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

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 × 10cells/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 × 10and 1.40 ± 0.79 × 10P0-SF-MSCs were isolated, and 1.34 ± 0.66 × 10and 0.94 ± 0.20 × 10passage 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 × 10P0-SF-MSCs, from which 0.85 ± 0.48 × 10of 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.