benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone has been researched along with Diabetes-Mellitus--Type-1* in 2 studies
2 other study(ies) available for benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Diabetes-Mellitus--Type-1
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Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application.
One challenge that must be overcome to allow transplantation of neonatal porcine islets (NPIs) to become a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our standard protocol, we routinely isolate NPIs from a maximum of four pancreases, requiring tissue culture in 16 Petri dishes (four per pancreas) in Ham's F10 and bovine serum albumin (BSA). We have now developed a scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes. For culture, BSA is replaced with human serum albumin and supplemented with Z-VAD-FMK general caspase inhibitor and a protease inhibitor cocktail. The caspase inhibitor was added to the media for only the first 90 min of culture. NPIs isolated using the scalable protocol had significantly more cellular insulin recovered (56.9 ± 1.4 µg) when compared to the standard protocol (15.0 ± 0.5 µg; p < 0.05). Compared to our standard protocol, recovery of β-cells (6.0 × 10(6) ± 0.2 vs. 10.0 × 10(6) ± 0.4; p < 0.05) and islet equivalents (35,135 ± 186 vs. 41,810 ± 226; p < 0.05) was significantly higher using the scalable protocol. During a static glucose stimulation assay, the SI of islets isolated by the standard protocol were significantly lower than the scale-up protocol (4.3 ± 0.2 vs. 5.5 ± 0.1; p < 0.05). Mice transplanted with NPIs using the scalable protocol had significantly lower blood glucose levels than the mice that receiving NPIs from the standard protocol (p < 0.01) and responded significantly better to a glucose tolerance test. Based on the above findings, this improved simpler scalable protocol is a significantly more efficient means for preparing therapeutic quantities of clinical grade NPIs. Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Caspase Inhibitors; Culture Media; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Humans; Insulin; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Mice; Serum Albumin; Swine; Tissue Culture Techniques; Transplantation, Heterologous | 2016 |
Recapitulation of normal and abnormal BioBreeding rat T cell development in adult thymus organ culture.
Congenitally lymphopenic diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous T cell-dependent autoimmunity. Coisogenic diabetes-resistant (DR) BB rats are not lymphopenic and are free of spontaneous autoimmune disease, but become diabetic in response to depletion of RT6+ T cells. The basis for the predisposition to autoimmunity in BB rats is unknown. Abnormal T cell development in DP-BB rats can be detected intrathymically, and thymocytes from DR-BB rats adoptively transfer diabetes. The mechanisms underlying these T cell developmental abnormalities are not known. To study these processes, we established adult thymus organ cultures (ATOC). We report that cultured DR- and DP-BB rat thymi generate mature CD4 and CD8 single-positive cells with up-regulated TCRs. DR-BB rat cultures also generate T cells that express RT6. In contrast, DP-BB rat cultures generate fewer CD4+, CD8+, and RT6+ T cells. Analysis of the cells obtained from ATOC suggested that the failure of cultured DP-BB rat thymi to generate T cells with a mature phenotype is due in part to an increased rate of apoptosis. Consistent with this inference, we observed that addition of the general caspase inhibitor Z-VAD-FMK substantially increases the number of both mature and immature T cells produced by DP-BB rat ATOC. We conclude that cultured DR-BB and DP-BB rat thymi, respectively, recapitulate the normal and abnormal T cell developmental kinetics and phenotypes observed in these animals in vivo. Such cultures should facilitate identification of the underlying pathological processes that lead to immune dysfunction and autoimmunity in BB rats. Topics: ADP Ribose Transferases; Amino Acid Chloromethyl Ketones; Animals; Antigens, Differentiation, T-Lymphocyte; Apoptosis; Cell Differentiation; Cell Division; Cellular Senescence; Diabetes Mellitus, Type 1; Female; Histocompatibility Antigens; Immunity, Innate; Lymphocyte Count; Male; Membrane Glycoproteins; Organ Culture Techniques; Rats; Rats, Inbred BB; T-Lymphocytes; Thy-1 Antigens; Thymus Gland | 1999 |