Search

A method is described for the production of highly cytotoxic effector T lymphocytes. The cells can be raised in vivo in healthy animals in 4--5 days, thus combining the short time period of sensitization obtained in MLC or radiation GVH, without any of the attendant risks of these two methods (infection or loss of cultures or animals). DNA synthesis and blastogenesis are maximal 4 days after sensitization and are insignificant by day 6. 20--30% of the effector cells from conjugates with the sensitizing cell strain on days 4 through 7. Cytotoxicity was specific for cells of the sensitizing strain; blast cells are cytotoxic at the peak of the reaction (day 4) but small lymphocytes are cytotoxic on subsequent days.

Human fetal pancreases were dissected into 1- to 2- mm3 fragments and frozen to -196 C using a modification of procedures previously used successfully for cryopreservation of the rat fetal pancreas. Freeze-recovered pancreatic pieces were able to incorporate radioactive amino acids into proteins. When placed into organ culture, nonfrozen control and freeze-recovered pancreas tissue secreted radioimmoassayable insulin in response to glucose plus theophylline, but not to glucose alone. Freeze-killed pancreatic pieces were totally inactive functionally. With respect to those parameters tested, freeze-recovered pancreas tissues were equivalent to nonfrozen tissue.

To determine the optimal conditions for successful cryopreservation of the human fetal pancreas, techniques developed for the rat organ were modified. The parameters studied were the conditions of exposure to the cryopreservation agent dimethyl sulfoxide (DMSO), the rate of cooling and thawing, and the effect of in vitro culture. A total of 33 pancreases, obtained after fetal death due to prostaglandin-induced abortion, was studied. Survival of the pancreas is based on incorporation of 3H-amino acids into protein by pancreas pieces (2 mm3) during a 4-h incubation compared with nonfrozen control pieces from the same pancreas. Toxicity of DMSO at 37°C was found to be severe after a 4-h exposure. Varying the effects of temperature, time of exposure, and concentrations of DMSO on survival after freeze-thaw revealed that 1.5 M DMSO for 1 h at room temperature was optimal. The cooling rate was 0.22°C/min and thawing was at room temperature. Since these conditions resulted in only 50% survival, a period of culture before exposure to DMSO was added. The optimal duration of culture was 12–16 h. Using this method with addition of culture for 24 h after thawing, survival has varied from 70 to 120% of control. If a functional test for growth and insulin production by the frozen-thawed pancreas is positive, permanent shortage of the human fetal pancreas will be possible.