CN114807031A - Construction method of human peripheral blood immune cell bank and stem cell bank - Google Patents

Construction method of human peripheral blood immune cell bank and stem cell bank Download PDF

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CN114807031A
CN114807031A CN202210518999.3A CN202210518999A CN114807031A CN 114807031 A CN114807031 A CN 114807031A CN 202210518999 A CN202210518999 A CN 202210518999A CN 114807031 A CN114807031 A CN 114807031A
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cells
cell
peripheral blood
culture
freezing
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陈莉
邓长江
尚现岗
李春娜
夏冬梅
王范
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Shandong Saienfu Stem Cell Engineering Group Co ltd
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Shandong Saienfu Stem Cell Engineering Group Co ltd
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Abstract

The invention discloses a method for constructing a human peripheral blood immune cell bank and a stem cell bank. The method comprises the following steps: collecting human peripheral blood at one time, and obtaining peripheral blood mononuclear cells by adopting a density gradient centrifugation method and a differential centrifugation method; one part of the cells was directly frozen and the other part of the cells was separated by flow cytometry to give CD3 + Performing CIK cell amplification culture on the lymphocytes, and freezing and storing codes; sorting out CDs 3 + The remaining cells after the lymphocytes were sorted by flow cytometry for CD56 + Carrying out NK cell amplification culture on the lymphocytes, and carrying out coding cryopreservation; sorting out CDs 56 + The remaining cells after the lymphocytes were sorted by flow cytometry for CD34 ‑ CellsMSC cell culture is carried out, and then coding and freezing storage are carried out. The method collects peripheral blood once, separates mononuclear cells, can simultaneously separate NK cells, CIK cells and MSC cells, meets various requirements, and avoids the problem of collecting blood for multiple times.

Description

Construction method of human peripheral blood immune cell bank and stem cell bank
Technical Field
The invention relates to a method for constructing a human peripheral blood immune cell bank and a stem cell bank, belonging to the field of cell culture and cell bank construction.
Background
The cell therapy is to obtain living cells with specific functions from human autologous or allogeneic sources, and the living cells are returned to the human body after in vitro separation, purification, culture, amplification, cryopreservation, resuscitation and qualified quality inspection, so as to play roles in treating diseases and resisting aging. Cell therapy can be classified into stem cell therapy, immune cell therapy, somatic cell therapy, and the like, according to cell types.
Since the discovery of continuously proliferating and differentiable hematopoietic stem cells in mouse bone marrow in 1960, the existence of stem cells was gradually recognized, and by the fact that the british scientists cloned dori sheep in 1997, realized the regeneration of stem cells, and extended to the mountains, the successful induction of pluripotent stem cells (ipscs) by using somatic cells was achieved, the development of stem cell technology was rapid within 50 years, and the hope of being able to treat various major diseases and regenerate various tissues and organs by using stem cells was started. With the promulgation of Nobel medicine to Induced Pluripotent Stem Cells (iPSCs) in 2012, the potential medical application of stem cells is more widely regarded in countries around the world, and with the acceleration of the aging process of China, the prospect of utilizing stored stem cells to perform individualized treatment is closer to reality. Stem cell therapy is the most advanced clinical treatment technology of the regenerative medicine revolution, and the technology is to propagate brand new, normal and even younger cells, tissues or organs in vitro through the processes of stem cell separation, in vitro culture, directional induction, even gene modification and the like, and finally realize the treatment of clinical diseases through the transplantation of cell tissues or organs. The immunotherapy is based on the principle and method of immunology, and is characterized by that the immune cells from human peripheral blood or umbilical blood, etc. are collected, cultured and expanded in vitro in large scale so as to raise target killing function, and then the obtained immune cells are returned into human body, and the pathogen, cancer cell and mutant cell in blood and tissue are killed and killed by means of regulating immune system of human body so as to inhibit tumor growth and raise immunity of human body. At present, the immune cells which are clinically applied mostly comprise CIK cells, NK cells, NKT cells and the like.
Cell cryopreservation is one of the most effective methods for long-term preservation of cells. Everybody has own unique cells, and if in young and healthy state, the cells with high quality are frozen in a deep low temperature liquid nitrogen tank at-196 ℃ in advance, the aging of the cells can be stopped, and the cells can be permanently kept in the youngest and most viable state. In the future, the seed cells reserved in advance can be used for culturing more high-quality cells once needed by the body, and the cells are used for improving the immunity of the body, improving/enhancing the physical condition, resisting cancer and even saving life. In order to store various cell resources conveniently, a plurality of cell banks or biological sample banks are established worldwide at present, including umbilical cord blood banks, umbilical cord mesenchymal stem cell banks, immune cell banks and the like.
Peripheral blood is one of important sources of immune cells and stem cells, and cells such as NK (natural killer cells), CIK (cytokine-induced killer cells) and iPSC (induced pluripotent stem cells) can be amplified and cultured under certain induction conditions. Collecting 100mL of human peripheral blood, and separating to obtain 0.6-1.5 × 10 8 At present, through literature and patent search, most methods are found that peripheral blood mononuclear cells are directly frozen and preserved, and when the peripheral blood mononuclear cells are used, the peripheral blood mononuclear cells are cultured again. Moreover, the yield of the peripheral blood mononuclear cells after the recovery of the peripheral blood mononuclear cells is only 50% approximately, the number of the peripheral blood mononuclear cells is only enough for one cell, such as CIK (cytokine induced killer) cell amplification culture, and the problems of secondary blood collection and tertiary blood collection are often faced. Therefore, a new method is urgently needed,the freeze storage requirements of different types of cells including stem cells and immune cells can be met by one-time blood collection, multiple blood collection is not needed, the stem cells and the immune cells are respectively frozen and stored, and then different diseases can be treated.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a method for constructing a human peripheral blood immune cell bank and a stem cell bank, the invention collects human peripheral blood at one time, a density gradient centrifugation method is combined with a differential centrifugation method to obtain mononuclear cells, and a part of cells are directly frozen; another part is separated out CD3 by flow cytometry + Performing CIK cell amplification culture on the cells; sorting out CD3 + Sorting of the remaining cells to CD56 + Performing NK cell amplification culture on the cells; sorting out CD56 + The remaining cells of the cells were sorted by flow cytometry for CD34 - Cells were cultured with MSC cells (mesenchymal stem cells) and then individually encoded for cryopreservation. The method can realize the cryopreservation requirements of different types of cells including stem cells and immune cells by taking blood once, and avoids the problem of taking blood for many times.
The technical scheme of the invention is as follows: a method for constructing a human peripheral blood immune cell bank and a stem cell bank is characterized by comprising the following steps (as shown in figure 1):
s1: collecting human peripheral blood at one time, and obtaining peripheral blood mononuclear cells by adopting a density gradient centrifugation method combined with a differential centrifugation method; directly freezing and storing a part of cells;
s2: step S1 separating CD3 from the other part of the cells except for the frozen cells by flow cytometry + Performing CIK cell amplification culture on the lymphocytes, and freezing and storing codes;
s3: step S2 sorting out CD3 + The remaining cells after the lymphocytes were sorted by flow cytometry for CD56 + Carrying out NK cell amplification culture on the lymphocytes, and carrying out coding cryopreservation;
s4: step S3 sorting out CD56 + The remaining cells after the lymphocytes were sorted by flow cytometry for CD34 - Cells were cultured for MSC cells and then encoded for cryopreservation.
Further, the step S1 is to collect human peripheral blood at a time, and obtain peripheral blood mononuclear cells by using a density gradient centrifugation method in combination with a differential centrifugation method, specifically:
1) transferring peripheral blood in the heparin sodium blood collection tube into a centrifugal tube, and centrifuging; after the centrifugation is finished, preparing the upper layer plasma into autologous plasma for later use;
2) adding physiological saline into the centrifuged lower peripheral blood, blowing, uniformly mixing, adding into the lymphocyte separation liquid, and centrifuging again;
3) adding normal saline into the leukocyte layer cells in the centrifuge tube, reversing and mixing uniformly, centrifuging, removing the supernatant, repeating for 1-3 times (adding normal saline, reversing and mixing uniformly, centrifuging, removing the supernatant), and precipitating to obtain peripheral blood PBMC.
Further, the partial cells of step S1 are directly frozen as follows: adding peripheral blood PBMC into immune cell freezing medium to resuspend cells for freezing, wherein the cell density is 2-3 multiplied by 10 6 one/mL. The immune cell frozen stock solution comprises the following formula: the adhesive comprises the following components in parts by volume: 1-5% of autologous plasma, 10% of DMSO (dimethyl sulfoxide), 10% of dextran 40 (6% dextran-40 glucose injection), and 75-79% of hydroxyethyl starch (colloidal solution), wherein the total amount is 100%.
Further, the step S2 performs CIK cell expansion culture by:
1) sorted CD3 + Adding KBM581 or X-VIVO15 culture medium into the cells for resuspension, adding INF-gamma, and culturing in incubator;
2) on day 2, IL-2 and CD3 Ab were added and the culture continued; on days 4-5, the complete medium was supplemented,
3) on day 6, counting, if the cell density reaches 1-2 × 10 6 Transferring all cells into a cell culture bag, supplementing a complete culture medium, and continuing to supplement the complete culture medium in 8-10 days;
4) on day 14, centrifugation was carried out, the supernatant was discarded, and physiological saline was added for washing to collect CIK cells.
The final concentration of the added INF-gamma is 100-1000IU/mL, the final concentration of IL-2 is 1000 +/-200 IU/mL, and the final concentration of CD3 Ab is 20-200 ng/mL.
Wherein, the complete culture medium is: KBM581 or X-VIVO15 medium containing 1000 + -200 IU/mL IL-2.
Further, the step S3 performs NK cell expansion culture by:
1) sorted CD56 + Adding KBM581 or X-VIVO15 culture medium for resuspension, and adding IL-2, IL-21 and IL-15 to stimulate NK cell activation and expansion;
2) the cells were placed in the incubator for the first 3 days without moving; on day 4, adding KBM581 or X-VIVO15 culture medium and cytokines IL-2, IL-21, and IL-15 for culture;
2) on day 6, counting, if the cell density reaches 1-2 × 10 6 Transferring all the cells into a cell culture bag, and supplementing KBM581 or X-VIVO15 culture medium and cytokines IL-2, IL-21 and IL-15; then, on 8-10 days, continuously supplementing a KBM581 or X-VIVO15 culture medium and cytokines IL-2, IL-21 and IL-15;
4) on day 14, centrifugation was carried out, the supernatant was discarded, and the NK cells were collected by washing with physiological saline.
Furthermore, the final concentration of IL-2 used is 1000 + -200 IU/mL, the final concentration of IL-21 is 100 IU/mL, and the final concentration of IL-15 is 10-500 ng/mL.
Further, the frozen stock warehouse of the steps S2 and S3 is: adding immune cell freezing medium, and adjusting the cell density to 2-3 × 10 7 one/mL. The immune cell frozen stock solution comprises the following formula: the adhesive comprises the following components in parts by volume: 1-5% of autologous plasma, 10% of DMSO, 10% of dextran 40 (6% of dextran-40 glucose injection), 75-79% of KBM581 culture medium, and the total amount is 100%.
Further, the MSC cell culture of step S4 specifically includes:
1) sorting out CDs 34 - Adjusting the cell density of the cells by using a serum-free culture medium, and culturing the cells in an incubator;
2) and on the 2 nd day, removing floating cells by changing the liquid, adding a fresh serum-free culture medium, continuously culturing for 5-7 days, and obtaining the MSC cells by a trypsin digestion method when the cells grow to 80-90% fusion.
Furthermore, the serum-free culture medium is an alpha-MEM culture medium containing 2-200 ng/mL VEGF (cell growth factor), 2-200 ng/mL PDGF (growth factor), 0.1-10 mu g/mL EGF (epidermal growth factor), 1-100 mu g/mL recombinant human insulin and 5-50 mu g/mL transferrin.
Further, the freezing storage of step S4 is: adding a freezing medium to freeze and store cells, wherein the freezing density is 0.5-1 multiplied by 10 7 And (2) freezing and storing in a warehouse, wherein the formula (by volume ratio) of the freezing solution is as follows: 10% DMSO, 20% platelet lysate, 70% α -MEM medium.
The method has the advantages that:
1. 100mL of peripheral blood is collected at one time, mononuclear cells are separated, and NK cells, CIK cells and MSC cells can be simultaneously amplified and cultured to meet various requirements.
2. Sorting CD3 using flow cytometry + Cell culture CIK, CD56 + Cell culture of NK cells, CIK cell purity CD3 + CD56 + Can reach more than 85 percent, and the purity of NK cells is CD3 - CD56 + Can reach more than 90 percent, and is higher than the traditional method; cell proliferation is faster: cells can proliferate nearly 1000-fold at day 14.
3. Sorting CD34 using flow cytometry - The cell culture of MSC cell has faster cell proliferation and purity up to more than 98%.
4. All reagents used in the culture process and the cryopreservation do not contain animal-derived bovine serum, human serum albumin and the like; plasma used was plasma from a client's own peripheral blood separation.
Drawings
FIG. 1 is a flow chart of the present invention for one-time collection of peripheral blood to isolate NK cells, CIK cells and MSC cells to construct cell banks;
FIG. 2 is a morphological diagram of CIK cells cultured by flow-sorted PBMC induction observed under an inverted microscope: a is day 3 after induction (× 40 times); b is day 5 after induction (× 40 times); c is day 6 after induction (× 40 times);
FIG. 3 is a photograph of NK cell morphology observed under an inverted microscope in flow sorted PBMC induction cultures: a is day 4 after induction (× 40 times); b is day 7 after induction (× 40 fold);
FIG. 4 is a photograph of MSC cell morphology observed under an inverted microscope in flow sorted PBMC induction cultures: a is day 1 after passage (× 40 times); b is day 2 after passage (× 40 times);
FIG. 5 is a flow cytometer detecting cell phenotype of flow sorted cord blood mesenchymal stem cells.
Detailed Description
The embodiment of the present invention will be described in detail below with reference to the following examples and the flowchart of FIG. 1 of the present specification for isolating NK cells, CIK cells and MSC cells in one collection of peripheral blood to construct a cell bank. Word annotation: PBMC: peripheral blood mononuclear cells; IL-2: interleukin 2; IL-15: interleukin 15; IL-21: interleukin 21; CD3 Ab: a CD3 monoclonal antibody; INF-gamma: an interferon gamma; MSC: mesenchymal stem cells; NK: a natural killer cell; and (3) CIK: cytokine-induced killer cells; CD 3-PE: a PE-labeled CD3 monoclonal antibody; CD 56-FITC: FITC-labeled CD56 monoclonal antibody.
Example 1:
first, separating and freezing and storing the mononuclear cells into a warehouse
1. Transferring peripheral blood in a heparin sodium blood collection tube into a 50mL centrifuge tube, freezing the centrifuge at a low temperature of 4 ℃ and 1500rpm, centrifuging for 10 minutes, and increasing 9 and decreasing 3 (increasing 9 and decreasing 3, the same below);
2. after centrifugation, transferring the upper plasma into a 50mL centrifuge tube, carrying out water bath at 56 ℃ for 30 minutes, centrifuging at 4 ℃ and 3000rpm for 10 minutes, lifting 9 and reducing 9, collecting supernatant, and preserving at-20 ℃ for later use as autologous plasma;
3. adding physiological saline into the peripheral blood (the part left after collecting the supernatant) left in the step 2 according to the volume ratio of 1:1 for dilution, and uniformly mixing by blowing;
4. slowly adding the peripheral blood uniformly diluted in the step 3 to the upper layer of a centrifuge tube of prepared 1.077g/mL lymphocyte separation solution (15 mL/tube, manufacturer GE, product number 17-5442-02), wherein 30mL of the peripheral blood is added to each centrifuge tube;
5. centrifuging at 1800-2500 rpm/min for 20min by using a low-temperature refrigerated centrifuge, and increasing 7 and reducing 3 at the temperature of 20 ℃;
6. taking the leukocyte in the middle tunica albuginea layer of the centrifuge tube to a new 50mL centrifuge tube, adding physiological saline to 50mL, reversing and uniformly mixing, performing centrifugation at 1800rpm/min for 10 min;
7. discarding the supernatant, adding physiological saline to 50mL, resuspending the cells, and centrifuging at 1600rpm/min for 8 min;
8. discarding the supernatant, adding physiological saline to 50mL, resuspending cells, mixing well, counting 0.5mL cell suspension, 1200rpm/min, and centrifuging for 8 min. Discarding the supernatant, and obtaining a precipitate as peripheral blood PBMC; part of the quality test is qualified (quality test standard: sterile, mycoplasma-free, endotoxin less than or equal to 0.25EU/mL, cell number more than or equal to 1 × 10 8 Then frozen and stored in a warehouse in the following step 9; the other part is sorted by a flow cytometer, and the following operations of two, three and four are sequentially carried out;
9. freezing and storing in a warehouse: adding immune cell freezing medium to resuspend cells with the cell density of 2-3 multiplied by 10 6 one/mL. The formula of the frozen stock solution is as follows: the composite material comprises the following components in percentage by volume: 1-5% of autologous plasma, 10% of DMSO, 10% of dextran 40 (6% of dextran-40 glucose injection), 75-79% of hydroxyethyl starch (colloidal solution), and the total amount is 100%.
II, culturing and freezing CIK cells for storage
1. CD3 in PBMC cells using flow cytometry + Separating out cells;
2. sorted CD3 + 3X 10 of the cells were removed 7 Adding 30mL KBM581 or X-VIVO15 culture medium into each cell for resuspension, adding INF-gamma (final concentration 500-1000 IU/mL), placing at 37 deg.C and 5% CO 2 Culturing in an incubator;
3. on the 2 nd day, IL-2 (final concentration 1000IU/mL) and CD3 Ab (final concentration 50-200 ng/mL) are added for continuous culture;
4. on day 4, 30mL of complete medium was supplemented, wherein the complete medium was: KBM581 or X-VIVO15 medium containing 1000IU/mL IL-2;
5. on day 5, 90mL of complete medium was supplemented;
6. on the 6 th day, counting is carried out, if the cell density reaches 1-2 × 10 6 Transferring all cells into a cell culture bag, and supplementing 450ml of complete culture medium;
7. continuously supplementing 1200mL of complete culture medium (1200 mL in total in 3 days) on 8-10 days;
8. on day 11, samples were taken for detection of bacteria, fungi and mycoplasma (quality control criteria: no bacterial fungi, no mycoplasma infection);
9. on day 14, centrifuging at 2000rpm for 5min, removing supernatant, collecting cells, washing with 0.9% physiological saline for three times, and centrifuging at 2000rpm for 5 min;
10. freezing and storing in a warehouse: removing supernatant, adding immune cell freezing medium with cell density of 2-3 × 10 7 one/mL. The formula of the frozen stock solution is as follows: the material comprises the following components in percentage by volume: 1-5% of autologous plasma, 10% of DMSO, 10% of dextran 40 (6% of dextran-40 glucose injection), 75-79% of KBM581 culture medium, and the total amount is 100%.
The morphology of CIK cells cultured by flow sorted PBMC induction observed under an inverted microscope in this example is shown in FIG. 2, where A is day 3 (x 40 fold) after induction; b is day 5 after induction (× 40 times); c is day 6 after induction (x 40 fold), as can be seen in figure 2: the proliferation speed of CIK cells is high, cells are in a suspension state on day 3, cells begin to aggregate on day 5, cells proliferate rapidly on day 6, and cell aggregates increase obviously.
And thirdly, culturing NK cells and freezing and storing in a warehouse
1. Coating a culture flask: adding a recombinant anti-human CD16 monoclonal antibody (5-50 mu g/mL) into a T75 culture bottle, and standing at 37 ℃ for 1h or storing at 4 ℃ overnight;
2. sorting out CD56 by flow cytometry + Cells, 2X 10 removal 7 Adding 20mL of KBM581 or X-VIVO15 culture medium to resuspend the cells, transferring the cells into a coated T75 culture flask, and adding IL-2 (final concentration 1000IU/mL), IL-21 (final concentration 100-1000IU/mL) and IL-15 (final concentration 50-500 ng/mL) to stimulate NK cell activation and amplification; the cells were incubated at 37 ℃ with 5% CO for the first 3 days 2 Incubator, do not move;
3. on day 4, adding 40mL of KBM581 or X-VIVO15 culture medium, cytokine IL-2 (final concentration 1000IU/mL), IL-21 (final concentration 100-;
4. on the 6 th day, counting is carried out, if the cell density reaches 1-2 × 10 6 Transferring all the cells into a cell culture bag, and supplementing 300mL KBM581 or X-VIVO15 culture medium and cytokines IL-2 (the final concentration is 1000IU/mL), IL-21 (the final concentration is 100 IU/mL) and IL-15 (the final concentration is 50-500 ng/mL);
5. on 8-10 days, 1200mL of KBM581 or X-VIVO15 culture medium (1200 mL in total in 3 days) and cytokines IL-2 (1000 IU/mL in final concentration), IL-21 (1000 IU/mL in final concentration) and IL-15 (50-500 ng/mL in final concentration) are continuously added;
6. on day 11, samples were taken for detection of bacteria, fungi and mycoplasma (quality control criteria: no bacterial fungi, no mycoplasma infection);
7. on day 14, centrifuging at 2000rpm for 5min, removing supernatant, washing with 0.9% physiological saline twice, sampling, counting, centrifuging at 2000rpm for 5min, removing supernatant, and collecting NK cells;
8. flow cytometry detection of cell phenotype: taking NK cells harvested in the step 7 (total number of cells is 5X 10) 6 ) Supplementing physiological saline to 50mL in a 50mL centrifuge tube, centrifuging at 2000rpm for 5min, discarding the supernatant, adding 500 μ L physiological saline to resuspend cells, subpackaging 100 μ L/EP tube and 5 tube, adding 10 μ L CD3-PE/CD56-FITC monoclonal antibody, adding 10 μ L CD3-PE in 1 tube, adding 10 μ L CD56-FITC in 1 tube, adding isotype control in 1 tube, using one tube as a blank control, mixing uniformly, and incubating at 37 ℃ for 30min in a dark place. Centrifuging at 2000rpm for 5min, discarding supernatant, adding 1 mL/tube of physiological saline to wash cells for 2 times, centrifuging at 2000rpm for 5min, discarding supernatant, adding 200 μ L of physiological saline per tube to resuspend cells, storing at 4 deg.C, and detecting on machine with flow cytometer.
9. Freezing and storing in a warehouse: taking NK cells harvested in the step 7, supplementing physiological saline to 50mL, centrifuging at 2000rpm for 5min, removing supernate, adding immune cell freezing solution, wherein the cell density is 2-3 multiplied by 10 7 one/mL. The formula of the frozen stock solution is as follows: the material comprises the following components in percentage by volume: 1-5% of autologous plasma, 10% of DMSO, 10% of dextran 40 (6% of dextran-40 glucose injection) and 75-79% of KBM581 culture medium.
Comparison data of proliferation fold and cell phenotype of NK cells induced by flow sorted PBMC and unsorted PBMSC are shown in table 1, and morphology of NK cells induced by flow sorted PBMC under inverted microscope is shown in fig. 3, where a is day 4 (x 40) after induction; b is day 7 after induction (× 40 fold).
Table 1: comparative data on NK cell proliferation fold and cell phenotype
Figure BDA0003642491540000071
As shown in Table 1, the purity of NK cells CD3 was induced and cultured by PBMC flow sorting according to the present invention - CD56 + The expression level can reach more than 90%, which is about 10% higher than that of unsorted PBMC, and as can be seen from figure 3: cell clusters appeared on day 4 of culture, and cells were greatly expanded on day 7, with cell clusters significantly increasing.
Fourthly, MSC cell culture and frozen storage and warehousing
1. Sorting out CD34 by flow cytometry - Cells were cultured in serum-free medium (. alpha. -MEM medium containing 20ng/mL VEGF, 20ng/mL PDGF, 1. mu.g/mL EGF, 10. mu.g/mL recombinant human insulin, 10. mu.g/mL transferrin) to adjust the cell density to 5X 10 5 One cell/mL, inoculated in a T75 flask (15mL serum-free medium), placed at 37 ℃ in 5% CO 2 Culturing in an incubator;
2. on the 2 nd day, removing floating cells by changing the liquid, adding 15mL of fresh serum-free culture medium, continuously culturing for 5-7 days, and observing the cell morphology and the fusion degree every day under an inverted microscope;
3. subculturing when the cells grow to 80-90% of fusion, and subculturing for 1 time every 3 days;
the cell passage is specifically as follows: harvesting cells by a trypsinization method (when the cell growth reaches 80-90% fusion, culture solution is removed, 3 mL/bottle of normal saline is added to clean the cells twice, 1 mL/bottle of 0.05% trypsin is added to digest for 1min, 15 mL/bottle of fresh culture medium is added to stop digestion after the cells are shrunk and rounded, the cells are collected into a 50mL centrifuge tube, centrifuged for 5min at 1200rpm, supernatant is removed, andresuspending the cells in 50mL of physiological saline, counting 0.5mL of the cell suspension, centrifuging at 1200rpm for 5min, discarding the supernatant), adding fresh medium to resuspend the cells at 8000 cells/cm 2 The density of (a) to seed the cells.
4. Flow cytometry detection of cell phenotype: MSC cells were collected at P4 passages in logarithmic growth phase (total number of cells 8X 10) 6 ) Centrifuging at 1200rpm for 5min in a 50mL centrifuge tube, discarding the supernatant, adding 50mL physiological saline for resuspension and washing twice, centrifuging and discarding the supernatant, retaining the cells, adding 800 μ L physiological saline for resuspension of the cells, subpackaging with 100 μ L/EP tube and 8 tubes, adding 10 μ L PE-labeled CD34, CD45, CD73, CD90, CD105, HLA-DR and PE-IgG antibodies, using the other tube as a blank control, mixing uniformly, and incubating at 37 ℃ for 30min in a dark place. Centrifuging at 1200rpm for 5min, discarding the supernatant, adding 1mL/EP tube of physiological saline to wash the cells for 2 times, centrifuging at 1200rpm for 5min, discarding the supernatant, adding 200 μ L of physiological saline per tube to resuspend the cells, storing at 4 deg.C, and detecting on a flow cytometer. The cell phenotype of the umbilical cord blood mesenchymal stem cells subjected to flow sorting by the flow cytometry is shown in figure 5, and can be seen from figure 5: the umbilical blood mesenchymal stem cells have high expression of CD73, CD90 and CD105, the positive rate is more than 99 percent, the negative expression of CD34, CD45 and HLA-DR, the positive rate is less than 0.5 percent, and completely accord with the characteristics of the mesenchymal stem cells.
6. Freezing and storing in a warehouse: and (2) harvesting cells by a trypsinization method when the cells grow to 80-90% and are fused (the step is the same as the step 3), adding a freezing medium to freeze and store the cells, wherein the freezing density is 0.5-1 multiplied by 10 7 And (5) freezing and storing in a warehouse. The formula of the frozen stock solution is as follows: 10% DMSO, 20% platelet lysate, 70% α -MEM medium.
The morphology of MSC cells induced by flow sorted PBMCs observed under an inverted microscope in this example is shown in FIG. 4, where A is day 1 after passage (x 40-fold); b is day 2 after passage (x 40 times); as shown in fig. 4: cells grow adherent to the walls on the 2 nd day after passage under an inverted microscope, have uniform shape, are in a long fusiform shape or a fusiform shape, accord with the typical shape of the mesenchymal stem cells, proliferate rapidly on the 3 rd day, have the fusion degree of 80-90 percent, and can be subjected to passage and cryopreservation operation.

Claims (11)

1. A method for constructing a human peripheral blood immune cell bank and a stem cell bank is characterized by comprising the following steps:
s1: collecting human peripheral blood at one time, and obtaining peripheral blood mononuclear cells by adopting a density gradient centrifugation method and a differential centrifugation method; directly freezing and storing a part of cells;
s2: step S1 separating CD3 from the other part of the cells except for the frozen cells by flow cytometry + Performing CIK cell amplification culture on the lymphocytes, and freezing and storing codes;
s3: step S2 sorting out CD3 + The remaining cells after the lymphocytes were sorted by flow cytometry for CD56 + Carrying out NK cell amplification culture on the lymphocytes, and carrying out coding cryopreservation;
s4: step S3 sorting out CD56 + The remaining cells after the lymphocytes were sorted by flow cytometry for CD34 - Cells were cultured for MSC cells and then encoded for cryopreservation.
2. The method according to claim 1, wherein the step S1 of collecting human peripheral blood at a time is performed to obtain mononuclear cells from peripheral blood by a density gradient centrifugation method in combination with a differential centrifugation method, and specifically comprises:
1) transferring peripheral blood in the heparin sodium blood collection tube into a centrifugal tube, and centrifuging; after the centrifugation is finished, preparing the upper layer plasma into autologous plasma for later use;
2) adding physiological saline into the centrifuged lower peripheral blood, blowing, uniformly mixing, adding into the lymphocyte separation liquid, and centrifuging again;
3) and adding physiological saline into the leucocyte layer cells in the centrifugal tube, reversing and uniformly mixing, centrifuging, discarding the supernatant, repeating for 1-3 times, and obtaining the sediment which is the PBMC of the peripheral blood.
3. The method according to claim 1, wherein the partial cells of step S1 are directly frozen as follows: adding part of peripheral blood PBMC into immune cell freezing solution for freezing and storing the resuspended cells with the cell density of 2 to c3×10 6 Per mL; the immune cell frozen stock solution comprises the following components in parts by volume: 1-5% of autologous plasma, 10% of DMSO, 10% of 6% of dextran-40 glucose injection, 75-79% of hydroxyethyl starch, and the total amount is 100%.
4. The method according to claim 1, wherein the step S2 of performing CIK cell expansion culture comprises:
1) sorted CD3 + Adding KBM581 or X-VIVO15 culture medium into the cells for resuspension, adding INF-gamma, and culturing in incubator;
2) on day 2, IL-2 and CD3 Ab were added and the culture continued; on days 4-5, the complete medium was supplemented,
3) on day 6, counting, if the cell density reaches 1-2 × 10 6 Transferring all the cells into a cell culture bag, supplementing a complete culture medium, and continuing to supplement the complete culture medium for 8-10 days;
4) on day 14, centrifugation was carried out, the supernatant was discarded, and physiological saline was added for washing to collect CIK cells.
5. The method for constructing a human peripheral blood immune cell bank and a stem cell bank according to claim 4, wherein the final concentration of the added INF-gamma is 100-1000IU/mL, the final concentration of IL-2 is 1000 +/-200 IU/mL, and the final concentration of CD3 Ab is 20-200 ng/mL; the complete culture medium is as follows: KBM581 or X-VIVO15 medium containing 1000 + -200 IU/mL IL-2.
6. The method according to claim 1, wherein the step S3 of performing NK cell expansion culture comprises:
1) sorted CD56 + Adding KBM581 or X-VIVO15 culture medium for resuspension, and adding IL-2, IL-21 and IL-15 to stimulate NK cell activation and expansion;
2) the cells were placed in the incubator for the first 3 days without moving; on day 4, the culture medium KBM581 or X-VIVO15 and cytokines IL-2, IL-21 and IL-15 are added for culture;
2) on day 6, counting, if the cell density reaches 1-2 × 10 6 Transferring all the cells into a cell culture bag, and supplementing KBM581 or X-VIVO15 culture medium and cytokines IL-2, IL-21 and IL-15; then, on 8-10 days, continuously supplementing a KBM581 or X-VIVO15 culture medium and cytokines IL-2, IL-21 and IL-15;
4) on day 14, centrifugation was carried out, the supernatant was discarded, and the NK cells were collected by washing with physiological saline.
7. The method as claimed in claim 6, wherein the final concentration of IL-2 is 1000 + -200 IU/mL, the final concentration of IL-21 is 100-1000IU/mL, and the final concentration of IL-15 is 10-500 ng/mL.
8. The method for constructing the human peripheral blood immune cell bank and the stem cell bank according to claim 4 or 6, wherein the frozen stock banks of the steps S2 and S3 are: adding immune cell freezing medium, and adjusting the cell density to 2-3 × 10 7 Per mL; the immune cell frozen stock solution comprises the following components in percentage by volume: 1-5% of autologous plasma, 10% of DMSO, 10% of 6% of dextran-40 glucose injection and 75-79% of KBM581 culture medium, wherein the total amount is 100%.
9. The method according to claim 1, wherein the MSC cell culture of step S4 is specifically:
1) sorting out CDs 34 - Adjusting the cell density of the cells by using a serum-free culture medium, and culturing the cells in an incubator;
2) and on the 2 nd day, removing floating cells by changing the liquid, adding a fresh serum-free culture medium, continuously culturing for 5-7 days, and obtaining the MSC cells by a trypsin digestion method when the cells grow to 80-90% fusion.
10. The method for constructing human peripheral blood immune cell bank and stem cell bank according to claim 9, wherein the serum-free medium is: an alpha-MEM medium containing 2-200 ng/mL VEGF, 2-200 ng/mL PDGF, 0.1-10 μ g/mL EGF, 1-100 μ g/mL recombinant human insulin, 5-50 μ g/mL transferrin.
11. The method according to claim 9 or 10, wherein the cryopreservation in step S4 is as follows: adding a freezing medium to freeze and store cells, wherein the freezing density is 0.5-1 multiplied by 10 7 And (2) freezing and storing in a warehouse, wherein the frozen stock solution comprises the following components in volume ratio: 10% DMSO, 20% platelet lysate, 70% α -MEM medium.
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