Skip to main content
SpringerLink
Log in

A Simple Pipetting-based Method for Encapsulating Live Cells into Multi-layered Hydrogel Droplets

  • Original Article
  • Published:
BioChip Journal Aims and scope Submit manuscript

Abstract

Here, we present a simple pipetting-based approach for generating multi-layered core-shell hydrogel droplets composed of alginate for outer shell and collagen for culturing cells inside. By using a multi-hole plastic substrate and by pipetting, multi-layered hydrogel droplets were generated in a simple and rapid manner. HEK293 cells, which are human embryonic kidney cells, were cultured for 14 days in double-layered hydrogel droplet with high viability. Cancer cells were co-cultured with epithelial cells in multi-layered hydrogel droplets and applied for drug tests with curcumin. As epithelial cells protect cancer cells from anti-cancer drugs, co-cultured cells showed lower sensitivity to curcumin. We developed a simple and easy method for creating complex hydrogel particles for 3D multicellular co-culture and developed an alternative method for drug testing in vivo.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Griffith, L.G. & Swartz, M.A. Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 7, 211–224 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Pampaloni, F., Reynaud, E.G. & Stelzer, E.H.K. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839–845 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Breslin, S. & O’Driscoll, L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov. Today 18, 240–249 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Bhadriraju, K. & Chen, C.S. Engineering cellular microenvironments to improve cell-based drug testing. Drug Discov. Today 7, 612–620 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Elliott, N.T. & Yuan, F. A review of three-dimensional in vitro tissue models for drug discovery and transport studies. J. Pharmaceutical Sci. 100, 59–74 (2011).

    Article  CAS  Google Scholar 

  6. Breslin, S. & O’Driscoll, L. The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance. Oncotarget 7, 45745–45756 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Petersen, O.W., Ronnovjessen, L., Howlett, A.R. & Bissell, M.J. Interaction with basement-membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc. Natl. Acad. Sci. USA 89, 9064–9068 (1992).

    Article  CAS  PubMed  Google Scholar 

  8. Tanaka, H. et al. Chondrogenic differentiation of murine embryonic stem cells: effects of culture conditions and dexamethasone. J. Cell Biochem. 93, 454–462 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Abbott, A. Cell culture: biology’s new dimension. Nature 424, 870–872 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Nelson, C.M. & Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 22, 287–309 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee, J., Cuddihy, M.J. & Kotov, N.A. Three-Dimensional Cell Culture Matrices: State of the Art. Tissue Eng. 14, 61–86 (2008).

    Article  CAS  Google Scholar 

  12. Pageau, S.C., Sazonova, O.V., Wong, J.Y., Soto, A.M. & Sonnenschein, C. The effect of stromal components on the modulation of the phenotype of human bronchial epithelial cells in 3D culture. Biomaterials 32, 7169–7180 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Luca, A.C. et al. Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines. PLoS One 8, e59689 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tibbitt, M.W. & Anesth, K.S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng 103, 655–663 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Saha, K., Pollock, J.F., Schaffer, D.V. & Healy, K.E. Designing synthetic materials to control stem cell phenotype. Curr. Opin. Chem. Biol. 11, 381–387 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nguyen, K.T. & West, J.L. Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 23, 4307–4314 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Lutolf, M.P. & Hubbell, J.A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23, 47–55 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Jongpaiboonkit, L. et al. An adaptable hydrogel array format for 3-dimensional cell culture and analysis. Biomaterials. 29, 3346–3356 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Smrdel, P. et al. Characterization of calcium alginate beads containing structurally similar drugs. Drug Dev. Ind. Pharm. 32, 623–633 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Rowley, J.A., Madlambayan, G. & Mooney, D.J. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20, 45–53 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Lee, K.Y. & Mooney, D.J. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37, 106–126 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Annan, N.T., Borza, A.D. & Hansen, L.T. Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions. Food Res. Int. 41, 184–193 (2008).

    Article  CAS  Google Scholar 

  23. Butcher, J.T. & Nerem, R.M. Porcine aortic valve interstitial cells in three-dimensional culture: comparison of phenotype with aortic smooth muscle cells. J. Heart Valve Dis. 13, 478–485 (2004).

    PubMed  Google Scholar 

  24. Golden, A.P. & Tien, J. Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial element. Lab Chip 7, 720–725 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Lee, W. et al. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. Biomaterials 30, 1587–1595 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Jeong, S.H., Lee, D.W., Kim, S., Kim, J. & Ku, B. A study of electrochemical biosensor for analysis of three-dimensional (3D) cell culture. Biosens. Bioelectron. 35, 128–133 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Hwang, H., Park, J., Shin, C., Do, Y. & Cho, Y.-K. Three dimensional multicellular co-cultures and anti-cancer drug assays in rapid prototyped multilevel microfluidic devices. Biomed. Microdevices 15, 627–634 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Sung, K.E. et al. Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices. Biomaterials 30, 4833–4841 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Shamloo, A., Mohammadaliha, N., Heilshorn, S.C. & Bauer, A.L. A comparative study of collagen matrix density effect on endothelial sprout formation using experimental and computational approaches. Ann. Biomed. Eng. 44, 929–941 (2016).

    Article  PubMed  Google Scholar 

  30. Weaver, V.M. et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol. 137, 231–245 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bissell, M.J., Rizki, A. & Mian, I.S. Tissue architecture: the ultimate regulator of breast epithelial function. Curr. Opin. Cell Biol. 15, 753–762 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhang, K. et al. The collagen receptor discodin domain receptor 2 stabilizes SNAIL1 to facilitate breast cancer metastasis. Nat. Cell Biol. 15, 677–687 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Riching, K.M. et al. 3D collagen alignment limits protrusions to enhance breast cancer cell persistence. Biophys. J. 107, 2546–2558 (2014).

    CAS  Google Scholar 

  34. Koh, W.G., Revzin, A. & Pishko, M.V. Poly(ethylene glycol) hydrogel microstructures encapsulating living cells. Langmuir 18, 2459–2462 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Liu, V.A. & Bhatia, S.N. Three-dimensional photopatterning of hydrogels containing living cells. Biomed. Microdevices 4, 257–266 (2002).

    Article  CAS  Google Scholar 

  36. Albrecht, D.R., Tsang, V.L., Sah, R.L. & Bhatia, S.N. Photo- and electropatterning of hydrogelencapsulated living cell arrays. Lab Chip 5, 111–118 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Smeds, K.A. et al. Photocrosslinkable polysaccharides for in situ hydrogel formation. J. Biomed. Mater. Res. 54, 115–121 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Chung, C. & Burdick, J.A. Influence of Three-dimensional hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis. Tissue Eng. A 15, 243–254 (2009).

    Article  CAS  Google Scholar 

  39. Nichol, J.W. et al. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 31, 5536–5544 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tan, W.-H. & Takeuchi, S. Monodisperse alginate hydrogel microbeads for cell encapsulation. Adv. Mater. 19, 2696–2701 (2007).

    Article  CAS  Google Scholar 

  41. Tsuda, Y., Morimoto, Y. & Takeuchi, S. Monodisperse cell-encapsulating peptide microgel beads for 3D cell culture. Langmuir 26, 2645–2649 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Franco, C.L., Price, J. & West, J.L. Development and optimization of a dual-photoinitiator, emulsion-based technique for rapid generation of cell-laden hydrogel microspheres. Acta Biomater. 7, 3267–3276 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Koh, W.-G. & Pishko, M.V. Fabrication of cell-containing hydrogel microstructures inside microfluidic devices that can be used as cell-based biosensors. Anal. Bioanal. Chem. 385, 1389–1397 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Yu, L. et al. Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation. Biomicrofluidics 9, 024118 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Chen, Q. et al. Controlled assembly of heterotypic cells in a core-shell scaffold: organ in a droplet. Lab Chip. 16, 1346–1349 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lu, Y.-C. et al. Designing compartmentalized hydrogel microparticles for cell encapsulation and scalable 3D cell culture. J. Mater. Chem. B 3, 353–360 (2015).

    Article  CAS  Google Scholar 

  47. Mahow, R., Meier, R.P.H., Bühler, L.H. & Wandrey, C. Alginate-Poly(ethylene glycol) Hybrid Microspheres for Primary Cell Microencapsulation. Materials 7, 275–286 (2014).

    Article  CAS  Google Scholar 

  48. Anderson, T., Auk-Emblem, P. & Dornish, M. 3D Cell Culture in Alginate Hydrogels. Microarrays 4, 133–161 (2015).

    Article  CAS  Google Scholar 

  49. Dietmair, S. et al. A Muiti-Omics Analysis of Recombinant protein Production in Hek293 cells. PLoS One 7, e43394 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K.-L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signaling. Nat. Cell Biol. 4, 648–657 (2002).

    Article  CAS  PubMed  Google Scholar 

  51. Seth, R.B., Sun, L., Ea, C.-K. & Chen, Z.J. Identification and Characterization of MAVS, a Mitochondrial Antiviral Signaling Protein the Activates NF-κB and IRF3. Cell 122, 669–682 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Mai, A. et al. Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1. J. Cell Sci. 127, 1938–1952 (2014).

    Article  CAS  PubMed  Google Scholar 

  53. Shao, N. et al. Hydrogen-bonding dramatically modulates the gene transfection efficacy of surface-engineered dendrimers. Biomater. Sci. 3, 500–508 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Yoon, M.J., Kim, E.H., Kim, J.H., Kwon, T.K. & Choi, K.S. Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells. Free Radic. Biol. Med. 48, 713–726 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Liu, Q., Loo, W.T.Y., Sze, S.C.W. & Tong, Y. Curcumin inhibits cell proliferation of MDA-MB-231 and BT-483 breast cancer cells mediated by down-regulation of NFB, cyclinD and MMP-1 transcription. Phytomedicine 16, 916–922 (2009).

    Article  CAS  PubMed  Google Scholar 

  56. Tate, T. On the magnitude of a drop of liquid formed under diffferent circumstances. Phil. Mag. 22, 176–180 (1864).

    Article  Google Scholar 

  57. Perez, R.A. et al. Utilizing core-shell fibrous collagen-alginate hydrogel cell delivery system for bone tissue engineering. Tissue Eng. A 20, 103–114 (2014).

    Article  CAS  Google Scholar 

  58. Carey, S.P., Starchenko, A., Mcgregor, A.L. & Reinhart-King, C.A. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model. Clin. Exp. Metastasis 30, 615–630 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Integrative Biosciences, University of Brain Education, Cheonan, Republic of Korea

    Ju Hun Yeon

  2. Korea Institute of Brain Science, Seoul, Republic of Korea

    Ju Hun Yeon

  3. School of Integrative Engineering, Chung-Ang University, Seoul, Republic of Korea

    Sung Hee Chung, Changyoon Baek & Junhong Min

  4. BBB Inc., Seoul, Republic of Korea

    Hyundoo Hwang

Authors
  1. Ju Hun Yeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sung Hee Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Changyoon Baek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hyundoo Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Junhong Min
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Junhong Min.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yeon, J.H., Chung, S.H., Baek, C. et al. A Simple Pipetting-based Method for Encapsulating Live Cells into Multi-layered Hydrogel Droplets. BioChip J 12, 184–192 (2018). https://doi.org/10.1007/s13206-018-2307-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-018-2307-z

Keywords

Search

Navigation