CN113862221B - Culture solution and culture method of peripheral blood mononuclear cells - Google Patents

Culture solution and culture method of peripheral blood mononuclear cells Download PDF

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CN113862221B
CN113862221B CN202111257689.2A CN202111257689A CN113862221B CN 113862221 B CN113862221 B CN 113862221B CN 202111257689 A CN202111257689 A CN 202111257689A CN 113862221 B CN113862221 B CN 113862221B
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cells
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CN113862221A (en
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高歌
周安宇
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Ixcell Biotechnology Co ltd
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Abstract

The invention discloses a Peripheral Blood Mononuclear Cell (PBMC) culture solution and a culture method. The culture solution of the invention comprises: the volume ratio is 1:0.9-1.3 DMEM/F12 medium and blood cell culture medium StemPro-34, as well as cell growth factors, L-ascorbic acid, beta-mercaptoethanol, sodium nitrite, ethanolamine, ROCK inhibitors, human serum albumin, transferrin, heparin sodium, AHR inhibitors, MEK inhibitors and GSK-3 inhibitors. The culture solution and the culture method disclosed by the invention greatly improve the efficiency of in-vitro amplification culture of PBMC cells, shorten the culture time, simultaneously maintain the population characteristics of the PBMC cells, and are more beneficial to inducing reprogramming into pluripotent stem cells.

Description

Culture solution and culture method of peripheral blood mononuclear cells
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a PBMC (PBMC) culture solution and a culture method.
Background
Induced pluripotent stem cells (Induced Pluripotent STEM CELLS, IPSCS) are cells that function similarly to embryonic stem cells obtained using reprogramming techniques. The method has the potential of differentiating into all cell types including three germ layers, is a great breakthrough in the field of the current research on stem cells and tissue regeneration, solves the difficult problems of limited sources and ethical barriers of embryonic stem cells, and has wide application prospect in the medical field. At present, human iPSC cells are mainly reprogrammed from skin fibroblasts, and sampling from the skin is subject to long tissue biopsy time, inconvenient material taking, strong pain of a patient, risk of bacterial infection and the like, and the obtained cells need to be expanded in vitro to enough fibroblasts to induce iPSC, so that the time consumption is long. In 2010, scientists induced iPSC from peripheral blood cells, and the appearance of the technology brought a qualitative leap to the research field of iPSC. Compared with skin fibroblasts, the peripheral blood is convenient to obtain and has rich sources, is an ideal iPSC donor cell, and has wide application prospect.
Peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMC), which refer to cells with a single nucleus in peripheral blood, include lymphocytes, monocytes, dendritic cells and other small numbers of progenitor cells (e.g., hematopoietic stem cells, etc.). The most commonly used method for separating peripheral blood mononuclear cells is Ficoll density gradient centrifugation, and the method has the advantages of good cell purity and small red blood cell proportion. Mononuclear cells separated from peripheral blood are generally cultured in vitro by using FBS or components (such as BSA) containing animal sources, and the PBMC culture solution has poor amplification culture efficiency, low cell activity and adverse clinical application.
Therefore, to allow the reprogramming of PBMCs into human induced pluripotent stem cells to be widely used, it is necessary to develop a serum-free, animal-derived component-free PBMC culture solution that can rapidly and efficiently amplify PBMCs while having high reprogramming efficiency.
Disclosure of Invention
The invention provides a Peripheral Blood Mononuclear Cell (PBMC) culture solution and a culture method, which can greatly improve the PBMC cell culture and stem cell reprogramming induction efficiency while well maintaining the population characteristics of PBMC cells.
In one aspect, the invention provides a Peripheral Blood Mononuclear Cell (PBMC) culture broth comprising: basal medium DMEM/F12 and blood cell culture medium StemPro-34, cell growth factor, L-ascorbic acid, beta-mercaptoethanol, sodium nitrite, ethanolamine, ROCK inhibitor, human serum albumin, transferrin, heparin sodium and AHR inhibitor.
In some embodiments, the ROCK inhibitor is Thiazovivin, Y-27632 or KD-025; more preferably, the concentration of the ROCK inhibitor is 1-10 mu M/mL, and more preferably 5 mu M/mL.
In some embodiments, the AHR inhibitor is SR1, PD98059, CH-223191, AHR antagolist 2, AHR antagolist 4, GNF351, or PDM2; more preferably, the AHR inhibitor concentration is 0.5-5. Mu.M/mL, more preferably 2. Mu.M/mL.
In some embodiments, the culture broth further comprises a deacetylase inhibitor.
In some embodiments, the deacetylase inhibitor is VPA, naBu, TSA or SAHA; more preferably, the concentration of the deacetylase inhibitor is 0.5-5. Mu.M/mL, more preferably 2. Mu.M/mL.
In some embodiments, the culture broth further comprises a GSK-3 inhibitor.
In some embodiments, the GSK-3 inhibitor is CHIR99021, SB 216763, TWS119, LY2090314, tideglusib, or a small molecule compound BIO; more preferably, the GSK-3 inhibitor concentration is 0.1-1. Mu.M/mL, more preferably 0.5. Mu.M/mL.
In some embodiments, the cell growth factors included in the culture broth include those including SCF, IGF-1, FLT-3L, TPO, EPO, and GM-CSF.
In some embodiments, the concentration of growth factor SCF in the culture broth is 10-100ng/mL, preferably 10-50ng/mL, more preferably 25ng/mL.
In some embodiments, the concentration of growth factor IGF-1 in the culture broth is between 10 and 100ng/mL, preferably between 10 and 50ng/mL, and more preferably 25ng/mL.
In some embodiments, the concentration of growth factor FLT-3L in the culture broth is 10-100ng/mL, preferably 10-50ng/mL, more preferably 20ng/mL.
In some embodiments, the concentration of growth factor TPO in the culture broth is 10-100ng/mL, preferably 10-50ng/mL, more preferably 25ng/mL.
In some embodiments, the concentration of growth factor EPO in the culture broth is 10-100ng/mL, preferably 10-50ng/mL, more preferably 30ng/mL.
In some embodiments, the concentration of the growth factor GM-CSF in the culture broth is between 10 and 100ng/mL, preferably between 10 and 50ng/mL, and more preferably 25ng/mL.
In some embodiments, the L-ascorbic acid concentration in the culture broth is 100. Mu.M-1 mM/mL, preferably 500. Mu.M/mL.
In some embodiments, the concentration of beta-mercaptoethanol in the culture broth is 1-10. Mu.M/mL, preferably 5. Mu.M/mL.
In some embodiments, the concentration of sodium nitrite in the culture broth is 1-10. Mu.M/mL, preferably 5. Mu.M/mL.
In some embodiments, the concentration of ethanolamine in the culture broth is 1-10. Mu.M/mL, preferably 5. Mu.M/mL.
In some embodiments, the concentration of human serum albumin in the culture broth is 5-50g/L, preferably 10g/L.
In some embodiments, the concentration of transferrin in the culture broth is 50-500 μg/mL, preferably 75 μg/mL.
In some embodiments, the heparin sodium concentration in the culture broth is 50-500. Mu.g/mL, preferably 250. Mu.g/mL.
In some embodiments, the volume ratio of DMEM/F12 medium to blood cell medium StemPro-34 in the culture broth is 1:0.9-1.3.
In another aspect, the invention provides a method of culturing Peripheral Blood Mononuclear Cells (PBMCs), the method comprising culturing peripheral blood mononuclear cells in a culture broth as described above.
In some embodiments, the method comprises the steps of:
Step 1) Peripheral Blood Mononuclear Cells (PBMC) are inoculated into a cell culture container according to the ratio of 0.005-0.05X10 6 cells/hole; culturing cells inoculated in a cell culture vessel with the PBMC culture solution of any one of claims 1-8 at 35-39 ℃ for 3-5 days with 3-7% CO 2; changing culture solution every 1-3 days during culture;
Step 2) when the cell density is 80% -100%, transferring the cells to a new cell culture container, wherein the transfer ratio is 1:3-1:10; the culture of the peripheral blood mononuclear cell culture broth according to any of claims 1-8 is continued for 2-5 days, preferably 3 days, at 35-39 ℃, preferably 37 ℃, 3-7% co 2.
In some embodiments, the seeding cell density in step 1) is 0.015×10 6 cells/well.
In some embodiments, the temperature of the culturing in step 1) is 37 ℃.
In some embodiments, the PBMCs are cultured in step 1) under 5% co 2 conditions.
In some embodiments, the PBMC cells are cultured for 4 days in step 1).
In some embodiments, the culture medium is replaced every 2 days in step 1).
In some embodiments, the ratio of cell passages in step 2) is 1:5.
In some embodiments, the temperature of the culturing in step 2) is 37 ℃.
In some embodiments, the PBMCs are cultured in step 2) under 5% co 2 conditions.
In some embodiments, the PBMC cells are cultured for 3 days in step 2).
In some embodiments, the cell culture vessel may be a multi-well plate.
Another aspect of the invention provides the use of the aforementioned PBMC culture fluid in the culture of peripheral blood mononuclear cells.
In some embodiments, the culturing is to expand peripheral blood mononuclear cells, i.e., to promote proliferation and increase the number of peripheral blood mononuclear cells.
In some embodiments, the cultured peripheral blood mononuclear cells of the invention are used to reprogram into induced pluripotent stem cells (ipscs).
The term "Peripheral Blood Mononuclear Cells (PBMC)" as used herein refers to mononuclear cells isolated from peripheral blood, mainly including lymphocytes, but also including a few monocytes, plasma cells, hematopoietic stem cells, other progenitor cells, etc., and can be obtained by isolating frozen or freshly collected blood samples by Ficoll-Paque density gradient centrifugation.
The "basal medium DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)" described herein is a 1:1 mixture of DMEM and Ham's F-12, a widely used basal medium that can be used to support a variety of mammalian cell growth and can be purchased from a variety of suppliers (e.g., thermo FISHER SCIENTIFIC).
The "blood cell culture medium StemPro-34" described herein is a serum-free medium that can be obtained from a variety of suppliers (e.g., gibco,-34 SFM) purchased.
As used herein, "cell growth factor" refers to a cytokine that promotes cell survival, growth and/or expansion in vitro, including but not limited to SCF, IGF-1, FLT-3L, TPO, EPO, and GM-CSF. Where SCF is Stem Cell Factor, IGF-1 is insulin-like growth Factor-1 (Insulin Growth Factor-1), FLT-3L is Fms-related tyrosine kinase 3ligand (Fms-related tyrosine kinase 3 ligand), TPO is thrombopoietin (Thrombopoietin, also called platelet growth Factor), EPO is erythropoietin (Erythropoietin, also called erythrocyte growth Factor), and GM-CSF is granulocyte-macrophage colony stimulating Factor (Granulocyte-Macrophage Colony-Stimulating Factor). One or more of the cell growth factors may be of human origin and/or recombinantly produced. The cell growth factor is commercially available from a variety of suppliers.
As used herein, "ROCK inhibitor" refers to inhibitors of Rho-associated protein kinase (Rho-associated protein kinase, ROCK), including, but not limited to, thiazovivin, Y-27632 and KD-025 (also known as SLx-2119). One or more ROCK inhibitors may be included in the culture broth of the present invention.
As used herein, "AHR inhibitor" refers to an inhibitor of the aromatic hydrocarbon receptor (Aryl hydrocarbon receptor, AHR) and includes, but is not limited to, SR1 (STEMREGENIN-1), PD98059, CH-223191, AHR antagonst 2 (see, for example, WO2019101641A1, component example 1, CAS No. 2338747-54-7), AHR antagonst 4 (see, for example, WO2018146010A1, example 293, CAS No. 2242465-58-1), GNF351, and PDM2. One or more AHR inhibitors may be included in the culture broth of the present invention.
The "deacetylase inhibitor" according to the present invention, which may also be referred to as histone deacetylase inhibitor (Histone deacetylase inhibitor, HDACI), is an agent capable of inhibiting Histone Deacetylase (HDAC) activity, including but not limited to VPA (valproic acid ), naBu (sodium butyrate), TSA (Trichostatin a) and SAHA (suberoylanilide hydroxamic acid ). One or more deacetylase inhibitors may be included in the culture broth of the present invention.
The "GSK-3 inhibitor" refers to inhibitors of Glycogen synthase kinase-3 (Glycogen SYNTHASE KINASE-3), including but not limited to CHIR99021, SB 216763, TWS119, LY2090314, tideglusib and small molecule compound BIO (6-bromoindirubin-3-oxime ). One or more GSK-3 inhibitors may be included in the culture broth of the invention. ROCK inhibitors, AHR inhibitors, deacetylase inhibitors and GSK-3 inhibitors of the present invention are all commercially available from a variety of suppliers.
The components in the culture solution can synergistically and rapidly activate the peripheral blood cells extracted in vitro, and the amplification culture efficiency of Peripheral Blood Mononuclear Cells (PBMC) is improved. Wherein DMEM/F12 and blood cell culture medium StemPro-34 are used as serum-free basal medium, can provide abundant nutrition sources for cell growth and proliferation, and can maintain the phenotypic characteristics and cell activity of PBMC. L-ascorbic acid and beta-mercaptoethanol have strong reducibility, can neutralize oxygen free radicals accumulated in a culture medium in the cell culture process, and promote the growth and proliferation of cells. Human serum albumin, transferrin and ethanolamine can provide a sufficient nutritional supply for cells in serum-free medium. The anticoagulation effect of heparin sodium can better maintain the activity of PBMC, and is beneficial to the reprogramming of subsequent cells. AHR inhibitors (e.g., SR1, PD98059, CH-223191, AHR antagonst 2, AHR antagonst 4, GNF351 or PDM 2) have the effects of promoting PBMC growth, promoting CD34+ cell proliferation, reducing VentX expression level, and maintaining cell phenotype function. Deacetylase inhibitors (e.g., VPA, naBu, TSA or SAHA) promote expansion of hematopoietic stem cells during in vitro culture, and can increase the intensity of action of other cytokines such as SCF, IGF-1 on cells in culture by modulating the degree of chromatin open structure. GSK-3 inhibitor (such as CHIR99021, SB 216763, TWS119, LY2090314, tideglusib or small molecule compound BIO) can inhibit apoptosis, thereby promoting proliferation of cells and increasing cell expansion efficiency. ROCK inhibitors (e.g., thiazovivin, Y-27632 or KD-025) can increase cell-ECM adhesion mediated beta 1 integrin activity, promote cell survival by cooperating with other growth factors, and promote in vitro expansion of PBMC cells. The synergistic effect of cytokines SCF, GM-CSF and TPO can promote the expansion of cells in vitro and continuously produce a large number of peripheral blood cells. EPO, IGF-1 and FLT-3L can promote cell growth, improve the in vitro expansion efficiency of PBMC cells and protect cells.
PBMC cells cultured with the culture fluid of the invention may be used to reprogram the induced pluripotent stem cells (Induced pluripotent stem cell), for example PBMC cells cultured with the culture fluid of the invention may be cultured with the induced pluripotent stem cell culture fluid and reprogramming factors (e.g. OCT-4, klf-4 and C-MYC transcription factors may be added, for example the PBMC cells may be transfected with plasmids carrying these transcription factors, preferably the transfected amount of each plasmid DNA is OCT-4:klf-4:c-myc=1:1:1, respectively), followed by culturing with the reprogramming induction culture fluid.
The PBMC culture solution disclosed by the invention is based on a DMEM/F12 culture medium and a blood cell culture medium StemPro-34, and is added with an AHR inhibitor, an acetylase inhibitor, a GSK-3 inhibitor, a ROCK inhibitor, and a combination of cytokines such as SCF, IGF-1, FLT-3L, TPO, EPO, GM-CSF and the like, so that the PBMC culture solution can be synergistic in vitro, promote the rapid proliferation of peripheral blood mononuclear cells, maintain the cell characteristics while being efficiently expanded in vitro, and provide a sufficient cell source for the subsequent reprogramming of iPSC cells. The method can enable the PBMC cells to be rapidly amplified in a short time to reach the quantity required by cell reprogramming, and can efficiently reprogram the iPSC cells.
The terms "comprising," "including," "having," and similar referents used herein do not exclude the presence of unrecited elements and may also include other unrecited elements, as well as consist of the elements recited alone.
Drawings
FIG. 1 is a photograph of PBMC microscopes obtained from different culture solutions;
FIG. 2 shows the number of PBMC obtained from different cultures;
FIG. 3 shows the PBMC activities obtained with different culture solutions;
FIG. 4 shows the cloning after reprogramming of PBMC obtained from different cultures;
Examples
The technical scheme of the present invention will be described in further detail below by way of examples with reference to the accompanying drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 Effect of different culture fluid compositions on the proliferation Rate of PBMC
The implementation steps are as follows: peripheral Blood Mononuclear Cells (PBMC) were isolated from frozen or freshly collected blood samples by Ficoll-Paque density gradient centrifugation, seeded in 12-well plates at 0.015X 10 6 cells/well density, cultured at 37℃for 4 days in 5% CO 2 per cell culture broth designed for each experimental group, and the culture broth was replaced every 2 days during the culture period to remove non-adherent cells, and the cell numbers of each experimental group were counted after digestion.
The culture fluids of each experimental group were designed as follows, wherein:
Control PBMC media composition as basal medium -34SFM (available from Gibco under the trade designation 10639-011), IL-3 at 20ng/mL, IL-6 at 20ng/mL, SCF at 100ng/mL and FL-3 at 100ng/mL were added.
The basic components of the experimental culture solution are DMEM/F12 culture medium and StemPro-34 in volume ratio of 1:1, wherein the concentration of added growth factor SCF is 25ng/mL, the concentration of IGF-1 is 25ng/mL, the concentration of FLT-3L is 20ng/mL, the concentration of TPO is 25ng/mL, the concentration of EPO is 30ng/mL, the concentration of GM-CSF is 25ng/mL, the concentration of L-ascorbic acid is 500 mu M/mL, the concentration of beta-mercaptoethanol is 5 mu M/mL, the concentration of sodium nitrite is 5 mu M/mL, the concentration of ethanolamine is 5 mu M/mL, the concentration of human serum albumin is 10g/L, and the concentration of transferrin is 75 mu g/mL.
FIGS. 1A-D are bright field photographs of cells obtained after culturing PBMC with different media. FIG. 2 shows the number of cells obtained after culturing PBMC with different culture solutions. As can be seen from fig. 1 and 2, in comparison of experimental groups 1 and 2, heparin sodium, ROCK inhibitor and AHR inhibitor were added to the basic culture components, and the cell numbers were greater than those obtained by culturing PBMCs with the control culture solution; the addition of the deacetylase inhibitor VPA is continued on the basis of the component of the experimental group 2, and the cell number is further increased, which is possibly related to the effect intensity of other cytokines such as SCF and IGF-1 in the cell indirectly regulated culture solution by the VPA; the experimental group 4 is characterized in that the GSK-3 inhibitor CHIR99021 is further added on the basis of the culture solution composition of the experimental group 3, the number of PBMC is obviously increased, and the obtained cell number is the largest, which shows that the GSK-3 inhibitor has obvious influence on the proliferation of PBMC. The results show that the signal path control components in the culture solutions of the experimental groups 2-4 can effectively promote the proliferation of PBMC, and compared with the culture solution component of the experimental group 1, the signal path control components can remarkably improve the amplification efficiency of PBM, and can be used for PBMC amplification.
EXAMPLE 2 Effect of culture fluid composition on PBMC Activity
The design and implementation steps of each experimental group are the same as in example 1, and the cell viability obtained by different experimental groups is counted by adopting a counter.
FIG. 3 shows the cell viability obtained after culturing PBMC with different culture solutions. Comparing experimental groups 2,3 and 4, the results show that along with the gradual addition of heparin sodium, ROCK inhibitor and AHR inhibitor, the deacetylase inhibitor and the GSK-3 inhibitor in the culture solution, the cell activity rate is also increased, which indicates that the heparin sodium, the ROCK inhibitor, the AHR inhibitor, the deacetylase inhibitor and the GSK-3 inhibitor can well maintain the metabolic demands of cells, inhibit apoptosis and increase the cell activity rate. Compared with the experimental group 1, the culture solution of the experimental groups 2-4 adopts DMEM/F12 culture medium and StemPro-34, SCF, IGF-1, FLT-3L, TPO, EPO, GM-CSF cell factor and the combination of AHR inhibitor, acetylase inhibitor, GSK-3 inhibitor and/or ROCK inhibitor are added, so that the cell viability is remarkably improved, the effectiveness of the components of the culture solution is shown, and the culture solution can be used for maintaining the activity of PBMC.
EXAMPLE 3 cloning number obtained after reprogramming of PBMC obtained from different culture broths
The specific implementation steps are as follows: PBMCs were isolated from frozen or freshly collected blood samples by Ficoll-Paque density gradient centrifugation, inoculated into 12 well plates, cultured for 4 days at 37 ℃ with 5% co 2 using the PBMC culture fluid described in each experimental group in example 1, and once every two days, passaged into new 12 well plates until the cell density became 80-100%, at a passaging ratio of 1:5, culturing with PBMC culture solution corresponding to each experimental group at 37 ℃ for 3 days by 5% CO 2; the reprogramming experiment was performed with cultured PBMC cells 0.03x10 6. Plasmids carrying OCT-4, KLF-4 and C-MYC transcription factors are electrically transfected into human peripheral blood mononuclear cells, and the transfection contents of the plasmid DNA are respectively OCT-4: KLF-4: c-myc=1:1:1; cells were subjected to alternate Day plating with PBMC medium from the four experimental groups of example 1 at Day0-Day2 stage after transfection, and cells were subjected to alternate Day half plating with electrotransfer reprogramming induction medium ESEENTIAL 6 TM (Thermofisher) at Day3-Day5 stage after transfection; after transfection, day5-Day10 stage, the cells are completely changed every other Day by using electrotransfection reprogramming induction culture solution; clones appeared from Day10-Day15 after transfection, and the number of clones obtained for each experimental group was counted.
FIG. 4 shows statistics of the number of clones obtained with different culture solutions. As can be seen from the graph, the number of clones obtained under the same culture conditions by using different culture solutions, the number of cells obtained by using the control culture solution in the experimental group 1 is 10 or so, and the number of clones obtained after the culture of the experimental groups 2 to 4 by sequentially adding the signal path control agents is gradually increased, which indicates that each signal path control agent can effectively act. The increase in cell numbers obtained from the culture fluid compositions of experimental groups 2-4 indicates that the culture fluid compositions are effective, and maintain the cell characteristics of PBMC while efficiently expanding PBMC in vitro, thereby providing a sufficient cell source for subsequent reprogramming of iPSC cells.
In conclusion, the PBMC culture solution provided by the invention is innovatively added with small molecular inhibitors such as AHR inhibitor, MEK inhibitor, GSK-3 inhibitor and/or ROCK inhibitor and the like and the combination of cytokines such as SCF, IGF-1 and FLT-3L, TPO, EPO, GM-CSF, and can be used for obviously improving the proliferation of hematopoietic stem cells and progenitor cells in peripheral blood through in vitro synergism, so that PBMC cells can be rapidly amplified in a short time, the number required by cell reprogramming is reached, iPSC cell reprogramming can be efficiently carried out, and the PBMC culture solution can be applied to the directions of disease mechanism research, drug screening, cell treatment and the like.

Claims (16)

1. A Peripheral Blood Mononuclear Cell (PBMC) culture broth, comprising: basic culture medium DMEM/F12 and blood cell culture medium StemPro-34, cell growth factor, L-ascorbic acid, beta-mercaptoethanol, sodium nitrite, ethanolamine, ROCK inhibitor, human serum albumin, transferrin, heparin sodium and AHR inhibitor;
Wherein the ROCK inhibitor is Thiazovivin, and the concentration of the ROCK inhibitor is 1-10 mu M/mL;
Wherein the AHR inhibitor is SR1, and the concentration of the AHR inhibitor is 0.5-5 mu M/mL;
Wherein the cell growth factors comprise SCF, IGF-1, FLT-3L, TPO, EPO and GM-CSF, the concentration of SCF is 10-100ng/mL, the concentration of IGF-1 is 10-100ng/mL, the concentration of FLT-3L is 10-100ng/mL, the concentration of TPO is 10-100ng/mL, the concentration of EPO is 10-100ng/mL, and the concentration of GM-CSF is 10-100ng/mL; and
Wherein the concentration of L-ascorbic acid is 100 mu M-1mM/mL; the concentration of the beta-mercaptoethanol is 1-10 mu M/mL; the concentration of sodium nitrite is 1-10 mu M/mL; the concentration of the ethanolamine is 1-10 mu M/mL; the concentration of the human serum albumin is 5-50g/L; transferrin concentration is 50-500 μg/mL; heparin sodium concentration is 50-500 mug/mL.
2. The peripheral blood mononuclear cell culture broth of claim 1, wherein the concentration of ROCK inhibitor is 5 μΜ/mL and the concentration of AHR inhibitor is 2 μΜ/mL.
3. The peripheral blood mononuclear cell culture broth of claim 1, further comprising a deacetylase inhibitor VPA at a concentration of 0.5-5 μm/mL.
4. The peripheral blood mononuclear cell culture broth of claim 3, wherein the concentration of the deacetylase inhibitor is 2 μm/mL.
5. The peripheral blood mononuclear cell culture broth of claim 3, further comprising GSK-3 inhibitor CHIR99021 at a concentration of 0.1-1 μm/mL.
6. The peripheral blood mononuclear cell culture broth of claim 5, wherein the concentration of GSK-3 inhibitor is 0.5 μΜ/mL.
7. The peripheral blood mononuclear cell culture broth of any of claims 1-6, wherein the cytokine has a SCF concentration of 25ng/mL, an IGF-1 concentration of 25ng/mL, an FLT-3L concentration of 20ng/mL, a TPO concentration of 25ng/mL, an EPO concentration of 30ng/mL, and a GM-CSF concentration of 25ng/mL.
8. The peripheral blood mononuclear cell culture broth of any of claims 1-6, wherein the L-ascorbic acid concentration is 500 μΜ/mL; the concentration of the beta-mercaptoethanol is 5 mu M/mL; the concentration of sodium nitrite is 5 mu M/mL; the concentration of ethanolamine is 5 mu M/mL; the concentration of human serum albumin is 10 g/L; transferrin concentration was 75 μg/mL; heparin sodium concentration was 250. Mu.g/mL.
9. The peripheral blood mononuclear cell culture broth of any of claims 1-6, wherein the volume ratio of DMEM/F12 medium to blood cell medium StemPro-34 is 1:0.9-1.3.
10. The peripheral blood mononuclear cell culture broth of claim 9, wherein the volume ratio of DMEM/F12 medium to blood cell medium StemPro-34 is 1:1.
11. A method of culturing Peripheral Blood Mononuclear Cells (PBMCs), the method comprising: culturing peripheral blood mononuclear cells in the peripheral blood mononuclear cell culture broth of any of claims 1-10.
12. The culture method according to claim 11, comprising the steps of:
Step 1) Peripheral Blood Mononuclear Cells (PBMC) are inoculated into a cell culture container according to the cell density of 0.005-0.05X10 6 cells/hole; culturing cells inoculated in a cell culture container with the peripheral blood mononuclear cell culture solution according to any one of claims 1 to 10 at 35 to 39 ℃ and 3 to 7% CO 2 for 3 to 5 days; changing culture solution every 1-3 days during culture;
Step 2) when the cell density is 80% -100%, transferring the cells to a new cell culture container, wherein the transfer ratio is 1:3-1:10; culturing the peripheral blood mononuclear cell culture fluid according to any one of claims 1-10 at 35-39 ℃ for 2-5 days with 3-7% CO 2.
13. The culture method according to claim 12, wherein in the step 1), the inoculated cells have a cell density of 0.015X 10 6 cells/well, the culture conditions are 37℃and 5% CO 2, and the culture is carried out for 4 days with the culture medium being replaced every 2 days during the culture; wherein in step 2), the cell passage ratio is 1:5, the continuous culture condition is 37 ℃, 5% CO 2, and the culture is carried out for 3 days.
14. The culture method of any one of claims 11-13, wherein the cultured peripheral blood mononuclear cells are used for reprogramming to induce pluripotent stem cells.
15. Use of the PBMC culture liquid of any one of claims 1-10 for culturing peripheral blood mononuclear cells.
16. The use of claim 15, wherein the cultured peripheral blood mononuclear cells are used for reprogramming to induced pluripotent stem cells.
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CN102625837A (en) * 2009-08-07 2012-08-01 国立大学法人京都大学 Method of efficiently establishing induced pluripotent stem cells
CN109642212A (en) * 2016-06-16 2019-04-16 西达-赛奈医疗中心 Blood is reprogrammed into the novel and effective method induced multi-potent stem cell
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