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Improved method for separation an purification of rat islet cells

A technology for separation and purification of islet cells, applied in the field of cell biology, can solve the problems of operator's wrist fatigue, tendon injury, high price, etc.

Inactive Publication Date: 2005-11-23
NANJING UNIV
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Problems solved by technology

This method is only suitable for experiments with a small number of islets (≤1000), because the picking process is a great challenge to the physical strength and energy of the operator
In particular, hundreds of times of repeated absorption process can easily cause fatigue of the operator's wrist and even tendon damage, and the applicant suffers greatly from it.
Although some automated separation instruments have been introduced, such as flow cytometry and Cell Processor, they are mainly used in clinical operations of human islet transplantation (the amount of islets required is ≥ 100,000), and the price is extremely expensive, such as the RMB of Cell Processor. The price is more than 8 million yuan, and it needs a special person to learn to operate

Method used

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  • Improved method for separation an purification of rat islet cells
  • Improved method for separation an purification of rat islet cells
  • Improved method for separation an purification of rat islet cells

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Embodiment

[0028] 1. Blunt isolation and digestion of rat pancreas The rats used in the experiment are clean SD male rats, weighing 150-250 g, provided by Nanjing Qinglongshan Animal Breeding Center. Rats were anesthetized with 10% (w / v) chloral hydrate and fixed on a foam board in a supine position. Open the abdomen, clamp the common bile duct into the duodenum with an arterial clamp. Liver upturned, free common bile duct. Cut open the chest cavity, break the heart and let the blood out, and immediately perfuse about 10ml of pre-cooled LiberaseRI enzyme working solution (0.167mg / ml) into the pancreas through the common bile duct to fully expand the pancreas ( figure 1 ). The complete pancreas was bluntly separated, put into a 50ml centrifuge tube, then added 5ml of Liberase RI enzyme working solution, and placed in a 37°C water bath for static digestion for 27.5min. After digestion, place the tube on a vortex shaker for 10 seconds. At this time, the pancreas has been decomposed into ...

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Abstract

Disclosed is an improved method for separation an purification of rat islet cells, which comprises pouring rat pancreas with novel Liberase RI enzyme, and centrifugalizing rat pancreas cells by employing Ficoll density gradient. The initially purified pancreas cells are cultured over night, and are selected one by one by means of glass needles connected with rubber pipes through the control of the mouth cavity and tongue of the operator.

Description

technical field [0001] The invention relates to the technical field of cell biology, and includes a method for perfusing and digesting rat pancreas with novel Liberase RI enzyme, and using polysucrose Ficoll density gradient centrifugation to separate and purify rat islet cells. technical background [0002] The pancreas is an important organ in the animal body and is composed of endocrine glands and exocrine glands. Endocrine gland tissue is what we usually call islet cells, also known as Langerhans cells, which account for about 1% of the total weight of the pancreas, and are scattered and embedded in exocrine gland tissue. Islet cells are cell clusters of different sizes composed of α, β and δ cells, among which β cells are the only source of insulin secretion, and the loss of its number and function will cause abnormal blood sugar concentration inside the body, leading to diabetes. Therefore, islet cells are an important research object of molecular pathology of diabete...

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Application Information

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IPC IPC(8): C12N5/071
Inventor 刘畅张春妮王军玲张辰宇项阳张燕朱凌云戚小强
Owner NANJING UNIV
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