Adult Pig Islet Isolation
Summary
This detailed protocol covers the methodological steps of adult pig islet isolation from the digestion phase via purification to the final functional assessment of the islets. This outline can be used as a guideline for adult pig islet isolation in research institutions.
Abstract
Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic β cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.
Introduction
Type 1 diabetes mellitus (T1DM) is a serious disease in which autoimmune destruction of beta cells results in little or no insulin production1,2,3. A substantial group of patients with T1DM cannot stabilize glycemic lability with insulin therapy and experience life-threatening hypoglycemic episodes. Islet transplantation, when successful, can achieve so. Over 1,500 diabetic patients have undergone successful islet transplantation worldwide, showing lower risk yet long-term outcome success than pancreas transplantation4.
Compared with insulin therapy, islet transplantation has better results in reducing the progression of complications5. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM6,7. Islet transplantation may be the best therapeutic option currently available for patients with T1DM who experience life-threatening hypoglycemic episodes.
However, the shortage of human allogeneic donor islets limits the widespread use of islet transplantation8,9. Therefore, the use of animal islets as a replacement is desirable10. The pig has been chosen as a donor for islet cells in preclinical xenotransplantation, and it is of potential translatability to the clinic due to 1) availability, 2) metabolic similarities with humans, 3) rather large beta-cell mass, and 4) possibility of genetically engineering to improve immunological compatibility to humans11.
High purity and viability of islets are key steps for the success of xenotransplantation. However, the procedure to isolate islets from adult pig donors is challenging because of the architecture of the pancreas itself, which differs from the islets of mice or humans12. Generally speaking, the shape of porcine pancreatic islets is not compact12. Compared with human and rodent pancreatic islets, pig islets more easily dissociate12. However, the spontaneous dissociation of the outer layer of islet cells, accompanied by a long culture time, leads to a substantial reduction in pancreatic islet size10.
During the islet isolation process, many factors influence the quality of islets, such as the donor's age, the warm ischemia time, enzymatic activity, the distension by enzymatic injection13,14. Although many previous studies provided methods for pig islet isolation, there is no detailed step-by-step video protocol for researchers as an effective instruction10,15,16,17,18,19,20,21,22,23.
For this purpose, this detailed protocol covers all isolation steps, from organ retrieval to the post-isolation functional assessment of the islets, hoping to offer a simple and understandable overview of the process for easy applicability. This protocol is based on the previously published methods with modifications10,11.
Protocol
Representative Results
Discussion
Islet xenotransplantation, using pigs as a source of islets, is a promising approach to overcome the shortage of human islets. Although the isolation of adult pig islets is challenging, several groups established protocols to successfully isolate islets consistently10,11. Regardless of the method, islet viability and functional properties are to be demonstrated to confirm the high quality of the products. This protocol is based on those published<sup class="xref"…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Professor David K. C. Cooper (Center for Transplantation Sciences, Massachusetts General Hospital) for helping us set up the whole xenotransplantation system. We thank Miss Xingling Hu (Shenzhen Second People's Hospital), Miss Xiaohe Tian (University of California, Berkeley), Mr. Bo Zhou (Boston University) for helpful discussion and suggestions. This work was supported by grants from the National Key R&D Program of China (2017YFC1103701, 2017YFC1103704), Special Funds for the Construction of High-Level Hospitals in Guangdong Province (2019), and Sanming Project of Medicine in Shenzhen (SZSM201812079).
Materials
| 0.22 µm 500 mL disposable filter | Corning | 431097 | |
| 1 L Plastic blue cap bottle | Celltrans | YKBH1 | |
| 10 mL, 25 mL disposable pipette | CORNING | 4488 | |
| 150 mm patri dish | BIOLGIX | 66-1515 | |
| 16 G angiocath | Hongda | 20193141874 | |
| Epidural catheter | Aoocn | No. 20163661148 | |
| 1x HBSS basic | GIBCO | C14175500BT | |
| 200 mL conical centrifuge bottle | Falcon | 352075 | |
| 50 mL centrifuge tube | NEST | 602052/430829 | |
| 500 mL Ricordi Chamber | Biorep | 600-MDUR-03 | |
| 500-micron mesh | Yikang | YKBE | |
| 6 well-plate | COSTAR | 3511 | |
| Anesthesia Machine | RWD | R620-S1 | |
| Anesthetics A: Lumianning (2.5–5 mg/kg) | Huamu, China | Animal Drugs GMP (2015) 070011777 |
|
| Anesthetics B: Propofol (2–3 mg/kg) | Sigma Aldrich | S30930-100g | |
| Beaker (500 mL, 1000 mL) | Shuniu | SB500ml, SB1000ml | |
| Blood glucose meter | Sinocare | 2JJA0R05232 | |
| Blood glucose test strips | Sinocare | 41120 | |
| Calcein/PI cell viability assay kit | Beyotime | C2015M | |
| CMRL 1066 | Thermo Fisher scientific | 11530037 | |
| Collagenase V | Sigma Aldrich | C9263 | |
| CyQUANT cell proliferation assay kit | Molecular Probes | C7026 | |
| Digestive tract | Celltrans | YKBAO | |
| Disposable blood collection needle | FKE | 20153152149 | |
| Dithizone | Sigma Aldrich | D5130 | |
| Drapes | Xinwei | 20182640332 | |
| Flat chassis | Jinzhong | R0B010 | |
| Heparin Sodium | Chinawanbang | 99070 | |
| HEPES | GIBCO | 15630-080 | |
| Histopaque 1077 | Sigma Aldrich | 10771-100ml | 1077 Polysucrose solution |
| Histopaque 1083 | Sigma Aldrich | 10831-100ml | 1083 Polysucrose solution |
| Histopaque 1119 | Sigma Aldrich | 11191-100ml | 1119 Polysucrose solution |
| Infusion tube | BOON | 20163660440 | |
| Iodophor | LIRCON | Q/1400ALX002 | |
| Isoflurane | Rwdls | R510-22-16 | |
| No. 0-2 suture | Jinhuan | No. 20142650770 | |
| No. 22 surgical blade | Lianhui | 2011126 | |
| Penicillin/streptomycin | GIBCO | 15140-122 | |
| Peristaltic pump | LongerPump | BT300-2J | |
| Pig serum | Kangyuan | 20210601 | |
| RPMI-1640 Medium | GIBCO | C1875500BT | |
| Sampling syringe | Yikang | YKBB0 | |
| Scalpel | Jinzhong | J11030 | |
| Silicon nitride beads | Celltrans | YKBI0 | |
| Straight blood-vessel forceps | Jinzhong | J31120 | |
| Straight Sided Jar | Nalgene | 2118-0001 | |
| Tissue forceps | Jinzhong | J41010 | |
| Tissue scissors | Jinzhong | J21210 | |
| Toothed forceps | Jinzhong | JD1060 | |
| Towel forceps | Shinva | 154285 | |
| Vacutainer blood collection tube | Sanli | 20150049 | |
| Water bath | Yiheng | HWS-12 |
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