Directional EPC (endothelial progenitor cell) sample cell, preparation method and application

A cell and adipocyte technology, which is applied in the field of preparation and directional EPC-like cells, can solve the problems of unclear role in regenerative medicine, difference in in vitro differentiation ability, and short survival period in vivo, so as to restore sensory and motor functions and reduce neuronal apoptosis. Death, the effect of restoring blood supply

Inactive Publication Date: 2018-05-04
北京再生生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficacy of MSCs transplantation has been controversial: 1) heterogeneous MSCs cell population, what are the effector cells; 2) the immunoregulatory function of MSCs has two sides; 3) very little engraftment potential, relying on short-term paracrine effects Induces intrinsic tissue repair with unclear role in regenerative medicine; 4) short in vivo survival; 5) large discrepancy between in vivo developmental behavior and in vitro differentiation capacity

Method used

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  • Directional EPC (endothelial progenitor cell) sample cell, preparation method and application
  • Directional EPC (endothelial progenitor cell) sample cell, preparation method and application
  • Directional EPC (endothelial progenitor cell) sample cell, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1: Preparation of autologous ADSCs

[0096] 1.1 Fat collection

[0097] Donor physical examination before collection, should have no tumor history, no virus infection, no mycoplasma infection. Fat collection is collected in professional medical collection institutions. 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.

[0098] The preservation solution is DMEM / F12 with the following components added:

[0099] 50ug / mL gentamicin sulfate;

[0100] 10% heparin sodium anticoagulant.

[0101] 1.2 Isolation of fat cells

[0102] Remove small blood vessels and connective tissue, cut 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 the fat layer and sediment laye...

Embodiment 2

[0109] Embodiment two: CEL preparation

[0110] Resuspend P2 generation ADSCs in ADSCs medium, adjust the cell density to 10000 / cm 2 , inoculated to 175cm 2 In a tissue culture flask, culture for 24 hours, replace the EPCs medium, and culture for 3 days; add a final concentration of 10 μg / ml DiI-Ac-LDL to the medium, incubate at 37°C for 4 hours, wash 3 times with DMEM / F12, and harvest DIL Labeled CEL (ie DIL-CEL). Resuspend DIL-CEL with 0.5% PuraMatrix™ Peptide Hydrogel, adjust the cell density to 1×10 8 / mL is the CIL preparation.

[0111] The EPCs culture medium is DMEM / F12 containing the following components:

[0112] 5% animal-derived component-free serum substitute;

[0113] 10−8 mol / L dexamethasone;

[0114] 20 ng / mL recombinant human vascular endothelial growth factor;

[0115] 5ng / mL recombinant human basic fibroblast growth factor;

[0116] 5ng / mL insulin-like growth factor-1.

Embodiment 3

[0117] Example Three: Flow Cytometry Detection

[0118] Direct staining on the cell surface, that is, washing the cells with staining buffer, adding fluorescently labeled antibodies, incubating at 4°C for 30 minutes, washing twice with cold saline, resuspending the cells with 500uL cold saline, and testing on the machine; indirect staining in the cells, namely Wash cells with staining buffer, fix with 4% paraformaldehyde solution at 4°C for 30 minutes, wash once with cold saline, permeabilize with 0.25% Triton™ X-100 for 20 minutes, wash once with cold saline, add a Antibody, incubated at 4°C for 2 hours, washed once with cold saline, added secondary antibody, incubated at 4°C for 30 minutes, washed twice with cold saline, resuspended cells in 500uL cold saline, and tested on the machine.

[0119] Antibodies used include:

[0120] Mouse anti-human CD11b-FITC;

[0121] Mouse anti-human CD19-FITC;

[0122] Mouse anti-human CD34-FITC;

[0123] Mouse anti-human CD45-FITC;

[...

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PUM

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Abstract

The invention discloses a preparation method of a directional EPC (endothelial progenitor cell) sample cell. The preparation method comprises the following steps: S1, collecting fat, and separating fat cells; S2, culturing the fat cells by virtue an ADSCs (adipose derived stem cells) culture medium to obtain a P2-generation ADSCs; and S3, culturing the P2-generation ADSCs by virtue of an EPCs (endothelial progenitor cells) culture medium to obtain a directional EPC sample cell. The directional EPC sample cell, the preparation method and the application have the beneficial effects adipose-derived stromal cells (ADSCs) are pre-induced for three days by adopting an endothelial progenitor cells culture medium to obtain heterogeneous CEL (committed EPC-like) with CD133 expression of 47.63+/-12.50 percent and vWF expression of 31.10+/-5.32 percent, and the CEL can increase the blood supply of an injured part, can alleviate ischemic symptom, and is not only beneficial for the structural restoration of an injured area, but also beneficial for alleviating the functional injury of a neurological function; and the CEL restores the blood supply by rebuilding a tissue structure of the injured area, thereby inhibiting the infection environment, reducing the neuronal apoptosis, promoting axonal regeneration and synaptic connection, and restoring partial feeling and movement function.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a directional EPC-like cell, a preparation method and an application thereof. Background technique [0002] Nervous system degenerative changes and regeneration repair and functional reconstruction after injury have always been a major topic in the field of neuroscience. Structural remodeling includes neuronal neogenesis and regeneration, axon outgrowth and synaptic connections. In recent years, several stem cell types have been used in regenerative medicine research for brain injury, including embryonic stem cells (ESCs), induced pluripotent stem cells (IPSs), neural stem cells (NSCs) , glial cells, mesenchymal stem cells (mesenchymal stem cells, MSCs), endothelial progenitor cells (endothelial progenitor cells, EPCs), etc. Early studies mostly used ESCs, iPCs-derived neural precursor cells or adult NSCs transplantation to treat nerve injury and repair neurons. Although...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61K35/44A61P25/00
CPCA61K35/44C12N5/0692C12N2501/105C12N2501/115C12N2501/165
Inventor 张洪钿苑春慧
Owner 北京再生生物科技研究院有限公司
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