S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) Theses (Ph.D. / Sc.D._의학과)
Effect of an oxygen-generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters (NPCCs)
산소 발생 기능성 구조체가 신생돼지 췌도의 생존율과 인슐린 분비기능에 미치는 영향에 관한 연구
- 의과대학 의학과
- Issue Date
- 서울대학교 대학원
- oxygen-generating scaffold; macroencapsulation; neonatal pancreatic cell clusters; insulin secretion; microfluidic system
- 학위논문 (박사)-- 서울대학교 대학원 : 의학과, 2016. 8. 안규리.
- Effect of an oxygen-generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters (NPCCs)
Eun Mi Lee
Major in Immunology, Department of Medicine
The Graduate School
Seoul National University
Diabetes is a chronic metabolic disease that occurs when the body cannot produce enough insulin or cannot use insulin and is diagnosed by observing elevated levels of glucose in the blood. Diabetes therapies can be classified into oral medication, insulin injection, and pancreas or pancreatic islet transplantation. In case of final stage of diabetes, pancreatic islet transplantation would be consider as a best therapeutic strategy. Most successful diabetes therapies through intraportal islet transplantation are considered as the Edmonton protocol
however, pancreatic islet transplantation into the hepatic venous vessels results in induction of instant blood-mediated inflammatory reaction (IBMIR). To overcome this limitation, many researchers have tried to apply an encapsulation technique for islet transplantation. It has been widely considered as a promising method for this purpose because it blocks host antibody-mediated or cellular immune responses to the donor islets. However, it is critical to maintain the survival and function of the islets because macroencapsulation reduces the oxygen supply to the islets. Therefore, in this study, to ameliorate hypoxic damage, an oxygen-generating scaffold was used to investigate whether it can improve the viability and insulin secretion function of the islets.
Porcine neonatal pancreatic cell clusters (NPCCs) were isolated from ~5-day-old piglets and used after 7 days of culture for their maturation. A CCK-8 assay revealed that the PDMS-CaO2 scaffold group had higher viability than the other groups. In addition, the PDMS-CaO2 group showed reduced activity of apoptosis-related enzymes such as Caspase 3 and 7, which resulted in lower hypoxia-induced NPCCs death. Moreover, the reactive oxygen species (ROS) level in the PDMS-CaO2 group was lower than that in the control group. Since the goal of using oxygen-generating scaffolds is to provide supplemental oxygen for the NPCCs, the oxygen consumption rate (OCR) was determined in the NPCCs of the PDMS-CaO2 group. The PDMS-CaO2 group mostly showed higher OCR than the control group. The insulin secretion index of the NPCCs was higher in the PDMS-CaO2 group than in the PDMS or control group.
To investigate the impact of the oxygen-generating scaffolds ex vivo, a microfluidic system was established using a BioFlux 200 instrument and microfluidic device-pump system for representing the physical conditions. After injection of the NPCCs into the PDMS or PDMS-CaO2 scaffolds, they were embedded on the hole of microfluidic device (A2) and cultured under normoxic and hypoxic conditions. The viability of the NPCCs was higher in the PDMS-CaO2 group than in the PDMS group.
In addition, a biocompatibility test of the oxygen-generating scaffolds was conducted through subcutaneous implantation in mice. The scaffolds showed stability until 8 months and the immunogenicity was very low in the spleens of the recipient mice. Thus, this test can be used to confirm the viability or function of NPCCs delivered using oxygen-generating scaffolds in animal models.
This study present that NPCCs encapsulated in PDMS-CaO2 scaffolds showed higher viability and insulin secretion compared to those encapsulated in PDMS scaffolds or the controls in vitro or ex vivo. In addition, a biocompatibility test of the oxygen-generating scaffold was performed by subcutaneous implantation in mice, where the scaffold showed stability until 8 months and very low immunogenicity in the spleens of the recipient mice. Thus, the oxygen-generating scaffold has potential for application in transplantation studies in the future.