CN117660336A - T cell activation method and application thereof - Google Patents
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Abstract
The invention discloses a T cell activation method and application thereof, provides a T cell activation method, provides an activated T cell prepared by the T cell activation method, and also provides application of the activation method in preparing a high-activation T cell product, and application of the activation method in improving T cell activation efficiency, improving T cell survival rate and reducing T cell depletion degree.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a T cell activation method and application thereof.
Background
In recent years, immunotherapy of tumors has been progressed, and T cells are the main effector cells. T cell immunoregulatory antibodies represented by PD 1 antibodies rapidly enter the clinic, are widely used for tumor treatment, but have lower proportion of effective population, and have about 18% of effective rate in lung cancer of maximum indication. The combined immunotherapy is a development direction for improving curative effect, comprises PD-1 antibody combined chemotherapy, radiotherapy, anti-angiogenesis drugs and combined treatment strategies, has practical significance for enlarging income crowd, prolonging survival of patients and even curing tumors, and is also an internationally recognized research direction.
CAR-T and TCR-T therapies are currently the focus of research for adoptive immune cell therapies, in which both CAR-T and TCR-T cells are cultured in vitro, and T cells need to be activated before subsequent steps of operation can be performed, so it is important to find an efficient activation method for T cells.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the following technical scheme:
the first aspect of the present invention provides a method for activating T cells, the method comprising the steps of:
1) Adding a lymph separating liquid into a sample, centrifuging, and separating to obtain PBMC;
2) Adding normal saline into the PBMC in the step 1), centrifuging, and collecting the PBMC; small amounts of PBMCs were taken and tested for CD3 + T cell ratio;
3) Taking magnetic beads, swirling, adding an equal volume of DPBS, uniformly mixing, placing on a magnetic frame for 1min, and discarding the supernatant;
4) Based on the measurement in step 2), CD3 is regulated + T cell concentration, step 3) adding magnetic beads into a cell suspension, uniformly mixing, incubating at room temperature, diluting with DPBS, placing the diluted mixture of the magnetic beads and the cells in a magnetic rack for 2min, and discarding the supernatant;
5) Resuspension of the beads and cell mixture with T cell expansion medium, adjustment of cell density, and exposure to 5% CO at 37deg.C 2 Culturing in an incubator, removing magnetic beads, and culturing for 14 days to obtain activated T cells.
As used herein, the term"T cells" or "T lymphocytes" are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cells may be T helper (Th) cells, such as T helper 1 (Th 1) or T helper 2 (Th 2) cells. T cells may be helper T cells (HTL; CD 4) + T cells), cytotoxic T cells (CTLs; CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, αβt cells expressing the α and β chains of the T Cell Receptor (TCR), and γδ T cells expressing the tcrγ and δ chains, or any other subset of T cells. Other illustrative T cell populations suitable for use in particular embodiments include memory T cells, suitably early memory T cells. The term "T cell" includes within its scope natural T cells (e.g., isolated from an organism, such as a mammal, e.g., a human, e.g., a subject), ex vivo grown T cells, and genetically engineered T cells. The term T cell also includes T cells comprising a T cell receptor (e.g., a native TCR, or a recombinant TCR) and T cells comprising an artificial T cell receptor (e.g., CAR-T cells).
Further, in the step 1), PBMC is separated by using a centrifuge to increase the speed 9 and decrease the speed 0.
Further, the sample in step 1) is from a mammal.
Further, the sample in step 1) is from a human or other non-human mammal.
Further, the sample comprises a blood sample.
The term "sample" is derived from any cell suspension that can carry T cells, including but not limited to blood, interstitial fluid, lymph fluid, and the like. The term "sample" is considered synonymous with "biological sample" in a broad sense, meaning a sample obtained or derived from a biological source of interest (e.g., tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or comprises biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; a cell-containing body fluid; a free floating nucleic acid; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological liquid; a skin swab; a vaginal swab; an oral swab; a nasal swab; irrigation or lavage fluid, such as catheter lavage fluid or bronchoalveolar lavage fluid; aspirate; scraping objects; a bone marrow sample; a tissue biopsy sample; a surgical sample; feces, other body fluids, secretions and/or excretions; and/or cells derived therefrom, etc. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the cells obtained are or include cells from an individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsies (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a formulation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and/or by adding one or more agents to the primary sample).
Further, the centrifugal force in the step 1) is 2000rpm, and the centrifugal time is 25min.
Further, the centrifugal force in the step 2) is 1800rpm, and the centrifugal time is 8min.
Further, the swirling time in the step 3) is 30s.
Further, the magnetic beads in the step 3) are CD3 sorting magnetic beads.
Further, the magnetic beads in the step 3) are magnetic beads for CD3/CD28 sorting.
Further, the step 4) of modulating CD3 + T cell concentration of 1X 10 7 And each mL.
Further, the ratio of the magnetic beads to the number of cells in the step 4) includes 1-2:1.
In some embodiments, the ratio of magnetic beads to cell number in step 4) comprises 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.
further, the incubation time at room temperature in the step 4) is 30min.
Further, the T cell expansion medium in the step 5) is X-VIVOTM 15 medium containing 100-500IU/mL IL-2.
In some embodiments, the T cell expansion medium in step 5) is 100IU/mL IL-2 in X-VIVOTM 15 medium, 150IU/mL IL-2 in X-VIVOTM 15 medium, 200IU/mL IL-2 in X-VIVOTM 15 medium, 250IU/mL IL-2 in X-VIVOTM 15 medium, 300IU/mL IL-2 in X-VIVOTM 15 medium, 350IU/mL IL-2 in X-VIVOTM 15 medium, 400IU/mL IL-2 in X-VIVOTM 15 medium, 450IU/mL IL-2 in X-VIVOTM 15 medium, 500IU/mL IL-2 in X-VIVOTM 15 medium.
Further, the step 5) adjusts the cell density to 1X 10 6 personal/mL-5X 10 6 And each mL.
In some embodiments, the step 5) adjusts the cell density to 1×10 6 individual/mL, 1.5X10) 6 Per mL, 2X 10 6 Per mL, 2.5X10 6 3X 10 per mL 6 3.5X10 g/mL 6 Per mL, 4X 10 6 individual/mL, 4.5X10 6 Per mL, 5X 10 6 And each mL.
Further, the step 5) of removing the magnetic beads comprises removing the magnetic beads on days 2-9 of cell culture.
In some embodiments, the time for removing the magnetic beads in step 5) comprises a day 2 removal of cell culture, a day 3 removal of cell culture, a day 4 removal of cell culture, a day 5 removal of cell culture, a day 6 removal of cell culture, a day 7 removal of cell culture, a day 8 removal of cell culture, a day 9 removal of cell culture.
In a specific embodiment, the method of activating T cells comprises the steps of: 1) The blood sample was added with the lymph separation solution, centrifuged at 2000rpm for 25min,then separating to obtain PBMC under the method of increasing the speed by 9 and decreasing the speed by 0; 2) Adding normal saline into the PBMC in the step 1), centrifuging for 10min at 1800rpm, collecting PBMC, collecting small amount of PBMC, and detecting CD3 + T cell ratio; 3) Taking a proper amount of magnetic beads, swirling for 30s, adding an equal volume of DPBS, uniformly mixing, then placing on a magnetic rack for 1min, and discarding the supernatant to obtain the magnetic beads for preparation; 4) According to CD3 in step 2) + Measurement of T cell ratio, modulation of CD3 + T cell concentration to 1X 10 7 Adding the prepared magnetic beads in the step 3) in a ratio of 1-2:1, uniformly mixing, incubating for 30min at room temperature, diluting the mixture of the magnetic beads and the cells, placing the mixture on a magnetic rack for 2min, and discarding the supernatant; 5) Resuspension of the supernatant from step 4) with 100-500IU/mL IL-2 in X-VIVOTM 15 medium, adjusting the cell concentration to 1X 10 6 personal/mL-5X 10 6 Individual/mL, then placed at 37℃at 5% CO 2 Culturing in an incubator for 14 days, removing magnetic beads 2-9 days after culturing, and finally culturing for 14 days to obtain activated T cells.
In a second aspect, the invention provides an activated T cell prepared by the activation method described above.
Further, the activation includes one or more of induced cytokine expression and secretion, phagocytosis, cell signaling, antigen processing and presentation, target cell killing function.
The term "activated" refers to a state in which monocytes and/or macrophages have been stimulated sufficiently to induce detectable cell proliferation and/or have been stimulated to exert their effector functions, such as induced cytokine expression and secretion, phagocytosis, cell signaling, antigen processing and presentation, and target cell killing functions.
In a third aspect the invention provides the use of an activation method as hereinbefore described for the preparation of a highly activated T cell product.
Further, the product includes genetically engineered cells, immune effector cells.
The term "product" refers to a composition comprising the manufacture of a genetically engineered cell (e.g., an immune effector cell), e.g., a population of cells, wherein a plurality of cells are engineered to express a CAR, e.g., a CAR described herein. The manufactured product can be any genetically engineered immune effector cell (e.g., T cell, NK cell), such as a genetically engineered immune effector cell obtained from the blood of a subject, such as a manufactured CAR-expressing cell product, such as a manufactured CD19 CAR-expressing cell product. In one embodiment, cells engineered to express the CAR (e.g., immune effector cells) can be obtained from an activated cryopreserved expanded population of cells (e.g., an expanded population of immune effector cells).
In a fourth aspect the invention provides a composition comprising an agent for use in the activation method described hereinbefore.
The term "composition" refers to reagents that can be used in the activation methods described above, including reagents used in the separation, purification, mixing, sorting of T cells, including chemical reaction reagents and washing reagents.
In a fifth aspect, the invention provides the use of an activation method as described above for increasing the efficiency of T cell activation, increasing the survival of T cells, and reducing the extent of T cell depletion.
The term "cell depletion" refers to a negatively selected process of separating desired cells from unwanted cells. As used herein, a cell sample may comprise blood-derived cells (e.g., PBMCs), cell culture-derived cells, or cells of tissue origin (e.g., brain or bone that may be isolated prior to use). Suitable methods for cell depletion include centrifugation, filtration, magnetic cell sorting, and fluorescent cell sorting. The terms "CD3 expressed" and "CD3 positive" (CD 3) + ) Cells have interchangeable meanings and describe cells expressing CD3 antigen.
A sixth aspect of the invention provides the use of the activation method described hereinbefore in a computer model or device.
The term "computer model" refers to a solution that employs hardware, software, or a combination thereof to carry out a purpose. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as one or more controllers that control the above-discussed functions. The one or more controllers can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.
It should be appreciated that one implementation includes at least one computer-readable storage medium (i.e., at least one tangible, non-transitory computer-readable medium) encoded with a computer program (i.e., a plurality of instructions), such as a computer memory (e.g., hard disk drive, flash memory, processor working memory, etc.), a floppy disk, an optical disk, a magnetic tape, or other tangible, non-transitory computer-readable medium, that when executed on one or more processors performs the functions discussed above. The computer-readable storage medium may be removable so that the program stored thereon can be loaded onto any computer resource to implement the techniques discussed herein. In addition, it should be understood that references to a computer program that when executed performs the functions discussed above are not limited to application programs running on a host computer. Rather, the term "computer program" is used herein in a generic sense to refer to any type of computer code (e.g., software or microcode) that can be employed to program one or more processors to implement the above techniques.
A seventh aspect of the invention provides an apparatus, the apparatus comprising: a single or multiple processors, and a memory for storing a single or multiple computer programs that when executed by the single or multiple processors implement the steps of the activation method described previously.
The term "apparatus" refers to an apparatus, device, means, processor, sensor, externally or internally applied, that transmits, transmits or receives signals, energy or data and is used as part of the treatment of the present invention. The apparatus may include, but is not limited to, physical, mechanical, digital, ultrasonic, electrical, or magnetic devices.
Further, the processor may perform the steps of the activation method described above singly, or a plurality of processors may perform the steps of the activation method described above together.
The term "processor" may refer to a single processor or multiple processors, which collectively implement the various steps of the process. Similarly, a "storage device" or "database" may refer to a single device or database or multiple devices or databases distributed with programming instructions and/or data.
The term "memory" is used herein in its conventional sense to refer to a device that stores information for later retrieval by a processor, and may include magnetic or optical devices (e.g., hard disk, floppy disk, CD, or DVD) or solid state storage devices (e.g., volatile or non-volatile RAM). The memory or storage unit may have more than one physical storage device of the same or different types (e.g., the memory may have multiple storage devices, such as multiple hard disk drives or multiple solid state storage devices, or some combination of hard disk drives and solid state storage devices). The memory may be a computer readable medium or a persistent memory. In an embodiment, the memory may include one or more data sets having a plurality of stored identities corresponding to each of the labeled biomolecules, labels, activated biomolecules, activated labels, and reactive linkers in the system database.
In an eighth aspect the invention provides the use of an activated T cell as hereinbefore described in the preparation of a CAR-T cell or TCR-T cell.
The term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds an antigen or target, a transmembrane domain, and an intracellular signaling domain. Binding of the extracellular domain of the CAR to the target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activation stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and/or production of molecules capable of mediating cell death of expressing target antigens in a manner independent of Major Histocompatibility (MHC).
The term "CAR-T" or "CAR-T cell" generally refers to a T cell capable of expressing a CAR (also known as a "chimeric antigen receptor"). The CAR generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain. CARs are core components of chimeric antigen receptor T cells (CAR-T), which may include a targeting moiety (e.g., a moiety that binds a tumor-associated antigen (TAA)), a hinge region, a transmembrane region, and an intracellular domain.
The term "T cell receptor" or "TCR" or "engineered TCR" refers to a molecule present on the surface of a T cell that is responsible for recognizing an antigen displayed on the surface of an Antigen Presenting Cell (APC). Each T cell expresses a unique TCR, which is produced by randomly classifying genes, thereby ensuring that the T cell can respond to almost any infection. TCRs are also capable of recognizing tumor-specific proteins (antigens) from within cells. The structural formula of TCRs is composed of two different protein chains comprising an alpha (alpha) chain and a beta (beta) chain. In certain embodiments, a TCR may have one or more amino acid substitutions, deletions, insertions, or modifications compared to a naturally occurring sequence, so long as the TCR retains its ability to form a TCR and retains the ability to recognize an antigen, as well as being involved in immunologically relevant cytokine signaling in transfected T cells. The engineered TCRs also bind with high affinity to target cells that display related tumor-associated peptides.
In a ninth aspect, the invention provides a method of activation as hereinbefore described, an activated T cell as hereinbefore described, a use of a composition as hereinbefore described for increasing proliferation capacity of T cells, and increasing T cell conversion efficiency.
The term "proliferative capacity" refers to the process of cell proliferation, i.e. the process that causes an increase in the number of cells, and is defined by the balance between cell division and loss of cells caused by cell death or differentiation.
The invention has the advantages and beneficial effects that:
the method greatly improves the activation efficiency of the T cells, increases the proliferation capacity of the T cells, reduces the technical requirement of operation, and improves the safety and the use possibility of clinical application of the T cells prepared by using the technology.
Drawings
FIG. 1 is a statistical graph of the results of the degree of cell depletion in different experimental examples.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Process for activating T cells by magnetic beads
1. Experimental protocol
1) Experimental example 1
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio of 1.5:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
Magnetic beads and fines were resuspended in X-VIVOTM 15 medium with 200IU/mL IL-2 in T cell expansion MediumCell mixture, sampling count, and cell density was adjusted to 1.0X10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 2 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
Cell numbers were measured on day 3, day 5, day 7, day 9, day 11, and day 14, respectively, and cell viability and cell depletion were measured on day 14.
2) Experimental example 2
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio 1:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
The beads and cell mixture were resuspended in 300IU/mL IL-2X-VIVOTM 15 medium, the counts were sampled and the cell density was adjusted to 1.5X10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 3 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
Cell numbers were measured on day 3, day 5, day 7, day 9, day 11, and day 14, respectively, and cell viability and cell depletion were measured on day 14.
3) Experimental example 3
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio was 2:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
The beads and cell mixture were resuspended in 300IU/mL IL-2X-VIVOTM 15 medium, the counts were sampled and the cell density was adjusted to 2.0X10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 4 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
Cell numbers were measured on day 3, day 5, day 7, day 9, day 11, and day 14, respectively, and cell viability and cell depletion were measured on day 14.
4) Experimental example 4
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio of 2.0:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
The beads and cell mixture were resuspended in 500IU/mL IL-2X-VIVOTM 15 medium, the counts were sampled and the cell density was adjusted to 5X 10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 5 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
The culture was continued for 14 days, and cell numbers were measured on days 3, 5, 7, 9, 11, and 14, and cell viability and cell depletion were measured on 14 days, respectively.
5) Experimental example 5
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio of 2.0:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
The beads and cell mixture were resuspended in 500IU/mL IL-2X-VIVOTM 15 medium, the counts were sampled and the cell density was adjusted to 5X 10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 6 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
The culture was continued for 14 days, and cell numbers were measured on days 3, 5, 7, 9, 11, and 14, and cell viability and cell depletion were measured on 14 days, respectively.
6) Experimental example 6
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio of 2.0:1. will beThe sample was placed on a mixing instrument and incubated at room temperature for 30min. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
The beads and cell mixture were resuspended in 500IU/mL IL-2X-VIVOTM 15 medium, the counts were sampled and the cell density was adjusted to 5X 10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 7 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
The culture was continued for 14 days, and cell numbers were measured on days 3, 5, 7, 9, 11, and 14, and cell viability and cell depletion were measured on 14 days, respectively.
7) Experimental example 7
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio of 2.0:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
IL-2 500IU/mL in T cell expansion mediumThe X-VIVOTM 15 medium resuspended the beads and cell mixture, the samples were counted and the cell density was adjusted to 5X 10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 8 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
The culture was continued for 14 days, and cell numbers were measured on days 3, 5, 7, 9, 11, and 14, and cell viability and cell depletion were measured on 14 days, respectively.
8) Experimental example 8
The experimental example optimizes the process of activating T cells by using magnetic beads, and comprises the following steps:
(1) Separation of PBMC from blood sample
The blood sample was added to the lymph separation solution, centrifuged at 2000rpm for 25min, the centrifuge was run up at 9 and run down at 0 to separate PBMCs, the PBMCs were supplemented with an appropriate amount of physiological saline, centrifuged at 1800rpm for 8min, and PBMCs were collected. Taking a proper amount of PBMC, and performing flow detection on CD3 + T cell ratio.
(2) Magnetic bead sorting T cells
Taking the amount of the magnetic beads to be used, and vortexing for 30s to resuspend the magnetic beads; adding an equal volume of DPBS and uniformly mixing; placing on a magnetic rack for 1min, and discarding supernatant. Modulation of CD3 based on streaming results + T cell concentration of 1X 10 7 An appropriate amount of cell suspension was added per tube per mL. Magnetic beads and CD3 + T cell ratio of 2.0:1. the samples were placed on a mixing instrument and incubated for 30min at room temperature. The magnetic beads and cell mixture was diluted with DPBS to ensure the sorting volume of the magnetomotive force frame. The centrifuge tube was placed on a magnetic rack for 2min and the supernatant was discarded.
(3) T cell activation and removal of magnetic beads
The beads and cell mixture were resuspended in 500IU/mL IL-2X-VIVOTM 15 medium, the counts were sampled and the cell density was adjusted to 5X 10 6 Every mL, put at 37 ℃ 5% CO 2 Culturing in an incubator. The magnetic beads were removed on day 9 of cell culture, and samples were taken to examine the activation efficiency of T cells (CD 69) + CD25 + Percentage of cells);
(4) T cell expansion culture
The culture was continued for 14 days, and cell numbers were measured on days 3, 5, 7, 9, 11, and 14, and cell viability and cell depletion were measured on 14 days, respectively.
2. Experimental results
1) The results of the T cell activation efficiency and the cell activation rate of 8 groups of experimental examples are shown in Table 1, and the contents of Table 1 show that the activation efficiency of activated T cells obtained in the experimental example method of 8 groups of optimized conditions is quite high, wherein the T cell activation efficiency of experimental example 1 reaches 95.6%; the cell viability was also excellent, and the cell viability of examples 1, 2 and 6 reached 96.19%, 96.17% and 96.72%, indicating the excellent T cell activation method of examples provided by the present invention.
TABLE 1T cell activation efficiency and cell Activity
| Activation efficiency | Cell viability | |
| Experimental example 1 | 95.60％ | 96.19％ |
| Experimental example 2 | 93.21％ | 96.17％ |
| Experimental example 3 | 93.12％ | 95.17％ |
| Experimental example 4 | 92.87％ | 94.44％ |
| Experimental example 5 | 92.21％ | 95.54％ |
| Experimental example 6 | 90.20％ | 96.72％ |
| Experimental example 7 | 90.32％ | 92.28％ |
| Experimental example 8 | 89.89％ | 91.71％ |
2) The results of the test of the proliferation capacity of T cells in 8 groups of test cases are shown in Table 2, and the results in Table 2 show that the proliferation times of the T cells in each group are very excellent, especially in test cases 1-5, the proliferation times of the T cells on 14 th day are more than 600 times, especially in the activation method of the T cells in test case 1, the proliferation times of the T cells on 14 th day are 660.48 times, and the T cell activation method provided by the invention can greatly improve the proliferation times of the T cells.
TABLE 2T cell proliferation fold
| For 3 days | For 5 days | For 7 days | 9 days | 11 days | 14 days | |
| Experimental example 1 | 4.62 | 8.85 | 18.42 | 47.76 | 165.12 | 660.48 |
| Experimental example 2 | 4.07 | 10.83 | 20.84 | 49.00 | 161.48 | 649.92 |
| Experimental example 3 | 3.39 | 8.79 | 23.60 | 51.16 | 159.44 | 637.76 |
| Experimental example 4 | 5.82 | 10.36 | 16.20 | 54.80 | 155.24 | 628.96 |
| Experimental example 5 | 4.58 | 8.82 | 23.76 | 49.92 | 158.04 | 616.16 |
| Experimental example 6 | 5.66 | 10.94 | 18.42 | 50.84 | 154.40 | 595.60 |
| Experimental example 7 | 4.07 | 7.83 | 18.84 | 52.00 | 157.51 | 587.92 |
| Experimental example 8 | 4.39 | 8.79 | 17.60 | 45.16 | 156.41 | 560.76 |
3) The results of cell depletion experiments on T cells cultured for 14 days in 8 groups of experimental examples are shown in FIG. 1, and the results show that the cell depletion degrees of different experimental examples are different, and particularly the experimental example 1 is most excellent, and almost no cell depletion exists, so that the T cell activation method provided by the invention can ensure that the T cells maintain excellent cell performance when cultured for 14 days.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. A method of activating T cells, the method comprising the steps of:
1) Adding a lymph separating liquid into a sample, centrifuging, and separating to obtain PBMC;
2) Adding normal saline into the PBMC in the step 1), centrifuging, and collecting the PBMC; small amounts of PBMCs were taken and tested for CD3 + T cell ratio;
3) Taking magnetic beads, swirling, adding an equal volume of DPBS, uniformly mixing, placing on a magnetic frame for 1min, and discarding the supernatant;
4) Based on the measurement in step 2), CD3 is regulated + T cell concentration, step 3) adding magnetic beads into a cell suspension, uniformly mixing, incubating at room temperature, diluting with DPBS, placing the diluted mixture of the magnetic beads and the cells in a magnetic rack for 2min, and discarding the supernatant;
5) Resuspension of the beads and cell mixture with T cell expansion medium, adjustment of cell density, and exposure to 5% CO at 37deg.C 2 Culturing in an incubator, removing magnetic beads, and culturing for 14 days to obtain activated T cells.
2. The method of claim 2, wherein the sample in step 1) is from a mammal;
preferably, the sample in step 1) is from a human or other non-human mammal;
preferably, the centrifugal force in the step 1) is 2000rpm, and the centrifugal time is 25min;
preferably, the centrifugal force in the step 2) is 1800rpm, and the centrifugal time is 8min;
preferably, the vortex time in the step 3) is 30s;
preferably, the step 4) is the modulation of CD3 + T cell concentration of 1X 10 7 individual/mL;
preferably, the ratio of the magnetic beads to the number of cells in step 4) comprises 1-2:1;
preferably, the incubation time at room temperature in the step 4) is 30min;
preferably, the T cell expansion medium in the step 5) is X-VIVOTM 15 medium containing 100-500IU/mL IL-2;
preferably, the cell density is adjusted to 1X 10 in the step 5) 6 personal/mL-5X 10 6 individual/mL;
preferably, the step 5) of removing the magnetic beads comprises removing the magnetic beads on days 2-9 of cell culture.
3. An activated T cell prepared by the activation method of claim 1 or 2;
preferably, the activation comprises one or more of induced cytokine expression and secretion, phagocytosis, cell signaling, antigen processing and presentation, target cell killing function.
4. Use of the activation method of claim 1 or 2 for the preparation of a highly activated T cell product;
preferably, the product comprises genetically engineered cells, immune effector cells.
5. A composition comprising the agent used in the activation method of claim 1 or 2.
6. Use of the activation method of claim 1 or 2 for increasing the efficiency of T cell activation, increasing the survival of T cells, decreasing the extent of T cell depletion.
7. Use of the activation method of claim 1 or 2 in a computer model or device.
8. An apparatus, the apparatus comprising: a single or multiple processors, and a memory for storing a single or multiple computer programs that when executed by the single or multiple processors implement the steps of the activation method of claim 1 or 2;
preferably, the processor may perform the steps of the activation method of claim 1 or 2 singly, or a plurality of processors may jointly perform the steps of the activation method of claim 1 or 2.
9. Use of the activated T cell of claim 3 in the preparation of a CAR-T cell or a TCR-T cell.
10. Use of the activation method of claim 1 or 2, the activated T cell of claim 3, the composition of claim 5 for increasing proliferation capacity of T cells, increasing T cell transformation efficiency.
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| CN117136231A (en) * | 2021-04-08 | 2023-11-28 | 武田药品工业株式会社 | Methods for activating T cells |
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