Hostname: page-component-8448b6f56d-jr42d Total loading time: 0 Render date: 2024-04-16T22:11:16.744Z Has data issue: false hasContentIssue false
  • English
  • Français

Dynamic bioengineered hydrogels as scaffolds for advanced stem cell and organoid culture

Published online by Cambridge University Press:  29 August 2017

Laura C. Bahlmann ,
Ana Fokina  and
Molly S. Shoichet
Laura C. Bahlmann
Affiliation:
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S3E1, Canada
Ana Fokina
Affiliation:
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada
Molly S. Shoichet*
Affiliation:
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S3E1, Canada Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada
*
Address all correspondence to Molly S. Shoichet at molly.shoichet@utoronto.ca
Get access

Abstract

Bioengineered hydrogels enable systematic variation of mechanical and biochemical properties, resulting in the identification of optimal in vitro three-dimensional culture conditions for individual cell types. As the scientific community attempts to mimic and study more complex biologic processes, hydrogel design has become multi-faceted. To mimic organ and tissue heterogeneity in terms of spatial arrangement and temporal changes, hydrogels with spatiotemporal control over mechanical and biochemical properties are needed. In this prospective article, we present studies that focus on the development of hydrogels with dynamic mechanical and biochemical properties, highlighting the discoveries made using these scaffolds.

Type
Biomaterials for 3D Cell Biology Prospective Article
Information
MRS Communications , Volume 7 , Issue 3 , September 2017 , pp. 472 - 486
Copyright
Copyright © Materials Research Society 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

These authors contributed equally to this work.

References

1.Nowak, M., Freudenberg, U., Tsurkan, M.V., Werner, C., and Levental, K.R.: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro. Biomaterials 112, 20 (2017).Google Scholar
2.Ranga, A., Girgin, M., Meinhardt, A., Eberle, D., Caiazzo, M., Tanaka, E.M., and Lutolf, M.P.: Neural tube morphogenesis in synthetic 3D microenvironments. Proc. Natl. Acad. Sci. U.S.A. 113, E6831 (2016).Google Scholar
3.Moeendarbary, E., Weber, I.P., Sheridan, G.K., Koser, D.E., Soleman, S., Haenzi, B., Bradbury, E.J., Fawcett, J., and Franze, K.: The soft mechanical signature of glial scars in the central nervous system. Nat. Commun. 8, 14787 (2017).Google Scholar
4.Mammoto, T., Mammoto, A., and Ingber, D.E.: Mechanobiology and developmental control. Annu. Rev. Cell Dev. Biol. 29, 27 (2013).Google Scholar
5.Heisenberg, C.-P. and Bellaïche, Y.: Forces in tissue morphogenesis and patterning. Cell 153, 948 (2013).Google Scholar
6.Bouchonville, N., Meyer, M., Gaude, C., Gay, E., Ratel, D., Nicolas, A., Reinhart-King, C.A., Margulies, S.S., Dembo, M., Boettiger, D., Hammer, D.A., Weaver, V.M., Shi, Y., and Robinson, S.P.: AFM mapping of the elastic properties of brain tissue reveals kPa μm−1 gradients of rigidity. Soft Mat. 12, 6232 (2016).Google Scholar
7.Budday, S., Sommer, G., Birkl, C., Langkammer, C., Haybaeck, J., Kohnert, J., Bauer, M., Paulsen, F., Steinmann, P., Kuhl, E., and Holzapfel, G.A.: Mechanical characterization of human brain tissue. Acta Biomater. 48, 319 (2017).Google Scholar
8.Rho, J.Y., Ashman, R.B., and Turner, C.H.: Young's modulus of trabecular and cortical bone material: Ultrasonic and microtensile measurements. J. Biomech. 26, 111 (1993).Google Scholar
9.Hoenig, E., Leicht, U., Winkler, T., Mielke, G., Beck, K., Peters, F., Schilling, A.F., and Morlock, M.M.: Mechanical properties of native and tissue-engineered cartilage depend on carrier permeability: a bioreactor study. Tissue Eng. A 19, 1534 (2013).Google Scholar
10.Bilston, L.E.: Brain Tissue Mechanical Properties in Biological and Medical Physics, Biomedical Engineering (Springer, New York, USA, 2011, vol. 69).Google Scholar
11.Engler, A.J., Sen, S., Sweeney, H.L., and Discher, D.E.: Matrix elasticity directs stem cell lineage specification. Cell 126, 677 (2006).Google Scholar
12.Trappmann, B., Gautrot, J.E., Connelly, J.T., Strange, D.G.T., Li, Y., Oyen, M.L., Cohen Stuart, M.A., Boehm, H., Li, B., Vogel, V., Spatz, J.P., Watt, F.M., and Huck, W.T.S.: Extracellular-matrix tethering regulates stem-cell fate. Nat. Mater. 11, 642 (2012).Google Scholar
13.Wen, J.H., Vincent, L.G., Fuhrmann, A., Choi, Y.S., Hribar, K.C., Taylor-Weiner, H., Chen, S., and Engler, A.J.: Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nat. Mater. 13, 979 (2014).Google Scholar
14.Leipzig, N.D. and Shoichet, M.S.: The effect of substrate stiffness on adult neural stem cell behavior. Biomaterials 30, 6867 (2009).Google Scholar
15.You, J., Park, S.-A., Shin, D.-S., Patel, D., Raghunathan, V.K., Kim, M., Murphy, C.J., Tae, G., and Revzin, A.: Characterizing the effects of heparin gel stiffness on function of primary hepatocytes. Tissue Eng. A 19, 2655 (2013).Google Scholar
16.Mittal, N., Tasnim, F., Yue, C., Qu, Y., Phan, D., Choudhury, Y., Tan, M.-H., and Yu, H.: Substrate stiffness modulates the maturation of human pluripotent stem-cell-derived hepatocytes. ACS Biomater. Sci. Eng. 2, 1649 (2016).Google Scholar
17.Nava, A., Mazza, E., Furrer, M., Villiger, P., and Reinhart, W.H.: In vivo mechanical characterization of human liver. Med. Image Anal. 12, 203 (2008).Google Scholar
18.Huwart, L., Peeters, F., Sinkus, R., Annet, L., Salameh, N., ter Beek, L.C., Horsmans, Y., and Van Beers, B.E.: Liver fibrosis: non-invasive assessment with MR elastography. NMR Biomed.. 19, 173 (2006).Google Scholar
19.Hanjaya-Putra, D., Yee, J., Ceci, D., Truitt, R., Yee, D., and Gerecht, S.: Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells. J. Cell. Mol. Med. 14, 2436 (2010).Google Scholar
20.Caliari, S.R., Vega, S.L., Kwon, M., Soulas, E.M., and Burdick, J.A.: Dimensionality and spreading influence MSC YAP/TAZ signaling in hydrogel environments. Biomaterials 103, 314 (2016).Google Scholar
21.Khetan, S., Guvendiren, M., Legant, W.R., Cohen, D.M., Chen, C.S., and Burdick, J.A.: Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nat. Mater. 12, 458 (2013).Google Scholar
22.Huebsch, N., Arany, P.R., Mao, A.S., Shvartsman, D., Ali, O.A., Bencherif, S.A., Rivera-feliciano, J., and Mooney, D.J.: Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9, 518 (2010).Google Scholar
23.Gjorevski, N., Sachs, N., Manfrin, A., Giger, S., Bragina, M.E., Ordóñez-Morán, P., Clevers, H., and Lutolf, M.P.: Designer matrices for intestinal stem cell and organoid culture. Nat. Publ. Gr. 539, 560 (2016).Google Scholar
24.Caliari, S.R., Perepelyuk, M., Soulas, E.M., Lee, G.Y., Wells, R.G., and Burdick, J.A.: Gradually softening hydrogels for modeling hepatic stellate cell behavior during fibrosis regression. Integr. Biol. 8, 720 (2016).Google Scholar
25.Boehnke, N., Cam, C., Bat, E., Segura, T., and Maynard, H.D.: Imine hydrogels with tunable degradability for tissue engineering. Biomacromolecules 16, 2101 (2015).Google Scholar
26.Kharkar, P.M., Kloxin, A.M., and Kiick, K.L.: Dually degradable click hydrogels for controlled degradation and protein release. J. Mater. Chem. B, Mater. Biol. Med. 2, 5511 (2014).Google Scholar
27.Young, J.L. and Engler, A.J.: Hydrogels with time-dependent material properties enhance cardiomyocyte differentiation in vitro. Biomaterials 32, 1002 (2011).Google Scholar
28.Yang, C., Tibbitt, M.W., Basta, L., and Anseth, K.S.: Mechanical memory and dosing influence stem cell fate. Nat. Mater. 13, 645 (2014).Google Scholar
29.Guvendiren, M. and Burdick, J.A.: Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics. Nat. Commun. 3, 792 (2012).Google Scholar
30.Stowers, R.S., Allen, S.C., and Suggs, L.J.: Dynamic phototuning of 3D hydrogel stiffness. Proc. Natl. Acad. Sci. U.S.A. 112, 1953 (2015).Google Scholar
31.Abdeen, A.A., Lee, J., Bharadwaj, N.A., Ewoldt, R.H., and Kilian, K.A.: Temporal modulation of stem cell activity using magnetoactive hydrogels. Adv. Healthc. Mater. 5, 2536 (2016).Google Scholar
32.Ubaidillah, U., Sutrisno, J., Purwanto, A., and Mazlan, S.A.: Recent progress on magnetorheological solids: materials, fabrication, testing, and applications. Adv. Eng. Mater. 17, 563 (2015).Google Scholar
33.Mitsumata, T., Ohori, S., Honda, A., and Kawai, M.: Magnetism and viscoelasticity of magnetic elastomers with wide range modulation of dynamic modulus. Soft Matter 9, 904 (2013).Google Scholar
34.Mayer, M., Rabindranath, R., Boerner, J., Hoerner, E., Bentz, A., Salgado, J., Han, H., Boese, H., Probst, J., Shamonin, M., Monkman, G.J., and Schlunck, G.: Ultra-soft PDMS-based magnetoactive elastomers as dynamic cell culture substrata. PLoS ONE 8, e76196 (2013).Google Scholar
35.Lei, Y. and Schaffer, D.V.: A fully defined and scalable 3D culture system for human stem cell expansion and differentiation. Proc. Natl. Acad. Sci. U. S. A. 110, E5039 (2013).Google Scholar
36.Li, Q., Lin, H., Wang, O., Qiu, X., Kidambi, S., Deleyrolle, L.P., Reynolds, B.A., and Lei, Y.: Scalable production of glioblastoma tumor-initiating cells in 3 dimension thermoreversible hydrogels. Sci. Rep. 6, 31915 (2016).Google Scholar
37.Cho, M.O., Li, Z., Shim, H.-E., Cho, I.-S., Nurunnabi, M., Park, H., Lee, K.Y., Moon, S.-H., Kim, K.-S., Kang, S.-W., and Huh, K.M.: Bioinspired tuning of glycol chitosan for 3D cell culture. NPG Asia Mater. 8, e309 (2016).Google Scholar
38.Wolfenson, H., Meacci, G., Liu, S., Stachowiak, M.R., Iskratsch, T., Ghassemi, S., Roca-Cusachs, P., O'Shaughnessy, B., Hone, J., and Sheetz, M.P.: Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices. Nat. Cell Biol. 18, 33 (2015).Google Scholar
39.Meacci, G., Wolfenson, H., Liu, S., Stachowiak, M.R., Iskratsch, T., Mathur, A., Ghassemi, S., Gauthier, N., Tabdanov, E., Lohner, J., Gondarenko, A., Chander, A.C., Roca-Cusachs, P., O'Shaughnessy, B., Hone, J., and Sheetz, M.P.: α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions. Mol. Biol. Cell 27, 3471 (2016).Google Scholar
40.Whang, M. and Kim, J.: Synthetic hydrogels with stiffness gradients for durotaxis study and tissue engineering scaffolds. Tissue Eng. Regen. Med. 13, 126 (2016).Google Scholar
41.Tingting, X., Wanqian, L., and Li, Y.: A review of gradient stiffness hydrogels used in tissue engineering and regenerative medicine. J. Biomed. Mater. Res. A 105, 1799 (2017).Google Scholar
42.Tse, J.R. and Engler, A.J.: Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate. PLoS ONE 6, e15978 (2011).Google Scholar
43.Yang, C., DelRio, F.W., Ma, H., Killaars, A.R., Basta, L.P., Kyburz, K.A., and Anseth, K.S.: Spatially patterned matrix elasticity directs stem cell fate. Proc. Natl. Acad. Sci. U.S.A. 113, E4439 (2016).Google Scholar
44.Lutolf, M.P., Lauer-Fields, J.L., Schmoekel, H.G., Metters, A.T., Weber, F.E., Fields, G.B., and Hubbell, J.A.: Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc. Natl. Acad. Sci. U.S.A. 100, 5413 (2003).Google Scholar
45.Fisher, S.A., Anandakumaran, P.N., Owen, S.C., and Shoichet, M.S.: Tuning the microenvironment: click-crosslinked hyaluronic acid-based hydrogels provide a platform for studying breast cancer cell invasion. Adv. Funct. Mater. 25, 7163 (2015).Google Scholar
46.Schultz, K.M., Kyburz, K.A., and Anseth, K.S.: Measuring dynamic cell–material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels. Proc. Natl. Acad. Sci. U.S.A. 112, E3757 (2015).Google Scholar
47.Tong, X. and Yang, F.: Sliding hydrogels with mobile molecular ligands and crosslinks as 3D stem cell niche. Adv. Mater. 28, 7257 (2016).Google Scholar
48.Liu, Z. and Bilston, L.: On the viscoelastic character of liver tissue: experiments and modelling of the linear behaviour. Biorheology 37, 191 (2000).Google Scholar
49.Geerligs, M., Peters, G.W.M., Ackermans, P.A.J., Oomens, C.W.J., and Baaijens, F.P.T.: Linear viscoelastic behavior of subcutaneous adipose tissue. Biorheology 45, 677 (2008).Google Scholar
50.McDonald, S.J., Dooley, P.C., McDonald, A.C., Schuijers, J.A., Ward, A.R., and Grills, B.L.: Early fracture callus displays smooth muscle-like viscoelastic properties ex vivo: implications for fracture healing. J. Orthop. Res. 27, 1508 (2009).Google Scholar
51.Chaudhuri, O., Gu, L., Darnell, M., Klumpers, D., Bencherif, S.A., Weaver, J.C., Huebsch, N., and Mooney, D.J.: Substrate stress relaxation regulates cell spreading. Nat. Commun. 6, 6365 (2015).Google Scholar
52.Chaudhuri, O., Gu, L., Klumpers, D., Darnell, M., Bencherif, S.A., Weaver, J.C., Huebsch, N., Lee, H.-P., Lippens, E., Duda, G.N., and Mooney, D.J.: Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15, 326 (2015).Google Scholar
53.Tao, J., Wang, H., Lin, Q., Shen, H., and Li, L.S.: Quantum-dot-based light-emitting diodes with improved brightness and stability by using sulfuric acid-treated PEDOT:PSS as efficient hole injection layer. IEEE Trans. Nanotechnol. 14, 57 (2015).Google Scholar
54.Burdick, J.A. and Prestwich, G.D.: Hyaluronic acid hydrogels for biomedical applications. Adv. Mater. 23, H41 (2011).Google Scholar
55.Fisher, S.A., Tam, R.Y., and Shoichet, M.S.: Tissue mimetics: engineered hydrogel matrices provide biomimetic environments for cell growth. Tissue Eng. A 20, 895 (2014).Google Scholar
56.Nie, T., Akins, R.E., and Kiick, K.L.: Production of heparin-containing hydrogels for modulating cell responses. Acta Biomater.. 5, 865 (2009).Google Scholar
57.Nguyen, H.H., Payré, B., Fitremann, J., Lauth-De Viguerie, N., and Marty, J.D.: Thermoresponsive properties of PNIPAM-based hydrogels: effect of molecular architecture and embedded gold nanoparticles. Langmuir 31, 4761 (2015).Google Scholar
58.Chwalek, K., Tsurkan, M.V., Freudenberg, U., and Werner, C.: Glycosaminoglycan-based hydrogels to modulate heterocellular communication in in vitro angiogenesis models. Sci. Rep. 4, 4414 (2014).Google Scholar
59.Hettiaratchi, M.H., Chou, C., Servies, N., Smeekens, J.M., Cheng, A., Esancy, C., Wu, R., McDevitt, T.C., Guldberg, R.E., and Krishnan, L.: Competitive protein binding influences heparin-based modulation of spatial growth factor delivery for bone regeneration. Tissue Eng. A 23, 683 (2017).Google Scholar
60.Hettiaratchi, M.H., Miller, T., Temenoff, J.S., Guldberg, R.E., and McDevitt, T.C.: Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2. Biomaterials 35, 7228 (2014).Google Scholar
61.Chalovich, J.M. and Eisenberg, E.: Heparin mimicking polymer promotes myogenic differentiation of muscle progenitor cells. Biophys. Chem. 257, 2432 (2005).Google Scholar
62.Christman, K.L., Vázquez-Dorbatt, V., Schopf, E., Kolodziej, C.M., Li, R.C., Broyer, R.M., Chen, Y., and Maynard, H.D.: Nanoscale growth factor patterns by immobilization on a heparin-mimicking polymer. J. Am. Chem. Soc. 130, 16585 (2008).Google Scholar
63.Lin, L., Marchant, R.E., Zhu, J., and Kottke-Marchant, K.: Extracellular matrix-mimetic poly(ethylene glycol) hydrogels engineered to regulate smooth muscle cell proliferation in 3-D. Acta Biomater. 10, 5106 (2014).Google Scholar
64.Tian, Y.F., Ahn, H., Schneider, R.S., Yang, S.N., Roman-Gonzalez, L., Melnick, A.M., Cerchietti, L., and Singh, A.: Integrin-specific hydrogels as adaptable tumor organoids for malignant B and T cells. Biomaterials 73, 110 (2015).Google Scholar
65.Gould, S.T. and Anseth, K.S.: Role of cell-matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels. J. Tissue Eng. Regen. Med. 10, E443 (2016).Google Scholar
66.Enemchukwu, N.O., Cruz-Acuna, R., Bongiorno, T., Johnson, C.T., Garcia, J.R., Sulchek, T., and Garcia, A.J.: Synthetic matrices reveal contributions of ECM biophysical and biochemical properties to epithelial morphogenesis. J. Cell Biol. 212, 113 (2016).Google Scholar
67.Lee, T.T., García, J.R., Paez, J.I., Singh, A., Phelps, E.A., Weis, S., Shafiq, Z., Shekaran, A., Del Campo, A., and García, A.J.: Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials. Nat. Mater. 14, 352 (2015).Google Scholar
68.Kloxin, A.M., Kasko, A.M., Salinas, C.N., and Anseth, K.S.: Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59 (2009).Google Scholar
69.DeForest, C.A. and Tirrell, D.A.: A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels. Nat. Mater. 14, 523 (2015).Google Scholar
70.Goubko, C.A., Majumdar, S., Basak, A., and Cao, X.: Hydrogel cell patterning incorporating photocaged RGDS peptides. Biomed. Microdevices 12, 555 (2010).Google Scholar
71.Luo, Y. and Shoichet, M.S.: Light-activated immobilization of biomolecules to agarose hydrogels for controlled cellular response. Biomacromolecules 5, 2315 (2004).Google Scholar
72.Wegner, S.V., Sentürk, O.I., and Spatz, J.P.: Photocleavable linker for the patterning of bioactive molecules. Sci. Rep. 5, 18309 (2015).Google Scholar
73.Tsurkan, M.V., Wetzel, R., Pérez-Hernández, H.R., Chwalek, K., Kozlova, A., Freudenberg, U., Kempermann, G., Zhang, Y., Lasagni, A.F., and Werner, C.: Photopatterning of multifunctional hydrogels to direct adult neural precursor cells. Adv. Healthc. Mater. 4, 516 (2015).Google Scholar
74.Mosiewicz, K.A., Kolb, L., van der Vlies, A.J., Martino, M.M., Lienemann, P.S., Hubbell, J.A., Ehrbar, M., and Lutolf, M.P.: In situ cell manipulation through enzymatic hydrogel photopatterning. Nat. Mater. 12, 1072 (2013).Google Scholar
75.Owen, S.C., Fisher, S.A., Tam, R.Y., Nimmo, C.M., and Shoichet, M.S.: Hyaluronic acid click hydrogels emulate the extracellular matrix. Langmuir 29, 7393 (2013).Google Scholar
76.Gao, B., Konno, T., and Ishihara, K.: Building cell-containing multilayered phospholipid polymer hydrogels for controlling the diffusion of a bioactive reagent. RSC Adv. 5, 44408 (2015).Google Scholar
77.Wosnick, J.H. and Shoichet, M.S.: Three-dimensional chemical patterning of transparent hydrogels. Chem. Mater. 20, 55 (2008).Google Scholar
78.Wylie, R.G., Ahsan, S., Aizawa, Y., Maxwell, K.L., Morshead, C.M., and Shoichet, M.S.: Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nat. Mater. 10, 799 (2011).Google Scholar
79.Aizawa, Y., Wylie, R., and Shoichet, M.: Endothelial cell guidance in 3D patterned scaffolds. Adv. Mater. 22, 4831 (2010).Google Scholar
80.Mahmoodi, M.M., Fisher, S.A., Tam, R.Y., Goff, P.C., Anderson, R.B., Wissinger, J.E., Blank, D.A., Shoichet, M.S., Distefano, M.D.: 6-Bromo-7-hydroxy-3-methylcoumarin (mBhc) is an efficient multi-photon labile protecting group for thiol caging and three-dimensional chemical patterning. Org. Biomol. Chem. 14, 8289 (2016).Google Scholar
81.Souza, G.R., Molina, J.R., Raphael, R.M., Ozawa, M.G., Stark, D.J., Levin, C.S., Bronk, L.F., Ananta, J.S., Mandelin, J., Georgescu, M.-M., Bankson, J.A., Gelovani, J.G., Killian, T.C., Arap, W., and Pasqualini, R.: Three-dimensional tissue culture based on magnetic cell levitation. Nat. Nanotechnol. 5, 291 (2010).Google Scholar
82.Bratt-Leal, A.M., Kepple, K.L., Carpenedo, R.L., Cooke, M.T., and McDevitt, T.C.: Magnetic manipulation and spatial patterning of multi-cellular stem cell aggregates. Integr. Biol. 3, 1224 (2011).Google Scholar
83.Mabry, K.M., Schroeder, M.E., Payne, S.Z., and Anseth, K.S.: Three-dimensional high-throughput cell encapsulation platform to study changes in cell-matrix interactions. ACS Appl. Mater. Interfaces 8, 21914 (2016).Google Scholar
84.Chan, H.F., Zhang, Y., Ho, Y.-P., Chiu, Y.-L., Jung, Y., and Leong, K.W.: Rapid formation of multicellular spheroids in double-emulsion droplets with controllable microenvironment. Sci. Rep. 3, 3462 (2013).Google Scholar