Improved stem/progenitor cell and regenerative porcine islet cell co-culturing method

A technology of co-cultivation of islet cells, applied in blood/immune system cells, bone/connective tissue cells, animal cells, etc., can solve the problem of not involving newborn pig islet cells, low efficiency, complicated procedure of stem cell encapsulation of islet cells, etc. problems, to achieve the effect of being suitable for transplantation, increasing efficiency, and high coating efficiency

Inactive Publication Date: 2015-02-25
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current literature reports mostly focus on co-culture studies between MSCs and EPCs and human islet cells, adult porcine islet cells, and mouse islet cells, and do not involve the study of newborn porcine islet cells, and the traditional method of encapsulating islet cells with stem cells is complicated and inefficient.

Method used

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  • Improved stem/progenitor cell and regenerative porcine islet cell co-culturing method
  • Improved stem/progenitor cell and regenerative porcine islet cell co-culturing method
  • Improved stem/progenitor cell and regenerative porcine islet cell co-culturing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Separation and purification of neonatal porcine islet cells

[0030]1. The experiment was carried out in accordance with the requirements of the 2006 "Guiding Opinions on Treating Experimental Animals" issued by the Ministry of Science and Technology. Newborn pigs born 3-5 days old were adequately anesthetized with 3% pentobarbital, and the pancreas was taken by sterilized laparotomy. Place the removed pancreatic tissue in a pre-cooled D-Hanks liquid glass dish, remove the capsule, fat and blood vessels around the pancreatic tissue block, weigh them, and then cut the pancreatic tissue block into a tissue size of about 0.5-1mm3 piece.

[0031] 2. Add an appropriate amount of type V collagenase, place in a 37°C water bath for shaking digestion, and obtain islet cell suspension.

[0032] 3. Add the islet cell suspension to the culture dish with complete medium (FAMS / F-10, containing 10% porcine serum, streptomycin 100U / mL, penicillin 100U / mL, 0.1% glutamine) ...

Embodiment 2

[0033] The cultivation and identification of embodiment 2.MSCs

[0034] 1. Subculture of MSCs

[0035] The isolated and purified umbilical cord-derived mesenchymal stem cells were resuscitated from the liquid nitrogen tank, cultured with MSC medium, and the medium was changed every three days, and the culture was continued until 4-5 days. It can be seen that the cells are more than 90% confluent. 1x10 4 cells / cm 2 (2.5x10 5 cells / T25 culture flask) for subculture, and the P3-5 passage MSCs were taken for experiments.

[0036] 2. Identification of MSCs

[0037] Take the umbilical cord-derived mesenchymal stem cells of the P3-5 generation, remove the medium, wash twice with PBS, and digest with trypsin, stop the digestion with medium containing serum, wash with PBS, resuspend the cells, and adjust the cell concentration to 1×10 6 / mL single cell suspension, take the flow tube and add 0.1ml cell suspension to each tube, add mouse anti-human monoclonal antibodies FITC-CD34, ...

Embodiment 3

[0039] The cultivation and identification of embodiment 3.EPCs

[0040] 1. Subculture of EPCs

[0041] The isolated and purified umbilical cord blood-derived endothelial progenitor cells were resuscitated from the liquid nitrogen tank, inoculated into a culture dish covered with a special layer (such as fibronectin), and cultured by adding special growth factors (such as vascular endothelial growth factor). factor, epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, etc.) in EGM-2 medium, cultured for 4-5 days, cell colonies appeared in the adherent cells. 8-10 days, more than 90% cell confluence can be seen, according to 5x10 4 cells / cm 2 (10 6 cells / T25 culture flask) for subculture, and P3-5 generation EPCs for experiments ( figure 1 ).

[0042] 2. Identification of EPCs

[0043] Take cord blood-derived endothelial progenitor cells of P3-5 generation, remove the medium, wash twice with PBS, digest with trypsin, stop digestion with medium...

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Abstract

The invention discloses an improved stem/progenitor cell and regenerative porcine islet cell co-culturing method. The method comprises the following steps: culturing NICCs by using a complete medium for 3d, re-suspending three kinds of cells by using the complete medium according to a cell quantity ratio of NICCs (IEQ): EPCs (P3-P5): MSCs (P3-P5) of 1:8:8-1:12:12, placing in an anchorage-independent culture dish or culture bottle, co-culturing in a 37DEG C thermotank for 8-12h without shaking, transferring the cells into a culture dish or culture bottle with the medium containing area about 6 times the above culture dish or culture bottle, continuously culturing, and replacing a culture solution once every other day. The method has the advantages of increase of the NICC coating efficiency of the MSCs and the EPCs, reduction of the cell apoptosis rate, increase of the cell viability, and improvement of the cell culture yield, and is an improved simple, stable and efficient stem cell and regenerative porcine islet cell co-culturing method.

Description

technical field [0001] The present invention relates to neonatal pig islet cells that are isolated, purified and cultured for 3 days by simply and efficiently coating human umbilical cord-derived mesenchymal stem cells (mesenchymal stem cells, MSCs) and human umbilical cord blood-derived endothelial progenitor cells (endothelial progenitor cells, EPCs) Improved method of co-cultivation. Background technique [0002] In the in vitro culture of neonatal porcine islets, reducing the loss of islets and obtaining high-quality islets is the premise and basis for ensuring the success of porcine islet transplantation in the treatment of diabetes. Regarding the research on the protection of islet cell transplantation, the current main methods focus on how to reduce the damage of mechanical damage, enzyme damage and ischemia and hypoxia in the process of islet isolation and purification. Islets are rich in blood vessels and vascular network. During the process of islet cell digestion...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0775C12N5/0789
Inventor 王维易受南马小倩张娟
Owner HUNAN XENO LIFE SCI
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