Slow cooling cryopreservation of cell-microcarrier constructs

Ideally, tissue engineered constructs should be readily available to meet the need for fast intervention in complex bone defects. To circumvent the long culture period of these constructs before implantation, we investigated the possibility of cryopreserving cell-loaded constructs. Goat bone marrow-derived mesenchymal stem cells (BMSC) and mouse osteoblast-like cells from the MC3T3-E1 cell line were cultured on gelatin CultiSpher-S(R) microcarriers. These constructs were cryopreserved using the slow cooling technique, i.e. cooling to -80 degrees C at a rate of -1.5 degrees C/min, and were then stored in liquid nitrogen for 1 week. Four different cryomedia were tested, i.e. 90 vol% serum with 10 vol% dimethylsulphoxide (Me(2)SO) with or without ascorbic acid (AA) and 90 vol% serum supplemented with 5 vol% Me(2)SO and 5 vol% hydroxyethyl starch or 5 vol% sucrose (60 mM). Cell viability on the constructs was assessed with fluorescent live/dead staining and the colorimetric MTS assay. Cell viability was compared before freezing and at fixed time points after thawing. Immediately after thawing, the viability percentages in all groups were significantly lower than before cryopreservation (p = 0.0369). No significant differences were observed between the viability percentages on the cell constructs cryopreserved in the different media; however, there was a general tendency for higher cell survival and faster recolonization of constructs cryopreserved in Me(2)SO with or without AA than of the constructs cryopreserved in the other media. For constructs cryopreserved in 10 vol% Me(2)SO with or without AA, the recolonization period was 3 days for the BMSC constructs and 3.6 and 3.8 days, respectively, for the MC3T3-E1 constructs.