Laudes M, Gaumann A, Laue C, Schrezenmeir J
III Department of Internal Medicine-Endocrinology, Federal Research Center, Kiel, Germany.
J Endocrinol Invest. 2001 Feb;24(2):98-103. doi: 10.1007/BF03343821.
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.
从实体器官中制备细胞时，由于所应用的消化酶（如胶原酶、胰蛋白酶或分散酶）对细胞的蛋白水解损伤，常常会导致功能受损。为了保存组织并提高细胞产量，劳厄等人报道了一种胰岛细胞分离方法，即在4℃下进行预孵育，使酶能够扩散到组织中，并在整个颗粒中均匀地发挥活性。本研究的目的是探讨该方法是否可应用于甲状旁腺。因此，将猪甲状旁腺切成1立方毫米的小块。随后，将其中一组小块在4℃下于2毫克/毫升的胶原酶中孵育22小时，然后将温度升至37℃激活酶30分钟。另一组则直接在37℃下孵育30分钟。通过光学显微镜和台盼蓝染色评估细胞产量及其活力。在将细胞固定在钡藻酸盐中并培养7天后，通过在不同钙浓度下孵育并测量甲状旁腺激素（PTH）来测试其功能。最后，通过组织学评估活力。与未进行预孵育的胶原酶消化相比，采用胶原酶冷预孵育可获得显著更多的分离细胞。两组的活力均约为100%，无差异。固定和培养后，无论有无预孵育，活力均降至30%以下。与活力不同，两种方法处理的细胞PTH分泌存在显著差异。通过在4℃下用胶原酶进行预孵育，提出了一种温和的方法，可提高甲状旁腺单细胞的产量和功能。
