細胞異質性存在於所有細胞群中，包括組織、器官和培養細胞，尤其在腫瘤組織內。分析群體細胞通常會丟失群體中稀有細胞的關鍵信息，因為它們的信號將被總群體細胞平均。因此，有必要識別細胞與細胞之間的不同特徵，以全面了解異質性疾病的調節機制。單細胞分析提供了一種方式能夠分析細胞群中個別的細胞特徵，以揭示異質細胞群中細胞之間的生理工作機制。儘管已經建立了各種單細胞操作方法，但它們存在效率低、操作困難、單細胞確效困難或成本高等問題。因此，單細胞相關研究尚未在一般生物實驗室中廣泛開展。在這項研究中，我們設計易於操作的便攜式裝置，允許使用一般實驗室中現成的工具來進行單細胞操作和分析。我們開發了兩項便攜式裝置，包括（1）便攜式可控壓應力裝置以單細胞分辨率監測細胞突起。此裝置能夠在一般實驗室中簡單設置來達到對細胞施加可控壓力，允許及時觀察，並且可直接收取裝置上的細胞進行後續核酸或蛋白分析，此過程不需要酵素作用可以避免影響細胞蛋白表現。（2）單細胞克隆芯片建立單克隆細胞系。此裝置透過簡單的操作與傳統稀釋法相比可顯著提升單細胞獲取效率，並在單克隆培養初期時確效單細胞，不須進行重複克隆，節省克隆獲取時間。這些便攜式設備都具有簡單操作，低成本，省時(直接收取細胞不須使用膠原酶水解或不需重複克隆)等優點。未來探討壓力對癌細胞影響之機制或進行單株細胞株建立都可使用這些方便的裝置。與過去其他方法相比，它們可大幅降低勞動力和成本，使單細胞研究在普通生物實驗室也能順利執行。

Cellular heterogeneity presents in all cell populations, including tissues, organs, and cultured cells. Analyzing a bulk of cells usually causes losing critical information of rare cells, which play key roles, in the population because their signals would be averaged by total population cells. Single cell analysis provides distinguishing inter-cell characterizations in cell populations to reveal the cell-to-cell physiological working mechanisms in heterogeneous cell populations. Although various single cell operation methods have been established, they have several issues, including low efficiency, difficulty in operation, difficulty in validating single cells, or high cost. Thus, single cell-related studies have not been widely performed in general biological laboratories. In this study, we have designed portable devices that allow single-cell manipulation and analysis using readily available tools in general laboratories. These devices include (1) Monitor cellular protrusions at single-cell resolution with a portable controllable compressive stress device. The device can be easily operated in general laboratories to apply controllable pressure on cells and then allowed real-time observation。Besides, the experimental cells can be straightforwardly harvested from the device for RNA or protein analysis without cell damage by collagenase. (2) Monoclonal cell lines establishment with a single-cell cloning chip. The device allows obtaining high efficient single cell events through several simple steps compared to the traditional limiting dilution method and provides validation of single cells at the initial stage omitting re-cloning procedures to save time. There are several advantages of our devices, including easy operation, low cost, and time-saving (straightforward harvesting of cells without collagenase hydrolysis or re-cloning processes) that promise robustly decreasing labor and costs compared with other previous methods in common biological laboratories.

Table of content
Chapter 1: Overview of Single-Cell Analysis 1
1.1 Importance of Single-Cell Analysis 1
1.2 Clinical Applications of Single-Cell Analysis 2
1.3 Techniques of Single Cell Manipulation 3
1.4 Thesis Overview 5
Chapter 2: Monitor Cellular Protrusions at Single-cell Resolution with A Portable Controllable Compressive Stress Device 7
2.1 Introduction 7
2.1.1 Study compressive stress with an in vitro device 7
2.1.2 Development of in vitro devices 7
2.1.2 Improvement of in vitro devices 8
2.1.3 The design of our compressive pressure device 8
2.2 Materials and Methods 9
2.2.1 Cell maintenance and transfection 9
2.2.2 Preparation of agarose gel 9
2.2.3 In vitro compressive stress device 11
2.2.4 Evaluation of cell viability and apoptosis 11
2.2.5 Cell area measurement 11
2.2.6 Time-lapse microscopy 12
2.2.7 Cell tracking and statistical analysis 12
2.2.8 Staining of actin filaments 12
2.2.9 Immunofluorescence 13
2.3 Results 13
2.3.1 Conception and manipulation of the pressure controllable device 13
2.3.2 High compressive stress does not affect cell viability or apoptosis on the device 14
2.3.3 Compressive pressure triggers cellular morphological changes 16
2.3.4 Compressive pressure changes the ratio of blebs or lamellipodia protrusion on a low-adhesive surface 18
2.3.5 Breast cancer cells present highly locomotion on a low-adhesive surface 20
2.3.6 Compressive pressure inhibits cell locomotion on a low-adhesive surface 21
2.3.7 Phosphorylation of Rac-1 involved in compressive-pressure-induced lamellipodia formation on the low-adhesive surface 22
2.4 Discussion 24
2.4.1 Advantages of the device 24
2.4.2 Our finding and hypothesis 25
2.5 Conclusion 28
Chapter 3: Monoclonal Cells Lines Establishment with a Single-Cell Cloning Chip 29
3.1 Introduction 29
3.1.1 Importance and challenges of monoclonal cell line 29
3.1.2 Techniques and challenges of single cell cloning 30
3.1.3 Design of the device 31
3.2 Materials and Methods 32
3.2.1. Conception and Fabrication of the Device 32
3.2.2 Cell Culture and Maintenance 32
3.2.3 SCC Device Preparation for Single-Cell Capture 33
3.2.4 Single-Cell Capture and Cloning in SCC Device 33
3.2.5 Identification of Single-Cell Events by Limiting Dilution, SCC Device, and FACS 34
3.2.6 Transferring and Releasing Cell Colonies from the SCC Device 35
3.2.7 Quantitation of transfer and release of cells from PDMS of chips to 96-well plates 35
3.2.8 Single-Cell Growth 36
3.2.9 Single-Cell Growth after Release from PDMS Plug 36
3.2.10 LifeAct Plasmid Transfection 36
3.2.11 Clonality-Validated A549 LifeAct Cell Line Established by the SCC Device 37
3.2.12 A549 LifeAct Monoclonal Cell Line Characterization 37
3.2.13 Acquisition of Cell Images 37
3.2.14 Statistical Analyses 37
3.3 Results and Discussion 38
3.3.1 Conception and Manipulation of the SCC Device 38
3.3.2 High-Efficiency Single-Cell Isolation and Identification with SCC Device 43
3.3.3 Culture Wells of chip provide Single Cells to Divide and retain their Monoclonality 46
3.3.4 Expanded Monoclonal Cells Can Be Transferred and Maintain Their Purity and Viability 48
3.3.5 Establishing Monoclonal Genetically Modified Cells with SSC Device 52
3.4 Conclusions 55
Chapter 4: Conclusions and Future perspectives of single cell analysis platforms 56
4.1 Summary 56
4.2 Future Perspectives 56
4.3 Publication Lists 59
References: 61

References:
1. Fattore, L.; Ruggiero, C.F.; Liguoro, D.; Mancini, R.; Ciliberto, G. Single cell analysis to dissect molecular heterogeneity and disease evolution in metastatic melanoma. Cell Death Dis 2019, 10, 827, doi:10.1038/s41419-019-2048-5.
2. Schreier, S.; Triampo, W. The Blood Circulating Rare Cell Population. What Is It and What Is It Good for? Cells 2020, 9, doi:ARTN 79010.3390/cells9040790.
3. Proserpio, V.; Lonnberg, T. Single-cell technologies are revolutionizing the approach to rare cells. Immunol Cell Biol 2016, 94, 225-229, doi:10.1038/icb.2015.106.
4. Cho, J.H.; Kim, S.Y.; Lee, J.; Park, S.H.; Park, J.O.; Park, Y.S.; Lim, H.Y.; Kang, W.K.; Kim, S.T. Detection of circulating tumor cells (CTCs) in cerebrospinal fluid of a patient with HER2-overexpressing gastric cancer and single cell analysis of intra-patient heterogeneity of CTCs. Transl Cancer Res 2019, 8, 2107-2112, doi:10.21037/tcr.2019.09.27.
5. Powell, A.A.; Talasaz, A.H.; Zhang, H.; Coram, M.A.; Reddy, A.; Deng, G.; Telli, M.L.; Advani, R.H.; Carlson, R.W.; Mollick, J.A.; et al. Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 2012, 7, e33788, doi:10.1371/journal.pone.0033788.
6. Bernard, V.; Semaan, A.; Huang, J.; San Lucas, F.A.; Mulu, F.C.; Stephens, B.M.; Guerrero, P.A.; Huang, Y.; Zhao, J.; Kamyabi, N.; et al. Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression. Clin Cancer Res 2019, 25, 2194-2205, doi:10.1158/1078-0432.CCR-18-1955.
7. Wisdom, A.J.; Mowery, Y.M.; Hong, C.S.; Himes, J.E.; Nabet, B.Y.; Qin, X.; Zhang, D.; Chen, L.; Fradin, H.; Patel, R.; et al. Single cell analysis reveals distinct immune landscapes in transplant and primary sarcomas that determine response or resistance to immunotherapy. Nat Commun 2020, 11, 6410, doi:10.1038/s41467-020-19917-0.
8. Gross, A.; Schoendube, J.; Zimmermann, S.; Steeb, M.; Zengerle, R.; Koltay, P. Technologies for Single-Cell Isolation. Int J Mol Sci 2015, 16, 16897-16919, doi:10.3390/ijms160816897.
9. Shieh, A.C. Biomechanical forces shape the tumor microenvironment. Ann Biomed Eng 2011, 39, 1379-1389, doi:10.1007/s10439-011-0252-2.
10. Chaudhuri, P.K.; Low, B.C.; Lim, C.T. Mechanobiology of Tumor Growth. Chem Rev 2018, 118, 6499-6515, doi:10.1021/acs.chemrev.8b00042.
11. Kalli, M.; Papageorgis, P.; Gkretsi, V.; Stylianopoulos, T. Solid Stress Facilitates Fibroblasts Activation to Promote Pancreatic Cancer Cell Migration. Annals of Biomedical Engineering 2018, 46, 657-669, doi:10.1007/s10439-018-1997-7.
12. Sachdeva, U.M.; Shimonosono, M.; Flashner, S.; Cruz-Acuna, R.; Gabre, J.T.; Nakagawa, H. Understanding the cellular origin and progression of esophageal cancer using esophageal organoids. Cancer Lett 2021, 509, 39-52, doi:10.1016/j.canlet.2021.03.031.
13. Tse, J.M.; Cheng, G.; Tyrrell, J.A.; Wilcox-Adelman, S.A.; Boucher, Y.; Jain, R.K.; Munn, L.L. Mechanical compression drives cancer cells toward invasive phenotype. Proc Natl Acad Sci U S A 2012, 109, 911-916, doi:10.1073/pnas.1118910109.
14. Kim, B.G.; Gao, M.Q.; Kang, S.; Choi, Y.P.; Lee, J.H.; Kim, J.E.; Han, H.H.; Mun, S.G.; Cho, N.H. Mechanical compression induces VEGFA overexpression in breast cancer via DNMT3A-dependent miR-9 downregulation. Cell Death Dis 2017, 8, e2646, doi:10.1038/cddis.2017.73.
15. Kalli, M.; Voutouri, C.; Minia, A.; Pliaka, V.; Fotis, C.; Alexopoulos, L.G.; Stylianopoulos, T. Mechanical Compression Regulates Brain Cancer Cell Migration Through MEK1/Erk1 Pathway Activation and GDF15 Expression. Front Oncol 2019, 9, doi:ARTN 99210.3389/fonc.2019.00992.
16. Morikura, T.; Miyata, S. Effect of Mechanical Compression on Invasion Process of Malignant Melanoma Using In Vitro Three-Dimensional Cell Culture Device. Micromachines-Basel 2019, 10, doi:ARTN 66610.3390/mi10100666.
17. Morikura, T.; Miyata, S. Mechanical Intermittent Compression Affects the Progression Rate of Malignant Melanoma Cells in a Cycle Period-Dependent Manner. Diagnostics 2021, 11, doi:ARTN 111210.3390/diagnostics11061112.
18. Novak, C.M.; Horst, E.N.; Lin, E.; Mehta, G. Compressive Stimulation Enhances Ovarian Cancer Proliferation, Invasion, Chemoresistance, and Mechanotransduction via CDC42 in a 3D Bioreactor. Cancers (Basel) 2020, 12, doi:10.3390/cancers12061521.
19. Busser, H.; De Bruyn, C.; Urbain, F.; Najar, M.; Pieters, K.; Raicevic, G.; Meuleman, N.; Bron, D.; Lagneaux, L. Isolation of Adipose-Derived Stromal Cells Without Enzymatic Treatment: Expansion, Phenotypical, and Functional Characterization. Stem Cells Dev 2014, 23, 2390-2400, doi:10.1089/scd.2014.0071.
20. Lee, J.T.Y.; Cheung, K.M.C.; Leung, V.Y.L. Systematic study of cell isolation from bovine nucleus pulposus: Improving cell yield and experiment reliability. J Orthop Res 2015, 33, 1743-1755, doi:10.1002/jor.22942.
21. Claridge, B.; Rai, A.; Fang, H.Y.; Matsumoto, A.; Luo, J.T.; McMullen, J.R.; Greening, D.W. Proteome characterisation of extracellular vesicles isolated from heart. Proteomics 2021, 21, doi:ARTN e210002610.1002/pmic.202100026.
22. Hooshmand, S.; Ghaderi, A.; Yusoff, K.; Karrupiah, T.; Rosli, R.; Mojtahedi, Z. Downregulation of RhoGDIalpha increased migration and invasion of ER (+) MCF7 and ER (-) MDA-MB-231 breast cancer cells. Cell Adh Migr 2013, 7, 297-303, doi:10.4161/cam.24204.
23. Aumsuwan, P.; Khan, S.I.; Khan, I.A.; Ali, Z.; Avula, B.; Walker, L.A.; Shariat-Madar, Z.; Helferich, W.G.; Katzenellenbogen, B.S.; Dasmahapatra, A.K. The anticancer potential of steroidal saponin, dioscin, isolated from wild yam (Dioscorea villosa) root extract in invasive human breast cancer cell line MDA-MB-231 in vitro. Arch Biochem Biophys 2016, 591, 98-110, doi:10.1016/j.abb.2015.12.001.
24. Kwiatkowska, E.; Wojtala, M.; Gajewska, A.; Soszynski, M.; Bartosz, G.; Sadowska-Bartosz, I. Effect of 3-bromopyruvate acid on the redox equilibrium in non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells. J Bioenerg Biomembr 2016, 48, 23-32, doi:10.1007/s10863-015-9637-5.
25. Samandari, M.; Rafiee, L.; Alipanah, F.; Sanati-Nezhad, A.; Javanmard, S.H. A simple, low cost and reusable microfluidic gradient strategy and its application in modeling cancer invasion. Sci Rep 2021, 11, 10310, doi:10.1038/s41598-021-89635-0.
26. Ryu, H.H.; Jung, S.; Jung, T.Y.; Moon, K.S.; Kim, I.Y.; Jeong, Y.I.; Jin, S.G.; Pei, J.; Wen, M.; Jang, W.Y. Role of metallothionein 1E in the migration and invasion of human glioma cell lines. Int J Oncol 2012, 41, 1305-1313, doi:10.3892/ijo.2012.1570.
27. Torka, R.; Thuma, F.; Herzog, V.; Kirfel, G. ROCK signaling mediates the adoption of different modes of migration and invasion in human mammary epithelial tumor cells. Exp Cell Res 2006, 312, 3857-3871, doi:10.1016/j.yexcr.2006.08.025.
28. Lammermann, T.; Sixt, M. Mechanical modes of 'amoeboid' cell migration. Curr Opin Cell Biol 2009, 21, 636-644, doi:10.1016/j.ceb.2009.05.003.
29. Seetharaman, S.; Etienne-Manneville, S. Cytoskeletal Crosstalk in Cell Migration. Trends Cell Biol 2020, 30, 720-735, doi:10.1016/j.tcb.2020.06.004.
30. Shellard, A.; Mayor, R. All Roads Lead to Directional Cell Migration. Trends Cell Biol 2020, 30, 852-868, doi:10.1016/j.tcb.2020.08.002.
31. Paluch, E.K.; Aspalter, I.M.; Sixt, M. Focal Adhesion-Independent Cell Migration. Annu Rev Cell Dev Biol 2016, 32, 469-490, doi:10.1146/annurev-cellbio-111315-125341.
32. Mgrditchian, T.; Sakalauskaite, G.; Muller, T.; Hoffmann, C.; Thomas, C. The multiple roles of actin-binding proteins at invadopodia. Int Rev Cel Mol Bio 2021, 360, 99-132, doi:10.1016/bs.ircmb.2021.03.004.
33. Thompson, S.B.; Waldman, M.M.; Jacobelli, J. Polymerization power: effectors of actin polymerization as regulators of T lymphocyte migration through complex environments. Febs J 2021, doi:10.1111/febs.16130.
34. Mehidi, A.; Rossier, O.; Schaks, M.; Chazeau, A.; Biname, F.; Remorino, A.; Coppey, M.; Karatas, Z.; Sibarita, J.B.; Rottner, K.; et al. Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion. Curr Biol 2019, 29, 2852-+, doi:10.1016/j.cub.2019.07.035.
35. Schaks, M.; Doring, H.; Kage, F.; Steffen, A.; Klunemann, T.; Blankenfeldt, W.; Stradal, T.; Rottner, K. RhoG and Cdc42 can contribute to Rac-dependent lamellipodia formation through WAVE regulatory complex-binding. Small GTPases 2021, 12, 122-132, doi:10.1080/21541248.2019.1657755.
36. Meyer, A.S.; Hughes-Alford, S.K.; Kay, J.E.; Castillo, A.; Wells, A.; Gertler, F.B.; Lauffenburger, D.A. 2D protrusion but not motility predicts growth factor-induced cancer cell migration in 3D collagen. J Cell Biol 2012, 197, 721-729, doi:10.1083/jcb.201201003.
37. Fort, L.; Batista, J.M.; Thomason, P.A.; Spence, H.J.; Whitelaw, J.A.; Tweedy, L.; Greaves, J.; Martin, K.J.; Anderson, K.I.; Brown, P.; et al. Fam49/CYRI interacts with Rac1 and locally suppresses protrusions. Nat Cell Biol 2018, 20, 1159-1171, doi:10.1038/s41556-018-0198-9.
38. Alexandrova, A.Y.; Chikina, A.S.; Svitkina, T.M. Actin cytoskeleton in mesenchymal-to-amoeboid transition of cancer cells. Int Rev Cell Mol Biol 2020, 356, 197-256, doi:10.1016/bs.ircmb.2020.06.002.
39. Broders-Bondon, F.; Nguyen Ho-Bouldoires, T.H.; Fernandez-Sanchez, M.E.; Farge, E. Mechanotransduction in tumor progression: The dark side of the force. J Cell Biol 2018, 217, 1571-1587, doi:10.1083/jcb.201701039.
40. Supplement to the points to consider in the production and testing of new drugs and biologicals produced by recombinant DNA technology: nucleic acid characterization and genetic stability. Biologicals 1993, 21, 81-83, doi:10.1006/biol.1993.1050.
41. Hochedlinger, K.; Jaenisch, R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 2002, 415, 1035-1038, doi:10.1038/nature718.
42. Kilmartin, J.V.; Wright, B.; Milstein, C. Rat Monoclonal Antitubulin Antibodies Derived by Using a New Non-Secreting Rat-Cell Line. J Cell Biol 1982, 93, 576-582, doi:DOI 10.1083/jcb.93.3.576.
43. McLaughlin, P.; Grillo-Lopez, A.J.; Link, B.K.; Levy, R.; Czuczman, M.S.; Williams, M.E.; Heyman, M.R.; Bence-Bruckler, I.; White, C.A.; Cabanillas, F.; et al. Rituximab chimeric Anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: Half of patients respond to a four-dose treatment program. J Clin Oncol 1998, 16, 2825-2833, doi:Doi 10.1200/Jco.1998.16.8.2825.
44. Trikha, M.; Corringham, R.; Klein, B.; Rossi, J.F. Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: A review of the rationale and clinical evidence. Clin Cancer Res 2003, 9, 4653-4665.
45. Goldstein, G. A Randomized Clinical-Trial of Okt3 Monoclonal-Antibody for Acute Rejection of Cadaveric Renal-Transplants. New Engl J Med 1985, 313, 337-342.
46. Andreeff, M.; Bartal, A.; Feit, C.; Hirshaut, Y. Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry. Hybridoma 1985, 4, 277-287, doi:10.1089/hyb.1985.4.277.
47. Lattenmayer, C.; Loeschel, M.; Schriebl, K.; Steinfellner, W.; Sterovsky, T.; Trummer, E.; Vorauer-Uhl, K.; Muller, D.; Katinger, H.; Kunert, R. Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones. Biotechnol Bioeng 2007, 96, 1118-1126, doi:10.1002/bit.21183.
48. Kim, N.S.; Byun, T.H.; Lee, G.M. Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification. Biotechnol Prog 2001, 17, 69-75, doi:10.1021/bp000144h.
49. Chusainow, J.; Yang, Y.S.; Yeo, J.H.; Toh, P.C.; Asvadi, P.; Wong, N.S.; Yap, M.G. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 2009, 102, 1182-1196, doi:10.1002/bit.22158.
50. Hunter, M.; Yuan, P.; Vavilala, D.; Fox, M. Optimization of Protein Expression in Mammalian Cells. Curr Protoc Protein Sci 2019, 95, e77, doi:10.1002/cpps.77.
51. Noh, S.M.; Shin, S.; Lee, G.M. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 2018, 8, 5361, doi:10.1038/s41598-018-23720-9.
52. Lai, T.; Yang, Y.; Ng, S.K. Advances in Mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel) 2013, 6, 579-603, doi:10.3390/ph6050579.
53. Li, F.; Vijayasankaran, N.; Shen, A.Y.; Kiss, R.; Amanullah, A. Cell culture processes for monoclonal antibody production. MAbs 2010, 2, 466-479, doi:10.4161/mabs.2.5.12720.
54. Mao, S.J.; France, D.S. Enhancement of limiting dilution in cloning mouse myeloma-spleen hybridomas by human low density lipoproteins. J Immunol Methods 1984, 75, 309-316, doi:10.1016/0022-1759(84)90114-5.
55. Staszewski, R. Cloning by Limiting Dilution - an Improved Estimate That an Interesting Culture Is Monoclonal. Yale J Biol Med 1984, 57, 865-868.
56. McFarland, D.C. Preparation of pure cell cultures by cloning. Methods Cell Sci 2000, 22, 63-66, doi:10.1023/a:1009838416621.
57. Greenfield, E.A. Single-Cell Cloning of Hybridoma Cells by Limiting Dilution. Cold Spring Harb Protoc 2019, 2019, doi:10.1101/pdb.prot103192.
58. Mathupala, S.P.; Sloan, A.E. An agarose-based cloning-ring anchoring method for isolation of viable cell clones. Biotechniques 2009, 46, 305-307, doi:10.2144/000113079.
59. Underwood, P.A.; Bean, P.A. Hazards of the Limiting-Dilution Method of Cloning Hybridomas. Journal of Immunological Methods 1988, 107, 119-128.
60. Evans, K.; Albanetti, T.; Venkat, R.; Schoner, R.; Savery, J.; Miro-Quesada, G.; Rajan, B.; Groves, C. Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging. Biotechnol Progr 2015, 31, 1172-1178, doi:10.1002/btpr.2145.
61. Pai, J.H.; Xu, W.; Sims, C.E.; Allbritton, N.L. Microtable arrays for culture and isolation of cell colonies. Anal Bioanal Chem 2010, 398, 2595-2604, doi:10.1007/s00216-010-3984-1.
62. Matsumura, T.; Tatsumi, K.; Noda, Y.; Nakanishi, N.; Okonogi, A.; Hirano, K.; Li, L.; Osumi, T.; Tada, T.; Kotera, H. Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device. Biochem Bioph Res Co 2014, 453, 131-137, doi:10.1016/j.bbrc.2014.09.081.
63. Yoshimoto, N.; Kida, A.; Jie, X.; Kurokawa, M.; Iijima, M.; Niimi, T.; Maturana, A.D.; Nikaido, I.; Ueda, H.R.; Tatematsu, K.; et al. An automated system for high-throughput single cell-based breeding. Sci Rep-Uk 2013, 3, doi:ARTN 119110.1038/srep01191.
64. Lin, C.H.; Hsiao, Y.H.; Chang, H.C.; Yeh, C.F.; He, C.K.; Salm, E.M.; Chen, C.; Chiu, I.M.; Hsu, C.H. A microfluidic dual-well device for high-throughput single-cell capture and culture. Lab Chip 2015, 15, 2928-2938, doi:10.1039/c5lc00541h.
65. Mcdonald, A.R.; Garbary, D.J.; Duckett, J.G. Rhodamine-Phalloidin Staining of F-Actin in Rhodophyta. Biotech Histochem 1993, 68, 91-98, doi:Doi 10.3109/10520299309104673.
66. Chazotte, B. Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging. Cold Spring Harb Protoc 2010, 2010, pdb prot4947, doi:10.1101/pdb.prot4947.
67. Espulgar, W.; Yamaguchi, Y.; Aoki, W.; Mita, D.; Saito, M.; Lee, J.K.; Tamiya, E. Single cell trapping and cell-cell interaction monitoring of cardiomyocytes in a designed microfluidic chip. Sensor Actuat B-Chem 2015, 207, 43-50, doi:10.1016/j.snb.2014.09.068.
68. Nguyen, T.A.; Yin, T.I.; Reyes, D.; Urban, G.A. Microfluidic Chip with Integrated Electrical Cell-Impedance Sensing for Monitoring Single Cancer Cell Migration in Three-Dimensional Matrixes. Analytical Chemistry 2013, 85, 11068-11076, doi:10.1021/ac402761s.
69. 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, doi:10.3390/cells9061482.
70. Shen, F.M.; Zhu, L.; Ye, H.; Yang, Y.J.; Pang, D.W.; Zhang, Z.L. A High Throughput Micro-Chamber Array Device for Single Cell Clonal Cultivation and Tumor Heterogeneity Analysis. Sci Rep-Uk 2015, 5, doi:ARTN 1193710.1038/srep11937.
71. Pei, H.M.; Li, L.; Han, Z.J.; Wang, Y.G.; Tang, B. Recent advances in microfluidic technologies for circulating tumor cells: enrichment, single-cell analysis, and liquid biopsy for clinical applications. Lab on a Chip 2020, 20, 3854-3875, doi:10.1039/d0lc00577k.
72. Huang, X.W.; Yue, W.Q.; Liu, D.D.; Yue, J.B.; Li, J.Q.; Sun, D.; Yang, M.S.; Wang, Z.K. Monitoring the intracellular calcium response to a dynamic hypertonic environment. Sci Rep-Uk 2016, 6, doi:ARTN 2359110.1038/srep23591.
73. Altemose, N.; Maslan, A.; Rios-Martinez, C.; Lai, A.R.; White, J.A.; Streets, A. mu DamID: A Microfluidic Approach for Joint Imaging and Sequencing of Protein-DNA Interactions in Single Cells. Cell Systems 2020, 11, 354-+, doi:10.1016/j.cels.2020.08.015.
74. Lamanna, J.; Scott, E.Y.; Edwards, H.S.; Chamberlain, M.D.; Dryden, M.D.M.; Peng, J.X.; Mair, B.; Lee, A.; Chan, C.; Sklavounos, A.A.; et al. Digital microfluidic isolation of single cells for -Omics. Nature Communications 2020, 11, doi:ARTN 563210.1038/s41467-020-19394-5.
75. Sesen, M.; Whyte, G. Image-Based Single Cell Sorting Automation in Droplet Microfluidics. Sci Rep-Uk 2020, 10, doi:ARTN 873610.1038/s41598-020-65483-2.