Improved yield and functionality of parathyroid cells separated by using collagenase-digestion with cold pre-incubation

Preparation of cells from solid organs often induces a functional impairmen t due to the proteolytic cell damage by the applied digestive enzyme like c ollagenase, trypsin or dispase. To preserve the tissue and to enhance the y ield of cells, Laue et al. reported an islet cell isolation with pre-incuba tion at 4 C permitting the enzyme to diffuse into the tissue and explicite activity equally throughout the whole particle. The aim of this study was t o investigate whether this procedure can be applied to parathyroid glands. Therefore porcine parathyroid glands were dissected into 1 mm(3) pieces. Su bsequently one group of these pieces was incubated 22 h at 4 C in 2 mg/ml c ollagenase before activating the enzyme by elevating the temperature to 37 C for 30 min. The other group was incubated directly at 37 C for 30 min. Th e yield of cells and their viability was assessed by light-microscopy and s taining with trypan-blue. After the cells were immobilized in barium-algina te and cultivated for 7 days, the function was tested by incubation in diff erent calcium concentrations and PTH-measurement. Finally, the viability wa s assessed by histology. Using a cold pre-incubation with collagenase, a si gnificantly higher number of isolated cells was retrieved compared with col lagenase-digestion without pre-incubation. The viability was about 100% and did not differ between both groups. After immobilization and cultivation t he viability decreased to less than 30%, with and without pre-incubation. I n contrast to viability the PTH-secretion of the cells differed significant ly between both procedures. By preincubation with collagenase at 4 C a gent le method is presented resulting in an enhancement of yield and function of single cells of parathyroid glands. (C)2001, Editrice Kurtis .