Cryopreservation of κ-carrageenan hydrogels laden with stem cells aimed at cartilage regeneration strategies

A promising scenario for cartilage tissue engineering is the development of individual autologous products. One of the main prospects of cartilage tissue engineering is the possibility of developing custom-made regenerative medicine solutions on an individual patient basis. The efficient preservation and storage procedure will provide products available as needed which could be adapted to an autologous immediate solution. Thus, this study aims to examine the effects of cryopreservation on the chondrogenic differentiation of human adipose derived stem cells (hASCs) encapsulated in κ-carrageenan (κCR) hydrogels. hASCs (5 x10⁶ cell/cm³) were encapsulated in hydrogels discs (5 mm dia. x 3 mm height) produced using an ionotropic gelation method, cultured in standard basal (BM) or chondrogenic media (ChM) and later cryopreserved in liquid nitrogen for up to one month.

The morphology of hASCs-hydrogel was observed under light microscope and viability/proliferation rate was determined by DNA quantification. Additionally, chondrogenic differentiation of hASCs was characterized by histological staining and qPCR (Sox9, aggrecan, collagen: type I, type II and type X). Furthermore, we determine hASCs-hydrogel mechanical stability by DMA analysis. The characterization assays were performed both before and after cryopreservation.

The developed system was mechanically stable to maintain cellular viability when exposed to low temperatures. The assessment of chondrogenic features indicated stable chondrocyte phenotype after cryopreservation.

In summary, hASCs-hydrogel constructs have high long term storage potential and a ready-to-use bioengineered tissue substitutes for cartilage regeneration strategies. Thus, cell encapsulation systems of natural based hydrogels may be an interesting approach for the long term preservation of cartilage tissue engineered products.