CN113008891B - Construction method and application for screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential - Google Patents
Construction method and application for screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential Download PDFInfo
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Abstract
The invention discloses a construction method and application for screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential, wherein the construction method comprises the following steps: step one, primary separation and expansion of umbilical cord mesenchymal stem cells from a plurality of male human umbilical cords, and subculturing to P5 generation; step two, respectively adding osteogenic differentiation liquid to induce differentiation into osteoblasts; step three, respectively carrying out alizarin red staining after 21 days of induction culture, and collecting pictures; and step four, analyzing the picture obtained in the step three by adopting image J software to obtain alizarin red coloring area percentage in the picture, and taking average value of the alizarin red coloring area percentage of a plurality of umbilical cord mesenchymal stem cells to obtain quantitative standard of the mesenchymal stem cells for screening high-quality osteogenic differentiation potential. The invention provides a cell selection standard for basic research and clinical transformation of stem cell treatment bone tissue regeneration and repair, so as to improve the treatment effect.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a quantification standard for screening stem cells, and particularly relates to a construction method and application for screening high-quality mesenchymal stem cells with osteogenic differentiation potential.
Background
Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) can be isolated and cultured from a range of tissues including, but not limited to, bone marrow, fat, umbilical cord, amniotic membrane, placenta, etc., have self-renewal and multipotent differentiation potential, and are multipotent cells capable of differentiating into a variety of mesenchymal tissue cells such as adipocytes, osteoblasts, chondroblasts, hepatocytes, and neural cells. Therefore, the mesenchymal stem cells become a hot spot for research in the fields of basic medicine and clinical tissue organ injury repair and regeneration.
Numerous studies have demonstrated that MSCs induce immunosuppression in vivo and can exert anti-inflammatory regulatory capacity through immunomodulation for the treatment of immune disorders, e.g., to reduce graft versus host and rejection during hematopoietic stem cell transplantation therapy. The immunosuppressive ability of MSCs is demonstrated in a number of animal experiments and clinical studies, suggesting that mesenchymal stem cells have a very broad prospect in the treatment of immune diseases. The human umbilical cord mesenchymal stem cells (human umbilical cord derived MSCs, HUCMSCs) are wide in source, the materials are available in noninvasive operation, the cell use is in ethical dispute and the like, and can be subjected to large-scale industrialized amplification and storage, and meanwhile, the human umbilical cord mesenchymal stem cells have stronger proliferation capability and lower immunogenicity, can be used for allograft transplantation and are widely concerned. However, there are problems that, at present, there are great differences in the quality of HUCMSCs due to the difference in the methods of cell separation and expansion among different sources of cell donors, and thus, there are great differences in the regulatory immune effects of MSCs in different laboratories. In addition, due to the characteristics of cell therapy, MSCs have uncertainty in the effect of treating certain autoimmune diseases, such as large variability in the therapeutic effect of transplantation after HUCMSCs derived from different donors are transplanted into recipients. There are no reports of HUCMSCs quantification standard that is independent of good quality immunomodulatory capacity.
Disclosure of Invention
The invention provides a construction method and application for screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential, so as to overcome the defects of the prior art.
In order to achieve the above object, the present invention provides a construction method for screening mesenchymal stem cell quantification standard of high quality osteogenic differentiation potential, which has the following characteristics: the method comprises the following steps:
step one, primary separation and expansion of umbilical cord mesenchymal stem cells from a plurality of male human umbilical cords, and subculturing to P5 generation;
step two, respectively adding osteogenic differentiation liquid to induce differentiation into osteoblasts;
step three, respectively carrying out alizarin red staining after 21 days of induction culture, and collecting pictures;
and step four, analyzing the picture obtained in the step three by adopting image J software to obtain alizarin red coloring area percentage in the picture, and taking average value of the alizarin red coloring area percentage of a plurality of umbilical cord mesenchymal stem cells to obtain quantitative standard of the mesenchymal stem cells for screening high-quality osteogenic differentiation potential.
Furthermore, the invention provides a construction method for screening mesenchymal stem cell quantification standards with high quality and osteogenic differentiation potential, which can also have the following characteristics:
in the first step, the separation and expansion method of the human umbilical cord mesenchymal stem cells comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord blood trace, removing umbilical cord artery and vein blood vessel, cutting, adherence culturing, digesting, centrifuging, re-suspending, culturing to 80-90% fusion after inoculation, cell digesting, passage amplifying culturing, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of 5 th generation for later use.
Furthermore, the invention provides a construction method for screening mesenchymal stem cell quantification standards with high quality and osteogenic differentiation potential, which can also have the following characteristics: in the second step, a plurality of P5 generation umbilical cord mesenchymal stem cells are added into a 6-hole plate for culture after digestion, centrifugation, resuspension and counting, and the quantitative cells in each hole are 1 multiplied by 10 5 The method comprises the steps of carrying out a first treatment on the surface of the 6 holes are plated on each cell, when the cell density reaches 60-70% fusion degree, 3 holes are set as a control group, and the complete culture medium of the umbilical cord mesenchymal stem cells is always replaced in the culture process; the complete medium was 1L of low sugar MEM added: 10% fetal bovine serum, 100U/mL penicillin, 100g/mL streptomycin; in addition, 3 holes are set as experimental groups, and the culture medium is always replaced by an osteodifferentiation induction culture medium in the culture process; the osteogenic differentiation induction medium was 1L of low sugar MEM added: 10% fetal bovine serum, 100U/mL penicillin, 100g/mL streptomycin, 10 mu mol/L ascorbic acid, 1.8mmol/L potassium dihydrogen phosphate and 10nmol/L dexamethasone; the medium was changed every three days and co-cultured for 21 days.
Furthermore, the invention provides a construction method for screening mesenchymal stem cell quantification standards with high quality and osteogenic differentiation potential, which can also have the following characteristics: in the third step, all cells in the holes are stained with alizarin red, and the specific method is as follows: all wells were discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde for 30min, then washed 3 times with PBS, added with alizarin dye solution 1 mL/Kong Ranse min, and then washed 3 times with PBS; after the dyeing is finished, a photo is acquired under a microscope, 5 pictures are acquired per hole, and 15 pictures are acquired per cell experiment group and control group respectively.
Furthermore, the invention provides a construction method for screening mesenchymal stem cell quantification standards with high quality and osteogenic differentiation potential, which can also have the following characteristics: analyzing the acquired pictures by adopting image J software to obtain alizarin red coloring area percentage of each picture, taking the average value of all pictures of each hole, and taking the average value of all holes of each cell to obtain alizarin red coloring area percentage of each cell; and averaging the percentage of the alizarin red coloring area of a plurality of strains of cells to obtain the quantitative standard for screening the mesenchymal stem cells with high-quality osteogenic differentiation potential.
Furthermore, the invention provides a construction method for screening mesenchymal stem cell quantification standards with high quality and osteogenic differentiation potential, which can also have the following characteristics: in the fourth step, the number of umbilical cord mesenchymal stem cells is 6, and the percentage of alizarin red coloring area of the 6 male umbilical cord mesenchymal stem cells is 40 percent.
The invention also provides application of the mesenchymal stem cell quantification standard for screening high-quality osteogenic differentiation potential, and the mesenchymal stem cell quantification standard has the following characteristics: inducing cells to be screened to differentiate into osteoblasts, performing alizarin red staining after induced culture, collecting pictures, and analyzing the pictures by using image J software to obtain the alizarin red staining area percentage in the pictures; comparing the alizarin red coloring area percentage of the cells to be screened with the quantitative standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential, and obtaining the mesenchymal stem cells with high-quality osteogenic differentiation potential when the cells larger than the quantitative standard.
The invention has the beneficial effects that: the invention utilizes the mesenchymal stem cells of multiple human umbilical cords to induce the osteogenic differentiation, and statistical analysis discovers that the mesenchymal stem cells from different donors have obvious heterogeneity of the osteogenic differentiation capability, has large difference, and provides a screening standard of high-quality stem cells with the osteogenic differentiation capability based on the heterogeneity, which is a HUCMSCs quantification standard capable of screening high-quality immunoregulation capability, provides a cell selection standard for basic research and clinical transformation of stem cell treatment bone tissue regeneration and repair, improves the treatment effect, can be used for advancing the technical field of the osteogenic differentiation of the mesenchymal stem cells, and overcomes the blindness of basic research and clinical transformation cell selection of HUCMSCs treatment bone tissue regeneration and repair.
Drawings
FIG. 1 is an osteogenic differentiated alizarin red staining chart of 6 male human umbilical cord mesenchymal stem cells of example 1 and 6 female human umbilical cord mesenchymal stem cells of a control example;
FIG. 2 is a statistical chart of the osteogenic differentiation capacities of 6 male human umbilical cord mesenchymal stem cells of example 1 and 6 female human umbilical cord mesenchymal stem cells of control example;
FIG. 3 is a statistical chart of sex difference in the osteoblast differentiation ability of umbilical cord-derived mesenchymal stem cells.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1
The embodiment provides a construction method for screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential, which comprises the following steps:
step one, primary separation and expansion of umbilical cord mesenchymal stem cells from 6 male human umbilical cords, and subculturing to the generation P5.
The method for separating and amplifying the human umbilical cord mesenchymal stem cells comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord blood trace, removing umbilical cord artery and vein blood vessel, cutting, adherence culturing, digesting, centrifuging, re-suspending, culturing to 80-90% fusion after inoculation, cell digesting, passage amplifying culturing, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of 5 th generation for later use. The method comprises the following specific steps:
1) The parturient meeting the health requirements of biological sample donors is screened to collect samples, so that the safety of the biological samples is ensured, viruses including HIV, HBV, HCV, CMV, HIV-1&2, EBV and other possible detection are eliminated, and the risk of spreading infectious diseases is avoided.
2) Umbilical cord source: the experimental scheme is approved by the ethical committee of stem cell research of the drummer hospital, and the acquisition requirements meet the following two points: (1) apgar scoring is carried out on healthy birth control infants or after caesarean section, and the total score is 8-10 points; (2) taking the umbilical cord near the fetal end under the aseptic condition, wherein the length of the umbilical cord is about 10-20 cm. The two ends are cut off by 2cm respectively, the umbilical cord is soaked in DPBS containing 2% of green streptomycin, and the umbilical cord is transported by a medical incubator and treated within 4 hours.
3) The staff enters the facility and operates in the biosafety cabinet in the whole course.
4) The umbilical cord was cut into small pieces of about 2cm, and washed with DPBS containing 2% of penicillin streptomycin until no blood was present.
5) 3 blood vessels in the umbilical cord, namely two arteries and one vein are removed. The umbilical cord after removing the blood vessel is sheared into about 1mm by the ophthalmology scissors 3 Is a small tissue mass.
6) The tissue is cut and crushed and directly attached to a T75 culture flask, the flask is inverted for 4 hours, then the flask is arranged right, 10mL of human mesenchymal stem cells complete medium is added, and the complete medium formula is as follows: 10% MSC grade fetal bovine serum+DMEM low-sugar medium.
7) The cells climb out after about 14 days, so that colonies CFU-F can be formed, the culture flask is gently tapped, the tissue blocks are separated, and the tissue blocks are discarded.
8) The cell surface is gently washed by PBS, 10mL of fresh mesenchymal stem cell complete culture medium is replaced, the culture is continued in an incubator, and the primary cells are passaged when the primary cells grow to 40% -50% of fusion.
9) The old culture solution was discarded, DPBS was added and washed once with DPBS left at room temperature, and the DPBS was aspirated. Proper amount of Tryple was added and incubated at 37℃for digestion for 3min.
10 Gently beating cells, collecting cell suspension into a centrifuge tube, centrifuging at room temperature of 1200r/min for 5min, and discarding the supernatant. Adding human mesenchymal stem cell culture medium cell culture solution to re-suspend cells, gently blowing and beating uniformly, and inoculating at a density of about 1.5X10 4 /cm 2 。
11 At 37 ℃,5% CO 2 And (3) standing and culturing in an incubator until 80-90% of the umbilical cord mesenchymal stem cells are fused, and then performing a new round of digestion and passage, thereby obtaining the umbilical cord mesenchymal stem cells.
And step two, respectively adding the osteoblast differentiation liquid to induce differentiation into osteoblasts.
The specific method comprises the following steps: the 6P 5 generation umbilical cord mesenchymal stem cells are added into a 6-hole plate for culture after digestion, centrifugation, resuspension and counting, and the quantitative cells of each hole are 1 multiplied by 10 5 The method comprises the steps of carrying out a first treatment on the surface of the 6 holes are plated on each cell, when the cell density reaches 60-70% fusion degree, 3 holes are set as a control group, and the complete culture medium of the umbilical cord mesenchymal stem cells is always replaced in the culture process; the complete medium was 1L of low sugar MEM added: 10% fetal bovine serum, 100U/mL penicillin, 100g/mL streptomycin; in addition, 3 holes are set as experimental groups, and the culture medium is always replaced by an osteodifferentiation induction culture medium in the culture process;the osteogenic differentiation induction medium was 1L of low sugar MEM added: 10% fetal bovine serum, 100U/mL penicillin, 100g/mL streptomycin, 10 mu mol/L ascorbic acid, 1.8mmol/L potassium dihydrogen phosphate and 10nmol/L dexamethasone; the medium was changed every three days and co-cultured for 21 days.
And thirdly, respectively carrying out alizarin red staining after 21 days of induction culture, and collecting pictures.
The specific method comprises the following steps: all wells were discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde for 30min, then washed 3 times with PBS, added with alizarin dye solution 1 mL/Kong Ranse min, and then washed 3 times with PBS; after the dyeing is finished, a photo is acquired under a microscope, 5 pictures are acquired per hole, and 15 pictures are acquired per cell experiment group and control group respectively. The pictures of the individual cells are shown in FIG. 1.
And step four, analyzing the pictures obtained in the step three by adopting image J software to obtain the alizarin red coloring area percentage of each picture, taking the average value of all pictures of each hole, and taking the average value of all holes of each cell to obtain the alizarin red coloring area percentage of each cell, namely the osteogenic differentiation capacity of each cell, as shown in figure 2. And averaging the alizarin red coloring area percentages of the 6 umbilical cord mesenchymal stem cells to obtain the quantitative standard for screening the mesenchymal stem cells with high-quality osteogenic differentiation potential.
And taking the average value of alizarin red coloring area percentage of 6 male umbilical cord mesenchymal stem cells as 40%, namely screening the quantitative standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential.
Control example: the same method is adopted to sequentially and respectively induce osteoblast differentiation to 6 female umbilical cord mesenchymal stem cells, alize and dye alizarin red after induction culture, collect pictures (shown in figure 1), analyze each picture by image J software, and obtain the alizarin red coloring area percentage (shown in figure 2) in the pictures, wherein the average value of the alizarin red coloring area percentage of the 6 female umbilical cord mesenchymal stem cells is 3%.
Fig. 3 is a statistical graph showing the difference in the osteoblast differentiation capacity of human umbilical cord-derived mesenchymal stem cells between example 1 and comparative example, and it is understood that there is a difference in the osteoblast differentiation capacity of male umbilical cord mesenchymal stem cells from female umbilical cord mesenchymal stem cells, and that the statistical significance of the osteoblast differentiation capacity of male umbilical cord mesenchymal stem cells is higher than that of female stem cells (about 10-fold).
Example 2
The embodiment provides an application of screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential:
inducing the cells to be screened to differentiate into osteoblasts, performing alizarin red staining after induced culture, collecting pictures, and analyzing the pictures by using image J software to obtain the alizarin red staining area percentage in the pictures.
Comparing the alizarin red coloring area percentage of the cells to be screened with the quantitative standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential, and obtaining the mesenchymal stem cells with high-quality osteogenic differentiation potential when the cells larger than the quantitative standard.
Specifically, if the alizarin red staining area percentage of the cells to be screened is more than 40%, the cells are high-quality mesenchymal stem cells with osteogenic differentiation potential.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (5)
1. A construction method for screening high-quality mesenchymal stem cell quantification standard with osteogenic differentiation potential is characterized in that:
the method comprises the following steps:
step one, primary separation and expansion of umbilical cord mesenchymal stem cells from a plurality of male human umbilical cords, and subculturing to P5 generation;
step two, respectively adding osteogenic differentiation liquid to induce differentiation of osteoblasts: several P5 generation umbilical cord mesenchymal stem cells are added into a 6-hole plate for culture after digestion, centrifugation, resuspension and counting, and the quantitative cells of each hole are 1 multiplied by 10 5 The method comprises the steps of carrying out a first treatment on the surface of the 6 holes are plated on each cell, when the cell density reaches 60-70% fusion degree, 3 holes are set as a control group, and one cell is culturedDirectly replacing a complete culture medium of umbilical cord mesenchymal stem cells; the complete medium was 1L of low sugar MEM added: 10% foetal calf serum, 100U/mL penicillin, 100g/mL streptomycin; in addition, 3 holes are set as experimental groups, and the culture medium is always replaced by an osteodifferentiation induction culture medium in the culture process; the osteogenic differentiation induction medium was 1L of low sugar MEM added: 10% fetal bovine serum, 100U/mL penicillin, 100g/mL streptomycin, 10 mu mol/L ascorbic acid, 1.8mmol/L monobasic potassium phosphate, 10nmol/L dexamethasone; changing the culture medium every three days, and co-culturing for 21 days;
step three, respectively carrying out alizarin red staining after 21 days of induction culture, and collecting pictures;
analyzing the picture obtained in the step three by adopting image J software to obtain alizarin red coloring area percentage in the picture, and taking average value of the alizarin red coloring area percentage of a plurality of umbilical cord mesenchymal stem cells to obtain quantitative standard of the mesenchymal stem cells for screening high-quality osteogenic differentiation potential;
the application method for screening the mesenchymal stem cell quantification standard with high-quality osteogenic differentiation potential comprises the following steps: inducing cells to be screened to differentiate into osteoblasts, performing alizarin red staining after induced culture, collecting pictures, and analyzing the pictures by using image J software to obtain the alizarin red staining area percentage in the pictures; comparing the alizarin red coloring area percentage of the cells to be screened with the quantitative standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential, and obtaining the mesenchymal stem cells with high-quality osteogenic differentiation potential when the cells larger than the quantitative standard.
2. The method for constructing the quantitative standard for screening high-quality osteoblastic differentiation potential mesenchymal stem cells according to claim 1, wherein the method comprises the following steps:
in the first step, the separation and expansion method of the human umbilical cord mesenchymal stem cells comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord blood trace, removing umbilical cord artery and vein blood vessel, cutting, adherence culturing, digesting, centrifuging, re-suspending, culturing to 80-90% fusion after inoculation, cell digesting, passage amplifying culturing, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of 5 th generation for later use.
3. The method for constructing the quantitative standard for screening high-quality osteoblastic differentiation potential mesenchymal stem cells according to claim 1, wherein the method comprises the following steps:
in the third step, all cells in the holes are stained with alizarin red, and the specific method is as follows: all wells were discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde for 30min, then washed 3 times with PBS, added with alizarin dye solution 1 mL/Kong Ranse min, and then washed 3 times with PBS;
after the dyeing is finished, a photo is acquired under a microscope, 5 pictures are acquired per hole, and 15 pictures are acquired per cell experiment group and control group respectively.
4. The method for constructing the quantitative standard for screening high-quality osteoblastic differentiation potential mesenchymal stem cells according to claim 2, wherein the method comprises the following steps:
analyzing the acquired pictures by adopting image J software to obtain alizarin red coloring area percentage of each picture, taking the average value of all pictures of each hole, and taking the average value of all holes of each cell to obtain alizarin red coloring area percentage of each cell;
and averaging the percentage of the alizarin red coloring area of a plurality of strains of cells to obtain the quantitative standard for screening the mesenchymal stem cells with high-quality osteogenic differentiation potential.
5. The method for constructing the quantitative standard for screening high-quality osteoblastic differentiation potential mesenchymal stem cells according to claim 1, wherein the method comprises the following steps:
in the fourth step, the number of umbilical cord mesenchymal stem cells is 6, and the percentage of alizarin red coloring area of the 6 male umbilical cord mesenchymal stem cells is 40 percent.
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