CN108866147B - Dynamic optical detection method for cell deformation and cell activity - Google Patents
Dynamic optical detection method for cell deformation and cell activity Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
Abstract
The invention discloses a dynamic optical detection method for cell deformation and cell activity, which is characterized in that: preparing a cell deformation culture plate; observing the cultured cells with a microscope, confirming that the growth state is good, carrying out trypsinization treatment on the cultured cells, centrifuging, and removing the original culture medium on the upper layer; resuspending and diluting cells by using a new culture medium, taking single cells and a certain volume of the new culture medium, transferring the single cells and the new culture medium into a cell deformation culture plate for continuous culture, and allowing the cultured cells to generate different deformations in the cell deformation culture plate according to different cell activities; observing the morphological change of the same cell by using a microscope at different time according to the requirement, recording the observation result, and analyzing to obtain the cell activity; the advantages are that: the method is simple and rapid to operate, and can continuously, dynamically and visually monitor the deformation and activity characteristics of the cells in different time periods.
Description
Technical Field
The invention relates to the field of biotechnology application, in particular to a dynamic optical detection method for cell deformation and cell activity.
Background
Cell activity is an important index for determining whether cells cultured in vitro can normally grow under some conditions, such as drug treatment, irradiation with radioactivity or ultraviolet light, change of culture conditions, and the like. The detection of cell activity is a common means for relevant scientific research, and is one of the important methods for screening antitumor drugs in vitro and clinical tumor drug sensitivity tests. There are many methods for detecting cell activity, such as staining, colony formation, colorimetry, and radioisotope incorporation.
The staining method is the most commonly used method for checking cell death and viability in cell culture, and is divided into a chemical staining method and a fluorescent staining method, namely, the cell viability is checked by using different affinities of dead cells and live cells to a dye, and the result can be observed under an optical or fluorescent microscope after staining. However, in the chemical staining method, if the staining time is too long, living cells are also stained, which interferes with the determination of the result, and the cells are not fixed and have unclear morphology during the chemical staining process. Fluorescent dyes used in fluorescent staining generally have certain toxicity, which affects the health of operators.
The clone (colony) formation method is one of the effective methods for measuring the proliferation capacity of single cells, and the basic principle is that the single cells continuously divide and proliferate for more than 6 times in vitro, and cell groups formed by the descendants are called clones or colonies. By counting the colony formation rate, the proliferation potential of single cells can be quantitatively analyzed. Common methods are plate clone formation assay, soft agar formation assay, etc. The method is commonly used in sensitivity tests of antitumor drugs, radioactive biology tests of tumors and the like. However, this method is cumbersome and time consuming, is not suitable for monitoring a large number of samples simultaneously, and has a very large uncertainty in the manual counting process, especially when the size of the formed cell clones varies greatly, making it difficult to obtain more efficient and accurate data.
MTT colorimetry, XTT colorimetry and CCK-8 colorimetry are commonly used in the colorimetry. The principle of the MTT colorimetric method is that amber dehydrogenase in mitochondria in living cells can reduce MTT [3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide ] which is yellowish in appearance into blue-purple crystals and precipitate the crystals in the cells, but dead cells do not have the function. The dimethyl sulfoxide can dissolve purple crystals in cells, the depth of the solution is in direct proportion to the content of the crystals in the cells, and the activity of the cells can be judged by measuring the OD value by using an enzyme-labeling instrument. However, the MTT staining method is not suitable for suspending cells, because the culture medium needs to be sucked out before dissolving the purple crystals, and the step is easy to cause crystal loss, thereby causing deviation of experimental results. The XTT staining method and the CKK-8 staining method are optimized on the basis of the MTT staining method, the XTT and the CKK-8 are tetrazole nitrogen derivatives of new contracts, belong to the same class of substances as the MTT, can be degraded by mitochondrial dehydrogenase in living cells to generate brown yellow water-soluble substances, and can directly measure OD values through spectral absorption so as to predict the activity of the cells. Compared with MTT method, XTT and CKK-8 method does not need lysate to dissolve precipitate, and is suitable for adherent and suspension growing cells. However, the disadvantages are high cost, unstable XTT aqueous solution, need to be stored at low temperature or used at present, and CKK-8 has color close to that of the culture medium containing phenol red, which is easy to cause missing or excessive addition without attention, thereby causing deviation of detection results.
The radioisotope incorporation method is a method of determining the activity of a cell by measuring the anabolic state of DNA or protein in the cell. Detection of protein synthesis is generally marked with the radionuclide 35S-methionine, and detection of DNA replication is generally marked with 3H-TdR. The radioisotope can be absorbed by living cells after being added to a cell culture solution, and the more the cells proliferate, the greater the amount of the radioisotope incorporated. Cell viability is reflected by measuring the radioactivity intensity as the cells pass through a liquid scintillation counter. The method has accurate detection result, but the method needs a specific liquid scintillation counter, so the equipment price is high, the raw material cost is high, and the radioactive nuclide is easy to cause environmental pollution.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a dynamic optical detection method for cell deformation and cell activity, which is simple and rapid to operate and can continuously, dynamically and visually monitor the deformation and activity characteristics of cells in different time periods.
The technical scheme adopted by the invention for solving the technical problems is as follows: a dynamic optical detection method for cell deformation and cell activity comprises the following steps:
firstly, preparing a cell deformation culture plate;
observing the cultured cells with a microscope, confirming that the growth state is good, removing the original culture medium, carrying out trypsinization treatment on the cultured cells, adding a new culture medium to stop digestion, centrifuging for 2-5 min under the centrifugal force condition of 150-250 g, and removing the culture medium on the upper layer;
suspending and diluting cells by using a new culture medium, taking single cells and a certain volume of new culture medium, transferring the single cells and the new culture medium into a cell deformation culture plate for continuous culture, and enabling the cultured cells to generate different deformations in the cell deformation culture plate according to different cell activities;
fourthly, observing the morphological change of the same cell by using a microscope at different time according to the requirement, and recording the observation result;
judging that the cell has activity if the cell grows in the cell deformation culture plate, and obtaining the activity degree of the cell according to the degree, direction and speed of deformation of the cell in the cell deformation culture plate; if the observed cell shrinks or does not change in the deformed cell culture plate, the cell is judged to be inactive or less active.
In some embodiments, the cell deformation culture plate comprises a substrate, wherein an elastic composite material layer with uniform thickness is coated on the upper surface of the substrate, and a plurality of uniformly arranged cell deformation culture holes are formed in the elastic composite material layer. Therefore, the cell deformation culture hole has certain deformation capacity, and provides a foundation for the growth form change of cells in the culture process.
In some embodiments, the thickness of the elastic composite material layer is 5 to 10 μm, and the depth of the cell deformation culture hole is less than or equal to the thickness of the elastic composite material layer, thereby having a superior effect.
In some embodiments, the elastic composite layer is made of polydimethylsiloxane or hydrogel.
In some embodiments, the shape of the deformed cell culture well is selected from at least one of the following: round, rice-shaped and X-shaped.
In some embodiments, the deformed cell culture well has an X-shape or a mi-shape, and the deformed cell culture well includes a central portion and a plurality of deformed end portions that communicate with the central portion and extend outward. Therefore, the deformation condition can be displayed more obviously, and the observation is further facilitated.
In some embodiments, said step of taking single cells and a volume of new medium, transferring to a cell deformation plate for culturing further comprises: and (3) taking a single cell and putting the single cell into the central part of the cell deformation culture hole, and then adding a certain volume of new culture medium into the cell deformation culture hole until the cell deformation culture hole is filled.
In some embodiments, the growing the cells in the cell shape-changing culture plate comprises: the cells continuously expand from the central part of the cell deformation culture hole to a plurality of deformation end parts; the shrinking of the cells in the cell deformation culture plate specifically comprises the following steps: the cells are continuously shrunk to the central part from a plurality of deformed end parts filled with the deformed culture holes of the cells, or the extending distance of the deformed end parts is shortened.
In some embodiments, the step of culturing the cells comprises: adherent cells and non-adherent cells.
In some embodiments, the step of incubating the cultured cells further comprises a step of performing fluorescent staining on the cultured cells.
Compared with the prior art, the invention has the advantages that: the activity of the cells can be judged by observing the morphological change of the cells in the culture holes by using the cell deformation culture plate and the culture holes with special structures. Compared with other traditional cell activity detection methods, the method has the advantages of simple and quick operation, no toxic or side effect, visual detection result and capability of realizing the dynamic monitoring process of cell deformation and cell activity of the same cell in different culture periods. Provides a new research method for detecting the in vitro culture growth state of cells, in vitro screening of tumor drugs and clinical drug sensitivity test of the tumor drugs.
Drawings
FIG. 1 is a schematic structural diagram of a cell deformation culture plate according to an embodiment of the present invention;
FIG. 2 is a schematic view showing the state of cultured cells in a cell deformation culture well;
FIG. 3 is a microscopic view of the cells of the experimental group of the fourth example on days 1 and 3;
FIG. 4 is a microscopic image of group 1 cells of example five, before and after dosing.
The cell deformation culture plate comprises a cell deformation culture plate 1, a base plate 11, an elastic composite material layer 12, a cell deformation culture hole 13, a central part 14 and a deformation end part 15.
Detailed Description
The dynamic optical detection method of cell deformation and cell activity of the present invention will be described in further detail with reference to the accompanying drawings, but the present invention is not limited thereto.
Example one
A dynamic optical detection method for cell deformation and cell activity comprises the following steps:
firstly, preparing a cell deformation culture plate 1;
observing the cultured cells with a microscope, confirming the growth state is good, removing the original culture medium, carrying out trypsinization treatment on the cultured cells, adding a new culture medium to stop digestion, centrifuging for 3min under the centrifugal force of 200g, and discarding the culture medium on the upper layer;
suspending and diluting cells by using a new culture medium, taking single cells and a certain volume of new culture medium, transferring the single cells and the new culture medium into the cell deformation culture plate 1 for continuous culture, and enabling the cultured cells to generate different deformations in the cell deformation culture plate 1 according to different cell activities;
fourthly, observing the morphological change of the same cell by using a microscope at different time according to the requirement, and recording the observation result;
the deformation and mechanics of the cell have strong correlation with the cell physiology such as activity, and the like, and the cell is judged to have activity as time goes on if the observed cell grows in the cell deformation culture plate 1, and the activity degree of the cell is obtained according to the deformation degree, direction and speed of the cell in the cell deformation culture plate 1; if the observed cell shrinks or does not change in the deformed cell culture plate 1, the cell is judged to be inactive or less active.
In this embodiment, the cell deformation culture plate 1 includes a substrate 11, a layer of elastic composite material layer 12 with uniform thickness is coated on the upper surface of the substrate 11, and a plurality of cell deformation culture holes 13 are formed in the elastic composite material layer 12 and are uniformly arranged. Therefore, the cell deformation culture holes 13 have certain deformation capacity, and provide a foundation for the growth form change of cells in the culture process.
In this embodiment, the thickness of the elastic composite layer 12 is 8 μm, and the depth of the cell deformation culture hole 13 is slightly less than the thickness of the elastic composite layer 12, thereby having a better effect. In other embodiments, the thickness of the elastic composite layer 12 may be 5 μm, 10 μm.
In this embodiment, the material of the elastic composite material layer 12 is Polydimethylsiloxane (PDMS), and in other embodiments, the material of the elastic composite material layer 12 may be hydrogel (hydrogel) or other materials.
In this embodiment, the shape of the cell shape-changing culture hole 13 is an X-shape or a rice-shape, and in other embodiments, the shape of the cell shape-changing culture hole 13 may be a circular shape. The cell-deforming culture hole 13 includes a central portion 14 and a plurality of deforming end portions 15 communicating with the central portion and extending outward. Therefore, the deformation condition can be displayed more obviously, and the observation is further facilitated.
In this embodiment, the step of taking a single cell and a certain volume of new culture medium, transferring the single cell and the new culture medium to a cell deformation culture plate, and continuing the culture specifically includes: and (3) taking a single cell and putting the single cell into the central part of the cell deformation culture hole, and then adding a certain volume of new culture medium into the cell deformation culture hole until the cell deformation culture hole is filled.
In this embodiment, the step of growing the cells in the cell shape change culture plate specifically includes: the cells continuously expand from the central part of the cell deformation culture hole to a plurality of deformation end parts; the shrinking of the cells in the cell deformation culture plate specifically comprises the following steps: the cells are continuously shrunk to the central part from a plurality of deformed end parts filled with the deformed culture holes of the cells, or the extending distance of the deformed end parts is shortened.
In this example, the cells cultured in the step two include: adherent cells and non-adherent cells. The method has wide applicability and is suitable for adherent cells, suspension cells and the like.
In this embodiment, the step (c) further includes a step of performing fluorescent staining on the cultured cells. This enables a more clear observation of the result.
Example two
The rest is the same as the first embodiment, except that: ② centrifuging for 5min under the centrifugal force of 150g, discarding the culture medium on the upper layer;
EXAMPLE III
The rest is the same as the first embodiment, except that: ② centrifuging for 2min under the centrifugal force of 250g, and removing the culture medium on the upper layer;
example four
In order to show the morphological change of cells in the process of culturing in a cell deformation culture plate, taking blood vessel smooth muscle cells expressing green fluorescent protein by transfection as an example, taking single cells in the blood vessel smooth muscle cells to culture in the cell deformation culture plate, and observing the morphological change of the cells with different culture time under a microscope.
The experimental method comprises the following steps:
(1) transfection of the green fluorescent protein gene of vascular smooth muscle cells: cloning and recombining a green fluorescent protein gene to a eukaryotic expression vector, and culturing vascular smooth muscle cells to cover 60-80% of a culture dish; adding liposome into the eukaryotic expression recombinant vector, incubating for 15Min at room temperature, and simultaneously replacing a fresh vascular smooth muscle cell culture medium; thirdly, adding the mixture of the recombinant expression vector and the liposome into a cell culture dish, slightly shaking the culture dish, uniformly mixing, and incubating for 6 hours at 37 ℃; and fourthly, replacing the transfection medium after incubation, adding a fresh growth medium, culturing for 24 hours at 37 ℃, and then adding antibiotics for screening culture.
(2) The selected transfected cells are expanded and cultured until the cells cover 60-80% of a culture dish, then all culture media are discarded, 1ml of pancreatin is added, the cells are digested at 37 ℃ for 2min, cell suspension is sucked, and the cells are centrifuged at 4000rmp for 3min, and supernatant is discarded.
(3) Resuspending and diluting the cells by using a new culture medium, and putting single transfected vascular smooth muscle cells into culture holes of a cell deformation culture plate for continuous culture to serve as an experimental group; putting another part of transfected vascular smooth muscle cells into a common cell culture plate for continuous culture to serve as a control group; the experimental group and the control group have the same culture environment and culture condition.
(4) On the 1 st day of culture, the transfected vascular smooth muscle cells in the experimental group and the control group are respectively placed under an optical microscope to observe the morphology of the cells, and the results are recorded; on the 3 rd day of culture, the same cells in the experimental group were placed under an optical microscope to observe the morphology of the cells, and since the cells in the common culture plate (control group) were observed only once, a single transfected vascular smooth muscle cell in the control group was placed under an optical microscope to observe the morphology of the cells, and the results were recorded.
The experimental results are as follows:
the observation results showed that transfected vascular smooth muscle cells cultured in the normal cell culture plate (control group) proliferated continuously with the increase of culture time, and gradually filled the culture plate. Transfected vascular smooth muscle cells cultured in the deformed cell culture plate (experimental group) also increased with time, with increasing volume in the culture wells, extending towards the deformed ends of the "X-shaped" culture wells from day 1, concentrated in the center of the culture wells, to day 3, as shown in fig. 3. The experimental result shows that whether in the experimental group or the control group, the transfected vascular smooth muscle cells continuously grow and proliferate, the cell deformation culture plate has the same effect as the control group in the aspects of expressing cell deformation and cell activity, and in addition, the invention also has the advantages of simple and rapid operation and capability of realizing dynamic monitoring on the cell deformation process and the cell activity of the same cell in different culture time periods.
EXAMPLE five
In this example, lung cancer tumor cell line a549 cell was used as drug sensitive test object, Carboplatin (CAB) was used as experimental antitumor drug, and the feasibility and effectiveness of the method of the present invention in dynamic monitoring of cell deformation and cell activity were demonstrated by contrast to the conventional MTT staining method.
The experimental method comprises the following steps:
(1) firstly, diluting carboplatin with Phosphate Buffer Solution (PBS) to prepare carboplatin mother liquor, and storing for later use.
(2) And (3) recovering A549 cells: culturing A549 cells in a culture dish until the culture dish is approximately 80% full, discarding all culture medium, adding 1ml of pancreatin, digesting at 37 ℃ for 2min, sucking cell suspension, centrifuging at 4000rmp for 3min, and discarding supernatant.
(3) Group 1 (cell deformation culture plate group): resuspending and diluting the cells to a concentration of 105 cells/ml by using a new culture medium, respectively placing 100 single cells into 100 culture wells of a cell deformation culture plate, and continuously culturing at 37 ℃, wherein the cell deformation culture plate with 100 wells and an X-type culture well are selected in the embodiment, but not limited to the embodiment;
another 96-well common culture plate is taken, and 3 experimental areas I, II and III are divided on the plate, wherein the experimental area I comprises 6 parallel holes, the experimental area II comprises 6 parallel holes, and the experimental area III comprises 1 parallel hole;
group 2 (common plate addition group): taking single cells and 200 mu L of cell suspension liquid respectively to 6 parallel culture wells of the experimental area I, and continuing to culture at 37 ℃;
group 3 (common plate control): taking single cells and 200 mu L of cell suspension liquid respectively, putting the single cells and the cell suspension liquid into 6 parallel culture holes of an experimental area II, and continuously culturing at 37 ℃;
group 4 (common plate blank): only 200. mu.L of cell suspension without cells were taken in 1 parallel well of experiment zone III and incubation was continued at 37 ℃.
(4) The carboplatin stock solution was diluted to a concentration of 3X 10 with fresh medium-5nmol/L liquid medicine culture medium;
(5) the morphology of the cells of group 1 was observed by optical microscopy and recorded, showing the results of group 1 before the drug was added, and then the medium in the culture wells was completely discarded and replaced with the one containing carboplatin at a concentration of 3X 10-5Continuously culturing the nmol/L liquid medicine culture medium;
simultaneously observing group 2, group 3 and group 4 with optical microscope, completely discarding culture medium in group 2, group 3 and group 4 after cells are fully paved in each culture well, and adding 200 μ L of carboplatin with concentration of 3 × 10 into each culture well of group 2 and group 4-5In nmol/L of the liquid culture medium, 200. mu.L of a fresh medium containing no carboplatin was added to each well of group 3, and the culture was continued.
(6) After culturing for 24 hours, observing the shapes of the cells in the group 1 cell deformation culture plate under an optical microscope and recording;
simultaneously, 20 mu L of MTT solution with the concentration of 5mg/ml is respectively added into each hole of the 2 nd group, the 3 rd group and the 4 th group, the culture is continued for 4 hours, the culture medium is absorbed, 200 mu L of dimethyl sulfoxide (DMSO) is added, the shaking is carried out for 10min, the mixture is put into an automatic enzyme linked immunosorbent assay, the 4 th group is taken as a blank control, the light absorption value (OD value) of each hole of the 2 nd group and the 3 rd group is respectively measured, and then the average OD value of the 2 nd group and the 3 rd group is respectively calculated.
The experimental results are as follows:
the average OD value of 6 wells in group 3 on a 96-well common culture plate was measured to be 0.618, and the average OD value of 6 wells in group 2 was measured to be 0.237, so that it was found that some of the cells in group 2 after drug addition were apoptotic due to the action of the drug. By observing the cell morphology in the cell deformation culture plate, as shown in fig. 4, the cells also shrink continuously from filling the whole culture well before adding the drug, reflecting the continuous apoptosis process of the cells, and the cell behavior is consistent with that of the cells stained by MTT in a 96-well common culture plate. However, the detection method is simpler and more convenient to operate, more intuitive to detect, does not need to sample for many times, and can realize dynamic and continuous observation on cell deformation and cell activity of the same cell.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereby, and the present invention may be modified in materials and structures, or replaced with technical equivalents, in the constructions of the above-mentioned various components. Therefore, structural equivalents made by using the description and drawings of the present invention or by directly or indirectly applying to other related arts are also encompassed within the scope of the present invention.
Claims (8)
1. A dynamic optical detection method for cell deformation and cell activity is characterized by comprising the following steps:
firstly, preparing a cell deformation culture plate;
observing the cultured cells with a microscope, confirming that the growth state is good, removing the original culture medium, carrying out trypsinization treatment on the cultured cells, adding a new culture medium to stop digestion, centrifuging for 2-5 min under the centrifugal force condition of 150-250 g, and removing the culture medium on the upper layer;
suspending and diluting cells by using a new culture medium, taking single cells and a certain volume of new culture medium, transferring the single cells and the new culture medium into a cell deformation culture plate for continuous culture, and enabling the cultured cells to generate different deformations in the cell deformation culture plate according to different cell activities;
fourthly, observing the morphological change of the same cell by using a microscope at different time according to the requirement, and recording the observation result;
judging that the cell has activity if the cell grows in the cell deformation culture plate, and obtaining the activity degree of the cell according to the degree, direction and speed of deformation of the cell in the cell deformation culture plate; if the observed cell is atrophied or unchanged in the cell deformation culture plate, judging that the cell loses activity or is poor in activity;
the cell deformation culture plate comprises a substrate, wherein the upper surface of the substrate is coated with an elastic composite material layer with uniform thickness, and the elastic composite material layer is provided with a plurality of uniformly arranged cell deformation culture holes;
the shape of the cell deformation culture hole is selected from at least one of the following shapes: rice character type and X type.
2. The method according to claim 1, wherein the thickness of the elastic composite material layer is 5-10 μm, and the depth of the cell deformation culture hole is less than or equal to the thickness of the elastic composite material layer.
3. The method according to claim 1 or 2, wherein the elastic composite material layer is made of polydimethylsiloxane or hydrogel.
4. A method according to claim 3, wherein the deformable culture well includes a central portion and a plurality of deformable end portions extending outwardly from and communicating with the central portion.
5. A dynamic optical detection method of cell deformation and cell activity as claimed in claim 4, wherein said step (c) of taking single cells and a volume of new culture medium, transferring to a cell deformation culture plate for continuous culture specifically comprises: and (3) taking a single cell and putting the single cell into the central part of the cell deformation culture hole, and then adding a certain volume of new culture medium into the cell deformation culture hole until the cell deformation culture hole is filled.
6. A method as claimed in claim 5, wherein the step of growing the cells in the cell shape change culture plate includes: the cells continuously expand from the central part of the cell deformation culture hole to a plurality of deformation end parts; the shrinking of the cells in the cell deformation culture plate specifically comprises the following steps: the cells are continuously shrunk to the central part from a plurality of deformed end parts filled with the deformed culture holes of the cells, or the extending distance of the deformed end parts is shortened.
7. The method of claim 1, wherein the cells cultured in step (II) comprise: adherent cells and non-adherent cells.
8. The method of claim 1, wherein the step of staining the cultured cells with fluorescence is further performed before the step of detecting the cell shape change and the cell activity.
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