Cryopreservation of Human Pancreatic Islets from Non-Heart-Beating Donors Using Hydroxyethyl Starch and Dimethyl Sulfoxide as Cryoprotectants

Abstract

Although widely used, DMSO is toxic for pancreatic islets. We combined hydroxyethyl starch (HES) with DMSO to simplify the procedure of freezing and thawing, and to decrease the toxicity of DMSO. A preclinical study was performed using islets from beagle dogs. After storage for 4 weeks, the islets were thawed and examined. The islet structure was well maintained after thawing. Although the number of the islets decreased to 71.2 +/- 20.1%, the function of the islets was evaluated by static incubation after thawing and showed a 1.80 +/- 0.78 stimulation index. We have introduced this technique for the cryopreservation of human islets from non-heart-beating donors. Twelve cases of human islet cryopreservation were performed. The sample tube of each human cryopreservation was thawed to evaluate the morphology, contamination, and endocrine function. Although fragmentation was observed in five samples (41.6%), the other seven (58.4%) showed a normal structure when evaluated by microscopic and electron microscopic study. The stimulation index (SI) of static incubation deteriorated from 3.37 +/- 3.02 to 1.34 +/- 0.28 after thawing. We divided the thawed islets into two groups: group 1 (n=8), SI > 1.2; group 2 (n=4), SI < 1.2. The group 1 islets showed a higher rate of normal structure (87%) than did group 2 (25%). Moreover, the SI before cryopreservation was 4.01 +/- 3.57 in group 1, which was higher than the SI of 2.11 +/- 0.72 in group 2. Based on the good results from the preclinical study using a large-animal model, this method was introduced for clinical application. Even from the pancreata of non-heart-beating donors, a successful islet cryopreservation was achieved. However, the isolated islets with poor function should not be cryopreserved for transplantation.

Optimal method for short-term or long-term islet preservation: comparison of islet culture, cold preservation and cryopreservation

“…Poor recovery rate, low insulin secretion, and in vivo function of islets in 37°C culture medium have been reported recently [6][7][8]. Meanwhile, islet cold preservation in 4°C University of Wisconsin (UW) solution [9][10][11] and islet cryopreservation at -80°C [5,12,13] were applied in research and clinical islets transplantation. Even though some researchers have compared the in vitro and in vivo function of islet that were cultured in either 37°C culture medium or preserved in 4°C UW solution [6,7,9,11], the function of cryopreserved islets has not been compared with cultured and cold preserved islet as of today.…”

Sensing and Sensibility: Single‐Islet‐based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules

“…[ 19 ] These results demonstrated that suboptimal cryopreservation process can cause cryoinjuries (i.e., ice injury and solute injury) to the cells and loss of islet functionalities, which warranted the need of further improvements of the islet cryopreservation protocols. [ 3,4,45 ] To understand how cryopreservation and microencapsulation affect the islet functionality, we fi rst measured the ATP and static insulin release values of bare islets (B), encapsulated islets (E), bare islets cryopreserved and stored at −80 °C for 1 or 7 d (B1d or B7d), encapsulated islets cryopreserved and stored at −80 °C for 1 or 7 d (E1d or E7d), and encapsulated islets cryopreserved with trehalose (additional CPA to the routinely used DMSO medium) and stored at −80 °C for 1 or 7 d (ET1d or ET7d) for comparison studies. Figure 3 shows the ATP value of islets under different protocols for encapsulated/bare islets with or without cryopreservation.…”

“…[ 1 ] In the last decade, transplantation of pancreatic islets, such as the well-known "Edmonton protocol", has been considered as the most physiologically advantageous solution for glucose homeostasis in clinical treatments of selected patients with type 1 diabetes mellitus, which can eliminate insulin injection, reverse neurovascular complications, and even prevent end-stage organ failure. [2][3][4][5][6] In such treatments, suffi cient islet equivalents with high quality are critical for positive clinical outcomes. For example, the Edmonton protocol requires a cumulative islet mass of 10 000 islet equivalents or more per kilogram of body weight of the recipient.…”

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“…4 The major obstacle associated with cryopreservation of pancreatic islets is loss of function of the frozen/thawed islets compared with freshly isolated ones. 2 This disadvantage might be overcome by islet microencapsulation in a semipermeable membrane. Application of effective methods for immunoprotection to avoid the shortcomings related to longterm banking of islets is important for islet transplant therapy.…”

“…Cryostorage allows for the accumulation of sufficient amounts of donor tissue and shipping to other institutions. 2 Moreover, cryopreservation can decrease immunogenicity of islet allograft by reducing major histocompatibility complex class I antigen expression 3 or by depleting "passenger" lymphoid cells. 4 The major obstacle associated with cryopreservation of pancreatic islets is loss of function of the frozen/thawed islets compared with freshly isolated ones.…”