vials and the basal medium bottle by spraying
them with 70% ethanol.
5. Using aseptic technique, and working in a laminar flow hood
or biosafety cabinet, transfer the volume of each growth kit
ATCC Primary Cell Solutions aortic
smooth muscle cells (ATCC PCS-100-012)
dual stained for von Willebrand factor
(green) as a marker for endothelial cells
and smooth muscle -actin (red) after
dierentiation.
Primary Aortic Smooth Muscle Cells,
Normal, Human (ATCC PCS-100-012)
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Smooth Muscle Cells
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component, as indicated in the following table to the bottle of basal medium using a separate
sterile pipette for each transfer.
Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-042)
Component Volume Final Concentration
rh FGF-basic 0.5 mL 5 ng/mL
rh Insulin 0.5 mL 5 g/mL
Ascorbic acid 0.5 mL 50 g/mL
L-glutamine 25.0 mL 10 mM
rh EGF 0.5 mL 5 ng/mL
Fetal Bovine Serum 25.0 mL 5%
6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored
under these conditions, complete growth media is stable for 30 days.
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last page of the product information
sheet for the total number of viable cells recovered from each lot of ATCC Primary Human
Smooth Muscle Cells.
2. Using the total number of viable cells, determine how much surface area can be inoculated to
achieve an initial seeding density between 2,500 and 5,000 cells per cm
2
.
3. Prepare the desired combination of flasks. Add 5 mL of complete growth media per 25 cm
2
of
surface area. Place the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-
equilibrate to temperature and pH for 30 minutes prior to adding cells.
4. While the culture flasks equilibrate, remove one vial of ATCC Primary Human Smooth Muscle
Cells from storage and thaw the cells by gentle agitation in a 37C water bath. To reduce the
possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid
(approximately 1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth media [volume = (1 mL x number of flasks to
be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1.0 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C with a 5% CO
2
atmosphere. Incubate for
at least 24 hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
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Smooth Muscle Cells
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multiple times.
2. 24 to 36 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth media per 25 cm
2
of surface area and return the
flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to
determine percent cellular confluence. If not ready to passage,
repeat steps 3 and 4 as described above. When cultures have reached
approximately 80% to 90% confluence, and are actively proliferating
(many mitotic gures are visible), it is time to subculture. Vascular
smooth muscle cells are contact inhibited; post-confluent vascular
smooth muscle cells may not proliferate after passaging.
Subculture
1. Passage normal vascular smooth muscle cells when the culture has reached approximately 80%
confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing
Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm complete growth
medium to 37C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual traces of
serum.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away from each other and round up
(typically within 1 to 3 minutes), remove the flask from the microscope and gently tap it from
several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of Trypsin
Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to
ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the flask to collect any additional cells that might have
been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to
8 mL fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new flasks at a density of 2,500 to 5,000 cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
, incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
Note:
Cells are typically ready to
passage after 7 to 9 days in
culture when inoculated with
2,500 cells/cm2.
www.atcc.org page 12
Epithelial Cells
www.atcc.org page 12
ATCC PRIMARY HUMAN EPITHELIAL CELL SOLUTIONS
Introduction
Epithelia are tissues found throughout the body. They line the
cavities of glands and organs in the body and also cover flat surfaces,
such as skin. Epithelial cells are polarized cells that discriminate
between an apical and basolateral compartment. They perform
a variety of functions depending on their location, including
boundary and protection, sensory, secretion, transportation,
absorption and diusion. One challenging aspect of culturing
primary epithelial cells is overgrowth of stromal cells. Stromal
broblasts can be inhibited by culturing explants in low serum or serum-free culture media, limiting
the calcium concentration in the growth medium, selectively inhibiting broblasts with agents, or
by targeting with antimesodermal antibodies
14
. ATCC Primary Cell Solutions oers epithelial cells
isolated from human bronchi, trachea and small airways, as well as the cornea, prostate and kidneys.
The usefulness of such cultures has been found in studies related to inflammation, microbial infection
and pathogenesis including influenza, cancer, toxicology, gene regulation and tissue development,
cell-matrix interactions, as well as application in toxicology testing and drug screening/development.
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Epithelial Cells Growth Kit Options* Basal Medium
Small Airway Epithelial Cells; Normal, Human
(ATCC PCS-301-010)
Small Airway Epithelial Cell
Growth Kit (ATCC PCS-301-
040)
Airway Epithelial Cell Basal
Medium (ATCC PCS-300-
030)
Bronchial/Tracheal Epithelial Cells; Normal,
Human (ATCC PCS-300-010)
Bronchial Epithelial Cell
Growth Kit (ATCC PCS-300-
040)
Renal Proximal Tubule Epithelial Cells; Normal,
Human (ATCC PCS-400-010)
Renal Epithelial Cell Growth Kit
(ATCC PCS-400-040)
Renal Epithelial Cell Basal
Medium (ATCC PCS-400-
030)
Renal Cortical Epithelial Cells; Normal, Human
(ATCC PCS-400-011)
Renal Mixed Epithelial Cells; Normal, Human
(ATCC PCS-400-012)
Prostate Epithelial Cells; Normal, Human (ATCC
PCS-440-010)
Prostate Epithelial Cell Growth
Kit (ATCC PCS-440-040)
Prostate Epithelial Cell
Basal Medium (ATCC PCS-
440-030)
Corneal Epithelial Cells; Normal, Human (ATCC
PCS-700-010)
Corneal Epithelial Cell Growth
Kit (ATCC PCS-700-040)
Corneal Epithelial Cell Basal
Medium (ATCC PCS-700-
030)
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
*Phenol red and antibiotics may be added if desired, and are listed in the appendix.
Primary Prostate Epithelial Cells; Normal,
Human (ATCC PCS-440-010)
Email [email protected] Phone 800.638.6597 page 13
Epithelial Cells
Email [email protected] Phone 800.638.6597 page 13
Preparation of Complete Growth Media
1. Obtain one Epithelial Cell Growth Kit (corresponding to the epithelial cell type being used) from
the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium.
3. Obtain one bottle of Epithelial Cell Basal Medium (corresponding to the epithelial cell being used)
from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium
bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the
indicated volume of each growth kit component as indicated in the cultures corresponding
product information sheet, to the bottle of basal medium using a separate sterile pipette for
each transfer.
6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored
under these conditions, complete growth media is stable for 30 days.
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last page of the product information
sheet for the total number of viable cells recovered from each lot of ATCC Primary Human
Epithelial Cells.
2. Using the total number of viable cells, determine how much surface area can be inoculated to
achieve an initial seeding density of 5,000 cells per cm
2
.
3. Prepare the desired combination of flasks. Add 5 mL of complete growth medium per 25 cm
2
of
surface area. Place the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-
equilibrate to temperature and pH for 30 minutes prior to adding cells.
4. While the culture flasks equilibrate, remove one vial of ATCC Primary Human Epithelial Cells from
storage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility of
contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately
1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of flasks
to be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1.0 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C, 5% CO
2
atmosphere. Incubate for at least
24 hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
www.atcc.org page 14
Epithelial Cells
www.atcc.org page 14
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
multiple times.
2. 24 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth medium per 25 cm
2
of surface area and return
the flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular
confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have
reached about 80% confluence, and are actively proliferating (many mitotic gures are visible), it
is time to subculture.
Subculture
1. Passage normal human epithelial cells when the culture has reached about 80% confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing
Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete
growth medium to 37C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away from each other and round up
(typically within 1 to 3 minutes), remove the flask from the microscope and gently tap it from
several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin
Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to
ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the culture flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that
might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL
fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
Email [email protected] Phone 800.638.6597 page 15
Fibroblasts
Email [email protected] Phone 800.638.6597 page 15
ATCC PRIMARY HUMAN FIBROBLAST SOLUTIONS
Introduction
Fibroblasts play an important role in maintaining the structural
integrity of connective tissue, and synthesize extracellular
matrix proteins such as collagens, glycosaminoglycans, and
glycoproteins
15,16
. Fibroblasts are morphologically heterogeneous,
and their appearance is dependent on in vivo location and activity.
Injury of tissue displays a proliferative stimulus for broblasts and
induces them to produce wound healing proteins. Fibroblasts used
in primary cell culture are commonly isolated from the dermis
layer of human neonatal foreskin or adult skin. They are frequently
used in studies related to wound healing, tissue engineering and
regeneration applications, and the induction of pluripotent stem
(iPS) cells. Fibroblasts, treated with mitomycin C to inhibit proliferation, have been extensively used as
feeder layers to enhance the cultivation of human stem cells and keratinocytes in vitro.
ATCC Primary Human Fibroblasts can be cultured in complete growth medium with or without serum.
The use of Fibroblast Growth KitSerum-Free creates a completely dened medium for the serum-free
culture of human broblasts. The rate of proliferation is equal to or greater than media supplemented
using FBS (at concentrations ranging from 2% to 10%) through 10 population doublings.
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Fibroblasts Growth Kit Options* Basal Medium
Dermal Fiboblasts; Normal, Human Neonatal
(ATCC PCS-201-010)
Choose ONE:
Fibroblast Growth Kit Serum
Free (ATCC PCS-201-040)
Fibroblast Growth Kit Low
Serum (ATCC PCS-201-041)
Fibroblast Basal Medium
(ATCC PCS-201-030)
Dermal Fiboblasts; Normal, Human, Adult
(ATCC PCS-201-012)
Dermal Fiboblasts; Normal, Human Neonatal,
Mitomycin C Treated (ATCC PCS-201-011)**
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
*Phenol red and antibiotics may be added if desired, and are listed in the appendix.
**0.1% Gelatin Solution (ATCC PCS-999-027) is also needed for culturing Mitomycin C Treated Dermal Fibroblasts, Normal,
Human Neonatal (ATCC PCS-201-011).
Preparation of Complete Growth Media
1. Obtain one growth kit from the freezer; make sure that the caps of all containers are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is
necessary to warm the L-glutamine component in a 37C water bath, and shake to dissolve any
precipitates prior to adding to the basal medium.
3. Obtain one bottle of Fibroblast Basal Medium from cold storage.
Primary adult human dermal broblasts
(ATCC PCS-201-012) cultured in serum-
free medium.
www.atcc.org page 16
Fibroblasts
www.atcc.org page 16
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium
bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the
volume of each growth kit component, as indicated in the following tables, to the bottle of basal
medium using a separate sterile pipette for each transfer.
Fibroblast Growth KitSerum-Free (ATCC PCS-201-040)
Component Volume Final Concentration
L-glutamine 18.75 mL 7.5 mM
Hydrocortisone Hemisuccinate 0.5 mL 1 g/mL
HLL Supplement 1.25 mL
HSA 500 g/mL
Linoleic Acid 0.6 M
Lecithin 0.6 g/mL
rh FGF 0.5 mL 5 ng/mL
rh EGF / TGF -1 Supplement 0.5 mL
5 ng/mL
30 pg/mL
rh Insulin 0.5 mL 5 g/mL
Ascorbic acid 0.5 mL 50 g/mL
Fibroblast Growth KitLow Serum (ATCC PCS-201-041)
Component Volume Final Concentration
rh FGF 0.5 mL 5 ng/mL
L-glutamine 18.75 mL 7.5 mM
Ascorbic acid 0.5 mL 50 g/mL
Hydrocortisone Hemisuccinate 0.5 mL 1 g/mL
rh Insulin 0.5 mL 5 g/mL
Fetal Bovine Serum 10.0 mL 2%
6. Tightly cap the bottle of complete growth
medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to
avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark
at 2C to 8C (do not freeze). When stored under
these conditions, complete growth media is stable
for 30 days.
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last page of the product information
sheet for the total number of viable cells recovered from each lot of ATCC Primary Human
Fibroblasts.
2. Using the total number of viable cells reported, determine how much surface area can be
inoculated to achieve an initial seeding density of 2,500 to 5,000 cells per cm
2
for untreated
broblasts. Note: Mitomycin C treated broblasts should be seeded at a density of 20,000 to
40,000 cells per cm
2
for use with stem cells.
Note:
Gelatin-Coated Tissue Culture Flasks are needed
to culture Mitomycin C Treated Dermal Fibroblasts
(ATCC PCS-201-011) in order to achieve optimal
cell attachment. Culture flasks should be prepared
before thawing cells. Non-coated tissue culture
flasks can also be used, if needed.
Email [email protected] Phone 800.638.6597 page 17
Fibroblasts
Email [email protected] Phone 800.638.6597 page 17
3. Prepare the desired combination of flasks. Add 5mL of complete growth medium per 25 cm
2
of
surface area. Place the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-
equilibrate to temperature and pH for 30 minutes prior to adding cells.
4. While the culture flasks equilibrate, remove one vial of corresponding ATCC Primary Human
Fibroblasts from storage and thaw the cells by gentle agitation in a 37C water bath. To reduce
the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be
rapid (approximately 1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth media [volume = (1 mL x number of flasks to
be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Cultures. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C, 5% CO
2
atmosphere. Incubate at least 24
hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
multiple times.
2. 24 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth media per 25 cm
2
of surface area and return the
flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular
confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have
reached 80% to 100% confluence, and are actively proliferating (many mitotic gures are visible),
it is time to subculture. Fibroblasts are not a contact inhibited cell type.
Subculture
1. Passage normal human broblasts when the cells have reached
approximately 80% to 100% confluence and are actively
proliferating.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-
003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004)
to room temperature prior to dissociation. Warm the complete growth medium to 37C prior to
use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer two times with 3 to 5 mL of D-PBS per 25 cm
2
of surface area (ATCC 30-2200)
to remove any residual traces of serum. Rinse the cell layer one time with 3 to 5 mL of D-PBS if
Note:
Mitomycin C treated broblasts
are mitotically arrested and
cannot be subcultured.
www.atcc.org page 18
Fibroblasts
www.atcc.org page 18
serum-free culture conditions are used.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away from each other and round up
(typically within about 3 to 5 minutes), remove the flask from the microscope and gently tap it
from several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells appear to have detached, quickly add to each flask, a volume of the
Trypsin Neutralizing Solution (ATCC PCS-999-004) equal to the volume of trypsin-EDTA solution
used previously. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has
been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the culture flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that
might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to
8 mL fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 2,500 to 5,000 cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
Inoculation of Mitomycin-C-treated Fibroblast Feeder Layers with Co-culture Cells of Interest
1. Aspirate the complete broblast growth medium from the feeder cell layer.
2. Add pre-warmed medium appropriate for the cell type of interest (e.g., embryonic stem cell).
3. Return the culture flask to the incubator at 37C, 5% CO
2
. Allow to equilibrate for at least 30
minutes before seeding the cells of interest.
For more information regarding the use of feeder cells with stem cell lines, refer to
ATCC Human ES/iPS Cell Culture Guide: Protocols for Feeder-free and Feeder-dependent Systems available
online at www.atcc.org.
Email [email protected] Phone 800.638.6597 page 19
Keratinocytes
Email [email protected] Phone 800.638.6597 page 19
ATCC PRIMARY HUMAN KERATINOCYTE SOLUTIONS
Introduction
Keratinocytes are the most common type of skin cells making up
the majority of the epidermis. Dividing keratinocytes produce
keratin (a protein that provides strength to skin, hair and nails) and
migrate to the surface of the skin, the stratum corneum, where they
serve as the most abundant cells in the skins outermost layer
17
.
Once present in the stratum corneum, keratinocytes dierentiate,
stop dividing as cornied cells
18
and eventually undergo apoptosis.
Keratinocytes can be isolated from dierent locations of the body.
However, the most utilized keratinocytes in primary cell culture
have been isolated from the epidermis of human juvenile foreskin
or human adult skin, the latter of which is signicant in the study of adult diseases such as psoriasis
and skin cancer
14
. Keratinocytes cultured in serum-free, low calcium growth medium will maintain the
cells in an un-dierentiated, proliferative state while inhibiting broblast outgrowth. Keratinocytes
are useful for a number of scientic applications including the study of growth factor behavior, wound
healing, toxicity/irritancy studies, and use as target cells for derivation of induced pluripotent stem
cells.
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Keratinocytes Growth Kit Options* Basal Medium
Epidermal Keratinocyte; Normal, Human,
Neonatal Foreskin (ATCC PCS-200-010)
Keratinocyte Growth Kit
(ATCC PCS-200-040)
Dermal Cell Basal Medium
(ATCC PCS-200-030)
Epidermal Keratinocyte; Normal, Human, Adult
(ATCC PCS-200-011)
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
*Phenol red and antibiotics may be added if desired, and are listed in the appendix.
Preparation of Complete Growth Media
1. Obtain one Keratinocyte Growth Kit from the freezer; make sure that the caps of all components
are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is
necessary to warm the L-glutamine component in a 37C water bath and shake to dissolve any
precipitates prior to adding to the basal medium.
3. Obtain one bottle of Dermal Cell Basal Medium from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium
bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the
indicated volume of each growth kit component, as indicated in the following table, to the bottle
of basal medium using a separate sterile pipette for each transfer.
Primary Epidermal Keratinocytes; Normal,
Human, Adult (ATCC PCS-200-011)
www.atcc.org page 20
Keratinocytes
www.atcc.org page 20
Keratinocyte Growth Kit (ATCC PCS-200-040)
Component Volume Final Concentration
Bovine Pituitary Extract (BPE) 2.0 mL 0.4%
rh TGF- 0.5 mL 0.5 ng/mL
L-Glutamine 15.0 mL 6 mM
Hydrocortisone Hemisuccinate 0.5 mL 100 ng/mL
rh Insulin 0.5 mL 5 g/ml
Epinephrine 0.5 mL 1.0 M
Apo-Transferrin 0.5 mL 5 g/ml
6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored
under these conditions, complete growth media is stable for 30 days.
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last page of the product information
sheet for the total number of viable cells recovered from each lot of ATCC Primary Human
Keratinocytes.
2. Using the total number of viable cells, determine how much surface area can be inoculated to
achieve an initial seeding density between 2,500 and 5,000 cells per cm
2
.
3. Prepare the desired combination of flasks. Add 5 mL of complete growth medium per 25 cm
2
of
surface area. Place the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-
equilibrate to temperature and pH for 30 minutes prior to adding cells.
4. While the culture flasks equilibrate, remove one vial of ATCC Primary Human Keratinocytes from
storage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility of
contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately
1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of flasks
to be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1.0 ml of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C, 5% CO
2
atmosphere. Incubate for at least
24 hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
Email [email protected] Phone 800.638.6597 page 21
Keratinocytes
Email [email protected] Phone 800.638.6597 page 21
multiple times.
2. 24 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth medium per 25 cm
2
of surface area and return
the flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular
confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have
reached approximately 80% confluence, and are actively proliferating (many mitotic gures are
visible), it is time to subculture. Keratinocytes will begin to terminally dierentiate once they become
100% confluent.
Subculture
1. Passage normal keratinocytes when the culture has reached approximately 70% to 80%
confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing
Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete
growth medium to 37C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away
from each other and round up (typically within 3 to 6 minutes),
remove the flask from the microscope and gently tap it from
several sides to promote detachment of the cells from the flask
surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin
Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to
ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the culture flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that
might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL
fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 2,500 to 5,000 cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
Note:
If cells are difficult to detach,
incubate each flask containing
cel l s and the trypsi n- EDTA
solution at 37C to facilitate
dispersal.
www.atcc.org page 22
Melanocytes
www.atcc.org page 22
ATCC PRIMARY HUMAN MELANOCYTE SOLUTIONS
Introduction
Melanocytes are found mainly in the epidermis, but may occur
elsewhere in the body such as in the matrix of the hair. They are
specialized skin cells that produce the pigment melanin, which
gives skin its color and protects it from the hazardous eects of
UV radiation. The most useful melanocytes for research are isolated
from the epidermis of human juvenile foreskin or human adult skin.
Special care must be taken to prevent contamination of melanocyte
cultures with keratinocytes or broblasts found in the epidermis.
Lowering the concentration of calcium in the medium to 60 M and
selecting for adherent melanocytes in hormone-supplemented
medium aids in the isolation of melanocytes from epidermal tissue
14
. Melanocytes are frequently used
in the in vitro study of wound healing, and as testing models for toxicity/irritancy studies, melanoma,
dermal response to UV radiation, psoriasis and other skin diseases, and cosmetic research (e.g., skin
lightening compounds, skin protecting compounds).
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Melanocytes Growth Kit Options Basal Medium
Epidermal Melanocytes; Normal, Human,
Neonatal (ATCC PCS-200-012)
Melanocyte Growth Kit (ATCC
PCS-200-041)
Dermal Cell Basal Medium
(ATCC PCS-200-030)
Epidermal Melanocytes; Normal, Human, Adult
(ATCC PCS-200-013)
Adult Melanocyte Growth Kit
(ATCC PCS-200-042)
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
*Phenol red and antibiotics may be added if desired, and are listed in the appendix.
Preparation of Complete Growth Media
1. Obtain one Melanocyte Growth Kit (corresponding to the melanocyte being used) from the
freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is
necessary to warm the L-glutamine component in a 37C water bath and shake to dissolve any
precipitates prior to adding to the basal medium.
3. Obtain one bottle of Dermal Cell Basal Medium from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium
bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the
indicated volume of each growth kit component, as indicated in the following table, to the bottle
of basal medium using a separate sterile pipette for each transfer.
Adult human melanocytes (ATCC PCS-
200-013).
Email [email protected] Phone 800.638.6597 page 23
Melanocytes
Email [email protected] Phone 800.638.6597 page 23
Melanocyte Growth Kit (ATCC PCS-200-041)
Component Volume Final Concentration
rh Insulin 0.5 mL 5 g/ml
Ascorbic Acid 0.5 mL 50 g/ml
L-Glutamine 15.0 mL 6 mM
Epinephrine 0.5 mL 1.0 M
Calcium Chloride 80 l 0.2 mM
M8 Supplement 5 ml Proprietary formulation
6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored
under these conditions, complete growth media is stable for 30 days.
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last page of the product information
sheet for the total number of viable cells recovered from each lot of ATCC Primary Human
Melanocytes.
2. Using the total number of viable cells, determine how much surface area can be inoculated to
achieve an initial seeding density of 5,000 cells per cm
2
.
3. Prepare the desired combination of flasks. Add 5 mL of complete growth medium per 25 cm
2
of
surface area. Place the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-
equilibrate to temperature and pH for 30 minutes prior to adding cells.
4. While the culture flasks equilibrate, remove one vial of ATCC Primary Human Melanocytes from
storage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility of
contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately
1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of flasks
to be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1.0 ml of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C, 5% CO
2
atmosphere. Incubate for at least
24 hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
multiple times.
www.atcc.org page 24
Melanocytes
www.atcc.org page 24
2. 24 to 36 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth medium per 25 cm
2
of surface area and return
the flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular
confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have
reached 80% to 90% confluence, and are actively proliferating (many mitotic gures are visible),
it is time to subculture. Melanocytes are not contact inhibited; however, they will proliferate best
if the cells are passaged prior to 100% confluence. Melanocytes from lightly pigmented tissue
should reach 80% confluence in 7 to 9 days.
Subculture
1. Passage normal melanocytes when the culture has reached approximately 80% to 90% confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing
Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete
growth medium to 37C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away
from each other and round up (typically within 1 to 3 minutes),
remove the flask from the microscope and gently tap it from
several sides to promote detachment of the cells from the flask
surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin
Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to
ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the culture flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that
might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL
fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 2,500 to 5,000 cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
Note:
Melanocytes are sensitive to over-
trypsinization.
Email [email protected] Phone 800.638.6597 page 25
Human Mesenchymal Stem Cells
page 25 Email [email protected] Phone 800.638.6597 Email [email protected] Phone 800.638.6597 page 25
ATCC HUMAN MESENCHYMAL STEM CELL AND
DIFFERENTIATION SOLUTIONS
Introduction
ATCC Human Mesenchymal Stem Cells (MSCs) are self-renewing
multipotent adult stem cells. MSCs are traditionally found in the
bone marrow, but can also be isolated from other tissues including
adipose, human umbilical cord or cord blood, and peripheral
blood. Adipose-derived mesenchymal stem cells are isolated from
human adipose (fat) tissues by lipoaspiration or biopsy. Umbilical
cord derived mesenchymal stem cells are isolated from Whartons
Jelly found in human umbilical cord. Although MSCs from primary
adipose tissue and Whartons Jelly are considered to be relatively
easy to obtain, isolation of consistent stem cell populations from
such material is costly and time-consuming. Lipoaspirates and umbilical cord tissue represent a
heterogeneous mixture of cell types, including adipocytes, endothelial cells, smooth muscle, pericytes
and progenitor cells
2
. The inherent heterogeneity of this source material, then, yields a high potential
for cellular contamination to predominate cultures.
To ensure the purity of ATCC Primary Cell Solutions Normal Human Mesenchymal Stem Cells, each
batch is isolated from single-donor tissue, cryopreserved in the second passage, and tested for:
1. Authentication of growth and morphology, including adherence to plastic when cultured in
optimized growth medium consisting of Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-
030) supplemented with Mesenchymal Stem Cell Growth Kit-Low Serum (ATCC PCS-500-040)
2. Verication of surface antigen expression
13
, including a total of 10 markers: Positive ( 95%) for
CD29, CD44, CD73, CD90, CD105, and CD166; and, negative ( 2%) for CD14, CD31, CD34, and
CD45
3. Conrmation of multi-lineage dierentiation into osteoblasts, adipocytes, and chondrocytes
using optimized dierentiation kits and protocols
MSCs are useful tools for non-controversial stem cell dierentiation research and for the creation of
induced pluripotent stem (iPS) cell lines
8
. MSCs have also found applications in tissue engineering,
3,14,5,11
,
cell therapy
12
, and regenerative medicine
6,7,9,10
.
Adipose-Derived Mesenchymal Stem
Cells, Normal, Human (ATCC PCS-500-
011).

0%
20%
40%
60%
80%
100%
120%
CD29 CD44 CD73 CD90 CD105 CD166 CD14 CD31 CD34 CD45
Surface Antigen
P
e
r
c
e
n
t

C
e
l
l
s

P
o
s
i
t
i
v
e
ATCC Primary Cell Solutions adipose-derived
mesenchymal stem cells were taken from
liquid nitrogen and cultures initiated. A
sample for analysis by flow cytometry was
taken when the culture was initiated and
then after 48-hours of growth. The cells must
test positive for CD29, CD44, CD73, CD90,
CD105, and CD166 (greater than 95% of the
cell population expresses these markers by
flow cytometry). The cells must test negative
for CD14, CD31, CD34, and CD45 (less than
2% of cell population expresses these
markers by flow cytometry).
Fresh from Cryo 48 Hours Growth
www.atcc.org page 26
Human Mesenchymal Stem Cells
www.atcc.org page 26
Cell Culture Protocols
Materials Needed
Primary cells and complete growth medium
Mesenchymal Stem Cells Growth Kit Options Basal Medium
Adipose-Derived Mesenchymal Stem Cells,
Normal, Human (ATCC PCS-500-011)
Mesenchymal Stem Cell
Growth Kit-Low Serum(ATCC
PCS-500-040)
Mesenchymal Stem Cell
Basal Medium (ATCC PCS-
500-030)
Umbilical Cord-Derived Mesenchymal Stem
Cells; Normal, Human (ATCC PCS-500-010)
Reagents for Subculture
D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003)
Trypsin Neutralizing Solution (ATCC PCS-999-004)
Preparation of Complete Growth Media
1. Obtain one Mesenchymal Stem Cell Growth KitLow Serum from the freezer; make sure that the
caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium.
3. Obtain one bottle of Mesenchymal Stem Cell Basal Medium from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium
bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the
indicated volume of each growth kit component, as indicated in the following table, to the bottle
of basal medium using a separate sterile pipette for each transfer.
Mesenchymal Stem Cell Growth KitLow Serum (ATCC PCS-500-040)
Component Volume Final Concentration
MSC Supplement 10 mL
2% FBS
5 ng/mL rh FGF basic
5 ng/mL rh FGF acidic
5 ng/mL rh EGF
L-Alanyl-L-Glutamine 6 mL 2.4 mM
6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a
homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored
under these conditions, complete growth media is stable for two weeks.
Handling Procedure for Frozen Cells and Initiation of Cultures
1. Refer to the batch specic information provided on the last page of the product information
sheet for the total number of viable cells recovered from each lot of ATCC Human Mesenchymal
Stem Cells.
2. Using the total number of viable cells, determine how much surface area can be inoculated to
achieve an initial seeding density of 5,000 cells per cm
2
.
3. Prepare the desired combination of flasks. Add 5 mL of complete growth medium per 25 cm
2
of
Email [email protected] Phone 800.638.6597 page 27
Human Mesenchymal Stem Cells
page 27 Email [email protected] Phone 800.638.6597 Email [email protected] Phone 800.638.6597 page 27
surface area. Place the flasks in a 37C, 5% CO
2
, humidied incubator and allow the media to pre-
equilibrate to temperature and pH for 30 minutes prior to adding cells.
4. While the culture flasks equilibrate, remove one vial of ATCC Human Mesenchymal Stem Cells from
storage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility of
contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately
1 to 2 minutes).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All operations from this point onward should be carried
out under strict aseptic conditions.
6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of flasks
to be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from
the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not
centrifuge.
7. Transfer 1.0 ml of the cell suspension to each of the pre-equilibrated culture flasks prepared in
steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times,
then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37C, 5% CO
2
atmosphere. Incubate for at least
24 hours before processing the cells further.
Maintenance
1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between
10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),
warm only the volume needed in a sterile conical tube. Avoid warming complete growth media
multiple times.
2. 24 hours after seeding, remove the cells from the incubator and view each flask under the
microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth medium per 25
cm
2
of surface area and return the flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to
determine percent cellular confluence. If not ready to passage,
repeat steps 3 and 4 as described above. When cultures have
reached approximately 70% to 80% confluence, and are actively
proliferating (many mitotic gures are visible), it is time to
subculture.
Subculture
1. Passage normal mesenchymal stem cells when the culture has reached approximately 70% to
80% confluence.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing
Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete
growth medium to 37C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm
2
) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells,
Note:
Adipose- and umbilical cord-
derived stem cells are contact
inhibited. It is essential that the
cells be subcultured BEFORE
reaching confluence as post-
confluent cells exhibit changes in
morphology, slower proliferation,
and reduced di fferenti ati on
capacity after passaging.
www.atcc.org page 28 www.atcc.org page 28
Human Mesenchymal Stem Cell Dierentiation
and then aspirate the excess fluid o of the monolayer.
7. Observe the cells under the microscope. When the cells pull away from each other and round up
(typically within 1 to 3 minutes), remove the flask from the microscope and gently tap it from
several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin
Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to
ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any
remaining cells in the culture flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that
might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-
dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL
fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm
2
.
16. Place newly seeded flasks in a 37C, 5% CO
2
incubator for at least 24 to 48 hours before processing
the cells further. Refer to Maintenance for guidelines on feeding.
Adipose-derived Mesenchymal Stem Cell Dierentiation Protocols
Adipocyte Dierentiation
Materials Needed
The Adipocyte Dierentiation Toolkit (ATCC PCS-500-050) contains medium and reagents designed
to induce adipogenesis in actively proliferating Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-
500-011) with high eciency, and support maturation of derived adipocytes during lipid accumulation.
Preparing Cells for Adipocyte Dierentiation
1. Follow the instructions for the growth of Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-
500-011). It is recommended that the cells not be passaged more than four (4) times before
initiating adipocyte dierentiation.
2. When cells are 70-80% confluent, passage them into a tissue culture plate at a density of 18,000
cells/cm
2
. Adjust the number of cells and volume of media according to the tissue culture plate
used.
Example: For a 6-well tissue culture plate with a surface area of 9.5 cm
2
/well, add a total of
171,000 viable cells to each well containing 2 mL of Mesenchymal Stem Cell Basal Medium (ATCC
PCS-500-030) supplemented with Mesenchymal Stem Cell Growth KitLow Serum (ATCC PCS-
500-040) components.
3. Gently rock the plate back and forth and side to side to evenly distribute cells before incubation.
Do not swirl.
4. Incubate the cells at 37C with 5% CO
2
for 48 hours before initiating adipocyte dierentiation.
Adipocyte Dierentiation Media Preparation
The adipocyte dierentiation process requires two separate media preparations: one for initiation and
Email [email protected] Phone 800.638.6597 page 29 page 29 Email [email protected] Phone 800.638.6597 Email [email protected] Phone 800.638.6597 page 29
Human Mesenchymal Stem Cell Dierentiation
one for maintenance. Stock solutions of these media can be prepared in tandem in advance as follows:
1. Thaw all three components of the dierentiation kit and warm to
37C in a water bath.
2. Decontaminate the external surfaces of all three kit components
by spraying them with 70% ethanol.
3. Using aseptic technique and working in a laminar flow hood or
biosafety cabinet:
a. Transfer 15 mL of Adipocyte Basal Medium and 1 mL of AD Supplement to a sterile 50 mL
conical tube, using a separate sterile pipette for each transfer. This is your working stock of
Adipocyte Dierentiation Initiation Medium used during the rst 96 hours of dierentiation.
b. Add 5 mL of ADM Supplement to the remaining 85 mL of Adipocyte Basal Medium. This is
your working stock of Adipocyte Dierentiation Maintenance Medium.
4. Tightly cap the each container of media and swirl the contents gently to assure a homogeneous
solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
5. Each container of dierentiation medium should be stored in the dark at 2C to 8C (do not
freeze). When stored under these conditions, the dierentiation media is stable for up to three
weeks.
Adipocyte Dierentiation Procedure
A. Initiation Phase
1. After incubating the prepared Adipose-Derived Mesenchymal Stem Cells for (as described
above), carefully aspirate the media from the wells.
2. Immediately rinse the cells once by adding 2 mL of room-temperature D-PBS (ATCC 30-
2200) to each well, then carefully aspirate the PBS from the wells.
3. Add 2 mL of pre-warmed (37C) Adipocyte Dierentiation
Initiation Medium to each well to begin the adipocyte
dierentiation process.
4. Incubate the cells at 37C with 5% CO
2
for 48 hours.
5. Feed the cells by carefully removing half the volume of media
(1 mL) from each well and adding another 2 mL of pre-warmed
(37C) Adipocyte Dierentiation Initiation Medium to each
well.
Important: DO NOT TILT plate during aspiration. It is important that the cell monolayer is not
exposed to air during this and subsequent steps to ensure that developing lipid vesicles do
not burst.
B. Maintenance Phase
6. Incubate the cells at 37C with 5% CO
2
for 48 hours.
7. Carefully remove 2 mL of media from each well (leaving
1 mL) and replace with 2 mL of pre-warmed (37C)
Adipocyte Dierentiation Maintenance Medium in each
well.
Important: DO NOT TILT plate during aspiration. It is
important that the cell monolayer is not exposed to
air during this and subsequent steps to ensure that
Adipocytes dierentiated from Adipose-
derived Mesenchymal Stem Cells (PCS-
500-011) and stained with Oil Red O to
detect the formation of lipid droplets.
Note:
It is recommended that you
transfer the required volume of
media to a sterile tube for pre-
warming prior to each feeding
rather than repeatedly re-warming
the entire working stock.
Note:
It may be necessary to shake the
AD Supplement and the ADM
Supplement upon warming to
help re-dissolve any components
that may have precipitated out of
solution upon freezing.
www.atcc.org page 30 www.atcc.org page 30
Human Mesenchymal Stem Cell Dierentiation
developing lipid vesicles do not burst.
8. Repeat Steps 6 and 7 every 3-4 days for another 11 days until adipocytes reach full maturity.
(Full maturity will be reached 15 days after the beginning of initiation phase, or 17 days from
initial plating of cells.)
9. Cells can be used at any phase of adipocyte dierentiation as predicated upon experimental
design. To conrm lipid accumulation, cells can be xed and stained with Oil Red O.
Additional differentiation protocols, including
Chondrocyte and Osteocyte differentiation
of Adipose-derived Mesenchymal Stem Cells,
are available online at www.atcc.org
ATCC Adipose-Derived Mesenchymal
Stem Cells were expanded in Complete
Growth Medium and then induced to
dierentiate to chondrocytes using
the Chondrocyte Dierentiation Tool.
Passage 3 cells, day 21 following
dierentiation (100X magnication),
stained with Alcian Blue.
Email [email protected] Phone 800.638.6597 page 31
REFERENCES
1. Stein GS, et. al. Human Stem Cell Technology and Biology: A Research Guide and Laboratory
Manual. Wiley & Sons, Hoboken, New Jersey, 2011.
2. Zuk PA, et al. Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies.
Tissue Eng. 7(2):211-228), 2001.
3. Zuk PA, et al. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell. 13:4279-
4295, 2002.
4. Ogawa R. The Importance of Adipose-Derived Stem Cell and Vascularized Tissue Regeneration in
the Field of Tissue Transplantation. Cur. Stem Cell Res. 1:13-20, 2006.
5. Shaefler A & Buechler C. Concise review: adipose tissue-derived stromal cellsbasic and clinical
applications for novel cell-based therapies. Stem Cells 25:818-827, 2007.
6. Kang SK, et al. Neurogenesis of Rhesus adipose stromal cells. J. Cell Sci. 117:4289-4299, 2004.
7. Saord KM, et al. Stem cell therepy for neurologic disorders: Therapeutic potential of adipose-
derived stem cells. Current Drug Targets 6:57-62, 2005.
8. Saord KM, et al. Characterization of neuronal/glial dierentiation of murine adipose-derived
adult stromal cells. Exp. Neurol. 187:319-328, 2004.
9. Kingham PJ, et al. Adipose-derived stem cells dierentiate into a Schwann cell phenotype and
promote neurite outgrowth in vitro. Exp. Neurol. 207:267-274, 2007.
10. Xu, et al. Myelin-forming ability of Schwann cell-like cells induced from rat adipose-derived stem
cells in vitro. Brain Res. 1239:49-55, 2008.
11. Seo MJ, et al. Dierentiation of human adipose stromal cells into hepatic lineage in vitro and in
vivo. Biochem. Biophys. Res. Commun. 328:258-264, 2005.
12. Timper K, et al. Human adipose tissue-derived mesenchymal stem cells dierentiate into insulin,
somatostatin, and glucagon expressing cells. Biochem. Biophys. Res. Commun. 341:1135-1140,
2006.
13. Dominici M, et al. Minimal criteria for dening multipotent mesenchymal stromal cells: The
International Society for Cellular Therapy position statement. Cytotherapy 8(4):315-317, 2006.
14. Freshney, RI. Culture of Animal Cells: A Manual of Basic Technique, 6th Ed. John Wiley & Sons,
Hoboken, New Jersey, 2010.
15. Histology Text Atlas Book. Chapter 3: Connective Tissue. Downloaded from Visual Histology.com
at http://www.visualhistology.com/products/atlas/VHA_Chpt3_Connective_Tissue.html on March
26, 2012.
16. Elias JA, et. al. Regulation of Human Lung Fibroblast Glycosaminoglycan Production by Recombinant
Interferons, Tumor Necrosis Factor, and Lymphotoxin. J. Clin. Invest. 81:325-333, 1988.
17. Rieger MM. Keratinocyte function and skin health. Cosmetics and Toiletries, July 01, 1992.
Downloaded from the WWW on March 26, 2012 from Access My Library at http://www.
accessmylibrary.com/article/print/1G1-12484573.
18. Bensouilah J, et. al. Aromadermatology: Aromatherapy in the Treatment and Care of Common Skin
Conditions. Blackwell Publishers, 2006. Chapter 1 downloaded from the WWW on March 26, 2012
at http://courses.washington.edu/bioen327/Labs/Lit_SkinStruct_Bensouillah_Ch01.pdf.
Have Questions?
Search ATCC Frequently Asked Questions online at www.atcc.org for answers related to primary cells,
or contact ATCC Technical Services at [email protected] for additional information.
Cell
Type
Product Name ATCC No. Species
Number of viable
cells-post thaw
Passage at
freezing
Cells tested upon
thaw to achieve
Basal media Growth kit Reagents and supplements Additional Reagents Applications
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Umbilical Vein Endothelial Cells;
Normal, Human
PCS-100-010 Human 5 x 10
5
1 15 PDL
Vascular Cell Basal
Medium (ATCC PCS-
100-030)
Endothelial Cell Growth
Kit-BBE (ATCC PCS-100-040)
or Endothelial Cell Growth Kit-
VEGF (ATCC PCS-100-041)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
NA
Physiological and pharmacological investigations, such as
macromolecule transport, blood coagulation, angiogenesis,
and fbrinolysis.
Umbilical Vein Endothelial Cells;
Normal, Human, Pooled
PCS-100-013 Human 5 x 10
5
2 15 PDL
Aortic Endothelial Cells; Normal,
Human
PCS-100-011 Human 5 x 10
5
2 15 PDL
Studies of vascular diseases such as thrombosis,
atherosclerosis, metabolism, and hypertension; stent-graft
compatibility testing; membrane conductance models.
Coronary Artery Endothelial Cells;
Normal, Human
PCS-100-020 Human 5 x 10
5
3 15 PDL
Pulmonary Artery Endothelial Cells;
Normal, Human
PCS-100-022 Human 5 x 10
5
3 15 PDL
Dermal Microvascular Endothelial
Cells; Normal, Human, Neonatal
PCS-110-010 Human 5 x 10
5
3 15 PDL
Vascular Cell Basal
Medium (ATCC PCS-
100-030)
Microvascular Endothelial
Cell Growth Kit-BBE (ATCC
PCS-110-040) or Microvascular
Endothelial Cell Growth Kit-
VEGF (ATCC PCS-110-041)
Studies of microvascular functions and cutaneous
infammation
S
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Aortic Smooth Muscle Cells, Normal,
Human
PCS-100-012 Human 5 x 10
5
2 15 PDL
Vascular Cell Basal
Medium (ATCC PCS-
100-030)
Vascular Smooth Muscle
Cell Growth Kit (ATCC PCS-
100-042)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
NA
Studies of vascular diseases such as thrombosis, and
atherosclerosis
Coronary Artery Smooth Muscle Cells,
Normal, Human
PCS-100-021 Human 5 x 10
5
2 15 PDL
Pulmonary Artery Smooth Muscle
Cells, Normal, Human
PCS-100-023 Human 5 x 10
5
2 15 PDL
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Small Airway Epithelial Cells; Normal,
Human
PCS-301-010 Human 5 x 10
5
1 15 PDL
Airway Epithelial Cell
Basal Medium (ATCC
PCS-300-030)
Small Airway Epithelial Cell
Growth Kit (ATCC PCS-301-
040) or Bronchial Epithelial
Cell Growth kit (ATCC PCS-
300-040)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
NA
Asthma, airway infammation, and would healing,
pulmonary fbrosis, COPD, Cancer, Toxicology, intracellular
pH regulations, IL-1b efect to stimulate airway epithelial
cell growth, ICAM-1 expression.
Bronchial/Tracheal Epithelial Cells;
Normal, Human
PCS-300-010 Human 5 x 10
5
1 15 PDL
Renal Proximal Tubule Epithelial Cells;
Normal, Human
PCS-400-010 Human 5 x 10
5
2 15 PDL
Renal Epithelial Cell
Basal Medium (ATCC
PCS-400-030)
Renal Epithelial Cell Growth Kit
(ATCC PCS-400-040)
In vitro studies of osmoregualtion and excretion, renal
fbrosis, infammation, as well as applications in drug
screening/development, e.g. hypertension, diabetes,
oncology, autoimmune disease, and toxicology screening.
Renal Cortical Epithelial Cells; Normal,
Human
PCS-400-011 Human 5 x 10
5
1 15 PDL
Renal Mixed Epithelial Cells; Normal,
Human
PCS-400-012 Human 5 x 10
5
1 15 PDL
Prostate Epithelial Cells; Normal,
Human
PCS-440-010 Human 5 x 10
5
2 15 PDL
Prostate Epithelial
Cell Basal Medium
(ATCC PCS-440-030)
Prostate Epithelial Cell Growth
Kit (ATCC PCS-440-040)
Hormonal regulation of the prostate, the secretory function
of prostate cells, and prostate cancer
Corneal Epithelial Cells; Normal,
Human
PCS-700-010 Human 5 x 10
5
2 3 passages
Corneal Epithelial
Cell Basal Medium
(ATCC PCS-700-030)
Corneal Epithelial Cell Growth
Kit (ATCC PCS-700-040)
Cell de-diferentiation, toxicology testing and drug
development.
F
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Dermal Fibroblasts, Normal, Human
Neonatal
PCS-201-010 Human 5 x 10
5
1
10 PDL in
serum-free
medium
Fibroblast Basal
Medium (ATCC PCS-
201-030)
Fibroblast Growth KitSerum-
Free (ATCC PCS-201-040) or
Fibroblast Growth KitLow
Serum (ATCC PCS-201-041)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
0.1%Gelatin Solution (ATCC
PCS-999-027) only for use
with Mitomicin C treated
Dermal Fibroblasts
Wound healing studies, tissue engineering and
regeneration applications, as well as induction of
pluripotent stem (iPSCs).
Dermal Fibroblasts, Normal, Human
Neonatal, Mitomicin C treated
PCS-201-011 Human 3 x 10
6
2
No growth or
divison beyond 4
weeks
Feeder cells for use with human stem cells and
keratinocytes
Dermal Fibroblasts, Normal, Human
Adult
PCS-201-012 Human 5 x 10
5
1
10 PDL in
serum-free
medium
Wound healing studies, tissue engineering and
regeneration applications, as well as induction of
pluripotent stem (iPSCs).
K
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Epidermal Keratinocytes, Normal,
Human, Neonatal Foreskin
PCS-200-010 Human 5 x 10
5
1 15 PDL
Dermal Cell Basal
Medium (ATCC PCS-
200-030)
Keratinocyte Growth Kit (ATCC
PCS-200-040)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
NA
Studies of growth factor activity, wound healing, toxicity/
irritancy studies, and use as target cells for derivation of
induced pluripotent stem cells
Epidermal Keratinocytes, Normal,
Human, Adult
PCS-200-011 Human 5 x 10
5
1 15 PDL
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Epidermal Melanocytes, Normal,
Human, Neonatal Foreskin
PCS-200-012 Human 5 x 10
5
2 15 PDL
Dermal Cell Basal
Medium (ATCC PCS-
200-030)
Melanocyte Growth Kit (ATCC
PCS-200-041)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
NA
Wound healing, testing models for toxicity/irritancy studies,
melanoma, dermal response to UV radiation, psoriasis and
other skin diseases, and cosmetic research Epidermal Melanocytes, Normal,
Human, Adult
PCS-200-013 Human 5 x 10
5
2 15 PDL
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Umbilical Cord-Derived Mesenchymal
Stem Cells, Normal, Human
PCS-500-010 Human 5 x 10
5
2 10 PDL
Mesenchymal Stem
Cell Basal Medium
(ATCC PCS-500-030)
Mesenchymal Stem Cell
Growth Kit-Low Serum (ATCC
PCS-500-040)
Phenol Red (ATCC PCS-999-001); D-PBS
(ATCC 30-2200); Trypsin-EDTA for Primary
Cells (ATCC PCS-999-003); Trypsin
Neutralizing Solution (ATCC PCS-999-004)
Adipocyte Diferentiation Tool
(ATCC PCS-500-050)
Adult stem cell diferentiation research, induced pluripotent
stem cell lines, tissue engineering, cell therapy, and
regenerative medicine
Chondrocyte Diferentiation
Tool (ATCC PCS-500-051)
Adipose-Derived Mesenchymal Stem
Cells, Normal, Human
PCS-500-011 Human 1 x 10
6
2 10 PDL
Osteocyte Diferentiation Tool
(ATCC PCS-500-052)
Tel
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PC-0312-01
2012 American Type Culture Collection.
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