CN117660325B - Culture medium for preparing umbilical cord blood MSC and method thereof - Google Patents
Culture medium for preparing umbilical cord blood MSC and method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a culture medium for preparing umbilical cord blood MSC and a method thereof. The invention provides a serum-free culture medium, which comprises MEM-alpha basal medium, component A and component B; the component A consists of the following components: elaoretin and thrombin-sensitive proteins; the component B consists of human platelet lysate. The umbilical cord blood mesenchymal stem cells cultured by the serum-free culture medium have the advantages of quick adherence, multiple cell numbers, stable phenotype and aging resistance after multiple passages, and overcome the defects of the traditional method for culturing cells by using fetal bovine serum.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a culture medium for preparing umbilical cord blood MSC and a method thereof.
Background
Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) have the potential to self-renew and differentiate in multiple directions, and are an attractive source of stem cells in tissue engineering. Although adult Bone Marrow (BM) and Adipose Tissue (AT) are the main sources of clinical use, their use is limited due to invasive procedures required for harvesting and stringent requirements on donor age, and may be clinically ineffective for mesenchymal stem cells from elderly patients. Thus, researchers began to seek alternative sources in raw or neonatal tissues, including placenta, umbilical cord, and amniotic membrane. However, these tissues require complex cell separation processes. Compared with mesenchymal stem cells of other sources, umbilical cord blood-derived mesenchymal stem cells have a number of advantages: the content is rich, the cells are more original, the sources are not invasive (from umbilical cord discarded after delivery of puerpera), the damage to the donor is avoided, the immunogenicity is avoided, the rejection reaction is avoided, the patient is easy to accept, the social ethical problem is avoided, and the like.
However, stem cell culture systems mainly use culture media containing animal serum (e.g., fetal bovine serum), however, bovine serum, human serum, or other animal serum may contain blood-borne pathogens, bovine serum may also elicit the production of antibodies against heterologous biomass proteins that may elicit immune responses in recipient patients, and bovine serum may also exhibit batch-to-batch differences, resulting in inconsistent performance. Some umbilical cord mesenchymal stem cells are also selected to be free from serum in the culture process, but the culture effect is not ideal, and the technical defects of low cell proliferation speed, low expression, limited cell number obtained for proliferation and the like exist.
Secondly, there is also a problem of cell senescence after expansion of umbilical cord blood mesenchymal stem cells in a wide range of cell culture, which is manifested by growth arrest and loss of differentiation potential of the mesenchymal stem cells; during the cell expansion process, cells enter senescence along with passage, so that the efficacy of the cells is gradually reduced, and the protein expression is obviously changed. However, in vitro mesenchymal stem cell expansion requires the generation of a sufficient number of pure cell populations to meet clinical needs. For therapeutic purposes, large scale expansion and anti-aging strategies are an urgent issue to be addressed.
With the increasing number and variety of clinical trials of adult cells for regenerative therapy, in vitro mesenchymal stem cell expansion requires the generation of a sufficient number of pure cell populations to meet clinical needs. Therefore, development of a preparation method of umbilical cord blood mesenchymal stem cells is needed, which can avoid animal serum to excite immune response and pathogen transmission, and can delay cell aging so as to expand clinical application requirements.
Disclosure of Invention
In order to solve the problems, the invention provides a culture method which is beneficial to promoting cell adhesion, keeping self-renewal and proliferation capacity of cells, delaying cell aging and ensuring cell activity, yield and long-term expansion.
In one aspect, the invention provides a serum-free medium comprising MEM-alpha basal medium, component a and component B; the component A consists of the following components: elaoretin and thrombin-sensitive proteins; the component B consists of human platelet lysate.
Specifically, the volume ratio of the elaoretin to the basal medium is 0.1-5%; the thrombin sensitive protein accounts for 0.1-5% of the volume of the basal culture medium; the platelet lysate accounts for 5-20% of the volume of the basal medium.
In some embodiments, the volume ratio of the elaorexin to the basal medium is 0.1%; the thrombin sensitive protein accounts for 0.1% of the volume of the basal medium; the platelet lysate accounts for 20% of the volume of the basal medium.
In other embodiments, the volume fraction of the base medium of the elaorexin is 0.1%; the thrombin sensitive protein accounts for 5% of the volume of the basal medium; the platelet lysate accounts for 5% of the volume of the basal medium.
In still other embodiments, the volume fraction of the base medium is 5% of the volume fraction of the elaireotide; the thrombin sensitive protein accounts for 0.1% of the volume of the basal medium; the platelet lysate accounts for 5% of the volume of the basal medium.
Specifically, the preparation method of the serum-free culture medium comprises the following steps:
S1, mixing a component A with MEM-alpha basal medium;
s2, adding the component B into the S1 when in use.
In yet another aspect, the invention provides the use of the serum-free medium described above for the preparation of mesenchymal stem cells.
In particular, the mesenchymal stem cells are isolated from one or more of bone marrow, fat, umbilical cord blood or placenta.
Preferably, the mesenchymal stem cells are umbilical cord blood mesenchymal stem cells.
In yet another aspect, the present invention provides a method for preparing mesenchymal stem cells, comprising the steps of:
(1) Isolation of mesenchymal stem cells: diluting umbilical cord blood, settling red blood cells, and sucking a white membrane layer;
(2) Performing inoculation culture and liquid exchange culture on the mesenchymal stem cells in the step (1);
(3) Performing subculture on the mesenchymal stem cells in the step (2) by using the serum-free culture medium;
(4) Collecting cells: discarding the culture medium, washing, enzyme digestion, stopping digestion and collecting.
Specifically, the dilution solution used in the dilution in the step (1) is PBS buffer solution.
Preferably, the dilution ratio is 1:1.
Specifically, the culture medium of the inoculation culture and the liquid change culture in the step (2) is the serum-free culture medium.
Specifically, the culture conditions of the inoculation culture and the liquid change culture in the step (2) are as follows: 4% -6% CO 2 and 35-40 ℃.
Further specifically, the 4% -6% CO 2 may be any other value from 4%, 5%, 6% or 4% -6%; the 35-40deg.C may be 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C or any other value of 35-40deg.C.
Preferably, the culture conditions of the inoculation culture and the liquid change culture in the step (2) are as follows: 5% CO 2, 37 ℃.
Specifically, the liquid-changing culture in the step (2) is that the liquid is changed for the second half of 4-6 days, and then the liquid is changed once every 2-3 days.
Preferably, the liquid change culture in the step (2) is a half liquid change after 5 days, and then the liquid change is performed every 2 days.
Specifically, the subculture conditions in the step (3) are as follows: 4% -6% CO 2 and 35-40 ℃.
Further specifically, the 4% -6% CO 2 may be any other value from 4%, 5%, 6% or 4% -6%; the 35-40deg.C may be 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C or any other value of 35-40deg.C.
Preferably, the culture conditions of the subculture in step (3) are: 5% CO 2, 37 ℃.
Specifically, the subculture ratio of the subculture in the step (3) is 1:2-3.
Preferably, the subculture in step (3) is performed at a passaging ratio of 1:3.
Specifically, the washing solution in the step (4) is PBS buffer solution.
Further specifically, the number of times of washing is 1 to 2, preferably 1.
Specifically, the enzyme in the step (4) is trypsin; preferably 0.25% trypsin.
Specifically, the termination described in step (4) is digested into the addition of serum-free medium.
Preferably, the terminating digestion described in step (4) is the addition of an equal amount of serum-free medium.
In particular, the mesenchymal stem cells include, but are not limited to: isolated from one or more of bone marrow, fat, umbilical cord, cord blood, or placenta.
Preferably, the mesenchymal stem cells are umbilical cord blood mesenchymal stem cells.
In yet another aspect, the present invention provides the use of the aforementioned preparation method for preparing mesenchymal stem cells.
The invention has the technical effects that:
(1) The mesenchymal stem cells of the umbilical cord blood are adhered to the wall quickly, and can keep cell proliferation for a long time, and the number of the cells is large.
(2) The umbilical cord blood mesenchymal stem cells are phenotypically stable.
(3) The umbilical cord blood mesenchymal stem cells can still keep the aging-free phenomenon after multiple passages.
(4) PRP is from human, and infusion into human body will not cause foreign protein immune reaction, thus solving the defect of traditional method for culturing cells by using fetal bovine serum.
Drawings
FIG. 1 is a graph showing the results of an aging test of beta-galactosidase.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Terminology
Elaoretin (ELAMIPRETIDE TFA) (MTP-131) is a cardiolipin peroxidase inhibitor and a mitochondrially targeted peptide that reduces mitochondrial dysfunction and oxidative damage in human trabecular meshwork cells.
Thrombin-sensitive protein (thrombospondins, TSP) is an extracellular matrix protein that can mediate a variety of biological processes, as an extracellular structural substance, that can transmit a series of signals, mediating cell-to-cell and cell-to-cell matrix interactions.
Example 1
1.1 Preparation of serum-free Medium
MEM-alpha basal medium (GIBCO, 12561-056), elaorexin (AbMole, MTP-131), thrombin sensitive protein (Sigma), human platelet lysate (PALL, 15950-017).
The culture medium consists of MEM-alpha basal medium and an additive, wherein the additive comprises a component A and a component B, and the volume ratio of the component A to the basal medium is as follows: the preparation method comprises the steps of (1) uniformly mixing an A component with a basic culture medium, wherein the A component is 0.1% of the elaireotide and 0.1% of thrombin-sensitive protein; when in use, component B is added, wherein the component B is human platelet lysate (PRP) and accounts for 20% of the volume of the basal medium.
1.2 Preparation of umbilical cord blood mesenchymal Stem cells
1.2.1 Umbilical cord blood sample collection
Selecting healthy pregnant women naturally produced in term of obstetrics and gynecology department in a hospital, requesting agreement of the pregnant women and family members thereof, obtaining umbilical cord blood 50 mL by aseptic operation, gently shaking a blood collection tube (heparin anticoagulation) to prevent blood coagulation, collecting, placing in a transport case, delivering to a GMP laboratory within 2 hours, preserving in a refrigerator at 4 ℃ and carrying out separation culture within 12 hours.
1.2.2 Isolation and culture of Human Umbilical Cord Blood Mesenchymal Stem Cells (HUCBMSCs)
(1) Dilution of
The mixture was diluted 1:1 with PBS buffer.
(2) Sedimentation red blood cells
Adding hydroxyethyl starch according to a ratio of 1:5, standing at room temperature for 30min, and settling red blood cells; sucking the supernatant, and slowly adding the supernatant onto a percoll separating liquid (GE, 17089102) according to a volume of 1:1; centrifuging at 2500 r/min for 10 min.
(3) Suction is carried out
Sucking the cloudy milky white cell layer (white membrane layer) and transferring into a new sterile centrifuge tube; centrifugation and washing, washing the cells 2 times with PBS, and centrifuging at 1200r/min for 5 minutes.
(4) Inoculating culture
Cell pellet was resuspended in serum-free medium, cell density adjusted to approximately 1X 10 6 cells per ml, inoculated into T175 flasks and incubated in 5% CO 2 at 37 ℃.
(5) Liquid-changing culture
Half the liquid change (serum-free medium) was performed after 5 days, after which the liquid change was performed every 2 days.
(6) Passage of
When the cells grew close to confluence (80-90% long), adherent cells were gently digested with 0.25% trypsin-0.02% EDTA for 2-5 min, stopped, transferred to a new sterile centrifuge tube and centrifuged at 1200 r/min for 5 min.
Subculturing is carried out according to the ratio of 1:3, and morphological characteristics of HUCB-MSCs are observed under a daily mirror.
1.2.3 Passage
When the strain is transferred to the generation P3, inoculating the strain into a T175 culture flask according to the density of 5000-10000cells/cm 2, and adding 30mL of prepared serum-free culture medium; the flask was placed horizontally in a 37℃and 5% CO 2 incubator for cultivation.
1.2.4 Collection of P5, P10, P15 cells
(1) Discarding the culture medium when the cells grow to 70-90% and washing the cell surface 1 time with 10mL PBS buffer solution;
(2) Discarding PBS, adding 6mL trypsin for digestion, gently shaking the culture flask to enable the trypsin to cover the bottom of the flask, rapidly observing under a mirror, and gently beating the outer side of the culture flask to enable the cells to float when the cells shrink into spheres;
(3) Adding an equal amount of serum-free medium, and stopping digestion;
(4) Collecting cell suspension, 1200rpm, 6L, centrifuging for 5min;
(5) Discarding the supernatant, adding 10mL of PBS to resuspend the cells, taking care of gently blowing, avoiding generating bubbles, and centrifuging;
(6) After the last centrifugation, the supernatant was discarded and the cells were resuspended in 10mL of PBS.
1.3 Detection
1.3.1 Cell count
0.1ML of cell suspensions of P5, P10 and P15 cells were taken and counted by a blood cell counting plate.
1.3.2 Cell phenotype flow identification
P5, P10 and P15 cells are collected, the cell concentration is adjusted to be 1X 10 7/mL, antibodies CD73, CD90, CD105, CD34 and CD45 are added, the cells combined with IgG1 are incubated for 30min at room temperature and in a dark place, PBS is used as isotype control, the cells are washed for 2 times after incubation, unbound antibodies are washed away, and a flow cytometer is used for detecting the expression of cell surface antigens.
1.3.3 Beta-galactosidase aging detection
Culturing P5, P10 and P15 generation umbilical cord blood mesenchymal stem cells until the cell fusion degree reaches 80%, digesting with pancreatin, inoculating the umbilical cord blood mesenchymal stem cells into a 24-well plate at a density of 1X 10 4/cm2, culturing 5X d, detecting cell aging conditions by using a beta-galactosidase staining kit, sucking cell culture solution in the 24-well plate, washing for 1 time by using PBS, adding 250 mu L of beta-galactosidase staining fixative solution, fixing 15 and min at room temperature, sucking cell fixative solution, washing for 3 times by using PBS, adding 250 mu L of staining working solution into each well, incubating overnight at 37 ℃, removing the working solution, washing three times by using PBS, placing the 24-well plate under a common optical microscope, randomly selecting 6 fields, and observing and recording experimental results. Quantitative statistics were performed on the stained sections using software Image J.
1.3.4 Total antioxidant Capacity T-AOC detection
P5, P10 and P15 cells were taken and tested for T-AOC (Nanjing established biosciences).
The activity level of the T-AOC can be measured to directly reflect the activity of antioxidant enzyme and the functional state of an antioxidant system of the organism, and the T-AOC can be used for reflecting the overall oxidative stress level in the organism; the lipid peroxidation damage degree of the organism can be indirectly reflected, and the level of the lipid peroxidation damage degree is positively correlated with the antioxidant capacity of cells and negatively correlated with the lipid peroxidation of the organism.
The T-AOC level was determined using Fe 3＋/Fe2＋ chemistry, with the specific procedure being exactly as that of the kit (Nanjing's established Biotechnology Co., ltd.).
The experimental methods are shown in table 1:
TABLE 1
Unit definition and calculation formula
(1) Definition: the absorbance (OD) value of the reaction system was increased by 0.01 per ml of supernatant at 37 ℃ in one total antioxidant capacity unit.
(2) The calculation formula is as follows:
total antioxidant capacity = (measured OD value-control OD value)/0.01/30× (total reaction solution/sample amount) x dilution of sample before test (unit/ml supernatant).
Example 2
The serum-free medium had the following composition:
the culture medium consists of MEM-alpha basal medium and an additive, wherein the additive comprises a component A and a component B, and the volume ratio of the component A to the basal medium is as follows: the method comprises the steps of (1) uniformly mixing an A component with a basic culture medium, wherein the A component is 0.1% of the elaireotide and 5% of thrombin-sensitive protein; when in use, component B is added, wherein the component B is human platelet lysate (PRP) and accounts for 5% of the volume of the basal medium.
Example 3
The serum-free medium had the following composition:
The culture medium consists of MEM-alpha basal medium and an additive, wherein the additive comprises a component A and a component B, and the volume ratio of the component A to the basal medium is as follows: 5% of elaoretin and 0.1% of thrombin-sensitive protein, and uniformly mixing the component A with a basic culture medium; when in use, component B is added, wherein the component B is human platelet lysate (PRP) and accounts for 5% of the volume of the basal medium.
Comparative example 1
The serum-free medium of comparative example 1 was free of thrombin-sensitive protein, and the same as in example 1.
Comparative example 2
The serum-free medium of comparative example 2 was free of elaorexin, and the same as in example 1 was repeated.
Comparative example 3
The serum-free medium of comparative example 3 was free of thrombin-sensitive protein and elaireotide, as in example 1.
Effect data:
(1) Observing morphological results of hUCB-MSCs under a lens
The cells of examples 1-3 attached significantly more than the cells of comparative examples 1-3 on day 3.
(2) Cell phenotype flow identification structure
The results are shown in Table 2:
TABLE 2 phenotypic flow results of cells
As can be seen from Table 2, the technical scheme of the invention has no influence on the immunophenotype of the mesenchymal stem cells of the umbilical cord blood, each group of cell surface antigens CD73, CD90 and CD105 are positively expressed, and CD34 and CD45 are negatively expressed, wherein the positive rates of the cells CD73, CD90 and CD105 obtained by culturing in the examples 1-3 are above 95%, the positive rates of the CD45 and CD34 are lower than 1.4%, the characteristics of the human mesenchymal stem cells are met, and the method is superior to those of the comparative examples 1-3. The technical scheme of the invention is proved to not change the 'stem property' of the cells and accord with the identification standard of mesenchymal stem cells. The serum-free culture medium provided by the invention is suitable for culturing human umbilical cord blood mesenchymal stem cells. Meanwhile, the composition of the culture medium has a certain influence on the performance of the culture medium.
(3) Beta-galactosidase aging detection result
Examples 1-3 umbilical cord blood mesenchymal stem cell senescent cell count was significantly less than comparative examples 1-3. Quantitative analysis found a significant difference in staining ratios for examples 1-3 and comparative examples 1-3 (FIG. 1). In conclusion, the technical scheme of the invention can delay the aging of the mesenchymal stem cells.
(4) Total antioxidant capacity T-AOC detection
The detection results are shown in Table 3:
TABLE 3T-AOC results
As can be seen from Table 3, the MSCs prepared in examples 1-3 have significantly higher total antioxidant capacity than those of comparative examples 1-3, indicating that the media formulations of the present protocol enhance cell antioxidant capacity. The serum-free culture medium provided by the invention has the characteristic of obviously enhancing the antioxidant capacity of umbilical cord blood mesenchymal stem cells. In conclusion, the total antioxidant capacity of the MSC prepared by the invention is obviously improved.
Claims (9)
1. A serum-free medium, wherein the serum-free medium comprises MEM-alpha basal medium, component a and component B; the component A consists of the following components: elaoretin and thrombin-sensitive proteins; the component B consists of human platelet lysate; the volume ratio of the elaireotide to the basal medium is 0.1-5%; the thrombin sensitive protein accounts for 0.1-5% of the volume of the basal culture medium; the platelet lysate accounts for 5-20% of the volume of the basal medium.
2. The serum-free medium according to claim 1, wherein the preparation method of the serum-free medium comprises the following steps:
S1, mixing a component A with MEM-alpha basal medium;
s2, adding the component B into the S1 when in use.
3. Use of a serum-free medium according to any one of claims 1-2 for the preparation of mesenchymal stem cells.
4. The use of claim 3, wherein said mesenchymal stem cells comprise one or more isolated from bone marrow, fat, umbilical cord blood or placenta.
5. A method for preparing mesenchymal stem cells, comprising the steps of:
(1) Isolation of mesenchymal stem cells: diluting umbilical cord blood, settling red blood cells, and sucking a white membrane layer;
(2) Performing inoculation culture and liquid exchange culture on the mesenchymal stem cells in the step (1);
(3) Subculturing the mesenchymal stem cells in step (2) with the serum-free medium of any one of claims 1 to 2;
(4) Collecting cells: discarding the culture medium, washing, enzyme digestion, stopping digestion and collecting.
6. The method according to claim 5, wherein the medium for the inoculation and liquid change culture in the step (2) is the serum-free medium according to any one of claims 1 to 2.
7. The method according to claim 5, wherein the conditions of the inoculation and liquid change culture of step (2) and the subculture of step (3) are as follows: 4% -6% CO 2 and 35-40 ℃.
8. The method according to claim 7, wherein the conditions of the inoculation and liquid change culture of step (2) and the subculture of step (3) are: 5% CO 2, 37 ℃.
9. The method according to claim 5, wherein the subculture in step (3) is carried out at a passaging ratio of 1:2-3.
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| CN104781394A (en) * | 2012-09-03 | 2015-07-15 | Medipost株式会社 | Method for culturing mesenchymal stem cells |
| CN109952112A (en) * | 2016-09-21 | 2019-06-28 | 阿帕特夫研究和发展有限公司 | CD123 binding protein and relevant composition and method |
| CN110337490A (en) * | 2017-01-11 | 2019-10-15 | 脊核细胞有限责任公司 | Enhance the method for fibroblast therapeutic activity |
| CN113728094A (en) * | 2019-05-02 | 2021-11-30 | Scm生命科学有限公司 | Cosmetic composition comprising mesenchymal stem cell culture fluid cultured in medium containing human platelet lysate |
| CN114085810A (en) * | 2020-06-30 | 2022-02-25 | 株式会社未来细胞生物 | Preparation method of similar mesenchymal stem cells and similar mesenchymal stem cells prepared by same |
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| CN104781394A (en) * | 2012-09-03 | 2015-07-15 | Medipost株式会社 | Method for culturing mesenchymal stem cells |
| CN109952112A (en) * | 2016-09-21 | 2019-06-28 | 阿帕特夫研究和发展有限公司 | CD123 binding protein and relevant composition and method |
| CN110337490A (en) * | 2017-01-11 | 2019-10-15 | 脊核细胞有限责任公司 | Enhance the method for fibroblast therapeutic activity |
| CN113728094A (en) * | 2019-05-02 | 2021-11-30 | Scm生命科学有限公司 | Cosmetic composition comprising mesenchymal stem cell culture fluid cultured in medium containing human platelet lysate |
| CN114085810A (en) * | 2020-06-30 | 2022-02-25 | 株式会社未来细胞生物 | Preparation method of similar mesenchymal stem cells and similar mesenchymal stem cells prepared by same |
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