Separation and in-vitro culture method of mesenchymal stem cells
Through the use of serum-free culture medium and a variety of additives, combined with oxygen content control, the problem of quickly obtaining large-batch, stable quality mesenchymal stem cells is solved, and efficient cell isolation and culture effects are achieved.
Patent Information
- Application Number
- CN202510744784.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-08-08
AI Technical Summary
It is difficult to quickly obtain large batches of mesenchymal stem cells with stable quality in the prior art, and the application of autologous mesenchymal stem cells has problems such as large individual differences in amplification capabilities, potential risk of tumor cell contamination and insufficient culture time.
Serum-free culture medium and combined with a variety of additives are used to control the oxygen content at different stages, simulate the microenvironment in the body, and separate and culture mesenchymal stem cells.
It has achieved rapid acquisition of large-batch, stable quality mesenchymal stem cells, improved cell yield and viability, and met the needs of clinical applications.
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell culture, and in particular to a method for separating and culturing mesenchymal stem cells in vitro. Background Art
[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells that possess all the common properties of stem cells: self-renewal and multidirectional differentiation. They are also the most widely used in clinical practice. Their combined use with hematopoietic stem cells can improve transplant success rates and accelerate hematopoietic reconstitution. When administered together with hematopoietic stem cells after high-dose chemotherapy, MSCs can significantly accelerate blood cell recovery, with no adverse reactions.
[0003] Mesenchymal stem cells are found not only in bone marrow but also in skeletal muscle, periosteum, and trabecular bone. Their wide range of tissue differentiation makes them highly valuable for clinical applications. However, the application of autologous mesenchymal stem cells has gradually become exposed to inconveniences, such as significant individual variability in proliferation capacity, potential risk of tumor cell contamination, and the time required for cultivation, which can hinder timely adaptation to patient needs. These factors have hindered the use of autologous mesenchymal stem cells.
[0004] Therefore, rapidly obtaining large quantities of mesenchymal stem cells with stable quality is an important way to solve the problems faced in current applications. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for isolating and culturing mesenchymal stem cells in vitro, so as to quickly obtain large quantities of mesenchymal stem cells with stable quality.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for isolating and culturing mesenchymal stem cells in vitro, comprising the following steps: (1) Obtaining primary mesenchymal stem cells; (2) inoculating primary mesenchymal stem cells into a preliminary culture medium, and obtaining preliminary cultured mesenchymal stem cells after culture; (3) The initially cultured mesenchymal stem cells are inoculated into an expansion culture medium for expansion culture to obtain in vitro cultured mesenchymal stem cells.
[0007] Preferably, the primary mesenchymal stem cells in step (1) are derived from fat, bone marrow, umbilical cord blood or placenta.
[0008] As a preference, the density of primary mesenchymal stem cells at the time of inoculation in step (2) is 3-6×10 5 pieces / mL.
[0009] Preferably, the preliminary culture medium in step (2) includes a basal culture medium and additives; The additives include platelet lysate accounting for 3-5% of the volume of the basic culture medium, 4-6 g / L of recombinant human albumin, and 10-15 mg / L of recombinant insulin.
[0010] Preferably, the basal culture medium is DMEM culture medium, DMEM-F12 culture medium or 1640 culture medium.
[0011] Preferably, the oxygen content during the culture in step (2) is 4-6%, and the culture time is 60-84 hours.
[0012] Preferably, the density of the mesenchymal stem cells initially cultured during the inoculation in step (3) is 4.0-8.0×10 4 pieces / mL.
[0013] Preferably, the expansion medium in step (3) includes a basal medium and additives; The additives include 8-12 mg / L recombinant human transferrin, 4-6 g / L recombinant human albumin, 0.5-5 mg / L ethanolamine, 2-4 g / L sodium bicarbonate, 1-10 mg / L nicotinamide mononucleotide, and 1.5-2.5 mg / L reduced glutathione.
[0014] Preferably, the basal culture medium is DMEM culture medium, DMEM-F12 culture medium or 1640 culture medium.
[0015] Preferably, the oxygen content during the expansion culture in step (3) is 19-22%, the cells are passaged every 2-3 days, and the density during passage is 4.0-8.0×10 4 pieces / mL.
[0016] The present invention provides a method for isolating and culturing mesenchymal stem cells in vitro, comprising the following steps: (1) obtaining primary mesenchymal stem cells; (2) inoculating the primary mesenchymal stem cells into a preliminary culture medium, and obtaining primary cultured mesenchymal stem cells after culturing; and (3) inoculating the primary cultured mesenchymal stem cells into an expansion culture medium, and performing expansion culture to obtain in vitro cultured mesenchymal stem cells. The present invention uses a serum-free culture medium to isolate and culture mesenchymal stem cells, resulting in a high cell yield and high cell viability. The introduction of multiple additives effectively promotes the culture and proliferation effects, and the control of oxygen content at different stages simulates the in vivo microenvironment to enhance cell activity. Large quantities of mesenchymal stem cells with stable quality can be rapidly obtained. DETAILED DESCRIPTION
[0017] The present invention provides a method for isolating and culturing mesenchymal stem cells in vitro, comprising the following steps: (1) Obtaining primary mesenchymal stem cells; (2) inoculating primary mesenchymal stem cells into a preliminary culture medium, and obtaining preliminary cultured mesenchymal stem cells after culture; (3) The initially cultured mesenchymal stem cells are inoculated into an expansion culture medium for expansion culture to obtain in vitro cultured mesenchymal stem cells.
[0018] In the present invention, the primary mesenchymal stem cells in step (1) are preferably derived from fat, bone marrow, umbilical cord blood or placenta.
[0019] In the present invention, the density of primary mesenchymal stem cells at the time of inoculation in step (2) is preferably 3-6×10 5 cells / mL, more preferably 4-5×10 6 pieces / mL.
[0020] In the present invention, the preliminary culture medium in step (2) preferably includes a basal culture medium and additives; The additive preferably includes 3-5% of the volume of the basal culture medium platelet lysate, 4-6 g / L of recombinant human albumin, and 10-15 mg / L of recombinant insulin, and more preferably includes 4% of the volume of the basal culture medium platelet lysate, 5 g / L of recombinant human albumin, and 13 mg / L of recombinant insulin.
[0021] In the present invention, the basal culture medium is preferably DMEM culture medium, DMEM-F12 culture medium or 1640 culture medium.
[0022] In the present invention, the oxygen content during the culture in step (2) is preferably 4-6%, more preferably 5%, and the culture time is preferably 60-84 h, more preferably 72 h.
[0023] In the present invention, the density of the mesenchymal stem cells initially cultured during the inoculation in step (3) is preferably 4.0-8.0×10 4 cells / mL, more preferably 6.0×10 4 pieces / mL.
[0024] In the present invention, the expansion medium in step (3) preferably includes a basal medium and additives; The additive preferably includes 8-12 mg / L recombinant human transferrin, 4-6 g / L recombinant human albumin, 0.5-5 mg / L ethanolamine, 2-4 g / L sodium bicarbonate, 1-10 mg / L nicotinamide mononucleotide, and 1.5-2.5 mg / L reduced glutathione. More preferably, it includes 10 mg / L recombinant human transferrin, 5 g / L recombinant human albumin, 2.5-3 mg / L ethanolamine, 3 g / L sodium bicarbonate, 4-5 mg / L nicotinamide mononucleotide, and 2 mg / L reduced glutathione.
[0025] In the present invention, the basal culture medium is preferably DMEM culture medium, DMEM-F12 culture medium or 1640 culture medium.
[0026] In the present invention, the oxygen content during the expansion culture in step (3) is preferably 19-22%, more preferably 20-21%, and the cells are preferably passaged every 2-3 days, and the density during passage is preferably 4.0-8.0×10 4 cells / mL, more preferably 6.0×10 4 pieces / mL.
[0027] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Example 1
[0029] A method for isolating and culturing mesenchymal stem cells in vitro comprises the following steps: (1) Using 1640 medium as the base, add platelet lysate (5% of the volume of the basal medium), recombinant human albumin (6 g / L), and recombinant insulin (10 mg / L) to prepare the initial culture medium for later use; To 1640 medium, add 8 mg / L recombinant human transferrin, 6 g / L recombinant human albumin, 0.5 mg / L ethanolamine, 4 g / L sodium bicarbonate, 1 mg / L nicotinamide mononucleotide, and 2.5 mg / L reduced glutathione to prepare expansion medium for later use; (2) Obtaining primary mesenchymal stem cells; (3) The primary mesenchymal stem cells were cultured at a rate of 6×10 5 The cells were inoculated into a preliminary culture medium at a density of 100 cells / mL and cultured at 38°C and 4% oxygen for 84 hours to obtain the primary cultured mesenchymal stem cells. (4) The mesenchymal stem cells were cultured at a rate of 8.0×10 4 The cells were inoculated into the expansion medium at a density of 8.0×10 / mL and cultured at 35°C with an oxygen content of 22% for expansion. The cells were passaged every 3 days.4 The cells were inoculated into a new expansion medium at a density of 100 cells / mL to obtain in vitro cultured mesenchymal stem cells.
[0030] Example 2
[0031] A method for isolating and culturing mesenchymal stem cells in vitro comprises the following steps: (1) Based on DMEM culture medium, add 3% of the volume of basal culture medium of platelet lysate, 4g / L recombinant human albumin, and 15mg / L recombinant insulin to prepare the preliminary culture medium for use; DMEM medium was used as the base, and 12 mg / L recombinant human transferrin, 4 g / L recombinant human albumin, 5 mg / L ethanolamine, 2 g / L sodium bicarbonate, 10 mg / L nicotinamide mononucleotide, and 1.5 mg / L reduced glutathione were added to prepare an expansion medium for later use; (2) Obtaining primary mesenchymal stem cells; (3) The primary mesenchymal stem cells were cultured at a rate of 3×10 5 The cells were inoculated into a preliminary culture medium at a density of 100 cells / mL and cultured at 35°C and 6% oxygen for 60 hours to obtain the primary cultured mesenchymal stem cells. (4) The mesenchymal stem cells were cultured at a rate of 4.0×10 4 The cells were inoculated into the expansion medium at a density of 10 cells / mL and cultured at 38°C with an oxygen content of 19% for expansion. The cells were passaged every 2 days at a density of 4.0×10 4 The cells were inoculated into a new expansion medium at a density of 100 cells / mL to obtain in vitro cultured mesenchymal stem cells.
[0032] Example 3
[0033] A method for isolating and culturing mesenchymal stem cells in vitro comprises the following steps: (1) Using DMEM-F12 medium as the base, add platelet lysate (4% of the volume of the basal medium), 5 g / L recombinant human albumin, and 13 mg / L recombinant insulin to prepare the initial culture medium for later use; DMEM-F12 medium was used as the base, and 10 mg / L recombinant human transferrin, 5 g / L recombinant human albumin, 2 mg / L ethanolamine, 3 g / L sodium bicarbonate, 5 mg / L nicotinamide mononucleotide, and 2 mg / L reduced glutathione were added to prepare an expansion medium for later use; (2) Obtaining primary mesenchymal stem cells; (3) The primary mesenchymal stem cells were cultured at a rate of 5×10 5The cells were inoculated into the preliminary culture medium at a density of 100 cells / mL and cultured at 37°C and 5% oxygen for 72 hours to obtain the primary cultured mesenchymal stem cells. (4) The mesenchymal stem cells were cultured at a rate of 6.0×10 4 The cells were inoculated into the expansion medium at a density of 100 / mL and cultured at 37°C with an oxygen content of 21% for expansion. The cells were passaged every 3 days at a density of 6.0×10 4 The cells were inoculated into a new expansion medium at a density of 100 cells / mL to obtain in vitro cultured mesenchymal stem cells.
[0034] Comparative Example 1
[0035] In steps (3) and (4), the primary culture medium was used for cell culture, and the remaining steps and conditions were the same as those in Example 3.
[0036] Comparative Example 2
[0037] The oxygen content was not controlled in steps (3) and (4), and the remaining steps and conditions were the same as those in Example 3.
[0038] Test example
[0039] 1. Obtaining primary mesenchymal stem cells The adipose tissue was washed with physiological saline to remove fascia and blood vessels, then minced and digested with an equal volume of 0.1% collagenase solution at 37°C for 30 min. The cells were then centrifuged at 280g for 5 min, resuspended in DMEM-F12 medium containing 5% serum replacement (Thermo Fisher Scientific), passed through a 40μM cell sieve, and centrifuged and resuspended once more to obtain isolated cells. The separated cells were cultured at 37°C and 5% CO2. After the cells adhered to the wall and grew for 48 hours, the medium was changed to remove the non-adherent cells. Thereafter, half the volume of the medium was changed every 3 days. When the cells reached 80% confluence, they were digested with 0.25% trypsin and passaged at a ratio of 1:4. The second generation cells were used for subsequent culture.
[0040] 2. Cell Expansion The primary mesenchymal stem cells obtained above were cultured in vitro using the methods of Examples 1 to 3 and Comparative Examples 1 to 2, respectively. Each experimental group was passaged twice, the cell number after each passage was detected, and the proliferation multiples of each passage were calculated. The results are shown in Table 1.
[0041] Table 1 Group First passage proliferation multiples First passage proliferation multiples Experimental group 1 9.57 9.14 Experimental Group 2 9.06 9.55 Experimental Group 3 10.12 9.74 Control group 1 4.01 3.38 Control group 2 6.77 5.32 As can be seen from the above examples, the present invention provides a method for isolating and culturing mesenchymal stem cells in vitro, comprising the following steps: (1) obtaining primary mesenchymal stem cells; (2) inoculating the primary mesenchymal stem cells into a preliminary culture medium, and obtaining primary cultured mesenchymal stem cells after culturing; (3) inoculating the primary cultured mesenchymal stem cells into an expansion culture medium, and performing expansion culture to obtain in vitro cultured mesenchymal stem cells. The present invention uses serum-free culture medium to isolate and culture mesenchymal stem cells, resulting in a high cell yield and high cell viability. The introduction of multiple additives effectively promotes the culture and proliferation effects, and the control of oxygen content at different stages simulates the in vivo microenvironment to enhance cell activity. Large quantities of mesenchymal stem cells with stable quality can be rapidly obtained.
[0042] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A method for isolating and culturing mesenchymal stem cells in vitro, characterized in that: The steps include: (1) Obtaining primary mesenchymal stem cells; (2) inoculating primary mesenchymal stem cells into a preliminary culture medium, and obtaining preliminary cultured mesenchymal stem cells after culture; (3) The initially cultured mesenchymal stem cells are inoculated into an expansion culture medium for expansion culture to obtain in vitro cultured mesenchymal stem cells.
2. The method according to claim 1, characterized in that The primary mesenchymal stem cells in step (1) are derived from fat, bone marrow, umbilical cord blood or placenta.
3. The method according to claim 2, characterized in that The density of primary mesenchymal stem cells at the time of inoculation in step (2) is 3~6×10 5 pieces / mL.
4. The method according to claim 3, characterized in that The preliminary culture medium in step (2) includes a basal culture medium and additives; The additives include platelet lysate accounting for 3-5% of the volume of the basic culture medium, 4-6 g / L of recombinant human albumin, and 10-15 mg / L of recombinant insulin.
5. The method according to claim 4, characterized in that The basic culture medium is DMEM culture medium, DMEM-F12 culture medium or 1640 culture medium.
6. The method according to claim 1, characterized in that The oxygen content during the culture in step (2) is 4-6%, and the culture time is 60-84 hours.
7. The method according to claim 6, characterized in that The density of the mesenchymal stem cells initially cultured during the inoculation step (3) is 4.0-8.0×10 4 pieces / mL.
8. The method according to claim 7, characterized in that The expansion medium in step (3) includes a basal medium and additives; The additives include 8-12 mg / L recombinant human transferrin, 4-6 g / L recombinant human albumin, 0.5-5 mg / L ethanolamine, 2-4 g / L sodium bicarbonate, 1-10 mg / L nicotinamide mononucleotide, and 1.5-2.5 mg / L reduced glutathione.
9. The method according to claim 8, characterized in that The basic culture medium is DMEM culture medium, DMEM-F12 culture medium or 1640 culture medium.
10. The method according to claim 1, characterized in that The oxygen content during the expansion culture in step (3) is 19-22%, and the cells are passaged every 2-3 days, with a density of 4.0-8.0×10 4 pieces / mL.