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

Abstract

The invention discloses a construction method of a human peripheral blood immune cell bank and a stem cell bank. The method includes: collecting human peripheral blood at one time, and obtaining peripheral blood mononuclear cells by density gradient centrifugation combined with differential centrifugation; a part of the cells are directly frozen, and another part of the cells is sorted by flow cytometry CD3 ⁺ lymphocytes, Carry out CIK cell expansion and culture, coding and cryopreservation; the remaining cells after sorting CD3 ⁺ lymphocytes, CD56 ⁺ lymphocytes are sorted by flow cytometry, NK cells are expanded and cultured, and coding cryopreservation; sorting The remaining cells after CD56 ⁺ lymphocytes were sorted by flow cytometry for CD34 ^‑ cells for MSC cell culture, and then encoded and cryopreserved. The method collects peripheral blood at one time, separates mononuclear cells, can separate NK cells, CIK cells and MSC cells at the same time, meets various needs, and avoids the problem of multiple blood collection.

Description

A construction method of human peripheral blood immune cell bank and stem cell bank

technical field

The invention relates to a construction method of a human peripheral blood immune cell bank and a stem cell bank, and belongs to the field of cell culture and cell bank construction.

Background technique

Cell therapy refers to obtaining living cells with specific functions from human autologous or allogeneic sources, which are isolated, purified, cultured, expanded, cryopreserved, resuscitated, and qualified in vitro, and then returned to the human body to treat diseases and resist aging. effect. According to the cell type, cell therapy can be divided into stem cell therapy, immune cell therapy and somatic cell therapy.

From 1960, the discovery of proliferating and differentiated hematopoietic stem cells in the bone marrow of mice, people gradually realized the existence of stem cells, until British scientists cloned Dolly the sheep in 1997, realizing the regeneration effect of stem cells, and then to Shinya Yamanaka to use Somatic cells have successfully induced pluripotent stem cells (iPSCs). In the past 50 years, stem cell technology has developed rapidly, and people have begun to hope that stem cells can be used to treat various major diseases and regenerate various tissues and organs. With the 2012 Nobel Prize in Medicine awarded to induced pluripotent stem cells (iPSCs), the potential medical applications of stem cells have been more widely recognized in countries around the world, and with the acceleration of China's aging process, the use of stored stem cells for individual The prospect of chemotherapy is also getting closer to reality. Stem cell therapy is the most advanced clinical treatment technology in the regenerative medicine revolution. Tissue or organ, and finally achieve the treatment of clinical disease through the transplantation of cell tissue or organ. Immunotherapy refers to the collection of immune cells from human peripheral blood or umbilical cord blood based on the principles and methods of immunology, large-scale culture and expansion in vitro to enhance the targeted killing function, and then returned to the human body. The immune system kills pathogens, cancer cells and mutant cells in the blood and tissues, inhibits tumor growth, and enhances the body's immunity. Immune cells with more clinical applications now include CIK cells, NK cells, and NKT cells.

Cryopreservation of cells is one of the most effective methods for long-term preservation of cells. Everyone has their own unique cells. If you freeze high-quality cells in a liquid nitrogen tank at -196°C in advance when you are young and healthy, you can stop the aging of the cells and keep them at the youngest and most vigorous forever. status. Once the body needs it in the future, the seed cells stored in advance can be used to cultivate more high-quality cells, which can be used to improve the immunity of the body, improve/enhance physical condition, fight cancer, and even save lives. In order to facilitate the storage of various cell resources, a number of cell banks or biological sample banks have been established around the world, including umbilical cord blood banks, umbilical cord mesenchymal stem cell banks, and immune cell banks.

Peripheral blood is one of the important sources of immune cells and stem cells. Under certain induction conditions, NK (natural killer cells, natural killer cells), CIK (cytokine-induced killer, cytokine-induced killer cells) and iPSCs can be expanded and cultured. (induced pluripotent stem cells, induced pluripotent stem cells) and other cells. Collect 100mL of human peripheral blood, and generally 0.6-1.5×10 ⁸ mononuclear cells can be isolated. At present, through literature and patent searches, it is found that most methods also directly freeze peripheral blood mononuclear cells. For culture, CIK cells and NK cells cultured by this method have low expansion efficiency and poor cell killing activity, which cannot meet clinical needs. Moreover, the recovery rate of cryopreserved peripheral blood mononuclear cells after recovery is only about 50%, and the number is only enough for one type of cell, such as the expansion and culture of CIK cells. Therefore, there is an urgent need for a new method that can achieve the cryopreservation requirements of different types of cells, including stem cells and immune cells, in one blood collection. For treatment, stem cells can be used for the treatment of chronic diseases including diabetes, liver disease, cardiovascular and cerebrovascular diseases, and nervous system diseases. Immune cells can be used for tumor treatment, immunity enhancement, sub-health conditioning, etc.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned deficiencies in the prior art, the present invention provides a method for constructing a human peripheral blood immune cell bank and a stem cell bank. The present invention collects human peripheral blood at one time, and uses density gradient centrifugation combined with differential centrifugation to obtain single nuclei. Cells, some cells were directly frozen; another part was sorted by flow cytometry CD3 ⁺ cells, and the CIK cells were expanded and cultured; the remaining cells of the CD3 ⁺ cells were sorted in the previous step, and CD56 ⁺ cells were sorted for NK cell expansion. Proliferate the culture; the remaining cells of the CD56 ⁺ cells sorted in the previous step are sorted by flow cytometry for CD34 ^- cells for MSC cell (mesenchymal stem cell) culture, and then encoded and cryopreserved. In this method, the need for cryopreservation of different types of cells, including stem cells and immune cells, can be achieved in one blood collection, avoiding the problem of multiple blood collections.

The technical scheme of the present invention is: a method for constructing a human peripheral blood immune cell bank and a stem cell bank, characterized in that it comprises the following steps (as shown in Figure 1 ):

S1: Collect human peripheral blood once, and obtain peripheral blood mononuclear cells by density gradient centrifugation combined with differential centrifugation; some cells are directly frozen;

S2: In step S1, in addition to the other part of the cells other than cryopreservation, the CD3 ⁺ lymphocytes are sorted by flow cytometry, and the CIK cells are expanded and cultured, and the code is cryopreserved;

S3: the remaining cells after the CD3 ⁺ lymphocytes are sorted in step S2, the CD56 ⁺ lymphocytes are sorted by flow cytometry, the NK cells are expanded and cultured, and the coding is cryopreserved;

S4: the remaining cells after the CD56 ⁺ lymphocytes are sorted in step S3 ^, the CD34- cells are sorted by flow cytometry for MSC cell culture, and then encoded and cryopreserved.

Further, in the first collection of human peripheral blood in step S1, a density gradient centrifugation method combined with a differential centrifugation method is used to obtain peripheral blood mononuclear cells, specifically:

1) Transfer the peripheral blood in the heparin sodium blood collection tube into a centrifuge tube and centrifuge; after centrifugation, prepare the upper plasma into autologous plasma for later use;

2) The centrifuged lower peripheral blood is added with physiological saline, pipetting and mixing, then added to the lymphocyte separation solution, and centrifuged again;

3) Take the buffy coat cells in the middle of the centrifuge tube and add normal saline, invert and mix, centrifuge, discard the supernatant, repeat 1-3 times (add normal saline, invert and mix, centrifuge, discard the supernatant), the precipitate is the peripheral blood PBMC .

Further, the direct cryopreservation of part of the cells in the step S1 is as follows: adding peripheral blood PBMCs into an immune cell cryopreservation solution to resuspend the cells for cryopreservation, and the cell density is 2-3×10 ⁶ cells/mL. The formula of immune cell cryopreservation solution is: by volume, including the following components: 1-5% autologous plasma, 10% DMSO, 10% dextran 40 (6% dextran-40 glucose injection), 75%-79% % of hydroxyethyl starch (colloidal solution), total 100%.

Further, in the step S2, the expansion and cultivation of CIK cells is carried out as follows:

1) KBM581 or X-VIVO15 medium was added to the sorted CD3 ⁺ cells to resuspend, INF-γ was added, and cultured in an incubator;

2) On the 2nd day, add IL-2 and CD3 Ab, and continue to cultivate; on the 4th to 5th day, supplement the complete medium,

3) On the 6th day, count, if the cell density reaches 1～2×10 ⁶ , transfer all the cells to the cell culture bag, add complete medium, and then continue to add complete medium on the 8th to 10th day;

4) On the 14th day, centrifuge, discard the supernatant, add normal saline to wash, and collect CIK cells.

The final concentration of INF-γ added above is 100-1000 IU/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 medium is: KBM581 or X-VIVO15 medium containing 1000±200 IU/mL IL-2.

Further, in the step S3, the NK cells are expanded and cultured as follows:

1) Selected CD56 ⁺ cells, add KBM581 or X-VIVO15 medium to resuspend, add IL-2, IL-21 and IL-15 to stimulate NK cell activation and expansion;

2) For the first 3 days, the cells were placed in the incubator and do not move; on the 4th day, continue to add KBM581 or X-VIVO15 medium and cytokines IL-2, IL-21, and IL-15 for culture;

2) On the 6th day, count, if the cell density reaches 1～2×10 ⁶ , transfer all cells to a cell culture bag, add KBM581 or X-VIVO15 medium and cytokines IL-2, IL-21, IL -15; then on the 8th to 10th day, continue to supplement KBM581 or X-VIVO15 medium and cytokines IL-2, IL-21, IL-15;

4) On the 14th day, centrifuge, discard the supernatant, add normal saline to wash, and collect NK cells.

Further, the final concentration of IL-2 used above is 1000±200 IU/mL, the final concentration of IL-21 is 100-1000 IU/mL, and the final concentration of IL-15 is 10-500 ng/mL.

Further, the cryopreservation in the steps S2 and S3 is as follows: adding an immune cell cryopreservation solution, and adjusting the cell density to 2-3×10 ⁷ cells/mL. The formula of immune cell cryopreservation solution is: including the following components by volume: 1-5% autologous plasma, 10% DMSO, 10% dextran 40 (6% dextran-40 glucose injection), 75%-79% KBM581 medium, total 100%.

Further, the MSC cell culture in step S4 is specifically:

1) Sort out CD34- cells, adjust the cell density with serum ^- free medium, and cultivate in an incubator;

2) On the second day, the medium was changed to remove the floating cells, fresh serum-free medium was added, and the culture was continued for 5-7 days. When the cells grew to 80-90% confluence, MSC cells were obtained by trypsinization.

Further, the serum-free medium contains 2-200 ng/mL VEGF (cell growth factor), 2-200 ng/mL PDGF (growth factor), 0.1-10 μg/mL EGF (epidermal growth factor), 1-100 μg/mL ml recombinant human insulin, 5-50 μg/ml transferrin α-MEM medium.

Further, the cryopreservation in step S4 is as follows: adding a cryopreservation solution for cryopreservation, the cryopreservation density is 0.5-1×10 ⁷ cells/mL, and the cryopreservation solution is stored in a warehouse. The cryopreservation solution formula (by volume ratio) is: 10 % DMSO, 20% platelet lysate, 70% α-MEM medium.

The advantages of the method of the present invention:

1. Collect 100mL of peripheral blood at a time, separate mononuclear cells, and simultaneously expand and culture NK cells, CIK cells and MSC cells to meet various needs.

2. Using flow cytometry to sort CD3 ⁺ cells to culture CIK, CD56 ⁺ cells to culture NK cells, the purity of CIK cells CD3 ⁺ CD56 ⁺ can reach more than 85%, and the purity of NK cells CD3 ^- CD56 ⁺ can reach more than 90%, which is more traditional The method is higher; cells proliferate faster: cells can proliferate nearly 1000 times in 14 days.

3. Using flow cytometry to sort CD34 ^- cells to culture MSC cells, the cells proliferate faster and the purity can reach more than 98%.

4. All reagents used in the culture process and cryopreservation do not contain animal-derived bovine serum, human albumin, etc.; the plasma used is from the customer's plasma isolated from peripheral blood.

Description of drawings

Fig. 1 is a flow chart of the present invention to collect peripheral blood to separate NK cells, CIK cells and MSC cells to construct a cell bank;

Figure 2 shows the morphology of CIK cells induced and cultured by flow-sorted PBMCs under an inverted microscope: A is the 3rd day after induction (×40 times); B is the 5th day after induction (×40 times); C is the 6th day after induction (×40 times);

Figure 3 is the morphological diagram of NK cells induced and cultured by flow-sorted PBMC observed under an inverted microscope: A is the 4th day after induction (×40 times); B is the 7th day after induction (×40 times);

Figure 4 is a graph showing the morphology of MSC cells induced and cultured by flow-sorted PBMC under an inverted microscope: A is the first day after passage (×40 times); B is the second day after passage (×40 times);

Fig. 5 is the cell phenotype of flow-sorted cord blood mesenchymal stem cells detected by flow cytometry.

Detailed ways

The scheme of the present invention will be described in detail below with reference to the example and the flow chart of one-time collection of peripheral blood to separate NK cells, CIK cells and MSC cells to construct a cell bank in Figure 1 of the description. Word Notes: PBMC: peripheral blood mononuclear cells; IL-2: interleukin 2; IL-15: interleukin 15; IL-21: interleukin 21; CD3 Ab: CD3 monoclonal antibody; INF-γ: Interferon γ; MSC: mesenchymal stem cells; NK: natural killer cells; CIK: cytokine-induced killer cells; CD3-PE: PE-labeled CD3 monoclonal antibody; CD56-FITC: FITC-labeled CD56 monoclonal antibody.

Example 1:

1. Isolation and cryopreservation of mononuclear cells

1. Transfer the peripheral blood in the heparin sodium blood collection tube into a 50mL centrifuge tube, centrifuge in a low-temperature refrigerated centrifuge at 4°C, 1500rpm, for 10 minutes, increase by 9 and decrease by 3 (speed 9, decrease 3, the same below);

2. After centrifugation, the upper plasma was transferred to a 50mL centrifuge tube, centrifuged at 56°C for 30 minutes, 4°C, 3000rpm for 10 minutes, raising 9 to 9, collecting the supernatant and storing it at -20°C for use as autologous plasma;

3. To the remaining peripheral blood in step 2 (the remaining part after collecting the supernatant), add normal saline to the volume ratio of 1:1 to dilute, and mix by pipetting;

4. The peripheral blood mixed and diluted in step 3 was slowly added to the upper layer of the centrifuge tube of the prepared 1.077g/mL lymphocyte separation solution (15mL/tube, manufacturer GE, product number 17-5442-02). Add 30mL;

5. Centrifuge at 1800～2500rpm/min for 20min in a low-temperature refrigerated centrifuge, increase 7 to 3, and the temperature is 20℃;

6. Take the buffy coat cells in the middle of the centrifuge tube into a new 50mL centrifuge tube, add normal saline to 50mL, invert and mix, centrifuge at 1800rpm/min for 10min;

7. Discard the supernatant, add normal saline to 50mL, resuspend the cells, centrifuge at 1600rpm/min for 8min;

8. Discard the supernatant, add normal saline to 50 mL, resuspend the cells, take 0.5 mL of the cell suspension after mixing and count, centrifuge at 1200 rpm/min for 8 min. Discard the supernatant, and the precipitation is the peripheral blood PBMC; a part of the quality inspection is qualified (quality inspection standard: sterility, no mycoplasma, endotoxin ≤ 0.25EU/mL, the number of cells ≥ 1 × 10 ⁸ ) and then proceed to the following step 9 The other part is sorted by flow cytometry, and the following two, three and four operations are performed in sequence;

9. Cryopreservation and storage: add immune cell cryopreservation solution to resuspend the cells, and the cell density is 2-3×10 ⁶ cells/mL. The formula of the freezing solution is: by volume fraction, including the following components: 1-5% autologous plasma, 10% DMSO, 10% dextran 40 (6% dextran-40 glucose injection), 75%-79% hydroxyl Ethyl starch (colloidal solution), total 100%.

2. CIK cell culture and cryopreservation

1. Use flow cytometry to sort out CD3 ⁺ cells in PBMC cells;

2. From the sorted CD3 ⁺ cells, take out 3×10 ⁷ cells, add 30 mL KBM581 or X-VIVO15 medium to resuspend, add INF-γ (final concentration 500-1000 IU/mL), and place at 37°C, Cultured in a 5% CO ₂ incubator;

3. On the second day, add IL-2 (final concentration 1000IU/mL), CD3 Ab (final concentration 50-200ng/mL), and continue to culture;

4. On the 4th day, add 30 mL of complete medium, wherein the complete medium is: KBM581 or X-VIVO15 medium containing 1000 IU/mL IL-2;

5. On the 5th day, add 90 mL of complete medium;

6. On the 6th day, count, if the cell density reaches 1～2×10 ⁶ , transfer all the cells to the cell culture bag, and add 450ml of complete medium;

7. On the 8th to 10th day, continue to add 1200mL of complete medium (a total of 1200ml for 3 days);

8. On the 11th day, take samples to detect bacteria, fungi and mycoplasma (quality control standard: no bacterial fungi, no mycoplasma infection);

9. On the 14th day, centrifuge at 2000rpm for 5min, discard the supernatant, collect the cells, add 0.9% physiological saline and wash three times, and centrifuge at 2000rpm for 5min;

10. Cryopreservation and storage: discard the supernatant, add the immune cell cryopreservation solution, and the cell density is 2-3×10 ⁷ cells/mL. The formula of cryopreservation solution is: including the following components by volume fraction: 1-5% autologous plasma, 10% DMSO, 10% dextran 40 (6% dextran-40 glucose injection), 75%-79% KBM581 culture basis, 100% of the total.

Figure 2 shows the morphological diagram of CIK cells induced and cultured by flow-sorted PBMCs observed under an inverted microscope in this example, where A is the 3rd day after induction (×40 times); B is the 5th day after induction (×40 times). 40 times); C is the 6th day after induction (×40 times), it can be seen from Figure 2: CIK cells proliferate faster, the cells are in suspension on the 3rd day, the cells begin to aggregate into clusters on the 5th day, and the On the 6th day, the cells proliferated rapidly, and the cell mass increased significantly.

3. NK cell culture and cryopreservation

1. Coating the culture flask: Add recombinant anti-human CD16 monoclonal antibody (5-50 μg/mL) to the T75 culture flask, place at 37°C for 1 hour or store at 4°C overnight;

2. Sort CD56 ⁺ cells by flow cytometry, take out 2 × 10 ⁷ cells, add 20 mL of KBM581 or X-VIVO15 medium to resuspend, transfer to a coated T75 culture flask, add IL-2 ( The final concentration of 1000IU/mL), IL-21 (final concentration of 100-1000IU/mL), and IL-15 (final concentration of 50 to 500ng/mL) stimulated NK cell activation and expansion; cells were placed at 37°C for the first 3 days, 5 %CO ₂ incubator, do not move;

3. On the 4th day, continue to add KBM581 or X-VIVO15 medium 40mL and cytokines IL-2 (final concentration 1000IU/mL), IL-21 (final concentration 100-1000IU/mL), IL-15 (final concentration 10 ~100ng/mL);

4. On the 6th day, count, if the cell density reaches 1～2×10 ⁶ , transfer all cells to a cell culture bag, add 300 mL of KBM581 or X-VIVO15 medium and cytokine IL-2 (final concentration 1000 IU/ mL), IL-21 (final concentration 100-1000IU/mL), IL-15 (final concentration 50-500ng/mL);

5. On the 8th to 10th day, continue to add 1200mL KBM581 or X-VIVO15 medium (1200ml in total for 3 days) and cytokines IL-2 (final concentration 1000IU/mL), IL-21 (final concentration 100-1000IU/mL) ), IL-15 (final concentration 50～500ng/mL);

6. On the 11th day, take samples to detect bacteria, fungi and mycoplasma (quality control standard: no bacterial fungi, no mycoplasma infection);

7. On the 14th day, centrifuge at 2000 rpm for 5 min, discard the supernatant, add 0.9% saline to wash twice, sample and count, centrifuge at 2000 rpm for 5 min, discard the supernatant, and collect NK cells;

8. Detection of cell phenotype by flow cytometry: Take the NK cells harvested in step 7 (the total number of cells is 5×10 ⁶ ) in a 50 mL centrifuge tube, add normal saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, add Resuspend the cells in 500 μL of normal saline, dispense 100 μL/EP tubes, 5 tubes, add 10 μL CD3-PE/CD56-FITC monoclonal antibody, add 10 μL CD3-PE to 1 tube, add 10 μL CD56-FITC to 1 tube, 1 tube Add isotype control, one tube as blank control, mix well, and incubate at 37°C for 30min in the dark. Centrifuge at 2000 rpm for 5 min, discard the supernatant, add 1 mL/tube of normal saline to wash the cells twice, centrifuge at 2000 rpm for 5 min, discard the supernatant, add 200 μL of normal saline to each tube to resuspend the cells, store at 4°C, and detect on a flow cytometer.

9. Cryopreservation and storage: Take the NK cells harvested in step 7, add normal saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, and add the immune cell cryopreservation solution, the cell density is 2-3×10 ⁷ cells/ mL. The formula of cryopreservation solution is: including the following components by volume fraction: 1-5% autologous plasma, 10% DMSO, 10% dextran 40 (6% dextran-40 glucose injection), 75%-79% KBM581 culture base.

The comparison data of the proliferation fold and cell phenotype of NK cells induced and cultured by flow-sorted PBMC and unsorted PBMSC-induced culture are shown in Table 1. The flow-sorted PBMC-induced culture was observed under an inverted microscope. The morphological diagram of cultured NK cells is shown in Figure 3, where A is the 4th day after induction (×40 times); B is the 7th day after induction (×40 times).

Table 1: Comparative data of NK cell proliferation fold and cell phenotype

As shown in Table 1, the purity of CD3 ^- CD56 ⁺ expression of NK cells induced and cultured by PBMC flow sorting in the present invention can reach more than 90%, which is about 10% higher than that of unsorted PBMC induced and cultured. It can be seen from Figure 3 : Cell clusters appeared on the 4th day of culture, and on the 7th day, the cells expanded greatly, and the cell clusters increased significantly.

4. MSC cell culture and cryopreservation

1. CD34- cells were sorted by flow cytometry and treated with serum ^- free medium (containing 20ng/mL VEGF, 20ng/mL PDGF, 1 μg/mL EGF, 10 μg/ml recombinant human insulin, 10 μg/ml transferrin alpha -MEM medium) to adjust the cell density to 5×10 ⁵ cells/mL, inoculate it in a T75 flask (15 mL of serum-free medium), and place it in a 37°C, 5% CO ₂ incubator;

2. On the second day, change the medium to remove floating cells, add 15 mL of fresh serum-free medium, continue to culture for 5-7 days, and observe the cell morphology and degree of fusion under an inverted microscope every day;

3. Subculture when the cells grow to 80-90% confluence, and subculture once every 3 days;

The cell passages are as follows: harvest the cells by trypsin digestion (when the cells grow to 80-90% confluence, discard the culture medium, add 3 mL/bottle of normal saline to wash the cells twice, add 1 mL/bottle of 0.05% trypsin to digest for 1 min, wait for After the cells shrunken and rounded, add 15 mL/bottle of fresh medium to terminate the digestion, collect the cells into a 50 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 50 mL of normal saline to resuspend the cells, and count 0.5 mL of the cell suspension. Centrifuge at 1200 rpm for 5 min, discard the supernatant), then add fresh medium to resuspend the cells, and seed the cells at a density of 8000 cells/cm ² .

4. Detection of cell phenotype by flow cytometry: collect P4 generation MSC cells in logarithmic growth phase (the total number of cells is 8×10 ⁶ ) in a 50 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 50 mL of normal saline Resuspend and wash twice, centrifuge to discard the supernatant, keep the cells, add 800 μL of normal saline to resuspend the cells, dispense 100 μL/EP tubes, 8 tubes, add 10 μL PE-labeled CD34, CD45, CD73, CD90, CD105, HLA-DR and PE-IgG antibody, another tube as blank control, mix well, and incubate at 37°C for 30min in the dark. Centrifuge at 1200 rpm for 5 min, discard the supernatant, add 1 mL/EP tube of normal saline to wash the cells twice, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 200 μL of normal saline to each tube to resuspend the cells, store at 4°C, and test on a flow cytometer. . The cell phenotype of the flow-sorted cord blood mesenchymal stem cells detected by flow cytometry is shown in Figure 5. It can be seen from Figure 5 that the cord blood mesenchymal stem cells highly express CD73, CD90, CD105, and the positive rate is >99%, low expression of CD34, CD45, HLA-DR, positive rate <0.5%, fully consistent with the characteristics of mesenchymal stem cells.

6. Cryopreservation and storage: After the second step, when the cell growth reaches 80-90% confluence, harvest the cells by trypsin digestion (the steps are the same as the third step), add the freezing solution to freeze the cells, and the cryopreservation density is 0.5 ~1×10 ⁷ cells/mL, stored frozen for storage. The freezing solution formula is: 10% DMSO, 20% platelet lysate, 70% α-MEM medium.

Figure 4 shows the morphological diagram of MSC cells induced and cultured by flow-sorted PBMCs observed under an inverted microscope in this example, where A is the first day after passage (×40 times); B is the second day after passage (×40 times). 40 times); as shown in Figure 4: under the inverted microscope, the cells adhered to the wall on the 2nd day after passage, and the shape was uniform, in the shape of a long spindle or spindle, which was in line with the typical shape of mesenchymal stem cells, and the cells proliferated rapidly on the 3rd day. , the degree of fusion reaches 80-90%, and it can be passaged and cryopreserved.

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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