Cryopreservation of equine mesenchymal stem cells in 95% autologous serum and 5% DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95% and DMSO at 10 or 5.

INTRODUCTION

Equine superficial digital flexor tendon injury is a well-accepted model of human tendon injury and is routinely treated with local injections of autologous mesenchymal stem cells (MSCs). Identification of a clinically safe medium for short-term cryopreservation of MSCs prior to cell implantation would streamline laboratory and clinical procedures for autologous regenerative therapies. Veterinary experience with short-term (MSCs prepared after the injury has occurred) cryopreserved MSCs in naturally occurring injury in the horse will be of value to human practitioners.

METHODS

Equine bone marrow derived MSCs were cryopreserved in 6 different solutions consisting of 20% serum, 10% DMSO and 70% media or 95% serum and 5% DMSO. Serum was autologous serum, commercially available pooled equine serum or fetal bovine serum (FBS). Cell survival, morphology and growth kinetics were assessed by total cell number, measurement of growth kinetics, colony-forming-unit-assay and morphology of MSCs after monolayer culture post-thaw.

RESULTS

There were no significant differences in post-thaw viability, total cell number, morphology scores or growth kinetics among the 6 solutions. Post thaw viabilities from each group ranged from 80-90%. In all solutions, there were significantly fewer MSCs and the majority (99%) of MSCs remained in the original generation 24 hours post-thaw. Seventy two hours post-thaw, the majority of MSCs (50%) were proliferating in the fourth generation. Mean colony count in the CFU-F assay ranged from 72 to 115 colonies.

CONCLUSIONS

Each of the serum sources could be used for short-term cryopreservation of equine bone marrow derived MSCs. Prior to clinical use, clinicians may prefer autologous serum and a lower concentration of DMSO.