Establishing protocols for the efficient expansion of canine and feline adipose tissue-derived mesenchymal stromal cells following cell isolation

Abstract

Background Novel treatments using autologous or allogeneic mesenchymal stromal cells (MSC) are gaining traction in both human and veterinary medicine. MSC secrete multiple factors with immunomodulatory properties, making them promising for treating immune-mediated and inflammatory conditions. However, their application is challenged by donor variability, inconsistent tissue sources and lack of standardized manufacturing protocols, especially in veterinary medicine approaches exploiting the use of canine and feline MSC. Adipose tissue (AT), in particular, is a readily accessible MSC source for both species. This study applied a previously established enzymatic isolation method and focused on developing an efficient ex vivo expansion protocol for canine and feline adipose tissue-derived MSC, emphasizing the selection of a cost-effective culture medium that ensures high viability and proliferation. Subcutaneous AT was collected from 5 female dogs and 5 female cats during ovariohysterectomy. Samples were processed enzymatically, and isolated cells were cultured in basal medium supplemented with different concentrations of fetal bovine serum (FBS) (either standard FBS or FBS pre-screened for MSC-culture, hereon FBS-MSC). MSC identity was confirmed by flow cytometry (CD90+, CD44+, MHC-II−) and multilineage differentiation assays. Results Upon isolation, cells showed typical MSC morphology and ≥ 95% viability at the end of P1. For canine-derived cells, maximum average densities were 1.5 × 10⁵ (± 0.47) and 1.3 × 10⁵ (± 0.50) cells/cm² at P2 when using 10% FBS-MSC and 10% standard FBS supplementation, respectively. Concerning feline-derived cells, peak densities occurred earlier at P1, reaching 0.8 × 10⁵ (± 0.52) cells/cm² and 0.3 × 10⁵ (± 0.15) cells/cm² using 20% and 10% FBS-MSC supplementation, respectively. Expanded cells expressed the characteristic MSC immunophenotype and successfully differentiated into adipogenic, osteogenic and chondrogenic lineages. Conclusions Optimal cell expansion was achieved with 10% FBS-MSC supplementation for canine-derived cells and 20% FBS-MSC for feline-derived cells. P2 allowed a reasonable compromise of culture time vs. cell yield towards potential clinical use, achieving an average expected total cell yield from maximum expansion of 15.7 × 109 and 1.3 × 109 cells for canine- and feline-derived cells, respectively. This study sets critical parameters for the manufacturing of canine- and feline-derived MSC for regenerative medicine settings.