Effect of cooling rate and its interaction with pre-freeze and post-thaw tissue culture on the in vitro and in vivo function of cryopreserved pancreatic islets.

Rapid cooling destroys passenger lymphoid cells during cryopreservation. We now describe improved in vivo survival of rat islets after rapid cooling by adding pre-freeze tissue culture. Islets were equilibrated with 2M dimethylsulphoxide, cooled at 0.3 degrees, 20 degrees, or 70 degrees C/min, and stored at -196 degrees C. The culture was kept at 37 degrees C for 2 or 72 h before and/or after preservation. When cooled at 0.3 degrees C/min, keeping the culture for 72 h gave the highest proportion of dye-excluding cells, but more than 50% were viable under all culture conditions. Islets cooled at 20 degrees or 70 degrees C/min (rapidly) required 72 h of culture for a survival rate of more than 50%. When islets were cultured for 72 h before cryopreservation, their in vitro insulin secretory ability was similar to that of slowly cooled islets and they were able to sustain normoglycaemia in diabetic animals, although more islets were needed. Extended tissue culture before freezing improves the survival of rapidly cooled islets and is therefore important for combined immunomodulation and cryopreservation.