In Vitro Analysis of Co-cultured Pancreatic Islet with Lymphocyte

Kang, Choong Hoon; Kim, Song Cheol; Jang, Hyuk Jae; We, You Me; Park, Hee Young; Cho, Kyung Min; Han, Duck Jong

Abstract

BACKGROUND: Transplantation of Pancreatic islet represents one of the most exciting treatment modalities for Type I diabetes mellitus. To achieve better graft survival, it is important to protect the graft from alloantigen-specific immune response. It was emphasized that islets, as with other forms of cellular transplants, have the potential advantage of being immunologically altered before transplantation, resulting in tolerance to the host without using long-term, nonspecific immunosuppression. The mixed islet-lymphocyte culture is an excellent tool to evaluate the immunogenicity of a pancreatic islets. Therefore, we co-cultured pancreatic islet and lymphocyte to investigate cytokine gene expression from the lymphocyte, and to investigate pancreatic islet viability and functional changes after co-culture.
MATERIALS AND METHODS
Lewis rat islets were purified from pancreas by collagenase type XI and dextran gradient method. Afterwards, Lewis rat islets were co-cultured as a stimulator and Wistar rat lymphocyte as a reponder. As a control group lymphocyte alone and islet alone were cultured in a same condition. For estimating islets viability, we counted islet viability as an IEQ (Islet equivalent). For evaluation of islets function, insulin release assay was perfomed by RIA under the glucose challenge. used by RIA. We studied cytokines gene expression of cultured cells by reverse transcriptase polymerase chain reaction (RT-PCR). RESULT AND CONCLUSION: 1) Islet yield was 1229.8 420.1 per rat, and counted as 2615.4 548.2 IEQ per rat. 2) Islet viability was decreased gradually with the lapse of time, more rapid in allogenic co-culture group than islet alone or isogenic co-culture group. 3) Release of insulin increased until day 3, and then decreased gradually. Insulin release was positively correlated with glucose gradient. The amount of released insulin was greater in co-cultured group than islet alone group. 4) Interleukine-2 and interferron-gamma gene expression increased in allo co-culture group, however transforming growth factor-beta gene expression was not affected by co-culture.