기능성 췌장소도 클러스터 개발 및 적용

Abstract

Type 1 diabetes mellitus(T1DM) is an autoimmune disease, which results in the permanent destruction of β-cells. T1DM leads to complication such as cardiovascular risk, autonomic neuropathy, and the degree of renal dyfunction. Therefore, T1DM must be treated. Since type 1 diabetic patients only need pancreatic islets, which contain the insulin-secreting beta cells. To transplant pancreatic islets, they are isolated from pancreas organ by using enzyme digestion. However, the viability of isolated islets having large size is affected due to the hypoxia state since the microvessels around islets are destroyed during procedure of islet isolation. Therefore, the size-controllable islet cell clusters (ICCs) are recently focused on to overcome this limitation. The ICCs were prepared during re-aggregation of islet single cells after dispersion of intact islets. Unfortunately, the weak-point in this ICC procedure is that the functionality of ICCs was lost because the interaction of receptors and integrins on the surface of islet single cells was not restored. Therefore, new strategies are required to generate functional ICCs having improved cell-cell interaction. In chapter two, to overcome this limitation, we re-aggregated dispersed islet single cells at the concave micro-well array that was fabricated with poly(dimethylsiloxane) (PDMS) by using soft-lithography and mold-replication technology. The aggregated ICCs were uniform in size, and the amount of their insulin secretion in response to the change of low and high glucose concentration was similar to those of intact islets. This result indicated that the cell-cell interaction of islet single cells was strongly induced by the shape of concave micro-well. Also, after cultivation for 2 weeks, the expression of hypoxia-induced factor-1α (HIF-1α; a hypoxia marker protein) in the ICC was rare, meaning that hypoxia was attenuated in the central area of ICC. On the other hand, the ICC generated in the flat-bottomed dish (control group) had abnormal insulin secretion caused by weak cell-cell interactions. In chapter three, the formation of ICCs was optimized by using the concave-patterned micro-well with different diameters (300, 500, and 700㎛). Aggregated ICCs in different-sized concave microwells had similar morphology of intact islets with narrow size distribution and size dependency according to the diameter of concave microwell. However, ICCs in the flat-bottomed dish (control) had a pattern of random aggregation with a broad size distribution. Interestingly, the pattern of insulin secretion of ICC aggregated in 300 and 500㎛ concave patterned-microwells was similar that of intact islets. This was achieved by the increased cell-cell interactions which were confirmed by electron microscope and strong FITC-phalloidin immunostain. However, ICCs in the concave patterned-microwell with a 700 ㎛ diameter and in the flat-bottomed dish did not show a normal insulin secretion in accordance with varying glucose concentrations. In chapter four, we fabricated the embossing-type ICC sheet using concave microwells. To this end, collagen-mixed alginate solution was treated after ICC formation on the concave microwell. After gelation using CaCl2 solution, collagen-mixed alginate ICC (CAC) sheet was prepared. When CAC-encapsulated ICCs were transplanted into diabetic mice, they normally regulated blood glucose levels below 200 mg/dL for more than one month. In addition, from the result of intraperitoneal glucose tolerance test (IPGTT), the normal glucose responsiveness of CAC-encapsulated ICCs was confirmed. In chapter five, to overcome the hypoxia-induced islet death, heme oxygenase-1 (HO-1) gene was delivered into islets. The main role of HO-1 is the formation of three kinds of products: carbon monoxide, free iron, and biliverdin. It is reported that these products can attenuate the hypoxia-induced islet death. Therefore, HO-1 gene was delivered into islets by using Lentivirus system. From the viability study using hypoxia chamber, HO-1 expressing pancreatic islets could be overcome. Also, when HO-1-expressing pancreatic islets were transplanted into subcutaneous space in diabetic mice, they could normally regulate the blood glucose levels, meaning that HO-1 gene could give the transplanted islets the hypoxia-resistance. In conclusion, the functional islet cell cluster (ICC) could be designed by using concave microwell array system and HO-1 gene delivery. Therefore, it may be a promising alternative for the treatment of type 1 diabetes using islet transplantation.