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

마이코 페놀산에 의한 췌도세포주 사멸의 기전

목적: Mycophenolic acid (MPA)는 췌장이식을 포함한 다양한 종류의 장기이식에 사용되는 면역억제제로 inosine monophosphate dehydrogenase (IMPDH)의 선택적이고 비경쟁적인 억제제이나 췌도세포주에서는 세포 사멸을 유도한다고 알려져 있다. 본 연구에서는 인슐린을 분비하는 췌도 세포주인 HIT-T15 세포를 사용하여, MPA가 세포 사멸을 일으키는 기전을 규명하고자 하였다. 방법: 세포주는 American Type Culture Collection에서 구입하였으며 10% fetal bovine serum이 포함된 RPMI-1640을 사용하여 배양하였다. 세포 활성은 methylthiazoletetrazolium (MTT) assay, 세포 사멸은 annexin V와 PI 염색법, mitogen-activated protein kinase (MAPK) 활성화와 caspase-3 분절은 Western blot 분석으로 측정하였다. 결과: MPA 1μM과 10μM을 처리하였을 때 MTT, caspase-3 분절 그리고 annexin V 염색이 24시간에 농도 의존적으로 증가하였으며, 이는 외부에서 함께 투여한 guanosine 500μM에 의하여 부분적으로 회복되었으나 adenosine 500μM 투여에서는 변화가 없었다. 또한 MPA는 extracellular-regulated protein kinase (ERK), p38 MAPK 그리고 c-jun N-terminal protein kinase (JNK)의 활성화를 8시간과 24시간에서 증가시켰고 guanosine 투여는 이를 부분적으로 회복시켰다. ERK의 억제제인 PD98059, p38 MAPK 억제제인 SB203580 그리고 JNK 억제제인 SP600125는 MPA와 함께 처리하였을 때 각 시간에 증가된 MAPK 활성을 감소시켰지만 MTT와 caspase-3 분절을 살펴본 결과 PD98059는 영향이 없었으며 SB203580은 세포사멸을 증가시켰고, SP600125만이 MPA가 일으킨 세포 사멸을 일부 환원시켰다. Pan-caspase 억제제인 Z-VAD-FMK 또한 세포 사멸을 환원시켰다. 결론: MPA는 MAPK 활성을 IMPDH 의존적으로 증가시키지만, ERKl와 p38 MAPK와는 상관없이, JNK 활성화를 통한 caspase-3 증가의 경로로 췌도 세포 사멸을 유도함을 알 수 있었다.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-κB 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-κB, 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 ± 20.41 μm 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

AEB-071 versus tacrolimus monotherapy to prevent acute cardiac allograft rejection in the rat: a preliminary report

Inhibition of T-cell activation is the most efficient way to prevent transplant rejection. Protein kinase C (PKC) is an important signaling enzyme in the activation and regulation of T lymphocytes. AEB-071 (AEB) is a low-molecular-weight compound that blocks early T-cell activation via selective inhibition of PKC, a mechanism that differs from that of the calcineurin inhibitors. The present study sought to compare the effects of AEB versus tacrolimus (Tac) to prevent acute rejection in rats that had undergone heterotopic heart transplantation. We investigated the Brown Norway-Lewis rat strain combination for cardiac graft survival over 30 days after transplantation using varying doses of oral AEB and Tac monotherapy. Grafts were monitored by daily palpation; cessation of palpable ventricular contraction was considered to be rejection. Apart from necropsy, we performed histologic examinations of cardiac graft at 7 days after transplantation. In untreated recipients, allograft mean survival times (MST) was 6.83+/-0.41 days. AEB at 15, 30, or 60 mg/kg versus Tac at 1.2 mg/kg significantly prolonged graft survival to a MST of 12.33+/-1.21, 16.67+/-1.21, and 19.33+/-3.83, versus 17.00+/-6.90 days, respectively. Histologic assessment at 7 days after transplantation showed that high-dose AEB significantly decreased the histologic rejection score, indicative of decreased inflammatory cell infiltration into the graft. These results suggested that the administration of AEB (medium or high-dose), a PKC inhibitor, mitigated acute rejection and displayed significantly longer MST, similar to high-dose Tac after heterotopic heart transplantation in the rat.ope

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