A Xeno-Free Protocol for Umbilical Cord TissueCryopreservation and Subsequent Mesenchymal S tem CellIsolation

Objective: Umbilical cord (UC) tissue, which is once consideredas clinical was te, is recognized today a valuable source ofmesenchymal s tromal cells (MSCs). It has been demons tratedthat in vitro long-term expansion of cells could induce cellularsenescence which is associated with reduction of therapeuticproperties. Therefore, it may be prudent to cryopreserve MSCsin the UC format; so that, it would provide access to MSCsfrom the same donor at any future time point and the cells canbe culture expanded again from passage zero (P۰) s tage. In thiss tudy, we introduced an improved protracted cryo-conservationprotocol and evaluated the freezing impact on subsequent extractedMSCs in different time intervals, by four cryogeniccompositions and two principal isolation approaches: enzymaticdiges tion and explantation.Materials and Methods: UCMSCs were isolated from ۱۰ tissuesamples. For each fresh UC, a portion was designated forexplantation, the second was used for diges tion and the remainingthird portion was assigned for tissue cryopreservation. Differentcriteria of fresh and cryopreserved UC derived MSCsincluding, viability, immunophenotyping profile, proliferativeability, colony-forming potential, migration, and cellular senescencewere evaluated and compared.Results: A xeno-free and sugar containing formulation, withDMSO and autologous plasma, exhibited significantly greatercell yield/ gram of UC at different cryos torage intervals. Thisadvantage was present only for frozen explants after defros ting,while the utilization of enzymatic reaction on cryopreservedsamples resulted in significantly fewer viable cells.Conclusion: The findings of the present s tudy sugges t that explantprocedure is a safer and reasonable way to isolate MSCsafter UC cryopreservation; whereas, enzymatic model may bean optimal s trategy for fresh sys tems