Culture method of high-purity CD4 + T cells

Abstract

The invention aims to provide a culture method of high-purity CD4 + T cells, and relates to the technical field of cell culture, the method can efficiently extract a high-purity CD4 + T cell population, avoids interference of other cell types, is beneficial to accurate control and research of functions and metabolic characteristics of the cells in a laboratory, and has a good application prospect. The culture method for the high-purity CD4 + T cells has the advantages that the survival rate and functional performance of the CD4 + T cells can be enhanced, adverse effects in the cell culture process are reduced, and the culture method for the high-purity CD4 + T cells not only is related to the accuracy and repeatability of laboratory research, but also directly affects the effectiveness and safety of the high-purity CD4 + T cells in clinical research and application.

Description

Technical Field

[0001] The present invention relates to the technical field of culturing high-purity CD4+T cells, and in particular to a method for culturing high-purity CD4+T cells. Background Art

[0002] Human peripheral blood contains a variety of immune cells, among which lymphocytes are the main type of immune cells in the human body, including T cells, B cells and natural killer cells. At present, immune cell therapy in clinical research and application is mainly based on T cells, such as chimeric antigen receptor technology (CAR-T) or T cell receptor (TCR-T) technology. Autologous T cells can be directly infused for the treatment of diseases such as tumors due to their lack of immunogenicity. At the same time, their memory characteristics can also inhibit the recurrence of the disease to a certain extent. Adoptive immune cell therapy refers to the infusion of autologous or allogeneic immune effector cells activated in vitro into patients to kill tumor cells in the patient's body. Adoptive immune cell therapy is an emerging treatment method with significant efficacy in current tumor immunotherapy. Among them, T cell adoptive immune cell therapy is the most widely used and most effective immunotherapy method in the world. Its working principle is to use specific T cells activated by in vitro expansion to implement the killing function of antigens.

[0003] There are two main types of specific T cells, CD4+T cells and CD8+T cells. In adoptive immune cell therapy, CD4+T cells not only have their own killing ability, but can also assist the body's own immune cells and co-adopted immune cells. They have strong versatility and can complement each other with CD8+T cells. At present, there is no report on the culture method of high-purity CD4+T cells in the existing technology. Exploring a culture method of high-purity CD4+T cells has become a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing high-purity CD4+T cells to solve the problems raised in the background technology and facilitate promotion.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: A method for culturing high-purity CD4+T cells comprises the following steps: Step 1: Isolation of PBMCs; Step 2: Sorting CD4.

[0006] Furthermore, the separation of PBMCs comprises the following steps: Step 1: Draw 50 ml of whole blood into a custom sterile sodium heparin blood collection tube and mix by inverting several times; Step 2: Centrifuge the whole blood for 15 minutes at room temperature, 1800rpm / (757g), 9 up and 7 down. After the centrifugation, transfer the upper plasma into a centrifuge tube with a pipette, and put the plasma into a water bath at 56 degrees for half an hour to inactivate. Step 3: Place the inactivated plasma in a 4-degree refrigerator for half an hour to reduce the effect of platelets on cell activation. Take the plasma out of the refrigerator and centrifuge it at 3000 rpm (1057 g) for 5 minutes to take the supernatant for later use. Step 4: Resuspend the blood cell pellet after centrifugation with an equal volume of physiological saline and mix well. Slowly add the mixed blood cells to two 10 ml / tubes of lymphocyte separation solution. Place the centrifuge tubes symmetrically in a centrifuge and centrifuge for 20 minutes at room temperature, 2000 rpm / min (939 g), 9 for rise and 0 for drop. Step 5: After centrifugation, aspirate the middle PBMC layer (white film layer), aspirate the cell layer at the junction of the two liquid surfaces, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50ml, centrifuge at 2000 rpm for 5min, discard the supernatant and cover the centrifuge tube, pat it vigorously in the palm of your hand to fully resuspend the cells, add physiological saline to 10ml, take samples and count, repeat the wash once, and discard the supernatant for later use.

[0007] Furthermore, the CD4 sorting comprises the following steps: Step 1: Prepare buffer solution, PBS + 0.5% human albumin, add 1ml human albumin to 39ml PBS, and set aside the buffer solution; Step 2: According to the number of cells, add 100 μL buffer for every 10^7 monocytes and resuspend them. Add 10 μL CytoSinctCD magnetic beads for every 10^7 monocytes. After adding the buffer and magnetic beads, gently pipette to mix, and incubate at 2-8°C for 15 minutes. Step 3: Add 1-2 ml of buffer per 10^7 monocytes, wash the cells once, mix gently, centrifuge at 300g for 10 minutes, aspirate the supernatant completely, and resuspend in 500 μL buffer; Step 4: Magnetic separation, empty 500μL buffer once, prepare centrifuge tubes to collect unlabeled cells that flow through, add the prepared cell suspension to the column before the buffer is finished, wash the column with 500μL buffer three times, prepare new centrifuge tubes, and finally quickly punch with 1ml buffer. The cells that flow through at this time are CD4 cells, which are collected for later use; Step 5: Add gentamicin (1x) to 1L GIBCO culture medium and mix well to make complete culture medium; Step 6: Use a Pasteur pipette to add 12 ml of GIBCO complete culture medium, 1.2 ml of plasma, and solution A to the prepared resuspended cells and mix well. Then use a pipette to mix well and transfer to a T75 culture flask. Step 7: Tap the culture flask vigorously before each cell transfer to fully disperse the cells; Step 8: Add 15 ml to 30 ml of Gibco complete culture medium and 1.5 ml of plasma to the T75 culture flask; Step 9: Add 30 ml to 60 ml of Gibco complete culture medium to the T75 culture flask; Step 10: Transfer the cells to a T175 culture flask and add 20 ml of plasma and Gibco complete culture medium to 200 ml; Step 11: Divide the cells equally into two 175 ml culture flasks, add 100 ml Gibco complete medium to each culture flask to a final volume of about 200 ml, with a total volume of 400 ml. The next day, take 6 ml of the cell suspension from the culture flask for testing, and use a 5 ml syringe to draw the sample from the sampling port for sterility testing; Step 12: Add gentamicin (1x) to 1L of Icosai T cell culture medium and mix well to make complete culture medium; Step 13: Divide the two bottles of cells equally into four T175 culture flasks, add Icosel T cell complete medium to a volume of 200 ml per flask, and a total volume of 800 ml; Step 14: Place the four bottles of cells into two 2L culture bags respectively, and add Icosel T cell complete medium to a volume of 900 ml per bag; Step 15: Cell collection. Collect all the cells in the two culture bags into two 500ml centrifuge bottles, centrifuge three times to collect the cell precipitate, centrifuge at 2000rpm for 7 minutes, discard the supernatant each time, tap the bottom of the bottle vigorously to resuspend the cells after each centrifugation, add normal saline to one of the centrifuge bottles, transfer the cells to the other centrifuge bottle to merge the cells, add normal saline to 500ml, invert to mix, aspirate the cells for counting, centrifuge at 2000rpm for 7 minutes, discard the supernatant, tap the bottom of the bottle vigorously to resuspend the cells, add 500ml normal saline to resuspend the cells, centrifuge at 2000rpm for 7 minutes, discard the supernatant, tap the bottom of the bottle vigorously to resuspend the cells, take out a bag of 250ml normal saline for use, use a 20ml syringe to draw 10-20ml of normal saline and add it to the culture bottle, then aspirate the cell suspension together and inject it into the 200ml blood bag, and seal it with a sealing machine.

[0008] Furthermore, if the experiment cannot be performed immediately in step 1, the blood is placed in an environment of 10 degrees and brought to room temperature half an hour before the experiment.

[0009] Furthermore, in step three, at most 10^8 monocytes are resuspended in 500 μL buffer.

[0010] As an improvement, the beneficial effects of the present invention are: The present invention provides a method for culturing high-purity CD4+ T cells. The method can efficiently extract a CD4+ T cell population with a high purity, avoid interference from other cell types, and help to accurately control and study the functions and metabolic characteristics of these cells in the laboratory. At the same time, it can enhance the survival rate and functional performance of CD4+ T cells, while reducing adverse effects in the cell culture process. The method for culturing high-purity CD4+ T cells of the present invention is not only related to the accuracy and repeatability of laboratory research, but also directly affects its effectiveness and safety in clinical research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A positive lateral flow diagram of a method for culturing high-purity CD4+T cells of the present invention; Figure 2 A leukocyte flow cytometric diagram of a method for culturing high-purity CD4+T cells of the present invention; Figure 3 A T lymphocyte flow cytometric diagram of a method for culturing high-purity CD4+T cells of the present invention; Figure 4 A diagram showing the ratio of CD3+CD8+ cells in a method for culturing high-purity CD4+T cells of the present invention; Figure 5 A diagram showing the ratio of CD3+CD4+ cells in a method for culturing high-purity CD4+T cells of the present invention; Figure 6 A diagram showing the proportion of CD3-NK+ cells in a method for culturing high-purity CD4+T cells of the present invention; Figure 7 A diagram showing the ratio of CD3-CD19+ cells in a method for culturing high-purity CD4+T cells of the present invention; Figure 8 This is a data diagram of the detection of a method for culturing high-purity CD4+T cells of the present invention; DETAILED DESCRIPTION

[0012] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0013] This embodiment provides a method for culturing high-purity CD4+T cells, and the preferred scheme is as follows: Day 1 PBMC Isolation and CD4 Sorting Isolation of PBMCs The steps for PBMC isolation and CD4 sorting on the first day are as follows: Draw 50 ml of whole blood from the blood collection tube, put it into a sterile sodium heparin blood collection tube, and mix it by inverting several times. If the experiment cannot be carried out immediately, place the blood at an ambient temperature of about 10 degrees and bring it to room temperature half an hour before the experiment.

[0014] Centrifuge the whole blood at room temperature for 15 minutes at 1800 rpm (757 g). After centrifugation, use a pipette to transfer the upper plasma to a new centrifuge tube and place the plasma in a water bath at 56 degrees for half an hour to inactivate it.

[0015] Place the extinguished plasma in a 4-degree refrigerator for half an hour to reduce the effect of platelets on cell activation. After taking the plasma out of the refrigerator, centrifuge it for 5 minutes at 3000 rpm (1057 g), and take the supernatant for later use.

[0016] Resuspend the blood cell pellet after centrifugation in an equal volume of physiological saline and mix well. Slowly add the mixed blood cells into two 10ml centrifuge tubes on the lymphocyte separation medium. Place the centrifuge tubes symmetrically in the centrifuge and centrifuge for 20 minutes at room temperature, 2000rpm (939g).

[0017] After centrifugation, aspirate the PBMC layer (buffy coat layer) in the middle layer, and try to aspirate all the cells at the junction of the two liquid surfaces. Place the buffy coat layer in a new centrifuge tube, resuspend and mix with physiological saline to a total volume of 50 ml, and then centrifuge at 2000 rpm / 5 min, and discard the supernatant.

[0018] Cover the centrifuge tube cap, tap vigorously in the palm of your hand to fully resuspend the cells, add physiological saline to 10ml, take a sample and count. Repeat the washing process once, discard the supernatant and set aside.

[0019] Sorting CD4 First, prepare a buffer by adding 1 ml of human albumin to 39 ml of PBS (phosphate buffered saline) to prepare a buffer of PBS + 0.5% human albumin.

[0020] Depending on the number of cells, add 100 μL of buffer per 10^7 monocytes for resuspending. Next, add 10 μL of CytoSinct CD magnetic beads per 10^7 monocytes, pipette gently to mix, and then incubate at 2-8°C for 15 minutes.

[0021] Add 1-2 ml of buffer per 10^7 monocytes, mix gently, centrifuge at 300g for 10 minutes, and completely aspirate the supernatant. Then resuspend the cells with 500 μL of buffer (500 μL can resuspend up to 10^8 monocytes).

[0022] Perform magnetic separation: First, let 500 μL of buffer pass through once and collect the unlabeled cells that flow through. Before the buffer runs out, add the prepared cell suspension to pass through the column, and then wash the column three times with 500 μL of buffer. Prepare a new centrifuge tube, and finally quickly rinse the column with 1 ml of buffer. The cells that flow through are CD4 cells, which are collected for later use.

[0023] Add solution B to 1 L of GIBCO culture medium and mix well to make complete culture medium.

[0024] Use a Pasteur pipette to add 12 ml of GIBCO complete culture medium, 1.2 ml of plasma, and solution A to the centrifuge tube, mix well, and add to the prepared resuspended cells. Mix again and transfer to a T75 culture flask.

[0025] Before each transfer, tap the flask vigorously to ensure that the cells are well dispersed.

[0026] On the third day, add 15 to 30 ml of Gibco complete medium and 1.5 ml of plasma to the T75 flask.

[0027] On the fifth day, add 30 to 60 ml of Gibco complete medium to the T75 flask.

[0028] On the seventh day, the cells were transferred to a T175 culture flask, and 20 ml of plasma and an appropriate amount of Gibco complete medium were added to make the total volume reach 200 ml.

[0029] On the ninth day, the cells were divided equally into two T175 culture flasks, one of which had slightly more cells. Each flask was then added with 100 ml of Gibco complete medium to bring the total volume to 400 ml. The next day, 6 ml of cell suspension was removed from the culture flask for testing, and the sample was taken from the sampling port using aseptic technique.

[0030] Add gentamicin (1x) to 1 liter of Icosyl T cell culture medium and mix to prepare complete culture medium.

[0031] On the eleventh day, the two bottles of cells were equally divided into four T175 culture flasks, and each flask was filled with 200 ml of Icosai T cell complete medium, with a total volume of 800 ml.

[0032] On the thirteenth day, the four bottles of cells were placed in two 2-liter culture bags, and each bag was filled with 900 ml of Icosai T cell complete culture medium (the culture medium can be appropriately increased according to the cell density).

[0033] On the fifteenth day, collect cells. Collect all the cells in the two culture bags into two 500 ml centrifuge bottles and centrifuge three times, each time at 2000rpm for 7 minutes, and discard the supernatant. After each centrifugation, tap the bottom of the bottle to resuspend the cells. Add a small amount of saline to one of the centrifuge bottles, transfer the cells to another centrifuge bottle to merge the cells, add saline to 500 ml, invert and mix, and take a small amount of cells for counting. Centrifuge again at 2000rpm for 7 minutes, discard the supernatant, and tap the bottom of the bottle to resuspend the cells. Add 500 ml of saline to suspend the cells again, centrifuge at 2000rpm for 7 minutes, discard the supernatant, and tap the bottom of the bottle to resuspend the cells. Take out a bag of 250 ml saline for use, use a 20 ml syringe to draw a small amount of saline (10-20 ml) and add it to the culture bottle, mix the cell suspension with it, then suck it out and inject it into the 200 ml blood bag, and seal it with a sealer.

[0034] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for culturing high-purity CD4+T cells, The following steps are involved: Step 1: Isolation of PBMCs; Step 2: sorting CD4; The PBMC separation comprises the following steps: Step 1: Draw 50 ml of whole blood into a custom sterile sodium heparin blood collection tube and mix by inverting several times; Step 2: Centrifuge the whole blood for 15 minutes at room temperature, 1800rpm / (757g), 9 up and 7 down. After the centrifugation, transfer the upper plasma into a centrifuge tube with a pipette, and put the plasma into a water bath at 56 degrees for half an hour to inactivate. Step 3: Place the extinguished plasma in a 4-degree refrigerator for half an hour to reduce the effect of platelets on cell activation. Take the plasma out of the refrigerator and centrifuge it at 3000 rpm (1057 g) for 5 minutes to take the supernatant for later use. Step 4: Resuspend the blood cell pellet after centrifugation with an equal volume of physiological saline and mix well. Slowly add the mixed blood cells to two 10 ml / tubes of lymphocyte separation solution. Place the centrifuge tubes symmetrically in a centrifuge and centrifuge for 20 minutes at room temperature, 2000 rpm / min (939 g), 9 for rise and 0 for drop. Step 5: After centrifugation, aspirate the middle PBMC layer (white film layer), aspirate the cell layer at the junction of the two liquid surfaces, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50ml, centrifuge at 2000 rpm for 5min, discard the supernatant and cover the centrifuge tube, pat it vigorously in the palm of your hand to fully resuspend the cells, add physiological saline to 10ml, take samples and count, repeat the wash once, and discard the supernatant for later use; The CD4 sorting process includes the following steps: Step 1: Prepare buffer solution, PBS + 0.5% human albumin, add 1ml human albumin to 39ml PBS, and set aside the buffer solution; Step 2: According to the number of cells, add 100 μL buffer for every 10^7 monocytes and resuspend them. Add 10 μL CytoSinctCD magnetic beads for every 10^7 monocytes. After adding the buffer and magnetic beads, gently pipette to mix, and incubate at 2-8°C for 15 minutes. Step 3: Add 1-2 ml of buffer per 10^7 monocytes, wash the cells once, mix gently, centrifuge at 300g for 10 minutes, aspirate the supernatant completely, and resuspend in 500 μL buffer; Step 4: Magnetic separation, empty 500μL buffer once, prepare centrifuge tubes to collect unlabeled cells that flow through, add the prepared cell suspension to the column before the buffer is finished, wash the column with 500μL buffer three times, prepare new centrifuge tubes, and finally quickly punch with 1ml buffer. The cells that flow through at this time are CD4 cells, which are collected for later use; Step 5: Add one tube of solution B to 1L of GIBCO culture medium and mix well to make a complete culture medium; Step 6: Use a Pasteur pipette to add 12 ml of GIBCO complete culture medium, 1.2 ml of plasma, and solution A to the prepared resuspended cells and mix well. Then use a pipette to mix well and transfer to a T75 culture flask. Step 7: Tap the culture flask vigorously before each cell transfer to fully disperse the cells; Step 8: Add 15 ml to 30 ml of Gibco complete culture medium and 1.5 ml of plasma to the T75 culture flask; Step 9: Add 30 ml to 60 ml of Gibco complete culture medium to the T75 culture flask; Step 10: Transfer the cells to a T175 culture flask and add 20 ml of plasma and Gibco complete culture medium to 200 ml; Step 11: Divide the cells equally into two 175 ml culture flasks, add 100 ml of Gibco complete medium to each culture flask to a final volume of about 200 ml, with a total volume of 400 ml. The next day, take 6 ml of the cell suspension from the culture flask for testing, and use a 5 ml syringe to draw the sample from the sampling port for sterility testing; Step 12: Add gentamicin (1x) to 1L of Icosai T cell culture medium and mix well to make complete culture medium; Step 13: Divide the two bottles of cells equally into four T175 culture flasks, add Icosel T cell complete medium to a volume of 200 ml per flask, and a total volume of 800 ml; Step 14: Place the four bottles of cells into two 2L culture bags respectively, and add Icosel T cell complete medium to a volume of 900 ml per bag; Step 15: Cell collection. Collect all the cells in the two culture bags into two 500ml centrifuge bottles, centrifuge three times to collect the cell precipitate, centrifuge at 2000rpm for 7 minutes, discard the supernatant each time, tap the bottom of the bottle vigorously to resuspend the cells after each centrifugation, add normal saline to one of the centrifuge bottles, transfer the cells to the other centrifuge bottle to merge the cells, add normal saline to 500ml, invert to mix, aspirate the cells for counting, centrifuge at 2000rpm for 7 minutes, discard the supernatant, tap the bottom of the bottle vigorously to resuspend the cells, add 500ml normal saline to resuspend the cells, centrifuge at 2000rpm for 7 minutes, discard the supernatant, tap the bottom of the bottle vigorously to resuspend the cells, take out a bag of 250ml normal saline for use, use a 20ml syringe to draw 10-20ml of normal saline and add it to the culture bottle, then aspirate the cell suspension together and inject it into the 200ml blood bag, and seal it with a sealing machine.

2. A method for culturing high-purity CD4+T cells according to claim 1, It is characterized in that If the experiment cannot be performed immediately in step 1, place the blood in an environment of 10 degrees and bring it to room temperature half an hour before the experiment.

3. The method for culturing high-purity CD4+T cells according to claim 1, It is characterized in that In the step 3, a maximum of 10^8 monocytes are resuspended in 500 μL of buffer.

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