Experimental Study on Cryopreservation of Pancreas Islet Cell in Rat / 대한이식학회지

PURPOSE: Cryopreservation of pancreas islet cells can facilitate the clinical islet transplantation by giving a means of storage of islets and immunomodulation on pancreatic islet preparations. METHODS: Pancreatic islets were isolated by standard technique using collagenase in rat. Cryopreservation was performed by using DMSO as a cryoprotectant after one day or 48 hr culture. Recovery rate, viability and insulin release in assay in vitro and vivo were checked under the various conditions, such as concentration of DMSO (1.5 M or 2.0 M), culture condition (1 day or 2 day), and taurine treatment. RESULTS: Percentage of recovery of cryopreserved islet was 56.8+/-10% after thawing. Viability was decreased from 97.2+/-1.1% before cryopreservation to 82.7+/-9.9% after thawing. Glucose stimulation index was reduced from 1.7+/-0.2 before cryopreservation to 1.2+/-0.8 after thawing. Nucleation method by metal rod showed better viability than control (no nucleation) or chamber nucleation method. Mean viability and glucose stimulation index was 81% and 1.5 in one day culture, and 84.2% and 1.2 in 2 day culture before cryopreservtion. Islet treated with taurine showed better insulin release and intracellular insulin content compared with non treated islets before and after cryopreservation. When 4000 IEQ (Islet Equivalent) of islets treated with taurine and non treated cryopreserved islets were transplanted into syngenic streptozotocin induced diabetic rat, all showed normoglycemia over 60 days. CONCLUSION: Cryopreservation of islets could give a tool of storage with preservation of islet secretion function. However pertinent effort to improve the recovery is needed in order to be used in the clinical islet transplantation.

Comparative Study on Biologic and Immunologic Characteristics of the Pancreas Islet Cell between 24degrees C and 37degrees C Culture in the Rat / 대한이식학회지

PURPOSE: Islet cell transplantation, as an alternative approach to endocrine cell replacement to treat the diabetes mellitus, has received significant attention because it holds several advantages over whole gland transplantation. However cell damage from islet isolation and immunologic rejection after transplantation prevent from successful clinical application for diabetic patients. Culture of cells at low temperature has known to stabilize the cell viability, and to decrease the immunologic antigenicity. Aim of this study is to investigate the effect of culture at 24oC on cell viability, cellular function, immunogenicity and cytokine profiles in rat pancreas islet. METHODS: Pancreas islets were isolated from Lewis rat and cultured at 24oC or 37oC during 14 days. Islet recovery after culture period was counted as islet equivalent number, and islet viability was examined with fluorescent vital staining (FDA/PI). Islet function was measured with glucose stimulation test. Annexin V expression and MHC class I and II expression were measured with flow cytometric assay for apoptosis and immunogenicity respectively. Lymphocyte cell proliferation through mixed lymphocyte islet culture was examined with WST-1 proliferation assay. Cytokine profiles were analyzed with quantitative real time RT-PCR. All these parameters were measured on 1, 3, 5, 7, 14 culture days after islet isolation. RESULTS: Islet recovery was higher in islet cultured at 24oC than in islet cultured at 37oC without change of viability. Insulin secretion after glucose stimulation was more effective in 24oC culture condition. Decrease of apoptotic cell death was demonstrated in 24oC cultured islet. MHC class I and II expression on islets and lymphocyte proliferation when cocultured with islets were less prominent in 24oC cultured islet. TNF-alpha and IL-4 cytokine expression was higher in islet cultured at 24oC than in islet cultured at 37oC. IL-1beta and IL-10 cytokine expression were similar in both culture condition. CONCLUSION: This study demonstrated that cell recovery and function are increased in islet cultured at 24oC than in islet cultured at 37oC while antigenicity and proinflammatory cytokine expression are decreased. Low temperature culture can be a good approach to prevent the loss of islet mass, and to reduce the immunologic rejection of transplanted islet for successful clinical islet transplantation.