CN111690604A

CN111690604A - MSC in-vitro amplification method

Info

Publication number: CN111690604A
Application number: CN202010584282.XA
Authority: CN
Inventors: 曹哲厚; 郑海刚; 韦俊; 吴立前
Original assignee: Hangzhou Yuansheng Biotechnology Co ltd
Current assignee: Hangzhou Yuansheng Biotechnology Co ltd
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2020-09-22

Abstract

The invention provides an MSC in vitro amplification method, belonging to the technical field of cell amplification. The method solves the problems that the source of the existing mesenchymal stem cells is limited and the like, and the method for in vitro expansion of the MSC comprises the following steps: s01: soaking the MSPM membrane in water to prepare MSPM mixed liquor; resuspending the mesenchymal stem cells in a first culture medium to prepare an MSC cell suspension; s02: taking the MSPM mixed solution, discarding the supernatant, re-suspending with a second culture medium, then putting into an incubator for culture, discarding the supernatant again, infiltrating the MSPM carrier with the MSC cell suspension, then supplementing the first culture medium, and putting into the incubator for culture. The invention has the advantages of capability of in vitro amplification of MSC and the like.

Description

MSC in-vitro amplification method

Technical Field

The invention belongs to the technical field of cell amplification, and particularly relates to an MSC in-vitro amplification method.

Background

MSC refers to mesenchymal stem cells, one of stem cells, which can differentiate into mesenchymal tissues. The mesenchymal stem cells are named as mesenchymal tissues because of being capable of differentiating into the mesenchymal tissues, have sub-totipotent differentiation potential, and can be induced and differentiated into various histiocytes under specific in vivo and in vitro environments. Mesenchymal stem cells have the commonality of stem cells, namely the ability to self-renew, differentiate and home. Mesenchymal stem cells can differentiate into various cells such as nerve, heart, liver, bone, cartilage, tendon, fat, epithelium, and the like. This ability to differentiate in multiple directions provides an important starting material for the treatment of various diseases in humans.

At present, the main source of the mesenchymal stem cells is adult bone marrow, but the number and the proliferation and differentiation potential of the adult bone marrow-derived mesenchymal stem cells are reduced along with the increase of age, the virus infection rate is higher, and the source of the donor mesenchymal stem cells is limited by bone marrow puncture, so that the clinical application of the mesenchymal stem cells is limited. Therefore, it is necessary to invent a method for in vitro expansion of mesenchymal stem cells to solve the above problems.

Disclosure of Invention

The present invention is directed to the above problems in the prior art, and provides a method for in vitro amplification of MSCs.

The purpose of the invention can be realized by the following technical scheme: an in vitro MSC amplification method, comprising the following steps:

s01: soaking the MSPM membrane in water to prepare MSPM mixed liquor; resuspending the mesenchymal stem cells in a first culture medium to prepare an MSC cell suspension;

s02: taking the MSPM mixed solution, discarding the supernatant, re-suspending with a second culture medium, then putting into an incubator for culture, discarding the supernatant again, infiltrating the MSPM carrier with the MSC cell suspension, then supplementing the first culture medium, and putting into the incubator for culture.

Preferably, in step S02, the MSPM mixed solution is taken, the supernatant is discarded, the second culture medium is used for resuspending, the resuspended solution is added into the pore plate, the pore plate is placed into the incubator for culturing, the MSC cell suspension is added into each pore to infiltrate the MSPM carrier after the supernatant is discarded again, then the first culture medium is supplemented, and the pore plate is placed into the incubator for culturing the mesenchymal stem cells.

Preferably, in step S02, the well plate is taken out every 15min within 1 hour of the mesenchymal stem cell culture, and is shaken and mixed, and the first culture medium is added after 3 hours of the mesenchymal stem cell culture.

Preferably, in step S02, after three days of the mesenchymal stem cell culture, the original first culture solution in each well is aspirated, and a fresh first culture solution is newly added.

Preferably, the incubator is CO with 5% carbon dioxide content²An incubator.

Preferably, the first culture medium is a friend serum-free culture medium; the second culture medium is a DMEM HG culture medium.

Preferably, in step S01, the density of the MSC cell suspension is 1 x 10⁶cells/mL。

The working principle of the invention is as follows: the MSPM membrane is used as a carrier to culture the mesenchymal stem cells, and the method has the advantages of simple steps and high culture efficiency.

Compared with the prior art, the invention has the following advantages:

1. the MSPM film is used as a carrier, and the component of the MSPM film is parylene, so that the MSPM film is suitable for being implanted into a human body for a long time and is a biocompatible coating material. Good biostability, low cytotoxicity and also hydrolysis resistance.

2. The invention discloses an in vitro amplification method of MSC, which can effectively amplify MSC in vitro.

Drawings

FIG. 1 is a microscopic magnification of 4 times the objective lens for observing MSC cells on MSPM under microscope at day 1 according to the present invention;

FIG. 2 is a microscopic magnification of 4 times magnification of the objective lens for observing MSC cells on MSPM under microscope at day 6 of the present invention;

FIG. 3 is a histogram of the number of cells adsorbed for 3 hours according to the invention;

FIG. 4 is a bar graph of adsorption efficiency for 3 hours of adsorption time according to the present invention;

FIG. 5 is a bar graph of the fold increase of 5 days in culture according to the invention.

Detailed Description

The following are specific embodiments of the present invention and are further described with reference to the drawings, but the present invention is not limited to these embodiments.

Example 1

(1) Preparation of MSPM (carrier) mixture: cutting a MSPM film with an area of 1cm²The length and width of each 1cm are soaked in 4ml of purified water, and the mixture is sterilized at 121 ℃ for 30min, and can be stored for 3 months at 4 ℃ after being sterilized.

(2) Preparation of MSC cell suspension: obtaining MSC within P4 generation, re-suspending cells with Youkang serum-free culture medium (purchased from market), and making into cell suspension with cell suspension density of 1 × 10⁶cells/ml, volume 500ul, cell viability greater than 85%.

(3) Cell inoculation: taking 2ml of MSPM mixed solution (containing MSPM membrane in the mixed solution), sucking supernatant, re-suspending with 2ml of DMEMHG culture medium (DMEM high-sugar culture medium purchased from market), mixing, adding ultra-low adsorption 6-pore plate, and transferring into CO²Incubator (carbon dioxide concentration 5%). In CO²Incubating in an incubator for 30min, taking out a 6-pore plate, discarding supernatant with a pipette, adding 100ul of MSC cell suspension into each pore, slightly shaking the 6-pore plate for 10s, repeatedly infiltrating microcarrier, supplementing 400ul of Youkang serum-free culture medium into each pore, and adding CO²An incubator. Within the first hour, take out the 6-well plate every 15min and mix gently back and forth in the biosafety cabinet. And after 3 hours, replenishing Youkang serum-free culture medium. And taking a pore cracking count to calculate the adsorption rate.

(4) Cell observation and exchange: continued in CO²Culturing in an incubator, and on the third day, sucking away the old culture solution and adding the fresh culture solution.

(5) Cell harvesting: on day 6, sucking out all suspension and MSPM in each well of the 6-well plate together, putting into a 15ml centrifuge tube, adding 5ml PBS, and centrifuging at 1500rpm for 5 min; discarding the supernatant, adding 1ml of Trpsin (trypsin), gently mixing with a centrifuge tube, placing at room temperature (21 ℃), and standing for 10 min; after the cells are totally detached from the MSPM under the observation of a microscope, adding 4ml of PBS, fully and uniformly mixing, and centrifuging for 5min at the rotating speed of 1500rpm again; discarding the supernatant, adding 100PBS to resuspend the cells, taking 20ul of cell suspension and mixing with the fluorescent dye solution at a ratio of 1:1, counting, and calculating the cell survival rate and the total cell number, wherein the cell survival rate multiplied by the total cell number is the cell harvest number.

Example 1 parallel 3 experiments, group a, group B, group C. The adsorption rate after 3 hours of adsorption time is shown in fig. 4, the number of cells after 3 hours of adsorption time is shown in fig. 3, the proliferation factor of cells cultured for 5 days (the final harvest number divided by the initial number adsorbed for 3 hours after 5 days is the proliferation factor) is shown in fig. 5, the MSC cells on MSPM are observed under a microscope at day 1, and the magnification factor of the objective lens is 4 times, as shown in fig. 1; MSC cells on MSPM were observed under microscope at day 6 with 4 x magnification of objective lens as shown in figure 2. Low sorption 6-well plates were purchased directly from the market.

MSPM film: is a porous grid film from Zhejiang laboratories, and contains parylene. The parylene is suitable for long-term implantation in human body, and is a biocompatible coating material. It has good biological stability, low cytotoxicity and hydrolysis resistance.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Although terms are used more often herein, the possibility of using other terms is not excluded. These terms are used merely to more conveniently describe and explain the nature of the present invention; they are to be construed as being without limitation to any additional limitations that may be imposed by the spirit of the present invention.

Claims

1. An in vitro MSC amplification method is characterized by comprising the following steps:

2. The method according to claim 1, wherein in step S02, the MSPM mixture is taken, the supernatant is discarded, the MSPM mixture is resuspended in a second culture medium, the resuspended solution is added into the well plate and then placed into the incubator for culturing, after the supernatant is discarded again, the MSC cell suspension is added into each well to infiltrate the MSPM carrier, then the first culture medium is supplemented, and the well plate is placed into the incubator for culturing the mesenchymal stem cells.

3. The method according to claim 1, wherein in step S02, the well plate is removed every 15min within 1 hour of the mesenchymal stem cell culture, and is shaken and mixed, and the first culture medium is added after 3 hours of the mesenchymal stem cell culture.

4. The method of claim 1, wherein in step S02, after three days of culturing the mesenchymal stem cells, the original first culture solution in each well is aspirated and fresh first culture solution is added again.

5. The method of claim 1, wherein the culture chamber is CO with 5% carbon dioxide²An incubator.

6. The method for in vitro expansion of MSCs according to claim 1, wherein said first culture medium is a friend serum-free medium; the second culture medium is a DMEM HG culture medium.

7. The method according to claim 1, wherein the density of the MSC cell suspension in step S01 is 1 x 10⁶cells/mL。