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 production^1,2,3. 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 T1DM^6,7. 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 transplantation^8,9. 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 injection^13,14. Although many previous studies provided methods for pig islet isolation, there is no detailed step-by-step video protocol for researchers as an effective instruction^{10,15,16,17,18,19,20,21,22,23}.

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 modifications^10,11.

Protocol

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

1. Cleaning medium: To prepare 100 mL of cleaning medium, mix Hank's Balanced Salt Solution (HBSS), 2% porcine serum, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1% Penicillin-Streptomycin (P/S).
2. Collecting medium: Mix RPMI 1640 with 5% porcine serum and 1% Penicillin-Streptomycin (P/S).
3. Culture medium: Mix CMRL 1066 with 10% porcine serum, 10 mmol/L nicotinamide, 1% P/S, and 2 mmol/L L-glutamine.
4. Enzyme solution: Prepare 1 mg/mL Collagenase Type V (activity: 918 units/mg solid) solution in HBSS.
5. Low glucose concentration solution: To prepare a low glucose concentration solution, add 0.5% Bovine Serum Albumin (BSA) and 2.8 mM D-Glucose to 1 mL of Kreb's Ringer bicarbonate buffer (KRBB).
6. High glucose concentration solution: To prepare a high glucose concentration solution, add 0.5% Bovine Serum Albumin (BSA) and 28 mM D-Glucose to 1 mL of Kreb's Ringer bicarbonate buffer (KRBB).

2. Pancreas retrieval

1. Pig anesthesia and skin preparation
   1. Anesthetize the donor pig with intramuscular injection of anesthetics Lumianning (2.5-5 mg/kg) and Propofol (2-3 mg/kg) (see Table of Materials for details).
   2. Maintain anesthesia with 1%-3% isoflurane in a 1-1.5 L/min ventilation volume by gas-anesthesia masks.
   3. Monitor the heart rate to confirm the proper anesthetization. Use vet ointment on eyes to prevent dryness while under anesthesia.
   4. Shave the abdomen, clean it with iodophor, and cover the pig with sterile surgical drape appropriately.
2. Opening the abdomen
   1. After ensuring sterile conditions, make a midline laparotomy incision from the xiphoid to the umbilicus slightly near the tail.
   2. Incise the diaphragm.
   3. Isolate the suprahepatic aorta and suprahepatic inferior vena cava (IVC) using vascular forceps, and then isolate the infrarenal aorta and infrahepatic IVC with the same method.
   4. Administer intravenous heparin (100 U/kg) to the pig to prevent blood clotting.
   5. Exteriorize the small and large intestine, and push the liver and stomach toward the head to fully expose the pancreas.
3. Pancreas perfusion
   1. Clamp the aorta above the diaphragm with vascular forceps, intubate the aorta above the branch of the renal aorta via the aortic cannula (16 G), and tie with 2-0 suture thread.
   2. Infuse the animal with 2-3 liters of cold HBSS solution (previously bagged and maintained at 4 °C) through the aortic cannula (16 G). Infuse the HBSS solution through the aortic cannula by gravity, while the suprahepatic IVC and the IVC are transected to release the perfusion fluid.
      NOTE: Continue the infusion until the IVC outflow is clear.
4. Pancreas excision
   1. Peel off the omentum, find the pancreas and spleen lobes along with the spleen and separate them.
   2. Separate the connected lobe of the pancreas with adjacent tissue.
   3. Find the main pancreatic duct in the descending part of the duodenum. Ligate the bile duct with a suture (2-0) and transect it with scissors near the pancreatic duct.
   4. Clamp a section of the duodenum containing the ampulla of Vater on either side of the head of the pancreas with vascular forceps, and then resect the duodenum with scissors.
   5. Take out the entire pancreas, immerse it in cold HBSS, and immediately transport it to the islet isolation facility.
      ​NOTE: The pigs were euthanized using pentobarbital sodium (100-200 mg/kg). Intraductal infusion of HBSS or preservation solution can be used to distend the pancreas at the site of organ procurement if pancreases are procured at the local abattoir.

3. Prepare three biosafety cabinets for the following experiments

1. Set up biosafety cabinet #1 with kidney basin, surgical instruments, and beakers for cleaning the pancreas (section 4, Figure 1A).
2. Set up biosafety cabinet #2 with a water bath, peristaltic pump, tube stand with the recirculating tube, and a digestion chamber for islet digestion (section 5, Figure 1B).
3. Set up biosafety cabinet #3 with disposable filters and centrifuge tubes for enzyme preparation, islet purification, and the following steps (sections 6-8, Figure 1C).

4. Pancreas cleaning

1. Clean and weigh the pancreas.
   1. Rinse the pancreas in 200 mL of iodophor for 3 min in biosafety cabinet #1. Rinse the pancreas in 200 mL of cold HBSS twice, 2 min each.
   2. Put the cleaned pancreas into a kidney basin containing 100 mL of cleaning medium on ice.
   3. Keep the ampulla clamped, resect the duodenum. Clean the pancreas of external fat and connective tissue.
   4. Weigh the pancreas.

5. Pancreas digestion

1. Infusion of collagenase
   1. Cut a cross-section of one of the annular branches of the pancreas and insert an 18 G angiocath (if the duct diameters are smaller, consider a 20 G angiocath) into the main pancreatic duct and suture it into place with a 2-0 braided silk suture.
   2. Cut off the tissue bridge that passes through the connecting lobe near the head of the pancreas, and clamp the catheter on the side of the incision near the head of the pancreas to reduce enzyme leakage.
   3. Place multiple angiocaths for better intraductal enzyme infusion. For this, insert additional angiocaths (size 20-22 G) after sectioning the pancreatic organ to expose the duct.
   4. Install the filling pipeline, including a 1 m silicone tube (16#) with a Luer-lock connector. The middle section of the tube is embedded in the peristaltic pump head.
   5. Turn on the peristaltic pump to fill the tubing system (tube size 16#) 300 mL of 1 mg/mL, Collagenase Type V (activity: 918 units/mg) preheated to 24 °C and remove air bubbles in the piping.
   6. Connect the perfusion tubing to the angiocath, and turn on the peristaltic pump at the rate of 20 mL/min.
   7. Stop the perfusion when most of the enzyme has been infused and the pancreas is well distended (Figure 2B).
   8. Cut the clear gelatinous tissue that forms around the pancreas during the perfusion.
      NOTE: This step is very important because the gel can clog the filter in the chamber and make it difficult to collect the digested tissue (Figure 2B).
   9. Remove the angiocaths after infusion.
2. Tissue digestion
   1. Preheat 1 L of HBSS solution to 36 °C. Prepare the digestion system consisting of the digestion chamber and tubing connected into a closed system in advance, and transfer the heating coil of the circuit into a 36 °C water bath.
   2. Cut the pancreas into 3-4 pieces and pour them with Collagenase Type V into a 500 mL beaker.
   3. Cover the beaker with with a sterile petri dish lid, transfer it to biosafety cabinet #2 and put the beaker's contents in the digestion chamber containing four siliconized glass marbles and a 500 µm mesh.
   4. Close the chamber and tighten the screws. Put the remaining enzyme into the recirculating beaker and turn on the peristaltic pump at a speed of 80 mL/min.
   5. Add warm HBSS solution (typically around 150 mL) preheated at step 5.2.1 to fill the circuit to completion. Shake the chamber gently and turn it over so that the siliconized glass marbles hit the tissue.
      NOTE: Since pig islets are more delicate than human tissue, shakings should be even and slow to avoid damage. As the tissue digests, it will start to fall apart and flow out of the chamber.
   6. Take a sample (approximately 1 mL) from the circuit every 2 min to check for free islets after shaking for 1 min.
   7. Place the sample in a 6 well-plate with 80 µL of dithizone (DTZ). Add 2 mL of phosphate-buffered saline (PBS) and examine the sample under the microscope with 40x magnification. DTZ will stain islets in red.
3. Collection of the digest
   1. Continue the digestion until free islets are observed (even if many are still partially trapped in the exocrine).
      NOTE: Islet release takes about 10-15 min, but digestion time is often influenced by the donor's age, enzyme lots, the concentration of enzyme, etc.
   2. Open the circuit and collect the tissue released into 1 L bottles. Keep the bottles on ice while collecting to help to inactivate the Collagenase Type V.
   3. Add the remaining warm HBSS solution (~850 mL, step 5.2.1) to the recirculation beaker and collect the remaining digested tissue from the digestion chamber.
   4. Add cold collecting medium to the chamber (via the recirculating beaker).
      NOTE: Stop collection when tissue pellets get smaller. The collection phase (steps 5.3.2-5.3.3) could take up to 40-75 min and 5-8 L of collecting media.
   5. Transfer the tissue from the collection bottle to 250 mL conical centrifuge tubes, and centrifuge at 200 x g for 3 min at 4 °C.
   6. Discard the supernatant and resuspend the tissue in 500 mL of fresh collecting medium.
   7. Continue to combine the cell pellets into cold collecting medium (500 mL) until all digest is collected.
   8. Wash the cell pellet suspended in the cold medium by centrifugation (3 min at 200 x g, 4 °C).
   9. Discard the supernatant. Evaluate and quantify the cell pellet following the steps mentioned in sections 6-8.
      ​NOTE: The final volume depends on the pancreas size but is expected to be within 40-80 mL.

6. Islet purification

1. Purify the pig islets by density gradient separation.
   NOTE: Refer to the Table of Materials for the details of the density gradient solutions used.
   1. Add 2 mL of pancreatic tissue to 50 mL conical tubes.
   2. Add 12 mL of 1119 polysucrose gradient (1.119 g/cm³) to resuspend each 2 mL of pancreatic tissue.
   3. Add 10 mL of 1083 polysucrose solution (1.083 g/cm³), 10 mL of 1077 polysucrose solution (1.077 g/cm³), 10 mL of 1037 polysucrose solution (1.037 g/cm³), and 5 mL of HBSS, successively.
      NOTE: 1037 polysucrose solution (1.037 g/cm³) is prepared by mixing 1077 polysucrose solution (1.077 g/cm³) and hypertonic citrate purine solution (38.5 mL:11.5 mL).
   4. Centrifuge at 900 x g without brake for 10 min at 4 °C.
   5. Aspirate the islets in between the layers of 1077 polysucrose solution and 1037 polysucrose solution.
   6. Transfer the islets to a 50 mL centrifuge tube, evenly distribute the liquid in each tube, and balance before centrifugation.
2. Wash pig islets
   1. Centrifuge at 200 x g for 3 min at 4 °C, discard the supernatant. Add the collecting medium to bring up the volume to 45 mL.
   2. Centrifuge at 200 x g for 3 min at 4 °C, leaving about 2 mL of supernatant; shake gently to mix.
   3. Combine all the pellets into the same 50 mL centrifuge tube. Add the collecting medium to bring up the volume to 45 mL.

7. Counting islet equivalents (IEQ) and islet culture

1. DTZ staining of islets
   1. Pool the islets together, add more collecting medium to bring the volume up to 250 mL, and resuspend the islets.
   2. Take 500 µL of the representative sample and place it in a 35 mm dish with 80 µL of DTZ. Allow it to incubate at room temperature for 1-2 min, and then add 2 mL of PBS.
   3. Examine at 40x magnification under an inverted microscope. Islet aggregates are stained red by the DTZ.
2. Counting stained islets
   1. Count the stained islets by dividing them into the size categories listed in Table 1. Use the conversion factor to determine the islet equivalent (IEQ) according to size.
      NOTE: IEQ conversion is carried out when the majority of the DTZ-positive aggregates' diameter is greater than 50 µm (in adult (>2 years) and some young-adult (6-12 months) preparations proceed to step 7.2.4). In all other cases (in young pigs less than 6 months old and in the remaining young adult pigs), the number of islets was estimated based on particle volume or DNA content (according to steps 7.2.2-7.2.3).
   2. (OPTIONAL) Centrifuge at 200 x g at 4 °C for 2 min to measure the pallets.
   3. (OPTIONAL) Evaluate the DNA content of single islets, as well as of the islet cell pellets by the cell proliferation assay kit. Extrapolate pig beta cell numbers from the DNA content and immunocytochemical analysis.
   4. Add all the categories and determine the total IEQ for the sample. Multiply that number by the volume of the fraction. (e.g., 200 IEQs in 500 µL of a 250 mL fraction = 200 x 2 x 250 = 100,000 IEQs).
3. Islet culture
   1. Centrifuge the islets at 200 x g at 4 °C for 1 min; discard the supernatant.
   2. Add 5 mL of pre-warmed culture medium to resuspend islets and transfer to a 150 mm Petri dish.
      NOTE: Resuspend 10,000 islets/dish.
   3. Add more culture medium to bring the volume up to 30 mL in the 150 mm Petri dish.

8. Assessment of islet quality

1. Calcein AM (CA) -Propidium Iodide (PI) staining of islets
   1. Handpick 50 islets into a 48-well plate. Wash once with 1 mL of HBSS.
   2. Add 200 µL of the mixture containing 1x CA and 1x PI according to the instructions of the cell viability staining kit.
   3. Cover the Petri dish and place it in an incubator at 37 °C for 30 min.
   4. Visualize the islets using a fluorescence microscope and capture the images.
   5. Quantitate the viability by counting the live cells (green) and dead cells (red) after CA-PI staining.
2. Glucose-stimulated insulin secretion (GSIS)
   1. Add 1 mL of low glucose concentration solution to three wells of a 24-well plant.
   2. After at least overnight culture, handpick 100 islets of similar size in the range of 100-200 µm in diameter and transfer them into the three wells of the 24-well plate pre-filled with low glucose concentration solution (as in step 8.2.1).
      NOTE: Wash the islets in an intermediate well with HBSS before transferring them into the well to wash out the culture medium.
   3. Incubate the islets in a 5% CO₂ incubator at 37 °C for 30 min.
   4. Remove the supernatant carefully with a pipette without touching the islets.
   5. Add 1 mL of new low glucose concentration solution.
   6. Incubate the islets in a 5% CO₂ incubator at 37 °C for 60 min.
   7. Collect the supernatant (no islets) and transfer at least 500 µL of it into a 1.5 mL centrifuge tube. This will contain insulin secreted under low glucose stimulation.
   8. Add 1 mL of high glucose concentration solution carefully into the same wells.
   9. Incubate the islets in a 5% CO₂ incubator at 37 °C for 60 min.
   10. Transfer another 500 µL of the culture medium to a new 1.5 mL centrifuge tube. This will contain insulin secreted under high glucose stimulation.
   11. Use enzyme-linked immunosorbent assay (ELISA) kits for porcine insulin to measure the concentration of insulin released following low and high glucose stimulation (steps 8.2.7 and 8.2.10).
       NOTE: Stimulation Index (SI) is calculated as the ratio of the insulin concentration measured following high glucose stimulation divided by the insulin concentration following low glucose stimulation.

Results

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 filte...

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 consistently^10,11. 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

Materials

Name	Company	Catalog Number	Comments
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