Hydrogel and preparation method thereof

Abstract

The invention relates to hydrogel and a preparation method thereof, the hydrogel is especially used for in-vitro efficient amplification of primary T cells, and the hydrogel is prepared by the following steps: preparing a T cell amplification A solution containing IgG antibodies; preparing a T cell amplification solution B containing methacrylated hyaluronic acid; mixing the T cell amplification solution A and the T cell amplification solution B to obtain a mixed solution, and irradiating the mixed solution by using an ultraviolet light source to form the antibody modified maleimide-sulfydryl / HAMA composite hydrogel. The invention innovatively provides a'maleimide-sulfydryl / HAMA composite hydrogel system ', and compared with a single'maleimide-sulfydryl' click chemical gel, the addition of HAMA effectively regulates and controls the gelation rate, and provides the time required by uniform mixing for the hydrogel. In addition, the HAMA enhances the mechanical strength of the hydrogel, and various extracellular matrix ligand components are introduced, so that the hydrogel is more suitable for amplification of immune cells.

Description

Technical Field

[0001] The invention relates to the technical field of composite hydrogel materials, in particular to a hydrogel and a preparation method thereof, and in particular to a hydrogel for in vitro activation and expansion of primary T cells. Background Art

[0002] In recent years, immunotherapy designed based on the immune system has made significant progress in the treatment of tumors and autoimmune diseases. Cellular immunotherapy replaces or repairs other cells or tissues by expanding or modifying human cells. As a key component of the immune system, T cells play a fundamental and vital role in anti-tumor immune responses. Research and development of T cells have also made major breakthroughs, such as engineering T cells for adoptive cell therapy (ACT). Despite this, the effective activation and expansion of T cells remains one of the main pain points of adoptive T cell therapy. Specifically, primary T cells face many technical difficulties in the process of in vitro activation and expansion. For example, the activation of primary T cells requires dual signal stimulation, and traditional activation methods often find it difficult to provide these two signals at the same time, resulting in low activation efficiency. Even if successfully activated, the expansion rate of T cells is usually slow, especially in large-scale production. How to quickly and efficiently expand enough T cells to meet clinical needs is an important challenge. Traditional culture methods usually take a long time and are prone to cell exhaustion and loss of function. On the other hand, T cells are prone to differentiation and exhaustion during long-term in vitro culture, losing their ability to kill tumor cells. Traditional T cell expansion methods are not only time-consuming and costly, but also difficult to achieve cost-effective mass production. This limits their promotion and popularization in large-scale clinical applications. How to maintain the long-term functional effects of T cells during in vitro expansion is an urgent problem to be solved.

[0003] Therefore, the currently commonly used activation and amplification materials (such as antibody-coated plastic plates, magnetic beads, etc.) have certain limitations. Click chemistry based on maleimide groups and thiol groups provides a highly modular strategy for biomolecule modification, which makes it easy to introduce various functional groups and ligands by adjusting the reaction components, greatly enhancing the diversity and flexibility of chemical modification. More importantly, this reaction can be carried out under physiological conditions without the need for extreme pH, temperature or catalysts, which is particularly critical for maintaining the activity and stability of biomolecules. In addition, the reaction is known for its high reaction rate and excellent yield, which shows significant advantages in orthogonal ligand modification because it allows the rapid and efficient introduction of functional ligands on biomolecules. Nevertheless, this efficient reaction speed also brings some technical challenges. Specifically, due to the rapid completion of the reaction, the hydrogel precursor may not be uniform enough during the mixing process, which in turn causes problems such as uneven stiffness distribution inside the hydrogel and poor material uniformity. Areas with excessively high local stiffness can be toxic to cells and increase the risk of cell death. Therefore, although the click chemistry reaction based on maleimide and thiol has significant advantages in biomolecule modification, the problem of uneven mixing and the resulting inconsistent stiffness still needs to be solved in practical applications, and the uniformity of the material and the survival rate of cells cannot be ensured. Methacrylated Hyaluronic Acid (HAMA) is a photosensitive biomaterial. By introducing methacrylate groups into hyaluronic acid molecules, it has the ability to photocuring. Due to its excellent biocompatibility, hydrogel materials constructed based on HAMA have been widely used in many biomedical fields, such as tissue engineering, drug delivery, and cell culture. Compared with the click chemistry reaction based on maleimide groups and thiol groups, the photoinitiated polymerization method can generate more uniform hydrogel materials. Photoinitiated polymerization can cross-link rapidly and uniformly under mild conditions, ensure the consistency of the internal structure of the material, and avoid the problem of excessive local stiffness. In addition, sodium hyaluronate, as a natural extracellular matrix, can provide cells with a variety of extracellular matrix ligand signals to promote cell adhesion, migration and proliferation. However, the main modifiable groups on the HAMA molecule are carboxyl and amino groups, which limits its application in efficient, purification-free, modular and orthogonal modification strategies. Specifically, the types of functional groups of HAMA are limited, making it difficult to achieve flexible and diverse functional modifications, which to a certain extent restricts its versatility and customization potential in complex biomedical applications. Therefore, although HAMA has shown great potential in the biomedical field due to its good biocompatibility and uniformity, its limitations in modification flexibility still need to be further overcome.

[0004] CN113499322A discloses an injectable microsphere system for the preparation of a drug carrier for activating and amplifying tumor-infiltrating T cells. The preparation method of the injectable microsphere system provided in the patent application requires the preparation of porous microspheres, and then the nanoparticles encapsulating cytokines are grafted by amide bonds, and then the antibodies are grafted to the surface of the microspheres by the action of dopamine. Multiple steps involve interfacial bonding between different materials (such as amide bonds and dopamine-mediated covalent bonds), which may lead to problems of unstable or uneven interfaces. At the same time, this method is relatively fixed and lacks the flexibility of orthogonalization and modularization.

[0005] Despite the tremendous potential of T cells in immunotherapy, their efficient in vitro activation and expansion remains a major bottleneck.

[0006] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0007] In view of the shortcomings of the prior art, the first aspect of the present invention provides a hydrogel, which is particularly used for efficient in vitro expansion of primary T cells. The hydrogel is prepared by the following steps:

[0008] Prepare T cell expansion solution A containing IgG antibodies;

[0009] preparing T cell expansion B solution containing methacryloylated hyaluronic acid;

[0010] The T cell expansion solution A and the T cell expansion solution B prepared in the above steps are mixed to obtain a mixed solution, and the mixed solution is irradiated with an ultraviolet light source to form an antibody-modified maleimide-thiol / methacrylylated hyaluronic acid composite hydrogel.

[0011] According to a preferred embodiment, the IgG class antibody can be an anti-human CD3 / CD28 antibody.

[0012] According to a preferred embodiment, the method for preparing T cell expansion solution A comprises the following steps:

[0013] A coupling reagent, an affinity ligand, and an anti-human CD3 / CD28 antibody are sequentially added to a solution of a multi-arm polyethylene glycol derivative having a maleimide functional group at the end to prepare a T cell expansion solution A of a predetermined concentration.

[0014] The coupling agent is preferably a functionalized polyethylene glycol derivative with biotin and thiol, and more preferably a four-arm polyethylene glycol, such as 4ARM-SH-20K.

[0015] The affinity ligand is preferably a protein that can specifically bind to biotin, such as streptavidin.

[0016] According to a preferred embodiment, the method for preparing T cell expansion B solution comprises the following steps:

[0017] A multi-arm polyethylene glycol derivative having a maleimide functional group at the end is added to a methacryloyl hyaluronic acid solution of a predetermined concentration to prepare a T cell expansion B solution.

[0018] According to a preferred embodiment, the concentration of the anti-human CD3 / CD28 antibody solution is 2.6×10 -3 ~6.5×10 - 2 nM.

[0019] According to a preferred embodiment, the amount of streptavidin is 2.5×10 -4 ~6.4×10 -3 unit.

[0020] According to a preferred embodiment, Biotin-PEG 2000 The dosage of -SH is 7.6×10 -4 ~1.9×10 -2 unit.

[0021] According to a preferred embodiment, the final concentration of 4ARM-MAL-20K in the composite hydrogel is 1%.

[0022] According to a preferred embodiment, the final concentration of 4ARM-SH-20K in the composite hydrogel is 1%.

[0023] According to a preferred embodiment, the volume ratio of T cell expansion solution A to T cell expansion solution B is 1:0.

[0024] According to a preferred embodiment, the wavelength of the ultraviolet light source for photocuring is 405 nm.

[0025] According to a preferred embodiment, the light source intensity of the ultraviolet light source for photocuring is 5 to 50 mW / cm 2 .

[0026] According to a preferred embodiment, the irradiation time of the ultraviolet light source for photocuring is 5 to 60 seconds.

[0027] A second aspect of the present invention provides a method for preparing a hydrogel, the method comprising the following steps:

[0028] Prepare T cell expansion solution A containing IgG antibodies;

[0029] preparing T cell expansion B solution containing methacryloylated hyaluronic acid;

[0030] The T cell expansion solution A and the T cell expansion solution B prepared in the above steps are mixed to obtain a mixed solution, and the mixed solution is irradiated with an ultraviolet light source to form an antibody-modified maleimide-thiol / methacrylylated hyaluronic acid composite hydrogel.

[0031] The third aspect of the present invention provides a cell culture matrix material, which is particularly used for in vitro activation and expansion of primary T cells. The cell culture matrix material comprises the hydrogel provided by the first aspect of the present invention.

[0032] The fourth aspect of the present invention provides a cell culture device, which comprises the cell culture matrix material provided by the third aspect of the present invention.

[0033] According to a preferred embodiment, the cell culture device is prepared by the following steps:

[0034] The cell culture matrix material provided by the third aspect of the present invention is added to the bottom of a cell culture vessel to form a thin film, and finally a cell culture vessel coated with the cell culture matrix material is prepared.

[0035] The fifth aspect of the present invention provides the use of the hydrogel provided by the first aspect of the present invention, the preparation method of the hydrogel provided by the second aspect of the present invention, the cell culture matrix material provided by the third aspect of the present invention, and the cell culture device provided by the fourth aspect of the present invention in the activation and / or expansion of immune cells.

[0036] According to a preferred embodiment, the immune cells are T lymphocytes.

[0037] Technical effect of this technical solution: Although there are studies on drug carriers for tumor-infiltrating T cells, they are mainly studies on in vivo applications, and the flexibility and adjustability are poor. The present invention provides a hydrogel system for in vitro activation and amplification of primary T cells, which uses PEG / HAMA composite hydrogel as a macromolecular skeleton, and antibodies with T cell activation function (such as human CD3 antibodies, CD28 antibodies, etc.) are modified to this skeleton by simple, robust and efficient chemical methods, so as to achieve efficient activation and amplification of primary T cells. Specifically, the present invention uses a specific reaction between streptavidin and biotin and a click chemistry reaction between sulfhydryl (-SH) and maleimide (-Mal) to efficiently and specifically couple the antibody to the macromolecular skeleton of the hydrogel. The introduction of HAMA not only prevents the problem of excessive local rigidity of the material caused by the excessive reaction speed of thiol and maleimide, but also enhances the uniformity of the PEG hydrogel generated by the click chemistry reaction. The "maleimide-thiol / methacrylylated hyaluronic acid composite hydrogel" provided by the present invention has better uniformity, which is reflected in its more uniform microstructure, lower porosity, better cell attachment and growth conditions, more stable mechanical properties, higher biocompatibility and more uniform diffusion characteristics. In contrast, the simple "maleimide-thiol hydrogel" has more inhomogeneities and defects, which will affect its performance in applications such as T cell amplification. In addition, the introduction of HAMA provides an extracellular matrix ligand for the PEG material, further enhancing the material's ability to activate and amplify T cells. The hydrogel system designed in this way does not require subsequent removal of impurities, but can also provide optimized physical support and activation signals to promote the effective amplification of T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A graph showing a comparison of the microstructures of the maleimide-thiol / HAMA composite hydrogel and the maleimide-thiol hydrogel provided by the present invention;

[0039] Figure 2 A flow chart of the preparation method of the maleimide-thiol / HAMA composite hydrogel provided by the present invention;

[0040] Figure 3 The fluorescence staining results of 0×gel (indicated by 0× in the figure), 2×gel (indicated by 2× in the figure), 5×gel (indicated by 5× in the figure) and 10×gel (indicated by 10× in the figure) provided by the present invention;

[0041] Figure 4 The results of the growth of T cells under three different culture conditions: conventional culture plate, 0×gel (hydrogel without antibody modification) and 10×gel (hydrogel modified with high concentration of antibody);

[0042] Figure 5 The statistical graphs of human primary T cell expansion under three different culture conditions: conventional culture plate, 0×gel (hydrogel without antibody modification) and 10×gel (hydrogel modified with high concentration of antibody);

[0043] Figure 6 The results of cell status comparison when the maleimide-thiol / HAMA composite hydrogel and the maleimide-thiol PEG hydrogel provided by the present invention are used for 3D cell culture;

[0044] Figure 7 Live-dead staining images of the maleimide-thiol / HAMA composite hydrogel and the maleimide-thiol PEG hydrogel provided by the present invention when used for 3D cell culture. DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present disclosure. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is obvious that one or more embodiments may also be implemented without these specific details. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure.

[0046] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used here should be interpreted as having a meaning consistent with the context of this specification, and should not be interpreted in an idealized or overly rigid manner. The shapes and sizes in the accompanying drawings do not reflect the actual size and proportion, but only illustrate the contents of the embodiments of the present disclosure. In addition, in the claims, any reference symbols between brackets should not be constructed as limitations on the claims. The ordinal numbers used in the specification or claims, such as the words "S1", "S2", "S3", etc., to modify the steps of the claims, do not themselves imply and represent any previous ordinal number of the request step, nor do they represent the order of a request step and another request step, or the order in the manufacturing method. The use of these ordinals is only used to make a request step with a certain name clearly distinguishable from another request step.

[0047] The words "include" and "comprises" in the text do not exclude the existence of elements or steps not listed in the claims. In the following examples, various processes and methods that are not described in detail are all conventional methods or technical means in the art. The sources of the reagents used, the trade names, and the components that need to be listed are all indicated when they first appear. The same reagents used thereafter are the same as the sources indicated for the first time unless otherwise specified; the reagents and materials involved are all obtained for commercial purposes unless otherwise specified. The materials used in the present invention include: biotinylated human CD3 antibody (Biotin anti-human CD3; 317320; BioLegend), human CD28 antibody (Biotin anti-human CD28; 302904; BioLegend), streptavidin (S4762; Sigma), BIOTIN-PEG 2000 -SH (0624001-014; JenKem), 4ARM-MAL-20K (ZZ457P172; JenKem), HAMA (EFL-HAMA-150K / 400K; EFL), PBS (10010023; thermoFisher), 405nm ultraviolet light source (EFL-LS-1602; EFL). It is worth noting that the present invention is a protection for this experimental scheme and experimental design ideas. For example, self-preparation: biotinylated CD3 antibody, biotinylated CD28 antibody, HAMA, PBS, etc., cannot be protected by the present invention because of the different methods of obtaining the raw materials. In addition, the PBS buffer used in the present invention can also be replaced by pH buffer reagents such as HEPES with good biocompatibility, all of which are within the protection scope of the present invention.

[0048] The CD3 / CD28 antibody used in the present invention is a mouse anti-human CD3 / CD28 antibody, whose molecular weight is about 150KDa. The concentration of the purchased antibody is 0.5mg / ml, and the corresponding molar concentration is 13.3μM. The concentration of the anti-human CD3 / CD28 antibody indicated in the following examples is the final concentration of the CD3 / CD28 antibody in the hydrogel pre-gel solution, which is 3.92-98.05μg / ml, and the corresponding molar concentration is 2.6x10 -3 ~6.5x10 -2 nM.

[0049] In the present invention, methacrylated hyaluronic acid is also represented by HAMA; polyethylene glycol is also represented by PEG; thiol is also called thiol and represented by -SH; Biotin-PEG 2000-SH is a chemical reagent containing biotin, a polyethylene glycol (PEG) chain and a thiol (-SH) group; 4ARM-MAL-20K is a multi-arm polyethylene glycol (multi-arm PEG) derivative, specifically, it is a four-arm polyethylene glycol (4-arm PEG) with a maleimide (MAL) functional group at the end of each arm, and "20K" indicates that its molecular weight is approximately 20,000 Daltons (i.e. 20kDa); 4ARM-SH-20K is a four-arm polyethylene glycol (4-arm PEG) derivative with a thiol (-SH) group at the end of each arm, "4ARM" indicates that the PEG molecule has four branches or arms, "SH" represents the thiol functional group, and "20K" refers to its molecular weight is approximately 20,000 Daltons (i.e. 20kDa).

[0050] The present invention innovatively proposes a "maleimide-thiol / HAMA composite hydrogel system". Compared with a single "maleimide-thiol" click chemistry gel, the addition of HAMA effectively regulates the gelation rate and provides the hydrogel with the time required for uniform mixing. In addition, HAMA enhances the mechanical strength of the hydrogel and introduces a variety of extracellular matrix ligand components, making it more suitable for the expansion of immune cells. Compared with a single HAMA hydrogel, the addition of the "maleimide-thiol" click chemistry gel component solves the problem of limited HAMA modification space, allowing the material to orthogonally and modularly modify CD3 / CD28 antibodies, thereby more effectively used for T cell activation and expansion. This composite hydrogel system not only optimizes physical support and activation signals, but also improves the versatility and application flexibility of the material.

[0051] Example 1

[0052] This embodiment provides a three-dimensional culture method for T cells, and the reagent materials used include: PBS buffer, a biotinylated human CD3 / CD28 antibody solution with a concentration of 0.5 mg / mL, a streptavidin solution with a concentration of 200 units / mL, and a Biotin-PEG solution with a concentration of 500 μM. 2000 -SH solution, 10% mass concentration of 4ARM-MAL-20K solution, 10% mass concentration of 4ARM-SH-20K solution, 1.5% mass concentration of HAMA solution, and T cell suspension.

[0053] The specific steps of the three-dimensional culture method of T cells include:

[0054] Step S1: Preparation of T cell expansion medium A

[0055] Add 27 μL of PBS buffer to the centrifuge tube, add 7.84 μL of 0.5 mg / mL biotinylated human CD3 antibody solution and 7.84 μL of 0.5 mg / mL biotinylated human CD28 antibody solution to the centrifuge tube respectively; then add 1.006 μL of 200 unit / mL streptavidin solution, and react the mixture at room temperature for 30 minutes to ensure that streptavidin and antibody are fully bound. Add 1.25 μL of 500 μM Biotin-PEG to the above mixed solution. 2000 -SH solution, mix again, and react at room temperature for 30 minutes to make Biotin-PEG 2000 -SH binds to streptavidin. Finally, add 5 μL of 10% 4ARM-MAL-20K solution and react at room temperature for 30 minutes. According to this embodiment, in this step, the antibody, streptavidin, Biotin-PEG can be adjusted according to specific needs. 2000 -SH dosage, as well as selection of different types and proportions of antibodies, to obtain customized and personalized T cell expansion A fluid.

[0056] Step S2: Preparation of T cell expansion fluid containing HAMA

[0057] Add 5 μL of 10% 4ARM-SH-20K solution to the centrifuge tube, then add 33.333 μL of 1.5% HAMA solution to the centrifuge tube, then add 11.667 μL of T cell suspension, and adjust the final concentration of cells in B solution to 2×10 7 cells / mL, and gently mix to ensure that the cells are evenly distributed in the solution. According to this embodiment, in this step, the concentration of T cells in the HAMA solution and the T cell suspension can be adjusted according to specific needs to obtain a customized and personalized T cell expansion B solution.

[0058] Step S3: Preparation of gel

[0059] Take 2.5 μL of the prepared T cell expansion solution A and 2.5 μL of the prepared T cell expansion solution B, and quickly mix the T cell expansion solution A and T cell expansion solution B for 2 seconds to ensure that the two solutions are fully in contact and start the reaction. Use 405 nm ultraviolet light at 30 mW / cm 2 The mixed solution was irradiated with an intensity of 1000 nm and the cross-linking time was 30 seconds to form a HAMA / PEG composite hydrogel. According to this embodiment, in this step, the solution volume and UV irradiation conditions can be adjusted according to specific needs to obtain a customized and personalized T cell expansion gel. The results are shown in FIG. Figure 1 As shown, Figure 1 A is the microstructure of maleimide-thiol hydrogel; Figure 1B is the microscopic result of maleimide-thiol / HAMA composite hydrogel. Figure 1 The results show that the "maleimide-thiol / HAMA composite hydrogel" prepared in this embodiment is a uniformly distributed granular structure without obvious holes or irregular areas, indicating that the components of the composite hydrogel are mixed very evenly during the formation process; the porosity is low and the overall structure is more compact, which helps to maintain the stability of the cell culture environment. However, the simple "maleimide-thiol hydrogel" has obvious holes and irregular areas, high porosity, and large holes. In practical applications, it will lead to the unevenness of material exchange in the cell culture environment, and the uneven structure may cause cells to over-aggregate in some areas, while in other areas, they are sparsely distributed, affecting the overall growth and function of the cells. Since the "maleimide-thiol / HAMA composite hydrogel" prepared in this embodiment has good uniformity, the structure is conducive to uniform attachment and growth of cells. Uniform structure usually means better mechanical properties, can better withstand external stress, and maintain stable shape. Avoid deformation or rupture under stress due to unstable mechanical properties. In addition, the uniform structure of the "maleimide-thiol / HAMA composite hydrogel" prepared in this example helps to improve biocompatibility and reduce adverse reactions between cells and materials; the uniform structure is also conducive to the uniform diffusion of nutrients and metabolites, promoting the healthy growth of cells. It is worth noting that this example gives the empirical volume of the "maleimide-thiol / HAMA composite hydrogel" for T cell activation and expansion, and this volume can be adjusted.

[0060] Step S4: Add T cell culture medium for culture. The formula of the T cell culture medium used in this embodiment is advanced 1640 + 10% FBS + 200 IU / ml interleukin 2 + 1% double antibody + 1% glutamine + 50 μM β-mercaptoethanol. Taking a 96-well plate as an example, a "maleimide-thiol / HAMA composite hydrogel" loaded with T cells with a total volume of 5 μL can be prepared in each well, and the density of the T cells contained is 1×10 7 cells / mL, the required volume of T cell culture medium is 200 μL. The T cell concentration, the components of T cell culture medium, the T cell culture container, and the required volume of T cell culture medium can be adjusted individually.

[0061] Figure 6 This is a comparison result of the cell status when the maleimide-thiol / HAMA composite hydrogel and the maleimide-thiol PEG hydrogel provided in this example are used for 3D cell culture. Figure 6 It was shown that compared with maleimide-thiol PEG hydrogel, when maleimide-thiol / HAMA composite hydrogel was used for cell 3D culture, cells were evenly distributed without obvious aggregation or void areas.

[0062] Figure 7 Live-death staining diagram of maleimide-thiol / HAMA composite hydrogel and maleimide-thiol PEG hydrogel used for 3D cell culture (green: live cells; red: dead cells) provided in this example. Figure 7 It can be seen that when maleimide-thiol PEG hydrogel is used for cell 3D culture, the number of live cells is small and the number of dead cells is large, indicating that it is less effective in supporting cell survival and proliferation. When the maleimide-thiol / HAMA composite hydrogel provided in this example is used for cell 3D culture, the number of live cells increases significantly and the number of dead cells decreases, indicating that the composite hydrogel system can better support cell survival and proliferation and provide a more suitable 3D culture environment.

[0063] Example 2

[0064] This embodiment provides a two-dimensional culture and expansion method of T cells, specifically providing a hydrogel system for in vitro activation and expansion of primary T cells. Figure 2 This is a flow chart of the method for preparing the hydrogel system provided in this embodiment.

[0065] The reagent materials required for the preparation method of the hydrogel system for in vitro activation and expansion of primary T cells include: PBS buffer, 0.5 mg / mL biotinylated human CD3 antibody solution, 0.5 mg / mL biotinylated human CD28 antibody solution, 200 unit / mL streptavidin solution, 500 μM Biotin-PEG 2000 -SH solution, 10% mass concentration of 4ARM-MAL-20K solution, 10% mass concentration of 4ARM-SH-20K solution, 1.5% mass concentration of HAMA (methacryloylated hyaluronic acid) solution, anti-mouse IgG fluorescent secondary antibody (1:50 dilution).

[0066] The specific steps include:

[0067] Step S1: Preparation of 1× T cell expansion solution A (multi-arm PEG reaction solution with maleimide as the terminal group)

[0068] Add 43.2 μL of PBS buffer to the centrifuge tube, add 0.784 μL of 0.5 mg / mL human CD3 antibody solution and 0.784 μL of 0.5 mg / mL human CD28 antibody solution to the centrifuge tube, then add 0.1 μL of 200 unit / mL streptavidin solution to the centrifuge tube, and react the mixture at room temperature for 30 minutes to ensure that streptavidin and antibodies are fully bound. Then add 0.125 μL of 500 μM Biotin-PEG 2000 -SH solution, gently mix, and react at room temperature for 30 minutes to make Biotin-PEG 2000 -SH binds to streptavidin. Finally, 5 μL of 10% 4ARM-MAL-20K solution was added and reacted at room temperature for 30 minutes to complete the formation of the entire complex. The solution containing human CD3 / CD28 antibody prepared in the above steps was defined as 1×A solution.

[0069] Step S2: Prepare 2×, 5×, and 10× solution A (while keeping the total volume of solution A at 50 μL and not changing the amount of 4ARM-MAL-20K added, increase the amount of CD3 / CD28 antibody, streptavidin, and Biotin-PEG in equal proportions). 2000 -SH concentration was increased to 2, 5, and 10 times that of 1× A solution, thereby obtaining 2×, 5×, and 10× A solutions)

[0070] 2× T cell expansion solution A

[0071] CD3 antibody: 0.784 μL × 2 = 1.568 μL; CD28 antibody: 0.784 μL × 2 = 1.568 μL; streptavidin: 0.1 μL × 2 = 0.2 μL; Biotin-PEG 2000 -SH: 0.125μL×2=0.25μL; 4ARM-MAL-20K: 5μL (remain unchanged); PBS buffer: 50μL-(1.568μL+1.568μL+0.2μL+0.25μL+5μL)=41.414μL.

[0072] 5× T cell expansion solution A

[0073] CD3 antibody: 0.784 μL × 5 = 3.92 μL; CD28 antibody: 0.784 μL × 5 = 3.92 μL; streptavidin: 0.1 μL × 5 = 0.5 μL; Biotin-PEG 2000-SH: 0.125μL×5=0.625μL; 4ARM-MAL-20K: 5μL (remain unchanged); PBS buffer: 50μL-(3.92μL+3.92μL+0.5μL+0.625μL+5μL)=36.035μL.

[0074] 10× T cell expansion solution A

[0075] CD3 antibody: 0.784 μL × 10 = 7.84 μL; CD28 antibody: 0.784 μL × 10 = 7.84 μL; streptavidin: 0.1 μL × 10 = 1 μL; Biotin-PEG 2000 -SH: 0.125μL×10=1.25μL; 4ARM-MAL-20K: 5μL (remain unchanged); PBS buffer: 50μL-(7.84μL+7.84μL+1μL+1.25μL+5μL)=27.07μL.

[0076] Step S3: Preparation of T cell expansion solution B containing HAMA (multi-arm PEG / HAMA reaction solution configuration with thiol end groups)

[0077] 5 μL of 10% 4ARM-SH-20K solution was added to the centrifuge tube, 33.333 μL of 1.5% HAMA solution was added to the centrifuge tube, and then 11.667 μL of PBS buffer was added to make the final mixed solution volume reach 50 μL.

[0078] Step S4: Preparation of HAMA / PEG composite hydrogel

[0079] Take 15 μL of liquid A (select 1×, 2×, 5× or 10× according to the required type), and take 15 μL of liquid B at the same time, quickly mix 15 μL of liquid A with 15 μL of liquid B in a 96-well plate, and gently stir for about 2 seconds, and use a 405nm ultraviolet light source to cross-link the mixture for 30 seconds to form a HAMA / PEG composite hydrogel. According to the type of liquid A used, the corresponding hydrogels are defined as 1×gel, 2×gel, 5×gel and 10×gel respectively. The hydrogel without modified antibodies is defined as 0×gel. Select an anti-mouse IgG fluorescent secondary antibody diluted 1:50, incubate the hydrogel at room temperature for 4 hours, and fluorescently stain the antibodies modified on the hydrogel. Use a fluorescence microscope to observe the fluorescence intensity on the surface of the material to characterize the modification of the antibody on the hydrogel. In this embodiment, the volume of the solution can be appropriately adjusted according to different culture conditions to meet the experimental needs. Figure 3 The fluorescence staining results of 0×gel, 2×gel, 5×gel and 10×gel are shown, among which, Figure 3A is the fluorescence result of antibody modification efficiency of 0× gel; Figure 3 B is the fluorescence result of antibody modification efficiency of 2× gel; Figure 3 C is the fluorescence result of antibody modification efficiency of 5× gel; Figure 3 D is the fluorescence result of antibody modification efficiency of 10× gel; Figure 3 E is the statistical graph of fluorescence intensity of the center lines of 0×gel, 2×gel, 5×gel and 10×gel.

[0080] Figure 3 A shows that there is almost no fluorescent signal, that is, no antibody is modified on the hydrogel. Figure 3 The fluorescence signal intensity of B is lower, indicating that a small amount of antibody is modified onto the hydrogel. Figure 3 The fluorescence signal intensity of C is stronger than Figure 3 The fluorescence signal in B indicates that more antibodies are modified onto the hydrogel. Figure 3 The fluorescence signal of D was the strongest, indicating that a large amount of antibodies were modified on the hydrogel. Figure 3 In E, the curve representing the centerline fluorescence intensity of 0×gel is close to the baseline, indicating that there is almost no fluorescence signal; the curve representing the centerline fluorescence intensity of 2×gel is higher than the baseline, but the fluctuation is small, indicating that there is a certain fluorescence signal; the curve representing the centerline fluorescence intensity of 5×gel is significantly higher than the baseline and higher than the curve representing the centerline fluorescence intensity of 2×gel, with large fluctuations, indicating that the fluorescence signal is strong. The curve representing the centerline fluorescence intensity of 10×gel is the farthest from the baseline and has the largest fluctuation, indicating that the fluorescence signal is the strongest.

[0081] Figure 3 It was demonstrated that this method can positively modify anti-human CD3 / CD28 antibodies on "maleimide-thiol / HAMA composite hydrogel" without catalyst, physiological pH, ionic strength and temperature. In particular, as the antibody concentration increases, the fluorescence signal also increases accordingly, indicating that the antibody modification efficiency increases with the increase in concentration. The centerline fluorescence intensity statistical graph further quantifies this trend, showing that 0×gel has almost no fluorescence signal, while the fluorescence signals of 2×gel, 5×gel and 10×gel increase in turn. That is, the antibody modification efficiency is positively correlated with the antibody concentration.

[0082] Step S5: Expansion of primary T cells

[0083] Prepare 0×gel (hydrogel without antibody modification), 10×gel (hydrogel with high concentration of antibody modification), and prepare a conventional cell culture plate as a control group. Inoculate 2×10 4Primary T cells from the patient's draining lymph nodes were cultured in three groups: 0×gel, 10×gel and conventional culture plate groups. T cells in all groups were cultured under the same conditions for 3 days. Figure 4 The growth status of T cells under three different culture conditions: conventional culture plate, 0×gel (hydrogel without antibody modification) and 10×gel (hydrogel modified with high concentration of antibodies). Figure 4 The results showed that the primary T cells on conventional culture plates were sparsely distributed and had a low overall density. Similarly, the number of primary T cells on 0×gel (hydrogel without antibody modification) was small and the distribution was still relatively dispersed. However, the number of primary T cells on 10×gel (hydrogel modified with high concentration antibodies) increased significantly and formed dense aggregation areas, showing an obvious amplification effect. Figure 4 The results showed that 10×gel (hydrogel modified with high concentration of antibodies) could significantly promote the growth and expansion of primary T cells, which was superior to hydrogel without antibody modification (0×gel) and conventional culture plates. Figure 5 The graphs show the statistical expansion of primary human T cells under three different culture conditions: conventional culture plate, 0×gel (hydrogel without antibody modification) and 10×gel (hydrogel modified with high concentration of antibodies). Figure 5 The horizontal axis represents different culture conditions, and the vertical axis represents the OD value (Optical Density), which is measured at a wavelength of 405 nm and is used to quantify the degree of cell proliferation. The "**" symbol is marked in the figure to indicate a statistically significant difference (usually p<0.01). Figure 5 The OD value of the conventional culture plate was about 0.31, indicating that the cell proliferation effect on the conventional culture plate was average. The OD value of 0×gel was slightly lower than that of the conventional culture plate; while the OD value of 10×gel was significantly higher than that of the conventional culture plate and 0×gel group, showing a clear cell proliferation advantage. That is, the high-concentration antibody-modified hydrogel (10×gel) can effectively promote the growth and expansion of primary T cells.

[0084] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", both of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as being required. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A hydrogel, which is particularly used for efficient in vitro expansion of primary T cells, characterized in that: The hydrogel is prepared by the following steps: Prepare T cell expansion solution A containing IgG antibodies; preparing T cell expansion B solution containing methacryloylated hyaluronic acid; The T cell expansion solution A and the T cell expansion solution B are mixed to obtain a mixed solution, and the mixed solution is irradiated with an ultraviolet light source to form an antibody-modified maleimide-thiol / methacrylylated hyaluronic acid composite hydrogel.

2. The hydrogel according to claim 1, characterized in that The IgG class antibody can be an anti-human CD3 / CD28 antibody.

3. The hydrogel according to claim 1 or 2, characterized in that The method for preparing the T cell expansion solution A comprises the following steps: A coupling reagent, an affinity ligand, and an anti-human CD3 / CD28 antibody are sequentially added to a solution of a multi-arm polyethylene glycol derivative having a maleimide functional group at the end to prepare a T cell expansion solution A of a predetermined concentration.

4. The hydrogel according to claim 1 or 2, characterized in that The method for preparing T cell expansion B solution comprises the following steps: A multi-arm polyethylene glycol derivative having a maleimide functional group at the end is added to a methacryloyl hyaluronic acid solution of a predetermined concentration to prepare a T cell expansion B solution of a predetermined concentration.

5. The hydrogel according to claim 1, characterized in that The volume ratio of the T cell expansion solution A to the T cell expansion solution B is 1:

1.

6. The hydrogel according to claim 1, characterized in that The light intensity of the UV light source for light curing is 5 to 50 mW / cm 2 .

7. A method for preparing a hydrogel, characterized in that: The method comprises the following steps: Prepare T cell expansion solution A containing IgG antibodies; preparing T cell expansion B solution containing methacryloylated hyaluronic acid; The T cell expansion solution A and the T cell expansion solution B are mixed to obtain a mixed solution, and the mixed solution is irradiated with an ultraviolet light source to form an antibody-modified maleimide-thiol / methacrylylated hyaluronic acid composite hydrogel.

8. A cell culture matrix material, which is particularly used for in vitro activation and expansion of primary T cells, characterized in that: The cell culture matrix material comprises the hydrogel according to any one of claims 1 to 6.

9. A cell culture apparatus, characterized in that: The cell culture vessel comprises the cell culture matrix material according to claim 8.

10. Use of the hydrogel according to any one of claims 1 to 6, the method for preparing the hydrogel according to claim 7, the cell culture matrix material according to claim 8, and the cell culture device according to claim 9 in activating and / or expanding immune cells.

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