A cell sorting stimulation kit and its use method

Abstract

The present invention relates to a cell sorting stimulation kit and a method for using the same. The kit comprises a microcarrier and a microcarrier digestion solution, wherein the raw materials of the microcarrier comprise biopolymers, sorting antibodies and cytokines. The main raw material of the microcarrier is a biopolymer, and a porous structure is naturally formed inside the biopolymer during the preparation of the microcarrier, thereby having the function of loading sorting antibodies and cytokines. The sorting antibody can effectively bind to target cells in PBMC. The cytokines can continuously and effectively stimulate the target cells bound to the microcarrier. The microcarrier digestion solution can digest the microcarrier, so that the target cells captured by the microcarrier are released.

Description

Technical Field

[0001] The present invention relates to the technical field of human cell sorting stimulation, and in particular to a cell sorting stimulation kit and a method for using the kit. Background Art

[0002] Peripheral blood mononuclear cells (PBMC) mainly include lymphocytes (such as T cells, B cells and NK cells, etc.), monocytes and dendritic cells. Through sorting and stimulation treatment, specific cells such as NK cells or T cells can be obtained. Conventional sorting and stimulation methods include: embedding antibodies in magnetic beads or well plates for sorting, adding cytokines to cell culture medium for stimulation; or using cells treated with radiation or the like as feeder cells for stimulation; or directly using different stimulating culture media for stimulation at different time periods. However, the operation steps of the above sorting and stimulation methods are too cumbersome, and the proliferation rate of stimulated cells is slow. The stimulated cells are mixed with cells that do not require stimulation and are difficult to separate, resulting in an insufficient proportion of target cells. Summary of the invention

[0003] The purpose of the present invention is to disclose a cell sorting stimulation kit and a method of using the same, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0004] To achieve the above object, the present invention is implemented through the following technical solutions:

[0005] The first aspect of the present invention is to provide a cell sorting stimulation kit.

[0006] The second aspect of the present invention is to provide a method for using the kit described in the first aspect of the present invention.

[0007] The kit described in the first aspect of the present invention comprises a microcarrier and a microcarrier digestion solution, wherein the raw materials of the microcarrier include biopolymers, sorting antibodies and cytokines. The main raw material of the microcarrier is a biopolymer, and a porous structure is naturally formed inside the biopolymer during the preparation of the microcarrier, thereby having the function of loading sorting antibodies and cytokines. The sorting antibody can effectively bind to the target cells in PBMC. The cytokine can continuously and effectively stimulate the target cells bound to the microcarrier. The microcarrier digestion solution can digest the microcarrier, so that the target cells captured by the microcarrier are released.

[0008] In some embodiments of the present invention, the kit further comprises a cell filter.

[0009] In some embodiments of the present invention, the microcarrier is a porous sphere made of biopolymers, sorting antibodies and cytokines through a cross-linking agent.

[0010] Furthermore, the preparation method of the microcarrier comprises the following steps: A) preparing a biopolymer solution, cooling it to room temperature, adding sorting antibodies, cytokines and cross-linking agents and mixing them evenly to prepare an aqueous phase; B) dripping the aqueous phase into a magnetically stirred oil phase; C) placing the mixed aqueous and oil phases in an environment below -20°C to react; and D) taking out and removing the oil phase after the reaction is completed to obtain the microcarrier.

[0011] The aqueous phase mixed with biopolymers, sorting antibodies, cytokines and cross-linking agents is wrapped into droplets in the oil phase. When the water molecules freeze at low temperature, they solidify and expand. After returning to room temperature, the water molecules melt and lose, resulting in holes inside the microcarrier. The microcarrier with a porous structure has the advantage of a large specific surface area, and can more easily capture target cells in PBMC through the sorting antibodies it carries. The lower freezing temperature helps the water molecules to condense quickly, ensuring that the microcarrier can form a suitable porous structure. If the freezing temperature of the water-oil phase is too high, the water molecules will not be able to form ice crystal particles in time and will be gelled and solidified, resulting in a smooth surface of the microcarrier, fewer holes, and a lower specific surface area. Preferably, the freezing conditions of the water-oil phase are within the range of -80~-20°C to meet general production and experimental requirements.

[0012] In some embodiments of the present invention, the particle size of the microcarrier is 50-100 μm. The pore size of the cell filter provided in the kit is slightly smaller than the particle size of the microcarrier, so that the user can conveniently discard the foreign cells not captured by the microcarrier during the sorting process.

[0013] In some embodiments of the present invention, the biopolymer is selected from collagen, gelatin or pectin. More preferably, the collagen is recombinant human collagen; the gelatin is animal-derived gelatin; and the pectin is plant-derived pectin.

[0014] Accordingly, when the biopolymer is selected from collagen or gelatin, the microcarrier digestion solution is selected from collagenase; when the biopolymer is selected from pectin, the microcarrier digestion solution is selected from pectinase.

[0015] In some embodiments of the present invention, the sorting antibody is selected from at least one of CD16, CD56, CD3 and CD28. CD16 and CD56 are sorting antibodies for capturing NK cells, and CD3 and CD28 are sorting antibodies for capturing T cells.

[0016] In some embodiments of the present invention, the cytokine is selected from at least one of IL-2 and IL-18. IL-2 can stimulate the growth of NK cells and T cells at the same time, and IL-18 can stimulate the growth of NK cells.

[0017] In some embodiments of the present invention, the cross-linking agent is genipin.

[0018] The method of use described in the second aspect of the present invention comprises the following steps: 1) adding the microcarrier to the cell basal culture medium, centrifuging to remove the supernatant, adding the complete culture medium to mix evenly, and placing in a container; 2) mixing the peripheral blood mononuclear cells separated from whole blood with the complete culture medium evenly, injecting into the container described in step 1), and placing in a cell culture incubator for incubation; 3) filtering, retaining the microcarrier, resuspending with the complete culture medium and shaking for culture; 4) filtering after the culture reaches the expected time, retaining the microcarrier, adding the microcarrier digestion solution, adding the complete culture medium to terminate the digestion reaction after the microcarrier is degraded, washing and resuspending, and centrifuging to remove the supernatant, thereby obtaining the sorted stimulated cells.

[0019] The microcarriers are stirred and cultured in the culture medium, which increases the surface area of ​​the cells in a single container and can increase the culture density of the target cells. After the culture reaches the expected density, the suspended miscellaneous cells that are not bound to the microcarriers can be filtered out through a cell filter, and then the microcarriers can be degraded by the digestive fluid to release the target cells, thereby increasing the harvest ratio of the target cells.

[0020] In some embodiments of the present invention, the centrifugation condition is 480×g to 520×g, and the centrifugation time is 2 to 3 minutes.

[0021] In some embodiments of the present invention, the incubation is performed at 37±1° C. and 5±0.1% carbon dioxide concentration.

[0022] In some embodiments of the present invention, the digestion reaction refers to immersing the microcarrier in the microcarrier digestion solution at 37° C.; the time required for digestion is 5 to 10 minutes.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The microcarrier is loaded with the sorting antibody, so that the microcarrier can specifically recognize and bind to the target cells. Subsequently, only simple filtration is required to effectively remove the foreign cells that are not bound to the microcarrier, thereby increasing the proportion of target cells in the early stage of culture.

[0025] (2) The microcarriers carry specific cytokines that can stimulate the target cells in a continuous and stable manner, which is a great improvement compared to traditional suspension culture stimulation.

[0026] (3) The microcarrier has a higher surface area ratio than the culture flask in the same volume of culture medium, which can greatly increase the culture density of the target cells.

[0027] (4) The culture process is simpler, no feeder cells are required, and there is no need to adjust and replace different culture media from time to time. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a microscope photograph of the microcarrier prepared in Example 1;

[0029] Figure 2 Schematic diagram of the cell sorting stimulation kit described in Example 1; the markings in the figure include: 1, microcarrier; 5, bottle A; 6, cell filter; 7, bottle B; 8, microcarrier digestion solution;

[0030] Figure 3 This is a schematic diagram of the principle of the microcarrier binding to the target cell described in Example 1; the symbols in the figure include: 1, microcarrier; 2, sorting antibody; 3, cytokine; 4, target cell; 9, carrier hole;

[0031] Figure 4 is a flow analysis result diagram of PBMCs before sorting and stimulation in Example 2;

[0032] Figure 5 This is a fluorescence micrograph of the microcarriers adsorbing and binding T cells for 1 day in Example 2;

[0033] Figure 6 This is a fluorescence micrograph of the microcarriers adsorbing and binding T cells for 5 days in Example 2;

[0034] Figure 7 is a micrograph of the initial moment of harvesting T cells and digesting microcarriers in Example 2;

[0035] Figure 8 is a micrograph of the harvested T cells digesting the microcarriers for 10 minutes in Example 2;

[0036] Fig. 9 is a flow cytometry analysis result diagram of harvested T cells in Example 2;

[0037] Fig.10 This is a fluorescence micrograph of the microcarriers adsorbing and binding NK cells for 1 day in Example 3;

[0038] Fig.11 This is a fluorescence micrograph of the microcarriers adsorbed and bound to NK cells for 10 days in Example 3;

[0039] Fig.12 is a micrograph of the initial moment of harvesting NK cells and digesting microcarriers in Example 3;

[0040] Fig.13 is a micrograph of the harvested NK cells digesting the microcarriers for 10 minutes in Example 3;

[0041] Fig.14 This is a flow cytometry analysis result of NK cells harvested in Example 3. DETAILED DESCRIPTION

[0042] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the methods and product instructions; the biological materials described in the methods, unless otherwise specified, can be obtained from commercial channels.

[0043] Example 1: Construction and use of a cell sorting stimulation kit.

[0044] (1) Preparation of microcarriers:

[0045] (1-1) 1 g of biopolymer was added to 5 mL of sterile water and heated to dissolve, and then sterilized by filtration using a sterile syringe and a 0.22 μm filter membrane in an ultra-clean workbench. In this example, recombinant human collagen was selected as the biopolymer.

[0046] (1-2) When the recombinant human collagen aqueous solution is cooled to room temperature, the sorting antibodies, cytokines and cross-linking agents that have been sterilized by filtration are added and mixed evenly to form an aqueous phase. To meet the needs of subsequent experiments, the corresponding aqueous phase solutions are prepared according to Table 1.

[0047]

[0048] (1-3) Pour 20 mL of sterilized vegetable oil filtered through a 0.22 μm filter membrane into a 50 mL beaker, place it on a clean bench, and use a magnetic stirrer to adjust the speed to 900 rpm. Add the aqueous phase solution dropwise into the oil phase under uniform magnetic stirring and stir evenly for 30 minutes. Seal the mixed aqueous and oil phases with a sterile sealing film and place in a -20°C refrigerator for reaction for 2 days.

[0049] (1-4) Remove the beaker, centrifuge at 4000×g for 10 minutes to remove the liquid, and add sterile water to resuspend.

[0050] (1-5) Add 5% SDS, mix and shake to wash, and centrifuge again at 4000×g for 10 minutes to remove the liquid. Finally, the precipitate is obtained to obtain the sterile microcarrier. The prepared microcarrier is as follows Figure 1 As shown, it can be observed that the diameter of the microcarrier ranges from 50 to 100 μm, presenting a porous spherical structure.

[0051] During the centrifugation operation, sterile container reagents should be used. The microcarriers can be sterilely lyophilized and stored in a -20°C refrigerator for later use.

[0052] (2) Construction of cell sorting stimulation kit:

[0053] like Figure 2 As shown, the cell sorting stimulation kit includes an A bottle 5 encapsulating the microcarrier 1 , a B bottle 7 encapsulating the microcarrier digestion solution 8 , and a cell screen 6 .

[0054] (3) Instructions for use of the cell sorting stimulation kit:

[0055] (2-1) Place the prepared microcarrier in a 15 mL centrifuge tube, inject the cell basal culture medium to the 12 mL mark, centrifuge at 500 × g for 2 minutes, remove the supernatant, then add complete culture medium, mix well, and pour into a cell shake flask.

[0056] (2-2) PBMC separated from whole blood are mixed evenly with complete culture medium and counted, and then poured into the cell shake flask described in step (2-1). The cell shake flask is placed in a cell culture incubator at 37°C and 5% carbon dioxide concentration for 12 hours to allow the target cells to recognize the sorting antibodies on the microcarriers and bind to the microcarriers.

[0057] (2-3) Remove the suspended foreign cells that are not bound to the microcarriers, rinse the microcarriers on the cell strainer with PBS into a centrifuge tube, centrifuge at 500×g for 2 minutes, remove the supernatant, resuspend with complete culture medium and pour into a new cell shake flask.

[0058] (2-4) Place the cell shaker on a horizontal shaker in a cell culture incubator and shake the culture medium regularly. Figure 3 As shown, the microcarrier 1 carries a large amount of sorting antibodies 2 and cytokines 3, and the sorting antibodies 2 are specifically bound to the target cells 4 to capture them on the surface of the microcarrier 1. At the same time, the cytokines 3 continuously stimulate the target cells 4 during the shaking culture process.

[0059] (2-5) After the culture reaches the expected time, filter the mixed solution in the cell shake flask with a 40 μm cell strainer, rinse the microcarriers on the cell strainer with PBS into a centrifuge tube, centrifuge at 500×g for 2 minutes to remove the supernatant, and repeat the rinsing with PBS and centrifugation to remove the supernatant.

[0060] (2-6) Add microcarrier digestion solution to the washed microcarriers and place in a 37°C constant temperature oven for digestion for 5-10 minutes. Remove and add complete medium to terminate the reaction. Rinse with PBS and centrifuge to remove the supernatant, repeat twice, and finally resuspend in complete medium for counting.

[0061] Example 2: Isolation of stimulated T cells from PBMCs.

[0062] The No. 1 microcarrier prepared in Example 1 was selected to cooperate with the cell sorting stimulation kit for T cell sorting stimulation.

[0063] PBMC separated from whole blood were analyzed by flow cytometry. Figure 4As shown, the analysis data shows that the CD3 positivity rate is 55.95%, and the CD56 positivity rate is 26.18%. It can be preliminarily inferred that T cells account for 55.95% and NK cells account for 26.18% of the CD45-positive lymphocytes in PBMCs before sorting stimulation.

[0064] Sorting and stimulating T cells:

[0065] (1) Place microcarrier No. 1 in a 15 mL centrifuge tube, inject 1640 cell basal medium to the 12 mL mark, centrifuge at 500×g for 2 minutes to remove the supernatant, then add complete medium and mix well before pouring into a cell shake flask. The complete medium is prepared from 1640 basal medium, 10% autologous plasma, 50 μmol / L β-mercaptoethanol, 1 mmol / L sodium pyruvate, and 10 mmol / L HEPES.

[0066] (2) PBMC separated from whole blood are mixed evenly with complete culture medium and counted, and then poured into the cell shake flask described in step (1). The cell shake flask is placed in a cell culture incubator at 37°C and 5% carbon dioxide concentration for 12 hours to allow the target cells to recognize the sorting antibody on the microcarrier and bind to the microcarrier.

[0067] (3) Use a 40 μm cell strainer to remove suspended foreign cells that are not bound to the microcarriers. Rinse the microcarriers on the cell strainer with PBS into a centrifuge tube. Centrifuge at 500 × g for 2 minutes to remove the supernatant. Resuspend the microcarriers with complete culture medium and pour into a new cell shake flask.

[0068] (4) Place the cell shaker on a horizontal shaker in the cell culture incubator and shake the culture. Set the horizontal speed to 90 rpm. After culturing for 1 day, take 50 μL of the sample and place it in a 96-well plate. Add 50 μL of AO / PI fluorescent dye and observe using a fluorescence microscope. The observation results are as follows: Figure 5 As shown in the figure, there are many green fluorescent spots of cells and very few red fluorescent spots of dead cells attached to the microcarriers, indicating that the cells recognize the antibodies on the microcarriers and bind to the microcarriers and grow well. On the third day of culture, an appropriate amount of complete culture medium was added to maintain continuous cell growth. After 5 days of culture, 50 μL of the sample was taken into a 96-well plate, 50 μL of AO / PI fluorescent dye was added, and the cells were observed under a fluorescence microscope. The observation results are shown in the figure below. Figure 6 As shown, the microcarriers are covered with a large number of green fluorescent spots of cells and a very small number of red fluorescent spots of dead cells, and the cells are obviously aggregated, indicating that the cells recognize the antibodies on the microcarriers and bind to the microcarriers and grow well.

[0069] (5) Filter the mixed solution in the cell shake flask with a 40 μm cell strainer to remove the suspended cells that are not bound to the microcarriers, rinse the microcarriers on the cell strainer with PBS into a centrifuge tube, centrifuge at 500 × g for 2 minutes to remove the supernatant, and repeat the rinsing with PBS and centrifugation to remove the supernatant.

[0070] (6) Add the microcarrier digestion solution in bottle B to the washed microcarriers and place them in a 37°C constant temperature oven for digestion. At the initial test time (0 minutes), sample 50 μL of microcarriers in a 96-well plate and observe them under a bright field microscope. The results are as follows: Figure 7 As shown in Figure 2, a large number of cell particles are attached to the surface of the transparent microcarrier. Samples were taken again after 10 minutes of digestion and observed under a bright field microscope. The results are as follows: Figure 8 As shown, the microcarriers were completely digested and the scattered cells covered the entire field of view. The digestion reaction was terminated by adding complete medium. The cells were rinsed with PBS and centrifuged to remove the supernatant, repeated twice, and finally resuspended and counted with complete medium.

[0071] The cells collected by digestion were identified by flow cytometry, and the results were as follows Fig. 9 As shown, the CD45 positive & CD3 positive rate is 98.7%, and it can be preliminarily determined that the proportion of T cells stimulated by PBMC sorting finally harvested is 98.7%, indicating that the cell sorting stimulation kit provided by the present invention has a significant function of PBMC cell sorting and stimulating T cells.

[0072] Example 3: PBMC sorting and stimulation of NK cells.

[0073] The No. 2 microcarrier prepared in Example 1 was used in combination with the cell sorting stimulation kit to perform NK cell sorting stimulation.

[0074] The PBMCs obtained from the same batch as Example 2 were used as sorting samples. Among the CD45-positive lymphocytes of the PBMCs before sorting stimulation, T cells accounted for 55.95% and NK cells accounted for 26.18%.

[0075] Sorting and stimulating NK cells:

[0076] (1) Place microcarrier No. 2 in a 15 mL centrifuge tube, inject AIM-V cell basal medium to the mark of 12 mL, centrifuge at 500×g for 2 minutes to remove the supernatant, then add complete medium and mix well, and then pour into a cell shake flask. The complete medium is prepared by AIM-V cell basal medium and 5% autologous plasma.

[0077] (2) PBMC separated from whole blood are mixed evenly with complete culture medium and counted, and then poured into the cell shake flask described in step (1). The cell shake flask is placed in a cell culture incubator at 37°C and 5% carbon dioxide concentration for 12 hours to allow the target cells to recognize the sorting antibody on the microcarrier and bind to the microcarrier.

[0078] (3) Use a 40 μm cell strainer to remove suspended foreign cells that are not bound to the microcarriers. Rinse the microcarriers on the cell strainer with PBS into a centrifuge tube. Centrifuge at 500 × g for 2 minutes to remove the supernatant. Resuspend the microcarriers with complete culture medium and pour into a new cell shake flask.

[0079] (4) Place the cell shaker on a horizontal shaker in the cell culture incubator and shake the culture. Set the horizontal speed to 90 rpm. After culturing for 1 day, take 50 μL of the sample and place it in a 96-well plate. Add 50 μL of AO / PI fluorescent dye and observe using a fluorescence microscope. The observation results are as follows: Fig.10 As shown in the figure, there are many green fluorescent spots of cells and very few red fluorescent spots of dead cells attached to the microcarriers, indicating that the cells recognize the antibodies on the microcarriers and bind to the microcarriers and grow well. On the third day of culture, an appropriate amount of complete culture medium was added to maintain continuous cell growth. After 10 days of culture, 50 μL of the sample was taken into a 96-well plate, 50 μL of AO / PI fluorescent dye was added, and observation was performed using a fluorescence microscope. The observation results are shown in the figure. Fig.11 As shown, the microcarriers are covered with a large number of green fluorescent spots of cells and a very small number of red fluorescent spots of dead cells, and the cells are obviously aggregated, indicating that the cells recognize the antibodies on the microcarriers and bind to the microcarriers and grow well.

[0080] (5) Filter the mixed solution in the cell shake flask with a 40 μm cell strainer to remove the suspended cells that are not bound to the microcarriers, rinse the microcarriers on the cell strainer with PBS into a centrifuge tube, centrifuge at 500 × g for 2 minutes to remove the supernatant, and repeat the rinsing with PBS and centrifugation to remove the supernatant.

[0081] (6) Add the microcarrier digestion solution in bottle B to the washed microcarriers and place them in a 37°C constant temperature oven for digestion. At the initial test time (0 minutes), sample 50 μL of microcarriers in a 96-well plate and observe them under a bright field microscope. The results are as follows: Fig.12 As shown in Figure 1, a large number of cell particles are attached to the surface of the transparent microcarrier. Samples were taken again after 10 minutes of incubation and observed under a bright field microscope. The results are as follows: Fig.13 As shown, the microcarriers were completely digested and the scattered cells covered the entire field of view. The digestion reaction was terminated by adding complete medium. The cells were rinsed with PBS and centrifuged to remove the supernatant, repeated twice, and finally resuspended and counted with complete medium.

[0082] The cells collected by digestion were identified by flow cytometry, and the results were as follows Fig.14 As shown, the CD3 negative & CD56 positive rate is 98.05%, and it can be preliminarily determined that the proportion of NK cells stimulated by PBMC sorting finally harvested is 98.05%, indicating that the cell sorting stimulation kit provided by the present invention has a significant function of PBMC cell sorting and stimulating NK cells.

[0083] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A cell sorting stimulation kit, characterized in that: The method comprises a microcarrier and a microcarrier digestion solution, wherein the raw materials of the microcarrier include biopolymers, sorting antibodies, cytokines and cross-linking agents, wherein the sorting antibodies are selected from at least one of CD16, CD56, CD3 and CD28, and the cytokines are selected from at least one of IL-2 and IL-18. The preparation method of the microcarrier comprises: The biopolymer is made into a solution, cooled to room temperature, and the sorting antibody, cytokine and cross-linking agent are added and mixed evenly to form an aqueous phase; Add the aqueous phase dropwise into the stirred oil phase; The mixed water-oil phase is placed in an environment below -20°C for reaction; After the reaction is completed, the oil phase is removed to obtain the microcarrier with a particle size of 50-100 μm.

2. The kit according to claim 1, characterized in that A cell strainer is also included.

3. The kit according to claim 1, characterized in that The biopolymer is selected from collagen, gelatin or pectin.

4. The kit according to claim 3, characterized in that: The microcarrier digestion solution is selected from collagenase or pectinase.

5. The kit according to claim 1, characterized in that The cross-linking agent is genipin.

6. The method for using the kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) adding the microcarrier to the cell basal culture medium, centrifuging to remove the supernatant, adding the complete culture medium and mixing evenly, and placing in a container; 2) Mixing the peripheral blood mononuclear cells separated from the whole blood with the complete culture medium, injecting them into the container of step 1), and placing them in a cell culture incubator for incubation; 3) filtering, retaining the microcarriers, resuspending them in complete culture medium, and then shaking and culturing; 4) After the expected culture time, filter and retain the microcarriers, add the microcarrier digestion solution, add complete culture medium to terminate the digestion reaction after the microcarriers are degraded, wash and resuspend, and then centrifuge to remove the supernatant, and then select the stimulated cells.

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