Cultured pancreatic cell, its culturing method and use

A technology of islet cells and islet stem cells, applied in the field of stem cells, can solve the problems of insufficient source of pancreatic tissue, toxicity, etc., and achieve the effect of solving the insufficient source of pancreatic tissue

Active Publication Date: 2007-06-20
BEIJING ZEPHYRM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method of autologous transplantation not only solves the problem of insufficient sources of pancreatic tissue in islet transplantation, but also solves the problem of toxicity caused by the use of immunosuppressants in allogeneic transplantation

Method used

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  • Cultured pancreatic cell, its culturing method and use
  • Cultured pancreatic cell, its culturing method and use
  • Cultured pancreatic cell, its culturing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Detection of nestin positive cells in pancreatic tissue of animal model of diabetes

[0059] Obtaining Pancreatic Tissue from Diabetic Cynomolgus Monkeys

[0060] Anesthesia: Ketamine 10mg / kg Atropine 0.04mg / kg Stability 1mg / kg

[0061] Abdominal skin preparation

[0062] Make a midline incision in the lower abdomen of the xiphoid process, cut the peritoneum into the abdominal cavity, and separate the omentum and intestinal tube. A slightly pink pancreas can be seen at the lower edge of the greater curvature of the stomach. The tail of the pancreas is mostly free in monkeys. Then a piece of pancreatic tissue about 0.5×1cm in size was sutured around it, put into ice-cold Hank's solution containing antibiotics and washed twice.

[0063] Detection of pancreatic stem cells

[0064] 1) Immunohistochemical method:

[0065] Pancreatic tissue was fixed in 4% paraformaldehyde at 4°C for 24 hours, then washed three times with 0.01M PBS (paraffin-embedded section ...

Embodiment 2

[0089] Example 2 Isolation of Residual Islet Cell Clusters from Pancreatic Tissues of Diabetic Animals

[0090] The pancreatic tissue obtained in Example 1 was shredded into 1mm 3Tissue pieces of different sizes were digested in 3mg / ml type V collagenase at 37°C for 10 minutes, then added an equal volume of ice-cold Hank's solution to stop the digestion, centrifuged at 1000rpm for 2-3 minutes, removed the supernatant, and washed in ice-cold Hank's solution for 3 minutes After filtering through 100-mesh stencil to remove large pieces of tissue, inoculate the isolated cell mass into a 60mm bacterial culture dish containing medium A for 3 days, and blow it once a day with a 10ml pipette to prevent cell mass from adhering The wall, and then pick out the islet cell mass under a dissecting microscope. Referring to FIG. 2A , the isolated islet cell mass is a floating round or oval islet cell mass in a bacterial culture dish.

[0091] In addition, according to the experimental data,...

Embodiment 3

[0092] Example 3 Isolation and cultivation of islet stem cells

[0093] The isolated nestin-positive islet cell cluster obtained in Example 2 was added to medium A, inoculated in a 60 mm Petri dish for 3 days, and the cells were blown once a day with a 10 ml pipette to prevent the islets from adhering to the wall. After 3 days, the picked islet cell clusters were inoculated in a common cell culture dish containing medium B and cultured for about 15 days. When the grown star-shaped islet stem cells were confluent, they were passaged, see Figure 2B and 2C.

[0094] The following are the components of medium A and medium B. In Example 3, the specific formula used is formula 4, but islet stem cells can also be obtained by using other formulas mentioned in the text.

[0095] Medium A

[0096] Composition Ratio Manufacturer

[0097] RPMI 1640 86% Invitrogen

[0098] FBS 10% Invitrogen

[0099] Formulation 1 HEPES(1M) 1% Invitrogen

[0100] Sodium...

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Abstract

A method for separating and culturing insulin dry cell from pancreatic tissue and inducing into pancreatic cell is carried out by separating pancreatic cell aggregate, adding it into culture dish with culture medium A for 2-5days, inoculating suspended pancreatic cell aggregate into culture dish with culture medium B, culturing for 6-20days to grow asteriform pancreatic dry cell, sub-culturing to obtain pancreatic dry cell, inoculating it culture dish with culture medium C, culturing for 2-5days, replacing culture medium C with culture medium D, and culturing for 4-15days to obtain the final product.

Description

technical field [0001] The present invention relates to stem cells, in particular to a method for culturing and expanding pancreatic islet stem cells from pancreatic islet cell clusters isolated from diabetic animals (especially mammals) and further inducing their differentiation into pancreatic endocrine cells. Background technique [0002] Since James Thomson successfully isolated and cultured human embryonic stem cells for the first time in 1998, since stem cells have important application prospects in the treatment of future diseases, stem cell technology has undoubtedly become one of the research hotspots in the field of biotechnology today. In China, there are currently more than 30 million diabetic patients, and the number of new patients is increasing at a rate of 750,000 per year. Diabetes has become the "third killer of human beings" after cardiovascular and cerebrovascular diseases and tumors, seriously endangering human health. Patients with Type I diabetes, whic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12Q1/68C12N5/071
Inventor 张愚邹春林陈彪
Owner BEIJING ZEPHYRM BIOTECHNOLOGY CO LTD
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