Research

Cryopreservation of parathyroid tissue has utility beyond autotransplantation for treatment of hypoparathyroidism. Research using parathyroid tissue can be facilitated by harvesting tissue on the day of resection for use at a time convenient to the investigator. Access to a "bank" of cryopreserved tissue frees the investigator from the vagaries of the operating room schedule and permits accumulation of masses of tissue sufficient to perform complex experiments. It also permits comparison of several patients' tissues in a single experiment as well as the same patient's tissue in several experiments. Cryopreserved tissue has been used in the investigation of several aspects of physiology: comparison of hormone release in adenoma versus hyperplasia,26 effect of lithium on thymidine incorporation,16 parathyroid immunology,27-28 effect of cimetidine on hormone secretion,29 effect of phorbol ester on hormone secretion,13 mitochondrial incorporation of sestamibi,17 and generation of microcapsules of parathyroid tissue.30 In general, cryopreservation appears to preserve parathyroid function, although there does appear to be a difference in preservation of estrogen receptors in fresh versus cryopreserved human parathyroid tissue (Table 60-4).31

For in vitro experiments, we have used the following protocol for preparation of cell suspensions. Thawed tissue is placed in 10 mL of a 0.5-mg/mL collagenase (Boehringer Mannheim, Indianapolis, IN) in RPMI-1640 solution. The culture tube containing the tissue is placed in a 37°C shaking water bath and agitated gently (92/min) for 30 to 60 minutes.

Because the viability of cryopreserved cells is variable, we have used an additional step to remove necrotic cells and enrich the proportion of viable cells. A stock solution of isotonic Percoll (Pharmacia, Uppsala, Sweden) is made by mixing Percoll with lOx phosphate-buffered saline (PBS). Working solutions of 25% and 75% stock solutions are made by dilution in PBS. In a 12- x 75-mm culture tube, 1.5-mL portions of the 25% and 75% stock solutions are added below the parathyroid cell suspension using a spinal needle, and the tube is centrifuged at 450 g for 15 minutes. The superficial

0.5.mL containing debris and necrotic cells is discarded. Of the remaining Percoll gradient, 1.5 mL is aspirated, diluted with 3.5 mL of culture medium, recentrifuged, and resus-pended in whatever solution is to be used for the experiment.

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