Background/Case Studies: Umbilical cord (UC) tissue is a rich source of mesenchymal stem cells (MSCs). Given their potential therapeutic value, many family stem cell banks have established UC storage programs; however, little has been reported on the stability of UC tissue after extended periods of cryostorage. We previously reported on the successful isolation of functional MSCs from UC tissue cryopreserved for 5 years [Skiles, 2017]. Here, we expand on those results and show recovery of functional MSCs from the same units 6 years later. These results demonstrate the stability of UC tissue in cryostorage for over a decade and support the suitability of cryopreserving UC tissue as a composite material for future isolation of MSCs.
Study
Design/Methods: UC units were cryopreserved as composite tissue pieces in LN2 in a DMSO-based cryoprotectant for 1.2-11.4 years (n=19). 12 units consisted of paired tissue from the same 6 donors tested 6 years ago and again in the present study [Skiles, 2017]. Units were rapidly thawed, rinsed in DPBS, and explanted onto plastic culture plates in MSC-supportive medium [Skiles, 2018]. Medium was exchanged on Day 7 and cells harvested and counted on Day 14. Isolated cells were subcultured to the end of passage 2, imaged, and collected for enumeration, viability determination, and flow cytometric immunophenotyping.
Results/Findings: Plastic-adherent, proliferative cells with fibroblastic morphology were obtained from 100% of UC units. There was no significant difference in explant cell yields between paired units thawed after 3.3(±0.2) years and again after 9.1(±0.3) years (p=0.3) or paired units thawed after 5.7(±0.3) years and again after 11.4(±0.5) years (p=0.6). Across all age groups, cells were positive for the MSC markers CD73, CD90, and CD105 (98.5% ± 1.6%, 98.0% ± 1.5%, and 91.7% ± 10%, respectively) and negative for the hematopoietic markers CD34/45 (1.3% ± 1.5%). Average passage 2 doubling time across all age groups was 1.8(±0.4) days. Theoretical cumulative passage 2 cell yield, calculated by [explant yield x passage 1 fold increase x passage 2 fold increase], was on the order of 1x107 cells or greater for each unit. Conclusions: To our knowledge, our work is the first to demonstrate the recovery of MSCs from UC tissue cryopreserved for over a decade. Results from this study suggest that long-term cryopreservation of UC tissue does not disrupt the ability to isolate functional MSCs from the tissue later. UC tissue analyzed from 1.2 to 11.4 years exhibited cellular growth and surface marker profiles consistent with MSCs, irrespective of cryostorage duration. This supports long term cryopreservation of UC as a composite material for future isolation of MSCs and subsequent expansion to clinically relevant doses.
Importance of research: Umbilical cord (UC) tissue is a rich source of mesenchymal stem cells (MSCs). Given their potential therapeutic value, many family stem cell banks have established UC storage programs; however, little has been reported on the stability of UC tissue after extended periods of cryostorage. To our knowledge, our work is the first to demonstrate the recovery of MSCs from UC tissue cryopreserved for over a decade.
