An effective purification method using large bottles for human pancreatic islet isolation

Figures & data

Figure 1. Bottle purification method. (A) The schema of the continuous gradient solution for the bottle purification method. (B) A “tip-bent” candy cane–shaped stainless pipe used for making the gradient (left panel). The pipe was useful to load the tissue into the top layer in the Top loading method (right panel). (C) Pictures of the bottles after loading and centrifugation. Left: Top loading. Right: Bottom loading. Islets exist in the upper layer in both methods (arrows).

Figure 2. Continuous density gradient was maintained in both the top- and bottom-loading methods during the purification. After centrifugation, the solution, including the digested tissue, was collected into 10 tubes, and the density of each tube was measured. The density of the low-density solution was 1.075, while that of the high density was 1.095. The experiments were performed three times independently.

Table 1. Human islet isolation variables before purification

Variable	Value (mean ± SE)
Age (y)	45.2 ± 4.8
Sex (F:M)	5:4
Body weight (kg)	86.4 ± 4.3
Body mass index (kg/m^2)	29.7 ± 1.6
Cold ischemic time (min)	191.1 ± 20.9
Pancreas weight (g)	100.1 ± 13.0
Islet equivalent of digest	1,065,012 ± 163,210
Islet equivalent/g	10,990 ± 977
Embedded islets (%)	33.4 ± 5.8
Pellet volume (mL)	51.6 ± 6.4
Undigested tissue (mL)	10.0 ± 3.1

Table 2. Postpurification variables based on purification method: COBE method, bottle method with top loading, or bottle method with bottom loading

Variable	COBE	Top	Bottom
Purity (%)	54.9 ± 4.8	53.6 ± 4.7	63.6 ± 3.4
Viability (%)	96.4 ± 1.1	97.0 ± 0.8	96.5 ± 0.9
Postpurification embedded islets (%)	15.1 ± 6.2	19.1 ± 6.4	15.6 ± 5.7
Final tissue volume rate (%)	16.3 ± 2.7	17.7 ± 2.4	10.4 ± 2.1
Stimulation Index	12.5 ± 2.8	13.5 ± 4.4	9.8 ± 2.7
Post-culture viability (%)	93.8 ± 0.9	93.7 ± 0.9	92.9 ± 1.0
Endotoxin (EU/ml)	< 0.250	< 0.250	< 0.250
Microbial contamination	ND	ND	ND

Data are expressed as mean ± SE.

Final tissue volume rate (%) indicates postpurification tissue volume/prepurification tissue volume × 100; ND, not detected.

Figure 3. Postpurification islet yield and size of islets in groups with different purification methods: COBE method, bottle method with top loading, or bottle method with bottom loading. (A) Postpurification Islet yield per pancreas weight (islet equivalent [IE]/g). (B) Postpurification recovery rate (postpurification IE/prepurification IE × 100). (C) Average islet diameter. *p < 0.025.

Figure 4. Normoglycemic rate of streptozotocin-induced diabetic nude mice after islet transplantation. COBE indicates COBE purification method (n = 19); top, bottle purification method with top loading (n = 18). *p < 0.05.