CN115838685A - Culture medium and culture method of bone marrow mesenchymal stem cells derived from leukemia patient - Google Patents
Culture medium and culture method of bone marrow mesenchymal stem cells derived from leukemia patient Download PDFInfo
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Abstract
The invention provides a culture medium and a culture method of bone marrow mesenchymal stem cells from leukemia patients, wherein the culture medium of the bone marrow mesenchymal stem cells from leukemia patients is prepared by adding the following components in final concentration in a basic culture medium: 0.5-1.5% L-glutamine, 0.05-0.15 mM beta-mercaptoethanol, 15-25 ng/ml bFGF, 0.5-1.5% autologous bone marrow blood-derived serum, 5-15% FBS, 0.8-1.2% penicillin and streptomycin. The method is simple, convenient and quick to operate, can greatly improve the success rate of in vitro culture of leukemia MSC and improve the cell state, and has the advantages of high culture anchorage rate, quick cell anchorage and the like.
Description
Technical Field
The embodiment of the invention belongs to the technical field of biomedicine, and relates to a culture medium and a culture method of bone marrow mesenchymal stem cells derived from leukemia patients.
Background
Mesenchymal Stem Cells (MSCs) are widely distributed in various tissues of the human body, including bone marrow, fat, placenta, skeletal muscle, etc., and among them, the mesenchymal stem cells derived from leukemia patients are considered to be the most easily available and abundant MSCs library. The mesenchymal stem cells from human leukemia patients have the characteristics of multidirectional differentiation potential, self-renewal, homing to pathological tissues and immunoregulation, and are widely applied to the clinical fields of tissue repair, transplantation immunoregulation and the like. BMMSC also has the characteristics of convenient material acquisition, easy amplification in vitro subculture, continuous replication, easy transfection of exogenous genes and the like, is excellent seed cells for carrying out various basic researches in a laboratory, and has wide application prospect. However, the problems of difficult acquisition of bone marrow specimens, rare M SC number in bone marrow, poor adaptability of cells in an in vitro environment, poor culture success rate of the traditional method and the like are bottlenecks which prevent the wide application of the method. Therefore, it is of great practical value to find a convenient and efficient culture method for obtaining BMMSCs.
Compared with normal mesenchymal stem cells from healthy people, the mesenchymal stem cells from leukemia patients have low culture success rate, poor proliferation capacity and anti-apoptosis capacity and easier aging. At present, most of MSCs (mesenchymal stem cells) from leukemia are cultured by a culture system the same as normal human MSCs, namely, the MSCs are cultured by an alpha-MEM (bovine serum albumin-MEM) culture medium of 10% fetal calf serum after a density gradient centrifugation method, however, by adopting the traditional culture mode, the successful culture rate of the MSCs from leukemia patients in vitro is very low, the cell viability is poor, the proliferation capacity is greatly reduced compared with that in vivo, the cell proliferation capacity is reduced along with the increase of passage times, the form is changed from long fusiform to wide flat form, and the multidirectional differentiation capacity is gradually lost, so that great difficulty is brought to experiments, and the requirement of follow-up research of scientific researchers is difficult to meet.
Therefore, there is a need to develop a culture medium and a culture method of leukemia patient-derived mesenchymal stem cells that can improve the success rate of leukemia MSC culture in vitro.
Disclosure of Invention
In order to solve the technical problems, the embodiment of the invention provides a culture medium and a culture method of bone marrow mesenchymal stem cells from leukemia patients, the method is simple, convenient and quick to operate, the success rate of in vitro culture of leukemia MSC can be greatly improved, the cell state is improved, and the culture medium and the culture method also have the advantages of high culture adherence rate, quick cell adherence and the like.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect of the embodiments of the present invention, there is provided a culture medium of leukemia patient-derived mesenchymal stem cells, wherein the culture medium of leukemia patient-derived mesenchymal stem cells is a basic culture medium supplemented with the following components at final concentrations:
0.5-1.5% L-glutamine, 0.05-0.15 mM beta-mercaptoethanol, 15-25 ng/ml bFGF, 0.5-1.5% autologous bone marrow blood-derived serum, 10-20% FBS, 0.8-1.2% penicillin and streptomycin.
Further, the culture medium of the bone marrow mesenchymal stem cells from the leukemia patient is prepared by adding the following components in final concentration in a basic culture medium:
1% L-glutamine, 0.1mM beta-mercaptoethanol, 20ng/ml bFGF, 1% autologous bone marrow blood-derived serum, 10% FBS, 1% penicillin and streptomycin.
Further, the basic culture medium is selected from one of alpha-MEM and DMEM-F12.
In a second aspect of the embodiments of the present invention, there is provided a method of culturing leukemia derived mesenchymal stem cells, the method including: the culture medium is adopted to culture the mesenchymal stem cells from leukemia.
Further, the method specifically comprises: and (2) inoculating the mesenchymal stem cells from leukemia sources into the culture medium, changing the culture medium after 2-4 days to remove non-adherent cells, changing the culture medium every 2-3 days, and digesting the cells for subculture when the confluence degree of the cells reaches 70-90%.
Further, the culture conditions are as follows: 5% of CO 2 The temperature was 37 ℃.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the culture medium and the culture method of the bone marrow mesenchymal stem cells from leukemia patients provided by the embodiment of the invention are simple, convenient, practical, rapid, efficient and strong in operability. The applicant finds out through experiments that: a method for amplifying MSC from leukemia patients is established by adding a small amount of autologous serum of the patients, L-glutamine, fibroblast growth factor (bFGF), beta-mercaptoethanol and other components into a basic culture medium, can greatly improve the success rate of in vitro culture of leukemia MSC and improve the cell state, and has the advantages of high culture adherence rate, quick cell adherence and the like.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is an identification diagram of MSCs; wherein FIG. 1A shows the detection result of mesenchymal stem cell surface markers; FIGS. 1B and 1C are results after cell staining; FIG. 1D is a bone marrow mesenchymal stem cell morphology;
FIG. 2 is a graph comparing the growth rate of MSC in example 1 and comparative example 1;
fig. 3 is a graph comparing MSC apoptosis in example 1 and comparative example 1.
Detailed Description
The embodiments of the present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
Interpretation of terms in the present invention:
MSCs are short for mesenchymal stem cells;
basic fibroblast growth factor (bFGF), available from Peprotech under the cat # 100-18B;
FBS: fetal bovine serum, purchased from Gibico under cat number 42F0282K;
beta-mercaptoethanol, purchased from Meck under stock number M917637;
l-glutamine, purchased from Biosharp, cat # BL549A;
P/S double antibody ("penicillin-streptomycin" or "penicillin and streptomycin" in this application) was purchased from Thermo Fisher Scientific under the cat # 15140122;
the effects of the present application will be described in detail below with reference to examples and experimental data.
Example 1 culture Medium and culture method of leukemia patient-derived mesenchymal Stem cells
1. A culture medium of bone marrow mesenchymal stem cells from leukemia patients is prepared by adding the following components in final concentration to a basic culture medium alpha-MEM:
1% L-glutamine, 0.1mM beta-mercaptoethanol, 20ng/ml bFGF, 1% autologous bone marrow blood-derived serum, 10% FBS, 1% penicillin and streptomycin.
2. A method of culturing leukemia patient-derived mesenchymal stem cells, the method comprising:
s1, obtaining serum from autologous bone marrow blood: specifically, 2-3ml of bone marrow blood of a patient is extracted and stored at low temperature, and is sent to a laboratory for isolated culture as soon as possible. Centrifuging at 1600rpm for 15min to extract serum for preparing improved culture medium;
s2, obtaining bone marrow mesenchymal stem cells from leukemia patients:
the bone marrow fluid was gently added to a 15ml sterile centrifuge tube containing an equal volume of Percoll working fluid (density 1.073 g/L) taking care that the procedure was as gentle and smooth as possible. Two distinct layers were then observed in the centrifuge tube, the upper layer being bone marrow and the lower layer being Percoll fluid.
And (3) putting the centrifugal tube into a centrifuge, setting the rotating speed to be 500g, centrifuging for 25min, and after centrifugation, dividing the centrifugal tube into four layers from top to bottom. The middle buffy coat layer was gently aspirated by a pipette and washed 2 times repeatedly with 2% FBS in PBS buffer. Sequentially centrifuging at 250g for 10min and 5min, discarding supernatant, collecting mononuclear cell precipitate,
s3, preparing culture medium components according to the formula, and performing heavy suspension inoculation on T25cm by using an improved bone marrow mesenchymal stem cell culture medium 2 Culturing in a culture bottle, removing non-adherent cells by changing the culture solution for the first time after three days, changing the culture solution every 2-3 days later, and digesting MSC for subculture when the cell confluency reaches 80%.
Example 2 culture Medium and culture method of leukemia patient-derived mesenchymal Stem cells
1. A culture medium of leukemia patient-derived mesenchymal stem cells, which is prepared by adding the following components in final concentration to a basic culture medium:
0.5% L-glutamine, 0.05 mM beta-mercaptoethanol, 15 ng/ml bFGF, 0.5% autologous bone marrow blood-derived serum, 5% FBS, 0.8% penicillin and streptomycin.
2. A method of inducing lumbosacral segment spinal cord neural stem cells by ipscs, the method being the same as in example 1.
Example 3 culture Medium and culture method of leukemia patient-derived mesenchymal Stem cells
1. A culture medium of leukemia patient-derived mesenchymal stem cells, which is prepared by adding the following components in final concentration to a basic culture medium:
1.5% L-glutamine, 0.15 mM beta-mercaptoethanol, 25 ng/ml bFGF, 1.5% autologous bone marrow blood-derived serum, 15% FBS, 1.2% penicillin and streptomycin.
2. A method of inducing lumbosacral segment spinal cord neural stem cells by ipscs, the method being the same as in example 1.
Comparative example 1 conventional unmodified Medium
The comparative example is a conventional unmodified medium and has the following formulation: α -MEM containing 10% of FBS, 1% of penicillin and streptomycin.
Experimental example 1 cell identification
1. Detection of mesenchymal stem cell surface markers
Taking the 3 rd-4 th generation human bone marrow mesenchymal stem cells obtained in example 1, and carrying out trypsinization to prepare 1 × 10 8 A cell suspension in ml; after PBS is washed once, 50 mu L of each suspension is taken, 2ul of each flow antibody marked by PE, namely CD34, CD44, CD45, CD11b and CD19 is respectively added, and the incubation is carried out for 30min at room temperature in a dark place; 1600rpm 10min PBS centrifugal washing 1 times, with 400ul PBS heavy suspension filtration after machine, using Beckman cytoflex flow cytometry machine for on-machine detection, cell CD34, CD44, CD45, CD11b, CD19 surface antigen expression. Data analysis was performed with cytoflex software.
The detection result is shown in fig. 1A, and the result shows that the mesenchymal stem cells cultured by using the improved culture medium of the embodiment 1 can well ensure that the biological characteristics of the mesenchymal stem cells are not changed, and the positive marker CD44 and the expression rate are more than 98%; the expression rate of negative markers CD34, CD45, CD11b and CD19 is less than 1%.
2. Bone marrow mesenchymal stem cell differentiation assay
Bone marrow mesenchymal stem cells were continuously cultured for 3 passages under the modified medium culture conditions of example 1, and then cell differentiation culture was performed using an osteogenic differentiation medium (cygen, HUXMX-90021) and an adipogenic differentiation medium (cygen, HUXMX-90031), respectively, and a cell differentiation test was performed in a conventional manner, and staining identification was performed in a conventional staining manner. The results after cell staining are shown in fig. 1b and c, and the results show that the mesenchymal stem cells can better maintain the differentiation potential of the stem cells and differentiate into osteoblasts and adipocytes at a higher proportion under the condition of the improved culture medium.
3. Detecting mesenchymal stem cell morphology
The cell culture was continuously passaged three times according to the above improved experimental method, and the cell morphology of each passage was followed, as shown in fig. 1D, and the results showed that the cell morphology was uniform and conformed to the spindle shape of the mesenchymal stem cell.
Experimental example 2, the effect of culturing MSC derived from leukemia patient according to the improved method of example 1 is compared with that of culturing MSC in the conventional manner of comparative example 1
1. Growth rate analysis of MSCs
The two cell culture experiments were carried out for 3 serial passages, and the time required for 80% of each cell fusion was recorded, as shown in FIG. 2, and the results showed that the MSC of leukemia patients cultured in the medium modified in example 1 took less time to fuse and grew faster. (see FIG. 2).
2. Apoptosis analysis of MSCs
After 3 serial passages according to the above experimental method of cell culture, the third generation of MSC was used for apoptosis detection, as shown in FIG. 3, the results showed that: the leukemia patients cultured by the culture medium with the improved mode in the embodiment 1 have less MSC apoptosis proportion and better cell viability. (as shown in fig. 3).
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.
Claims (7)
1. A culture medium of bone marrow mesenchymal stem cells from leukemia patients is characterized in that the culture medium of the bone marrow mesenchymal stem cells from leukemia patients is prepared by adding the following components in final concentration in a basic culture medium: 0.5-1.5% L-glutamine, 0.05-0.15 mM beta-mercaptoethanol, 15-25 ng/ml bFGF, 0.5-1.5% autologous bone marrow blood-derived serum, 10-20% FBS, 0.8-1.2% penicillin and streptomycin.
2. The culture medium of the leukemia patient-derived mesenchymal stem cells, according to claim 1, is prepared by adding the following components in the basic culture medium in final concentration: 1% L-glutamine, 0.1mM beta-mercaptoethanol, 20ng/ml bFGF, 1% autologous bone marrow blood-derived serum, 10% FBS, 1% penicillin and streptomycin.
3. The culture medium of the leukemia patient-derived bone marrow mesenchymal stem cells, according to claim 1, wherein the basic medium is one selected from the group consisting of alpha-MEM and DMEM-F12.
4. A method of culturing leukemia-derived mesenchymal stem cells, comprising: culturing leukemia derived mesenchymal stem cells using the culture medium according to any one of claims 1 to 3.
5. The method according to claim 4, characterized in that it comprises in particular: inoculating leukemia derived mesenchymal stem cells into the culture medium of any one of claims 1-3, removing non-adherent cells after 2-4 days, changing the medium every 2-3 days, and digesting the cells for subculture when the cell confluency reaches 70-90%.
6. The method according to claim 4 or 5, wherein the culture conditions are: 5% of CO 2 The temperature was 37 ℃.
7. A method according to claim 4 or 5, characterized in thatThe range of the inoculation amount of the mesenchymal stem cells from leukemia is the bone marrow mononuclear cells 10 6 -10 7 One per ml.
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| CN117210401B (en) * | 2023-11-09 | 2024-02-20 | 江苏睿源生物技术有限公司 | Preparation and culture medium for promoting mesenchymal stem cell adherent growth and preparation method thereof |
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