Effects and mechanism of single enzyme and compound enzyme digestive juice on pancreatic islet cell isolation of adult pigs

Chaoqi YAN; Ying FAN; Yunfu CUI; Xiaoming ZOU; Baibai ZHANG

ABSTRACT

BACKGROUND:

Recently,the technique of isolation procedure of viable islets cell from large animals has been mature and used in clinical islet cell transplantation. Some study results indicate that low yields of pig islet cell isolations is probably not due to the presence of higher or more aggressive enzyme activities during the porcine isolation procedure. Therefore, the causative factors resulting in the inconsistent results should be sought for the intrinsic properties of the pancreatic islet and pancreas. The purpose of the study is to get more pancreatic islet cells for diabetic patients.

OBJECTIVE:

To investigate the effect of single enzymatic solution (SES)and multi-enzymatic solution(MES) on adult porcine pancreatic islet cell isolation,and to provide the theory base for diabetic patients rehabilitation.

DESIGN:

A completely randomized and controlled experiment based on animals.

SETTING:

General surgery and gerontology department in a university hospital.MATERIALS The experiment was conducted in the Second Affiliated Hospital of Harbin Medical University from June 2003 to May 2004. Twenty consecutive random-bred adult pigs, 12 - 24 months old, weighing average 100 kg,were selected for experiment from Hada Slaughterhouse of Harbin.

INTERVENTIONS:

After slaughtering,the spleentic lobe of the pancreas was excised using sterile surgical gloves and surgical instruments. Each was immediately transported in 500 mL of sterile RPMI1640 solution at 4 ℃ to the laboratory for processing. After removal of fat peritoneum and superficial blood vessel,each pancreas was first immersed in 15 000 Liquor chlorhexidine for 3 minutes,then washed three times with cold RPMI1640, next mechanically minced into 1 mm×1 mm×1 mm fragments,weighed,and equally divided into two groups. Adult porcine islets were isolated with two different collagenase solutions(SES and MES).MAIN OUTCOME

MEASURES:

Islet count was performed with DTZ-staining. The viability was assessed by trypan-blue staining. Porcine islet insulin-secretory function was assessed by insulin content of cultured porcine islets and by insulin release. Islet morphological integrity was finally established by electron microscope examination.

RESULTS:

Overall islet cell content were statistically different between the two methods[(1 782 ±427) IE/g vs (1 293 ±451) IE/g,P<0.05]. No significant difference was found in viability,function or morphology of islet between MES and SES(P>0. 05).

CONCLUSION:

An average of isletisolated with MES method represents a uniquely massive yield in comparison with that by SES method. A good viability was also confirmed by static incubation and culture. The transplantation of those pancreatic islet cells can help to control the syndromes of diabetes.