Small Intestinal Submucosa Improves Islet Survival and Function in Vitro Culture

doi:10.1016/j.transproceed.2006.02.134

Transplantation Proceedings

Volume 38, Issue 5, June 2006, Pages 1552-1558

https://doi.org/10.1016/j.transproceed.2006.02.134 Get rights and content

Abstract

Introduction

Most centers maintain isolated human islet preparations in tissue culture to improve the safety as well as the practicality of islet transplantation. However, maintaining viability and recovery of islets remains a challenge. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell-matrix relationship seems to be necessary to sustain the structure and function of differentiated islets. Small intestinal submucosa (SIS), a natural ECM, is well known to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate recovery and function of isolated rat pancreatic islets during in vitro culture with SIS.

Methods

Pancreatic islets isolated from Wistar rats following intraductal collagenase distension, mechanical dissociation, and EuroFicoll purification were cultured in plates coated with multilayer SIS (SIS-treated group) or without (standard cultured group) for 7 or 14 days in an islet culture media of RPMI 1640 (Gibco). The islets from both experimental groups were stained and counted with dithizone. Islet recovery following culture was determined by the ratio of counts after culture to the yield of islets immediately following islet isolation. The viability of the islets was assessed by a glucose challenge test with low glucose (2.7 mmol/L), high glucose (16.7 mmol/L), and high glucose solution supplemented with 50 μmol/L 3-isobutyl-1-methylxanthine solution. The apoptosis of islet cells was measured by relative quantification of histone-complexed DNA fragments by using enzyme-linked immunosorbent assay.

Results

After 7 or 14 days of in vitro tissue culture, the recovery in SIS-treated islets group was about double of that cultured in the plates without SIS coating. In the SIS-treated group, there was no significant difference between the short- and the long-term periods of culture (95.8% ± 1.0% vs 90.8% ± 1.5%, P > .05). Following incubation with high glucose (16.7 mmol/L) solution, the insulin secretion in the SIS-treated group showed a greater increase than the control group after 14 days of culture (20.7 ± 1.1 mU/L vs 11.8 ± 1.1 mU/L, P < .05). When islets were placed in the high glucose solution containing IBMX, the stimulated insulin secretion was more increased in the SIS-treated than in the control group despite the duration of the culture. The calculated stimulation index of SIS-treated group was about two to three times greater than the control group. In addition, the stimulation index of the SIS-treated group remained constant regardless of short-term versus long-term culture (9.5 ± 0.2 vs 10.2 ± 1.2, P > .05). Much less apoptosis of islet cells occurred in the SIS-treated than in the control group.

Conclusion

Coculture of isolated rat islets with native sheetlike small intestinal submucosa seemed to build an ECM for islets providing possible biotrophic and growth factors that promote the recovery and subsequent function of islets.

Section snippets

Animals

Male Wistar rats (250 to 400 g body weight, Animal Lab of Xi’an Jiaotong University, China) were used for islet isolation. All animals received care in compliance with the guidelines of the local Animal Care and Use Committee following National Institutes of Health guidelines.

Design of the Study

Islets were isolated by using collagenase and purified by discontinuous Euroficoll gradients. The purified islets were separated into two experimental groups: the study group were islets cultured in plates coated with SIS,

Results

After 7 or 14 days of in vitro tissue culture, the group of islets cultured with SIS showed a twofold greater (P < .05) recovery than the control group (Table 1). In the SIS-treated group, there was no statistical difference between the short and the long periods of culture (95.8% ± 1.0% vs 90.8% ± 1.5%, P > .05). Most islets cultured in the control group lost their initial morphology, becoming “loose” in appearance at the end of the culture, where as islets cultured with SIS exhibited

Discussion

Islet transplantation is now a viable option for the treatment of insulin-dependent diabetes mellitus. Maintaining islets in culture is important to increase the safety, practicality, and efficacy imperative for successful clinical results. In addition to reducing the immunogenicity of islets,3, 17 culture simplifies the logistics of transplantation—the procedure can be scheduled during regular hospital hours, and the patient does not have to move from a distant location to the transplantation

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: This work was supported by the Key Program of Science & Technique of Ministry of Education of the People’s Republic of China (No. 104169).

View full text

Pancreas and islet transplantationSmall Intestinal Submucosa Improves Islet Survival and Function in Vitro Culture

Abstract

Introduction

Methods

Results

Conclusion

Section snippets

Animals

Design of the Study

Results

Discussion

J Surg Res

Surg Neurol

Urology

J Surg Res

J Pediatr Surg

Transplant Proc

Surgery

Transplant Proc

Cell

Current situation and prospect of the islet transplantation

Chinese Journal of Diabetes

Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen

N Engl J Med

Major histocompatibility complex antigens and murine islet allograft survival

Transplantation

A comparison of media supplement methods for the extended culture of human islet tissue

Transplantation

Insulin independence following isolated islet transplantation and single islet infusions

Ann Surg

Functional impact of attachment and purification in the short term culture of human pancreatic islets

J Clin Endocrinol Metab

Compliance, elastic modulus, and burst pressure of small-intestine submucosa (SIS), small diameter vascular grafts

J Biomed Mater Res

Preparation and characteristics of small intestinal submucosa

Chinese Journal of Reparative and Reconstructive Surgery

Replacement of large veins with free inverted segments of small bowelautografts of submucosal membrane in dogs and clinical use

Ann Surg

Pancreas and islet transplantation
Small Intestinal Submucosa Improves Islet Survival and Function in Vitro Culture