Mass culture of equine synovial fluid‐derived mesenchymal stromal cells using nonwoven polyethylene terephthalate fabrics

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Abstract Background Culture protocols need to yield 100 million equine synovial fluid (SF)‐derived mesenchymal stromal cells (SF‐MSCs) in around 3 weeks are needed, before these cells can be evaluated as agents of articular repair in clinical trials. Objectives To investigate mass culture of equine SF‐MSC culture protocols using nonwoven polyethylene terephthalate (PET) fabrics for the potential to meet the targets for clinical trials. Study Design In vitro experiments. Methods SF samples were collected from the carpal joints in thoroughbred racehorses ( n = 21) undergoing arthroscopic surgery and cultured in 10% fetal bovine serum (FBS‐protocol; n = 7) or 10% equine serum (ES‐protocol; n = 7)‐supplemented medium. Primary (P0) SF‐MSCs were isolated by dish culture and then passaged on nonwoven PET fabrics in culture bottles at a seeding density of 1 × 10 4 cells/strip. The other 7 SF samples were cultured in ES‐supplemented medium, and P0‐SF‐MSCs were passaged at a higher density using an automated culture device (ACD‐protocol; n = 7). Results Under the FBS and ES protocols, 0.84 ± 0.82 × 10 7 and 1.40 ± 0.79 × 10 7 P0‐SF‐MSCs were isolated, and 1.34 ± 0.66 × 10 8 and 0.94 ± 0.20 × 10 8 passage 1 (P1)‐SF‐MSCs were harvested. The respective total durations of primary and passage culture were 21.4 ± 1.8 and 27.6 ± 2.1 days duration. An ACD‐protocol yielded 0.71 ± 0.32 × 10 7 P0‐SF‐MSCs, from which 0.85 ± 0.48 × 10 8 of P1‐SF‐MSCs were cultured in 20.6 ± 1.1 days. Main Limitations Visualisation of SF‐MSC attached to PET nonwoven fabric, counting of SF‐MSCs attached to and detached from PET nonwoven fabric, and daily quantification of SF‐MSC proliferating on fabrics. Conclusions To support progress to clinical trials, of a sufficient number (100 million) of equine SF‐MSCs need to be harvested in around 3 weeks. These goals were achieved with the FBS protocol, but further optimisation is needed for the ES protocol. Our findings on in vitro MSCs amplification suggest that SF, a potential source for non‐invasively collected MSCs, is available as a promising matrix for therapeutic strategies using autologous transplantation.

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