Cell culture

By
HARIDHAR K
1ST M.PHARM
PHARMACEUTICAL ANALYSIS
• Equipmets used in cell culture lab

Essential  equipments Beneficial equipments Useful additional equipments

Incubator Laminar flow hood Low-temperature freezer

Microscope Cell counter Glassware washing machine
Sterilizer Vacuum Pump Colony counter
Washing up instrument CO2 incubator Closed-circuit machine
Sterilizing and drying oven Preparation and quality control Cell sizing
Centrifuge Temperature recording Time-lapse
Water purification Bulk culture Controlled-Rate cooler
Pipette aids  and automatic
Cell freezing Cinemicrography  
pipetting
    Centrifugal elutriator
Fluorescence activated Cell
   
sorter
• 1. Sterile work area required for cell culture:
• A HEPA filtered air is appropriate but is not economical.
• A laminar flow hood is supposed to the simplest and the most cost effective way
to supply aseptic conditions
• In order to meet the diversified research and clinical needs, Three kinds of laminar
flow hoods, have been designated as Class I, II and III.
• When used with proper microbiological techniques, Class I laminar flow hoods
supplies essential levels of protection to laboratory workers and to the
environment, but they do not protect cultures  from contamination. 
•  Class II laminar flow hoods are designed and they also allow an aseptic
environment essential for cell culture experiments.
•  Class III biosafety cabinets are gas-tight, and they supply the highest achievable
level of protection to personnel and the environment. 
• 2. Incubator:
• An incubator will be needed in order to supply the suitable
temperature environment for cell growth at 30-400 C
• Dry incubators are relatively cost-effective, but the cell cultures are
needed to be incubated in sealed flasks to avoid evaporation.
• In a dry incubator, if the water dish is placed, it can supply some
humidity however, they do not provide appropriate control of
atmospheric conditions in the incubator
• They can be used to incubate cells that are cultured
in petri-dishes or multiwell plates that needs a
regulated atmosphere of high humidity and
increased CO2 tension
•  Refrigerators and freezer for specimen storage:
• Both refrigerators and freezer are very essential for storage of liquid
media at 2–8°C and for enzymes (e.g. trypsin) and some media
components (e.g., glutamine and serum) at –5°C to –20°C.
• A freezer is required for keeping pre-aliquoted stocks of serum,
nutrients and antibiotics. 
• Cryogenic Storage
• There is high possibility for genetic instability in cell lines of
continuous culture as their passage number increases, hence, it is
necessary to prepare working stocks of the cells and preserve in
cryogenic storage
• The possibility of leaky vials or ampules exploding during removal is
highly reduced by use of vapor phase storage
•  Microscopes:
• In order to examine the cultures in flasks and dishes, a simple inverted
microscope is needed.
• The morphological changes in cultures should be recognized as they
are the first indicators for the identification of deterioration of a
culture.
• Although, a microscope of very high quality will be needed for
chromosome analysis or autoradiography work, a very simple light
microscope with X100 magnification will
suffice for routine cell counts in a hemocytometer.
• Reverse osmosis apparatus:
• For preparation of media, and rinsing glassware, a double distilled or
reverse osmosis water supply is required.
• The pH of the double distilled water should be checked regularly, as
this can vary in some instances.
• Variations in the quality of water used may account for variations in
outcomes, so it is necessary to use water from one source.
• Water is sterilized for 20 minutes at 121 °C by autoclaving.
• Centrifuge
• Periodically, to increase the concentration of cells or to wash off a
reagent, cell suspensions require centrifugation.
• For most purposes, a small bench-top centrifuge, preferably with
proportionally controlled braking, is enough.
• When experiments are concluded, centrifugation is often used
to support sample characterization and analysis.
• At 80 to 100 g, cells sediment satisfactorily;
higher g may cause damage and encourage
pellet agglutination.
• Media used in cell culture

• Artificial media-
• That contains all fully defined media components. so also called defined media.
the media should be maintained to physiological pH at 7 .also should be sterilized
before use.
• Natural media-
• In this include body fluid like serum, plasma, amniotic fluid, etc. but before
required to test toxicity. in this also include tissue extracts like liver, spleen etc.
• Serum-free media- the main advantage of this media id can control to growth by
changes in the composition of media. that also has thy factory witch help in cell
differentiation.
• Components-
• A culture media has many constituents in the different amount which
are essential to the growth of cells.
• Natural buffer- HEPES (chemical buffer) and phenol red as pH
indicator.
• Inorganic salts- help in balance to the osmotic pressure of cells, like
potassium, calcium etc.
• Amino acids– included for synthesizing proteins by cells.
• Carbohydrates– work as an energy source like glucose.
• Vitamins- for the proliferation and growth of cultured cells.
• Antibiotics– to avoid cells by infection through microbes like bacteria
and viruses.
• ANIMAL CELL CULTURE MEDIA COMPOSITION
• Cell culture is a great process to culture to animal cells but for this media is
an important step in the process. all type of media composes of many
essential components which used by cells for growth proliferation and
differentiate during culture. media constitutes are-
• Carbohydrates
• Vitamins
• Inorganic salt
• Amino acids
• Antibiotics
• Trace elements
• Proteins and peptides
• Buffers (control pH)
• Carbohydrates and Vitamins- Carbohydrate is the natural energy
source for media is used in form of glucose, galactose mostly. vitamins
in media are essential for cell proliferation and differentiation and not
be synthesized by cells during culture.
• Inorganic Salt and Amino Acids- Inorganic salts help to retain the
osmotic balance of cells for this used potassium, and calcium ions in
media. and amino acids they include for proteins synthesis by cells
because of cell unable to synthesize them mostly L- glutamine used in
media.
• Antibiotics and Trace Elements- antibiotics used to avoid to culture
from microbial contaminants, like viruses, fungal, bacterial. etc. trace
elements used mostly for serum-free media in this include copper,
selenium, zinc etc
• Proteins and Peptides- most proteins and peptide-like transferrin,
fibronectin, and albumin used in cell culture media.
• Buffers- Natural buffer, HEPES, phenol red comes under this. witch
balanced to gaseous content of the medium. and some for regulate pH
level, phenol redwork as pH indicator in the medium.
• Preparation of Medium– For this, all components should be in the
appropriate quantity and may be sterilized. and prepared liquid
medium by using sterilized water
• Enriched media – It is created when a basic medium is supplemented with
nutrients like eggs, blood or serum. For example, a blood agar medium is used for
the growth of bacteria like Streptococcus which specifically requires blood for its
proliferation.
• Selective media – Selective media contain components that prevent the growth of
all but a small number of bacterial species and make it easier to isolate a specific
species. When mixed bacterial flora is anticipated in specimens, these media are
utilised to isolate specific bacteria from those specimens. For example, bile salt
acts as a selective agent in BSA or bile salt agar. While preventing the growth of
other intestinal organisms, it favours the growth of Vibrio cholerae.
• Enrichment media – This media contains several ingredients that either stimulate
the bacteria being grown or suppress their competitors. Examples – Alkaline
peptone water and tetrathionate broth.
• Transport media – These are employed when dealing with delicate organisms
that might not make it through the transit period or might become covered with
non-pathogenic germs. Special media are developed for the transportation of such
bacteria to laboratories and these are known as transport media. Example –
Stuart’s transport medium.
• Indicator media – When bacteria multiply in these media containing an indicator,
they tend to change their colour. MacConkey’s medium is also an example of an
indicator medium. Another classic example is the black colonies of Salmonella
typhi that develop on sulphite-containing Wilson and Blair media.
• Differential media – It is a term used to describe a medium that has components
that aid in identifying the various properties of bacteria. Peptone, agar, lactose,
neutral red and sodium taurocholate are all ingredients in MacConkey’s medium.
Here, the colonies made by lactose fermenters are pink, but those made by non-
lactose fermenters are pale or colourless.
• Sugar media – It contains 1% sugar, which can be any fermentable substance like
glucose, mannitol, sucrose and lactose. The generation of acid following the
fermentation of sugar transforms the medium into pink due to the presence of an
indicator. Also to show that gas is produced, Durham’s tube is kept inverted inside
the sugar tube and gas bubbles are observed.