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A compound cell preparation, preparation method and use thereof

A cell preparation, cell technology, applied in cell culture active agents, biochemical equipment and methods, animal cells, etc., to reduce limb pain and coldness, and prolong amputation time.

Active Publication Date: 2020-08-04
北广再生医学科技(广东)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of animal experiments showed that when transplanted with autologous bone marrow-MSCs and fat-MSCs to treat DM-LEASO, they had stronger hypoxic environment tolerance, longer survival period, and the potential to induce angiogenesis was lower than or equivalent to that of BMSCs, but further Studies have shown that MSCs play a paracrine role in the process of angiogenesis, rather than directly participating in angiogenesis, and their role is more likely to induce the construction of an angiogenic microenvironment by regulating immune function.

Method used

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  • A compound cell preparation, preparation method and use thereof
  • A compound cell preparation, preparation method and use thereof
  • A compound cell preparation, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Preparation of autologous ADSCs

[0063] 1.1 Fat collection

[0064] Before the collection, the donor should have a physical examination, no tumor history, no virus infection, no mycoplasma infection, and the fat collection should be collected in a professional medical collection institution; the specific method is abdominal subcutaneous liposuction 50mL. Immediately after collection, place them in a 125mL ice-bath sterile bottle (model 2019-0250, manufacturer Nalgene), which contains 50mL of preservation solution.

[0065] The preservation solution is DMEM / F12 with the following components added:

[0066] 50ug / mL gentamicin sulfate;

[0067] 10% sodium heparin anticoagulant.

[0068] 1.2 Isolation of fat cells

[0069] Remove small blood vessels and connective tissue, cut them into fluid state, and rarely see particles; resuspend in 2 times the volume of medical saline (containing 50ug / mL gentamicin sulfate), centrifuge at 500g for 10 minutes, and take t...

Embodiment 2

[0076] Example 2: Preparation of EPCs derived from ADSCs

[0077] Resuspend P2 generation ADSCs in ADSCs medium, adjust the cell density to 10000 / cm2, inoculate into 175 cm2 tissue culture flask, culture for 24 hours, change EPCs medium, continue to culture for 6 days, and harvest P0 generation EPCs.

[0078] The EPCs culture medium is DMEM / F12 containing the following components:

[0079] 5% animal-derived component-free serum replacement;

[0080] 10-8mol / L dexamethasone;

[0081] 20ng / mL recombinant human vascular endothelial growth factor;

[0082] 5ng / mL recombinant human basic fibroblast growth factor;

[0083] 5ng / mL insulin-like growth factor-1.

Embodiment 3

[0084] Embodiment three: PRP preparation

[0085]Clinically collect 100mL of the recipient’s own sodium citrate anticoagulated peripheral blood, centrifuge at 150g for 10 minutes, collect the supernatant and some red blood cells; take another 240g and centrifuge for 5 minutes, discard the upper 30% of the supernatant, and collect the middle layer and the upper part of the red blood cell layer For the buffy coat, about 40mL of PRP is reserved.

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Abstract

The invention discloses a compound cell preparation, a preparation method and application of the preparation. The preparation comprises autologous platelet rich plasma (PRP), autologous adipose derived stromal cells (ADSCs) and endothelial progenitor cells (EPCs) of ADSC sources. The preparation method comprises the following steps that fat is collected, and adipocyte is separated; the adipocyte is cultured through an ADSC culture medium to obtain P2-generation autologous ADSCs; the P2-generation autologous ADSCs are cultured through an EPC culture medium to obtain EPCs of P0-generation ADSC sources; anticoagulation peripheral blood of a receptor is collected and centrifuged to obtain PRP, and the application of the compound cell preparation in the medicine for treating lower extremity arteriosclerosis obliterans due to diabetes is achieved. The compound cell preparation has the advantages that the EPC culture medium is adopted for inducing the ADSCs to be cultured, the EPCs with the expression percentage of CD133 as 68.72+ / -11.60% and the expression percentage of vWF as 75.91+ / -9.83% are obtained, bone marrow or mobilized peripheral blood does not need to be collected, and after affected parts of patients with lower extremity arteriosclerosis obliterans due to diabetes are injected with the preparation, the limb pain feeling and cold feeling can be lowered, and the new generation rate of collateral vessels is 100%; the total survival rate is 78.12%, the limb salvage survival rate is 68.00%, and the survival life of artificial limbs of the patients can be effectively prolonged.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a composite cell preparation, a preparation method and an application thereof. Background technique [0002] Lower extremity arteriosclerosis occlusive disease (LEASO) is a chronic disease in which atherosclerosis affects the arteries of the lower extremities, leading to arterial stenosis or occlusion, and causes limb ischemia symptoms. , fibrous matrix, abnormal deposition of lipids and tissue fragments, complex pathological changes in the process of hyperplasia in the arterial intima or media. The clinical symptoms of LEASO mainly depend on the development speed and degree of limb ischemia. No matter how wide the range of occlusive lesions is, as long as the development of arterial occlusive lesions is slow, collateral circulation can be effectively established, branch blood flow increases accordingly, and blood supply can be compensated, so that the tissue suffers from i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K35/44A61P3/10A61P9/10C12N5/071A61K35/28A61K35/16
CPCA61K35/16A61K35/28A61K35/44C12N5/0692C12N2501/105C12N2501/115C12N2501/165C12N2506/1384A61K2300/00
Inventor 张洪钿苑春慧
Owner 北广再生医学科技(广东)有限公司
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