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Chondrogenesis of Adipose-Derived Stem Cells Using an Arrayed Spheroid Format

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Abstract

Objective

The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling these systems up for high-throughput testing is technically challenging and wasteful given the necessary cell numbers and reagent volumes. However, adopting microscale spheroid cultures for this purpose shows promise. Spheroid systems work with only thousands of cells and microliters of medium.

Methods

Molded agarose microwells were fabricated using 2% w/v molten agarose and then equilibrated in medium prior to introducing cells. ASCs were seeded at 50, 500, 5k cells/microwell; 5k, 50k, cells/well plate; and 50k and 250k cells/15 mL centrifuge tube to compare chondrogenic responses across spheroid and micromass sizes. Cells were cultured in control or chondrogenic induction media. ASCs coalesced into spheroids/pellets and were cultured at 37 °C and 5% CO2 for 21 days with media changes every other day.

Results

All culture conditions supported growth of ASCs and formation of viable cell spheroids/micromasses. More robust growth was observed in chondrogenic conditions. Sulfated glycosaminoglycans and collagen II, molecules characteristics of chondrogenesis, were prevalent in both 5000-cell spheroids and 250,000-cell micromasses. Deposition of collagen I, characteristic of fibrocartilage, was more prevalent in the large micromasses than small spheroids.

Conclusions

Chondrogenic differentiation was consistently induced using high-throughput spheroid formats, particularly when seeding at cell densities of 5000 cells/spheroid. This opens possibilities for highly arrayed experiments investigating tissue repair and remodeling during or after exposure to drugs, toxins, or other chemicals.

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Acknowledgments

The authors would like to thank the Center for Alternatives to Animals in Testing for use of their imaging facility and David Silverberg in the Molecular Pathology Core for helping with paraffin histology.

Author Contributions

EMD, VCF, and RAG conceived and created the experimental design. VCF and RAG carried out the experiments. EMD, VCF, and RAG analyzed and interpreted the results. RAG drafted the initial manuscript. EMD, VCF, and RAG read, revised, and approved the final submitted manuscript.

Funding

This research was supported by the National Institutes of Health (NIH) (P30 GM122732 to EMD), National Science Foundation (CMMI 2054193 to EMD, GRFP 2018260690 to RAG).

Conflict of interest

Robert A. Gutierrez, Vera C. Fonseca, and Eric M. Darling confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Ethical approval

No human or animals were used in this study.

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Authors and Affiliations

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

    Robert A. Gutierrez & Eric M. Darling

  2. Department of Pathology and Laboratory Medicine, Providence, USA

    Vera C. Fonseca & Eric M. Darling

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

    Eric M. Darling

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

    Eric M. Darling

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  1. Robert A. Gutierrez
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  2. Vera C. Fonseca
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Correspondence to Eric M. Darling.

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Gutierrez, R.A., Fonseca, V.C. & Darling, E.M. Chondrogenesis of Adipose-Derived Stem Cells Using an Arrayed Spheroid Format. Cel. Mol. Bioeng. 15, 587–597 (2022). https://doi.org/10.1007/s12195-022-00746-8

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