WO2022040979A1 - 一种体外分化和扩增t细胞的方法及其应用 - Google Patents

一种体外分化和扩增t细胞的方法及其应用 Download PDF

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WO2022040979A1
WO2022040979A1 PCT/CN2020/111376 CN2020111376W WO2022040979A1 WO 2022040979 A1 WO2022040979 A1 WO 2022040979A1 CN 2020111376 W CN2020111376 W CN 2020111376W WO 2022040979 A1 WO2022040979 A1 WO 2022040979A1
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cells
colloidal particles
micro
particles
nano structure
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French (fr)
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王鹏元
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present application relates to the field of biomedical materials, in particular to a method for in vitro differentiation and expansion of T cells and applications thereof.
  • the immune system has two branches, the innate immune system and the adaptive immune system, which are composed of a large number and very rich types of immune cells or lymphocytes.
  • Human adaptive immunity is a powerful defense system against new external pathogens. It has the memory of antigens and enables autoimmune cells to recognize the next antigen invasion.
  • Immune cells include different subgroups, and the immune cells of different subgroups have different functions and regulate each other in the process of immune response.
  • T cells are divided into several subgroups: according to the difference of T cell surface differentiation antigen (CD), it can be mainly divided into two subgroups, CD4 and CD8; according to the difference of cell surface receptor (TCR), it can be mainly divided into ⁇ T Cell ⁇ T cells; can be divided into helper T cells (Th cells), regulatory T cells (Treg cells), cytotoxic T cells (CTL or Tc cells) and delayed-type hypersensitivity T cells according to their functions; according to their response to antigens It is mainly divided into naive T cells, activated T cells and memory T cells, etc., each of which has a more refined subgroup division.
  • CD T cell surface differentiation antigen
  • TCR cell surface receptor
  • Th cells helper T cells
  • Treg cells regulatory T cells
  • CTL or Tc cells cytotoxic T cells
  • delayed-type hypersensitivity T cells according to their functions
  • Th1 cells promote cellular immunity and Th2 cells promote humoral immunity; while Th17 cells have been found to be associated with autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. Due to the numerous subtypes of immune cells, the interactions between them are complex, and new cell subgroups will be discovered with the rapid development of technology. Studying the emergence of different subtypes of immune cells and the emergence of novel subtypes is of great significance for biomedical research and clinical applications.
  • interferon gamma IGF-gamma
  • IL4 the main secretion factor and differentiation factor of Th2 cells. Therefore, to obtain immune cells with a specific phenotype in vitro, the use of these cytokines and precise concentrations is required.
  • CD4/CD8 double positive (or double phenotype) T cells DPTC
  • This microbial factor stimulates CD4 cells and downregulates the expression of the transcription factor ThpoK protein, thereby stimulating DPTC production.
  • the study of DPTC and its formation mechanism is of great significance in biomedicine. However, due to the very small number of DPTC and its short existence, it is not easy to be found and there are not many related studies.
  • the methods of stimulating immune cells in vitro mainly rely on biochemical methods; for example, combining anti-CD3 and anti-CD28 antibodies for in vitro T cell activation; using IL4 to stimulate the Th2 cell differentiation of CD4 cells.
  • biochemical methods for example, combining anti-CD3 and anti-CD28 antibodies for in vitro T cell activation; using IL4 to stimulate the Th2 cell differentiation of CD4 cells.
  • these methods all use cytokines or receptor-inhibiting antibodies, which are expensive and complicated.
  • the present application provides a method for in vitro differentiation and expansion of T cells and its application.
  • This method of in vitro differentiation and expansion of T cells does not require the use of biochemical factors, but the biophysical signals generated after short-term contact with T cells through the special micro-nano structure coating on the culture dish are converted inside the cells into Biochemical signals to promote the differentiation and expansion of T cells in vitro; to solve the problems of high cost and complex operation in existing methods.
  • This approach will not be limited to T cells, but also applies to other immune cells, such as chimeric antigen receptor (CAR)-T cells and natural killer (NK) cells.
  • CAR chimeric antigen receptor
  • NK natural killer
  • the application provides a method for in vitro differentiation and expansion of T cells, comprising the following steps:
  • step (2) transferring the T cells in step (1) to a culture dish with a micro-nano structure coating on the surface to stimulate the differentiation and expansion of the T cells; wherein the micro-nano structure coating It comprises first colloidal particles, second colloidal particles and a surfactant, and the particle size ratio of the first colloidal particles to the second colloidal particles is greater than 2;
  • the surface of the side of the micro-nano structure coating away from the petri dish has a concave-convex structure.
  • the process of isolating the T cells further includes performing activation treatment on the T cells, and the process of the activation treatment includes: transferring the isolated T cells. to a surface coated with an activator; the activator includes one or both of an anti-CD3 antibody and an anti-CD28 antibody.
  • the activation treatment time is 6-72 hours.
  • the concentration of the activator is 1-20 mg/mL.
  • the culture time of the T cells in the culture dish with the micro-nano structure coating on the surface is 4-96 hours.
  • the first colloidal particles include SiO 2 particles, ZnO particles, TiO 2 particles, Fe 2 O 3 particles, Ta 2 O 5 particles, and Al 2 O 3 particles , at least one of PMMA (polymethyl methacrylate) colloidal particles and PS (polymethyl methacrylate) colloidal particles;
  • the second colloidal particles include PMMA colloidal particles, PS colloidal particles, PLGA (polylactic acid- Glycolic acid copolymer) colloidal particles, PCL (polycaprolactone) colloidal particles, PDMS (polydimethylsiloxane) colloidal particles, AGS (sodium alginate) colloidal particles and PNIPAM (poly(N-isopropyl propylene) amide)) at least one of the colloidal particles.
  • the particle size of the first colloidal particles is 0.5 ⁇ m-10 ⁇ m, and the particle size ratio of the first colloidal particles to the second colloidal particles is (2-100): 1.
  • the distance between any two adjacent first colloidal particles is 0.001 ⁇ m-5 ⁇ m.
  • the surface of the first colloidal particles is modified with a first functional group, and the first functional group includes a hydroxyl group, an aldehyde group, a carboxyl group, a sulfur group and an amine group.
  • the surface of the second colloidal particles is modified with a second functional group, and the second functional group includes at least one of a hydroxyl group, an aldehyde group, a carboxyl group, a sulfur group and an amine group.
  • the process of detecting the cultured T cells includes:
  • the micro-nano structure coating laid on the surface of the culture dish stimulates the T cells to express transcription factors
  • the transcription factors include interleukin 4, chemokine receptor 6, interferon ⁇ , One or more of L-selectin, tumor necrosis factor, xanthate receptor-related orphan receptor ⁇ t, CD44 protein, CD25 protein, aryl hydrocarbon receptor protein, Runx3 protein and ThPOK protein.
  • the micro-nano structure coating laid on the surface of the culture dish stimulates the T cells to differentiate into CD4/CD8 double-positive T cells.
  • promoting the differentiation or expansion of T cells into different subpopulations requires additionally adding expensive cytokines or other stimulating reagents to the medium for culturing T cells, resulting in high cost; and adding cytokines or other stimulating agents It is difficult to effectively control the concentration and activity of reagents, resulting in significantly lower efficiency of obtaining specific T cell subsets.
  • the method for in vitro differentiation and expansion of T cells described in this application is streamlined and stable. In vitro differentiation and expansion; this process requires no additional cytokines or other stimulating reagents, or only needs to add far less than the amount of existing reagents, can make T cells develop in a predetermined direction of differentiation or expand against differentiation increase.
  • the method for in vitro differentiation and expansion of T cells greatly reduces the preparation cost of T cells, and can also be widely applied to the culture of other immune cells.
  • a culture dish coated with a micro-nano structure on the surface to culture T cells is expected to replace reagents such as cytokines (or growth factors) to stimulate the growth of T cells and the expression of specific cell phenotypes.
  • reagents such as cytokines (or growth factors) to stimulate the growth of T cells and the expression of specific cell phenotypes.
  • the steps in the method can also be used for subsequent studies on the functions of different subtypes of T cells and how cells are activated and regulated by specific signals, which is of great significance to biomedical research and clinical applications.
  • the present application also provides an application of the method for in vitro differentiation and expansion of T cells described in the first aspect of the present application in regulating the phenotype and quantity of T cells as an anti-tumor immune drug.
  • T cells by adjusting the specific structure of the micro-nano structure coating on the surface of the culture dish, and the time for stimulating the T cells, it can be used to adjust the phenotype of T cells; for example, T cells
  • the phenotype is of great significance in immunotherapy.
  • immune cells T cells also play an important role in the treatment of hematological tumors and solid tumors, and have great application value.
  • T cells, CAR-T cells or NK cells obtained by the method for in vitro differentiation and expansion of T cells can increase the effect and success rate of immunotherapy.
  • FIG. 1 is a process flow diagram of a method for in vitro differentiation and expansion of T cells provided by an embodiment of the present application
  • FIG. 2 is a scanning electron microscope image of the micro-nano structure coatings BCC1-BCC10 on the surface of the culture dish provided by an embodiment of the application;
  • FIG. 3 is a scanning electron microscope image of the BCC4 of the micro-nano structure coating on the surface of the culture dish provided by an embodiment of the application forming a 50-degree shooting angle along the horizontal, and the cells are attached to the coating surface;
  • FIG. 4 is a flow data diagram of activated T cells obtained by the method provided in an embodiment of the present application.
  • FIG. 6 is a graph of surface antigen expression data of CD4 cells stimulated by the method provided in an embodiment of the present application.
  • Figure 7 is a data diagram of stimulating CD4 cells to express CCR6 and IL-4 by the method provided in an embodiment of the present application;
  • FIG. 8 is a graph of cell activity of cells in an activated treatment group and an unactivated treatment cell in the method provided in an embodiment of the present application;
  • FIG. 9 is a composition diagram of CD4/CD8 double-positive T cells, CD4 cells and CD8 cells in the activated treatment group and the non-activated treatment group in the method provided in an embodiment of the present application;
  • Figure 10 shows the ratio of cells expressing both CCR6 and TNFa in coated BCC1-BCC10 cells after T cells are stimulated by the method provided in an embodiment of the application;
  • FIG. 11 is a heat map of the surface antigen ratio of T cells obtained by the method provided by an embodiment of the present application through the activation treatment group and the non-activation treatment group, and the antigen expression of CD4 and CD8 cells after separation.
  • a method for in vitro differentiation and expansion of T cells comprising the following steps:
  • T cells are isolated from tissue or peripheral blood samples;
  • the micro-nano structure coating includes the first a colloidal particle, a second colloidal particle and a surfactant, wherein the ratio of the particle size of the first colloidal particle to the second colloidal particle is greater than 2;
  • the tissue or peripheral blood sample may be, but not limited to, origin from mammals.
  • the T cells are isolated and collected from mouse spleen or other lymphoid tissue, or peripheral blood.
  • the process of isolating the T cells further includes purifying the T cells.
  • step (1) after the process of isolating the T cells, further includes performing an activation treatment on the T cells, and the process of the activation treatment includes: transferring the isolated T cells to a coating Surface activation with an activator; the activator includes one or more of anti-CD3 antibody and anti-CD28 antibody.
  • the isolated T cells are transferred to the surface of a common petri dish coated with an activator, and the activator consists of an anti-CD3 antibody and an anti-CD28 antibody.
  • the activation treatment time is 6-72 hours. In one embodiment, the activation treatment time is 12-72 hours. In another embodiment, the time for the activation treatment is 24-48 hours; or, the time for the activation treatment is 20-24 hours.
  • the activation treatment time may be, but not limited to, 6 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 40 hours, or 48 hours.
  • the concentration of the activator is 0.5-10 mg/mL.
  • the activating agent can be, but is not limited to, a buffer solution containing anti-CD3 antibody and anti-CD28 antibody, wherein the solubility of both anti-CD3 antibody and anti-CD28 antibody is 2.5 mg/mL.
  • the buffer solution can be, but is not limited to, PBS buffer.
  • the isolated T cells before the cultured T cells are transferred to a culture dish with a micro-nano structure coating on the surface, the isolated T cells may or may not be activated. . Activating T cells is beneficial to prolong the survival time of cells and reduce the death rate of T cells. In the embodiment of the present application, the isolated T cells can be activated once, twice or three times. The activated and unactivated T cells can obtain the same or different differentiation and expansion effects under the method of in vitro differentiation and expansion of T cells.
  • the surface of the side of the micro-nano structure coating away from the culture dish has a concave-convex structure and morphology.
  • the first colloidal particles and the second colloidal particles are spherical or nearly spherical in shape.
  • the structure morphology with concave-convex undulations on the surface of one side of the micro-nano structure coating refers to a structure with micro-scale and nano-scale concave-convex undulations on its surface.
  • the micro-nano structure coating is composed of the first colloidal particles, the second colloidal particles and the surfactant, and is laid on the surface of the petri dish; therefore, the micro-nano structure coating has a surface on the side away from the petri dish.
  • the micro-nano-scale concave-convex structure morphology The structure and topography of the concave-convex and ridges extend all the way to the surrounding edges of the micro-nano structure coating.
  • the structure and morphology of the concave-convex volts may be regular and/or irregular.
  • the first colloidal particles are uniformly distributed in the micro-nano structured coating, and a plurality of second colloidal particles are uniformly distributed around each of the first colloidal particles , at this time, the surface of the micro-nano structure coating is formed into a regular concave-convex structure.
  • the first colloidal particles are distributed in the micro-nano structured coating in a non-uniform and orderly manner, and a plurality of second colloidal particles are randomly distributed around each of the first colloidal particles.
  • the surface of the micro-nano structure coating is formed into an irregular structure with concave-convex undulations.
  • the micro-nano structured coating described in the present application may also have, but is not limited to, a structure and morphology with regular concave-convex undulations in some regions, and a structure and morphology with irregular concave-convex undulations in some regions.
  • the first colloidal particles include at least SiO 2 particles, ZnO particles, TiO 2 particles, Fe 2 O 3 particles, Ta 2 O 5 particles, Al 2 O 3 particles, PMMA colloidal particles and PS colloidal particles A;
  • the second colloidal particles include at least one of PMMA colloidal particles, PS colloidal particles, PLGA colloidal particles, PCL colloidal particles, PDMS colloidal particles, AGS colloidal particles and PNIPAM colloidal particles.
  • the particle size of the first colloidal particles is 0.5 ⁇ m-10 ⁇ m, and the particle size ratio of the first colloidal particles to the second colloidal particles is (2-100):1.
  • the particle size of the first colloidal particles is 1 ⁇ m-8 ⁇ m.
  • the particle size of the first colloidal particles is 1 ⁇ m-5 ⁇ m.
  • the particle size of the first colloidal particles is 0.5 ⁇ m, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m or 10 ⁇ m.
  • the particle size of the second colloidal particles is 0.01 ⁇ m-0.7 ⁇ m.
  • the particle size of the second colloidal particles is 0.1 ⁇ m-0.4 ⁇ m.
  • the particle size of the second colloidal particles is 0.01 ⁇ m, or 0.1 ⁇ m, or 0.2 ⁇ m, or 0.3 ⁇ m, or 0.4 ⁇ m, or 0.5 ⁇ m, or 0.6 ⁇ m , or 0.7 ⁇ m.
  • the distance between any two adjacent first colloidal particles is 0.001 ⁇ m-5 ⁇ m.
  • the distance between any two adjacent first colloidal particles refers to the shortest distance between the outer surfaces of two adjacent first colloidal particles.
  • the distance between any two adjacent first colloidal particles is 0.001 ⁇ m-5 ⁇ m.
  • the distance between any two adjacent first colloidal particles is 0.1 ⁇ m-1 ⁇ m.
  • the distance between any two adjacent first colloidal particles may be, but not limited to, 0.001 ⁇ m, or 0.01 ⁇ m, or 0.1 ⁇ m, or 0.15 ⁇ m, or 0.2 ⁇ m , or 0.5 ⁇ m, or 0.6 ⁇ m, or 0.8 ⁇ m, or 1 ⁇ m, or 1.5 ⁇ m, or 2 ⁇ m, or 2.5 ⁇ m, or 3 ⁇ m, or 4 ⁇ m, or 5 ⁇ m.
  • the distance between any two adjacent second colloidal particles is very small, and it can be approximately considered that they are in contact with each other.
  • the distance between any two adjacent second colloidal particles is 0.0001-200 nm.
  • the distance between any two adjacent second colloidal particles is 1-10 nm.
  • the second colloidal particles are closely arranged around the first colloidal particles, and the distance between the second colloidal particles and the first colloidal particles is 0.1-100 nm.
  • the distance between the second colloidal particles and the first colloidal particles is 1-10 nm.
  • the surface of the first colloidal particles is modified with a first functional group, and the first functional group includes at least one of a hydroxyl group, an aldehyde group, a carboxyl group, a sulfur group and an amine group
  • the first functional group includes at least one of a hydroxyl group, an aldehyde group, a carboxyl group, a sulfur group and an amine group
  • the second functional group includes at least one of a hydroxyl group, an aldehyde group, a carboxyl group, a sulfur group and an amine group.
  • the first functional group is a hydroxyl group, or an aldehyde group, or a carboxyl group, or a sulfur group, or an amine group
  • the second functional group is a hydroxyl group, or an aldehyde group, or It is a carboxyl group, or a sulfur group or an amine group.
  • the first functional group or the second functional group modified on the surface of the first colloid or the second colloid in this application can affect the fixing strength between the first colloid and the surface of the contact object;
  • a functional group or a second functional group can also correspondingly change the surface charge of the first colloid or the second colloid, affect cell adhesion, and adjust the distribution distance of the first colloid or the second colloid.
  • the mass fraction of the surfactant in the micro-nano structure coating is 0.0001%-1%.
  • the mass fraction of the surfactant in the micro-nano structure coating is 0.001%-0.1%.
  • the mass fraction of the surfactant in the micro-nano structure coating may be, but not limited to, 0.0001%, or 0.0005%, or 0.001%, or 0.01%, or 0.005%, or 0.008%, or 0.01%, or 0.05%, or 0.08%, or 0.1%, or 0.5%, or 1%.
  • the surfactant includes a cationic surfactant, an anionic surfactant or a nonionic surfactant.
  • the surfactant described in the present application can be used to further adjust the amount of charge distribution on the surfaces of the first colloidal particles and the second colloidal particles, so as to make the distance between any two adjacent first colloidal particles in the micro-nano structured coating layer The distance between the second colloidal particles and the distance between the second colloidal particles are within a certain range, and a micro-nano structure coating of the corresponding structure is formed.
  • the material of the common petri dish or the petri dish covered with the micro-nano structure coating includes glass or plastic, and the material of the plastic includes one or more of PS, PMMA, PLGA or PCL.
  • the shape and size of the petri dish can be adjusted according to actual needs, for example, the petri dish has a well plate structure.
  • the culture dish is a 6-well plate or a 24-well plate or a 96-well plate.
  • the micro-nano structure coating can be, but not limited to, be laid on the bottom surface of the holes of the well plate.
  • the biophysical signals generated by the contact of the micro-nano structure coating on the surface of the culture dish with T cells can be converted into biochemical signals that promote the differentiation and expansion of T cells in vitro; There are certain differences in the differentiation and expansion of T cells promoted by the micro-nano structure coating in vitro; by adjusting the specific morphology and culture time of the micro-nano structure coating laid on the surface of the culture dish, it can directionally promote the in vitro differentiation and expansion of T cells. differentiation and expansion.
  • the culture time of the T cells in the culture dish with the micro-nano structure coating on the surface is 4-96 hours.
  • the culture time of the T cells in the culture dish with the micro-nano structure coating on the surface is 12-96 hours.
  • the culture time of the T cells in the culture dish coated with the micro-nano structure coating on the surface is 24-72 hours; or, the T cells are coated with the micro-nano structure coating on the surface.
  • the incubation time in the petri dishes of the layers is 48-72 hours.
  • the incubation time can be, but is not limited to, 4 hours, 6 hours, 10 hours, 12 hours, 20 hours, 24 hours, 36 hours, 40 hours, 48 hours, 56 hours, 72 hours, 80 hours, or 96 hours .
  • a method of first performing immunofluorescence staining on the cultured T cells, and then performing flow detection is performed on the surface or internal proteins of the T cells, and then the T cells are detected using a flow cytometer.
  • the detection here may refer to randomly extracting part of the cultured T cells for detection.
  • the culture medium used by the T cells in the culture dish with the micro-nano structure coating on the surface is not added with cytokines or growth factors.
  • the culture medium can be conventional T cell culture medium, and the culture medium includes minimal medium, fetal bovine serum and antibiotics.
  • the cytokines are polypeptide factors that immune cells and non-immune cells can synthesize and secrete small molecules, and they can regulate various cellular physiological functions.
  • cytokines or other stimulating reagents tend to exceed the cost.
  • the culture medium comprises RPMI-1640 minimal medium, 10% fetal bovine serum, 10 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 1 ⁇ non-essential amino acid solution, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin and 55 ⁇ M 2-mercaptoethanol.
  • the micro-nano structure coating laid on the surface of the culture dish stimulates the T cells to express transcription factors
  • the transcription factors include interleukin 4 (IL-4), chemokine receptor 6 (CCR6), interferon gamma (IFNg), L-selectin (CD62L), tumor necrosis factor (TNFa), xanthate receptor-related orphan receptor ⁇ t (ROR ⁇ t), CD44 protein, CD25 protein, aryl hydrocarbon receptor protein (Ahr), one or more of Runx3 protein and ThPOK protein.
  • the transcription factors are CCR6 and IL-4.
  • the micro-nano structure coating laid on the surface of the culture dish stimulates the T cells to form CD4/CD8 double positive T cells.
  • the vast majority of T cells isolated from tissue or peripheral blood samples are CD4 and CD8 cells.
  • the method described in the present application can increase the proportion of CD4/CD8 double positive T cells in the T cells in the T cells. Also, the method described in the present application can stimulate the specific surface antigen of CD4 cells, hardly changing the ratio and cell activity between CD4 cells and CD8 cells among the T cells.
  • a method for in vitro differentiation and expansion of T cells comprising the following steps:
  • the micro-nano structure coating includes the a first colloidal particle, a second colloidal particle and a surfactant, the ratio of the particle size of the first colloidal particle to the second colloidal particle is greater than 2;
  • step S201 except that in step S201, the separated T cells are activated, the remaining steps are the same as those described in the foregoing embodiments, and are not repeated in this embodiment.
  • using a culture dish coated with a micro-nano structure on the surface to culture T cells can replace reagents such as cytokines (or growth factors) to stimulate T cell growth and specific phenotype expression.
  • cytokines or growth factors
  • the steps in the method can also be used for subsequent studies on the functions of different subtypes of T cells and how they are activated and regulated by specific signals, which is of great significance to biomedical research and clinical applications.
  • a method for preparing a culture dish with a micro-nano structure coating on the surface comprising the following steps:
  • the order of particle addition can be the first particle and then the second particle, or the second particle and then the first particle, or the first particle and the second particle are mixed first and then the second particle is added.
  • the first colloidal particles include at least one of SiO 2 particles, ZnO particles, TiO 2 particles, Fe 2 O 3 particles, Ta 2 O 5 particles, Al 2 O 3 particles, PMMA colloidal particles and PS colloidal particles ;
  • the second colloidal particles include at least one of PMMA colloidal particles, PS colloidal particles, PLGA colloidal particles, PCL colloidal particles, PDMS colloidal particles, AGS colloidal particles and PNIPAM colloidal particles.
  • the culture dish body may include, but is not limited to, at least one groove, and a surface of the culture dish body may be, but not limited to, the bottom surface of the groove.
  • the culture dish body may be a 6-hole, 12-hole, 24-hole, or 96-hole culture dish, and each hole corresponds to a groove.
  • the surface of the petri dish body can also be coated before the micro-nano structure coating is applied.
  • the surface of the petri dish body may be coated with, but not limited to, a protein solution including a gelatin solution and/or a polydopamine solution.
  • a protein solution including a gelatin solution and/or a polydopamine solution.
  • the surface of the culture dish body after the coating treatment has stronger binding force with the micro-nano structure coating.
  • the particle size of the first colloidal particles is 0.5 ⁇ m-10 ⁇ m
  • the particle size ratio of the first colloidal particles to the second colloidal particles is (2-100): 1.
  • the organic solvent includes ethanol and toluene in a volume ratio of 1:(1-10).
  • the organic solvent includes ethanol and toluene in a volume ratio of 1:3.
  • the heating temperature may be 80°C, 84°C, 88°C, 92°C, 96°C, or 100°C.
  • the heating time is 0.5-48h. Further, optionally, the heating time can be but not limited to 1-24h.
  • the process in which the first colloidal particles and the second colloidal particles in the mixed solution form the micro-nano structure coating specifically includes the following three stages: the first stage, the sedimentation process, the The first colloidal particles and the second colloidal particles are in a disordered state in the mixed solution. After standing, the first colloidal particles first settle to the bottom and arrange at the bottom under the action of gravity.
  • the second colloidal particles are in contact with the first colloidal particles, and under the interaction of the surface charges of the particles, the second colloidal particles will fill around the first colloidal particles to form a micro-nano structured coating; in the second stage, the mixed solution After complete evaporation, the organic solution is used to make the micro-nano structure coating composed of the first colloidal particles and the second colloidal particles more attached to the surface of the petri dish body; in the third stage, the petri dish body is placed on the surface. Heating in an oven and cooling to obtain the Petri dish coated with the nanostructure coating.
  • the surface of the finally obtained micro-nano structured coating has a concave-convex structure.
  • the cell culture dish after the cell culture dish with the micro-nano structure coating on the surface is prepared, the cell culture dish can be cleaned and sterilized. For example, it can be washed with buffer or deionized water and sterilized by means of UV light. Because the positions of the first colloidal particles and the second colloidal particles are relatively fixed after the micro-nano structure coating is fabricated; therefore, the surface structure of the micro-nano structure coating will not change after cleaning.
  • the content and type of the surfactant can be used to obtain a micro-nano structured coating with a variety of concave-convex structure and morphology.
  • its specific morphology can be obtained by inspection with scanning electron microscope (SEM).
  • SEM scanning electron microscope
  • the surface of the group of Example 1-1 was prepared with a micro-nano structure coating.
  • Petri dishes, the corresponding micro- and nanostructured coatings were named BCC1-BCC10, respectively.
  • the culture plates prepared based on the above-mentioned examples 1-10 were respectively coated with the micro-nano structure coating BCC1-10, and the culture plate without the micro-nano structure coating was used as a blank control group, and after gold spraying treatment ( A film of about 10 nm thick was formed), and the specific surface structure of the micro-nanostructured coating in the cell culture plate was analyzed using a field emission scanning electron microscope (FE-SEM) at 20 keV, as shown in FIG. 2 .
  • FE-SEM field emission scanning electron microscope
  • the distance between adjacent first colloidal particles is 1.5 ⁇ m
  • the spherical diameter of the first colloidal particles is about 2 ⁇ m
  • the arrangement shape is hexagonal
  • adjacent first colloidal particles are hexagonal.
  • the distance between colloidal particles is 1.2 ⁇ m, the spherical diameter of the first colloidal particles is about 1.7 ⁇ m, and the shape of the arrangement is hexagonal; in BCC3 of Example 3, the distance between adjacent first colloidal particles is 1.2 ⁇ m, and the first The spherical diameter is about 1.85 ⁇ m, and its arrangement shape is hexagonal; in BCC4 of Example 4, the distance between adjacent first colloidal particles is 1.3 ⁇ m, the spherical diameter of the first colloidal particles is about 1.8 ⁇ m, and its arrangement shape is six.
  • BCC5 of Example 5 the distance between adjacent first colloidal particles is 1.2 ⁇ m, the spherical diameter of the first colloidal particles is about 1.8 ⁇ m, and the arrangement shape is hexagonal; in BCC6 of Example 6, adjacent The distance between the first colloidal particles is 1.3 ⁇ m, the spherical diameter of the first colloidal particles is about 1.9 ⁇ m, and the arrangement shape is hexagonal; in BCC7 of Example 7, the distance between adjacent first colloidal particles is 1.0 ⁇ m, and the first colloidal particles The spherical diameter of the particles is about 2.05 ⁇ m, and the shape of the arrangement is hexagonal; in BCC8 of Example 8, the distance between adjacent first colloidal particles is 1.0 ⁇ m, and the spherical diameter of the first colloidal particles is about 2.05 ⁇ m, and the shape of the arrangement is about 2.05 ⁇ m.
  • T cells Detect the effect of in vitro differentiation and expansion of T cells on the cell phenotype of CD4 and CD8 cells.
  • the isolated and purified T cells were added to a common 96-well plate culture dish with anti-CD3 antibody and anti-CD28 antibody added. About 50,000 cells/200 ⁇ L culture medium, cultured for 24-48 hours; the cultured T cells were transferred to the culture dish containing the micro-nanostructure coating of Example 1-10 and the blank control 96-well plate culture dish, and cultured for 6 -72 hours.
  • the culture medium consists of: RPMI-1640 substrate medium, which includes 10% FBS, 10mM HEPES, 1x non-essential amino acid solution, 1mM sodium pyruvate, 2mM L-glutamine, 100U/mL penicillin, 100 ⁇ g/mL chain and 55 ⁇ M 2-mercaptoethanol; immunofluorescence staining was performed on the cultured T cells, and then flow cytometry was used for flow cytometry.
  • Figure 4 shows the flow cytometry results of T cells after activation, including blank control group and Examples BCC2, BCC6 and BCC10.
  • the results showed that the proportion of CD4/CD8 double phenotype T cells in the blank control group was 2.7%, while in the experimental group, the proportion of CD4/CD8 double phenotype cells was 3.51%, 2.62% And 7.95% were higher than the blank control group.
  • the proportion of CCR6+/CD62L+ double-positive cells in the blank control group was 6.77%, while in the experimental group, the proportion of CCR6+/CD62L+ double-positive cells was 9.49%, 7.32% and 22.2% were higher than those in the blank control group, and DP in the figure is CD4 /CD8 double-positive cell population, CCR6, CD62L subset is CCR6+/CD62L+ double-positive cell population.
  • Figure 5 shows the statistical results. The results show that the proportion of CD4/CD8 double phenotype T cells and CCR6+/CD62L+ double positive cells is higher than that of the blank control after the cells are stimulated by BCC10. #2, #6 and #10 in the figure correspond to BCC2, BCC6 and BCC10 experimental groups.
  • the method provided in the detection example stimulates the differentiation and expansion of T cells
  • the culture medium consists of: RPMI-1640 substrate medium, which includes 10% FBS, 10mM HEPES, 1x non-essential amino acid solution, 1mM sodium pyruvate, 2mM L-glutamine, 100U/mL penicillin, 100 ⁇ g/mL chain and 55 ⁇ M 2-mercaptoethanol; immunofluorescence staining was performed on the cultured T cells, and then flow cytometry was used for flow cytometry.
  • the expressions of TNFa, CXCR5 protein (CXC chemokine receptor type 5, CXCR5), CD44 protein, CD25 protein, interleukin 17A (IL-17A), CD62L, CCR6, IL-4 and IFNg were detected respectively.
  • the results are shown in Figure 6 and As shown in Figure 7, 1-10 in the figure respectively refer to the experimental groups in which T cells were cultured in culture dishes containing BCC1-BCC10.
  • the micro-nanostructure-coated culture dish of Example 1-5 and the blank control 96-well plate culture dish were used; respectively, the activation treatment group 1-5 and the non-activation treatment group 1-5 were obtained, and the corresponding blank control group. Then, the T cell activity of each group was detected, and the results were shown in Figure 8 .
  • CD4/CD8 double positive T cells Since the study of CD4/CD8 double positive T cells is of great significance for intestinal immunity and aging, the method described in this application has the potential to obtain more CD4/CD8 double positive T cells.
  • the T cells in the activation treatment groups 1-10 obtained by the method all express CCR6 and TNFa.
  • the expression of CCR6 and TNFa in T cells of materials 1-10 was not lower than that of the blank control group.
  • the number of CCR6 and TNFa double-expressing cells was significantly higher than that of the blank group control group; therefore, the method provided in the examples of this application can stimulate T cells to express CCR6 and TNFa at the same time.
  • CCR6 plays an important role in cell migration and memory T cells
  • TNFa plays an important role in killing autoreactive T cells.
  • the method provided in the detection example stimulates the expression of T cells in the activated treatment group and the non-activated treatment group
  • the isolated and purified T cells were transferred to a culture dish containing the micro-nanostructure coating of Example 1-5 and a blank control 96-well plate at about 50,000 cells/200 ⁇ L culture medium per well, and cultured for 6-72 hours ; Obtain the T cells of the non-activated treatment group.
  • immunofluorescence staining was performed, and flow cytometry was used for single cell detection to detect the expressions of TNFa, CCR6, CD62L, IL-17A, CD44, IL-4, and IFNg, as shown in Figure 11.
  • TNFa, CCR6, CD62L, IL-17A, CD44, IL-4, and IFNg of T cells in the activated treatment group and the non-activated treatment group are expressed to different degrees on different materials.
  • the material groups BCC2 and BCC3 can stimulate activated T cells, wherein CD4 cells express TNFa, CCR6, CD62L, IL-17A, CD44, IL-4, IFNg proteins Among them, CD8 cells express TNFa, CCR6, IL-17A, CD44, IL-4, and IFNg proteins; among unactivated T cells, CD4 cells express CCR6, and CD8 cells express CD44 and IFNg.

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Abstract

提供了一种体外分化和扩增T细胞的方法，包括以下步骤：从组织或外周血样本中分离T细胞；将所述T细胞转移至表面铺设有微纳米结构涂层的培养皿中培养，以刺激所述T细胞分化和扩增；其中，所述微纳米结构涂层包括第一胶体颗粒、第二胶体颗粒和界面活性剂，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比值大于2；检测和收集所述培养后的T细胞。该体外分化和扩增T细胞的方法，能够将培养皿上微纳米结构涂层与T细胞接触所产生的物理信号转换成生化信号，促进T细胞在体外的分化和扩增。

Description

一种体外分化和扩增T细胞的方法及其应用 技术领域
本申请涉及生物医学材料领域，具体涉及一种体外分化和扩增T细胞的方法及其应用。
背景技术
免疫系统有先天免疫系统和适应性免疫系统两个分支，由数量庞大以及类型非常丰富的免疫细胞或称淋巴细胞组成。人类的适应性免疫是一种针对新型外界病原的一种强大防御系统，其具有对抗原的记忆，使自身免疫细胞对下一次的抗原入侵产生辨识。免疫细胞包括不同亚群，不同亚群的免疫细胞具有不同的功能，在免疫应答过程中互相调控。例如，T细胞分为若干亚群：按T细胞表面分化抗原(CD)的不同，主要可分为CD4和CD8两大亚群；按细胞表面受体(TCR)的不同，主要可分为αβT细胞γδT细胞；按功能可分为辅助性T细胞(Th细胞)、调节性T细胞(Treg细胞)、细胞毒T细胞(CTL或Tc细胞)和迟发型超敏反应T细胞；按对抗原应答的不同，主要分为初始T细胞、活化的T细胞和记忆性T细胞等，其中每种又有更加细化的亚群划分。例如，辅助性T细胞中，Th1细胞促进细胞免疫，Th2细胞促进体液免疫；而Th17细胞被发现与自身免疫性疾病相关，如多发性硬化症、类风湿关节炎和牛皮癣。由于免疫细胞的亚型众多，彼此间互相作用复杂，且随着技术的日新月异持续会有新的细胞亚群被发现。研究不同亚型免疫细胞的出现以及新型亚型的出现，对生物医学研究和临床应用具有重要意义。
体内免疫细胞的激活需要透过不同的生物因子在精确的时间介入。例如，干扰素γ(IFG-gamma)是Th1细胞的主要分泌因子与分化因子，IL4是Th2细胞的主要分泌因子与分化因子。因此，要在体外得到具有特定表型的免疫细胞，需要使用这些细胞因子与精确浓度。并且，体内有一群非常少数的T细胞，同时具有CD4与CD8表型，称为CD4/CD8双阳性(或双表型)T细胞(DPTC)，只在特定条件下存在。有研究证实，DPTC的存在与罗伊氏乳杆菌的分泌因子相关。此微生物因子刺激了CD4细胞，并且调降了转录因子ThpoK蛋白的表达，因此刺激了DPTC的生成。DPTC的研究与其形成机制在生物医学具有重大意义。但由于DPTC数量极少，且存在时间短占，因此不易被发现且相关研究不多。
现有技术中，刺激体外免疫细胞的方法主要是依靠生物化学方法；例如，结合抗CD3、抗CD28抗体用于体外T细胞活化；利用IL4刺激了CD4细胞的Th2细胞分化。但这些方法均使用细胞因子或受体抑制抗体，费用昂贵且操作过程复杂。
发明内容
为解决上述问题，本申请提供了一种体外分化和扩增T细胞的方法及其应用。该体外分化和扩增T细胞的方法，不需要透过生化因子，而是透过培养皿上的特殊微纳米结构涂层与T细胞短暂接触后所产生的生物物理信号，在细胞内部转换成生化信号，促进T细胞在体外的分化和扩增；解决现有方法中存在的成本高、操作过程复杂的问题。本方法将不限于T细胞，也适用于其他免疫细胞，例如嵌合抗原受体(CAR)-T细胞与自然杀手(NK)细胞。
第一方面，本申请提供了一种体外分化和扩增T细胞的方法，包括以下步骤：
(1)从组织或外周血样本中分离T细胞；
(2)将步骤(1)中的所述T细胞转移至表面铺设有微纳米结构涂层的培养皿中培养，以刺激所述T细胞分化和扩增；其中，所述微纳米结构涂层包括第一胶体颗粒、第二胶体颗粒和界面活性剂，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比值大于2；
(3)检测和收集所述培养后的T细胞。
本申请实施方式中，所述微纳米结构涂层远离所述培养皿的一侧表面具有凹凸起伏的结构形貌。
本申请实施方式中，所述步骤(1)中，所述分离T细胞的过程之后还包括对所述T细胞进行活化处理，所述活化处理的过程包括：将所述分离后的T细胞转移至涂布有活化剂的表面活化；所述活化剂包括抗CD3抗体和抗CD28抗体中的一种或两种。
本申请实施方式中，所述活化处理的时间为6-72小时。
本申请实施方式中，所述活化剂的浓度为1-20mg/mL。
本申请实施方式中，所述步骤(3)中，所述T细胞在所述表面铺设有微纳米结构涂层的培养皿中的培养时间为4-96小时。
本申请实施方式中，所述微纳米结构涂层中，所述第一胶体颗粒包括SiO 2颗粒、ZnO颗粒、TiO 2颗粒、Fe 2O 3颗粒、Ta 2O 5颗粒、Al 2O 3颗粒、PMMA(聚甲基丙烯酸甲酯)胶体颗粒和PS(聚甲基丙烯酸甲酯)胶体颗粒中的至少一种；所述第二胶体颗粒包括PMMA胶体颗粒、PS胶体颗粒、PLGA(聚乳酸-羟基乙酸共聚物)胶体颗粒、PCL(聚己内酯)胶体颗粒、PDMS(聚二甲基硅氧烷)胶体颗粒、AGS(褐藻酸钠)胶体颗粒和PNIPAM(聚(N-异丙基丙烯酰胺))胶体颗粒中的至少一种。
本申请实施方式中，所述微纳米结构涂层中，所述第一胶体颗粒的粒径尺寸为0.5μm-10μm，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比例为(2-100):1。
本申请实施方式中，任意相邻两个所述第一胶体颗粒之间的间距为0.001μm-5μm。
本申请实施方式中，所述微纳米结构涂层中，第一胶体颗粒表面修饰有第一官能基团，所述第一官能基团包括羟基、醛基、羧基、硫基和胺基中的至少一种；所述第二胶体颗粒表面修饰有第二官能基团，所述第二官能基团包括羟基、醛基、羧基、硫基和胺基中的至少一种。
本申请实施方式中，所述检测所述培养后的T细胞的过程包括：
先对所述培养后的T细胞进行免疫荧光染色，然后流式检测。
本申请实施方式中，所述培养皿表面铺设的所述微纳米结构涂层刺激所述T细胞表达转录因子，所述转录因子包括白细胞介素4、趋化因子受体6、干扰素γ、L-选择素、肿瘤坏死因子、黄酸受体相关孤儿受体γt、CD44蛋白、CD25蛋白、芳香烃受体蛋白、Runx3蛋白和ThPOK蛋白中的一种或多种。
本申请实施方式中，所述培养皿表面铺设的所述微纳米结构涂层刺激所述T细胞分化为CD4/CD8双阳性T细胞。
由于现有技术中，促进T细胞向不同亚群分化或扩增需要在培养T细胞的培养基中额外添加价格昂贵的细胞因子或其他刺激试剂，造成成本过高；并且添加细胞因子或其他刺激试剂的浓度与活性很难得到有效控制，造成得到特定的T细胞亚群的效率明显较低。而本申请所述体外分化和扩增T细胞的方法，工艺精简且稳定，原理是将培养皿上微纳米结构涂层与T细胞接触所产生的生物物理信号转换成生物化学信号，促进T细胞在体外的分化和扩增；该过程无需再添加额外细胞因子或其他刺激试剂，或仅需添加远低于现有试剂用量，就可以使T细胞朝向预设的分化方向发展或逆分化的扩增。该体外分化和扩增T细胞的方法大大降低了T细胞制备成本，也可以广泛适用于其他免疫细胞的培养。
本申请所述方法中，使用表面铺设有微纳米结构涂层的培养皿培养T细胞，可望取代细胞因子(或生长因子)等试剂，用于刺激T细胞生长与特定细胞表型表达。所述方法中的步 骤过程，还可以为后续研究T细胞不同亚型的功能以及细胞如何被特定信号激活和调控，对生物医学研究和临床应用具有重要意义。
第二方面，本申请还提供了一种本申请第一方面所述体外分化和扩增T细胞的方法在调节T细胞表型与数量，作为制备抗肿瘤免疫药物中的应用。
本申请第二方面所述应用中，通过调节所述微纳米结构涂层在培养皿表面的具体结构，以及刺激所述T细胞的时间，可以用于调节T细胞的表型；例如T细胞胞内所发生的生物反应，或是T细胞表面的受体与细胞间相互反应等，其表型在免疫治疗具有重大意义。并且，作为免疫细胞，T细胞在血液肿瘤与实体肿瘤的治疗方面也发挥着重要作用，具有巨大应用价值。例如，通过所述体外分化和扩增T细胞的方法所得到的T细胞、CAR-T细胞或NK细胞，可以增加免疫治疗的效果与成功率。
本申请的优点将会在下面的说明书中部分阐明，一部分根据说明书是显而易见的，或者可以通过本申请实施例的实施而获知。
附图说明
为更清楚地阐述本申请的内容，下面结合附图与具体实施例来对其进行详细说明。
图1为本申请一实施例提供的体外分化和扩增T细胞的方法的工艺流程图；
图2为本申请一实施例提供的培养皿表面的微纳米结构涂层BCC1-BCC10的扫描电镜图；
图3为本申请一实施例提供的培养皿表面的微纳米结构涂层的BCC4沿水平成50度拍摄角度，细胞贴附于涂层表面的扫描电镜图；
图4为本申请一实施例提供的方法得到的活化T细胞的流式数据图；
图5为本申请一实施例提供的方法得到的活化T细胞表型的比例柱状图；
图6为本申请一实施例提供的方法刺激CD4细胞的表面抗原表达数据图；
图7为本申请一实施例提供的方法刺激CD4细胞表达CCR6和IL-4数据图；
图8为本申请一实施例提供的方法中经活化处理组和未活化处理细胞的细胞活性图；
图9为本申请一实施例提供的方法中经活化处理组和未活化处理组的CD4/CD8双阳性T细胞，CD4细胞和CD8细胞含量组成图；
图10为本申请一实施例提供的方法刺激T细胞后，在涂层BCC1-BCC10细胞中CCR6与TNFa双表达细胞的比例；
图11为本申请一实施例提供的方法经过活化处理组和未活化处理组得到的T细胞的表面抗原比例热图与分离后CD4与CD8细胞的抗原表达。
具体实施方式
以下所述是本申请的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本申请的保护范围。
参见图1，本申请一实施方式中，提供了一种体外分化和扩增T细胞的方法，包括以下步骤：
S101、从组织或外周血样本中分离T细胞；
S102、将S101中的所述T细胞转移至表面铺设有微纳米结构涂层的培养皿中培养，以刺 激所述T细胞分化和扩增；其中，所述微纳米结构涂层包括所述第一胶体颗粒、第二胶体颗粒和界面活性剂，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比值大于2；
S103、检测和收集所述培养后的T细胞。
其中，所述S101步骤中，所述组织或外周血样本可以但不限于来源于包括哺乳动物。例如，一实施方式中，所述T细胞从老鼠脾脏或其他淋巴组织，或外周血中经分离收集得到。所述分离T细胞的过程还包括对T细胞进行纯化。
可选地，所述步骤(1)中，所述分离T细胞的过程之后还包括对所述T细胞进行活化处理，所述活化处理的过程包括：将所述分离后的T细胞转移至涂布有活化剂的表面活化；所述活化剂包括抗CD3抗体和抗CD28抗体中的一种或两种以上。一实施方式中，将所述分离后的T细胞转移至涂布有活化剂的普通培养皿表面活化，所述活化剂由抗CD3抗体和抗CD28抗体组成。
可选地，所述活化处理的时间为6-72小时。一实施方式中，所述活化处理的时间为12-72小时。另一实施方式中，所述活化处理的时间为24-48小时；或者，所述活化处理的时间为20-24小时。例如，所述活化处理的时间可以但不限为6小时，10小时，12小时，18小时，24小时，36小时，40小时，或48小时。
可选地，所述活化剂的浓度为0.5-10mg/mL。例如，所述活化剂可以但不限为含有抗CD3抗体和抗CD28抗体的缓冲溶液，其中，抗CD3抗体和抗CD28抗体的溶度均为2.5mg/mL。所述缓冲溶液可以但不限于为PBS缓冲液。
本申请实施方式中，在将所述培养后的T细胞转移至表面铺设有微纳米结构涂层的培养皿中培养之前，既可以对分离的T细胞进行活化处理，也可以不对其进行活化处理。对T细胞进行活化，有利于延长细胞的存活时间，减小T细胞的死亡率。本申请实施方式中，可以对分离的T细胞进行一次、二次或者三次活化处理。经过活化处理和未活化处理的T细胞，在所述体外分化和扩增T细胞的方法下，获得相同或不同的分化和扩增效果。
其中，所述S102步骤中，所述微纳米结构涂层远离所述培养皿的一侧表面具有凹凸起伏的结构形貌。其中，所述微纳米结构涂层中，所述第一胶体颗粒和所述第二胶体颗粒均为球形或近球形形状。所述微纳米结构涂层一侧表面具有凹凸起伏的结构形貌是指其表面具有微米级和纳米级的凹凸起伏的结构。所述微纳米结构涂层铺由第一胶体颗粒、第二胶体颗粒和界面活性剂组成，且铺设在培养皿表面；因此，所述微纳米结构涂层在远离培养皿的一侧表面就具有微纳米级的凹凸起伏的结构形貌。所述凹凸起伏的结构形貌一直延伸至所述微纳米结构涂层的四周边缘。
本申请实施方式中，所述凹凸起伏的结构形貌可以为规则型和/或非规则型。例如，所述微纳米结构涂层中，所述第一胶体颗粒均匀有序分布在所述微纳米结构涂层中，围绕每个所述第一胶体颗粒周围均匀分布有多个第二胶体颗粒，此时，所述微纳米结构涂层的表面形成为规则型凹凸起伏的结构形貌。反之，当所述微纳米结构涂层中，第一胶体颗粒非均匀有序分布在所述微纳米结构涂层中，围绕每个所述第一胶体颗粒周围随机分布有多个第二胶体颗粒时，所述微纳米结构涂层的表面形成为非规则型凹凸起伏的结构形貌。本申请所述微纳米结构涂层还可以但不限于部分区域为规则型凹凸起伏的结构形貌，部分区域为非规则型凹凸起伏的结构形貌。
可选地，所述第一胶体颗粒包括SiO 2颗粒、ZnO颗粒、TiO 2颗粒、Fe 2O 3颗粒、Ta 2O 5颗粒、Al 2O 3颗粒、PMMA胶体颗粒和PS胶体颗粒中的至少一种；所述第二胶体颗粒包括PMMA胶 体颗粒、PS胶体颗粒、PLGA胶体颗粒、PCL胶体颗粒、PDMS胶体颗粒、AGS胶体颗粒和PNIPAM胶体颗粒中的至少一种。
可选地，所述第一胶体颗粒的粒径尺寸为0.5μm-10μm，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比例为(2-100):1。
可选地，所述第一胶体颗粒的粒径尺寸为1μm-8μm。
可选地，所述第一胶体颗粒的粒径尺寸为1μm-5μm。
本申请一实施方式中，所述第一胶体颗粒的粒径尺寸为0.5μm、1μm、2μm、3μm、4μm、5μm、6μm、7μm、8μm、9μm或10μm。
可选地，所述第二胶体颗粒的粒径尺寸为0.01μm-0.7μm。
可选地，所述第二胶体颗粒的粒径尺寸为0.1μm-0.4μm。
本申请一实施方式中，所述第二胶体颗粒的粒径尺寸为0.01μm，或为0.1μm，或为0.2μm，或为0.3μm，或为0.4μm，或为0.5μm，或为0.6μm，或为0.7μm。
可选地，任意相邻两个所述第一胶体颗粒之间的间距为0.001μm-5μm。本申请中，所述任意相邻两个所述第一胶体颗粒之间的间距是指相邻两个所述第一胶体颗粒的外表面之间的最短距离。
可选地，任意相邻两个所述第一胶体颗粒之间的间距为0.001μm-5μm。
可选地，任意相邻两个所述第一胶体颗粒之间的间距为0.1μm-1μm。
本申请一具体实施方式中，任意相邻两个所述第一胶体颗粒之间的间距可以但不限于为0.001μm，或为0.01μm，或为0.1μm，或为0.15μm，或为0.2μm，或为0.5μm，或为0.6μm，或为0.8μm，或为1μm，或为1.5μm，或为2μm，或为2.5μm，或为3μm，或为4μm，或为5μm。
本申请实施方式中，任意相邻两个所述第二胶体颗粒之间的间距很小，可以近似认为其相互接触。
可选地，任意相邻两个所述第二胶体颗粒之间的间距为0.0001-200nm。
进一步地，可选地，任意相邻两个所述第二胶体颗粒之间的间距为1-10nm。
本申请实施方式中，所述第二胶体颗粒围绕所述第一胶体颗粒紧密排布，所述第二胶体颗粒和所述第一胶体颗粒的间距为0.1-100nm。
可选地，所述第二胶体颗粒和所述第一胶体颗粒的间距为1-10nm。
可选地，所述微纳米结构涂层中，第一胶体颗粒表面修饰有第一官能基团，所述第一官能基团包括羟基、醛基、羧基、硫基和胺基中的至少一种；所述第二胶体颗粒表面修饰有第二官能基团，所述第二官能基团包括羟基、醛基、羧基、硫基和胺基中的至少一种。
一实施方式中，所述第一官能基团为羟基，或为醛基，或为羧基，或为硫基，或为胺基；所述第二官能基团为羟基，或为醛基，或为羧基，或为硫基或为胺基。本申请中所述第一胶体或所述第二胶体表面修饰的第一官能基团或第二官能基团一方面能影响其与接触物体表面之间的固定强度；另一方面，所述第一官能基团或第二官能基团还可以对应改变第一胶体或所述第二胶体表面电荷，影响细胞贴附，以及调节第一胶体或所述第二胶体的分布间距。
可选地，所述界面活性剂在所述微纳米结构涂层中的质量分数为0.0001％-1％。
可选地，所述界面活性剂在所述微纳米结构涂层中的质量分数为0.001％-0.1％。本申请一具体实施方式中，所述界面活性剂在所述微纳米结构涂层中的质量分数可以但不限于为0.0001％，或为0.0005％，或为0.001％，或为0.01％，或为0.005％，或为0.008％，或为0.01％，或为0.05％，或为0.08％，或为0.1％，或为0.5％，或为1％。
本申请实施方式中，所述界面活性剂包括阳离子表面活性剂、阴离子表面活性剂或非离子表面活性剂。本申请所述界面活性剂可以用于进一步调节第一胶体颗粒和第二胶体颗粒表面的电荷分布量，从而使所述微纳米结构涂层中任意相邻两个所述第一胶体颗粒之间的间距，以及第二胶体颗粒之间的间距在一定范围内，并形成相应结构的微纳米结构涂层。
可选地，所述普通培养皿或所述铺设有微纳米结构涂层的培养皿的材质包括玻璃或塑料，所述塑料的材质包括PS、PMMA、PLGA或PCL中的一种或多种。可选地，所述培养皿的形状、尺寸可以根据实际的需求进行调整，例如，所述培养皿为孔板结构。一实施方式中，所述培养皿为6孔板或24孔板或96孔板。当所述培养皿为孔板结构中，所述微纳米结构涂层可以但不限于铺设在所述孔板孔洞的底部表面。
本申请实施方式中，培养皿表面铺设的微纳米结构涂层与T细胞接触所产生的生物物理信号能转换成促进T细胞在体外的分化和扩增的生物化学信号；不同具体结构的所述微纳米结构涂层促进T细胞在体外的分化和扩增会存在一定的差异；通过调节铺设在培养皿表面的微纳米结构涂层的具体形貌以及培养时间，可以定向促进T细胞在体外的分化和扩增。
可选地，所述T细胞在所述表面铺设有微纳米结构涂层的培养皿中的培养时间为4-96小时。一实施方式中，所述T细胞在所述表面铺设有微纳米结构涂层的培养皿中的培养时间为12-96小时。另一实施方式中，所述T细胞在所述表面铺设有微纳米结构涂层的培养皿中的培养时间为24-72小时；或者，所述T细胞在所述表面铺设有微纳米结构涂层的培养皿中的培养时间为48-72小时。例如，所述培养时间可以但不限为4小时，6小时，10小时，12小时，20小时，24小时，36小时，40小时，48小时，56小时，72小时，80小时，或96小时。
可选地，所述检测和收集所述培养后的T细胞的步骤中，采用先对所述培养后的T细胞进行免疫荧光染色，然后流式检测的方式。例如，所述先对T细胞的表面或内部蛋白进行免疫荧光染色，然后使用流式细胞仪进行对所述T细胞进行检测。这里的检测可以是指随机抽取部分培养后的T细胞进行检测。
本申请实施方式中，所述T细胞在表面铺设有微纳米结构涂层的培养皿中使用的培养液未添加有细胞因子或生长因子。所述培养液可以是常规T细胞培养液，所述培养液包括基本培养基、胎牛血清和抗生素。其中，所述细胞因子是免疫细胞和非免疫细胞能合成和分泌小分子的多肽类因子，它们可以调节多种细胞生理功能。但在实际应用，细胞因子或其他刺激试剂容易超出成本过高。
一实施方式中，所述培养液包括RPMI-1640基本培养基，10％胎牛血清，10mM HEPES(4-羟乙基哌嗪乙磺酸)，1×非必需氨基酸溶液，1mM丙酮酸钠，2mM L-谷氨酰胺，100U/mL青霉素，100μg/mL链霉素和55μM 2-巯基乙醇。
本申请实施方式中，所述培养皿表面铺设的所述微纳米结构涂层刺激所述T细胞表达转录因子，所述转录因子包括白细胞介素4(IL-4)、趋化因子受体6(CCR6)、干扰素γ(IFNg)、L-选择素(CD62L)、肿瘤坏死因子(TNFa)、黄酸受体相关孤儿受体γt(RORγt)、CD44蛋白、CD25蛋白、芳香烃受体蛋白(Ahr)、Runx3蛋白和ThPOK蛋白中的一种或多种。例如，所述转录因子为CCR6和IL-4。
可选地，所述培养皿表面铺设的所述微纳米结构涂层刺激所述T细胞形成为CD4/CD8双阳性T细胞。从组织或外周血样本中分离T细胞绝大部分是CD4细胞和CD8细胞。本申请所述方法可以增加所述T细胞中CD4/CD8双阳性T细胞在所述T细胞中的占比。并且，本申请所述方法可以刺激CD4细胞的特异表面抗原，几乎不改变所述T细胞之中CD4细胞和CD8细胞之间 的比例与细胞活性。
本申请另一实施方式中，还提供了一种体外分化和扩增T细胞的方法，包括以下步骤：
S201、从组织或外周血样本中分离T细胞，对所述分离后的T细胞在传统方法下进行活化处理；
S202、将所述活化处理后的T细胞转移至表面铺设有微纳米结构涂层的培养皿中培养，以刺激所述T细胞分化和扩增；其中，所述微纳米结构涂层包括所述第一胶体颗粒、第二胶体颗粒和界面活性剂，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比值大于2；
S203、检测和收集所述培养后的T细胞。
本实施方式中，除S201步骤中，对所述分离后的T细胞进行活化处理外，其余的步骤与上述实施例的描述一致，本实施方式中不做再次赘述。
本申请所述方法中，使用表面铺设有微纳米结构涂层的培养皿培养T细胞，可以取代细胞因子(或生长因子)等试剂，用于刺激T细胞生长与特定表型表达。所述方法中的步骤过程，还可以为后续研究T细胞不同亚型的功能以及它们是如何被特定信号激活和调控的，对生物医学研究和临床应用具有重要意义。
本申请一实施方式中，提供了一种表面铺设有微纳米结构涂层的培养皿的制备方法，用于培养T细胞，包括以下步骤：
S301、使用模具制备培养皿本体；
S302、分别配制第一胶体颗粒溶液和第二胶体颗粒溶液，然后按预设比例混合形成混合溶液，并添加适量界面活性剂，混合均匀后，将所述混合溶液使用自动化设备加入至培养皿本体的一表面上，且至少覆盖所述表面；颗粒加入的顺序可以是第一颗粒然后第二颗粒，也可以是第二颗粒然后第一颗粒，也可以是第一颗粒与第二颗粒先混合再加入培养皿中；静置干燥直至所述混合溶液的溶剂蒸发完，然后于80-100℃下加热，加入有机溶剂，待所述有机溶剂挥发后，形成固定在所述培养皿本体的一表面上的微纳米结构涂层，得到所述培养皿；
其中，所述第一胶体颗粒包括SiO 2颗粒、ZnO颗粒、TiO 2颗粒、Fe 2O 3颗粒、Ta 2O 5颗粒、Al 2O 3颗粒、PMMA胶体颗粒和PS胶体颗粒中的至少一种；所述第二胶体颗粒包括PMMA胶体颗粒、PS胶体颗粒、PLGA胶体颗粒、PCL胶体颗粒、PDMS胶体颗粒、AGS胶体颗粒和PNIPAM胶体颗粒中的至少一种。
可选地，所述S301中，所述培养皿本体可以但不限于包括至少一个凹槽，所述培养皿本体的一表面可以但不限于为所述凹槽的底部表面。一实施方式中，所述培养皿本体可以为6孔、12孔、24孔、96孔培养皿，每个孔对应一个凹槽。
可选地，所述培养皿本体的表面在铺设微纳米结构涂层之前还可以进行包被处理。
一实施方式中，可以但不限于使用蛋白质溶液对培养皿本体的表面进行包被处理，所述蛋白质溶液包括明胶溶液和/或聚多巴胺溶液。经过包被处理后的培养皿本体的表面与微纳米结构涂层的结合力更强。
可选地，所述S302中，所述第一胶体颗粒的粒径尺寸为0.5μm-10μm，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比例为(2-100):1。
可选地，所述有机溶剂包括体积为1:(1-10)比例的乙醇和甲苯。本申请一具体实施方式中，所述有机溶剂包括体积为1:3比例的乙醇和甲苯。可选地，所述加热温度可以为80℃、84℃、88℃、92℃、96℃、或100℃。可选地，加热时间为0.5-48h。进一步地，可选地，加热时间可以但不限为1-24h。
本申请所述制备过程中，所述混合溶液中的第一胶体颗粒和第二胶体颗粒形成所述微纳米结构涂层的过程，具体包括以下三个阶段：第一阶段，沉降过程，所述第一胶体颗粒和第二胶体颗粒在混合溶液中是一种无序状态，静置后在重力作用下所述第一胶体颗粒先沉降到底部排列，随着混合溶液溶剂的减少，所述第二胶体颗粒与所述第一胶体颗粒接触，在颗粒表面电荷相互作用下，所述第二胶体颗粒会填充在第一胶体颗粒周围，形成微纳米结构涂层；第二阶段，所述混合溶液完全蒸发后，利用有机溶液使所述由第一胶体颗粒和第二胶体颗粒组成的微纳米结构涂层更加贴合于所述培养皿本体的表面；第三阶段，将所述培养皿本体置于烘箱中加热，冷却后得到铺设有纳米结构涂层的所述培养皿。上述过程中，当第一胶体颗粒均匀排布在培养皿表面，第二胶体可以规则有序填充在第一胶体颗粒周围时，最后得到的微纳米结构涂层表面具有凹凸起伏的结构形貌。
本申请实施方式中，所述制备方法中，制备得到表面上设有微纳米结构涂层的细胞培养皿后，可以对所述细胞培养皿进行清洗和杀菌处理。例如，可以用缓冲液或去离子水进行清洗，通过紫外光照的方式对其进行杀菌处理。由于微纳米结构涂层制作完成后，第一胶体颗粒和第二胶体颗粒的位置相对固定；因此，经清洗处理后，微纳米结构涂层表面的结构不会改变。
本申请实施方式中，通过改变第一胶体颗粒与第二胶体颗粒的种类、粒径尺寸、体积或质量比例，以及表面修饰的第一官能基团或第二官能基团的种类或数目，调节界面活性剂的含量及种类，可以获得多种凹凸起伏的结构形貌的微纳米结构涂层。例如，通过利用扫描电子显微镜(SEM)检测可以获得其具体形貌。上述第一胶体颗粒、第二胶体颗粒、界面活性剂和制备工艺的具体限定与前文的论述一致，本实施方式中不做再次赘述。
下面分多个实施例对本申请实施例进行进一步的说明。
实施例
基于上述实施例提供的培养皿制备方法，分别按照表1所述的第一胶体颗粒、第二胶体颗粒和界面活性剂等参数，制备实施例1-1组表面铺设有微纳米结构涂层的培养皿，对应的微纳米结构涂层分别命名为BCC1-BCC10。
表1实施例1-10中的制备微纳米结构涂层的材料选择
Figure PCTCN2020111376-appb-000001
基于上述实施例1-10制备得到的分别铺设有微纳米结构涂层BCC1-10的培养板，并以没有微纳米结构涂层的培养板为空白对照组，分别对其进行喷金处理后(形成约10nm厚的薄膜)，使用场发射扫描电子显微镜(FE-SEM)在20keV下分析所述细胞培养板中的微纳米结构涂层的具体表面结构，如图2所示。为了更直观地观察微纳米结构涂层表面的凹凸起伏的结构形貌，通过放大倍数与调整角度拍摄，结果如图3所示。从图3中(A)和(B)数据中可以看出，纳米结构涂层表面实施例4的表面为凹凸起伏的非平面形貌；其中，细胞边缘与涂层接触的细节清晰可见。
其中，实施例1的BCC1中，相邻第一胶体颗粒间距为1.5μm，第一胶体颗粒的球径约2μm，其排布形体为六边形；实施例2的BCC2中，相邻第一胶体颗粒间距为1.2μm，第一胶体颗粒的球径约1.7μm，其排布形体为六边形；实施例3的BCC3中，相邻第一胶体颗粒间距为1.2μm，第一胶体颗粒的球径约1.85μm，其排布形体为六边形；实施例4的BCC4中，相邻第一胶体颗粒间距为1.3μm，第一胶体颗粒的球径约1.8μm，其排布形体为六边形；实施例5的BCC5中，相邻第一胶体颗粒间距为1.2μm，第一胶体颗粒的球径约1.8μm，其排布形体为六边形；实施例6的BCC6中，相邻第一胶体颗粒间距为1.3μm，第一胶体颗粒的球径约1.9μm，其排布形体为六边形；实施例7的BCC7中，相邻第一胶体颗粒间距为1.0μm，第一胶体颗粒的球径约2.05μm，其排布形体为六边形；实施例8的BCC8中，相邻第一胶体颗粒间距为1.0μm，第一胶体颗粒的球径约2.05μm，其排布形体为六边形；实施例9的BCC9中，相邻第一胶体颗粒间距为0.2μm，第一胶体颗粒的球径约0.85μm，其排布形体为六边形；实施例10的BCC10中，相邻第一胶体颗粒间距为0.2μm，第一胶体颗粒的球径约0.9μm，其排布形体为六边形，具体结构参见图2。
效果实施例
1、检测体外分化和扩增T细胞的方法对CD4和CD8细胞的细胞表型影响先将分离纯化的T细胞加入到添加了抗CD3抗体和抗CD28抗体的普通96孔板培养皿，每孔约50,000细胞/200μL培养液，培养24-48小时；将培养得到的T细胞转移到含有实施例1-10的微纳米结构涂层的培养皿和空白对照的96孔板培养皿中，培养6-72小时。其中，培养液组成为：RPMI-1640基板培养基，其中包括10％FBS，10mM HEPES，1x非必需氨基酸溶液，1mM丙酮酸钠，2mM L-谷氨酰胺，100U/mL青霉素，100μg/mL链霉素和55μM 2-巯基乙醇；将培养得到的T细胞进行免疫荧光染色，然后使用流式细胞仪进行流式检测。图4为T细胞活化后的细胞流式结果，包含空白对照组和实施例BCC2、BCC6与BCC10。在其中一组实例中，结果显示，CD4/CD8双表型T细胞在空白对照组中的比例为2.7％，而在实验组中，CD4/CD8双表型细胞的比例为3.51％，2.62％与7.95％均高于空白对照组。CCR6+/CD62L+双阳性细胞在空白对照组中比例为6.77％，而在实验组中，CCR6+/CD62L+双阳性细胞比例为9.49％，7.32％与22.2％均高于空白对照组，图中DP为CD4/CD8双阳性细胞群，CCR6，CD62L subset为CCR6+/CD62L+双阳性细胞群。图5为统计结果，结果显示细胞受BCC10刺激后无论是CD4/CD8双表型T细胞与CCR6+/CD62L+双阳性细胞比例都较空白对照高，图中的#2、#6和#10分别对应BCC2、BCC6和BCC10实验组。
2、检测实施例提供的方法刺激T细胞的分化和扩增
先将分离的T细胞加入到添加了抗CD3抗体和抗CD28抗体的普通96孔板培养皿，每孔约50,000细胞/200μL培养液，培养24-48小时；将培养得到的T细胞转移到含有实施例1-10的微纳米结构涂层的培养皿和空白对照的96孔板培养皿中，培养6-72小时。其中，培养液组成为： RPMI-1640基板培养基，其中包括10％FBS，10mM HEPES，1x非必需氨基酸溶液，1mM丙酮酸钠，2mM L-谷氨酰胺，100U/mL青霉素，100μg/mL链霉素和55μM2-巯基乙醇；将培养得到的T细胞进行免疫荧光染色，然后使用流式细胞仪进行流式检测。分别检测TNFa、CXCR5蛋白(C-X-C chemokine receptor type 5，CXCR5)、CD44蛋白、CD25蛋白、白细胞介素17A(IL-17A)、CD62L、CCR6、IL-4和IFNg的表达情况，结果如图6和图7所示，图中的1-10分别指T细胞在含BCC1-BCC10的培养皿中培养的实验组。
从图6所示内容可以看出CD4T细胞表面表达TNFa、CXCR5、CD44、CD25、IL-17A、CD62L、CCR6、IL-4、IFNg的表达强度。根据图7可以看出，本申请所述方法与材料可以刺激CD4T细胞表面的CCR6与IL-4表达。
3、检测经过活化处理和未活化处理的体外分化和扩增T细胞的方法对T细胞的活性影响
按上述实施例提供的方法，使用实施例1-5的微纳米结构涂层的培养皿和空白对照的96孔板培养皿；分别得到活化处理组1-5、未活化处理组1-5，以及对应的空白对照组。然后分别对各组的T细胞活性进行检测，结果如图8所述。
从图8的结果显示，经过活化处理的T细胞活性约90％，且BCC1-BCC5组与空白对照组间没有差异；未活化处理的T细胞其活性约70％，且BCC1-BCC5组与空白对照组间没有明显差异；其中，材料不影响未激活T细胞中CD4细胞的活性，但对CD8细胞的活性会有抑制作用，代表材料对CD4细胞有选择性。
其次，检测上述经过活化处理组1-5和未活化处理组1-3中CD4细胞和和CD8细胞的表型组成。结果如图9所示，其中，图9中的(A)为经过活化处理的细胞在材料组1-5的组成，图9中的(B)为未活化处理细胞在材料组1-3的组成，图中的柱状图自上至下依次为CD4/CD8双阳性T细胞、CD8细胞和CD4细胞占比。结果显示，经过活化处理组1-5中的CD8细胞占比约80％，CD4细胞不到10％，CD4/CD8双阳性T细胞比例相比于空白对照组没有显著提高；但是，未活化处理组1-3中的CD4/CD8双阳性T细胞比例相比于空白对照组与4-5号材料有些微的提高，且CD4细胞比例有显著提高约占30％，CD8细胞比例约占60％。
由于研究CD4/CD8双阳性T细胞对于肠道免疫和老化方面具有重要意义，因此，本申请所述方法对于获得更多CD4/CD8双阳性T细胞数量具有潜力。
4、检测本申请所述实施例提供的方法刺激T细胞的CCR6和TNFa表达
先将分离纯化的T细胞加入到添加了抗CD3抗体和抗CD28抗体的普通96孔板培养皿，每孔约50,000细胞/200μL培养液，培养24-48小时；将培养得到的T细胞转移到含有实施例1-10的微纳米结构涂层的培养皿和空白对照的96孔板培养皿中，培养6-72小时；得到经过活化处理组1-10和空白对照组。然后对各组T细胞的CCR6和TNFa标志物进行免疫荧光染色，然后使用流式细胞仪进行单细胞检测，结果如图10。
从图10所示内容可以看出，由所述方法得到活化处理组1-10的T细胞均表达CCR6和TNFa。相比于空白对照组，材料1-10的T细胞表达CCR6和TNFa量均不低于空白对照组。其中，材料组BCC4、BCC10与BCC12上，CCR6和TNFa双表达细胞的数量皆显著高于空白组对照组；因此，本申请实施例提供的所述方法能够刺激T细胞同时表达CCR6和TNFa。其中，CCR6对于细胞迁移，以及在记忆T细胞中发挥重要作用；TNFa对于杀死自身反应性T细胞方面具有重要作用。
5、检测实施例提供的方法刺激活化处理组和未活化处理组T细胞的表达
将分离纯化的T细胞，以每孔约50,000细胞/200μL培养液转移到含有实施例1-5的微纳米结 构涂层的培养皿和空白对照的96孔板培养皿中，培养6-72小时；得到未活化处理组的T细胞。先将分离纯化的T细胞加入到添加了抗CD3抗体和抗CD28抗体的普通96孔板培养皿，每孔约50,000细胞/200μL培养液，培养24-48小时；将培养得到的T细胞转移到含有实施例1-5的微纳米结构涂层的培养皿和空白对照的96孔板培养皿中，培养6-72小时；得到活化处理组的T细胞。然后分别进行免疫荧光染色，使用流式细胞仪进行单细胞检测，检测TNFa、CCR6、CD62L、IL-17A、CD44、IL-4、IFNg的表达情况，参见图11所示。
从图11的数据中可以得知，活化处理组和未活化处理组的T细胞的TNFa、CCR6、CD62L、IL-17A、CD44、IL-4、IFNg在不同材料上均有不同程度的表达。本申请实施例提供的方法中的微纳米结构涂层，材料组BCC2与BCC3可以刺激活化后的T细胞，其中CD4细胞表达TNFa、CCR6、CD62L、IL-17A、CD44、IL-4、IFNg蛋白，其中，CD8细胞表达TNFa、CCR6、IL-17A、CD44、IL-4、IFNg蛋白；在未活化T细胞中，CD4细胞表达CCR6，CD8细胞表达CD44与IFNg。并且从图11的数据中可以得知，在未活化的CD4细胞(未受CD8影响)，BCC3依然可以刺激CCR6与IFNg表达，未活化的CD8细胞(未受CD4影响)则一定程度的刺激TNFa、CCR6、IL-17A、CD44、IL-4、IFNg蛋白表达。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (14)

  1. 一种体外分化和扩增T细胞的方法，其中，包括以下步骤：
    (1)从组织或外周血样本中分离T细胞；
    (2)将步骤(1)中的所述T细胞转移至表面铺设有微纳米结构涂层的培养皿中培养，以刺激所述T细胞分化和扩增；其中，所述微纳米结构涂层包括第一胶体颗粒、第二胶体颗粒和界面活性剂，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比值大于2；
    (3)检测和收集所述培养后的T细胞。
  2. 如权利要求1所述的方法，其中，所述微纳米结构涂层远离所述培养皿的一侧表面具有凹凸起伏的结构形貌。
  3. 如权利要求1所述的方法，其中，所述步骤(1)中，所述分离T细胞的过程之后还包括对所述T细胞进行活化处理，所述活化处理的过程包括：将所述分离后的T细胞转移至涂布有活化剂的表面活化；所述活化剂包括抗CD3抗体和抗CD28抗体中的一种或两种。
  4. 如权利要求3所述的方法，其中，所述活化处理的时间为6-72小时。
  5. 如权利要求3所述的方法，其中，所述活化剂的浓度为1-20mg/mL。
  6. 如权利要求1所述的方法，其中，所述步骤(3)中，所述T细胞在所述表面铺设有微纳米结构涂层的培养皿中的培养时间为4-96小时。
  7. 如权利要求1-6任一项所述的方法，其中，所述微纳米结构涂层中，所述第一胶体颗粒包括SiO 2颗粒、ZnO颗粒、TiO 2颗粒、Fe 2O 3颗粒、Ta 2O 5颗粒、Al 2O 3颗粒、PMMA胶体颗粒和PS胶体颗粒中的至少一种；所述第二胶体颗粒包括PMMA胶体颗粒、PS胶体颗粒、PLGA胶体颗粒、PCL胶体颗粒、PDMS胶体颗粒、AGS胶体颗粒和PNIPAM胶体颗粒中的至少一种。
  8. 如权利要求1-6任一项所述的方法，其中，所述微纳米结构涂层中，所述第一胶体颗粒的粒径尺寸为0.5μm-10μm，所述第一胶体颗粒与所述第二胶体颗粒的粒径尺寸比例为(2-100):1；
  9. 如权利要求8所述的方法，其中，任意相邻两个所述第一胶体颗粒之间的间距为0.001μm-5μm。
  10. 如权利要求1-6任一项所述的方法，其中，所述微纳米结构涂层中，第一胶体颗粒表面修饰有第一官能基团，所述第一官能基团包括羟基、醛基、羧基、硫基和胺基中的至少一种；所述第二胶体颗粒表面修饰有第二官能基团，所述第二官能基团包括羟基、醛基、羧基、硫基和胺基中的至少一种。
  11. 如权利要求1所述的方法，其中，所述检测所述培养后的T细胞的过程包括：
    先对所述培养后的T细胞进行免疫荧光染色，然后流式检测。
  12. 如权利要求1-11任一项所述的方法，其中，所述培养皿表面铺设的所述微纳米结构涂层刺激所述T细胞表达转录因子，所述转录因子包括白细胞介素4、趋化因子受体6、干扰素γ、L-选择素、肿瘤坏死因子、黄酸受体相关孤儿受体γt、CD44蛋白、CD25蛋白、芳香烃受体蛋白、Runx3蛋白和ThPOK蛋白中的一种或多种。
  13. 如权利要求1-11任一项所述的方法，其中，所述培养皿表面铺设的所述微纳米结构涂层刺激所述T细胞分化为CD4/CD8双阳性T细胞。
  14. 一种如权利要求1-13任一项所述体外分化和扩增T细胞的方法在调节T细胞反应和制备抗肿瘤药物中的应用。
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