Affinity column for sorting t lymphocytes, method for preparing the same and method for sorting

Abstract

The application discloses an affinity column for sorting T lymphocytes, a preparation method and a sorting method, and the affinity column comprises a polymer framework and PD-1 nucleic acid aptamer modified on the polymer framework. The affinity column for sorting T lymphocytes has the advantages of simple preparation, easy operation, high activity and high purity of separated T lymphocytes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to an affinity column for sorting T lymphocytes, a preparation method thereof and a sorting method. BACKGROUND

[0002] The separation and purification of cells play an important role in in vitro diagnosis, treatment and cytological basic research. In recent years, the sources and types of cells used for cell therapy are also constantly enriched, among which the cell therapy based on T cells dominates. T cell therapy is a new precise targeted therapy for treating tumors. The surface T cell receptor can recognize tumors and release cytokines and perforin to kill cancer cells. Compared with traditional radiotherapy and chemotherapy, it causes less damage to normal tissues and has fewer side effects. In addition, the treatment using the patient's own T cells requires the separation of a sufficient number of high-purity T cells, which are then modified to recognize and attack cancer cells, thereby providing personalized and customized treatment plans for patients. Therefore, it is of great significance to quickly and efficiently separate T cells on a large scale.

[0003] Currently, there are two main types of cell separation methods. One is based on the physical and chemical properties of cells such as size (centrifugal separation and density gradient centrifugation), density (density gradient centrifugation), light scattering characteristics (cell sorter), membrane potential, etc. This type is commonly used for pre-purification and concentration, and has low specificity, making it difficult to handle on a large scale. The other is based on the affinity interaction of cell surface molecules, such as affinity matrix, flow cytometry and magnetic bead method, etc. Flow cytometry has high cell purity, high recovery rate, and the operation environment is fully enclosed, which is not easy to be contaminated. However, this method requires expensive equipment, takes a long time, and is highly stimulating to the sorted cells. Moreover, only one cell sample can be sorted in the same period of time. Immunomagnetic bead cell sorting is a relatively efficient and simple cell sorting method. The required equipment and operation are relatively simple. The cells are basically unaffected in a low magnetic field. The recovery rate and cell activity of the separated cells are superior to those of flow sorting. However, this method is expensive in terms of consumables, difficult to scale up, and only suitable for simple labeling and sorting with low cell purity requirements.

[0004] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgment or any form of suggestion that this information forms prior art with respect to the present application. SUMMARY

[0005] The present application aims to provide an affinity column for sorting T lymphocytes, which can be used to separate (or sort) T lymphocytes. The affinity column has the advantages of simple preparation, easy operation, high activity and high purity of the separated T lymphocytes.

[0006] To achieve the above object, the embodiment of the present application provides an affinity column for sorting T lymphocytes, comprising a polymer framework and a PD-1 nucleic acid aptamer modified on the polymer framework.

[0007] In one or more embodiments of the present application, the polymer framework is any one of polyacrylamide gel, polymethacrylamide gel and polyethylene glycol gel.

[0008] In one or more embodiments of the present application, the PD-1 nucleic acid aptamer has the sequence shown in SEQ ID No. 1.

[0009] The embodiment of the present application provides a preparation method of the affinity column for sorting T lymphocytes as described above, comprising the following steps:

[0010] obtaining a PD-1 nucleic acid aptamer modified by a polymer monomer, the polymer monomer being a monomer of the polymer framework;

[0011] mixing the modified PD-1 nucleic acid aptamer with the polymer monomer to generate a polymerization reaction, and obtaining the affinity column for sorting T lymphocytes.

[0012] In one or more embodiments of the present application, the step of mixing the modified PD-1 nucleic acid aptamer with the polymer monomer to generate a polymerization reaction is:

[0013] mixing a solution of the modified PD-1 nucleic acid aptamer with a solution of the polymer monomer to generate a polymerization reaction.

[0014] In one or more embodiments of the present application, the temperature of the polymerization reaction is -20 to -10℃, and the time of the polymerization reaction is at least 10h.

[0015] In one or more embodiments of the present application, the preparation method further comprises:

[0016] thawing the product of the polymerization reaction, soaking in deionized water, separating, and obtaining the affinity column for sorting T lymphocytes.

[0017] The embodiment of the present application provides a method for sorting T lymphocytes, comprising the following steps:

[0018] culturing a cell solution containing T lymphocytes to obtain a cell suspension;

[0019] pretreating the affinity column as described above;

[0020] adding the cell suspension into the affinity column, incubating, and separating.

[0021] In one or more embodiments of the present application, the step of culturing the cell solution containing T lymphocytes to obtain the cell suspension comprises:

[0022] Add complete culture medium solution to the cell solution containing T lymphocytes, centrifuge, remove supernatant, add complete culture medium solution to resuspend the cell solution;

[0023] Incubate in an incubator, filter to obtain a cell suspension.

[0024] In one or more embodiments of the present application, the step of pretreatment comprises:

[0025] Rinse the affinity column with buffer, add complete culture medium solution, and equilibrate the environment in the column.

[0026] Compared with the prior art, the affinity column for sorting T lymphocytes according to the embodiments of the present application, the preparation method and the sorting method have the advantages of simple manufacturing, easy operation, high activity and high purity of the separated T lymphocytes. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a schematic diagram of the preparation principle of the affinity column for sorting T lymphocytes according to an embodiment of the present application;

[0028] Figure 2 is a scanning electron microscope picture of the sample flake cross section of the affinity column for sorting T lymphocytes according to Example 1 of the present application;

[0029] Figure 3 is a schematic diagram of the structural formula of the PD-1 nucleic acid aptamer modified by polymer monomers according to an embodiment of the present application. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments.

[0031] Unless otherwise explicitly indicated, throughout the specification and claims, the term "comprise" or its variants such as "comprises" or "comprising" will be understood to include the stated element or component, but not to exclude the presence of other elements or components.

[0032] As shown in Figure 1 According to a preferred embodiment of the present application, an affinity column for sorting T lymphocytes comprises a polymer framework and a PD-1 nucleic acid aptamer modified on the polymer framework.

[0033] It can be understood that the affinity column uses the PD-1 nucleic acid aptamer modified on the polymer framework to recognize the PD-1 protein on the T lymphocyte, that is, the PD-1 nucleic acid aptamer specifically binds to the PD-1 protein on the T lymphocyte, so as to bind the T lymphocyte on the surface and internal pores of the affinity column, and play a role in separating (sorting) and enriching the T lymphocyte.

[0034] Among them, the internal interconnected large pore structure of the affinity column does not produce large extrusion and shear effect on the transported cells, so as to well reserve the activity and surface functional protein of the cells, and the non-target cells are separated out by pre-purification and buffer flushing. Secondly, the release of T lymphocytes can be realized by extruding or using complementary oligonucleotide reverse agent to destroy the binding between the PD-1 protein on the T lymphocyte and the PD-1 nucleic acid aptamer. This method can release high-purity T lymphocytes from the affinity column carrier without trace, and plays a role in separating, enriching and purifying T lymphocytes.

[0035] It should be noted that, Figure 1 The PD-1 nucleic acid aptamer in the formula (I) is not only modified on the end of the polymer, but also can be located in the chain of the polymer.

[0036] Specifically, the polymer framework can be considered as a polymer formed by polymerizing monomers, a gel structure composed of polymer macromolecules, such as a hydrogel.

[0037] Further, the polymer framework is any one of polyacrylamide gel, polymethacrylamide gel and polyethylene glycol gel. The polyacrylamide gel can be considered as a gel structure formed by polymerizing acrylamide.

[0038] In a specific embodiment, the PD-1 nucleic acid aptamer has the sequence shown in SEQ ID No. 1. That is, the PD-1 nucleic acid aptamer has the sequence 5'-GACGATAGCGGTGACGGCACAGACGGTACAGTTCCCGTCCCTGCACTACACGTATGCCGCTTCCGTCCGTCGCTC-3'.

[0039] The embodiment of the present application provides a preparation method of the affinity column for sorting T lymphocytes as described above, comprising the following steps:

[0040] S101, obtaining a PD-1 nucleic acid aptamer modified by a polymer monomer, the polymer monomer being a monomer of a polymer framework.

[0041] It can be understood that the polymeric monomer modified PD-1 nucleic acid aptamer is obtained so that the polymeric monomer group is connected to the PD-1 nucleic acid aptamer, so that the polymeric monomer group on the modified PD-1 nucleic acid aptamer and the polymeric monomer in step S102 undergo a polymerization reaction, thereby modifying the PD-1 nucleic acid aptamer to the polymer framework.

[0042] Taking the polymer framework as a polyacrylamide gel, i.e. the polymeric monomer as acrylamide as an example. The polymeric monomer modified PD-1 nucleic acid aptamer is an acrylamide modified PD-1 nucleic acid aptamer. The specific preparation method of the acrylamide modified PD-1 nucleic acid aptamer can adopt the solid phase phosphoramidite triester method. The acrylamide modified PD-1 nucleic acid aptamer can be synthesized by a third-party synthesis company, such as the acrylamide modified PD-1 nucleic acid aptamer synthesized by Shenguo Biotechnology Co., Ltd. as shown in the following structure formula. Figure 3

[0043] In the structure formula, the structure in the left rectangular box is the structure with the acrylamide group, and the structure outside the right rectangular box is the nucleic acid aptamer (having the sequence shown in SEQ ID No. 1). Figure 3 In the structure formula, only the first base structure formula of the 5' end of the nucleic acid aptamer (having the sequence shown in SEQ ID No. 1) is shown. Figure 3

[0044] It should be noted that the polymeric monomer modified PD-1 nucleic acid aptamer can also be synthesized according to its own needs, or it can be synthesized by other synthesis companies on the market. The difference between the steps and methods of different synthesis companies or self-synthesis will lead to the difference between the structure (group) of the polymeric monomer group and the PD-1 nucleic acid aptamer in the polymeric monomer modified PD-1 nucleic acid aptamer, i.e. Figure 3 The structure (group) in the rectangular box in the structure formula may be slightly different, as long as it does not affect the subsequent steps, does not damage the PD-1 nucleic acid aptamer, and does not affect the recognition of the PD-1 protein on the T lymphocyte by the PD-1 nucleic acid aptamer.

[0045] S102, mixing the modified PD-1 nucleic acid aptamer and the polymeric monomer to occur a polymerization reaction, and obtaining an affinity column for sorting T lymphocytes.

[0046] Specifically, step S102 includes mixing the solution of the modified PD-1 nucleic acid aptamer and the polymeric monomer solution to occur a polymerization reaction. That is, the modified PD-1 nucleic acid aptamer is configured into a solution and mixed with the polymeric monomer solution for reaction. Different polymeric monomers may result in different solvents of the polymeric monomer solution.

[0047] ​​It can be understood that the solvent in the solution of the modified PD-1 nucleic acid aptamer can be the same or different, for example, the solution of the modified PD-1 nucleic acid aptamer synthesized by Shenguo Biotechnology Co., Ltd. can be a solution obtained by dissolving the acrylamide-modified PD-1 nucleic acid aptamer as shown in Figure 3 in deionized water. Among them, the concentration of the modified PD-1 nucleic acid aptamer in the solution of the modified PD-1 nucleic acid aptamer can be about 100 μM (in this application, μM represents μmol / L). In actual operation, those skilled in the art can adjust its concentration according to actual needs.

[0048] The solvent in the polymer monomer solution can also be different according to the different polymer monomers. For example, the solvent can be deionized water for acrylamide. Among them, the concentration of the polymer monomer in the polymer monomer solution can be 4-6% wt. In actual operation, those skilled in the art can adjust its concentration according to actual needs.

[0049] In addition, in step S102, in order to allow the polymerization reaction to occur, a crosslinking agent, an initiator, a promoter and the like can also be added. The crosslinking agent, the initiator and the promoter are all products that can be directly purchased on the market, for example, the crosslinking agent can be BIS, the initiator can be APS, and the promoter can be TEMED.

[0050] In a specific embodiment, the temperature of the polymerization reaction is -20 to -10°C, and the time of the polymerization reaction is at least 10 h. The temperature of the polymerization reaction is -20 to -10°C, which can be considered as low-temperature polymerization, that is, under frozen conditions, most of the solvent is frozen to form ice crystals, and the dissolved monomers are concentrated in the un-frozen area in the ice crystal gap, and the polymerization is completed. After complete polymerization, the solvent crystals as pores melt and leave a large, interconnected pore system. These pores act as capillaries and provide channels for the flow of the mobile phase. That is, a larger pore size can be obtained in the affinity column for sorting T lymphocytes.

[0051] Further, step S102 further comprises: thawing the product of the polymerization reaction, and soaking in deionized water, separating, and obtaining the affinity column for sorting T lymphocytes. The soaking is to remove the unreacted monomers in the column, and the separation is until the affinity column is separated from the deionized water.

[0052] It can be understood that, in order to obtain the affinity column for sorting T lymphocytes, the reactor for polymerization reaction can be a columnar mold, so as to obtain a columnar affinity column. Since the conventional separation mode on the market adopts a columnar structure, the reactor for polymerization reaction of the present application can be a columnar mold. In other embodiments, the shape of the affinity column for sorting T lymphocytes can be adjusted according to actual needs, so that the reactor for polymerization reaction can be adjusted to obtain the required shape, such as spherical, sheet type, dendritic, circular truncated cone, hexahedron and the like.

[0053] The embodiment of the present application provides a sorting method of T lymphocytes, comprising the following steps:

[0054] S201, culturing the cell liquid containing T lymphocytes to obtain a cell suspension.

[0055] Specifically, step S201 comprises: adding complete culture medium solution to the cell liquid containing T lymphocytes, centrifuging, removing supernatant, adding complete culture medium to resuspend the cells; incubating in a culture box, and filtering to obtain a cell suspension. Specifically, the cell density in the culture box is about 80%-90%, and the cell suspension is obtained by filtering into a clean centrifuge tube with a cell screen.

[0056] It can be understood that the cell liquid containing T lymphocytes can be considered that all the cells in the cell liquid are T lymphocytes.

[0057] S202, pretreating the affinity column as described above.

[0058] Specifically, step S202 comprises: the pretreatment step comprises flushing the affinity column with a buffer solution, adding complete culture medium solution, and balancing the environment in the column.

[0059] Specifically, the buffer solution is a PBS buffer solution, which can be directly purchased on the market

[0060] S203, adding the cell suspension to the affinity column, incubating, and separating.

[0061] Specifically, step S203 comprises: adding the cell suspension to the upper end of the affinity column for loading. The lower end of the affinity column is closed to make the cell suspension incubate in the column for a certain period of time (for example, about 10 minutes, and the specific time can be adjusted according to needs), so that the PD-1 protein on the T lymphocytes in the cell suspension is fully combined with the PD-1 aptamer on the affinity column.

[0062] The affinity column for sorting T lymphocytes, the preparation method and the sorting method of the present application will be described in detail below in combination with specific embodiments.

[0063] Embodiment 1

[0064] (1) 20 μL of acrylamide-modified PD-1 nucleic acid aptamer solution (100 μM) was mixed with 2 ml of polyacrylamide prepolymer solution (including 4% wt of acrylamide monomer, 0.05% wt of BIS, 0.01% wt of APS, 3 μL of TEMED, and the balance of deionized water) in a columnar mold, and polymerized at -15°C for 12 h; Figure 3

[0065] (2) The polymerized affinity column was removed and thawed at room temperature, and soaked in deionized water for 24 hours to remove unreacted monomers in the column. After separation, the affinity column for sorting T lymphocytes was obtained.

[0066] The sample slice cross-section of the affinity column was quickly frozen at -80°C for 24 hours, and then placed in a vacuum freeze dryer overnight. After platinum spraying, scanning electron microscope photos were taken. The results are shown in Figure 2 It can be seen that the pore size of the affinity column is in the range of 50-200 μm, and the pore wall surface is smooth. (The effect of platinum spraying makes the surface of the polymer conductive, so that the structure of the polymer surface can be observed more clearly.)

[0067] Example 2

[0068] (1) 20 μL of acrylamide-modified PD-1 nucleic acid aptamer solution (100 μM) was mixed with 2 ml of polyacrylamide prepolymer solution (including 4% wt of acrylamide monomer, 0.05% wt of BIS, 0.01% wt of APS, 3 μL of TEMED, and the balance of deionized water) in a columnar mold, and polymerized at -15°C for 12 h; Figure 3

[0069] (2) The polymerized affinity column was removed and thawed at room temperature, and soaked in deionized water for 24 hours to remove unreacted monomers in the column. After separation, the affinity column for sorting T lymphocytes was obtained. The structure of the obtained affinity column is similar to that obtained in Example 1.

[0070] Example 3

[0071] (1) 20 μL of acrylamide-modified PD-1 nucleic acid aptamer solution (100 μM) was mixed with 2 ml of polyacrylamide prepolymer solution (including 4% wt of acrylamide monomer, 0.05% wt of BIS, 0.01% wt of APS, 3 μL of TEMED, and the balance of deionized water) in a columnar mold, and polymerized at -15°C for 12 h; Figure 3

[0072] ​​​(2) Take out the affinity column after polymerization is completed and thaw at room temperature, and soak in deionized water for 24 hours to remove the unreacted monomers in the column. After separation, the affinity column for sorting T lymphocytes is obtained. The structure of the obtained affinity column is similar to that of the affinity column obtained in Example 1.

[0073] Example 4

[0074] (1) Culture of mouse T lymphocytes (CTLL2): A frozen tube containing 1 mL of cell suspension (cells in which are T lymphocytes (CTLL2)) is quickly shaken to thaw in a 37℃ water bath, and 4 mL of complete culture medium solution (TCM-G724 of Haixing Biology) is added and mixed uniformly. Centrifugation is performed at 1000 rpm for 4 minutes, the supernatant is discarded, and about 1 mL of complete culture medium solution is added and blown uniformly. Then all the cell solution is added to a culture bottle and cultured in a CO2 incubator at 37℃ overnight. The next day, the liquid is changed and the cell density is checked. If the cell density reaches 80%-90%, the cells are filtered with a cell screen into a clean centrifuge tube to obtain a cell suspension.

[0075] (2) The affinity column obtained in Example 1 is washed with 50 mL of PBS buffer, 20 mL of complete culture medium solution is added to balance the environment in the column, 2 mL of cell suspension is added to the upper end of the affinity column for loading. The lower end of the affinity column is closed to make the cell suspension incubate in the column for 10 minutes.

[0076] (3) The lower end of the affinity column is opened, and the cell suspension (total cell number is about 100 million) is made to flow through the affinity column at a rate of 1 mL / min by using a peristaltic pump, and is eluted with an eluent. The liquid flowing out of the lower end of the affinity column is collected, and the number of cells flowing out is calculated by using a cell counting plate. The enrichment efficiency is calculated according to the following formula: enrichment efficiency (%) = (total cell number-flowing out cell number) / total cell number x 100%.

[0077] Steps (1), (2) and (3) are repeated three times, and the number of cells flowing out is 1.54 million, 1.29 million and 1.06 million respectively. According to the above formula, the enrichment efficiency (i.e. cell capture efficiency) of the affinity column in the book example is about 84.6%, 87.1% and 89.4% respectively.

[0078] From the enrichment efficiency (i.e. cell capture efficiency) data of Example 4, it can be seen that the enrichment efficiency (i.e. cell capture efficiency) of the affinity column of the present application is between 80-90%. It has good separation (sorting) performance. In this example, the cells refer to T lymphocytes.

[0079] The advantages of the affinity column for sorting T lymphocytes of the present application are:

[0080] (1) The preparation method is simple and easy to operate.

[0081] (2) has good separation (sorting) effect.

[0082] (3) can prepare different specifications of affinity column according to sample amount, and can be reused after elution.

[0083] (4) lower cost.

[0084] (5) can separate T lymphocytes on a large scale.

[0085] (6) convenient storage and transportation, long storage time, and low storage requirement.

[0086] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application to the precise forms disclosed. Many modifications and variations are possible in light of the above teachings without departing from the spirit or essential characteristics of the application. The exemplary embodiments are chosen and described in order to explain the principles of the application and its practical application to thereby enable others skilled in the art to best utilize the application and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A method for preparing an affinity column for sorting T lymphocytes, characterized in that, Includes the following steps: PD-1 nucleic acid aptamers modified with polymeric monomers were obtained, wherein the polymeric monomers were monomers of the polymer framework; The modified PD-1 nucleic acid aptamer was mixed with a polymerizing monomer to undergo a polymerization reaction at a temperature of -20 to -10°C for at least 10 hours to obtain an affinity column for sorting T lymphocytes. The affinity column for sorting T lymphocytes includes a polymer framework and a PD-1 nucleic acid aptamer modified on the polymer framework; the polymer framework is any one of polyacrylamide gel, polymethacrylamide gel, and polyethylene glycol gel.

2. The method for preparing the affinity column for sorting T lymphocytes as described in claim 1, characterized in that, The step of mixing the modified PD-1 nucleic acid aptamer with the polymerizing monomer to carry out a polymerization reaction is as follows: The modified PD-1 nucleic acid aptamer solution was mixed with the monomer solution to carry out a polymerization reaction.

3. The method for preparing the affinity column for sorting T lymphocytes as described in claim 1, characterized in that, The preparation method further includes: The product of the polymerization reaction was thawed, soaked in deionized water, and separated to obtain an affinity column for sorting T lymphocytes.

4. An affinity column for sorting T lymphocytes, characterized in that, It is prepared by the method for preparing the affinity column for sorting T lymphocytes as described in any one of claims 1 to 3.

5. The affinity column for sorting T lymphocytes as described in claim 4, characterized in that, The PD-1 nucleic acid aptamer has the sequence shown in SEQ ID No.

1.

6. A method for sorting T lymphocytes, characterized in that, Includes the following steps: Cell suspensions were obtained by culturing cell slurry containing T lymphocytes. Pretreatment of the affinity column as described in claim 4 or 5; The cell suspension was added to the affinity column, incubated, and then separated.

7. The method for sorting T lymphocytes as described in claim 6, characterized in that, The step of culturing cell fluid containing T lymphocytes to obtain a cell suspension includes: Add complete culture medium solution to the cell solution containing T lymphocytes, centrifuge, remove the supernatant, and resuspend the cell solution by adding complete culture medium solution. The cells were incubated in an incubator and then filtered to obtain a cell suspension.

8. The method for sorting T lymphocytes as described in claim 7, characterized in that, The preprocessing steps include: Rinse the affinity column with buffer solution, add complete culture medium solution, and equilibrate the column environment.

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