Skip to main content
SpringerLink
Log in

Cell Mimicking Microparticles Influence the Organization, Growth, and Mechanophenotype of Stem Cell Spheroids

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

Substrate stiffness is known to alter cell behavior and drive stem cell differentiation, though most research in this area has been restricted to traditional, two-dimensional culture systems rather than more physiologically relevant, three-dimensional (3D) platforms. In this study, we utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to human adipose derived stem cells in 3D spheroid culture to examine changes in adipogenic differentiation response and mechanophenotype. After 21 days of adipogenic induction, spheroids containing CMMPs (composite spheroids) stiffened in accordance with CMMP elasticity such that spheroids containing the stiffest, ~ 10 kPa, CMMPs were over 27% stiffer than those incorporating the most compliant, ~ 0.25 kPa CMMPs. Adipogenically induced, cell-only spheroids were over 180% larger and 50% more compliant than matched controls. Interestingly, composite spheroids cultured without chemical induction factors dissociated when presented with CMMPs stiffer than ~ 1 kPa, while adipogenic induction factors mitigated this behavior. Gene expression for PPARG and FABP4 were upregulated more than 45-fold in adipogenically induced samples compared to controls but were unaffected by CMMP elasticity, attributed to insufficient cell-CMMP contacts throughout the composite spheroid. In summary, mechanically tuned CMMPs influenced whole-spheroid mechanophenotype and stability but minimally affected differentiation response.

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

Abbreviations

AFM:

Atomic force microscopy

APS:

Ammonium persulfate

ASCs:

Adipose derived stem cells

CMMP:

Cell mimicking microparticle

E elastic :

Young’s modulus/elastic modulus

E R :

Relaxed modulus

E 0 :

Instantaneous modulus

FABP4:

Fatty acid binding protein 4

IBMX:

3-Isobutyl-1-methylxanthine

PAAm:

Polyacrylamide

PBS:

Phosphate buffered saline

PPARG:

Peroxisome proliferator-activated receptor gamma

TEMED:

Tetramethylethylenediamine

µ :

Apparent viscosity

2D:

Two-dimensional

3D:

Three-dimensional

References

  1. Achilli, T.-M., J. Meyer, and J. R. Morgan. Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin. Biol. Therapy 12:1347–1360, 2012.

    Article  CAS  Google Scholar 

  2. Anderson, S. B., C. C. Lin, D. V. Kuntzler, and K. S. Anseth. The performance of human mesenchymal stem cells encapsulated in cell-degradable polymer-peptide hydrogels. Biomaterials 32:3564–3574, 2011.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Baraniak, P. R., M. T. Cooke, R. Saeed, M. A. Kinney, K. M. Fridley, and T. C. McDevitt. Stiffening of human mesenchymal stem cell spheroid microenvironments induced by incorporation of gelatin microparticles. J. Mech. Behav. Biomed. Mater. 11:63–71, 2012.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Baraniak, P. R., and T. C. McDevitt. Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res. 347:701–711, 2011.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Chaudhuri, O., L. Gu, D. Klumpers, M. Darnell, S. A. Bencherif, J. C. Weaver, N. Huebsch, H. P. Lee, E. Lippens, G. N. Duda, and D. J. Mooney. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15:326–334, 2016.

    Article  PubMed  CAS  Google Scholar 

  6. Cheng, N. C., S. Y. Chen, J. R. Li, and T. H. Young. Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl. Med. 2:584–594, 2013.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Darling, E. M., S. Zauscher, J. A. Block, and F. Guilak. A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential? Biophys. J . 92:1784–1791, 2007.

    Article  PubMed  CAS  Google Scholar 

  8. Darling, E. M., S. Zauscher, and F. Guilak. Viscoelastic properties of zonal articular chondrocytes measured by atomic force microscopy. Osteoarthr. Cartil. 14:571–579, 2006.

    Article  PubMed  CAS  Google Scholar 

  9. Dimitriadis, E. K., F. Horkay, J. Maresca, B. Kachar, and R. S. Chadwick. Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82:2798–2810, 2002.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Dolega, M. E., M. Delarue, F. Ingremeau, J. Prost, A. Delon, and G. Cappello. Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression. Nat. Commun. 8:14056, 2017.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Engler, A. J., S. Sen, H. L. Sweeney, and D. E. Discher. Matrix elasticity directs stem cell lineage specification. Cell 126:677–689, 2006.

    Article  PubMed  CAS  Google Scholar 

  12. Estes, B. T., B. O. Diekman, J. M. Gimble, and F. Guilak. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat. Protoc. 5:1294–1311, 2010.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Estes, B. T., B. O. Diekman, and F. Guilak. Monolayer cell expansion conditions affect the chondrogenic potential of adipose-derived stem cells. Biotechnol. Bioeng. 99:986–995, 2008.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Gonzalez-Cruz, R. D., V. C. Fonseca, and E. M. Darling. Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells. Proc. Natl. Acad. Sci. 109:E1523–E1529, 2012.

    Article  PubMed  Google Scholar 

  15. Guneta, V., Q. L. Loh, and C. Choong. Cell-secreted extracellular matrix formation and differentiation of adipose-derived stem cells in 3D alginate scaffolds with tunable properties. J. Biomed. Mater. Res. A 104:1090–1101, 2016.

    Article  PubMed  CAS  Google Scholar 

  16. Guo, W. H., M. T. Frey, N. A. Burnham, and Y. L. Wang. Substrate rigidity regulates the formation and maintenance of tissues. Biophys. J. 90:2213–2220, 2006.

    Article  PubMed  CAS  Google Scholar 

  17. Hayashi, K., and Y. Tabata. Preparation of stem cell aggregates with gelatin microspheres to enhance biological functions. Acta Biomater. 7:2797–2803, 2011.

    Article  PubMed  CAS  Google Scholar 

  18. Hielscher, A. H., J. R. Mourant, and I. J. Bigio. Influence of particle size and concentration on the diffuse backscattering of polarized light from tissue phantoms and biological cell suspensions. Appl. Opt. 36:125–135, 1997.

    Article  PubMed  CAS  Google Scholar 

  19. Huebsch, N., P. R. Arany, A. S. Mao, D. Shvartsman, O. A. Ali, S. A. Bencherif, J. Rivera-Feliciano, and D. J. Mooney. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9:518–526, 2010.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Kilian, K. A., B. Bugarija, B. T. Lahn, and M. Mrksich. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl. Acad. Sci. 107:4872–4877, 2010.

    Article  PubMed  Google Scholar 

  21. Kumachev, A., J. Greener, E. Tumarkin, E. Eiser, P. W. Zandstra, and E. Kumacheva. High-throughput generation of hydrogel microbeads with varying elasticity for cell encapsulation. Biomaterials 32:1477–1483, 2011.

    Article  PubMed  CAS  Google Scholar 

  22. Labriola, N. R., A. Azagury, R. Gutierrez, E. Mathiowitz, and E. M. Darling. Concise review: Fabrication, customization, and application of cell mimicking microparticles in stem cell science. Stem Cells Transl. Med. 7:232–240, 2018.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Labriola, N. R., and E. M. Darling. Temporal heterogeneity in single-cell gene expression and mechanical properties during adipogenic differentiation. J. Biomech. 48:1058–1066, 2015.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Labriola, N. R., E. Mathiowitz, and E. M. Darling. Fabricating polyacrylamide microbeads by inverse emulsification to mimic the size and elasticity of living cells. Biomater. Sci. 5:41–45, 2017.

    Article  CAS  Google Scholar 

  25. Lo Surdo, J., and S. R. Bauer. Quantitative approaches to detect donor and passage differences in adipogenic potential and clonogenicity in human bone marrow-derived mesenchymal stem cells. Tissue Eng. Part C Methods 18:877–889, 2012.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Loh, Q. L., and C. Choong. Three-dimensional scaffolds for tissue engineering applications: Role of porosity and pore size. Tissue Eng. Part B Rev. 19:485–502, 2013.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. McBeath, R., D. M. Pirone, C. M. Nelson, K. Bhadriraju, and C. S. Chen. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 6:483–495, 2004.

    Article  PubMed  CAS  Google Scholar 

  28. Napolitano, A., D. Dean, A. Man, J. Youssef, D. Ho, A. Rago, M. Lech, and J. Morgan. Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels. Biotechniques 43:494–500, 2007.

    Article  PubMed  CAS  Google Scholar 

  29. Nobusue, H., N. Onishi, T. Shimizu, E. Sugihara, Y. Oki, Y. Sumikawa, T. Chiyoda, K. Akashi, H. Saya, and K. Kano. Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation. Nat. Commun. 5:3368, 2014.

    Article  PubMed  CAS  Google Scholar 

  30. Parekh, S. H., K. Chatterjee, S. Lin-Gibson, N. M. Moore, M. T. Cicerone, M. F. Young, and C. G. Simon, Jr. Modulus-driven differentiation of marrow stromal cells in 3D scaffolds that is independent of myosin-based cytoskeletal tension. Biomaterials 32:2256–2264, 2011.

    Article  PubMed  CAS  Google Scholar 

  31. Shah, M. K., I. H. Garcia-Pak, and E. M. Darling. Influence of inherent mechanophenotype on competitive cellular adherence. Ann. Biomed. Eng. 45:2036–2046, 2017.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Silver, N., S. Best, J. Jiang, and S. Thein. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol. Biol. 7:33, 2006.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Singh, M., C. P. Morris, R. J. Ellis, M. S. Detamore, and C. Berkland. Microsphere-based seamless scaffolds containing macroscopic gradients of encapsulated factors for tissue engineering. Tissue Eng. Part C 14:299–309, 2008.

    Article  CAS  Google Scholar 

  34. Tse, J. R., and A. J. Engler. Preparation of hydrogel substrates with tunable mechanical properties. Curr Protoc Cell Biol 2010. https://doi.org/10.1002/0471143030.cb1016s47.

    Article  PubMed  Google Scholar 

  35. Yeung, T., P. C. Georges, L. A. Flanagan, B. Marg, M. Ortiz, M. Funaki, N. Zahir, W. Ming, V. Weaver, and P. A. Janmey. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskelet. 60:24–34, 2005.

    Article  Google Scholar 

  36. Young, D. A., Y. S. Choi, A. J. Engler, and K. L. Christman. Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue. Biomaterials 34:8581–8588, 2013.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Zheng, B., B. Cao, G. Li, and J. Huard. Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo. Tissue Eng. 12:1891–1901, 2006.

    Article  PubMed  CAS  Google Scholar 

  38. Zoldan, J., E. D. Karagiannis, C. Y. Lee, D. G. Anderson, R. Langer, and S. Levenberg. The influence of scaffold elasticity on germ layer specification of human embryonic stem cells. Biomaterials 32:9612–9621, 2011.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Manisha K. Shah for her assistance with confocal imaging. This work was supported by awards from the National Institute of General Medical Sciences (EMD, P20 GM104937), National Institute of Arthritis and Musculoskeletal and Skin Diseases (EMD, R01 AR063642), and the National Science Foundation (EMD, CAREER Award, CBET1253189). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation or National Institutes of Health.

Conflict of interest

NRL, EM, and EMD have patent filings relevant to the technology in this study. EMD owns MimicSphere, LLC, which focuses on the same technology.

Author information

Authors and Affiliations

  1. Center for Biomedical Engineering, Brown University, 175 Meeting Street, Box G-B397, Providence, RI, 02912, USA

    Nicholas R. Labriola, Jeffrey R. Morgan, Edith Mathiowitz & Eric M. Darling

  2. Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA

    Jessica S. Sadick, Jeffrey R. Morgan, Edith Mathiowitz & Eric M. Darling

  3. School of Engineering, Brown University, Providence, RI, USA

    Jeffrey R. Morgan, Edith Mathiowitz & Eric M. Darling

  4. Department of Orthopaedics, Brown University, Providence, RI, USA

    Eric M. Darling

Authors
  1. Nicholas R. Labriola
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jessica S. Sadick
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeffrey R. Morgan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Edith Mathiowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eric M. Darling
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

NRL and EMD designed the study. NRL performed all CMMP/substrate preparation, cell culture, mechanical testing, and imaging. NRL, EMD, and JSS analyzed the data. NRL, EMD, JSS, JRM, and EM wrote and edited the manuscript. JRM and EM provided materials and consultation on the design, execution, and interpretation of the experiment and data sets.

Corresponding author

Correspondence to Eric M. Darling.

Additional information

Associate Editor Debra T. Auguste oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1507 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Labriola, N.R., Sadick, J.S., Morgan, J.R. et al. Cell Mimicking Microparticles Influence the Organization, Growth, and Mechanophenotype of Stem Cell Spheroids. Ann Biomed Eng 46, 1146–1159 (2018). https://doi.org/10.1007/s10439-018-2028-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-018-2028-4

Keywords

Search

Navigation