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 impairment due to the proteolytic cell damage by the applied digestive enzyme like collagenase, trypsin or dispase. To preserve the tissue and to enhance the yield of cells, Laue et al. reported an islet cell isolation with pre-incubation 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 to investigate whether this procedure can be applied to parathyroid glands. Therefore porcine parathyroid glands were dissected into 1 mm3 pieces. Subsequently one group of these pieces was incubated 22 h at 4 C in 2 mg/ml collagenase 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. The yield of cells and their viability was assessed by light-microscopy and staining with trypan-blue. After the cells were immobilized in barium-alginate and cultivated for 7 days, the function was tested by incubation in different calcium concentrations and PTH-measurement. Finally, the viability was assessed by histology. Using a cold pre-incubation with collagenase, a significantly higher number of isolated cells was retrieved compared with collagenase-digestion without pre-incubation. The viability was about 100% and did not differ between both groups. After immobilization and cultivation the viability decreased to less than 30%, with and without pre-incubation. In contrast to viability the PTH-secretion of the cells differed significantly between both procedures. By pre-incubation with collagenase at 4 C a gentle method is presented resulting in an enhancement of yield and function of single cells of parathyroid glands.
III Department of Internal Medicine-Endocrinology, Federal Research Center, Kiel, Germany.
M
M
Laudes
A
A
Gaumann
C
C
Laue
J
J
Schrezenmeir
D000818:Animals; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003080:Cold Temperature; D017364:Collagenases / Q000378:metabolism*; D010280:Parathyroid Glands / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D010281:Parathyroid Hormone / Q000378:metabolism; D013552:Swine
D010281:Parathyroid Hormone; D017364:Collagenases; D002118:Calcium