CN114752560B - Umbilical cord mesenchymal stem cell culture medium - Google Patents

Umbilical cord mesenchymal stem cell culture medium Download PDF

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CN114752560B
CN114752560B CN202210671677.2A CN202210671677A CN114752560B CN 114752560 B CN114752560 B CN 114752560B CN 202210671677 A CN202210671677 A CN 202210671677A CN 114752560 B CN114752560 B CN 114752560B
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胡向兵
王珏
姬云
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SUZHOU KEBEI BIOTECHNOLOGY CO Ltd
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Abstract

The invention belongs to the technical field of culture medium preparation, and particularly relates to an umbilical cord mesenchymal stem cell culture medium. The umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, sulvastatin, edaravone, sodium zoledronate and human platelet lysate. The culture medium can be used for preparing umbilical cord mesenchymal stem cells, has the advantages of high proliferation speed, large quantity and stable phenotype, and can still maintain the multipotential differentiation capacity after multiple passages.

Description

Umbilical cord mesenchymal stem cell culture medium
Technical Field
The invention belongs to the technical field of culture medium preparation, and particularly relates to an umbilical cord mesenchymal stem cell culture medium.
Background
Under the induction condition in vivo or in vitro, umbilical cord Mesenchymal Stem Cells (MSCs) can be differentiated into a plurality of tissue cells such as fat, bone, cartilage, muscle, nerve, liver, cardiac muscle, endothelium and the like, continuous subculture still has multidirectional differentiation potential, the ideal seed cells can be used for repairing tissue and organ injuries caused by aging and pathological changes, the human umbilical cord mesenchymal stem cells become important candidate seed cells for cell therapy, and particularly have good application prospects in the aspects of tissue repair and immunoregulation.
The problems that the proliferation and adhesion capacity are reduced, the cell aging or apoptosis is easy to occur along with the extension of passage and culture time when the umbilical cord mesenchymal stem cells are prepared and cultured by a serum-free culture medium on the market at present, and the spontaneous differentiation is easy to occur in long-term in-vitro culture, so that the multipotential differentiation capacity is weakened or the multipotential differentiation potential is lost are solved. MSCs are very sensitive to culture density, can keep good proliferation capacity without differentiation under the condition of low-density inoculation, and are easy to differentiate due to contact inhibition after the MSCs grow to a certain density.
In vitro long-term culture of MSC is easy to cause DNA damage, accumulation, slow proliferation, weakened differentiation capacity, cell phenotype change and function reduction of MSC. In the process of in vitro amplification or radiotherapy, the intervention measures can reduce the accumulation of DNA damage in the MSC, are vital to obtaining the MSC with good function and have clinical significance. Meanwhile, after platelet-rich plasma is activated, more than 90% of presynthesized growth factors can be released within l hour, and the growth factors have short half-life and are easily degraded in vivo.
The invention aims to overcome the defects of the existing serum-free culture medium technology, and provides a preparation method of a culture medium required by stem cell preparation, which comprises the following steps: the preparation of the serum-free culture medium of the stem cells is suitable for a stable large-scale culture system of the mesenchymal stem cells; the invention can establish a stable stem cell preparation process; can maintain and promote the proliferation and multipotential differentiation capacity of the mesenchymal stem cells. Platelet Rich Plasma (PRP) contains rich composite growth factors, and the concentration and the proportion of various growth factors meet the physiological requirements, and can obviously promote the proliferation and the differentiation of MSCs and the formation of local new vessels. Meanwhile, by adjusting the composition of the culture medium, the technical problems that the growth factor has short half-life period and is easy to degrade in vivo are effectively solved.
Disclosure of Invention
In order to overcome the technical problems, the invention provides an optimized umbilical cord mesenchymal stem cell culture medium, which can be used for preparing umbilical cord mesenchymal stem cells, has high proliferation speed, large quantity and stable phenotype, and can still maintain the multipotential differentiation capacity after multiple passages.
In order to realize the purpose, the technical scheme provided by the invention is as follows:
an umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal Medium (Dulbecco's Modified Eagle Medium), F12 basal Medium (Ham's F12 nutrient Medium), vitamin C, rutin, algal polysaccharides, rosuvastatin, edaravone, zoledronic acid sodium, human platelet-rich plasma (PRP).
Preferably, the total concentration of the rutin, algal polysaccharide, rosuvastatin and edaravone in the mesenchymal stem cell culture medium is 1-200 uM.
Preferably, the mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 1-3:5-10:0.5-2: 1-2.
Preferably, the volume ratio of the DMEM basic medium to the F12 basic medium in the culture medium is 1-3:1, preferably 1: 1.
Preferably, the concentration of vitamin C in the mesenchymal stem cell culture medium is 10-500 uM.
Preferably, the concentration of zoledronic acid sodium in the mesenchymal stem cell culture medium is 0.1-50 uM.
Preferably, the human platelet lysate is 5-20% of the total volume of DMEM basal medium and F12 basal medium.
Another object of the present invention is to provide a method for preparing the culture medium, comprising the steps of:
firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and sodium zoledronate, and finally adding a human platelet lysate.
The invention also aims to provide application of the umbilical cord mesenchymal stem cell culture medium, wherein the application is to apply the culture medium to culture umbilical cord mesenchymal stem cells.
Another object of the present invention is to provide a method for preparing the umbilical cord mesenchymal stem cells, comprising the steps of:
(1) cleaning, soaking and sectionally treating the umbilical cord, and then adding the umbilical cord into the culture medium for culture;
(2) culturing until the tissue block cells climb out and grow to 70-80% fusion, and carrying out primary passage on the umbilical cord mesenchymal stem cells;
(3) p4, P10 and P15 cells were subcultured and collected.
Preferably, in (1), the washing is performed by disinfecting with alcohol and then washing with PBS.
Preferably, in (1), the soaking is performed for 0.5-3min by using alcohol.
Preferably, in (1), the segmentation is to divide the umbilical cord into 2-3cm umbilical cords.
Preferably, in (1) or (3), the culture is carried out at a temperature of 30-40 ℃ and a CO content of 3-7% by volume 2 Is carried out in an incubator.
Preferably, in (1) or (3), the culture is carried out at 37 ℃ and contains 5% CO by volume 2 Is carried out in an incubator.
Preferably, (3), the specific process of subculturing is as follows: when the strain is transferred to the P3 generation, the strain is inoculated into a culture bottle according to the density of 5000-10000 cells/square centimeter, and 30-60ml of prepared culture medium is added; horizontally putting the culture bottle into an incubator for culture; and (3) inoculating 96-well culture plates with P4, P10 and P15 generation cells, and performing cell growth curve detection.
Compared with the prior art, the invention has the technical advantages that:
(1) the invention provides a culture medium of umbilical cord mesenchymal stem cells, which can be used for preparing umbilical cord mesenchymal stem cells, has the advantages of high proliferation speed, large quantity and stable phenotype, and can still maintain the multipotential differentiation capacity after multiple passages.
(2) The invention effectively solves the technical problems that the growth factor has short half-life period and is easy to degrade in vivo by adjusting the composition of the culture medium.
(3) The serum-free culture medium provided by the invention is used by compounding low-concentration human platelet lysate with vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and sodium zoledronate, so that the in-vitro passage continuous culture of mesenchymal stem cells can be met; test results show that the human umbilical cord mesenchymal stem cells cultured by the serum-free culture medium have uniform morphology, can be induced and differentiated into osteoblasts and adipocytes, have cell surface marker expression meeting the standard, have high proliferation efficiency, and have no obvious difference in culture effect compared with fetal calf serum culture.
(4) In the culture medium provided by the invention, rutin, algal polysaccharide, rosuvastatin and edaravone have a good synergistic antioxidant effect, and the in-vitro subculture continuous culture of the culture medium on human umbilical mesenchymal stem cells is promoted.
Drawings
FIG. 1: counting by a human umbilical cord mesenchymal stem cell growth blood counting chamber (counting of cell growth condition);
FIG. 2: a human umbilical cord mesenchymal stem cell growth curve;
FIG. 3: multipotential differentiation (osteogenic differentiation) of human umbilical cord mesenchymal stem cells experiment alizarin red staining area;
FIG. 4: pluripotent differentiation (adipogenic differentiation) of human umbilical cord mesenchymal stem cells experiment oil red O staining area;
FIG. 5 is a schematic view of: pluripotent differentiation (chondrogenic differentiation) of human umbilical cord mesenchymal stem cells experiment Asin blue staining area;
the invention will now be further described with reference to the accompanying drawings and examples.
Detailed Description
The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise indicated, are commercially available; and different sources have no significant influence on the product performance.
Example 1
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone, sodium zoledronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 1: 1.
The concentration of vitamin C in the mesenchymal stem cell culture medium is 200 uM.
The total concentration of the rutin, algal polysaccharide, rosuvastatin and edaravone in the mesenchymal stem cell culture medium is 100 uM. The mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 2:8:1: 1.
The concentration of the sodium zoledronate in the mesenchymal stem cell culture medium is 30 uM.
The human platelet lysate was 10% of the total volume of DMEM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and zoledronate, and finally adding a human platelet lysate.
Example 2
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone, sodium zoledronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 3: 1.
The concentration of vitamin C in the mesenchymal stem cell culture medium is 10 uM.
The total concentration of the rutin, the algal polysaccharide, the rosuvastatin and the edaravone in the mesenchymal stem cell culture medium is 10 uM.
The mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 1:5:0.5: 1.
The concentration of zoledronic acid sodium in the mesenchymal stem cell culture medium is 50 uM.
The human platelet lysate was 5% of the total volume of DMEM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and sodium zoledronate, and finally adding a human platelet lysate.
Example 3
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone, sodium zoledronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 2: 1.
The concentration of vitamin C in the mesenchymal stem cell culture medium is 500 uM.
The total concentration of the rutin, the algal polysaccharide, the rosuvastatin and the edaravone in the mesenchymal stem cell culture medium is 50 uM.
The mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 3:10:2: 2.
The concentration of the sodium zoledronate in the mesenchymal stem cell culture medium is 0.1 uM.
The human platelet lysate was 20% of the total volume of DMEM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and zoledronate, and finally adding a human platelet lysate.
Comparative example 1
The difference from example 1 is that FBS was used instead of human platelet lysate.
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone, sodium zoledronate and Fetal Bovine Serum (FBS).
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 1: 1.
The total concentration of the rutin, the algal polysaccharide, the rosuvastatin and the edaravone in the mesenchymal stem cell culture medium is 100 uM. The mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 2:8:1: 1.
The concentration of the sodium zoledronate in the mesenchymal stem cell culture medium is 30 uM.
The FBS is 10 percent of the total volume of the DMEM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: mixing a DMEM basal medium and an F12 basal medium in proportion, sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and zoledronate, and finally adding FBS.
Comparative example 2
The difference compared to example 1 is the use of alendronate in place of zoledronate.
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone, sodium alendronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 1: 1.
The concentration of vitamin C in the mesenchymal stem cell culture medium is 200 uM.
The total concentration of the rutin, the algal polysaccharide, the rosuvastatin and the edaravone in the mesenchymal stem cell culture medium is 100 uM. The mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 2:8:1: 1.
The concentration of the sodium alendronate in the mesenchymal stem cell culture medium is 30 uM.
The human platelet lysate was 10% of the total volume of DMEM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and alendronate, and finally adding human platelet lysate.
Comparative example 3
Compared with example 1, the difference is that the seaweed polysaccharide is used to replace rutin.
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, algal polysaccharide, rosuvastatin, edaravone, sodium zoledronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 1: 1.
The concentration of vitamin C in the mesenchymal stem cell culture medium is 200 uM.
The total concentration of the algal polysaccharide, rosuvastatin and edaravone in the mesenchymal stem cell culture medium is 100 uM. The mass ratio of the algal polysaccharide to the rosuvastatin to the edaravone is 10:1: 1.
The concentration of the sodium zoledronate in the mesenchymal stem cell culture medium is 30 uM.
The human platelet lysate was 10% of the total volume of DMEM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, algal polysaccharides, rosuvastatin, edaravone and sodium zoledronate, and finally adding a human platelet lysate.
Comparative example 4
Compared with example 1, the difference is that rosuvastatin is used to replace edaravone.
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, rutin, algal polysaccharide, rosuvastatin, sodium zoledronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 1: 1.
The total concentration of the rutin, the algal polysaccharide and the rosuvastatin in the mesenchymal stem cell culture medium is 100 uM. The mass ratio of the rutin to the algal polysaccharide to the rosuvastatin is 2:8: 2.
The concentration of the sodium zoledronate in the mesenchymal stem cell culture medium is 30 uM.
The human platelet lysate is 10% of the total volume of the DMEM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding rutin, algal polysaccharide, rosuvastatin and zoledronate, and finally adding a human platelet lysate.
Comparative example 5
The difference compared to example 1 is the replacement of rosuvastatin with edaravone.
An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, edaravone, sodium zoledronate and human platelet lysate.
The volume ratio of the DMEM basic culture medium to the F12 basic culture medium in the culture medium is 1: 1.
The concentration of vitamin C in the mesenchymal stem cell culture medium is 200 uM.
The total concentration of the rutin, the algal polysaccharide and the edaravone in the mesenchymal stem cell culture medium is 100 uM. The mass ratio of the rutin to the algal polysaccharide to the edaravone is 2:8: 2.
The concentration of the sodium zoledronate in the mesenchymal stem cell culture medium is 30 uM.
The human platelet lysate was 10% of the total volume of DMEM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly mixing a DMEM basic culture medium and an F12 basic culture medium in proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, edaravone and zoledronate, and finally adding a human platelet lysate.
1. Isolated culture of human umbilical cord mesenchymal stem cells
(1) Cleaning and treatment of umbilical cord
Cleaning: after disinfecting the umbilical cord storage bottle with 75% alcohol, putting the umbilical cord storage bottle into a safety cabinet, taking out the umbilical cord, and washing the umbilical cord for 2 times by PBS;
soaking: adding 75% alcohol to soak the umbilical cord for 30 seconds;
segmenting: placing the umbilical cord in a sterile culture dish, and shearing into 2-3cm small sections by using a sterile surgical scissors;
and (3) processing: removing artery, vein and adventitia, and cutting;
bottle laying: evenly spreading the mixture to the bottom of a T75 culture bottle;
culturing: placing the culture bottle with the umbilical cord tissue blocks in a carbon dioxide incubator, and inverting for 6 hours;
(2) culturing
Slowly adding 5-10ml of prepared culture medium of each group into the culture bottle; the culture flask was horizontally placed in a 37 ℃ CO concentration of 5% by volume 2 Culturing in an incubator;
changing the liquid 1 time every 4 days, observing the growth condition of the cells at the edges of the tissue blocks, and carrying out primary passage on the umbilical cord mesenchymal stem cells when the cells of the tissue blocks climb out and grow to 70-80% fusion;
(3) passage of culture
When the strain is transferred to the P3 generation, the strain is inoculated into a T175 culture bottle according to the density of 5000-10000 cells/square centimeter, and 30ml of prepared culture medium is added; the flasks were horizontally placed in a 5% by volume CO2 incubator at 37 ℃.
(4) Collecting P4, P10, P15
When the cells grow to 70-90% and are fused, removing the culture medium, and washing the cell surface for 1 time by using 10mL of PBS buffer solution;
discarding PBS, adding 6mL trypsin for digestion, slightly shaking the culture flask to enable the trypsin to cover the bottom of the flask, quickly observing under a mirror, and slightly beating the outer side of the culture flask to enable the cells to float when the cells shrink into spheres;
adding the same amount of culture medium to terminate digestion;
collecting cell suspension, 1200rpm, 6 liters and 6 drops, and centrifuging for 5 min;
discarding supernatant, adding 10ml LPBS to resuspend cells, gently blowing and beating to avoid generating bubbles, and centrifuging;
after the final centrifugation, the supernatant was discarded and the cells were resuspended with 10ml PBS.
(5) Respectively taking 0.1mL of cell suspension of P4, P10 and P15 cells, counting by using a blood counting chamber, and counting the growth condition of the cells;
the culture media prepared in each example and comparative example were subjected to isolated culture of human umbilical cord mesenchymal stem cells according to the above method.
The results show that: example 1 the culture protocol was such that the cells were transferred to P15, and the proliferation capacity of the cells was still strong and was higher than that of comparative example 1 containing 10% FBS, which is better than that of comparative example 2 containing alendronate sodium and other groups. Cell growth was counted as shown in FIG. 1.
(6) Taking P4, P10 and P15 cells 1 x 10 respectively 7 Performing cell phenotype flow identification on the/ml cell suspension;
collecting P4, P10 and P15 cells, adjusting cell concentration to 1 × 10 7 Adding antibodies CD73, CD90, CD105, CD34 and CD45 into the mixture per ml, incubating the mixture for 30min at room temperature in a dark place, and replacing primary antibodies (primary antibodies refer to primary antibodies) with PBSAntibodies CD73, CD90, CD105, CD34 and CD 45) as a blank control, and the expression of cell surface antigens was detected using a flow cytometer, and the results are shown in table 1 below.
TABLE 1 phenotypic identification of human umbilical cord mesenchymal stem cells
Figure DEST_PATH_IMAGE001
As can be seen from the above table, each group of cell surface antigens CD73, CD90 and CD105 is positively expressed, and CD34 and CD45 are negatively expressed, wherein the positive rates of CD73, CD90 and CD105 of the cells obtained by the culture in example 1 are all above 95%, and the positive rates of CD45 and CD34 are lower than 2%, which are in line with the phenotypic characteristics of human umbilical cord mesenchymal stem cells, and have no obvious difference with the expression of the surface antigen of the human umbilical cord mesenchymal stem cells cultured in 10% FBS, and are better than that in comparative example 2 and better than that in comparative examples 3-5. The serum-free culture medium provided by the invention is suitable for culturing human umbilical cord mesenchymal stem cells. At the same time, the composition of the medium has a certain influence on its properties.
(7) And performing cell growth curve detection on P4, P10 and P15 respectively.
The culture media prepared in the examples and comparative examples were subjected to cell growth curve detection of human umbilical cord mesenchymal stem cells according to the following method.
Detecting the proliferation capacity of the human umbilical cord mesenchymal stem cells: collecting human umbilical cord mesenchymal stem cell suspension in logarithmic growth phase of P4, P10 and P15 generations, and adjusting cell concentration to 1 × 10 4 And each piece is inoculated in a 96-well plate, each piece is divided into 8 groups, and each group is provided with 3 multiple wells. Collecting one group every day after 24 hr, removing culture solution, adding CCK-8 solution, and continuously standing at 37 deg.C with 5% CO 2 The incubator is incubated for 4 hours in a dark place, the absorbance (A) value is measured at the 490nm wavelength of the microplate reader, the process is repeated for 3 times, and the average value is taken. The results of plotting a cell growth curve with the horizontal axis representing the cell culture time and the vertical axis representing the absorbance A value are shown in FIG. 2.
As can be seen from FIG. 2, the cell growth curves of example 1 and 10% fetal bovine serum culture group (comparative example 1) approximately exhibit "S" type, including typical incubation period, logarithmic growth period and plateau period; wherein, the example 1 is equivalent to the comparative example 1, and the growth is better than that of other groups, the cell proliferation is accelerated from the next day, the logarithmic growth phase is entered, and the plateau phase is entered from the 7 th day. The mesenchymal stem cells between the control groups had longer latency and proliferated slower.
(8) And (3) respectively taking P4, P10 and P15 cells, and carrying out pluripotent differentiation experiment detection.
Differentiation abilities of osteogenic induction, adipogenic induction and chondrogenic induction of mesenchymal stem cells obtained by isolation culture of human umbilical cord mesenchymal stem cells in the culture media prepared in each example and comparative example were evaluated according to the following methods.
Bone formation induction: osteogenic induction was performed using OriCell human umbilical cord mesenchymal stem cell osteogenic induction differentiation kit (Hipposhu Biotech Co., Ltd., HUXUC-90021, 200 ml/kit).
Osteogenic induction group: collecting P4, P10 and P15 cells at 1 × 10 4 /cm 2 The density of (A) was inoculated into 6-well plates, 2 ml/well, at 37 ℃ with 5% CO 2 The culture box is kept still for culture, when the cells grow to 80%, the original culture solution is removed, osteogenic induced differentiation solution (the components in the kit are also called osteogenic induced differentiation) is added, the solution is changed for 1 time every 3d, after 21 days of culture, alizarin red staining observation is carried out after 4% paraformaldehyde is adopted for fixation, the results are observed under a microscope, and the staining area is counted.
Meanwhile, a negative control group is prepared by the same method, the negative control group is only distinguished in that no osteogenic induced differentiation solution is added, and the result of the relative value of alizarin red staining areas of the osteogenic induced group and the negative control group is shown in figure 3.
② fat forming induction: OriCell human umbilical cord mesenchymal stem cell adipogenic induction differentiation kit (Hipposhu Biotech Co., Ltd., HUXUC-90031, 300 ml/kit) was subjected to adipogenic induction.
And (3) induction group: collecting P4, P10 and P15 cells at 1X 10 4 /cm 2 The density of (A) was inoculated into 6-well plates, 2 ml/well, at 37 ℃ with 5% CO 2 The culture box (2) is kept still for culture until the cells grow to 70%, the original culture solution is removed, and a adipogenic differentiation inducing solution (components in the kit, also called as the component of the kit) is addedLipid induced differentiation), changing the solution 1 time every 3d, culturing for 21 days, fixing by using 4% paraformaldehyde, performing oil red O staining observation, observing the result under a microscope, and counting the staining area.
Meanwhile, a negative control group was prepared in the same manner, and the difference between the negative control groups was only that no adipogenic differentiation medium was added, and the results of the relative values of the oil red O staining areas of the adipogenic induction group and the negative control group are shown in fig. 4.
③ inducing the formed cartilage: chondrogenic induction was performed using OriCell human umbilical cord mesenchymal stem cell chondrogenic induction differentiation kit (HUXUC-90041, 100ml/kit, seiko (guangzhou) biotechnology limited).
Chondrogenic induction group: collecting P4, P10 and P15 cells, and adjusting cell concentration to 1.5 × 10 5 Perml, 2ml of the suspension was inoculated into 6-well plates at 37 ℃ with 5% CO 2 The culture box is kept still for culture, preheated chondrogenic differentiation inducing liquid (components in the kit, also called chondrogenic differentiation inducing liquid) is added after 2 hours, the liquid is changed for 1 time every 3 days, after 14 days of culture, 4% paraformaldehyde is fixed, paraffin is embedded, the section is cut, and the staining area is counted by staining observation under a microscope.
Meanwhile, a negative control group was prepared in the same manner, and the difference between the negative control group and the chondrogenic differentiation medium was only that no chondrogenic differentiation medium was added, and the results of the staining area relative to the azure blue of the chondrogenic differentiation medium and the negative control group are shown in FIG. 5.
The results show that: the cells of each group are passed to P15, and the mesenchymal stem cells obtained by culturing in example 1 have no statistical difference in osteogenic induction, adipogenic induction and chondrogenic induction differentiation capacity compared with the human umbilical cord mesenchymal stem cells cultured in the group containing 10% fetal calf serum of comparative example 1. Better than comparative example 2, better than comparative examples 3-5.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.

Claims (7)

1. An umbilical cord mesenchymal stem cell culture medium comprises the following components: DMEM basal medium, F12 basal medium, vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone, sodium zoledronate and human platelet lysate;
the concentration of vitamin C in the mesenchymal stem cell culture medium is 10-500uM, and the concentration of zoledronic acid sodium is 0.1-50 uM;
the total concentration of the rutin, algal polysaccharide, rosuvastatin and edaravone in the mesenchymal stem cell culture medium is 1-200 uM;
the mass ratio of the rutin to the algal polysaccharide to the rosuvastatin to the edaravone is 1-3:5-10:0.5-2: 1-2;
the human platelet lysate is 5-20% of the total volume of the DMEM basal medium and the F12 basal medium.
2. The umbilical cord mesenchymal stem cell culture medium of claim 1, wherein a volume ratio of the DMEM basal medium to the F12 basal medium in the culture medium is 1-3: 1.
3. The method for preparing culture medium of umbilical cord mesenchymal stem cells according to any one of claims 1-2, comprising the steps of: firstly mixing a DMEM basic culture medium and an F12 basic culture medium according to a proportion, then sequentially adding vitamin C, rutin, algal polysaccharide, rosuvastatin, edaravone and sodium zoledronate, and finally adding a human platelet lysate.
4. The use of umbilical cord mesenchymal stem cell culture medium according to any one of claims 1 to 2, wherein the culture medium is used for culturing umbilical cord mesenchymal stem cells.
5. A preparation method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
(1) washing, soaking and sectionally treating an umbilical cord, and then adding the umbilical cord into the culture medium of any one of claims 1-2 for culture;
(2) culturing until the tissue block cells climb out and grow to 70-80% fusion, and carrying out passage on the umbilical cord mesenchymal stem cells;
(3) p4, P10 and P15 cells were subcultured and collected.
6. The method for preparing umbilical cord mesenchymal stem cells according to claim 5, wherein the culturing is performed at a temperature of 30-40 ℃ and a CO content of 3-7% by volume in step (1) or step (3) 2 Is carried out in an incubator.
7. The method for preparing umbilical cord mesenchymal stem cells according to claim 5, wherein in the step (3), the specific process of subculturing is as follows: when the strain is transferred to the P3 generation, the strain is inoculated into a culture bottle according to the density of 5000-10000 cells/square centimeter, and 30-60ml of prepared culture medium is added; horizontally putting the culture bottle into an incubator for culture; and (3) inoculating the P4, P10 and P15 generation cells into a 96-well culture plate, and performing cell growth curve detection.
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