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.

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.

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