Editorial
Single-Cell Analysis
Tuhin Subhra Santra 1,* and Fan-Gang Tseng 2
1 Department of Engineering Design, Indian Institute of Technology Madras, Tamil Nadu 600036, India
2 Department of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan;
[email protected]
* Correspondence: [email protected] or [email protected]; Tel.: +91-044-2257-4747
Received: 24 August 2020; Accepted: 25 August 2020; Published: 29 August 2020;
Cells are known to be the most fundamental building block of life. Cells form our bodies grow,
reproduce, process information, and they can respond to the stimuli and process different chemical
reactions [1]. However, to date, the interactions between cells, the responses of their organelles to
molecules and intracellular behaviour remain unknown. For the understanding of cellular
physiological interactions with different molecules and organelles, an average ensemble
measurement of millions of cells together is not enough to provide detailed information about cells
[2–4]. Again, bulk analysis of millions of cells cannot explore cellular heterogeneity characteristics
and dynamics at the molecular level, in a particular cell populations [2–4]. However, the
investigations on the progress of any disease have remained challenging because of the physiological
states of cells and heterogeneity nature of cells in a specific given population [2–5]. Compared to
the average analysis of millions of cells together in bulk, single-cell analysis (SCA), provides clear
information about each cell, such as specific biological factors of cell, which is helpful to understand
stem cells behaviour or tumour progression [2–8]. Moreover, SCA analysis can characterize in detail
the molecular contents of cells, related to cell state and type, spatial and temporal transformations,
and micro-environment. Single-cell omics such as genomics, proteomics, transcriptomics,
metabolomics/fluxomics can promote unrevealed information in regards to the functional mutation
and cells copy number variations [2,4,9–11]. All of these omics analyses may involve mathematical
and computational modelling to investigate cellular functions ranging from the complete genome to
living organisms. Single-cell genomics and gene expression profiling are used widely in medicine for
optimizing several biological, biomedical, and pathological conditions for various disease analyses
[2,9–11]. The single-cell proteomics and transcriptomics analyses play a significant role in the study
of cellular heterogeneity characteristics, complex diseases such as cancer, and evolve the dynamics
of differentiation and quantifying transcriptional stochasticity [2,4,10]. Moreover, these analyses help
us to study some biological phenomena such as tissue regeneration, immune response and embryonic
development. Single-cell RNA sequencing (scRNA-seq) can unlock the mystery of gene expression
and track the trajectories of distinct cell lineages in development. Single-cell metabolomics can
elucidate in detail about the phenotypical variations of cells [11]. Understanding of single-cell
metabolic reactions across organelles helps us to identify any disease pathologies. It also can
provide holistic and comprehensive insights at the cellular or even subcellular level to understand
the cellular organism responses, under different pathophysiological conditions. Furthermore single-
cell analysis helps us to understand cellular organelle responses to the environment and intracellular
interactions at a more profound level than bulk analysis, which would accelerate the developments
of therapeutics and diagnostic processes [2–4]. Due to the tremendous applications and prospects of
single-cell analysis, the research activity and scientific publications are increasing day by day. Figure
1 shows the worldwide single-cell related scientific article publications and their various applications,
Cells 2020, 9, 1993; doi:10.3390/cells9091993 www.mdpi.com/journal/cells
Cells 2020, 9, 1993 2 of 7
respectively. From this figure, it is clear that the research activity increases 3 to 4-fold in the last five
years compared to previous years.
2500
Web of science
Number of Publications
2000
1500
1000
500
0
2000 2005 2010 2015 2020
Year
Figure 1 Year wise single-cell related publications. The data are adapted from web of science. Data
until 25th August 2020.
The last two decades have seen a rapid advancement of micro and nanotechnologies and their
integration with life science, chemistry, chemical engineering, mechanical and biomedical
engineering; the new era has started based on the development of bio-microfluidics or Bio-MEMS
devices or µ-TAS (micro-total analysis systems). These devices have the ability to analyze single-cells
in the micro or nanofluidic environment. Moreover, the devices not only detect and manipulate bio-
samples in micro/nanofluidic environment but also they can consume a minimum amount of samples
for in-detail analysis [2–4,7,8,12]. The recent development of micro/nanofluidic devices can easily
detect and analyze different single-cell omics such as genomics, proteomics, metabolomics as well as
analyzing the different levels of disease progression [5]. Again, the devices have the ability to do
single-cell therapy, diagnostics[13] and their analysis by using different physical approaches such as
electroporation [14–18], mechanoporation[19], photoporation[20–23], microinjection [24,25] etc.
Besides the extensive applications for cell separation, isolation, manipulation, and lysis, these devices
are potentially helpful for studying cellular biochemical, mechanical and electrical characterization
at single-cell or in subcellular level [26]. The devices have the ability for single-cell sequencing, which
is potentially useful to study cellular heterogeneity, molecular mapping, immune infiltration and
epigenetic changes. Using these micro/nanofluidic technologies, the development of fluorescence-
activated cell sorting (FACS) will potentially act as a rapid, reliable, and sensitive immunodiagnostic
method in the clinical laboratory. It can analyze the content of the fluorophore and biomarkers at the
single-cell or even subcellular level and sort them based on the desired phenotype. Nowadays, the
multiplex biomarker analysis is of great interest using FACS, due to its isolation and identification
capabilities of immune cells to a specific phenotype.
This Special Issue of Cells entitled “Single-Cell Analysis” covers the recent advancements of
single-cell analysis using different approaches. Chio et al. [27] illustrated single-cell RNA sequencing
(sc-RNA-seq) in combination with protein and DNA analysis. The proposed techniques help us to
understand more details about cellular heterogeneity and biological information from a single-cell,
which is unobtainable from bulk analysis. The authors reviewed the recent advancement of scRNA-
seq profiles integrated along with proteomic or genomic data, which can reveal far better insights
into the complex cellular heterogeneity characteristics than transcriptome measurements alone. They
also briefly discussed scRNA-seq technology, together with conventional as well as advanced
microfluidic approaches with their advantages, limitations, future prospects and potential biological
and biomedical applications. The single nucleus RNA sequencing (snRNA-seq) of the whole
Cells 2020, 9, 1993 3 of 7
mammalian heart was emphasized by Wolfien et al. [28], which allows us to explore cellular
composition and cell features. The snRNA-seq quantification and clustering can convey precise cell
types affirmation. Their study revealed 24 individual clusters with 25.3% fibroblasts, 28.8%
endothelial, and 22.8% cardiomyocytes comprising the major cell populations. Again, they performed
the RNA velocity analysis, which allows to emphasize the transcription kinetics and unlock the
details of the dynamics as well as the identified cell clusters interconnectedness. They also identified
the cardiomyocytes subgroups with distinct marker profiles. The quantification of mRNA study at
single-cell level was illustrated by Jonasson et al. [29]. This quantification study can be widely
applicable for cellular characterization, immune responses, and mapping of intra-tumoral
heterogeneity etc. They developed an easy to use, fast and flexible process for total mRNA
quantification within a single-cell, using global reverse transcription mimicked by quantitative PCR
(polymer chain reaction). Moreover, the author performed human fibroblasts cells and three sarcoma
cell line analyses, which disclosed the variations of cell type and total quantification of mRNA. The
results showed that total mRNA levels among the cells have an eight-fold difference. The proposed
method does not prescribe sequencing, although it can be effortlessly associated with global RNA
sequencing and targeted qPCR and for additional cellular analysis.
The localized high-risk prostate cancer circulating tumour cells (CTC) genomic analysis was
discussed by Rangel-Pozzo et al. [30]. The tumour heterogeneity is one of the major causes of failure
in prostate cancer prediction and prognosis. The CTC analysis can provide individual patient-specific
clinical assessment, due to unlocking the mystery of tumour-derived and germline-specific genetic
information more precisely in comparison with a single diagnostic biopsy. In their study, the authors
combined a filtration-based CTC isolation method with prostate cancer CTC immunophenotyping to
identify the prostate cancer CTCs. They found that localized high-risk prostate cancer patient CTCs
can provide a high number of telomere signals with lower signal intensities. Whole-exome
sequencing of high-risk prostate cancer CTCs was carried out to gain information about cellular
genetic diversity or heterogeneity. The authors concluded that single-cell genome sequencing (whole-
exome sequencing and 3-D telomere profiling) approaches were useful for CTC heterogeneity
characteristics in a treatment-naïve prostate cancer patient risk group and copy number alternation
amplification, which visualized in a localized high-risk patient, could be playing a major role in the
therapeutic resistance in prostate cancer. Hepatocyte cells are the primary parenchymal cells of the
liver. These cells have a significant impact on liver homeostasis and disease process. Chang et al. [31]
discuss the heterogeneity characteristics of mouse hepatocytes and their distinctive functions during
cholestatic liver injury using single-cell transcriptome analysis. They reported single-cell RNA
sequencing and mouse liver injury model induced by Bile duct ligation (BDL). For their study, the
qPCR and Western blot were used for gene expression and immunofluorescence, to detect the gene
expression of hepatocytes.The detection of distinctive hepatocyte cluster (BDL-6) expressed
extracellular matrix gene, and it might be indicating hepatocytes undergo epithelial-mesenchymal
transition. Moreover, hepatocytes of BDL-6 can perform angiogenesis during cholestatic injury as a
function of tissue repair. Again, they reported BDL-2, BDL-3, BDL-4, and BDL-5 cholestatic
hepatocytes clusters were engaged in the inflammatory process in different ways.
Liu et al. [32] proposed various methods of single-cell proteome analysis and their potential
biomedical applications. The single-cell proteome analysis can help us to understand cellular
heterogeneity characteristics, the molecular dynamics of the cell, as well as the clinical applications
for tumour treatment and drug development. The authors initially discuss the conventional protein
characterization and analysis techniques, together with enzyme-linked immunospot assay,
fluorescence flow cytometry, mass cytometry, and capillary electrophoresis. Later they discuss
single-cell protein analysis using advanced microfluidic approaches such as microfluidic fluorescent
flow cytometry, microwell-based assay (micro engraving), droplet-based microfluidics, single-cell
Western blotting, and microchamber-based assay (barcoding microchips). The advantage, limitations
and future prospects of single-cell protein analysis are also elaborated . The extremely sensitive and
multiplexed in-situ protein analysis was performed by using cleavable fluorescent streptavidin, and
cleavable biotin-conjugated antibodies were discussed by Liao et al. [33]. Moreover, the author
Cells 2020, 9, 1993 4 of 7
successfully quantifies proteins in formalin-fixed paraffin-embedded tissues. The in-situ protein
analysis facilitates the understanding of normal physiological behaviour and disease pathogenesis.
In their study, first, they recognized protein targets by the cleavable biotin-labelled antibodies and
then used cleavable fluorescent streptavidin to stain the protein targets. The layer-by-layer signal
amplification was performed using cleavable biotin-conjugated orthogonal antibodies and cleavable
fluorescent streptavidin. As a result, the protein detection sensitivity enhanced at least 10-fold in
comparison with the current available in-situ proteomic methods. This approach is applicable for the
study of in-situ DNA, RNA and metabolic analysis. Yeh et al. [34] demonstrated the generation of a
monoclonal cell line from single-cell cloning using a disposable microfluidic device. The generation
of monoclonal cell lines is an important mean to produce proteins for basic biological research and
therapeutic drug development purposes. In their approach, they reduced the development time to
validate the generation of monoclonality of the cloned cells. Most of the monoclonal cell line
generation took the longest time (approximately one month) with complex techniques. Here, the
author produced monoclonal colonies of actin-GFP plasmid-transfected A549 cells using a simple
disposable microfluidic device within nine days. Later this cell line was transferred to wells within
plates to allow further expansion by using a tissue puncher.
Single-cell genomics and proteomics in integration with three-dimensional (3D) cell culture
technique can execute the new prospects for the unveiling of tumour heterogeneity. Alföld et al. [35]
distinguished the individuality of two-dimensional (2D), three-dimensional (3D) and in-vivo models
by using multi-parametric single-cell mass cytometry technique. The lung cancer markers were
characterized by using single-cell mass cytometry and compared with different in-vitro cell culture
techniques. Their research showed that by using an organic scaffold, A549 lung cancer cells could
grow, or scaffold-free 3D culturing techniques could express in-vivo tumours of gene and protein far
better than standard 2D cultures technique. Moreover, the authors showed the rate of heterogeneity
in 2D and 3D cultures and in solid-tumour. Lin et al. [5] investigated how single-cell technology has
been applied to inspect several infectious diseases. They studied how cellular heterogeneity is linked
substantially with the progression of infectious disease using fluorescence-activated cell sorting and
next-generation sequencing. The genomic and phenotypic biomarker characterization, as well as
host-pathogen interaction at the single-cell level, can help us to understand the unknown infection
mechanisms and potential diseases treatment. A piece of detailed single-cell information from
primary cells can identify rare but important cell subtypes, which help us to understand the complex
interplay between cells and the immune system. The advancement of single-cell analysis also can
help us for the development of drugs, vaccines and potential biomarkers.
Gupta et al. [36] reviewed various single-neuron models, single-neuron behaviour, and their
analysis. In comparison with the bulk analysis of millions of neurons together, the single-neuron
analysis can emphasize the detail of pathophysiology, electrophysiology, anatomical differences,
structural and functional features etc., at the single-neuron or even sub-neuron level. The single-
neuron analysis can play an important role in studying the in-between communications of neurons
and provide essential information of the brain activity. The author described the details of the single-
neuron mapping and the electrophysiological recording. They have highlighted the recent
advancement of single-neuron manipulation, isolation, and therapy using microfluidic devices
following different physical approaches such as electroporation, microinjection, microelectrode
array, optogenetic techniques, optical transfection, etc. Finally, the authors added the impact of
artificial intelligence in single-neuron and concluded the limitations and future prospects of single-
neuron analyses.
In conclusion, this Special Issue of Cells emphasizes in detail about single-cell analysis using
micro/nanofluidic devices, capillary electrophoresis, fluorescence-activated cell sorting (FACS),
single-cell Western blotting, single-cell mass cytometry, enzyme-linked immunospot assay study,
quantitative polymerase chain Reaction (qPCR) technique and next-generation sequencing (NGS)
etc. The single-cell RNA sequencing (scRNA-seq) technologies were briefly reviewed by using
microfluidic devices to understand cellular heterogeneity characteristics. The single nucleus RNA
sequencing (snRNA-seq) was used to investigate the cellular composition and cell features of an
Cells 2020, 9, 1993 5 of 7
entire adult mammalian heart. The mRNA levels in single-cell are quantified using global reverse
transcription method followed by quantitative PCR. This quantification technique can help us to
characterize cell molecules for different types and states of cells with the temporal variation of the
microenvironment. The single-cell transcriptome and analysis were performed using Western blot,
qPCR as well as immunofluorescence assay. The advancement of microfluidic technologies such as
microfluidic fluorescent flow cytometry, microwell-based assay (micro engraving), droplet-based
microfluidics, micro-chamber based assay (barcoding microchips), are used for protein analysis of
single-cells. The cleavable biotin-conjugated antibodies, as well as cleavable fluorescent streptavidin
(CFS), are employed for delicate in-situ protein analysis. The microfluidic single-cell cloning device
is used to generate monoclonal cells for producing proteins with high reproducibility, and it can be
applied for the production of therapeutic drugs. The single-cell mass cytometry was employed for
lung cancer markers characterization and comparing the results with in-vitro 2D and 3D cell culture
methods for drug screening applications. The importance of single-cell technologies has been well
emphasized, in particular, for controlling and investigating infectious diseases. Finally, the behaviour
of single-neuron and their analyses are discussed in detail by using single-neuron models, single-
neuron mapping and electrophysiological recording, single-neuron isolation, manipulation and
therapy. Further, the research area of artificial intelligence concerning single-neurons is highlighted.
Author Contributions Dr. Tuhin Subhra Santra wrote this editorial and Prof. Fan Gang Tseng corrected it.
Funding: This research was funded by the Department of Biotechnology (DBT)/Wellcome Trust India Alliance
Fellowship under the grant number IA/E//16/1/503062.
Acknowledgments The authors are greatly appreciatedto Dr. Pallavi Gupta (Bio-nano-µ-lab, Department of
Engineering Design, IIT Madras) to prepare Figure-1 from the web of science.
Conflicts of Interest The authors declare no conflict of interest.
References
1. Life: The Science of Biology, 7th ed.; William, K.P., David, E.S., Gordan, H.O., Craig Heller, H., Eds.; Sinauer
Associates and W.H. Freeman: Sunderland, MA, USA, 5 December 2003; ISBN 13 978-0716798569.
2. Fan-Gang Tseng, T.S.S. Essential of Single-Cell Analysis; Springer-Verlag GmbH: Berlin, Heidelberg,
Germany, 2016; ISBN 978-3-662-49118-8.
3. Microfluidics and Bio-MEMS: Devices and Applications, 1st ed.; Santra, T.S., Ed.; Jenny Stanford Publisher:
Singapore, Singapore, 2020; ISBN 9789814800853.
4. Tuhin, S.S.; Fan-Gang, T. Handbook of Single Cell Technologies, 1st ed.; Tuhin, S.S., Fan-Gang, T., Eds.;
Springer-Nature: Singapore, Singapore, 2021; ISBN 978-981-10-8952-7.
5. Lin, W.N.; Tay, M.Z.; Lu, R; Liu, Y.; Chen, C.-H.; Cheow, L.F. The Role of Single-Cell Technology in the
Study and Control of Infectious Diseases. Cells 2020, 9, 1440, doi:10.3390/cells9061440.
6. Shinde, P.; Mohan, L.; Kumar, A.; Dey, K.; Maddi, A.; Patananan, A.; Tseng, F.-G.; Chang, H.-Y.; Nagai, M.;
Santra, T. Current Trends of Microfluidic Single-Cell Technologies. Int. J. Mol. Sci. 2018, 19, 3143,
doi:10.3390/ijms19103143.
7. Fan-Gang, T.; Tuhin, S.S. Micro/Nano Fluidic Devices for Single Cell Analysis, 1st ed.; Fan-Gang, T., Tuhin,
S.S., Eds.; MDPI: Basel, Switzerland, 2015; ISBN 978-3-03842-090-3.
8. Fan-Gang, T.; Tuhin, S.S. Single Cell Analysis in Biotechnology and System Biology, 1st ed.; Fan-Gang, T., Tuhin,
S.S., Ed.; MDPI: Basel, Switzerland, 2016; ISBN 978-3-03842-194-8.
9. Li, L.; Guo, F.; Gao, Y.; Ren, Y.; Yuan, P.; Yan, L.; Li, R.; Lian, Y.; Li, J.; Hu, B.; et al. Single-cell multi-omics
sequencing of human early embryos. Nat. Cell Biol. 2018, doi:10.1038/s41556-018-0123-2.
10. Chappell, L.; Russell, A.J.C.; Voet, T. Single-Cell (Multi)omics Technologies. Annu. Rev. Genomics Hum.
Genet. 2018, doi:10.1146/annurev-genom-091416-035324.
11. Fritzsch, F.S.O.; Dusny, C.; Frick, O.; Schmid, A. Single-Cell Analysis in Biotechnology, Systems Biology,
and Biocatalysis. Annu. Rev. Chem. Biomol. Eng. 2012, doi:10.1146/annurev-chembioeng-062011-081056.
12. Santra, T.; Tseng, F. Micro/Nanofluidic Devices for Single Cell Analysis. Micromachines 2014, 5, 154–157,
doi:10.3390/mi5020154.
Cells 2020, 9, 1993 6 of 7
13. Shinde, P.; Kumar, A.; Illath, K.; Dey, K.; Mohan, L.; Kar, S.; Barik, T.K.; S-Rad, J.; Nagai, M.; Santra, T.S.
Physical Approaches for Drug Delivery-An Overview. In Delivery of Drugs; Elsevier: Amsterdam, The
Netherlands: 2020; Volume 2, ISBN 978-0-12-817776-1.
14. Fan-Gang, T.; Tuhin, S.S. Electroporation for single cell analysis. In Essentials of Single Cell Analysis; Fan-
Gang, T., Tuhin, S.S., Eds.; Springer-Verlag GmbH: Berlin, Heidelberg, Germany, 2016; ISBN 978-3-662-
49116-4.
15. Santra, T.S.; Chen, C.W.; Chang, H.Y.; Tseng, F.G. Dielectric passivation layer as a substratum on localized
single-cell electroporation. RSC Adv. 2016, doi:10.1039/c5ra18258a.
16. Santra, T.S.; Wang, P.C.; Chang, H.Y.; Tseng, F.G. Tuning nano electric field to affect restrictive membrane
area on localized single cell nano-electroporation. Appl. Phys. Lett. 2013, doi:10.1063/1.4833535.
17. Kar, S.; Loganathan, M.; Dey, K.; Shinde, P.; Chang, H.-Y.; Nagai, M.; Santra, T.S. Single-cell
electroporation: Current trends, applications and future prospects. J. Micromechanics Microengineering 2018,
28, 123002, doi:10.1088/1361-6439/aae5ae.
18. Santra, T.S.; Kar, S.; Borana, J.; Wang, P.-C.; Tseng, F.-G. Nanolocalized Single-Cell-Membrane
Nanoelectroporation: For higher efficiency with high cell viability. IEEE Nanotechnol. Mag. 2014, 8, 30–34,
doi:10.1109/MNANO.2014.2312031.
19. Kumar, A.; Mohan, L.; Shinde, P.; Chang, H.-Y.; Nagai, M.; Santra, T.S. Mechanoporation: Towards Single
Cell Approaches. In Handbook of Single Cell Technologies; Tuhin, S.S., Fan-Gang, T., Eds.; Springer-Nature:
Singapore, Singapore, 2021; p. 1200 ISBN 978-981-10-8954-1.
20. Kar, S.; Shinde, P.; Nagai, M.T.S.S. Optical Manipulation of Cells. In Microfluidics and Bio-MEMS:Devices
and Applications; Santra, T.S., Ed.; Jenny Stanford Publisher: Singapore, Singapore, 2020; p. 550, ISBN 978-
1-003-01493-5.
21. Santra, T.S.; Kar, S.; Chen, T.-C.; Chen, C.-W.; Borana, J.; Lee, M.-C.; Tseng, F.-G. Near-infrared nanosecond-
pulsed laser-activated highly efficient intracellular delivery mediated by nano-corrugated mushroom-
shaped gold-coated polystyrene nanoparticles. Nanoscale 2020, 12, 12057–12067, doi:10.1039/D0NR01792B.
22. Subhra Santra, Tuhin.; Wu, T.-H.; Chiou, P.-Y. Photothermal Microfluidics. In Optical MEMS for Chemical
Analysis and Biomedicine; Jiang, Ed.; Institution of Engineering and Technology: London, UK, 2016; ISBN
9781849198974.
23. Shinde, P.; Kar, S.; Mohan, L.; Chang, H.-Y.; Tseng, F.-G.; Nagai, M.; Santra, T.S. Infrared Pulse Laser
Activated Highly Efficient Intracellular Delivery Using Titanium Micro-Dish Device. ACS Biomater. Sci.
Eng. 2020, 00785, doi:10.1021/acsbiomaterials.0c00785.
24. Harshan, H.; Gupta, P.; Kiran, K.; S.Wankhar, T.S.S. Microneedles: Current Trends and Applications. In
Microfluidics and Bio-MEMS: Devices and Applications; Santra, T.S., Ed.; Jenny Stanford Publisher: Singapore,
Singapore, 2020; ISBN 978-1-003-01493-5.
25. Munish, M.S.; Santra, T.S. Microinjection for single cell analysis. In Series in BioEngineering; Tseng, F.-G.,
Santra, T.S., Eds.; Springer: Berlin/Heidelberg, 2016; ISBN 978-3-662-49116-4.
26. Kumar, A.; Shinde, P.; Mohan, L.; Mhapatra, P.S.T.S.S. Microfluidic Technologies for Cell Manipulation,
Therapeutics and Analysis. In Microfluidics and Bio-MEMS:Devices and Applications; Santra, T.S., Ed.; Jenny
Stanford Publisher: Singapore, Singapore, 2020; p. 550, ISBN 978-1-003-01493-5.
27. Choi, J.R.; Yong, K.W.; Choi, J.Y.; Cowie, A.C. Single-Cell RNA Sequencing and Its Combination with
Protein and DNA Analyses. Cells 2020, 9, 1130, doi:10.3390/cells9051130.
28. Wolfien, M.; Galow, A.-M.; Müller, P.; Bartsch, M.; Brunner, R.M.; Goldammer, T.; Wolkenhauer, O.;
Hoeflich, A.; David, R. Single-Nucleus Sequencing of an Entire Mammalian Heart: Cell Type Composition
and Velocity. Cells 2020, 9, 318, doi:10.3390/cells9020318.
29. Jonasson, E.; Andersson, L.; Dolatabadi, S.; Ghannoum, S.; Åman, P.; Ståhlberg, A. Total mRNA
Quantification in Single Cells: Sarcoma Cell Heterogeneity. Cells 2020, 9, 759, doi:10.3390/cells9030759.
30. Aline Rangel-Pozzo, Songyan Liu, Gabriel Wajnberg, Xuemei Wang, R.J.O.; Geoffrey, G.; Hicks, D.D. and
S.M. Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell
Level. Cells 2020, 9, 1863, doi:10.3390/cells9081863.
31. Chang; Tian; Ji; Zhou; Hou; Zhao; Yang; Yang; Li Single-Cell Transcriptomes Reveal Characteristic Features
of Mouse Hepatocytes with Liver Cholestatic Injury. Cells 2019, 8, 1069, doi:10.3390/cells8091069.
32. Liu, L.; Chen, D.; Wang, J.; Chen, J. Advances of Single-Cell Protein Analysis. Cells 2020, 9, 1271,
doi:10.3390/cells9051271.
Cells 2020, 9, 1993 7 of 7
33. Liao, R.; Pham, T.; Mastroeni, D.; Coleman, P.D.; Labaer, J.; Guo, J. Highly Sensitive and Multiplexed In-
Situ Protein Profiling with Cleavable Fluorescent Streptavidin. Cells 2020, 9, 852, doi:10.3390/cells9040852.
34. Yeh, C.-F.; Lin, C.-H.; Chang, H.-C.; Tang, C.-Y.; Lai, P.-T.; Hsu, C.-H. A Microfluidic Single-Cell Cloning
(SCC) Device for the Generation of Monoclonal Cells. Cells 2020, 9, 1482, doi:10.3390/cells9061482.
35. Alföldi; Balog; Faragó; Halmai; Kotogány; Neuperger; Nagy; Fehér; Szebeni; Puskás Single Cell Mass
Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In vivo And Three-Dimensional
Models over the Petri-dish. Cells 2019, 8, 1093, doi:10.3390/cells8091093.
36. Gupta, P.; Balasubramaniam, N.; Chang, H.-Y.; Tseng, F.-G.; Santra, T.S. A Single-Neuron: Current Trends
and Future Prospects. Cells 2020, 9, 1528, doi:10.3390/cells9061528.
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