CN-110229781-B - Cell separation method

CN110229781BCN 110229781 BCN110229781 BCN 110229781BCN-110229781-B

Abstract

The application discloses a cell separation method which comprises the steps of providing a cell sample liquid, mixing the cell sample liquid and first type magnetic beads in a separation cup, wherein the first type magnetic beads are combined with first type antibodies for being combined with the first type cells, the diameter of the first type magnetic beads is 500-4500 nanometers, placing the separation cup with the first type magnetic beads and the cell sample liquid above a separation seat with a magnet, so that the magnet adsorbs the first type magnetic beads to the bottom of the separation cup, and separating the first type magnetic beads from cell supernatant. The cell separation method of the application can improve the cell separation efficiency and purity.

Inventors

NIU HAITAO
LING JIANDONG
GUAN ZHIPING
XU AIHONG
YANG XUE
QIN XIAORUI

Assignees

深圳海思安生物技术有限公司
深圳海思安生物技术有限公司

Dates

Publication Date: 20260421
Application Date: 20190611
Priority Date: 20190611

Claims (8)

1. A cell separation method, characterized in that the cell separation method performs a separation step using a cell separation apparatus comprising: The separation seat comprises a partition plate and a side plate, a plurality of accommodating grooves are formed in the lower portion of the partition plate, a guide plate assembly perpendicular to the partition plate is arranged above the partition plate, the guide plate assembly comprises a first guide plate and a second guide plate which are arranged in parallel at intervals, the side plate is arranged on one side of the partition plate and perpendicular to the partition plate and the guide plate assembly, and a limiting hole is formed in the side plate; The separating cup comprises a plurality of separating cups, wherein two adjacent separating cups are connected through a connecting block to form a separating cup assembly, the separating cup assembly is integrally formed, the connecting block is of an inverted V shape, a first clamping groove and a second clamping groove are formed in the connecting positions of the connecting block and the two adjacent separating cups respectively, the first clamping groove is clamped with the first guide plate, the second clamping groove is clamped with the second guide plate, a limiting rod is arranged on the connecting block in an outward protruding mode, and the limiting hole is matched with the limiting rod; the magnets are correspondingly arranged in the accommodating grooves, and the magnets are in one-to-one correspondence with the separating cups; the cell separation method comprises the following steps: providing a cell sample fluid, wherein the cell sample fluid at least comprises a first type of cells; mixing the cell sample liquid and first type magnetic beads in a separation cup, wherein the first type magnetic beads are combined with first type antibodies and are used for being combined with the first type cells, and the diameters of the first type magnetic beads are 500-4500 nanometers; placing the separation cup with the first type of magnetic beads and the cell sample liquid over a separation seat with a magnet so that the magnet adsorbs the first type of magnetic beads to the bottom of the separation cup; inverting the separation cup and the separation seat, and pouring to remove the cell supernatant so as to separate the first type magnetic beads from the cell supernatant.
2. The method of claim 1, wherein separating the first type of magnetic beads from the cell supernatant further comprises: And removing the separation cup from the separation seat to obtain a first target cell, wherein the first target cell is a first type cell which is precipitated in the separation cup and is coupled with a first type magnetic bead.
3. The method of claim 2, wherein the removing the separation cup from the separation housing further comprises: And adding a cell culture solution into the separation cup to obtain the first target cell sample solution, wherein the first target cell sample solution can be directly used for the next operation.
4. The method of claim 1, wherein mixing the cell sample fluid and the first type of magnetic beads in a separation cup comprises: Adding the cell sample liquid into the separation cup, and adding 0.3-2.0 times of the first type magnetic beads into the cell sample liquid to obtain a cell mixed liquid, or Mixing the cell sample liquid with 0.3-2.0 times of the first type magnetic beads to obtain a cell mixed liquid, and adding the cell mixed liquid into the separation cup.
5. The method of claim 1, wherein separating the first type of magnetic beads from the cell supernatant further comprises: removing the separation cup from the separation seat, adding cell culture fluid into the separation cup, and re-suspending the first type magnetic beads and the first type cells; Placing the separating cup above the separating seat with the magnet again; separating the first type of magnetic beads from the cell supernatant.
6. The method according to claim 1, wherein the cell sample liquid further comprises a second type of cells, and the separating the first type of magnetic beads from the cell supernatant further comprises: collecting the cell supernatant, mixing the cell supernatant and a second type of magnetic beads in a separate cup, wherein the second type of magnetic beads are combined with a second type of antibody for combining with the second type of cells; Placing the separation cup with the second type of magnetic beads and the cell supernatant above the separation seat with the magnet; separating the second type of magnetic beads from the cell supernatant; And removing the separation cup from the separation seat to obtain second target cells, wherein the second target cells are second type cells which are precipitated in the separation cup and are coupled with second type magnetic beads.
7. The method of claim 1, wherein mixing the cell sample fluid and the first type of magnetic beads in the separation cup further comprises: and incubating the cell sample liquid mixed with the first type magnetic beads for 10-40 minutes at the temperature of 2-8 ℃.
8. The method of claim 1, wherein the first type of cells are positive cells and the first type of magnetic beads are magnetic beads with antibodies corresponding to the positive cells.

Description

Cell separation method Technical Field The application relates to the technical field of biology, in particular to a cell separation method. Background Lymphocytes (lymphocytes) are one of the white blood cells, the smallest volume of which is produced by lymphoid organs and which are important cellular components of the immune response function of the body. Lymphocytes are a class of cell lines with immune recognition function, and can be classified into T lymphocytes (also known as T cells), B lymphocytes (also known as B cells), and Natural Killer (NK) cells according to their migration, surface molecules, and functions. T cells and B cells are antigen-specific lymphocytes, which are initially identical in origin, and are derived from hematopoietic tissues. T lymphocytes circulate with blood to the thymus, mature under the action of thymus hormones and the like, while B cells differentiate and mature in the bone marrow. When stimulated by antigen, T lymphocyte is converted into lymphoblast, and then is subdivided into sensitized T lymphocyte to participate in cellular immunity, and its immune function is mainly resisting intracellular infection, tumor cell and foreign body cell, etc., while B lymphocyte is firstly converted into plasmablasts, then is subdivided into plasmablasts to produce and secrete immune globulin (antibody), and participates in humoral immunity, its function is to produce antibody, present antigen and secrete intracellular factor to participate in immune regulation, NK cell can spontaneously exert cytotoxity effect independent of antigen stimulation, and has the action of killing target cell. Twenty years ago, with the advent of recombinant hormone, soluble receptor and antibody based biological agents, an important revolution has emerged in the pharmaceutical industry, governed for a long time by small molecule drugs as the sole support. Now, along with the development of the multipotency of the treatment of microorganisms and human cells, biological medicine is pushed to the blast tip, and a new revolution is initiated. Continuous advances in cell engineering provide a systematic framework for developing safe, predictable cell therapies. Microorganisms and human cells are also used as therapeutic entities, potentially addressing some important, currently unmet needs, for the treatment of certain most deadly diseases, including cancer, autoimmune diseases, and the like. Researchers at the university of california, san francisco, in the journal of science-transformation medicine (ScienceTranslationalMedicine), column PERSPECTIVE, published an article on the overview of the prospect of cell therapy. The article states that cell therapy will become the "third largest medical stay" in the future, and will be used universally to treat patients as drugs now made with engineered proteins, antibodies or smaller chemicals. Researchers have come to the metaphor that if small molecules and biological products are tools, then the cells are carpenters, architects, and engineers. The cells can perform some functions that cannot be achieved by small molecule drugs and targeted drugs. For example, cells are adaptable, they can migrate to specific sites, sense the surrounding environment better than current drugs, then make their correct decisions themselves, change their responses, and adapt better to physiological conditions. First, cells are naturally responsible for many therapeutic tasks, such as phagocytosis of pathogens by macrophages, recruitment of adaptive immune cells, production of myeloid and lymphoid lineage cells by hematopoietic stem cells, production of chondrocyte extracellular matrix by chondrocytes, and the like. Second, the behavior of the cells is selective. For small molecules and biological agents, they do not have a switch, and exert their biological effects as long as they bind to the target. However, cells are able to sense the surrounding environment and only act when a specific signaling molecule is triggered. Therefore, the cell therapy can better avoid off-target effect, and also can play a better delivery role, thus being a good carrier for other therapies. Third, cell therapy can better adapt to human genetic diversity. For example, due to individual differences, drugs are metabolized differently in the human body and the therapeutic effects are also different. But through reforming cells, the concentration change can be automatically regulated like a resistance circuit, and the cell is suitable for metabolism of different hosts, so that better curative effect is exerted. Finally, the function of the cells can be regulated by altering the cellular genes. For example, T lymphocytes are modified by a biosynthesis technology to induce blood sugar and secrete insulin, so that the dependence of type 1 diabetics on blood sugar is relieved, and immune response to cancer is usually weaker, but anti-tumor response can be improved by manipulating and culturing immune cell popu