Conditioned umbilical cord tissue provides a natural three-dimensional storage compartment as in vitro stem cell...

The use of large amounts of human multipotent mesenchymal stroma/stem cells (MSC) for cell therapies represents a desirable property in tissue engineering and banking in the field of regenerative medicine.

Whereas cryo-storage of umbilical cord (UC) tissue pieces in liquid nitrogen without ingredients was associated with predominant appearance of apoptotic cells after thawing and re-culture, progressive growth of MSC was observed following use of cryo-medium. Moreover, conditioning of UC tissue pieces by initial explant culture and subsequent cryo-storage with cryo-medium accelerated a further MSC culture after thawing. These findings suggested that conditioning of UC tissue pieces provides an in vitro stem cell niche by maintenance of a 3-dimensional natural microenvironment for continuous MSC outgrowth and expansion. Indeed, culture of GFP-labeled UC tissue pieces was accompanied by increased outgrowth of GFP-labeled cells which was accelerated in conditioned UC tissue after cryo-storage. Moreover, cryopreserved conditioned UC tissue pieces in cryo-medium after thawing and explant culture could be cryopreserved again demonstrating renewed MSC outgrowth after repeated thawing with similar population doublings compared to the initial explant culture. Flow cytometry analysis of outgrowing cells revealed expression of the typical MSC markers CD73, CD90, and CD105. Furthermore, these cells demonstrated little if any senescence and cultures revealed stem cell-like characteristics by differentiation along the adipogenic, chondrogenic and osteogenic lineages.

Expression of MSC markers was maintained for at least 10 freeze/thaw/explant culture cycles demonstrating that repeated cryopreservation of conditioned UC tissue pieces provided a reproducible and enriched stem cell source.