WO2021238924A1 - Method for sorting t cells - Google Patents

Abstract

Provided is a method for sorting T cells. The method comprises the following steps of: a) mixing cells to be sorted with sorting magnetic beads, wherein the sorting magnetic beads comprise magnetic bead particles and a magnetic bead buffer solution; the sorting magnetic beads are selected from one or two of the following: sorting magnetic beads combined with CD4 and sorting magnetic beads combined with CD8, wherein the use amount of the sorting magnetic beads combined with the CD4 is such that every 107 cells to be sorted are mixed with every 1.25-5 μL of the sorting magnetic beads combined with the CD4, and the use amount of the sorting magnetic beads combined with the CD8 is such that every 107 cells to be sorted are mixed with every 1.25-5 μL of the sorting magnetic beads combined with the CD8; and b) sorting to obtain T cells.

Description

A T cell sorting method Technical field

This application relates to the field of biomedicine, in particular to a T cell sorting method.

Background technique

As an important component of immune cells, T cells are a very complex heterogeneous unit, which is constantly renewed in the body, and there can be subgroups of different developmental stages or co-energy at the same time. T cells, through their own or coordination with other immune cells, play an important role in recognizing antigens, generating immune responses, and maintaining the body's physiological balance. In recent years, T cell-based cellular immunotherapy has developed rapidly. As a new treatment method, it has played its unique advantages clinically compared to traditional therapies. For example, in the field of anti-tumor, T cells and adoptive cell therapy (ACT) based on T cells are increasingly showing good therapeutic effects. Adoptive cell therapy refers to the reinfusion of autologous or allogeneic lymphocytes (such as T cells, CAR-T cells, etc.) stimulated and expanded in vitro into the human body to achieve anti-tumor effects. In order to effectively implement CAR-T cell therapy, it is necessary to prepare and infuse autologous or allogeneic T cells or CAR-T cells in a timely and successful manner.

At present, there are a variety of methods for separating and obtaining T cells from biological tissues, for example, immunomagnetic bead separation methods or flow cytometric separation methods. Flow cytometry usually damages cells and may pollute the separated cells and affect subsequent operations. The immunomagnetic bead separation method also has low separation yield, insufficient cell purity, or biosafety. The problem of low sex. Therefore, there is a need to develop more efficient T cell sorting methods.

Summary of the invention

This application provides a method for separating and obtaining T cells by using magnetic beads capable of binding CD4 or CD8 and a small cell sorting device. The amount of the sorting magnetic beads is 1.25 μL to 5 μL per mixture of the sorting magnetic beads. For the 10 ⁷ cells to be sorted, the method has at least the following technical effects: 1) The obtained T cells have high purity, and the cell count has a cell purity of more than 90%; 2) The T cell sorting rate is high, and the cell Counting has a cell yield of more than 50%. The magnetic bead buffer solution may also not contain sodium azide, which has little damage to cells and has good biological safety.

This application provides a method for sorting T cells, which includes the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer;

The magnetic separation beads are selected from one or two of the following: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 is 1.25 μL-5 μL that bind CD4 The sorting magnetic beads are mixed for every 10 ⁷ cells to be sorted, and the amount of CD8-binding magnetic sorting beads is 1.25 μL-5 μL CD8-binding magnetic sorting beads for every 10 ⁷ cells to be sorted ；

b) Sorting to obtain T cells.

In some embodiments, the cells to be sorted include PBMC cells.

In some embodiments, the amount of the CD4-binding magnetic sorting beads is 1.25 μL-2.5 μL CD4-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD4-binding magnetic sorting beads is 2.25-2.75 μL CD4-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD8-binding magnetic sorting beads is 1.25 μL-2.5 μL CD8-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD8-binding magnetic sorting beads is 2.25-2.75 μL CD8-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In some embodiments, the magnetic separation beads are selected from the following two types: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8.

In some embodiments, every 2.25-2.75 μL of the CD4-binding magnetic sorting beads and every 2.25-2.75 μL of the CD8-binding sorting magnetic beads are mixed with every 10 ⁷ cells to be sorted; optional The ground is that every 2.5 μL of the CD4-binding magnetic sorting beads and every 2.5 μL of the CD8-binding magnetic sorting beads are mixed with every 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD4-binding magnetic separation beads and the CD8-binding magnetic separation beads is 1:1.

In some embodiments, the particle size of the magnetic bead particles is 40 nm-5 μm.

In some embodiments, the particle size of the magnetic bead particles is 40 nm-100 nm.

In some embodiments, the particle diameter of the magnetic bead particles is 50 nm.

In some embodiments, the magnetic bead particles include iron dextran beads.

In some embodiments, the magnetic bead buffer in the CD4-binding magnetic bead sorting is a PBS/EDTA buffer containing 0.03% (w/v) of poloxamer 188; CD8-binding sorting The magnetic bead buffer in the magnetic beads is a PBS/EDTA buffer containing 0.03% (w/v) poloxamer 188.

In some embodiments, the separation magnetic beads that bind CD4 are selected from one or two of the following: CliniMACS CD4 Reagent, CliniMACS CD4 GMP MicroBeads of Miltenyi Company; the separation magnetic beads that bind CD8 It can be selected from one or two of the following: Miltenyi's CliniMACS CD8 Reagent, CliniMACS CD8 GMP MicroBeads.

In some embodiments, the magnetic bead buffer does not contain sodium azide.

In some embodiments, the magnetic bead buffer contains bovine serum albumin.

In some embodiments, the method for sorting T cells includes the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer, and the magnetic bead buffer contains bovine serum albumin and does not contain sodium azide;

The magnetic separation beads are selected from one or two of the following: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 is 1.25 μL-5 μL that bind CD4 The sorting magnetic beads are mixed for every 10 ⁷ cells to be sorted, and the amount of CD8-binding magnetic sorting beads is 1.25 μL-5 μL CD8-binding magnetic sorting beads for every 10 ⁷ cells to be sorted ；

b) Sorting to obtain T cells.

In some embodiments, the content of the bovine serum albumin is 0.01-0.5 wt%.

In certain embodiments, wherein the sorting includes magnetic field sorting.

In some embodiments, wherein the sorting includes sorting using a small cell sorting device.

In some embodiments, the small cell sorting device includes a sorting column.

In some embodiments, the sorting column includes a Miltenyi Biotec LS sorting column.

In some embodiments, wherein the sorting includes centrifugation before the magnetic field sorting and/or after the magnetic field sorting.

In certain embodiments, wherein the sorting includes elution after the magnetic field sorting.

In some embodiments, wherein the purity of the T cells is at least 90%.

The method according to claim 1, wherein the sorting yield of the cells to be sorted is at least 45%.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Correspondingly, the drawings and descriptions in the specification of the present application are merely exemplary, rather than restrictive.

Detailed ways

The following specific examples illustrate the implementation of the invention of this application. Those familiar with this technology can easily understand the other advantages and effects of the invention of this application from the content disclosed in this specification.

Definition of Terms

In this application, the term "magnetic beads" generally refers to magnetic solid bead-shaped particles that can be prepared by methods well known to those skilled in the art. For example, colloidal beads, microspheres, nanoparticles, etc. Methods for generating such magnetic beads are well known in the art. The magnetic beads can be in a solution or suspension, or in a freeze-dried state before use. The lyophilized magnetic beads can be processed in an appropriate buffer before contact with the sample. For example, the diameter size of the particles may be a minimum of 20 nm and a maximum of 1400 nm; for example, the particles have a size of 50 to 100 nm in diameter. For example, there may be at least one antigen recognition portion coupled to the magnetic beads, wherein the magnetic beads having the at least one antigen recognition portion can specifically bind to at least one antigen specific to the corresponding cell component. The magnetic beads may include, for example, ferromagnetic particles, superparamagnetic particles, paramagnetic particles, and the like. "Ferromagnetic" materials generally refer to substances that maintain magnetic properties when the magnetic field is removed. "Paramagnetic" materials generally mean that they have only weak magnetic susceptibility and quickly lose their weak magnetic properties when the magnetic field is removed. "Superparamagnetic" materials are generally highly magnetically sensitive, or they become strongly magnetic when placed in a magnetic field, but like paramagnetic materials, they quickly lose their magnetism when the magnetic field is removed.

In this application, the term "sorting" generally refers to a process of separating a certain target cell (for example, T cell) from a mixture (for example, blood, lymph, PBMC, etc.). It does not have to be 100% separation, for example, it can be the separation of 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the target cells in the mixture; The obtained target cell does not need to be 100% pure, for example, it may be substantially pure, for example, it may be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. %, 96%, 97% or above cell purity.

In this application, the term "small cell sorting device" generally refers to the ability to separate cells (such as CD4 or CD8 cells in this application) with magnetic labels (such as the dextran iron beads of this application) and non-magnetic Equipment for cell separation. For example, the small cell sorting device may include a sorting column and a sorting tube, a tube rack, and a magnetic separator. For example, the sorting column may include the MS column, LS column, XS column, CliniMACS Tubing Sets TS and LS commercially available from Miltenyi Biotec, and the tube rack may include the acrylic tube rack commercially available from Miltenyi Biotec. The separator may include a MidiMACS ^TM separator, a QuadroMACS ^TM separator, a VarioMACS ^TM separator, a SuperMACS ^TM II separator, or a ^{MultiMACS TM} Cell24 separator Plus.

In this application, the term "CD4" usually refers to the CD4 receptor, the full name is "Cluster of Differentiation 4 receptors" (Cluster of Differentiation 4 receptors). In the field of biomedicine, CD4 is a glycoprotein molecule on the surface of immune cells (such as helper T cells, monocytes, macrophages, and dendritic cells). It was discovered in the late 1970s and was known as leu-3 and T4 before 1984. Generally, the CD4 receptor is one of the surface markers of helper T cells, and it is also an important receptor for helper T cells to perform their functions. When antigen-presenting cells (such as macrophages, dendritic cells, and B cells themselves) decompose foreign pathogens, combine the antigen with the major histocompatibility complex, and present it to helper T cells (that is, on the surface of helper T cells). CD4 receptor binding).

In this application, the term "CD8" generally refers to the CD8 (Cluster of Differentiation 8) receptor, which refers to a transmembrane glycoprotein that acts as a co-receptor for the T cell receptor (TCR). Normally, like TCR, CD8 binds to major histocompatibility complex (MHC) molecules, but it is specific for MHC class I proteins. There are two subtypes of CD8, namely α and β, which are coded by different genes. In humans, both of these genes are located at position 2p12 of chromosome 2. CD8 is mainly expressed on the surface of cytotoxic T cells, but can also be found in natural killer cells, cortical thymocytes and dendritic cells. CD8 molecule can be used as a marker of cytotoxic T cell population.

In this application, the term "PBMC" usually refers to peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC, refers to any peripheral blood cells with round or nearly round nuclei. These cells can include lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets do not belong to this category because they have no nucleus; granulocytes (including neutrophils, basophils and eosinophils) have leafy nuclei, It also does not fall into this category. In some embodiments, ficoll (a hydrophilic polysaccharide that can separate the blood layer) and gradient centrifugation can be used to extract PBMC from whole blood. For example, the blood is separated into the upper plasma layer and then A layer of PBMC and a layer of polymorphonuclear cells (such as neutrophils and eosinophils) and the bottom part of red blood cells.

In this application, the term "T cell" generally refers to thymus-derived cells that participate in various cell-mediated immune responses. For example, the T cells may include: helper T cells (Th), the helper T cells T cells can assist humoral immunity and cellular immunity; cytotoxic T cells (Cytotoxic T cells, Tc), the Cytotoxic T cells can kill target cells. For example, the T cell may have a cell surface marker CD8. For example, the T cell may have a cell surface marker CD4.

Detailed description of the invention

On the one hand, this application provides a method for sorting T cells, which may include the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer;

The magnetic separation beads are selected from one or two of the following: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 is 1.25 μL-5 μL (for example, , 1.25μL, 1.3μL, 1.35μL, 1.4μL, 1.45μL, 1.5μL, 1.55μL, 1.6μL, 1.65μL, 1.7μL, 1.75μL, 1.8μL, 1.85μL, 1.9μL, 1.95μL, 2.0μL, 2.05 μL, 2.1μL, 2.15μL, 2.2μL, 2.25μL, 2.3μL, 2.35μL, 2.4μL, 2.45μL, 2.5μL, 2.55μL, 2.6μL, 2.65μL, 2.7μL, 2.75μL, 2.8μL, 2.85μL, 2.9μL, 2.95μL, 3.0μL, 3.05μL, 3.1μL, 3.15μL, 3.2μL, 3.25μL, 3.3μL, 3.35μL, 3.4μL, 3.45μL, 3.5μL, 3.55μL, 3.6μL, 3.65μL, 3.7μL , 3.75μL, 3.8μL, 3.85μL, 3.9μL, 3.95μL, 4.0μL, 4.05μL, 4.1μL, 4.15μL, 4.2μL, 4.25μL, 4.3μL, 4.35μL, 4.4μL, 4.45μL, 4.5μL, 4.55 μL, 4.6μL, 4.65μL, 4.7μL, 4.75μL, 4.8μL, 4.85μL, 4.9μL, 4.95μL, 5.0μL) CD4-binding magnetic separation beads are mixed for every 10 ⁷ cells to be sorted, combined with CD8 The amount of sorting magnetic beads is 1.25μL-5μL (for example, 1.25μL, 1.3μL, 1.35μL, 1.4μL, 1.45μL, 1.5μL, 1.55μL, 1.6μL, 1.65μL, 1.7μL, 1.75μL, 1.8 μL, 1.85 μL, 1.9 μL, 1.95 μL, 2.0 μL, 2.05 μL, 2.1 μL, 2.15 μL, 2.2 μL, 2.25 μL, 2.3 μL, 2.35 μL, 2.4 μL, 2.45 μL, 2.5 μL, 2.55 μL, 2.6 μL, 2.65μL, 2.7μL, 2.75μL, 2.8μL, 2.85μL, 2.9μL, 2.95μL, 3.0μL, 3.05μL, 3.1μL, 3.15μL, 3.2μL, 3.25μL, 3.3μL, 3.35μL, 3.4μL, 3.45μL , 3.5μL, 3.55μL, 3.6μL, 3.65μL, 3.7μL, 3.75μL, 3.8μL, 3.85μL, 3.9μL, 3.95μL, 4.0μL, 4.05μL, 4.1μL, 4.1 5μL, 4.2μL, 4.25μL, 4.3μL, 4.35μL, 4.4μL, 4.45μL, 4.5μL, 4.55μL, 4.6μL, 4.65μL, 4.7μL, 4.75μL, 4.8μL, 4.85μL, 4.9μL, 4.95μL, 5.0 uL) binding sorted CD8 magnetic beads per 10 ⁷ of mixing the cells to be sorted;

b) Sorting to obtain T cells.

For example, the cells to be sorted may include cell suspensions or mixtures of different phenotypes or subpopulations in different amounts, such as in whole blood, peripheral blood, leukapheresis, buffy coat, and cord blood. And cells in the bone marrow. For example, the cells to be sorted include PBMC cells. For another example, the cells to be sorted may be PBMC cells.

For example, the cells to be sorted may include red blood cells, platelets and white blood cells, such as T cells, regulatory T cells, B-cells, NK cells, dendritic cells, monocytes, granulocytes and/or hematopoietic stem cells.

In some embodiments, the amount of the CD4-binding magnetic sorting beads is 1.25 μL-2.5 μL CD4-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In certain embodiments, wherein said binding to CD4 magnetic beads sorted in an amount of per-2.75μL 2.25μL beads sorted CD4 binding mixture per ¹⁰⁷ cells to be sorted.

In some embodiments, the amount of the CD4-binding magnetic sorting beads is 2.5 μL-5 μL CD4-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD8-binding magnetic sorting beads is 1.25 μL-2.5 μL CD8-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD8-binding magnetic sorting beads is 2.25 μL-2.75 μL CD8-binding magnetic sorting beads per 10 ⁷ cells to be sorted.

In certain embodiments, wherein said binding amount of CD8 magnetic bead sorting the binding 2.5μL-5μL per sorted CD8 magnetic beads mixed per ¹⁰⁷ cells to be sorted.

In some embodiments, the magnetic separation beads are selected from the following two types: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8.

For example, the method for sorting T cells may include the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer;

The magnetic separation beads can be selected from the following two types: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 can be 1.25 μL-5 μL of CD4 combination The separation magnetic beads are mixed for every 10 ⁷ cells to be sorted, and the amount of the CD8-binding magnetic separation beads can be 1.25 μL-5 μL CD8-binding magnetic separation beads for every 10 ⁷ cells to be sorted ；

b) Sorting to obtain T cells.

In some embodiments, every 2.25-2.75 μL of the CD4-binding magnetic sorting beads and every 2.25-2.75 μL of the CD8-binding sorting magnetic beads are mixed with every 10 ⁷ cells to be sorted; optional The ground is that every 2.5 μL of the CD4-binding magnetic sorting beads and every 2.5 μL of the CD8-binding magnetic sorting beads are mixed with every 10 ⁷ cells to be sorted.

In some embodiments, the amount of the CD4-binding magnetic separation beads and the CD8-binding magnetic separation beads is 1:1.

For example, the amount of the CD4-binding magnetic separation beads and the CD8-binding magnetic separation beads may be 2.25-2.75 μL of the CD4-binding magnetic separation beads and every 2.25-2.75 μL of the CD8-binding magnetic separation beads. The sorting magnetic beads are mixed with every 10 ⁷ cells to be sorted; and the amount of the CD4-binding sorting magnetic beads and the CD8-binding sorting magnetic beads is 1:1. For another example, the amount of the CD4-binding magnetic separation beads and the CD8-binding magnetic separation beads may be 2.5 μL of the CD4-binding magnetic separation beads and every 2.5 μL of the CD8-binding magnetic separation beads. The sorting magnetic beads are mixed with every 10 ⁷ cells to be sorted.

For example, the amount of the CD4-binding magnetic separation beads and the CD8-binding magnetic separation beads may be 1.25 μL of the CD4-binding magnetic separation beads and every 1.25 μL of the CD8-binding magnetic separation beads. The sorting magnetic beads are mixed with every 10 ⁷ cells to be sorted.

For example, the amount of the CD4-binding magnetic sorting beads and the CD8-binding magnetic sorting beads may be 5 μL of the CD4-binding magnetic sorting beads and every 5 μL of the CD8-binding sorting magnetic beads. The magnetic beads are mixed with every 10 ⁷ cells to be sorted.

For another example, the method for sorting T cells may include the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer;

The magnetic sorting beads may be CD4-binding magnetic sorting beads and CD8-binding magnetic sorting beads; wherein every 2.25-2.75 μL of the CD4-binding magnetic separation beads and every 2.25-2.75 μL of the CD8-binding magnetic sorting beads The magnetic sorting beads are mixed with every 10 ⁷ cells to be sorted; and the amount of the CD4-binding magnetic sorting beads and the CD8-binding magnetic sorting magnetic beads is 1:1; optionally, every 2.5 μL The CD4-binding magnetic sorting beads and every 2.5 μL of the CD8-binding magnetic sorting beads are mixed with every 10 ⁷ cells to be sorted;

b) Sorting to obtain T cells.

In some embodiments, the particle size of the magnetic bead particles is 40 nm-5 μm.

For example, the particle size of the magnetic bead particles may be 40nm-60nm, 40nm-70nm, 40nm-80nm, 40nm-90nm, 40nm-100nm, 50nm-100nm, 3μm-5μm. For example, the particle size of the magnetic bead particles may be 50 nm, 3.5 μm, or 4.5 μm.

For example, the magnetic bead particles may include ferromagnetic, superparamagnetic, or paramagnetic solid phases, such as colloidal particles, microspheres, and nanoparticles. The particles can be used in a buffer or in a lyophilized state.

For example, the magnetic properties of the magnetic bead particles depend on the iron content in the particles, which may exist in the form of iron oxide such as magnetite or maghemite.

For example, the magnetic bead particles may contain, for example, 0.5 pg-500 pg per particle and 1pg-50 pg iron per particle. For example, the magnetic bead particles may include MACSiBead particles, which may be retained by, for example, a standard magnet, such as a MACSiMAG separator (Miltenyi Biotec). For example, MACSiBeads particles with a diameter of 3.5 μm may contain 3%-10% w/w iron by dry weight.

For example, the magnetic bead particles may contain, for example, 0. lfg to 50 fg per particle and 0. lfg to 10 fg per particle of iron. For example, the magnetic bead particles may include MicroBead particles, which may be retained by, for example, a combination of a standard magnet and a sorting column, such as a QuadroMACS ^™ separator combined with an LS column available from Miltenyi Biotec. The MicroBead particles may have a diameter of 50-100 nm, and each particle contains 30%-60% w/w iron by dry weight. For example, the MicroBead particles may have a diameter of 50 nm.

For example, the magnetic bead particles of the present application can be characterized by a diameter outside of the iron content or instead of the iron content. For example, the size of the magnetic particles may be monodisperse, for example, the magnetic bead particles exhibit an average diameter of 1-5 μm, such as 2.5-3.5 μm, such as 3 μm-5 μm. For example, the cv of the diameter of the magnetic bead particles is less than 15%, for example, less than 10%. For example, the magnetic bead particles may have an average diameter of 10-250 nm, such as 30-150 nm, for example, 40-100 nm, for example, 40-60 nm, and the cv of the diameter thereof is, for example, less than 60%, for example, less than 30%.

For example, the determination of the iron content of the magnetic bead particles (dry) can be carried out by degrading iron oxide with acid (such as phosphoric acid), and quantifying the dissolved iron ions by an analytical tool, for example, using Merck Spectroquant detection reagent for iron test Box spectrophotometric determination of colored complexes. The particle count of the magnetic bead suspension can be determined by a particle counter or a Neubauer chamber using a microscope method. The mass concentration in the same magnetic bead suspension can be determined by: transferring a determined volume of the bead material of the magnetic bead suspension to water by washing, removing the water by drying and weighing the solid residue. Knowing the mass concentration of the dry bead material and the iron content of the dry bead material, knowing the number of particles in the bead suspension can calculate the iron content of the particles. For example, each particle of MACSiBead with 44.5 mg/ml dry weight beads, 1.36× ^{10 9} particles/mg and an iron content of 4.61% w/w in the dry bead material contains 1.51 pg of iron.

For example, the particle count can be obtained indirectly, but according to Aaron B. Kantor, Ian Gibbons, Stefan Miltenyi, and Jiirgen Schmitz in "Cell Separation Methods and Applications (Ed. Diether Recktenwald, Andreas Radbruch, Marcel Dekker)" , New York, 1998p.153-171), estimated by assuming a spherical shape and calculating the weight of a single particle using the hydrodynamic diameter measured by DLS and a particle density of 2.5mg/mL. For example, with 100nm The size of the microbead, the dry weight of 10 mg/mL, and the iron content of 40% w/w in the dry bead material contain 0.53 fg of iron per particle of Microbead.

For example, the magnetic bead particles may include dextran. For example, the magnetic bead particles may include iron dextran beads.

For example, magnetic beads can be manufactured with different process parameters to generate particles of different sizes. The particle size can be characterized by Beckman Coulter Delsa Nano and Coulter Counter Z2 instruments.

For example, the magnetic sorting beads comprise antibodies against CD4 or CD8, and the magnetic sorting beads bind CD4 or CD8 through the antibodies, respectively. The CD4 or CD8 may comprise CD4 or CD8 expressed on the surface of cells (for example, T cells). The antibodies include anti-CD4 murine monoclonal antibodies and anti-CD8 murine monoclonal antibodies. For example, the magnetic sorting beads comprise iron dextran beads coupled with a CD8 mouse monoclonal antibody and/or iron dextran beads coupled with a CD4 mouse monoclonal antibody.

In some embodiments, the magnetic bead buffer in the CD4-binding magnetic bead sorting is a PBS/EDTA buffer containing 0.03% (w/v) of poloxamer 188; CD8-binding sorting The magnetic bead buffer in the magnetic beads is a PBS/EDTA buffer containing 0.03% (w/v) poloxamer 188.

In some embodiments, the magnetic bead particles are commercially available through Miltenyi Biotec, for example, CliniMACS CD4 Reagent or CliniMACS CD4 GMP MicroBeads, CliniMACS CD8 Reagent, or CliniMACS CD8 GMP MicroBeads. For example, the magnetic bead particles can be commercially available through Stemcell, for example, EasySep ^TM Human CD4 Positive Selection Cocktail II, CliniMACS CD8 Reagent; EasySep ^TM Human CD8 Positive Selection Cocktail II. For example, the magnetic bead particles can be commercially available through Thermo, for example, Dynabeads ^™ CD4, Dynabeads ^™ CD8.

In some embodiments, the separation magnetic beads that bind CD4 are selected from one or two of the following: CliniMACS CD4 Reagent, CliniMACS CD4 GMP MicroBeads of Miltenyi Company; the separation magnetic beads that bind CD8 It can be selected from one or two of the following: Miltenyi's CliniMACS CD8 Reagent, CliniMACS CD8 GMP MicroBeads.

For example, the sorting magnetic beads that bind CD4 can be selected from one or two of the following: CliniMACS CD4 Reagent (CE; REF 276-01), CliniMACS CD4 GMP MicroBeads (REF 170-076-702) ); The separation magnetic beads combined with CD8 can be selected from one or two of the following: CliniMACS CD8 Reagent (CE; REF 275-01), CliniMACS CD8 GMP MicroBeads (REF 170-076-703) ).

In some embodiments, the magnetic bead buffer does not contain sodium azide.

In some embodiments, the magnetic bead buffer contains bovine serum albumin.

For example, the method for sorting T cells may include the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein, the sorting magnetic beads may include magnetic bead particles and a magnetic bead buffer, and the magnetic bead buffer may contain bovine serum albumin and does not contain sodium azide;

The magnetic separation beads can be selected from one or two of the following: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 is 1.25 μL-5 μL per binding The CD4 sorting magnetic beads are mixed for every 10 ⁷ cells to be sorted, and the amount of CD8-binding magnetic sorting beads is 1.25 μL to 5 μL CD8-binding sorting magnetic beads for every 10 ⁷ cells to be sorted cell;

b) Sorting to obtain T cells.

For another example, the method for sorting T cells may include the following steps:

a) Mix the cells to be sorted with the sorting magnetic beads;

Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer, and the magnetic bead buffer contains bovine serum albumin and does not contain sodium azide;

The magnetic sorting beads may be CD4-binding magnetic sorting beads and CD8-binding magnetic sorting beads; wherein every 2.25-2.75 μL of the CD4-binding magnetic separation beads and every 2.25-2.75 μL of the CD8-binding magnetic sorting beads The magnetic sorting beads are mixed with every 10 ⁷ cells to be sorted; and the amount of the CD4-binding magnetic sorting beads and the CD8-binding magnetic sorting magnetic beads is 1:1; optionally, every 2.5 μL The CD4-binding magnetic sorting beads and every 2.5 μL of the CD8-binding magnetic sorting beads are mixed with every 10 ⁷ cells to be sorted;

b) Sorting to obtain T cells.

For example, the content of the bovine serum protein may be 0.01-0.5 wt%. For example, the content of the bovine serum protein may be 0.01-0.1 wt%. For example, the content of the bovine serum protein may be 0.01-0.2 wt%. For example, the content of the bovine serum protein may be 0.01-0.3 wt%. For example, the content of the bovine serum protein may be 0.01-0.4 wt%. For example, the content of the bovine serum protein may be 0.1-0.5 wt%.

In some embodiments, the sorting includes magnetic field sorting. For example, the magnetic field sorting may include separating cells (such as CD4 or CD8 cells of the present application) with magnetic labels (such as the iron dextran beads of the present application) from non-magnetic cells in a high gradient magnetic field. For example, a suspension of cells (such as CD4 or CD8 cells of the present application) and non-magnetic cells with magnetic labels (such as the iron dextran beads of the present application) and non-magnetic cells is guided through a column equipped with a ferromagnetic matrix that is subjected to a strong magnetic field. The magnetically labeled cells remain on the ferromagnetic matrix, while the non-magnetic cells are removed, and then the magnetically labeled cells are harvested by removing the cylinder from the magnetic field and washing the cells from the ferromagnetic matrix.

For example, the sorting may include sorting using a small cell sorting device. For example, the small cell sorting device includes a sorting tube and a sorting column. For example, the sorting column may be placed in the sorting tube.

For example, the sorting column may include a matrix capable of generating a high gradient magnetic field. For example, the sorting column may contain a matrix capable of generating a high gradient magnetic field when it is placed in a magnetic separator.

For example, the matrix may comprise a ferromagnetic material. For example, the ferromagnetic material may be spherical, or other non-spherical particles, or an integrated three-dimensional grid with suitable porosity. For example, the ferromagnetic material can be coated with a coating that can maintain the relative position of the particles to each other. For example, the coating can be a varnish. For example, the size of the ferromagnetic material balls/particles is greater than about 100 μm, for example, greater than about 200 μm and less than about 2000 μm, for example, greater than about 200 μm and less than about 1000 μm, most for example, about 280 μm. The separation matrix that can be used for high gradient magnetic separation (HGMS) can be found in U.S. Patent Application 08/377,744 and U.S. Patent No. 5,235,235. For example, the void volume of the column, that is, the void volume of the filter portion not occupied by the matrix is less than about 800 μl, for example, less than about 700 μl, for example, less than about 500 μl, for example, about 400 μl. For example, the gravity flow rate is greater than about 200 μl/min, such as greater than about 400 μl/min, such as greater than about 700 μl/min.

For example, the sorting column may include MS column, LS column, XS column, CliniMACS Tubing Sets TS and LS commercially available from Miltenyi Biotec. For another example, the sorting column may include a Miltenyi Biotec LS sorting column.

For example, the sorting tube can be made of polyamide, polystyrene, polyolefins such as polyethylene and polypropylene, polycarbonate, polyoxymethylene, acrylics such as polymethylmethacrylate, PET, polylactic acid or polyamide, etc. production. For example, when the substrate is coated with lacquer paint ==, it can be made of plastic that can be combined with lacquer, such as resin, such as PCTG (polycyclohexamethylene terephthalate modified with ethylene glycol) . It can be made hydrophilic by being made of a hydrophilic material (for example, a column made of hydrophilic plastic), for example, the inner side of the sorting tube can be coated with a hydrophilic material such as polyvinylpyrrolidone.

For example, porous frit or grids can be positioned near the top of the matrix near the top of the matrix. The porous glass frit or grid can be made of glass or plastic or metal mesh, for example, and has a pore size greater than or equal to the pore size of the matrix and smaller than the particle size of the matrix.

For example, the gravity flow rate of the PBS buffer suitable for the sorting column may include 1.3-2.0 ml/min. For example, the PBS buffer may contain 0.4%-0.6% bovine serum albumin (BSA). For example, the PBS buffer may contain 0.5% bovine serum albumin (BSA). For example, the PBS buffer may contain EDTA. For example, the Ph value of the PBS buffer may be about 7.2. For example, the PBS buffer is a degassed buffer.

For example, the void volume of the sorting tube may include 300 μL-500 μL. For example, the void volume of the sorting tube may include 400 μL. For example, the reservoir volume of the sorting tube may include 8 mL.

For example, the cell sample amount of the sorting column may include 10 ⁵ -10 ⁸ labeled cells/10 ⁷ -2×10 ⁹ total cell number.

For example, the small cell sorting device may also include a magnetic separator. The magnetic separator is used to generate a magnetic field. For example, a MACS splitter can be included. For example, the MACS separator is selected from the following magnetic separators: MidiMACS ^™ separator, QuadroMACS ^™ separator, VarioMACS ^™ separator, SuperMACS ^™ II separator, or ^MultiMACS™ Cell24 separator Plus.

For example, the small cell sorting device may also include a MACS tube rack. For example, the MACS tube rack may include an acrylic tube rack. For example, the tube rack is a 15 mL tube rack. For example, MACS MultiStand.

For example, the sorting may include centrifugation before the magnetic field sorting and/or after the magnetic field sorting. For example, the centrifugation includes centrifugation after mixing the cells to be sorted with the sorting magnetic beads, and includes adding 1.5-3ml of sorting washing solution, such as 2ml sorting washing solution, 250-350g centrifugation, such as 300g centrifugation, and centrifugation. 8-12 minutes, for example, centrifuge for 10 minutes.

For example, the magnetic sorting may include placing a tube rack in a biological safety cabinet, using QuadroMACS Separator to set a magnetic field, one sorting tube in each group, placing the Miltenyibiotec LS sorting column in the magnetic field, and placing a 50ml centrifuge under the sorting tube The tube is used to receive CD4-CD8- cells. Add 1-5ml (for example, 1ml, 2ml, 3ml, 4ml or 5ml) of the sorting lotion to the sorting tube to rinse the adsorption column, and then add 500ul of the sorting lotion to the sorting tube after centrifugation. Add the mixture of the suspended cells to be sorted and the magnetic sorting beads into the sorting tube, and when the cell suspension has all flowed into the adsorption column of the sorting tube, add 1-5ml again (for example, 1ml, 2ml, 3ml, 4ml or 5ml) Separate the lotion for cleaning, cleaning 1-3 times. For example, 2 times, for example, 3 times.

For example, the sorting may include elution after the magnetic field sorting. For example, the elution includes removing the sorting column from the magnetic field, placing them on a 15 ml centrifuge tube, and adding 3-5 ml of sorting washing solution for elution. For example, add 4ml of sorting lotion.

For example, the sorting may include centrifugation after the magnetic field sorting. For example, the centrifugation includes centrifuging the collected eluate, which contains target cells (such as T cells), and adding 5-15ml of medium (such as 7ml, 8ml, 9ml, 10ml, 11ml, or 12ml) washing, 250-350g centrifugation, for example, 300g centrifugation, centrifugation for 8-12 minutes, for example, centrifugation for 10 minutes.

In some embodiments, the purity of the T cells may be at least 90%. For example, the purity of the T cell may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

In some embodiments, the sorting yield of the cells to be sorted may be at least 45%. For example, the sorting yield can be 48.2%, 48.7%, 55.2%, 53.8%, 63.5%, 70.5%, 71% or higher.

Without intending to be limited by any theory, the following examples are only used to illustrate the method of the present application, and are not used to limit the scope of the present application.

Example

Example 1

1. Peripheral blood mononuclear cells (PBMC) separated by Ficoll (purchased from GE, article number 17-1440-02) by density gradient centrifugation, counted 1x10 ⁷ cells per group, prepare each experimental group, and divide them into groups Centrifuge the PBMC cells at 1200 rpm, 4°C for 7 minutes. Respectively use 80ul sorting lotion (each 20ml of CliniMACS PBS/EDTA Buffer (purchased from Miltenyi (Miltenyi), item number: 200-070-025) and 0.5ml of human albumin injection containing 20% HSA) Hanging cells.

2. Add 20ul, 10ul, 5ul, 2.5ul and 1.25ul CD4 magnetic beads to each 1x10 ⁷ cells (purchased from Miltenyi, catalog number: 200-070-132 (276-01)) And CD8 magnetic beads (purchased from Miltenyi (Miltenyi), article number: 200-070-115 (275-01)), mix well, place at 2-8°C, let stand for 15 minutes, and mix well every 5 minutes during this period once.

3. Take out the cell suspension from 2-8°C, add 2ml of sorting lotion to each group, mix well, and centrifuge at 300g for 10 minutes.

4. Discard the supernatant, add 500ul sorting lotion to each group to resuspend the cells.

5. Place the tube rack (purchased from Miltenyibiotec, model: 130-042-303) in a biological safety cabinet (purchased from Heal Force, model: HFsafe 1200LCB2), and use a magnetic separator for sorting, which is purchased from Miltenyibiotec , Model: QuadroMACS Separator, each group uses a sorting tube, put the LS sorting column (purchased from Miltenyibiotec, model: 130-042-401) in a magnetic field, and place a 50ml centrifuge tube under the sorting tube to receive CD4 -CD8-cells, add 3ml of sorting lotion to the sorting tube to rinse the adsorption column.

6. Add all the cell suspensions of each group obtained in step 4 into the sorting tube, and when the cell suspensions have all flowed into the adsorption column of the sorting tube, add 3ml of sorting lotion again for washing, and repeat washing 3 times.

7. Remove all the sorting tubes from the magnetic field, put them on a 15ml centrifuge tube, and add 3-5ml sorting lotion for elution.

8. Add 10ml KBM581 medium (purchased from Corning, model: 88581-CM) to the eluted cell suspension, wash, 300g, and centrifuge for 10 minutes.

9. Discard the supernatant, add 3ml of sorting lotion to resuspend, sample and count for use, and calculate the T cell yield according to the following formula:

T cell yield = total number of cells after sorting/number of CD3+ cells in PBMC.

The results obtained are shown in Table 1.

10. Select peripheral blood mononuclear cells (PBMC) from another source, repeat steps 1-9, and the results are shown in Table 2.

Table 1 and Table 2 list the ratio of different cells to the total number of cells before and after sorting, as well as the cell yield of CD3+ T cells.

Table 1 Data results of CD4 and CD8 sorting magnetic beads with small cell sorting device (Sample 1)

Table 2 Data results of CD4 and CD8 sorting magnetic beads with small cell sorting device (Sample 2)

From the results of Table 1 and Table 2, CD3 + cell purity and yield of cells CD4 / CD8 magnetic bead sorting amount at 1.25 / 1.25ul / 10 ⁷ cells to 5 / 5ul / 10 ⁷ cells, obtained by sorting Both are relatively high and can sort and obtain T cells more efficiently.

Example 2

T cell sorting was performed on samples from different sources according to the method in Example 1, and the results are shown in Table 3.

Table 3 Data results of CD4/CD8 sorting magnetic beads dosage under ^{the condition of 2.5/2.5ul/10 7 cells}

Among them, (1) the content of CD3+ T cells, B cells, NK cells, monocytes, and granulocytes all refer to the proportion of CD45+ nucleated cells; (2) Sorting yield (T cells) = (cells after sorting) Total*T cell content after sorting)/(Total number of PBMC cells*T cell content in PBMC); Samples 13 to 22 are production data, and the T cell content in PBMC is not tested for official production, so the sorting yield (T Write N/A in the cell) column.

From the results of Table 3, for different samples, CD4 / CD8 magnetic beads sorted in an amount 1.25 / 1.25ul / 10 ⁷ cells under conditions 5 / 5ul / 10 ⁷ cell, CD3 + T cell purity substantially The above is above 95%, and the yield is above 50%, indicating that the purity and cell yield of CD3+ cells obtained by the method of the present application are relatively high, and it can sort and obtain T cells more efficiently.

Claims (27)

1. A method for sorting T cells, which includes the following steps:

   a) Mix the cells to be sorted with the sorting magnetic beads;

   Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer;

   The magnetic separation beads are selected from one or two of the following: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 is 1.25 μL-5 μL that bind CD4 The sorting magnetic beads are mixed for every 10 ⁷ cells to be sorted, and the amount of CD8-binding magnetic sorting beads is 1.25 μL-5 μL CD8-binding magnetic sorting beads for every 10 ⁷ cells to be sorted ；

   b) Sorting to obtain T cells.
2. The sorting method according to claim 1, wherein the cells to be sorted comprise PBMC cells.
3. The sorting method according to claim 1 or 2, wherein the amount of the CD4-binding magnetic sorting beads is 1.25 μL-2.5 μL CD4-binding magnetic sorting beads per 10 ⁷ cells to be sorted.
4. The sorting method according to claim 1 or 2, wherein the amount of the CD4-binding magnetic sorting beads is 2.25 μL-2.75 μL CD4-binding magnetic sorting beads per 10 ⁷ cells to be sorted.
5. The sorting method according to claim 1 or 2, wherein the amount of the CD8-binding magnetic sorting beads is 1.25 μL-2.5 μL of CD8-binding magnetic sorting beads per 10 ⁷ cells to be sorted.
6. The sorting method according to claim 1 or 2, wherein the amount of the CD8-binding magnetic sorting beads is 2.25 μL-2.75 μL CD8-binding magnetic sorting beads per 10 ⁷ cells to be sorted.
7. The sorting method according to claim 1, wherein the sorting magnetic beads are selected from the following two types: CD4-binding sorting magnetic beads and CD8-binding sorting magnetic beads.
8. The sorting method according to claim 7, wherein every 2.25-2.75 μL of the CD4-binding magnetic sorting beads and every 2.25-2.75 μL of the CD8-binding sorting magnetic beads and every 10 ⁷ cells to be sorted Mixing; Optionally, every 2.5 μL of the CD4-binding magnetic sorting beads and every 2.5 μL of the CD8-binding sorting magnetic beads are mixed with every 10 ⁷ cells to be sorted.
9. 8. The sorting method according to claim 8, wherein the amount of the CD4-bound magnetic separation beads and the CD8-bound magnetic separation beads is 1:1.
10. The sorting method according to claim 1, wherein the particle size of the magnetic bead particles is 40nm-5μm.
11. The sorting method according to claim 10, wherein the particle size of the magnetic bead particles is 40 nm-100 nm.
12. The sorting method according to claim 11, wherein the particle diameter of the magnetic bead particles is 50 nm.
13. The sorting method according to claim 1, wherein the magnetic bead particles comprise iron dextran beads.
14. The sorting method according to claim 1, wherein the magnetic bead buffer in the CD4-binding magnetic bead sorting is a PBS/EDTA buffer containing 0.03% (w/v) poloxamer 188; The magnetic bead buffer in the sorting magnetic beads of CD8 is a PBS/EDTA buffer containing 0.03% (w/v) poloxamer 188.
15. The sorting method according to claim 1, wherein the sorting magnetic beads that bind CD4 are selected from one or two of the following: CliniMACS CD4 Reagent, CliniMACS CD4 GMP MicroBeads of Miltenyi The magnetic separation beads are selected from one or two of the following: Miltenyi's CliniMACS CD8 Reagent, CliniMACS CD8 GMP MicroBeads.
16. The sorting method according to claim 1, wherein the magnetic bead buffer does not contain sodium azide.
17. The sorting method according to claim 1, wherein the magnetic bead buffer contains bovine serum albumin.
18. The sorting method according to any one of claims 16-17, which comprises the following steps:

    a) Mix the cells to be sorted with the sorting magnetic beads;

    Wherein the magnetic separation beads comprise magnetic bead particles and magnetic bead buffer, and the magnetic bead buffer contains bovine serum albumin and does not contain sodium azide;

    The magnetic separation beads are selected from one or two of the following: magnetic separation beads that bind CD4 and magnetic separation beads that bind CD8; wherein the amount of magnetic separation beads that bind CD4 is 1.25 μL-5 μL that bind CD4 The sorting magnetic beads are mixed for every 10 ⁷ cells to be sorted, and the amount of CD8-binding magnetic sorting beads is 1.25 μL-5 μL CD8-binding magnetic sorting beads for every 10 ⁷ cells to be sorted ；

    b) Sorting to obtain T cells.
19. The sorting method according to claim 18, wherein the content of the bovine serum protein is 0.01-0.5 wt%.
20. The sorting method according to claim 1, wherein the sorting includes magnetic field sorting.
21. The sorting method according to claim 1, wherein the sorting comprises sorting using a small cell sorting device.
22. The sorting method according to claim 21, wherein the small cell sorting device comprises a sorting column.
23. The sorting method according to claim 22, wherein the sorting column comprises a Miltenyi Biotec LS sorting column.
24. The sorting method according to claim 1, wherein the sorting comprises centrifugation before the magnetic field sorting and/or after the magnetic field sorting.
25. The sorting method according to claim 1, wherein the sorting includes elution after the magnetic field sorting.
26. The sorting method according to claim 1, wherein the purity of the T cells is at least 90%.
27. The sorting method according to claim 1, wherein the sorting yield of the cells to be sorted is at least 45%.