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Method for preparing high-purity menstrual-blood-derived stem cells

A stem cell and high-purity technology, which is applied in the field of in vitro culture expansion and cryopreservation, and the preparation of high-purity menstrual blood-derived stem cells. Clear and safe effect

Pending Publication Date: 2016-10-12
浙江奥比特生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] However, under the conditions of the existing collection and separation culture technology, due to the various types of cells in the collected menstrual blood samples and different functions, the cells in the primary culture stage are characterized by various shapes and poor uniformity, indicating that there are many miscellaneous cells and low purity of stem cells. It must take another 3-5 generations of culture time to obtain menstrual blood stem cells with higher purity. At the same time, there is a greater risk of contamination by bacteria and mycoplasma in the cultured cells under this technical method.

Method used

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  • Method for preparing high-purity menstrual-blood-derived stem cells
  • Method for preparing high-purity menstrual-blood-derived stem cells
  • Method for preparing high-purity menstrual-blood-derived stem cells

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Embodiment 1

[0035] Example 1: Preparation of highly pure menstrual blood-derived stem cells

[0036] The preparation of high-purity menstrual blood-derived stem cells in this embodiment specifically includes the following steps:

[0037] (1) Use a menstrual blood collection kit to collect menstrual blood into a menstrual blood collection tube containing antibiotic preservation solution. The mixed volume ratio of antibiotic preservation solution to menstrual blood is 1:1. Store at low temperature at 2-8°C, and the storage period is ≤72 hours;

[0038] The antibiotic preservation solution is based on HBSS (Hank's Balanced Salt Solution), containing 420 units of heparin sodium, 250 μg / mL of cephalexin, 120 μg / mL of gentamicin and 3 μg / mL of amphotericin B.

[0039] (2) Centrifuge the collection tube at 600×g for 10 minutes, discard the supernatant, and collect the cell pellet.

[0040] (3) After fully mixing the cell pellet and normal saline at a volume ratio of 1:1, slowly add it to the in...

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Abstract

The invention provides a method for preparing high-purity menstrual-blood-derived stem cells. Menstrual blood is collected by means of a menstrual blood collection sleeve; middle layer mononuclear cells are collected; a magnetic bead buffering solution is added, an FcR reagent is added after being fully and evenly mixed, then a cross-linking anti-human-antibody micro magnetic bead reagent is added after incubation, and incubation is conducted after uniform mixing; then a magnetic bead buffering solution is added, and after being washed and centrifuged, the mixture is resuspended in the magnetic bead buffering solution; the mixture is added into a separation column placed on a magnetic separation frame in advance, and then the column is washed by means of the magnetic bead buffering solution; the separation column is taken out, 1.5-2 ml of magnetic bead buffering solution is added, and cell suspension is collected; the obtained suspension is cultured by means of a menstrual blood stem cell primary culture medium, and the half of suspension is replaced every 2-3 days; after culture is conducted for 5-7 days, adherent cells are digested and collected, and multiplication culture continues by means of a menstrual blood stem cell subculture medium; the obtained cells are digested, and a cryopreservation solution is added for cryopreservation. The menstrual-blood-derived stem cells obtained through the method are high in purity, short in growth period, high in self-renewal capacity and low in contamination rate.

Description

technical field [0001] The invention relates to the technical field of separation and purification of stem cells, in particular to a method for the preparation, in vitro culture, expansion and cryopreservation of high-purity menstrual blood-derived stem cells. Background technique [0002] Menstrual blood stem cells are derived from the endometrial tissue shed with menstrual blood during women's menstruation. They belong to a type of adult stem cells and have multi-directional differentiation potential close to embryonic stem cells. They can be used in bone injury, spinal cord injury, nerve injury, liver disease, diabetes, heart disease It has broad clinical application prospects in major diseases such as vascular diseases. Studies have found that stem cells derived from menstrual blood have incomparable advantages over stem cells such as embryos, bone marrow, and umbilical cord blood. The number of stem cells is 30 times that of bone marrow, which can provide a rich source...

Claims

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Application Information

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IPC IPC(8): C12N5/074A01N1/02
CPCC12N5/0682A01N1/0221C12N2501/11C12N2501/115C12N2509/00
Inventor 葛剑云汪土根刘心星黄启明
Owner 浙江奥比特生物科技有限公司
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