EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Immunoisolation of pancreatic islets of Langerhans performed by the encapsulation process may be a method to avoid immunosuppressive therapy after transplant. The main problem related to islet transplant is shortage of human pancreata. Resolution of this obstacle may be cryopreservation of encapsulated islets, which enables collection of sufficient numbers of isolated islets required for transplant and long-term storage. Here, we assessed the ability of encapsulated islets to function after long-term banking at low temperature.

Materials and Methods: Islets of Langerhans isolated from rat, pig, and human pancreata were encapsulated within alginate-poly-L-lysine-alginate microcapsules. Cryopreservation was carried out using a controlled method of freezing (Kriomedpol freezer; Kriomedpol, Warsaw, Poland), and samples were stored in liquid nitrogen. After 10 years, the samples were thawed with the rapid method (with 0.75 M of sucrose) and then cultured.

Results: We observed that microcapsules containing islets maintained their shape and integrity after thawing. During culture, free islets were defragmented into single cells, whereas encapsulated islets were still round in shape and compact. After 1, 4, and 7 days of culture of encapsulated islets, the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests showed increased mitochondrial activity. After they were thawed, the insulin secretion capacity was comparable with that obtained with fresh islets.

Conclusions: Cryopreservation and storage of free and microencapsulated islets were possible for about 10 years, although only encapsulated islets retained viability and secretory properties.

Key words : Immunoisolation, Islet transplantation, Microencapsulation

Transplant of isolated islets of Langerhans persists as an efficient method for treatment of patients with type 1 diabetes. Effective treatment demands trans-plant of 8000 to 10 000 islet equivalents per kilogram of patient body weight.¹ The main problem related to islet transplant is shortage of human pancreata. Solutions for deficiency of organs from deceased donors may be cryopreservation of isolated islets, which involves tissue collection and long-term storage. 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 expression³ or by depleting “passenger” lymphoid cells.⁴

The major obstacle associated with cryopreser-vation 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 immuno-protection to avoid the shortcomings related to long-term banking of islets is important for islet transplant therapy.

An approach in terms of allogenic islet shortage is application of pig islets for clinical transplant. Human and porcine insulin is structurally similar, and there is an almost unlimited availability of pig donors, providing promise for this treatment in patients with type 1 diabetes. However, there are a number of limitations resulting from xenotransplant procedures. Rapid loss of transplanted islets during the instant blood-mediated inflammatory reaction⁵ and T-cell-mediated graft rejection6 have both been reported. Regarding the possibility of reduced pancreatic islet immunogenicity, the collection of sufficient numbers of encapsulated islets followed by cryopreservation and long-term banking was tested, showing that pig islet cells remained functional.⁷

The aim of our study was to assess the influence of long-term storage at low temperature on functional capabilities of encapsulated islets derived from rat, porcine, and human pancreata.

Materials and Methods

Human pancreatic islet isolation, purification, and viability process assessment
Human pancreata were harvested from brain-dead multiorgan donors. Donors were selected based on the Edmonton protocol for a clinical grade pancreas. All pancreata were procured using a standardized surgical technique to minimize warm ischemia time and were flushed with University of Wisconsin (UW) solution for pancreas perfusion, administered intra-ductally (approximately 1 mL/g pancreas weight). After the surrounding tissue was removed, organs were perfused with cold collagenase NB with neutral protease enzyme solution (Serva Electophoresis GmbH, Heidelberg, Germany) through a catheter placed in the pancreatic duct. The distended pancreas was subsequently cut into 10 pieces and placed in a Ricordi chamber and shaken gently at 37°C degree. The digested tissue was collected and washed with fresh medium (Mediatech solution; Mediatech Inc., Manassas, VA, USA) to remove the enzyme and then suspended in UW solution (ViaSpan, DuPont, Boston, MA, USA) before purification. Islets were isolated accordingly to the Ricordi method.⁸ After pancreas digestion, the suspended tissue was purified by continuous density gradient using standard purification method (COBE 2991 cell processor with Ficoll density gradient solution; COBE, Lakewood, CO, USA). Islet yield (IE/g), purity, and recovery rate after purification were evaluated in vitro. Islet viability was assessed using propidium iodide staining to visualize the distinction between the living islet cells and dead cells (apoptotic, degranulated); > 95% of cells were viable. The viable islets were identified by dithizone staining (3 mg/mL; Sigma, St. Louis, MO, USA). The endotoxin concentration was under detectable levels, and no bacteria or fungus growth was observed in the islet culture.

This study and the islet isolation protocol including clinical protocol were approved by our bioethics committee (182/2002) at Warsaw Medical University (Warsaw, Poland).

Isolation of pig islets
Porcine pancreata, as the waste material from market-weight pigs (sows weighing over 150 kg) were processed. The pancreata were dissected after pig exsanguination, hot water bathing (64°C), and shaving. Pancreata were subsequently flushed intraductally with cold collagenase P solution (1500-2000 U/mL in Hanks balanced salt solution; Roche Diagnostics GmbH, Penzberg, Germany) sup-plemented with 10% porcine serum. Instantly after distention (1-2 mL/g of pancreas), pancreata were dissected and transported to the laboratory in UW preservation solution (ViaSpan, DuPont, Boston, MA, USA). In the laboratory, islets were isolated using the semiautomated Ricordi method9 with minimal shaking at 37°C. The progress of digestion was monitored under a microscope by analyses of the collected suspension samples every 2 minutes and staining with dithizone (Sigma). The digestion was stopped when more than 50% of free islets were detected in the visual area. Islet purification was performed with the use of Histopaque 1.119/1.077 (Sigma) discontinuous density-gradient centrifu-gation (800g, 16 min).

Isolation of rat islets
Rat islets (from male Wistar albino Glaxo rats) were isolated as previously described10 using a method modified by Lacy and Kostianovsky.11 In brief, pancreata were intraductally injected with collagenase XI solution (1000 U/mL; Sigma), dissected, and digested at 37°C. Islet purification was performed with the Histopaque 1.083 (Sigma) discontinuous density-gradient centrifuge (800g, 16 min).

Islet microencapsulation
The isolated islets were encapsulated within alginate-poly-L-lysine-alginate (APA) membrane using the method of Lim and Sun.12 After overnight culture, islets were suspended in sodium alginate (1.5%; Sigma), and droplet formation was performed using an electrostatic droplet generator (IBIB PAN, Warsaw, Poland). After gelation, the beads were subjected to successive baths in a solution of 0.1% 2-(cyclohexylamino) ethanesulfonic acid (Sigma) in 0.9% sodium chloride (Polfa, Łódź, Poland), then in a 0.05% solution of poly-L-lysine (Sigma) in NaCl, and after that in a 0.15% sodium alginate solution (Sigma) in NaCl. Each stage was followed by rinsing with NaCl. The obtained microcapsules were approximately 300 μm in diameter and had a regular shape.

Cryopreservation and thawing
Cryopreservation was performed accordingly to the modified Rajotte method13 in the presence of 2 M dimethyl sulfoxide (DMSO). Cryopreservation tubes containing islets were placed in a programmable freezer (Kriomedpol, Warsaw, Poland). Islets were cooled at rates of 0.25 to 1.5°C/min to -40°C and at 1 to 3°C/min to -70°C. The frozen samples were stored in liquid nitrogen. After about 10 years of banking, samples were thawed using a rapid method (200°C/min) by agitation in water bath at 37°C. Suspensions were then centrifuged (550g, 5 min), and islets were incubated in culture thawing medium (RPMI 1640; Biogenet, Józefów, Poland), supple-mented with 10% fetal bovine serum (Sigma) and 0.75 M sucrose (from POCh, Gliwice, Poland) for 30 minutes and finally transferred into culture medium.

Evaluation of the islets
Islet morphology, insulin secretion capacity, and viability were assessed during a 7-day culture.

Static incubation assay
After 48-hour culture, samples of 20 human, pig, or rat islets (estimated size of 150 μm) were handpicked and then preincubated for 30 minutes in phosphate-buffered saline supplemented with 10% fetal calf serum. The samples were washed to eliminate the insulin released by the islets during culture. The islets were then incubated for 60 minutes in phosphate-buffered saline plus 10% fetal calf serum containing glucose at successive concentrations: 1.67 mM (primary), 16.7 mM (stimulatory), and 1.67 mM (repeat). Supernatant samples were collected and stored at -20°C. After islets were thawed, insulin presence was evaluated using the Insulin-CT radioimmunoassay kit (Cisbio, Codolet, France) and the Mini Gamma counter 1275 (Wallace, Turku, Finland).

Mitochondrial activity assay
We conducted the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests (Sigma) as previously described.14 In brief, aliquots of 10 islets (free or encapsulated) per well in triplicate 96-well plates were incubated in culture medium. We added 5 g/L MTT solution to culture medium at 1:10 dilution, which was followed by 2-hour incubation. The suspension was then centrifuged (1000 rpm, 3 min). The formazan crystals were dissolved by adding 100 μL DMSO to the pellet. After allowing for 10 minutes of shaking, absorbance was measured at 550 nm in a Hewlett Packard 8452 diode-array spectrophotometer (Palo Alto, CA, USA).

The morphologic parameters for human, pig, and rat islets were determined after cryopreservation and thawing. It was observed that the islets microen-capsulated in APA membrane maintained their shape and integrity. Moreover, the thawed free human islets and rat islets were compact and round in shape, in contrast to pig islets. All types of free islets had defragmented into single cells during culture. Comparisons of morphologic characteristics of free and encapsulated pig islets are presented in Figure 1.

Viability, represented by formazan production and expressed as absorbance, of encapsulated islets increased in all investigated groups after 1, 4, and 7 days of culture (Figure 2). Rat islets cryopreserved in microcapsules retained their viability properties at 43% compared with freshly isolated islets (data not shown). However, the mitochondrial activity of pig islets on day 7 of culture was 55% (mean) higher versus that shown in freshly isolated islets.

Insulin secretion results, expressed as stimulation index (stimulatory/primary secretion values), are presented in Table 1. All evaluated groups of encap-sulated islets responded to high glucose stimulation. The secretory reaction of encapsulated rat islets to stimulation was similar after 1 and 7 days of culture. Likewise, the stimulation index obtained for encapsulated pig islets was significantly higher (P < .05) versus the stimulation index for free islets after 1 day of culture (2.05 ± 0.03 vs 0.62 ± 0.04). The higher response to glucose stimulation observed for human islets (free and encapsulated) demonstrated their ability to maintain insulin secretion.

Successful islet transplant requires at least 8000 islet equivalents/kg of patient body weight to reverse type 1 diabetes mellitus.¹⁵ A shortage of human donors has made it necessary to find other specific methods for islet storage or to use alternative organ or tissue sources. The use of cryopreserved islets may be a way to resolve the dilemma of islet shortage for transplant. Furthermore, effective application of APA membrane before banking in terms of immuno-genicity reduction may increase the application potential of pig islets in xenotransplant to humans.

In the present study, the protective aspect of islet microencapsulation in semipermeable membrane during long-term cryopreservation was demonstrated. Islets from all tested species (human, pigs, and rats) maintained their insulin secretion capacity and their viability parameters. The preventive effect of the applied membrane was observed especially with pig islets. Encapsulated islets exhibited significantly higher stimulation index than free islets. This occurrence may be related to the necessity of improved insulin secretory response to glucose stimulation by cAMP-raising agent (eg, forskolin) used in other laboratories for freshly isolated pig islets.^16,17However, the poor insulin-secreting capacity of isolated pig islets in vitro does not necessarily imply a lack of function in vivo.¹⁸

The optimal condition for islet cryopreservation in freezing and thawing periods are still under investigation. Some authors demonstrated that the culture time before freezing^7,19or replacement of DMSO by other cryoprotectants4 may decrease the adverse effects of cryopreservation. In our study, islet microencapsulation and supplementation of the culture media with sucrose was applied for overcoming the toxicity of DMSO. This approach seemed to have a beneficial effect for islet protection.

Storage of cryopreserved free and microencapsulated human, pig, and rat islets was possible for at least 10 years, although only encapsulated islets retained viability. The protective effect of the APA membrane is of great importance, especially regarding pig islets, which are considered an alternative to human islets for transplant purposes in patients with type 1 diabetes.

Volume: 16 Issue: 4 August 2018

FULL TEXT