Viability of equine mesenchymal stem cells during transport and implantation

INTRODUCTION: Autologous mesenchymal stem cell (MSC) injection into naturally-occurring equine tendon injuries has been shown to be safe and efficacious and protocols inform translation of the technique into humans. Efficient transfer of cells from the laboratory into tissue requires well-validated transport and implantation techniques. METHODS: Cell viability in a range of media was determined over 72 hours and after injection through a 19G, 21G or 23G needle. Viability, proliferation and apoptosis were analysed using TrypanBlue, alamarBlue(®) and AnnexinV assays. RESULTS: Cell viability was similar in all re-suspension media following 24 hour storage, however cell death was most rapid in bone marrow aspirate, platelet-rich plasma and serum after longer storage. Cryogenic media exhibited greatest viability regardless of storage time. Cell proliferation after 24 and 72 hour storage was similar for all media, except after 24 hours in serum wherein proliferation was enhanced. MSC tri-lineage differentiation and viability did not significantly change when extruded through 19G, 21G or 23G needles, but 21G and 23G needles significantly increased apoptotic cells compared to 19G and non-injected controls. All gauges induced a decrease in metabolic activity immediately post-injection but cells recovered by 2 hours. CONCLUSIONS: These data indicate storage and injection influence viability and subsequent cell behaviour and provide recommendations for MSC therapy that implantation of cells should occur within 24 hours of recovery from culture, using larger needle bores.