Closed Vitrification System as A Platform for Cryopreservation of Tissue Engineered Constructs

Cryopreservation of mesenchymal stromal cells (MSCs) and MSCs-based tissue engineered constructs (TECs) is a promising strategy for regenerative medicine.

To examine vitrification system consisting of multicomponent vitreous solution, closed type container, human adult MSCs and two-step exposure procedure as a platform for cryopreservation of MSCs-based TECs.

Vitrification properties of solutions were studied by visual analysis and calorimetry. Viability (trypan blue, MTT-test), metabolic activity (Alamar Blue assay) and adhesion of cells were assessed both after exposure with vitreous solutions and following rapid cooling-thawing in standard cryovials.

The feasibility of the vitrification system was tested on MSCs suspensions (S-MSCs) and alginate encapsulated MSCs (AE-MSCs). The minimal concentrations of cryoprotectants, which allowed avoiding ice formation during rapid cooling and rewarming comprised 10 % for dimethylsulfoxide, 20 % for ethylene glycol, 20 % for 1.2-propanediol and 0.5 M sucrose. To achieve viability and metabolic activity rates of AE-MSCs comparable to S-MSCs after vitrification the extension of the exposure time within the same vitreous solution was sufficient. After vitrification both S-MSCs and AE-MSCs retained the capacity to osteogenic and adipogenic differentiation.

Data demonstrate that this vitrification system can be used as a platform for development of effective protocols for cryopreservation of MSCs-based TECs.