Preparations Containing Islet-like Cell Clusters

A technology of islet-like cells and preparations, applied in the field of biomedicine, can solve the problems of unsatisfactory clinical application, poor cell repair effect, etc.

Active Publication Date: 2020-12-11
长春万成生物电子工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current repair effect of cells in vivo is not good enough to meet clinical application.

Method used

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  • Preparations Containing Islet-like Cell Clusters
  • Preparations Containing Islet-like Cell Clusters
  • Preparations Containing Islet-like Cell Clusters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Plasmid preparation:

[0046] Artificially synthesize the DNA fragment shown in SEQ ID NO: 2, and connect it into the vector pRRLSIN.cPPT with BamH I, Sal I, restriction site BamH I: G^GATCC, 4021, Sal I: G^TCGAC, 4049. PGK-WPRE was constructed to obtain a plasmid vector expressing the fusion protein Pdx-1-Linker-IGF-1 (the complete sequence is shown in SEQ ID NO: 3).

[0047] 2. Virus preparation:

[0048] A: 293T cells are cultured in DMEM medium (complete medium) containing 10% FBS, + penicillin-streptomycin antibiotics, taking a 15ml culture dish as an example, add 8×10 6 Cells, 22.5ml complete medium, 37°C, 5% CO 2 To cultivate. B: After overnight culture, replace with serum-free medium DMEM. C: The three plasmids (pMD2G, pCMVR8.74, pRRLSIN.cPPT.PGK-WPRE-Pdx-1-Linker-IGF-1) were mixed according to a certain ratio, and the buffer was HeBS. D: Transfection method using CaCl 2 Transfection protocol, CaCl 2 Mix with HeBS buffer containing 3 plasmids in a cert...

Embodiment 2

[0054] Dissolve 90% deacetylated low molecular weight chitosan in 1% acetic acid to obtain a final solution with a specific gravity of 2%; sterilize at 120° C. for 20 minutes.

[0055] Take type 1 collagen, dissolve it in 1% acetic acid to obtain a 4% specific gravity solution, and sterilize at 120° C. for 20 minutes.

[0056] Mix chitosan solution and collagen solution according to the volume ratio of 1:1, use genipin as cross-linking agent, promote the cross-linking reaction of chitosan and collagen, obtain degradable material, cross-linking condition is room temperature (18 ~30°C) for 1 hour.

[0057] Take 1×10 6 The concentration of cell / ml is added to the degradable material after adding the islet-like cell mass prepared in Example 1 and mixing evenly at 4°C, adding to the sterile vegetable oil containing 15% Span80 at 0°C, and then adding the curing agent Beijing at 0°C Nippin, stir well for 3 minutes.

[0058] Then raise the temperature to room temperature, react for...

Embodiment 2

[0068] The microspheres prepared in Example 2 (MSCT group), Comparative Example 1 (MSC-1) and Comparative Example 2 (MSC-2) were tested.

[0069] 1. Identification of insulin secretion

[0070] With the microspheres that embodiment 2 (MSCT group), comparative example 1 (MSC-1) and comparative example 2 (MSC-2) make, during culturing, extract microsphere once every two days, be about total amount 5%, dithizone detection and microscopic detection were carried out, and the culture medium was extracted every day for identification of insulin secretion. The result is as Figure 2-4 .

[0071] The measurement results of the microspheres in the MSC-1 group showed that during the 10-day culture process, the insulin secretion continued to increase, and finally reached 65±20pg / ml, but the dithizone staining experiment showed that the conversion efficiency was not significantly improved and the clustering effect was not improved. constant. In the microsphere group of MSC-2 group, the...

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Abstract

The invention relates to the technical field of biological medicine and particularly relates to a preparation containing an islet-like cell cluster. The invention provides the preparation containing the islet-like cell cluster and a preparation method thereof. The preparation is degradable, and under a microscope, micro-spheres are intact in shape and uniform in particle size distribution. By in vitro culture of the micro-spheres, stem cells in the micro-capsules proliferate and gradually form embryonic tissues; and simultaneously, by detection, Pdx-1-Linker-IGF-1 and insulin can be detected in an extracapsular culture medium, thereby proving that the cells gradually evolve into pancreatic tissues, can release insulin and finally can be used for in vivo transplantation to repair islet functions.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation containing islet-like cell clusters. Background technique [0002] Diabetes Mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. The long-term high blood sugar in diabetes leads to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. [0003] According to the classification recommended by the World Health Organization, diabetes can be divided into two types: type I diabetes with absolute insulin deficiency and type II diabetes with relative insulin deficiency and insulin resistance. Among them, about 10% of patients belong to type I diabetes, and the importance of genetic factors in this type of diabetes is as high as 50%, and the onset is common in children and adolescents. In a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/51A61K35/28A61K38/28A61K38/30A61K47/36A61K47/42A61P3/10
CPCA61K9/5161A61K9/5169A61K35/28A61K38/28A61K38/30A61P3/10
Inventor 王立坚赵钢王竑婷
Owner 长春万成生物电子工程有限公司
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