15 research outputs found
Islet Encapsulation Using Chondrocyte
Diabetes mellitus is one of the leading metabolic diseases that cause an increasing rate of mortality and morbidity. Recently, rather than the current drug treatment, pancreatic islet transplantation has been regarded as a potentially promising strategy for insulin- dependent diabetes mellitus while preventing complications such as kidney damage, vascular damage, nerve damage, and blindness. Recently, a number of advanced islet encapsulation techniques have been designed to enhance the efficiency of islet transplantation, including cell sheet engineering and generation of 3D islet spheroids by high density suspension system (HDSS). Chondrocytes derived from cartilage sources have been used as an encapsulation biomaterial for islets not only for autograft but also for allograft and xenograft transplantation. Cartilage is an avascular, white connective tissue that is rich in extracellular matrix, and expandable in vitro. Hence, this tissue might have immunologically privileged properties that make it an intelligent cell source for manufacture of encapsulation biomaterials. However, cell sheet engineering and HDSS still have their respective limitations, which need to be elucidated. This review will describe the advantages and disadvantages of the current encapsulation techniques in order to provide a comprehensive foundation for further modifications and improvements of tissue engineering for islet transplantation.ope
๋ง์ด์ฝ ํ๋์ฐ์ ์ํ ์ท๋์ธํฌ์ฃผ ์ฌ๋ฉธ์ ๊ธฐ์
๋ชฉ์ : Mycophenolic acid (MPA)๋ ์ท์ฅ์ด์์ ํฌํจํ ๋ค์ํ ์ข
๋ฅ์ ์ฅ๊ธฐ์ด์์ ์ฌ์ฉ๋๋ ๋ฉด์ญ์ต์ ์ ๋ก inosine monophosphate dehydrogenase (IMPDH)์ ์ ํ์ ์ด๊ณ ๋น๊ฒฝ์์ ์ธ ์ต์ ์ ์ด๋ ์ท๋์ธํฌ์ฃผ์์๋ ์ธํฌ ์ฌ๋ฉธ์ ์ ๋ํ๋ค๊ณ ์๋ ค์ ธ ์๋ค. ๋ณธ ์ฐ๊ตฌ์์๋ ์ธ์๋ฆฐ์ ๋ถ๋นํ๋ ์ท๋ ์ธํฌ์ฃผ์ธ HIT-T15 ์ธํฌ๋ฅผ ์ฌ์ฉํ์ฌ, MPA๊ฐ ์ธํฌ ์ฌ๋ฉธ์ ์ผ์ผํค๋ ๊ธฐ์ ์ ๊ท๋ช
ํ๊ณ ์ ํ์๋ค. ๋ฐฉ๋ฒ: ์ธํฌ์ฃผ๋ American Type Culture Collection์์ ๊ตฌ์
ํ์์ผ๋ฉฐ 10% fetal bovine serum์ด ํฌํจ๋ RPMI-1640์ ์ฌ์ฉํ์ฌ ๋ฐฐ์ํ์๋ค. ์ธํฌ ํ์ฑ์ methylthiazoletetrazolium (MTT) assay, ์ธํฌ ์ฌ๋ฉธ์ annexin V์ PI ์ผ์๋ฒ, mitogen-activated protein kinase (MAPK) ํ์ฑํ์ caspase-3 ๋ถ์ ์ Western blot ๋ถ์์ผ๋ก ์ธก์ ํ์๋ค. ๊ฒฐ๊ณผ: MPA 1ฮผM๊ณผ 10ฮผM์ ์ฒ๋ฆฌํ์์ ๋ MTT, caspase-3 ๋ถ์ ๊ทธ๋ฆฌ๊ณ annexin V ์ผ์์ด 24์๊ฐ์ ๋๋ ์์กด์ ์ผ๋ก ์ฆ๊ฐํ์์ผ๋ฉฐ, ์ด๋ ์ธ๋ถ์์ ํจ๊ป ํฌ์ฌํ guanosine 500ฮผM์ ์ํ์ฌ ๋ถ๋ถ์ ์ผ๋ก ํ๋ณต๋์์ผ๋ adenosine 500ฮผM ํฌ์ฌ์์๋ ๋ณํ๊ฐ ์์๋ค. ๋ํ MPA๋ extracellular-regulated protein kinase (ERK), p38 MAPK ๊ทธ๋ฆฌ๊ณ c-jun N-terminal protein kinase (JNK)์ ํ์ฑํ๋ฅผ 8์๊ฐ๊ณผ 24์๊ฐ์์ ์ฆ๊ฐ์์ผฐ๊ณ guanosine ํฌ์ฌ๋ ์ด๋ฅผ ๋ถ๋ถ์ ์ผ๋ก ํ๋ณต์์ผฐ๋ค. ERK์ ์ต์ ์ ์ธ PD98059, p38 MAPK ์ต์ ์ ์ธ SB203580 ๊ทธ๋ฆฌ๊ณ JNK ์ต์ ์ ์ธ SP600125๋ MPA์ ํจ๊ป ์ฒ๋ฆฌํ์์ ๋ ๊ฐ ์๊ฐ์ ์ฆ๊ฐ๋ MAPK ํ์ฑ์ ๊ฐ์์์ผฐ์ง๋ง MTT์ caspase-3 ๋ถ์ ์ ์ดํด๋ณธ ๊ฒฐ๊ณผ PD98059๋ ์ํฅ์ด ์์์ผ๋ฉฐ SB203580์ ์ธํฌ์ฌ๋ฉธ์ ์ฆ๊ฐ์์ผฐ๊ณ , SP600125๋ง์ด MPA๊ฐ ์ผ์ผํจ ์ธํฌ ์ฌ๋ฉธ์ ์ผ๋ถ ํ์์์ผฐ๋ค. Pan-caspase ์ต์ ์ ์ธ Z-VAD-FMK ๋ํ ์ธํฌ ์ฌ๋ฉธ์ ํ์์์ผฐ๋ค. ๊ฒฐ๋ก : MPA๋ MAPK ํ์ฑ์ IMPDH ์์กด์ ์ผ๋ก ์ฆ๊ฐ์ํค์ง๋ง, ERKl์ p38 MAPK์๋ ์๊ด์์ด, JNK ํ์ฑํ๋ฅผ ํตํ caspase-3 ์ฆ๊ฐ์ ๊ฒฝ๋ก๋ก ์ท๋ ์ธํฌ ์ฌ๋ฉธ์ ์ ๋ํจ์ ์ ์ ์์๋ค.ope
The RhoGDI-alpha/JNK signaling pathway plays a significant role in mycophenolic acid-induced apoptosis in an insulin-secreting cell line
Mycophenolic acid (MPA)-induced beta-cell toxicity is an important factor for islet graft function. The signal transduction mechanisms underlying this process have not been fully explored. Using a proteomics approach, we examined protein expression patterns in MPA-treated RIN-5 cells and found that RhoGDI-alpha expression is altered by MPA-treatment. We examined the relationship between RhoGDI-alpha expression and activated JNK during MPA-induced apoptosis. Cells were treated with N-acetyl-cysteine (NAC), caspase inhibitor, JNK inhibitor, guanosine or GTP for 1 h before being treated with MPA. To investigate the regulatory effects of RhoGDI-alpha on JNK activity, we examined cells showing either elevated or reduced expression of RhoGDI-alpha as a result of transfection with cDNA or siRNA constructs, respectively. MPA significantly increased cell death, caspase-3 expression and JNK activation, but it decreased the expression of a protein spot 25 observed by two-dimensional electrophoresis. This protein 25 was identified as RhoGDI-alpha by mass spectrometry. MPA-induced cell death and down-regulation of RhoGDI-alpha were prevented by guanosine, GTP or a JNK inhibitor. However, MPA-induced cell death was partially restored by treatment with a caspase inhibitor, but not by NAC treatment. RhoGDI-alpha expression was not affected by treatment with NAC or caspase inhibitor. Over-expression of RhoGDI-alpha increased cell viability and decreased activated JNK expression following exposure to MPA, whereas knockdown of RhoGDI-alpha enhanced MPA-induced cell death and increased the activation of JNK. In conclusion, MPA induces significant apoptosis in insulin-secreting cells via down-regulation of RhoGDI-alpha linked with increased JNK expression. This RhoGDI-alpha/JNK pathway might be the focus of therapeutic target for the prevention of MPA-induced islet apoptosis.ope
Functional improvement of pig islet with exocrine encapsulation
Porcine-specific obstacles in islet isolation frequently result from the low purity or contamination with exocrine tissues. We implemented a new technique involving as capsulation of islets with excess exocrine tissue as a beneficial material to address those difficulties. Pig islets were hand-picked as high purity (HI) or low purity (LO) islets containing significant amounts of exocrine tissue. We performed static (ST) or shaking (SK) cultures of HI and LO islets. Islet function was examined after 24 hours by a glucose challenge test. Insulin secretion into the culture media was continuously measured using ELISA during a 6-day culture period. Islet function after 24 hours exhibited better maintenance under SK than ST culture as assessed by a stimulation index. The ideal islet morphology was seen in LO islets at 3 days of SK culture with typical islet shapes of a smooth surface and a spherical configuration. In contrast, typical islet morphology was not observed in HI islets under SK culture; maintenance of typical spherical appearances was difficult. Insulin secretion from LO islets under SK culture was higher than under other conditions during the 6-day period. Under SK culture conditions, exocrine-encapsulated LO islets showed enhanced islet function by condensing loose islet aggregates into firm spheroids with native exocrine tissues as a natural scaffold.ope
Hybrid cellular spheroids from hepatocellular carcinoma and insulin-secreting cell lines
During islet transplantation into the portal vein of the liver, the islet cells are expected to have complex interactions with hepatocytes. However, the mechanism underlying this interaction is not yet understood. Hence, we developed cellular complexes containing a mixture of human hepatocellular carcinoma cell line (Hep-G2) and rat insulin-secreting cell line (RIN-5F) by using a co-culture model and studied the function and morphology of the resultant hybrid cellular spheroids (HCSs). The RIN-5F and Hep-G2 cells were suspension cultured and, within 5 days of culture, the two types of cells aggregated to yield spheroids. The functionality of the thus formed HCSs was evaluated by measuring the levels of insulin and albumin in the culture supernatant. The HCSs retained their insulin- and albumin-secreting ability and their morphology, as revealed by immunohistological staining. The insulin and albumin levels secreted by the HCSs were considerably higher than those secreted by spheroids of single-cell origin. Generally, obtaining complexes from more than two types of cells is difficult. However, we were able to generate HCSs. We believe that this culture method could have various applications such as studying the in vitro cell-cell interactions and developing new cell transplantation models.ope
Impact of coculture with ischemic preconditioned hepatocellular carcinoma cell line (Hep-G2) cells on insulin secreting function of rat insulin-secreting cell line (RIN-5F) cells
INTRODUCTION: Although Islet cell isolation and culture have been well developed, there has been little progress to prolong transplanted islet survival. Hepatic ischemia and insufficient neovascularization of islets are considered to be the barriers to long-term survival, Hepatocytes that survive ischemic injury have been reported to protect themeslves and regenerate using the IL-6 interleukin 6 and STAT3 pathways.
MATERIALS AND METHODS: The hepatocellular carcinoma (Hep-G2) cell line preconditioned for 0, 2, 4, 6, and 24 hours in a hypoxic chamber, was cocultured with rat insulin-secreting celline (RIN-5F) cells. We measured cell viabilities, insulin secretion, and p-STAT3, IL-6, and NF-ฮบB levels.
RESULTS: Cocultured Hep-G2 and RIN-5F cells aggregated to form spheroids. Viabilities of Hep-G2 cells were no different after various ischemic preconditioning times, but insulin secretion increased in a time-dependent fashion with preconditioning. Western blotting showed p-STAT3, NF-ฮบB, and IL-6 levels to increase with preconditioning time.
CONCLUSION: The IL-6/STAT3 pathway of Hep-G2 cells after ischemic injury showed beneficial effects on insulin secretion of RIN-5f cells cocultured with themselves.ope
Manufacturing of insulin-secreting spheroids with the RIN-5F cell line using a shaking culture method
BACKGROUND: There have been many efforts to find methods to increase insulin production by islets or modified cells. Commercially available established cell lines can be a good source of artificial islets. We manufactured sphere-shaped cell clusters composed of insulin-secreting cells from the commercially available RIN-5F cell line.
METHODS: To generate artificial islets with insulin-secretion functions, we used the RIN-5F cell line. When cells cultured in RPMI-1640 medium containing 10% fetal bovine serum reached near confluency, they were trypsinized for suspension culture at high density, using a horizontal shaker. The cells were maintained for 5 days under 5% CO(2) with humidification. Next, the media from the RIN cell spheroid culture was collected over 5 consecutive days to test for insulin secretion.
RESULTS: Spheroids of artificial islets exhibited an oval shape with an approximate size of 94.13 ยฑ 20.41 ฮผm on day 5 during the shaking culture. Abnormal outgrowth of spheroids was not observed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were not detected among the overall spheroids, including the core position. Insulin secretion, measured by enzyme-linked immunosorbent assay, was well maintained in the culture media over 5 days after spheroid formation.
CONCLUSION: This result suggested that a culture method with shaking can be applied to commercially available established cell lines to generate artificial islets, which might be used for a bioartificial pancreasope
Change in serum lipid peroxide as an oxidative stress marker and its effects on kidney function after successful kidney transplantation
BACKGROUND: Reactive oxygen species are believed to be responsible for organ injury after reperfusion. We evaluated serial changes in lipid peroxide (LPO) as an oxidative stress marker after kidney transplantation and investigated its effects on graft function.
METHODS: Fifty-nine kidney transplant recipients were enrolled between September 2006 and March 2009. The control group consisted of kidney donors (n=40). Serum LPO concentrations were measured by a thiobarbituric acid reaction. The serum creatinine concentration and estimated glomerular filtration rate (eGFR) were used to evaluate graft function. Blood samples were obtained preoperatively, on postoperative day (POD) 5, and at 1 year posttransplantation. The median concentration of LPO on POD 5 was used as a cut-off.
RESULTS: The mean preoperative LPO concentration was greater than the control group. The mean LPO concentration on POD 5 was increased compared with the preoperative level. However, the mean LPO concentration at 1 year was significantly decreased compared with the preoperative day, but greater than the control group. On POD 5, the mean serum creatinine concentration in the low LPO group was lower than that in the high LPO group. The mean eGFR in the low LPO group was significantly higher than that in the high LPO group. There was no difference in mean serum creatinine concentrations and eGFR at 1 year between the groups.
CONCLUSION: Oxidative stress showed a significant impact on graft function in the immediate posttransplant periodope
Functional evaluation of chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas
In islet transplantation, encapsulation of immunoisolated islets may provide a way to protect the graft from immune attacks with no immunosuppression. To develop an immunodelusive immunoisolated bioartificial pancreas (BAP), chondrocyte sheets were prepared by cell sheet engineering. We made an immunoisolated BAP encapsulated with rodent-derived chondrocyte sheets and then evaluated its function. Sprague-Dawley rats were used as the source of auricular cartilage and chondrocytes were maintained and expanded by passages. Lewis rats were prepared for islet isolation. A 3-dimensional chondrocyte sheeting immunodelusive immunoisolated BAP (CSI-BAP) was created by multi-layering and unifying the chondrocyte sheets. Subsequently, islets were embedded between each multi-layer sheet. To evaluate the function of the CSI-BAP, a glucose challenge test was performed and secretion of insulin in the culture medium was measured by an enzyme-linked immunosorbent assay. When observed by phase-contrast microscopy, the CSI-BAP maintained close connections between chondrocyte sheets. Islets in the CSI-BAP maintained viability at day 10 and showed good insulin secretion, as revealed by a prompt reaction to increased concentrations of glucose at days 5 and 10. In long-term culture, the CSI-BAP maintained its ability to secrete insulin for 8 weeks. This BAP technology could be an important tool for successful islet transplantation without immunosuppressive drugs.ope
Microencapsulation of pancreatic islets with canine ear cartilage for immunoisolation
Improving human islet transplantation is often limited by the shortage of donors and the side effects of immunosuppressive agents. If immunoisolation is properly used, it can overcome these obstacles. Because artificial materials are adopted in this technique, however, there are still multiple issues with biocompatibility and foreign body reactions. We developed a chondrocyte microencapsulated immunoisolated islet (CMI-islet) that allows living cells to act as the immunoisolating material. To manufacture CMI-islets for xenotransplantation, isolated rat pancreatic islets were placed on low cell-binding culture dishes. Subsequently, expanded canine auricular cartiage primary cells were seeded on these dishes at a high density and maintained in a suspended state via a shaking culture system. Morphological evaluations showed good islet viability and a clear progression of the islet- encapsulation events. When the cells were challenged with glucose, they were able to secrete sufficient insulin according to glucose concentrations. The CMI-islets responded better to the glucose challenge than did nude pancreatic islets and created better glucose-insulin feedback regulation. Moreover, insulin secretion into the culture medium was confirmed over a period of 100 days, showing the survival and secretory capacity of the CMI-islet cells. By microencapsulating pancreatic islets with recipient ear cartilage cells, long-term insulin secretion can be maintained and the response to glucose challenges improved. This new immunodelusion technology differs from other immunoisolation techniques in that the donor tissue is enclosed with the recipient's tissue, thus allowing the transplanted cells to be recognized as recipient cells. This microencapsulation method may lead to developing viable xenotransplantation techniques that do not use immunosuppressive drugs.ope