CN115141805B - CIC cell simulating human tumor microenvironment, preparation method and sorting method - Google Patents
CIC cell simulating human tumor microenvironment, preparation method and sorting method Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
Abstract
The invention relates to the technical field of oncology, in particular to CIC cells simulating human tumor microenvironment, a preparation method and a sorting method. The preparation method comprises the following steps: the target ratio is 1: (1-3) providing human peripheral blood lymphocytes and tumor cells; co-incubating human peripheral blood lymphocytes and tumor cells in an incubator to obtain CIC cells; wherein the culture temperature of the incubator is 30-42 ℃, and the carbon dioxide concentration in the incubator is 1-10%. The method solves the problem that the CIC constructed manually in the prior art is difficult to simulate the tumor environment, is mainly suitable for constructing the CIC cell model between normal lymphocytes and tumor cells, and is more significant when the constructed CIC cells are closer to the tumor microenvironment in the human body and the obtained CIC cells are subjected to subsequent researches.
Description
Technical Field
The invention relates to the technical field of oncology, in particular to CIC cells simulating human tumor microenvironment, a preparation method and a sorting method.
Background
There is a phenomenon In which lymphocytes enter tumor cells to form Cell-In-Cell (CIC) In various tumor tissues, and CIC is associated with its malignancy In some tumors, suggesting poor prognosis. Therefore, the exploration of the effect of this heterogeneous CIC structure on colorectal cancer prognosis has a certain significance. CIC living cells are difficult to obtain and carry out in vitro and in vivo experiments, and CIC can be observed only in relevant pathological sections.
The existing method is to construct CIC cells through lymphocyte lines and tumor cell lines in blood tumor cell lines, so that the simulation of tumor environment is difficult, and the subsequent study of CIC cells is not facilitated.
Disclosure of Invention
In view of the above drawbacks of the prior art, the present invention is directed to a method for preparing CIC cells simulating human tumor microenvironment, which is used for solving the problem that CIC constructed manually in the prior art is difficult to simulate tumor environment, and simultaneously, the present invention also provides a method for sorting CIC cells simulating human tumor microenvironment; in addition, the invention also provides CIC cells simulating human tumor microenvironment.
To achieve the above-mentioned objects and other related objects,
in a first aspect of the present invention, a method for preparing CIC cells simulating human tumor microenvironment is provided, comprising the steps of:
the target ratio is 1: (1-3) providing human peripheral blood lymphocytes and tumor cells; and
co-incubating human peripheral blood lymphocytes and tumor cells in an incubator to obtain CIC cells;
wherein the culture temperature of the incubator is 30-42 ℃, and the carbon dioxide concentration in the incubator is 1-10%.
In one embodiment of the present invention, the effective target ratio of the human peripheral blood lymphocytes to the tumor cells is 1: (1.5-2.5); the culture temperature of the incubator is 34-39 ℃, and the carbon dioxide concentration in the incubator is 3-7%.
In one embodiment of the present invention, the effective target ratio of the human peripheral blood lymphocytes to the tumor cells is 1: (1.9-2.1); the culture temperature of the incubator is 36-38 ℃, and the concentration of carbon dioxide in the incubator is 4.5-5.5%.
In one embodiment of the present invention, the preparation process of the human peripheral blood lymphocytes comprises:
step one, mixing peripheral blood and lymphocyte separation liquid, and centrifuging to obtain human peripheral blood mononuclear cells;
step two, the human peripheral blood mononuclear cells are subjected to activation culture after being resuspended, and the human peripheral blood lymphocytes are obtained.
In one embodiment of the present invention, the preparation process of the human peripheral blood lymphocytes comprises:
step one, mixing peripheral blood, PBS buffer solution and lymphocyte separation solution, and centrifuging to obtain human peripheral blood mononuclear cells;
and step two, centrifuging the human peripheral blood mononuclear cells, adding the human peripheral blood mononuclear cells into a fetal bovine serum culture medium, and performing activated culture after resuspension to obtain the human peripheral blood lymphocytes.
In one embodiment of the invention, the volume ratio of the peripheral blood, the PBS buffer, and the lymphocyte separation liquid is 1: (0.8-1.2): (1.6-2.4).
In a second aspect of the present invention, a method for sorting CIC cells simulating human tumor microenvironment is provided, comprising the steps of:
staining human peripheral blood lymphocytes and tumor cells respectively;
preparing CIC cells from the stained human peripheral blood lymphocytes and tumor cells according to the preparation method; and
and preparing the cultured CIC cells into single cell suspension, and sorting the cells which are positive in double fluorescence by an upper machine to obtain the CIC cells.
In one embodiment of the invention, the human peripheral blood lymphocytes and tumor cells are stained with different colored fluorochromes.
In one embodiment of the invention, the human peripheral blood lymphocytes are stained with a green fluorescent dye; the tumor cells were stained with red fluorescent dye.
In a third aspect of the invention, a CIC cell simulating human tumor microenvironment is provided, wherein the CIC cell is prepared by the preparation method.
As described above, the CIC cell simulating human tumor microenvironment, the preparation method and the sorting method have the following beneficial effects: the invention provides a method for constructing CIC cells by using human normal lymphocytes and tumor cell lines, through the method, CIC cells can be prepared and screened for culture and subsequent related cell function tests, the method is mainly suitable for constructing CIC cell models between normal lymphocytes and tumor cells, the constructed CIC cells are closer to tumor microenvironments in human bodies, and the obtained CIC cells are more significant in subsequent researches.
Drawings
FIG. 1 shows a microscopic schematic of CIC cells at step S4 of example 6.
FIG. 2 is a microscopic schematic of cells of the control group in step S4 of example 6.
FIG. 3 shows microscopic schematic of CIC cells at step S5 of example 6.
FIG. 4 double positive CIC cells screened after flow sorting in example 6.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
1. Cell line:
human intestinal cancer cell line HCT-8 is purchased from the cell resource center of the national academy of medical science.
Human Peripheral Blood Mononuclear Cells (PBMCs) were extracted from peripheral blood of volunteers.
2. List of reagents:
3. list of instruments:
example 1
The preparation process of the human peripheral blood lymphocytes specifically comprises the following steps:
step one, 50ml of fresh peripheral blood from volunteers was collected and centrifuged with PBS buffer at 1:1, diluting and mixing uniformly, and mixing diluted blood according to the proportion of 1:1 was slowly added to the lymphocyte separation solution.
Step two, 800g, centrifuging for 30 minutes, wherein the speed increasing and decreasing are 0, and the stability is kept.
Step three, the inside of the centrifugal tube is divided into 4 layers according to the density, namely a platelet layer, an intermediate layer, a separated liquid layer and a red blood cell and granulocyte layer from top to bottom, wherein the density of the intermediate layer is similar to that of the separated liquid, the intermediate layer contains PBMC (human peripheral blood mononuclear cells), and the intermediate layer cells are carefully sucked into a new centrifugal tube.
Step four, 600g, centrifuging for 20 minutes, wherein the rising speed and the falling speed are 9.
And fifthly, obtaining the cell sediment in the centrifuge tube after centrifugation, namely human PBMC, re-suspending the cell sediment by using PBS buffer solution, centrifuging, and centrifuging for 5min at 1000 rpm.
Step six, after centrifugation, cell precipitation was performed using 104U/ml IL -2 The 10% fetal bovine serum RPMI-1640 culture medium is resuspended and then is activated and cultured, thus obtaining the human peripheral blood lymphocyte.
Example 2
A preparation method of CIC cells simulating human tumor microenvironment comprises the following steps:
s1, HCT-8 cells were cultured at 8X 10 5 The wells were plated into 6-well plates overnight for adherent growth.
S2, putting the real object into practiceThe human peripheral blood lymphocytes prepared in example 1 were activated with IL-2, and the activated human peripheral blood lymphocytes were added to the well-paved 2X 10 cells 7 In the culture dish of tumor cells, the stimulation of activated PBMCs was continued for 3 days.
S3, the human peripheral blood lymphocytes and tumor cells are subjected to the following steps of: 1.5 co-incubation at 35℃,
The culture is carried out for 14 to 16 hours in a 4 percent CO2 incubator.
Example 3
A preparation method of CIC cells simulating human tumor microenvironment comprises the following steps:
s1, HCT-8 cells were cultured at 8X 10 5 The wells were plated into 6-well plates overnight for adherent growth.
S2, activating the human peripheral blood lymphocytes prepared in the example 1 by IL-2, and adding the activated human peripheral blood lymphocytes into the paved 2X 10 7 In the culture dish of tumor cells, the stimulation of activated PBMCs was continued for 3 days.
S3, the human peripheral blood lymphocytes and tumor cells are subjected to the following steps of: 1.3 co-incubation at 36℃,
The culture is carried out for 14 to 16 hours in a 5 percent CO2 incubator.
Example 4
A preparation method of CIC cells simulating human tumor microenvironment comprises the following steps:
s1, HCT-8 cells were cultured at 8X 10 5 The wells were plated into 6-well plates overnight for adherent growth.
S2, activating the human peripheral blood lymphocytes prepared in the example 1 by IL-2, and adding the activated human peripheral blood lymphocytes into the paved 2X 10 7 In the culture dish of tumor cells, the stimulation of activated PBMCs was continued for 3 days.
S3, the human peripheral blood lymphocytes and tumor cells are subjected to the following steps of: 2.1 co-incubation at 37℃,
The culture is carried out for 14 to 16 hours in a 6 percent CO2 incubator.
Example 5
A preparation method of CIC cells simulating human tumor microenvironment comprises the following steps:
s1, HCT-8 cells were cultured at 8X 10 5 The wells were plated into 6-well plates overnight for adherent growth.
S2, activating the human peripheral blood lymphocytes prepared in the example 1 by IL-2, and adding the activated human peripheral blood lymphocytes into the paved 2X 10 7 In the culture dish of tumor cells, the stimulation of activated PBMCs was continued for 3 days.
S3, the human peripheral blood lymphocytes and tumor cells are subjected to the following steps of: 2.5 at 39℃,
The culture is carried out for 14 to 16 hours in a 6 percent CO2 incubator.
Example 6
A preparation and sorting method of CIC cells simulating human tumor microenvironment comprises the following steps:
s1, lymphocyte staining:
s11, human PBMC were cultured with 10% fetal bovine serum RPMI-1640 medium containing 104U IL-2 at 37℃with 5% CO 2 Is cultured in a cell culture incubator. The medium was changed every 2 days.
S12, after the cell growth state is good, the PBMC after stimulation and activation are collected and centrifuged, and the PBMC is washed 3 times with a serum-free RPMI-1640 medium for 10 minutes.
S13, preparing a final concentration of celltracker green fluorescent dye into 10ML of a serum-free RPMI-1640 culture medium with a final concentration of 2 mu M. The PBMC were resuspended in the prepared medium containing green fluorescent dye and then placed at 37℃in 5% CO 2 Incubate in incubator for 30 min protected from light. PBMC after completion of staining were centrifuged at 1000rpm for 5 minutes and washed 3 times for 30 minutes each with serum-free RPMI-1640 medium.
S2, tumor cell staining
S21, HCT-8 cell line Using 10% fetal bovine serum RPMI-1640 medium at 37℃and 5% CO 2 Is cultured in a cell culture incubator. The medium was changed every 2 days and the cells were passaged every 3-5 at a ratio of 1:3.
S22, removing the culture medium of the adherent tumor cells, and washing with PBS buffer solution for 3 times and 10 minutes.
S23, preparing a final concentration of a celltracker red fluorescent dye of 2 mu M serum-free RPMI-1640 medium 10ML. Adding dye-containing culture medium at 37deg.C and 5% CO 2 Incubate in incubator for 30 min protected from light. HCT8 after staining was washed 3 times for 30 minutes each with serum-free RPMI-1640 medium.
S3, intestinal cancer cells and human PBMC co-incubation simulation in-vivo CIC formation experiment
S31, HCT-8 cells were cultured at 8X 10 5 The wells were plated into 6-well plates overnight for adherent growth.
S32, activating the human peripheral blood lymphocytes prepared in example 1 by IL-2, and adding the activated human peripheral blood lymphocytes into the paved 2X 10 7 In the culture dish of tumor cells, the stimulation of activated PBMCs was continued for 3 days.
S33, respectively dyeing the dyed PBMC and the tumor cells according to the effective target ratio of 1:2 at 37℃in 5% CO 2 The incubator cultures for 14-16 hours.
S4, flow cell sorting
S41, discarding the co-incubated culture medium, performing pancreatin digestion, centrifuging at 1000rpm for 5 minutes, collecting cells, re-suspending the cells into single cell suspension by using PBS buffer, and adjusting the cell concentration to be 1 multiplied by 10 7 And (3) filtering by a 400-mesh filter screen, and then sorting by a machine.
S42, selecting two groups of cells after co-incubation by a flow cytometer according to fluorescence classification: tumor cells positive for bifluorescence are intestinal cancer cells (CIC group) drilled by PBMC, and specifically shown in FIG. 1; the single fluorescent tumor cells were intestinal cancer cells (control group) without PBMC penetration, as shown in fig. 2.
S5, culturing after flow cell sorting
The two groups of cells from the sorting were collected and centrifuged at 1000rpm for 5min, after removal of the PBS buffer, resuspended in complete medium, plated for adherent growth and CIC cells after incubation as shown in FIG. 3. FIG. 4 shows the double positive CIC cells selected after flow sorting, showing a tumor cell (largest cell) with multiple lymphocytes (green) drilled in.
In conclusion, the method is used for constructing the CIC cell model between normal lymphocytes and tumor cells, the constructed CIC cells are closer to the tumor microenvironment in the human body, and the obtained CIC cells are more significant in subsequent research. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (3)
1. The preparation method of the CIC cell simulating the human tumor microenvironment is characterized by comprising the following steps of:
mixing peripheral blood, PBS buffer solution and lymphocyte separation solution, and centrifuging to obtain human peripheral blood mononuclear cells;
centrifuging human peripheral blood mononuclear cells, adding the human peripheral blood mononuclear cells into a fetal bovine serum culture medium, and performing activated culture after resuspension to obtain human peripheral blood lymphocytes;
activating human peripheral blood lymphocytes by adopting IL-2, and then adding the activated human peripheral blood lymphocytes into a culture dish paved with HCT-8 cells for continuous stimulation and activation for 3 days to obtain activated human peripheral blood lymphocytes;
respectively staining the activated human peripheral blood lymphocytes and HCT-8 cells to obtain the respectively stained human peripheral blood lymphocytes and HCT-8 cells;
the target ratio of the human peripheral blood lymphocytes and HCT-8 cells respectively stained is 1: (1.9-2.1) co-incubating in an incubator, preparing single-cell suspension from the cells after co-incubation and culturing, and sorting cells positive to double fluorescence by an upper machine to obtain CIC cells;
wherein the culture temperature of the incubator is 34-39 ℃, and the carbon dioxide concentration in the incubator is 3-7%; the HCT-8 cells are dyed by adopting red fluorescent dyes, and the human peripheral blood lymphocytes and the HCT-8 cells are dyed by adopting fluorescent dyes with different colors.
2. The method for preparing CIC cells simulating human tumor microenvironment according to claim 1, wherein the steps of: the culture temperature of the incubator is 36-38 ℃, and the concentration of carbon dioxide in the incubator is 4.5-5.5%.
3. The method for preparing CIC cells simulating human tumor microenvironment according to claim 1, wherein the steps of: the volume ratio of the peripheral blood to the PBS buffer solution to the lymphocyte separation solution is 1: (0.8-1.2): (1.6-2.4).
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