Xenogen-Free Stem Cell Preparation Enhances Safety and Efficacy
Presented by: Aileen Rowland
Authors: Aileen L. Rowland(1), Madison E. Burns(1), Gwendolyn J. Levine(2), Ashlee E. Watts(1)
Affiliations: 1: Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, USA; 2: Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA
Introduction: Conventional methods for expansion of MSCs include the addition of fetal bovine serum (FBS) to culture media. We have previously shown the resultant intracellular contamination of FBS causes an adverse response after intra-articular injection of MSCs in horses. We hypothesized that this adverse response is secondary to immune recognition of FBS and destruction of injected MSCs because of intracellular FBS.
Hypothesis / Objectives: Our current objective was to evaluate horse response and MSC isolation from synovial fluid after intra-articular injection of autologous MSCs after culture expansion with (FBS) and without FBS (XENO-FREE) in the culture media.
Materials and Methods: We expanded autologous MSCs to passage 3 from 18 horses: MSCs were cultured using conventional technique (FBS in culture media; n=6) or with xenogen-free culture media (XENO-FREE; n=12). MSCs from both groups were extensively washed prior to intra-articular injection on days 0 and 29. On day 0, MSCs were injected alone and on day 29 MSCs were injected along with 2.5ng of lipopolysaccharide (LPS) to induce synovitis. Following injection, 8 drops of synovial fluid were collected for CFU assay and clinical exam was performed.
Results: There was less edema in XENO-FREE on day 1 (p=0.002), day 30 (p=0.001) and day 36 (p=0.02) and no difference on day 7. There was a smaller increase in limb circumference in the XENO-FREE on day 30 (p=0.002), but no difference on day 36. CFU-analysis was positive more often in horses injected with XENO-FREE on days 1 (12/12 vs. 3/6, p=0.02) 30 (11/12 vs. 4/6, p=0.02) and 36( 9/12 vs. 1/6, p=0.03). There were more colonies in XENO-FREE on day 7 (p=0.004) and day 36 (p=0.05), and no difference in number of colonies on day 1 or day 30
Conclusions: In this study, we have shown that FBS in the culture media results in mild adverse response (increased edema) and reduced synovial MSCs. Avoidance of immune destruction by expanding MSCs in xenogen-free media may limit adverse clinical reaction, but most importantly may result in greatly enhanced efficacy due to improved synovial MSC persistence. Our study is further evidence for immune recognition of FBS-contaminated MSCs that result in immune mediated rejection of autologous MSCs, despite minimal differences in observable clinical reaction.
Acknowledgements, Funding, and Conflicts of Interest: Funding provided by Link Endowment for Equine Research at Texas A&M University and the Dennis and Linda Clark Endowment for Equine Orthopedic Research from the Department of Large Animal Sciences at Texas A&M University. There are no conflicts of interest.