阅读说明：本技术 一种来源于发育早期中胚层的间充质干细胞制备方法 (Preparation method of mesenchymal stem cells derived from early-stage developmental mesoderm ) 是由 韦丹 朱学义 张云国 唐成 王迎耀 薛晓锋 于 2020-03-27 设计创作，主要内容包括：本发明公开了一种来源于发育早期中胚层的间充质干细胞制备方法,包括清洗、酶解、过滤、原代培养、传代培养、诱导分化和冷冻保存。本发明的有益效果是：提高干细胞的分离效率,使干细胞加热和冷藏过程中温度变化更加平和,避免细胞提取过程中的温度改变使细胞失活,降低细胞的提取率的问题,能够提高间充质干细胞的分离速度,多聚赖氨酸确保其在分离过程中保证纯度,且促进分离后的贴壁生长。(The invention discloses a preparation method of mesenchymal stem cells derived from early-stage developmental mesoderm, which comprises the steps of cleaning, enzymolysis, filtering, primary culture, subculture, induced differentiation and cryopreservation. The invention has the beneficial effects that: the separation efficiency of the stem cells is improved, the temperature change in the heating and refrigerating processes of the stem cells is smoother, the problems that the cells are inactivated due to the temperature change in the cell extraction process and the extraction rate of the cells is reduced are solved, the separation speed of the mesenchymal stem cells can be improved, the polylysine ensures that the purity of the mesenchymal stem cells is ensured in the separation process, and the adherent growth after separation is promoted.)

1. A preparation method of mesenchymal stem cells derived from early-stage developmental mesoderm is characterized by comprising the following steps: the method comprises the following steps:

step A: cleaning, extracting the umbilical cord, cleaning the umbilical cord with distilled water, and wiping with sterilized sponge;

and B: carrying out enzymolysis, taking out the umbilical cord, crushing, and carrying out enzymolysis by using collagenase with the concentration of 1% for 7 hours;

and C: filtering, namely bringing distilled water with 5 times of volume into the umbilical cord enzymolysis liquid, primarily filtering by using a screen, and then pouring into a differential separator for further filtering;

step D: primary culture, inoculating the filtered umbilical cord cells into a serum-free basal medium for culture, and preparing a novel culture solution by a cloning suspension method for in vitro amplification culture till the third generation;

step E: subculturing, replacing a serum-free culture medium, adding a serum-free nutrient additive into the serum-free culture medium, and periodically replacing the culture medium until cell clusters are generated;

step F: inducing differentiation, extracting the third generation cells, and inoculating the cells onto a differentiation medium by using a plate marking method for culture;

step G: and (4) performing cryopreservation on the prepared cells.

2. The method of claim 1, wherein the mesenchymal stem cell is derived from early-stage mesoderm development, and the method comprises: in the step A, the selected umbilical cord is cut into small pieces before enzymolysis, and the small pieces are placed into a heat-preservation tank for refrigeration, wherein the refrigeration duration is 2-6 months.

3. The method of claim 1, wherein the mesenchymal stem cell is derived from early-stage mesoderm development, and the method comprises: in the step D, a clone suspension method is adopted in the in-vitro amplification culture process of the stem cells, namely, an equal amount of cell suspension is added into each hole of each culture plate, the cells are mechanically separated and cloned again after the formation of neurospheres to prepare single cell suspension, the subculture size of the cloned neural stem cells is 200 m, the culture solution is DMEM/F12(1:1) and B27 and bFGF serum-free culture medium, and when the primary culture cells are fused to 85%, 0.25% trypsin is adopted to digest the cells for 30min at 37 ℃.

4. The method of claim 1, wherein the mesenchymal stem cell is derived from early-stage mesoderm development, and the method comprises: and C, separating the cells by using a desktop centrifuge and a constant-temperature magnetic stirrer of a biological safety cabinet, wherein the centrifugation method is lOOOrpm for 5min, and polylysine is added during separation.

5. The method of claim 1, wherein the mesenchymal stem cell is derived from early-stage mesoderm development, and the method comprises: and step F, mixing the pluripotent stem cell differentiation basal medium and the additive in the differentiation process, wherein every 10mL of the additive is thoroughly mixed with 500mL of the differentiation basal medium to obtain the human pluripotent stem cell differentiation medium (AC-1001002), and the human pluripotent stem cell differentiation medium can be stably stored at the temperature of 2-8 ℃ for 2-3 weeks.

Technical Field

The invention relates to a cell preparation method, in particular to a preparation method of mesenchymal stem cells derived from early-stage developmental mesoderm, and belongs to the technical field of cell preparation.

Background

The mesenchymal stem cells are stem cells which are derived from mesoderm and ectoderm in early development and have multidirectional differentiation potential, can be induced and differentiated into various cells such as skeletal muscle, smooth muscle, bone, cartilage, fat, cardiac muscle cells, nerve cells, hematopoietic stem cells and the like, are easy to introduce and express exogenous genes, are target cells for potential gene therapy and stem cell research, and can be used as novel seed cells due to the advantages of wide sources, easy acquisition, strong proliferation capacity, no ethical problems and the like of the human umbilical cord mesenchymal stem cells.

Research proves that the human umbilical cord mesenchymal stem cells and the induced islet-like cells thereof do not express cell surface markers CD80, CD86, CD40 and CD40L related to transplant rejection, which indicates that the human umbilical cord mesenchymal stem cells are low in immunogenicity before and after induction, and do not generate immune rejection reaction after cell transplantation, thereby providing a theoretical basis for transplantation treatment before and after induction of the human umbilical cord mesenchymal stem cells and being used as a cell source for transplantation treatment of diabetes.

The cells extracted by the conventional stem cell extraction method may have gene defects, which easily affect the health of a recipient, and the temperature change during the cell extraction process easily inactivates the cells, which causes a problem of reducing the extraction rate of the cells.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for preparing mesenchymal stem cells derived from early-developing mesoderm.

The invention realizes the purpose through the following technical scheme: a preparation method of mesenchymal stem cells derived from early-developing mesoderm comprises the following steps:

step A: cleaning, extracting the umbilical cord, cleaning the umbilical cord with distilled water, and wiping with sterilized sponge;

and B: carrying out enzymolysis, taking out the umbilical cord, crushing, and carrying out enzymolysis by using collagenase with the concentration of 1% for 7 hours;

and C: filtering, namely bringing distilled water with 5 times of volume into the umbilical cord enzymolysis liquid, primarily filtering by using a screen, and then pouring into a differential separator for further filtering;

step D: primary culture, inoculating the filtered umbilical cord cells into a serum-free basal medium for culture, and preparing a novel culture solution by a cloning suspension method for in vitro amplification culture till the third generation;

step E: subculturing, replacing a serum-free culture medium, adding a serum-free nutrient additive into the serum-free culture medium, and periodically replacing the culture medium until cell clusters are generated;

step F: inducing differentiation, extracting the third generation cells, and inoculating the cells onto a differentiation medium by using a plate marking method for culture;

step G: and (4) performing cryopreservation on the prepared cells.

As a still further scheme of the invention: in the step A, the selected umbilical cord is cut into small pieces before enzymolysis, and the small pieces are placed into a heat-preservation tank for refrigeration, wherein the refrigeration duration is 2-6 months.

As a still further scheme of the invention: in the step D, a clone suspension method is adopted in the in-vitro amplification culture process of the stem cells, namely, an equal amount of cell suspension is added into each hole of each culture plate, the cells are mechanically separated and cloned again after the formation of neurospheres to prepare single cell suspension, the subculture size of the cloned neural stem cells is 200 m, the culture solution is DMEM/F12(1:1) and B27 and bFGF serum-free culture medium, and when the primary culture cells are fused to 85%, 0.25% trypsin is adopted to digest the cells for 30min at 37 ℃.

As a still further scheme of the invention: and C, separating the cells by using a desktop centrifuge and a constant-temperature magnetic stirrer of a biological safety cabinet, wherein the centrifugation method is lOOOrpm for 5min, and polylysine is added during separation.

As a still further scheme of the invention: and step F, mixing the pluripotent stem cell differentiation basal medium and the additive in the differentiation process, wherein every 10mL of the additive is thoroughly mixed with 500mL of the differentiation basal medium to obtain the human pluripotent stem cell differentiation medium (AC-1001002), and the human pluripotent stem cell differentiation medium can be stably stored at the temperature of 2-8 ℃ for 2-3 weeks.

The invention has the beneficial effects that: the preparation method of the mesenchymal stem cells derived from the early developmental mesoderm has reasonable design:

1. the selected umbilical cord is cut into small pieces before enzymolysis and placed in a heat-preservation tank for refrigeration, the refrigeration duration is 2-6 months, the separation efficiency of stem cells is improved, the temperature change in the heating and refrigeration processes of the stem cells is smoother, and the problems that the cells are inactivated due to the temperature change in the cell extraction process and the cell extraction rate is reduced are solved;

2. the stem cells adopt a clone suspension method in the in vitro amplification culture process, namely, each hole on each culture plate is added with the same amount of cell suspension, the cell suspension is prepared by mechanically separating and cloning again after forming neurospheres, the subculture size after the neural stem cells are cloned is 200 m, the culture solution is DMEM/F12(1:1) and B27 and bFGF serum-free culture medium, when the primary culture cells are fused to 85 percent, 0.25 percent trypsin is adopted to digest for 30min at 37 ℃, the activity of the sorted cells can be prevented from being influenced, the culture process is simplified, and the culture cost is reduced;

3. separating cells by using a table centrifuge and a biological safety cabinet constant-temperature magnetic stirrer, wherein the method for centrifuging is lOOOrpm, the time is 5min, and polylysine is added during separation, so that the separation speed of the mesenchymal stem cells can be improved, the polylysine ensures that the purity of the mesenchymal stem cells is ensured during the separation process, and the adherent growth after separation is promoted;

4. the differentiation process adopts the pluripotent stem cell differentiation basal medium and the additive to mix, wherein every 10mL of the additive and 500mL of the differentiation basal medium are thoroughly mixed to obtain the human pluripotent stem cell differentiation medium (AC-1001002), the human pluripotent stem cell differentiation medium can be stably stored at the temperature of 2-8 ℃ for 2-3 weeks, the complete differentiation of the stem cells is facilitated, the expression of the cells is not influenced, the activity of the cells is ensured, and the health influence on a receiver is reduced.

Drawings

FIG. 1 is a schematic view of the structure of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, a method for preparing mesenchymal stem cells derived from early-developing mesoderm includes the steps of:

step A: cleaning, extracting the umbilical cord, cleaning the umbilical cord with distilled water, and wiping with sterilized sponge;

and B: carrying out enzymolysis, taking out the umbilical cord, crushing, and carrying out enzymolysis by using collagenase with the concentration of 1% for 7 hours;

and C: filtering, namely bringing distilled water with 5 times of volume into the umbilical cord enzymolysis liquid, primarily filtering by using a screen, and then pouring into a differential separator for further filtering;

step D: primary culture, inoculating the filtered umbilical cord cells into a serum-free basal medium for culture, and preparing a novel culture solution by a cloning suspension method for in vitro amplification culture till the third generation;

step E: subculturing, replacing a serum-free culture medium, adding a serum-free nutrient additive into the serum-free culture medium, and periodically replacing the culture medium until cell clusters are generated;

step F: inducing differentiation, extracting the third generation cells, and inoculating the cells onto a differentiation medium by using a plate marking method for culture;

step G: and (4) performing cryopreservation on the prepared cells.

Further, in the embodiment of the invention, in the step A, the selected umbilical cord is cut into small pieces before enzymolysis, and the small pieces are placed in a heat preservation tank for refrigeration, wherein the duration of the refrigeration is 2-6 months, so that the separation efficiency of stem cells is improved, the temperature change in the heating and refrigeration processes of the stem cells is smoother, and the problems that the cells are inactivated due to the temperature change in the cell extraction process and the cell extraction rate is reduced are avoided.

Further, in the embodiment of the present invention, in the step D, a clone suspension method is adopted during the in vitro amplification culture process of the stem cells, that is, an equal amount of cell suspension is added to each well of each culture plate, the single cell suspension is prepared by mechanically separating and cloning again after the neurosphere is formed, the subculture size after the neural stem cell cloning is 200 m, the culture solution is DMEM/F12(1:1) plus B27 and bFGF serum-free medium, and when the primary culture cells reach 85% fusion, 0.25% trypsin is adopted to digest for 30min at 37 ℃, so that the activity of the sorted cells can be prevented from being affected, the culture process is simplified, and the culture cost is reduced.

Further, in the embodiment of the present invention, in the step C, a desktop centrifuge and a constant temperature magnetic stirrer of a biological safety cabinet are used to separate the cells, the centrifugation method is lloorpm, 5min, and polylysine is added during the separation, so that the separation speed of the mesenchymal stem cells can be increased, and the polylysine ensures the purity of the mesenchymal stem cells during the separation process and promotes the adherent growth after the separation.

Further, in the embodiment of the present invention, in the step F, a pluripotent stem cell differentiation basic medium and an additive are mixed during the differentiation process, wherein each 10mL of the additive is thoroughly mixed with 500mL of the differentiation basic medium to form the human pluripotent stem cell differentiation medium (AC-1001002), and the human pluripotent stem cell differentiation medium can be stably stored at 2-8 ℃ for 2-3 weeks, so as to facilitate complete differentiation of stem cells, and does not affect the expression of cells, ensure the activity of cells, and reduce the health impact on recipients.

The working principle is as follows: when the preparation method of the mesenchymal stem cell derived from the mesoderm in the early development stage is used, firstly, the umbilical cord tissue is obtained, and is cleaned and enzymolyzed to obtain the umbilical cord cell, then the obtained umbilical cord cell is primarily cultured to be capable of being amplified and cultured in vitro till the third generation, and is subcultured till a cell cluster is generated, and finally, induced differentiation is carried out to prepare the mesenchymal stem cell,

it will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.