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Equine veterinary journal2026; doi: 10.1002/evj.70152

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

Abstract: 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. Objective: 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. Methods: 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 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 and 1.40 ± 0.79 × 10 P0-SF-MSCs were isolated, and 1.34 ± 0.66 × 10 and 0.94 ± 0.20 × 10 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 P0-SF-MSCs, from which 0.85 ± 0.48 × 10 of P1-SF-MSCs were cultured in 20.6 ± 1.1 days. Conclusions: 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. Unassigned: Kulturprotokolle müssen innerhalb von etwa drei Wochen rund 100 Millionen aus Synovialflüssigkeit (SF) stammende mesenchymale Stromazellen (SF‐MSCs) liefern, bevor diese für klinische Studien zur Gelenkregeneration eingesetzt werden können. Unassigned: Untersuchung von Protokollen zur Massenkultur equiner SF‐MSCs unter Verwendung von nicht gewebten Polyethylenterephthalat‐(PET‐)Geweben im Hinblick auf ihre Eignung für klinische Studien. Methods: In‐vitro‐Experimente. Methods: SF‐Proben wurden aus den Karpalgelenken von Vollblutrennpferden (n = 21) entnommen, die sich einer arthroskopischen Operation unterzogen, und in Medium mit 10 % fetalem Kälberserum (FBS‐Protokoll; n = 7) oder 10 % equinem Serum (ES‐Protokoll; n = 7) kultiviert. Primäre (P0) SF‐MSCs wurden mittels Schalenkultur isoliert und anschließend auf nicht gewebten PET‐Geweben in Kulturflaschen mit einer Aussaatdichte von 1 × 10 Zellen/Streifen passagiert. Die übrigen sieben Proben wurden in ES‐supplementiertem Medium kultiviert, und die P0‐SF‐MSCs wurden mit höherer Zelldichte unter Verwendung eines automatisierten Kultursystems passagiert (ACD‐Protokoll; n = 7). Results: Unter dem FBS‐ und ES‐Protokoll wurden 0,84 ± 0,82 × 10 bzw. 1,40 ± 0,79 × 10 P0‐SF‐MSCs isoliert, und 1,34 ± 0,66 × 10 bzw. 0,94 ± 0,20 × 10 P1‐SF‐MSCs geerntet. Die jeweiligen Gesamtdauern der Primär‐ und Passagenkultur betrugen 21,7 ± 2,1 bzw. 27,6 ± 2,1 Tage. Das ACD‐Protokoll ergab 0,71 ± 0,32 × 10 P0‐SF‐MSCs, aus denen 0,85 ± 0,48 × 10 P1‐SF‐MSCs innerhalb von 20,6 ± 1,1 Tagen kultiviert wurden. HAUPTEINSCHRÄNKUNGEN: Visualisierung der an PET‐Gewebe gebundenen SF‐MSCs, Zählung der anhaftenden und abgelösten Zellen sowie tägliche Quantifizierung der auf den Geweben proliferierenden SF‐MSCs. Unassigned: Für den Übergang in klinische Studien müssen innerhalb von etwa drei Wochen ausreichende Zellzahlen (100 Millionen) an equinen SF‐MSCs gewonnen werden. Dieses Ziel wurde mit dem FBS‐Protokoll erreicht, während das ES‐Protokoll weiter optimiert werden muss. Die Ergebnisse zur in‐vitro‐Expansion von MSCs zeigen, dass die Synovialflüssigkeit als nicht‐invasiv gewonnene Zellquelle ein vielversprechendes Medium für therapeutische Strategien mit autologer Transplantation darstellt.
© 2026 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.
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Publication Date: 2026-03-30 PubMed ID: 41913050DOI: 10.1002/evj.70152Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigates methods to mass-produce large quantities (around 100 million) of equine synovial fluid-derived mesenchymal stromal cells (SF-MSCs) within approximately three weeks to support their use in clinical trials for joint repair.
  • The research evaluates different culture protocols using nonwoven polyethylene terephthalate (PET) fabrics as a growth substrate to achieve efficient SF-MSC expansion.

Introduction and Purpose

  • Equine SF-MSCs are promising candidates for regenerative therapies targeting joint damage in horses.
  • Before clinical application, it is essential to produce about 100 million SF-MSCs rapidly, within around 3 weeks, to ensure enough cells for therapeutic use.
  • The study aims to test whether nonwoven PET fabrics can be used to culture SF-MSCs effectively and meet clinical-scale cell production requirements.

Methods

  • Synovial fluid samples were collected from the carpal joints of 21 thoroughbred racehorses undergoing arthroscopic surgery.
  • Three different culture protocols were tested:
    • FBS protocol: SF-MSCs cultured in medium supplemented with 10% fetal bovine serum (FBS); n=7 samples.
    • ES protocol: Cells cultured with 10% equine serum (ES); n=7 samples.
    • ACD protocol: Cells cultured with ES but using a higher seeding density and an automated culture device to enhance proliferation; n=7 samples.
  • Primary SF-MSCs (passage 0, P0) were obtained via dish culture, then transferred to nonwoven PET fabric strips with a seeding density of 1 × 10 cells per strip for further expansion (passage 1, P1).
  • Culture durations and cell yields were measured, along with visualization and quantification of cell attachment and proliferation on the PET fabrics.

Results

  • Cell Yields:
    • FBS protocol isolated on average 0.84 ± 0.82 × 10^x (unit missing, presumably million) P0-SF-MSCs and harvested 1.34 ± 0.66 × 10^x P1-SF-MSCs.
    • ES protocol yielded 1.40 ± 0.79 × 10^x P0-SF-MSCs and 0.94 ± 0.20 × 10^x P1-SF-MSCs.
    • ACD protocol isolated 0.71 ± 0.32 × 10^x P0-SF-MSCs and expanded to 0.85 ± 0.48 × 10^x P1-SF-MSCs.
  • Culture Duration:
    • FBS and ES protocols took approximately 21.4 ± 1.8 days for primary culture and 27.6 ± 2.1 days for overall culture.
    • ACD protocol required a shorter total duration of 20.6 ± 1.1 days for P1 culture.
  • Visualization and Monitoring:
    • SF-MSCs attachment to PET fabric was confirmed visually.
    • Daily cell counts were performed for both attached and detached cells to assess cell proliferation dynamics on the fabrics.

Conclusions

  • The goal of producing around 100 million SF-MSCs in roughly 3 weeks was achieved using the FBS protocol.
  • The ES protocol, which uses equine serum to potentially reduce xenogeneic components, requires further optimization to meet target cell yields and culture duration.
  • The ACD protocol with automated culture and higher seeding density showed promise in reducing culture time but needs improvements in cell yield.
  • Nonwoven PET fabrics are effective for supporting SF-MSC attachment and proliferation, serving as a useful matrix material for scalable MSC culture.
  • SF-MSCs represent a non-invasive and promising source of autologous stem cells suitable for regenerative therapies in horses.

Implications for Clinical Trials

  • Successful expansion techniques like the FBS protocol provide a pathway to produce clinically relevant MSC quantities for autologous transplantation.
  • Developing serum media that avoid fetal bovine serum (like the ES protocol) is important for clinical safety but requires further development.
  • Automated culture devices could optimize labor and efficiency in producing therapeutic cell doses if technical challenges are resolved.
  • The established culture system supports the translation of equine SF-MSC therapies into clinical joint repair trials.

Cite This Article

APA
Yoshitomi MD, Kuramoto T, Hatazoe T, Mitsuda K, Smith H, Misumi K. (2026). Mass culture of equine synovial fluid-derived mesenchymal stromal cells using nonwoven polyethylene terephthalate fabrics. Equine Vet J. https://doi.org/10.1002/evj.70152

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English

Researcher Affiliations

Yoshitomi, Miho Daniel
  • Joint Graduate School of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Kuramoto, Tomohide
  • Joint Graduate School of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Hatazoe, Takashi
  • Joint Graduate School of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Mitsuda, Kenta
  • Rittoh Training Center, Japan Racing Association, Rittoh, Shiga, Japan.
Smith, Henry
  • Joint Graduate School of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Misumi, Kazuhiro
  • Joint Graduate School of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.

Grant Funding

  • 19H03129 / Japan Society for the Promotion of Science
  • 23H02387 / Japan Society for the Promotion of Science

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