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Human amniotic membrane-derived mesenchymal stem cell pretreatment method and application thereof

A technology of stem cells and human amniotic membrane, applied in the fields of medicine and hematology, can solve the problems of tumorigenicity, unsafety, high immunogenicity, etc., to enhance the role of SDF-1/CXCR4 axis, improve the success rate and The effect of curative effect and cost reduction

Inactive Publication Date: 2018-11-20
FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral vectors have relatively high transfection efficiency, but they have high immunogenicity, poor targeting, and potential tumorigenicity
Non-viral vectors have better safety and targeting, but the transfection efficiency has been poor
And gene transfection technology takes a long time in vitro
[0011] In the early stage, the inventor used a combination of cytokines (Flt-3ligand, SCF, IL-6, HGF, IL-3) to increase the expression of BMSC to CXCR4, which was not only costly, but also could not guarantee the safety
Not all of these cytokines have been approved for clinical use, and due to the pleiotropic effects of cytokines, such a combination of cytokines may induce unpredictable and severe side effects when used in the human body

Method used

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  • Human amniotic membrane-derived mesenchymal stem cell pretreatment method and application thereof
  • Human amniotic membrane-derived mesenchymal stem cell pretreatment method and application thereof
  • Human amniotic membrane-derived mesenchymal stem cell pretreatment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The separation culture, purification and expansion of embodiment 1AMSC

[0067] (1) Material collection: Under sterile conditions, take the placenta of a healthy full-term cesarean section fetus (serological reactions such as hepatitis, HIV, and syphilis are all negative, with the informed consent of the parturient), and bluntly separate about 10 cm of the amniotic membrane on the fetal side of the placenta. ×10cm, rinse repeatedly to remove blood clots.

[0068] (2) Cutting: Cut the amniotic membrane into about 1mm 3 small pieces, in the shape of mince.

[0069] (3) Inoculation: Use a straw to suck up some small pieces of tissue, put them into a culture bottle, and inoculate the small pieces of tissue on the bottom of the culture bottle. The distance between the small pieces is about 0.5cm to 1cm. 2 The culture bottle can be inoculated with 45-60 pieces.

[0070](4) Culture: After the inoculation, add about 3ml of complete DMEM / F12 medium, gently turn the culture bo...

Embodiment 2

[0076] Isolation, cultivation, purification and expansion of AEC

[0077] (1) The method of amniotic membrane sampling is the same as before.

[0078] (2) Use tweezers to pick up several pieces of amniotic tissue into a centrifuge tube, add 5ml of 0.125% trypsin, and place it in a 37°C incubator for digestion for 3 times, each time for about 40 minutes. During the digestion period, shake the centrifuge tube once every 20 minutes. . Digestion was terminated with serum. Collect the digested liquid for 3 times, filter through a 200-mesh sieve, centrifuge at 1000r / min for 5min.

[0079] (3) Resuspend the cells with complete DMEM / F12 medium, with 6×10 5 / cm 2 Inoculated at 75cm 2 In the culture flask, add complete DMEM / F12 medium to 15ml.

[0080] (4) Change the medium for the first time after 48 hours, and then change the medium every 2 days. After about 5 to 7 days, the adherent cells have reached 80-90% fusion with each other. Pour off the old culture medium, wash it twice...

Embodiment 3 Embodiment 1

[0084] Embodiment 3 is identified to the AMSC that embodiment 1 obtains

[0085] The identification of AMSC mainly depends on the comprehensive identification of its morphology, cell surface molecular markers and differentiation ability. Obtaining high-quality AMSCs is a prerequisite for obtaining good clinical efficacy and ensuring patient safety.

[0086] 3.1 Immunophenotype of AMSC detected by flow cytometry

[0087] (1) Take the P3 passage cells and digest them with 0.125% trypsin, wash once with PBS containing 0.5% BSA, discard the supernatant, resuspend in PBS containing 0.5% BSA, and add the following primary antibodies: mouse anti-human The monoclonal antibodies CD11a, CD11b, CD29, CD31, CD34, CD44, CD45, CD105, HLA-DR, Pan-CK were incubated at 4°C for 30 min.

[0088] (2) Wash twice with PBS containing 0.5% BSA, add FITC-labeled goat anti-mouse secondary antibody, and incubate at 4°C for 30 min.

[0089] (3) Wash twice with PBS containing 0.5% BSA, suspend the cell...

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Abstract

The invention discloses a human amniotic membrane-derived mesenchymal stem cell pretreatment method which comprises the following steps: respectively performing separate subculture on an AMSC and an AEC and collecting the AMSC in a P10 generation and the AEC in a P5 generation; performing indirect co-culture treatment on the AMSC in the P10 generation and the AEC in the P5 generation; or enablingan AEC in a P3 generation to be used for preparing a conditioned culture medium and then performing condition culture on the AMSC in the P10 generation; collecting the treated AMSC to obtain the pre-treated AMSC. The human amniotic membrane-derived mesenchymal stem cell pretreatment method disclosed in the invention has the benefits that the stable cytological characteristic and the good proliferative activity of the AMSC can be maintained, so that the quality and the quantity of the MSCs required for clinical application are ensured; the AMSC homing and the number of implants in vivo are increased, so that the quantity of the cells required for treatment can be met; the method is simple and convenient, and due to the characteristic of low cost, the use cost is effectively reduced; meanwhile, the method is good in safety and free from obvious toxic and side effects and has important practical significance in the clinical application of the AMSC and the improvement in the success rate and the curative effect of HSCT.

Description

technical field [0001] The invention belongs to the technical field of medicine, relates to the technical field of hematology, in particular to a pretreatment method and application of human amniotic membrane-derived mesenchymal stem cells. Background technique [0002] Hematopoietic stem cell transplantation (HSCT) is one of the most effective treatments for hematological malignancies, severe aplastic anemia, severe immunodeficiency disease and some genetic diseases. At present, there are 40,000 new leukemia patients in my country every year, and nearly one million patients are waiting for life-saving hematopoietic stem cell transplantation. After HSCT, the survival, differentiation, proliferation and recovery of hematopoietic and immune functions of stem cells directly affect the effect of treatment. It is inevitable to encounter many problems during the transplantation process, such as the destruction of the hematopoietic microenvironment, the homing of stem cells and th...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2502/09C12N2509/00
Inventor 史明霞曾云洪敏李云涛武坤冉黎婧依香为张递思杨琼梅帅华洲陈锐憬刘茂兰粟靖
Owner FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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