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Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

ABSTRACT: Hydrogels have gained significant popularity as model platforms to study the reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within 96-well plates for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and was used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for cell culture applications was demonstrated by measuring both population-level and single cell-level metrics via microplate reader and high-content imaging. Finally, the 96-well hydrogel array was utilized for 3D cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adoptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

SUBMITTER: Skelton ML

PROVIDER: S-EPMC10592709 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

Skelton Mackenzie L ML Gentry James L JL Astrab Leilani R LR Goedert Joshua A JA Earl E Brynn EB Pham Emily L EL Bhat Tanvi T Caliari Steven R SR

bioRxiv : the preprint server for biology 20231012

Hydrogels have gained significant popularity as model platforms to study the reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within 96-well plates for increased throughput ...[more]

PMID: 37873098

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Project description:In vitro follicle growth is a promising technology to preserve fertility for cancer patients. We previously developed a three-dimensional (3-D) ovarian tissue culture system supported by mouse tumor cell-derived Matrigel. When murine ovarian tissues at 14 days old were cultured in Matrigel drops, antrum formation and oocyte competence were significantly enhanced compared with those cultured without Matrigel. In this study, we tested whether nonanimal-derived dextran hydrogels can support a 3-D ovarian tissue culture. We employed chemically defined dextran hydrogels consisting of dextran polymers crosslinked with polyethylene glycol (PEG)-based cell-degradable crosslinker. To determine the optimal gel elasticity for the 3-D tissue culture, we measured Young's modulus of dextran hydrogels at four concentrations (1.75, 2.25, 2.75, and 3.25 mmol/L), and cultured ovarian tissues in these gels for 7 days. As a result, 2.25 mmol/L dextran hydrogel with Young's modulus of 224 Pa was appropriate to provide physical support as well as to promote follicle expansion in the 3-D system. To mimic the natural extracellular matrix (ECM) environment, we modified the dextran hydrogels with two bioactive factors: ECM-derived Arg-Gly-Asp (RGD) peptides as a cell-adhesive factor, and activin A. The ovarian tissues were cultured in 2.25 mmol/L dextran hydrogels under four different conditions: Activin-/RGD- (A-R-), A + R-, A-R+, and A + R+. On Day 7 of culture, follicle and oocyte sizes were significantly increased in the RGD-modified conditions compared with those without RGD. The RGD-modified hydrogels also promoted mRNA levels of steroidogenic-related genes and estradiol production in the 3-D ovarian tissue culture. In vitro maturation and developmental competence of follicular oocytes were remarkably improved in the presence of RGD. In particular, blastocyst embryos were obtained only from A-R+ or A+R+ conditions after in vitro fertilization. We also determined synergistic effects of the RGD peptides and activin A on follicle growth and oocyte development in the 3-D tissue culture. In conclusion, our results suggest that RGD-modified dextran hydrogels provide an ECM-mimetic bioactive environment to support folliculogenesis in a 3-D ovarian tissue culture system.

Dataset Information

Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

Publications

Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

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