Microfluidics and Nanofluidics

, Volume 18, Issue 5–6, pp 955–966 | Cite as

A microfluidic method for the selection of undifferentiated human embryonic stem cells and in situ analysis

  • E. Jabart
  • S. Rangarajan
  • C. Lieu
  • J. Hack
  • I. Conboy
  • L. L. SohnEmail author
Research Paper


Conventional cell-sorting methods such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) can suffer from certain shortcomings such as lengthy sample preparation time, cell modification through antibody labeling, and cell damage due to exposure to high shear forces or to attachment of superparamagnetic Microbeads. In light of these drawbacks, we have recently developed a label-free, microfluidic platform that can not only select cells with minimal sample preparation but also enable analysis of cells in situ. We demonstrate the utility of our platform by successfully isolating undifferentiated human embryonic stem cells (hESCs) from a heterogeneous population based on the undifferentiated stem-cell marker SSEA-4. Importantly, we show that, in contrast to MACS or FACS, cells isolated by our method have very high viability (~90 %). Overall, our platform technology could likely be applied to other cell types beyond hESCs and to a variety of heterogeneous cell populations in order to select and analyze cells of interest.


Human embryonic stem cells (hESCs) Cell sorting Label-free In situ analysis 



We would like to thank the Siebel Scholars Foundation for their support of E. J. and the W. M. Keck Foundation, California Institute for Regenerative Medicine CIRM RN1-0032, and NIH/NIA AG 027252 for partial support of this work. We thank M. Mir, K. Balakrishnan, W. Cousin, and C. Elabd for a critical reading of this manuscript and helpful comments.


  1. Abcam (2014) Indirect flow cytometry (FACS) protocol.
  2. Abruzzese RV, Fekete RA (2013) Single cell gene expression analysis of pluripotent stem cells. In: Pluripotent stem cells, chapter 17, vol 997, pp 217–224. doi: 10.1007/978-1-62703-348-0
  3. Akerstroms B, Bjork L (1989) Protein L : an immunoglobulin light chain-binding bacterial protein. J Biol Chem 264(33):19740–19746Google Scholar
  4. Alix-Panabières C, Pantel K (2014) Technologies for detection of circulating tumor cells: facts and vision. Lab Chip. doi: 10.1039/c3lc50644d Google Scholar
  5. Armstrong JK, Wenby RB, Meiselman HJ, Fisher TC (2004) The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation. Biophys J 87(6):4259–4270. doi: 10.1529/biophysj.104.047746 CrossRefGoogle Scholar
  6. Balakrishnan KR, Anwar G, Chapman MR, Nguyen T, Kesavaraju A, Sohn LL (2013) Node-pore sensing: a robust, high-dynamic range method for detecting biological species. Lab chip 13:1302–1307. doi: 10.1039/c3lc41286e CrossRefGoogle Scholar
  7. Bronner V, Tabul M, Bravman T (2009) Rapid screening and selection of optimal antibody capturing agents using the ProteOn XPR36 protein interaction array system (Protein interaction analysis, Tech note 5820). Bio-Rad Laboratories, Inc. Hercules, CAGoogle Scholar
  8. Carbonaro A, Mohanty SK, Huang H, Godley LA, Sohn LL (2008) Cell characterization using a protein-functionalized pore. Lab Chip 8:1478–1485.
  9. Chapman MR, Balakrishnan KR, Li J, Conboy MJ, Huang H, Mohanty SK, Jabart E, Hack J, Conboy IM, Sohn, LL (2013). Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. Integr Biol Quant Biosci Nano Macro 692–702. doi: 10.1039/c3ib20290a
  10. Cheng X, Irimia D, Dixon M, Sekine K, Demirci U, Zamir L, Tompkins RG, Rodriguez W, Toner M (2007) A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects. Lab Chip 7:170–178.
  11. Cheung LS-L, Zheng X, Wang L, Baygents JC, Guzman R, Schroeder JA, Heimark RL, Zohar Y (2011) Adhesion dynamics of circulating tumor cells under shear flow in a bio-functionalized microchannel. J Micromech Microeng 21(5):054033. doi: 10.1088/0960-1317/21/5/054033 CrossRefGoogle Scholar
  12. Clausen J (1981) Laboratory techniques in biochemistry and molecular biology, vol 1 (T.S. Work). North-Holland Publishing Co., New YorkGoogle Scholar
  13. Cozens-Roberts C, Quinn JA, Lauffenberger DA (1990) Receptor-mediated adhesion phenomena. Model studies with the radical-flow detachment assay. Biophys J 58:107–125.
  14. De Château M, Nilson BH, Erntell M, Myhre E, Magnusson CG, Akerström B, Björck L (1993) On the interaction between protein L and immunoglobulins of various mammalian species. Scand J Immunol 37(4):399–405.
  15. Didar TF, Tabrizian M (2010) Adhesion based detection, sorting and enrichment of cells in microfluidic Lab-on-Chip devices. Lab Chip 10(22):3043–3053. doi: 10.1039/c0lc00130a CrossRefGoogle Scholar
  16. Draper JS, Pigott C, Thomson JA, Andrews PW (2002) Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 200(Pt 3):249–258.
  17. Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol CB 11(7):514–518.
  18. Emre N, Vidal JG, Elia J, O’Connor ED, Paramban RI, Hefferan MP, Navarro R, Goldberg DS, Varki NM, Marsala M, Carson CT (2010) The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers. PLoS One 5(8):e12148. doi: 10.1371/journal.pone.0012148 CrossRefGoogle Scholar
  19. Flanagan LA, Lu J, Wang L, Marchenko SA, Jeon NL, Lee AP, Monuki ES (2008) Unique dielectric properties distinguish stem cells and their differentiated progeny. Stem Cells (Dayton, Ohio) 26(3):656–665. doi: 10.1634/stemcells.2007-0810 CrossRefGoogle Scholar
  20. Fong CY, Peh GSL, Gauthaman K, Bongso A (2009) Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Stem Cell Rev 5(1):72–80. doi: 10.1007/s12015-009-9054-4 CrossRefGoogle Scholar
  21. Fong C-Y, Gauthaman K, Bongso A (2010) Teratomas from pluripotent stem cells: a clinical hurdle. J Cell Biochem 111(4):769–781. doi: 10.1002/jcb.22775 CrossRefGoogle Scholar
  22. Gleghorn JP, Pratt ED, Denning D, Liu H, Bander NH, Tagawa ST, Nanus DM, Giannakakou PA, Kirby BJ (2010) Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody. Lab Chip 10:27–29.
  23. Gu B, Zhang J, Wang W, Mo L, Zhou Y, Chen L, Liu Y, Zhang M (2010) Global expression of cell surface proteins in embryonic stem cells. PLoS One 5(12):e15795. doi: 10.1371/journal.pone.0015795 CrossRefGoogle Scholar
  24. Hewitt Z, Forsyth NR, Waterfall M, Wojtacha D, Thomson AJ, McWhir J (2006) Fluorescence-activated single cell sorting of human embryonic stem cells. Cloning Stem Cells 8(3):225–234. doi: 10.1089/clo.2006.8.225 CrossRefGoogle Scholar
  25. Holm F (2012) Cryopreservation of human embryonic stem cells and induced pluripotent stem cells. In: Ye K, Jin S (eds) Human embryonic and induced pluripotent stem cells. Totowa, NJ, Humana Press, pp 85–90. doi: 10.1007/978-1-61779-267-0
  26. Hwang NS, Varghese S, Elisseeff J (2007) Cartilage tissue engineering: directed differentiation of embryonic stem cells in three-dimensional hydrogel culture. In: Stem cell assays, Chapter 24, vol 407, pp 351–373Google Scholar
  27. Jabart EB, Balakrishnan KR, Sohn LL (2014) Label-free microfluidic techniques to isolate and screen single stem cells. In: Stem cells and tissue engineering, 2nd version. World Scientific Publishing, IncGoogle Scholar
  28. Lindström S, Andersson-Svahn H (2010) Overview of single-cell analyses: microdevices and applications. Lab Chip 10:3363–3372.
  29. Liu Y, Lakshmipathy U, Ozgenc A, Thyagarajan B, Lieu P, Fontes A, Xue H, Scheyhing K, MacArthur C, Chesnut JD (2010) hESC engineering by integrase-mediated chromosomal targeting. In: Turksen K (ed) Human embryonic stem cell protocols, vol 584. Totowa, NJ, Humana Press, pp 229–268. doi: 10.1007/978-1-60761-369-5
  30. Liu Y, Judd K, Lakshmipathy U (2013) Stable transfection using episomal vectors to create modified human embryonic stem cells. In: Lakshmipathy U, Vemuri MC (eds) Pluripotent stem cells, Chapter 21, vol 997. Totowa, NJ, Humana Press, pp 263–272. doi: 10.1007/978-1-62703-348-0
  31. Mantel C, Guo Y, Lee MR, Kim M-K, Han M-K, Shibayama H, Fukuda S, Yoder MC, Pelus LM, Kim K-S, Broxmeyer HE (2007) Checkpoint-apoptosis uncoupling in human and mouse embryonic stem cells: a source of karyotypic instability. Blood 109(10):4518–4527. doi: 10.1182/blood-2006-10-054247 CrossRefGoogle Scholar
  32. McCarty OJT, Jadhav S, Burdick MM, Bell WR, Konstantopoulos K (2002) Fluid shear regulates the kinetics and molecular mechanisms of activation-dependent platelet binding to colon carcinoma cells. Biophys J 83:836–848. doi: 10.1016/S0006-3495(02)75212-0 CrossRefGoogle Scholar
  33. Mittal S, Wong IY, Deen WM, Toner M (2012) Antibody-functionalized fluid-permeable surfaces for rolling cell capture at high flow rates. Biophys J 102(4):721–730. doi: 10.1016/j.bpj.2011.12.044 CrossRefGoogle Scholar
  34. Murthy SK, Sin A, Tompkins RG, Toner M (2004) Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers. Langmuir 20:11649–11655.
  35. Myung JH, Launiere CA, Eddington DT, Hong S (2010) Enhanced tumor cell isolation by a biomimetic combination of E-selectin and anti-EpCAM: implications for the effective separation of circulating tumor cells (CTCs). Langmuir 26(11):8589–8596. doi: 10.1021/la904678p CrossRefGoogle Scholar
  36. Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, Kwak EL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, Tompkins RG, Haber DA, Toner M (2007) Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450:1235–1239.
  37. Plouffe BD, Kniazeva T, Mayer JE, Murthy SK, Sales VL (2009) Development of microfluidics as endothelial progenitor cell capture technology for cardiovascular tissue engineering and diagnostic medicine. FASEB J 23:3309–3314. doi: 10.1096/fj.09-130260 CrossRefGoogle Scholar
  38. Pratt ED, Huang C, Hawkins BG, Gleghorn JP, Kirby BJ (2011) Rare cell capture in microfluidic devices. Chem Eng Sci 66(7):1508–1522. doi: 10.1016/j.ces.2010.09.012 CrossRefGoogle Scholar
  39. Qiu D, Xiang J, Li Z, Krishnamoorthy A, Chen L, Wang R (2008) Profiling TRA-1-81 antigen distribution on a human embryonic stem cell. Biochem Biophys Res Commun 369(2):735–740. doi: 10.1016/j.bbrc.2008.02.102 CrossRefGoogle Scholar
  40. Quinlan LR (2006) Phosphoinositides, inositol phosphates, and phospholipase C in embryonic stem cells. In: Methods in molecular biology: embryonic stem cell protocols, vol 329, pp 127–149Google Scholar
  41. Reverberi R, Reverberi L (2007) Factors affecting the antigen-antibody reaction. Blood transfusion Trasfusione del sangue 5(4):227–240. doi: 10.2450/2007.0047-07 Google Scholar
  42. Schriebl K, Satianegara G, Hwang A, Tan HL, Fong WJ, Yang HH, Jungbauer A, Choo A (2012) Selective removal of undifferentiated human embryonic stem cells using magnetic activated cell sorting followed by a cytotoxic antibody. Tissue Eng Part A 18. doi: 10.1089/ten.tea.2011.0311
  43. Sekine K, Revzin A, Tompkins RG, Toner M (2006) Panning of multiple subsets of leukocytes on antibody-decorated poly(ethylene) glycol-coated glass slides. J Immunol Methods 313:96–109.
  44. Sidhu KS, Tuch BE (2006) Cell lines by FACS sorting and their characterization. J Biol Chem 69:61–69Google Scholar
  45. Singh A, Suri S, Lee T, Chilton JM, Cooke MT, Chen W, Fu J, Stice SL, Lu H, McDevitt TC, García AJ (2013) Adhesion strength-based, label-free isolation of human pluripotent stem cells. Nat Methods 10(5):438–444. doi: 10.1038/nmeth.2437 CrossRefGoogle Scholar
  46. Stewart MH, Bossé M, Chadwick K, Menendez P, Bendall SC, Bhatia M (2006) Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nature 3(10):807–815. doi: 10.1038/NMETH939
  47. Stott SL, Hsu C-H, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, Rothenberg M, Shah AM, Smas ME, Korir GK, Floyd FP Jr., Gilman AJ, Lord JB, Winokur D, Springer S, Irimia D, Nagrath S, Sequist LV, Lee RJ, Isselbacher KJ, Maheswaran S, Haber DA, Toner M (2010) Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci USA 107:18392–18397. doi: 10.1073/pnas.1012539107 CrossRefGoogle Scholar
  48. Tárnok A, Ulrich H, Bocsi J (2010) Phenotypes of stem cells from diverse origin. Cytometry Part A 77:6–10.
  49. Thomson JA (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147. doi: 10.1126/science.282.5391.1145 CrossRefGoogle Scholar
  50. Toh YC, Voldman J (2011) Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction. FASEB J 25:1208–1217.
  51. Velugotla S, Pells S, Mjoseng HK, Duffy CRE, Smith S, De Sousa P, Pethig R (2012) Dielectrophoresis based discrimination of human embryonic stem cells from differentiating derivatives. Biomicrofluidics 6(4):44113. doi: 10.1063/1.4771316 CrossRefGoogle Scholar
  52. Wang S, Wang H, Jiao J, Chen KJ, Owens GE, Kamei K, Sun J, Sherman DJ, Behrenbruch CP, Wu H, Tseng HR (2009) Three-dimensional nanostructured substrates toward efficient capture of circulating tumor cells. Angew Chem Int Ed 48:8970–8973.
  53. Wang S, Liu K, Liu J, Yu ZT-F, Xu X, Zhao L, Lee T, Lee EK, Reiss J, Lee Y-K, Chung LWK, Huang J, Rettig M, Seligson D, Duraiswamy KN, Shen CK-F, Tseng H-R (2011a) Highly efficient capture of circulating tumor cells by using nanostructured silicon substrates with integrated chaotic micromixers. Angew Chem Int Ed 50:3084–3088. doi: 10.1002/anie.201005853 CrossRefGoogle Scholar
  54. Wang X, Chen S, Kong M, Wang Z, Costa KD, Li RA, Sun D (2011b) Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies. Lab Chip 11(21):3656–3662. doi: 10.1039/c1lc20653b CrossRefGoogle Scholar
  55. Zhao W, Ji X, Zhang F, Li L, Ma L (2012) Embryonic stem cell markers. Molecules (Basel, Switzerland) 17(6):6196–6236. doi: 10.3390/molecules17066196 CrossRefGoogle Scholar
  56. Zheng X, Cheung LS-L, Schroeder JA, Jiang L, Zohar Y (2011) Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells. Lab Chip 11(20):3431–3439. doi: 10.1039/c1lc20455f CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • E. Jabart
    • 1
  • S. Rangarajan
    • 1
  • C. Lieu
    • 2
  • J. Hack
    • 3
  • I. Conboy
    • 1
  • L. L. Sohn
    • 3
    Email author
  1. 1.Department of BioengineeringUniversity of CaliforniaBerkeleyUSA
  2. 2.School of MedicineCreighton UniversityOmahaUSA
  3. 3.Department of Mechanical EngineeringUniversity of CaliforniaBerkeleyUSA

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