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.
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.
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.
All procedures involving animals are approved by the Institutional animal care and use committee of Shenzhen Second People's Hospital and following all national regulations. In this protocol, Duroc-Landrace-Yorkshire swine (~6-months of age) purchased from the market were used as pancreatic donors. The weight of the pancreas collected was 123.63 g ± 22.50 g. Personal protective equipment, including protective clothing, masks, gloves, and caps, is worn during the experiments.
1. Media preparation
2. Pancreas retrieval
3. Prepare three biosafety cabinets for the following experiments
4. Pancreas cleaning
5. Pancreas digestion
6. Islet purification
7. Counting islet equivalents (IEQ) and islet culture
8. Assessment of islet quality
The preparation of the biosafety cabinet is shown in Figure 1. Three independent operating spaces are set up. Biosafety cabinet #1 is set with kidney basins, surgical instruments, and beakers for pancreas trimming (Figure 1A). Biosafety cabinet #2 is set with a water bath, peristaltic pump, tube stand with the recirculating tube, and digestion chamber for islet digestion (Figure 1B). Biosafety cabinet #3 is set with disposable filters and centrifuge tubes for enzyme preparation, islet purification, and the following steps (Figure 1C).
The pancreas (before and after enzyme perfusion) is shown in Figure 2. Collagenase Type V solution is perfused into the entire pancreas via the pancreatic duct starting from the head of the pancreas. If the connecting lobe is not perfused successfully, it needs to be cut into separate pieces, and each one is to be perfused.
Pancreas digestion is performed in the digestion chamber, as shown in Figure 3. The pancreatic tissue after the mechanical disruption, and the undigested pancreatic tissue remaining after digestion is shown in the figure. A small amount of undigested tissue indicates full digestion; however, it may also indicate over digestion; therefore, if 15%-25% of the pancreatic organ remains in the chamber, it is acceptable. The digested pancreatic tissue is then washed and centrifuged on discontinuous density gradients to separate the islets from the acinar cells (purification), as shown in Figure 4. Pancreatic islets are found in the middle layer.
DTZ staining of the islets is shown in Figure 5. The point at which digestion is stopped and collection begins (i.e., when free islets appear) is shown in Figure 5A. Purified pig islets after density gradient separation are shown in Figure 5B. Islets in brightfield are shown in Figure 6A. Islet quality is examined by Calcein AM (CA) -Propidium Iodide (PI) staining, as shown in Figure 6B. Live cells are green and dead cells are red. This protocol's average islet isolation yield is 360,935 ± 114,279 IEQ/pancreas and 2,439-3,252 IEQ/g of the pancreas, which is similar to the previous study (333,000 ± 129,000 IEQ/pancreas). The average viability of islet by this protocol is above 81%, which is slightly lower than the previous study (86.7%). One of the representative results of the stimulation index (SI = the ratio between insulin amounts (mU/L) released during high glucose over low glucose conditions) obtained by the glucose-stimulated insulin secretion test (GSIS) measured by ELISA by this protocol is 1.4 ± 0.3, which is similar to the previous study (1.75 ± 0.60)24. The above results are summarized in Table 2.
Figure 1: The preparation of the biosafety cabinet. (A) Biosafety cabinet #1 shows the kidney basin, surgical instruments, and beakers in the sterile field. (B) Biosafety cabinet #2 with (left to right) water bath, peristaltic pump, tube stand with recirculating tube and tubing, and digestion chamber. (C) Biosafety cabinet #3 with disposable filters and centrifuge tubes. Please click here to view a larger version of this figure.
Figure 2: The pancreas before and after enzyme perfusion. (A) Before enzyme perfusion. (B) Distended pancreas after enzyme perfusion. The red arrow indicates the flow of the solution of Collagenase Type V. Please click here to view a larger version of this figure.
Figure 3: The pancreas in the digestion chamber. (A) Pancreatic tissue after digestion and disruption with marbles. (B) Pancreatic tissue remaining after digestion. Please click here to view a larger version of this figure.
Figure 4: Pancreatic cell stratification after discontinuous density gradient centrifugation. After centrifugation, the islets will be concentrated between 1.077g/cm3 and the HBSS layer, and the bottom sediment is non-islet tissue. Please click here to view a larger version of this figure.
Figure 5: Dithizone (DTZ) staining of the islets during digestion. (A) Samples are collected from the digestive room. The islets in the sample are dyed red. The signal to start collection is when 1-2 islets are completely released from exocrine tissue. (B) Purified islets separated by discontinuous density gradient. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Figure 6: Live/dead islet cell viability staining. (A) Islets in bright field. (B) Calcein AM (CA) -Propidium Iodide (PI) staining of islets. Live cells are green and dead cells are red. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Category | Islet Diameter Range (μm) | IEQ Conversion Factor |
1 | 50–100 | x 0.167 |
2 | 101–150 | x 0.648 |
3 | 151–200 | x 1.685 |
4 | 201–250 | x 3.500 |
5 | 251–300 | x 6.315 |
6 | 301–350 | x 10.352 |
7 | >350 | x 15.833 |
Table 1: Conversion factors to calculate islet equivalents (IEQ).
This protocol | Previous studies24 | |
Islet yield (IEQ/pancreas) | 360,935 ± 114,279 | 333,000 ± 129,000 |
Islet viability | 81% | 86.70% |
Islet insulin stimulation index | 1.4 ± 0.3 | 1.75 ± 0.60 |
Table 2: Comparison of the results obtained by this protocol with previous studies.
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 published10,11 in a video format so as to be easy to understand and reproduce.
According to previous reports and our experience, several parameters are critical for the successful isolation of adult pig islets13,14. The critical parameters include (1) Donor pig age and sex: female pigs with more than two litters (so-called retired breeders) are preferred over younger pigs because they can easily provide a large number of high-quality islets25,26,27,28,29,30, (2) Warm ischemia time: limit to 10 min in order to reduce autodigestion14, (3) Digestion enzyme: Collagenase Type V is a valid option, (4) Digestion time: it is essential to stop the digestion early enough to avoid over digestion. As soon as the free islets are observed, the collection process is started even if they are partially trapped. It is very early in the process.
This protocol has several advantages. Compared with continuous or discontinuous density gradients using a COBE 2991 cell processor for islet purification, this protocol layer islets over density gradient solutions using conical tubes and a standard centrifuge. It is cost-effective and easy to master for beginners. Since this purification method requires more manual labor, bulk production and large pellets may still require a COBE cell processor to increase efficiency.
Some troubleshooting steps are also discussed here. (1) Suppose more than 25% of the pancreatic tissue is not digested, or most pancreatic islets are covered by acinar tissue. In that case, the possible reasons include poor perfusion, residual blood that affects the enzyme activity, the low enzyme concentration or activity, or low temperature during digestion. (2) Excessive digestion of the pancreatic tissue may lead to pancreatic islet fragmentation. The possible reason is that the warm ischemia time is not well controlled, high digestive enzyme concentration, longer exposures of digested islets to the digestive enzyme solution, or high digestion temperature. This can be prevented only by the standardization of enzyme/digestion parameters and optimization of the process. (3) Loss of pancreatic islet integrity may also occur during the culture process. Some reasons for this include acinar contamination leading to low pancreatic islet purity, too high islet culture density, insufficient nutrition, or mechanical damage. To overcome the loss of pancreatic islet integrity, increase the volume of the islet medium, increase the density of the medium, and centrifuge the islets more slowly and for a shorter time.
In summary, this protocol has successfully been employed to prepare adult pig islets to be transplanted in non-human primate recipients. It will be further used to obtain islets for future investigations.
The authors have nothing to disclose.
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).
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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