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Reasons for performing study Improvement has been reported following intra-articular (i.a.) injection of mesenchymal stromal cells ( MSCs) in several species. These observations have led to the use of i.a. MSCs in equine practice with little understanding of the mechanisms by which perceived improvement occurs. Objectives To evaluate the effect of i.a. allogeneic umbilical cord blood ( CB-) derived MSCs using a lipopolysaccharide ( LPS) induced synovitis model. We hypothesised that i.a. CB- MSCs would reduce the inflammatory response associated with LPS injection. Study design Randomised, blinded experimental study. Methods Feasibility studies evaluated i.a. LPS or CB- MSCs alone into the tarsocrural joint. In the principal study, middle carpal joint synovitis was induced bilaterally with LPS and then CB- MSCs were injected into one middle carpal joint. Lameness, routine synovial fluid analysis, and synovial fluid biomarkers were evaluated at 0, 8, 24, 48 and 72 h. Results LPS injection alone resulted in transient lameness and signs of inflammation. In joints untreated with LPS, injection of 30 million CB- MSCs resulted in mild synovitis that resolved without treatment. Mild ( grade 1-2) lameness in the CB- MSC-treated limb was observed in 2 horses and severe lameness ( grade 4) in the 3rd, 24 h post injection. Lameness did not correlate with synovitis induced by CB- MSC injection. Simultaneous injection of LPS and CB- MSCs resulted in significant reduction in synovial fluid total nucleated, neutrophil and mononuclear cell numbers compared with contralateral LPS-only joints. No difference was detected in other parameters associated with synovial fluid analysis or in synovial fluid biomarkers. The incidence of lameness was only different from baseline at 8 h, where horses were lame in CB- MSC limbs. Conclusions Allogeneic CB- MSCs reduced synovial fluid cell populations and stimulated mild self-limiting inflammation in the synovitis model. Continued evaluation of the effects of i.a. CB- MSC therapy on synovitis in horses is needed to evaluate anti- and proinflammatory properties of CB- MSCs. Immediate interests are dose, timing of treatment, and treatment frequency. [ABSTRACT FROM AUTHOR]

Reasons for performing study Multipotent mesenchymal stromal cells ( MSC) are often culture-expanded in vitro. Presently, expansion medium ( EM) for MSC is supplemented with fetal bovine serum ( FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate ( PL) has shown promise as an alternative supplement. Objectives To determine how equine umbilical cord blood ( CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Study design Randomised dose escalation study. Methods Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 1012 platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB- MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Results Cord blood- MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Conclusions Expansion medium enriched with PL can support short-term equine CB- MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB- MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB- MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB- MSC function. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1 L scale using a Vertical-Wheel ® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5 L bioreactor, scale-up from static culture to the 3 L computer-controlled bioreactor, and scale-up into the 3 L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3 L computer-controlled system resulted in a final cell densities of 1.5 × 10 5 cells/cm 2 on average, achieving 1.5 × 10 9 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3 L VW bioreactor., Competing Interests: Author TGK is the founder, CEO and a shareholder in eQcell. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Roberts, Abraham, Dang, Gysel, Mehrpouyan, Alizadeh, Koch and Kallos.)

Objective: Ongoing research is aimed at increasing cartilage tissue yield and quality from multipotent mesenchymal stromal cells (MSC) for the purpose of treating cartilage damage in horses. Low oxygen culture has been shown to enhance chondrogenesis, and novel membrane culture has been proposed to increase tissue yield and homogeneity. The objective of this study was to evaluate and compare the effect of reduced oxygen and membrane culture during in vitro chondrogenesis of equine cord blood (CB) MSC., Methods: CB-MSC (n = 5 foals) were expanded at 21% oxygen prior to 3-week differentiation in membrane or pellet culture at 5% and 21% oxygen. Assessment included histological examination (H&E, toluidine Blue, immunohistochemistry (IHC) for collagen type I and II), protein quantification by hydroxyproline assay and dimethylmethylene assay, and mRNA analysis for collagen IA1, collagen IIA1, collagen XA1, HIF1α and Sox9., Results: Among treatment groups, 5% membrane culture produced neocartilage most closely resembling hyaline cartilage. Membrane culture resulted in increased wet mass, homogenous matrix morphology and an increase in total collagen content, while 5% oxygen culture resulted in higher GAG and type II collagen content. No significant differences were observed for mRNA analysis., Conclusion: Membrane culture at 5% oxygen produces a comparatively larger amount of higher quality neocartilage. Matrix homogeneity is attributed to a uniform diffusion gradient and reduced surface tension. Membrane culture holds promise for scale-up for therapeutic purposes, for cellular preconditioning prior to cytotherapeutic applications, and for modeling system for gas-dependent chondrogenic differentiation studies., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)