CN114958755A - Method and device for grading enrichment of cancer cells and organoids - Google Patents
Method and device for grading enrichment of cancer cells and organoids Download PDFInfo
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Abstract
The invention discloses a method for enriching cancer cells and organoids in a grading way, which comprises the steps of mixing and incubating a cell recovery solution without digestive enzyme with hydrogel containing organoids and dispersed in cells to obtain a hydrogel liquefaction solution; then mixing DMEM cell culture solution or phosphate buffer solution containing 10% fetal calf serum with the hydrogel liquefied solution to obtain hydrogel liquefied diluent; slowly pouring the hydrogel liquefied diluent into a filter arranged in a container, and vibrating and sieving the diluent, wherein the filter is formed by vertically arranging a plurality of layers of screens at intervals and gradually increasing the pore diameter from bottom to top; taking out the screen from top to bottom layer by layer, respectively and reversely buckling the screen in different containers with cell culture solution, and eluting the filtered substance into the cell culture solution; centrifuging the cell culture solution containing the filtered substance, and discarding the supernatant to obtain cancer cells and organoids.
Description
Technical Field
The invention relates to a method and a device for collecting cancer cells and organoids, in particular to a method and a device for enriching the cancer cells and the organoids in a grading way.
Background
Organoids (organoids) are multicellular three-dimensional (3D) structures formed by stem, normal or tumor cells with proliferative capacity in a semi-solid matrix gel. Because the organoid well retains the morphological characteristics, biological functions and molecular characteristics of the source tissue on phenotype and function, obviously reduces unnecessary experimental animal loss due to the fact that the organoid can substitute the body reagent, and avoids the ethical problem of human body test, the organoid is used as a novel in vitro experimental model for biomedical research and drug research and development, and has very wide application prospect in the fields of disease model construction and prevention, tissue regeneration, injury repair, organ transplantation technology improvement, drug sensitivity screening and curative effect evaluation of cancer, drug safety evaluation and the like. Many biomedical studies require that the number of cells and/or organoids and volumes within the experimental system be as uniform as possible to avoid bias in the results, to obtain data that is reliable, reproducible, and amenable to high throughput analysis.
Most of the existing organoid construction methods are to inoculate cell suspension prepared from pluripotent stem cells, animal tissues and patient specimens into Engelbreth-Holm-swarm (EHS) mouse sarcoma basement membrane extract jelly/gel containing type IV collagen, laminin, Heparin Sulfate Proteoglycan (HSPG) and various growth factors for in vitro 3D culture (the ratio of the cell suspension to the gel is 1: 2); in the 3D culture process, cells can be continuously divided and proliferated to form cell clusters (sphenoids), and meanwhile, the cells can be integrated through mutual interaction and signal communication to form organoids with different sizes. With the gradual increase of the volume of the organoid, the organoid undergoes dynamic processes such as formation, growth, maturation stability and the like, if the organoid in the maturation stability stage is continuously increased, the internal cells of the organoid possibly have problems of hypoxia, malnutrition, autophagy, apoptosis and the like caused by hypoxia and malnutrition, so that the organoid enters the processes of degeneration, aging, even death and the like; in addition, when organoid culture is performed using a tissue, there is often a case where multiple types of cells are mixedly symbiotic in a culture system. This situation poses great difficulties in quantitative analysis (especially automated high-throughput analysis) of a particular cell type, such as cancer cells and organoids thereof, and it is difficult to ensure the reliability of the results of such medical research and detection.
At present, flow cell sorting and antibody coupling magnetic cell sorting based on cell biomarkers are used for detecting, qualitatively or quantitatively, according to differences of physical, physiological, biochemical, immunological, genetic and molecular biological properties and functional states of cells, but the method is only limited to sorting single cells and is not limited to sorting organoids with different shapes and sizes; the two methods are complex to operate and long in time consumption, meanwhile, the experiment cost is high due to the fact that instruments are not expensive, and more importantly, cell damage and bacterial pollution are easily caused, so that the success rate of in vitro experiment models is greatly reduced.
Disclosure of Invention
The present invention provides a method and a device for the hierarchical enrichment of cancer cells and organoids, which solves the above technical problems of the prior art.
The technical solution of the invention is as follows: a method for enriching cancer cells and organoids in a grading way sequentially comprises the following steps:
step 1, mixing cell recovery liquid without digestive enzyme with matrigel or hydrogel containing organoids and dispersed in cells according to the volume ratio of 1: 15-20, and incubating for 1-2 hours to obtain a gel liquefaction solution;
and 2, mixing the DMEM cell culture solution or the phosphate buffer solution containing 10% fetal calf serum and the hydrogel liquefied solution according to the volume ratio of 1: 20-50, and obtaining a hydrogel liquefaction diluent;
and 5, centrifuging the cell culture solution containing the filtered substances, and removing the supernatant to obtain cancer cells and organoids.
The device for the method for enriching the cancer cells and the organoids in a grading way is provided with a filter, the filter consists of a plurality of filter chambers which are vertically arranged and detachably connected, the filter chambers are formed by connecting a side wall and a screen positioned at the bottom, the apertures of the screen are gradually increased from bottom to top, and the apertures of each layer of the screen are uniform and respectively 10-20 μm, 20-30 μm, 45-55 μm, 75-85 μm, 115-155 μm, 145-155 μm and 195-205 μm.
And a handle is fixed outside the uppermost layer of the filter chamber.
The plurality of filter chambers are connected through screw threads.
Preferably, a container is arranged outside the filter, and a base is arranged below the container.
The invention can collect organoids with different sizes through one-time sieving so as to discard over-large organoids with poor health state or be used for other research purposes such as genome DNA extraction and high-flux detection, wherein the larger but still healthy organoids can be frozen to build a library or be digested for passage, the moderate organoids are used as an isolated model for experiments, and the smaller organoids are continuously cultured. Meanwhile, the cancer cells can be distinguished from the common cells through the sizes so as to meet the requirements of different experiments on the cancer cells. Has the advantages of simple method, short operation time, cell damage prevention, bacterial pollution prevention and the like.
Drawings
FIG. 1 is a schematic view of the structure of an apparatus used in example 1 of the present invention.
Detailed Description
The device used in the embodiment of the invention is provided with a filter 1 as shown in figure 1, wherein the filter 1 is composed of a plurality of filter chambers 2 which are vertically arranged and detachably connected through threads or splicing and the like, the filter chambers 2 are formed by connecting side walls 2-1 made of plastic and the like and a screen 2-2 positioned at the bottom, the apertures of the screen 2-2 are gradually increased from bottom to top, and the apertures of each layer of screen 2-2 are uniform and are respectively 15 micrometers, 25 micrometers, 50 micrometers, 80 micrometers, 120 micrometers, 150 micrometers and 200 micrometers. Preferably, a handle 3 (such as a clamp, etc.) is fixed outside the uppermost filter chamber 3, a container 4 is arranged outside the filter 1, and a base 5 is arranged below the container 4. It should be noted that, because the diameters of different types of tumor cells are different, and the sizes of the mature stable organoids formed by the tumor cells are different, the sizes (average diameters) of the tumor cells need to be measured in advance, and the mesh size needs to be adjusted within a corresponding range according to specific situations and experimental needs.
The method for enriching cancer cells and organoids in a grading way of the embodiment of the invention is sequentially carried out according to the following steps:
step 1, mixing a Corning Cell recovery Solution without digestive enzyme with hydrogel containing organoids and dispersed cells according to a volume ratio of 1: 20, mixing, and incubating at 4 ℃ or normal temperature for 1-2 hours (the specific time depends on the volume of the matrigel) to obtain a hydrogel liquefaction solution;
and 2, mixing DMEM cell culture solution containing 10% fetal calf serum or Phosphate Buffer Solution (PBS) with the hydrogel liquefied solution according to the volume ratio of 1: 20-50 to obtain a hydrogel liquefaction diluent, thereby achieving the purposes of reducing the viscosity of the hydrogel liquefaction liquid and fully releasing cells and organoids;
Claims (5)
1. A method for enriching cancer cells and organoids in a grading way is characterized by sequentially comprising the following steps:
step 1, mixing a cell recovery solution without digestive enzyme with hydrogel containing organoids and dispersed cells according to a volume ratio of 1: 15-20, and incubating for 1-2 hours to obtain a hydrogel liquefaction solution;
and 2, mixing the DMEM cell culture solution or the phosphate buffer solution containing 10% fetal calf serum and the hydrogel liquefied solution according to the volume ratio of 1: 20-50, and obtaining a hydrogel liquefaction diluent;
step 3, slowly pouring the hydrogel liquefied diluent into a filter arranged in a container and vibrating and sieving the hydrogel liquefied diluent, wherein the filter is formed by vertically arranging a plurality of layers of screens at intervals, the aperture of each layer of screen is gradually increased from bottom to top, the screen at the lowest layer is arranged at intervals with the bottom of the container, the liquid level of the cell culture solution in the container is over the screen at the top layer, and the apertures of the screens at all layers are uniform and respectively 10-20 μm, 20-30 μm, 45-55 μm, 75-85 μm, 115-125 μm, 145-150 μm and 195-205 μm;
step 4, taking out the screen from top to bottom layer by layer, respectively and reversely buckling the screen into different containers with cell culture solution inside, and eluting the filtered substance into the cell culture solution;
and 5, centrifuging the cell culture solution containing the filtered substances, and removing the supernatant to obtain cancer cells and organoids.
2. A device for use in the method of fractionally enriching cancer cells and organoids according to claim 1, wherein: the filter is provided with a filter (1), the filter (1) is composed of a plurality of vertically arranged and detachably connected filter chambers (2), the filter chambers (2) are formed by connecting side walls (2-1) and screens (2-2) positioned at the bottom, the apertures of the screens (2-2) are gradually increased from bottom to top, and the apertures of each layer of screens (2-2) are uniform and are respectively 10-20 μm, 20-30 μm, 45-55 μm, 75-85 μm, 115-125 μm, 145-150 μm and 195-205 μm.
3. The apparatus for the method of fractional enrichment of cancer cells and organoids according to claim 2, characterized in that a handle (3) is fixed to the outside of the uppermost filter chamber (2).
4. The apparatus for the fractional enrichment of organoids according to claim 3, characterized in that said plurality of filter chambers (2) are screwed together.
5. The apparatus for the method of fractional enrichment of cancer cells and organoids according to claim 3, characterized in that a container (4) is provided outside the filter (1), and a base (5) is provided below the container (4).
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| CN202210632273.2A CN114958755A (en) | 2022-06-07 | 2022-06-07 | Method and device for grading enrichment of cancer cells and organoids |
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| CN202210632273.2A CN114958755A (en) | 2022-06-07 | 2022-06-07 | Method and device for grading enrichment of cancer cells and organoids |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202482320U (en) * | 2012-01-09 | 2012-10-10 | 厚朴生物科技（苏州）有限公司 | Cell filter |
| CN111607495A (en) * | 2020-06-04 | 2020-09-01 | 奥格诺德生物科技(北京)有限公司 | Preparation method of tumor organoid, device and application thereof |
| CN112592897A (en) * | 2020-12-04 | 2021-04-02 | 广东普罗凯融生物医药科技有限公司 | Preparation method of tumor organoid |
| WO2021113924A1 (en) * | 2019-12-12 | 2021-06-17 | The Walter And Eliza Hall Institute Of Medical Research | Organoid cultures |
| WO2021260195A1 (en) * | 2020-06-26 | 2021-12-30 | Genome Research Limited | Culture of organoids |
| CN114540305A (en) * | 2022-01-07 | 2022-05-27 | 杭州海兰时生物科技有限责任公司 | Preparation method of organoid structure based on microfluidic technology high-flux culture |
-
2022
- 2022-06-07 CN CN202210632273.2A patent/CN114958755A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202482320U (en) * | 2012-01-09 | 2012-10-10 | 厚朴生物科技（苏州）有限公司 | Cell filter |
| WO2021113924A1 (en) * | 2019-12-12 | 2021-06-17 | The Walter And Eliza Hall Institute Of Medical Research | Organoid cultures |
| CN111607495A (en) * | 2020-06-04 | 2020-09-01 | 奥格诺德生物科技(北京)有限公司 | Preparation method of tumor organoid, device and application thereof |
| WO2021260195A1 (en) * | 2020-06-26 | 2021-12-30 | Genome Research Limited | Culture of organoids |
| CN112592897A (en) * | 2020-12-04 | 2021-04-02 | 广东普罗凯融生物医药科技有限公司 | Preparation method of tumor organoid |
| CN114540305A (en) * | 2022-01-07 | 2022-05-27 | 杭州海兰时生物科技有限责任公司 | Preparation method of organoid structure based on microfluidic technology high-flux culture |
Non-Patent Citations (1)
| Title |
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| NIKOLCE GJOREVSKI等: "Designer matrices for intestinal stem cell and organoid culture", NATURE ., vol. 539, no. 7630, XP055340803, DOI: 10.1038/nature20168 * |
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