The Purification Method Using Iodixanol (Optiprep)-Based Density Gradient Significantly Reduces Cytokine/Chemokine Production from Human Islet Preparations, Leading to Prolong β-Cell Survival During Culture

Abstract

Improved islet isolation has still been important to obtain adequate islet numbers for islet transplantation. Although Ficol-based density gradient is widely used for purification in most islet processing centers, OptiPrep-based density gradient is recently used in the limited centers and their clinical outcomes are excellent. Cytokine/chemokine production from islet preparations for transplantation widely varies. Some cytokines/chemokines have been reported to cause apoptosis in human islet preparations after isolation. Reducing cytokine/chemokine production is a key to improve islet numbers after isolation and islet transplantation outcome. The aim of current study is to investigate the variability of pro-inflammatory cytokine/chemokine production from islet preparations purified by different density gradient. After digestion of human pancreata, pre-purification digests were devided into two groups and purified using semi-automated cell processor with Ficoll-based and OptiPrep-based density gradient. Islet preparations were cultured for 2 days and assessed regarding islet cell viability (FDA/PI), fractional β-cell viability and β-cell content. Cytokine/chemokine production from islet preparations was also examined. After purification, purity, post-purification IEQ and islet recovery rate were comparable between two groups. Although FDA/PI and fractional β-cell viability showed no significant differences, β-cell survival during culture significantly increased in OptiPrep-based density gradient group when compared to Ficoll-based density gradient group. TNF-α, IL-1β, IFN-γ, IL-6 and MIP-1β production from OptiPrep-based density gradient group significantly decreased.

OptiPrep-based density gradient can reduce cytokine-production when compared to Ficoll-based density gradient, resulting in improvement of quantity of β-cell mass. Cytokine profiling could spot new light on assessment of islet preparations before transplantation.

Improved islet isolation techniques have contributed to the progression of islet transplantation as a treatment for patients with type 1 diabetes mellitus ^{1, 2}. However, many recipients require two or three times islet transplantation to achieve insulin independence. Improved islet isolation has still been important to obtain adequate islet numbers. Although Ficoll-based density gradient has been widely used for human islet purification in most islet processing centers, OptiPrep-based density gradient was recently used in the limited centers. It has been reported that Islet preparations purified using OptiPrep-based density gradient provided better clinical outcomes ³. It is well known that cytokine/chemokine production from islet preparations widely varies. Reducing cytokine/chemokine production from islet preparations may be a key to improve islet transplantation outcomes. The aim of current study is to investigate the variability of pro-inflammatory cytokine/chemokine production from human islet preparations purified using different density gradients.

Materials and Methods

Human islet isolations were performed using automated method ⁴. After digestion phase, pre-purification digests were divided into two groups and purified using semi-automated cell processor with Ficoll-based or OptiPrep-based density gradient. Human Islet preparations were cultured for 2 days, and assessed regarding islet cell viability (FDA/PI), fractional β-cell viability and β-cell content ⁵. Cytokine/chemokine production from islet preparations was also examined.

Results

Between Ficoll-based and OptiPrep-based density gradient groups, islet purity (80.0 ± 10.8 % and 74.3 ± 14.2 %, respectively, p=0.203), post-purification islet equivalent (IEQ) (128,504 ± 28,893 IEQ and 127,235 ± 16,970 IEQ, respectively, p=0.961) and islet recovery rate (46.4 ± 4.2 % and 57.3 ± 14.4 %, respectively, p=0.460) were comparable. There were no significant differences between these groups by FDA/PI (92.4 ± 2.5 % and 90.6 ± 3.6 %, respectively, p=0.550), and fractional β-cell viability in OptiPrep-based density gradient group was comparable when compared to Ficoll-based density gradient group (57.6 ± 8.3 % and 64.5 ± 5.7 %, respectively, p=0.178). β-cell survival during culture significantly increased 1.31 ± 0.02 times in OptiPrep-based density gradient group when compared to Ficoll-based density gradient group (p<0.001). TNF-α, IL-1β, IFN-γ, IL-6 and MIP-1β production from OptiPrep-based density gradient group significantly decreased to 22.8 ± 11.8 %, 27.7 ± 14.0 %, 37.3 ± 11.2 %, 45.3 ± 16.2 %, and 38.5 ± 18.6 % of each from Ficoll-based density gradient group (p<0.05).

Discussion

Current study clearly demonstrates that use of Ficoll, but not OptiPrep, for purification associates the release of pro-inflammatory cytokine/chemokine and could detrimentally affect islet survival during culture. These findings suggest the reduction of pro-inflammatory cytokine/chemokine production after purification by OptiPrep-based density gradient might indirectly contribute to improved stimulated β-cell survival. We obtained comparable purity, post-purification number of islet and recovery rate using the same density gradient made from either Ficoll or OptiPrep with the same procedure of purification. OptiPrep-based density gradient will be used Clinical Islet Transplant (CIT) trial in U.S.A. Although islets culture offers several advantages such as possible pre-transplant interventions including immunosuppression ^{1 3}, the number of islet equivalent reduces during culture ². Our results suggest that the purification method using OptiPrep-based density gradient may be of assistance in improving clinical outcomes, and that Cytokine/chemokine profiling from islet preparations could be helpful to develop better isolation methods.