CN116376833A - Construction method of colon cancer organoid with immune cells reserved - Google Patents
Construction method of colon cancer organoid with immune cells reserved Download PDFInfo
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- CN116376833A CN116376833A CN202211589615.3A CN202211589615A CN116376833A CN 116376833 A CN116376833 A CN 116376833A CN 202211589615 A CN202211589615 A CN 202211589615A CN 116376833 A CN116376833 A CN 116376833A
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Abstract
The invention provides a colon cancer organoid construction method for retaining immune cells, which comprises the following steps: cleaning and mechanically shredding fresh colon cancer tissue; placing the minced tissue blocks in a centrifuge tube, centrifuging to remove the supernatant and re-suspending the tissue with a special culture medium to obtain a tissue block suspension; adding special matrix into the bottom of the transwell chamber, and placing the matrix in a cell incubator until the matrix is solidified; mixing the tissue block suspension with the unfrozen matrigel, and inoculating the mixture to the solidified matrix upper layer at the bottom of the transwell chamber; after the matrigel is solidified, a special culture medium is added into the lower layer of the transwell chamber. The invention also provides application of the colon cancer organoids obtained by the method in colorectal cancer mouse model construction, colorectal mechanism research, drug screening and therapy evaluation.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a colon cancer organoid construction method for retaining immune cells.
Background
Colorectal cancer is the fourth most common cancer worldwide, and is also the cancer type with the third highest mortality. Colorectal cancers have a strong tumor heterogeneity, contain a variety of different gene types and epigenetic mutations, and therefore the therapeutic approaches for different types of colorectal cancer are also different. At present, colorectal cancer is treated mainly by surgery, radiotherapy and chemotherapy, and is assisted by targeted therapy and immunotherapy, but the treatment method selection and the combination of different schemes also show great differences according to the biological characteristics of tumors and different development stages. In the face of a wide variety of treatments, how to perform personalized, precise treatments for different patient characteristics is a critical issue.
To achieve personalized accurate treatment of tumors, scientists use a variety of preclinical models to simulate the actual efficacy of a particular treatment regimen in a patient. Among the most economical and common are two-dimensional culture models of tumor cells. The model is to culture tumor cell line or primary tumor cell in cell culture dish, add different chemotherapeutics and targeted therapeutic drugs into cell culture medium or irradiate the cell with ray, observe the change of cell morphology and activity and judge the response of tumor to specific treatment or combination of treatments. However, the two-dimensional culture system has a plurality of defects, firstly, cells in a culture dish form a single-layer two-dimensional structure, and in vivo distribution of the cells is polar, the cells form a specific structure, and functions of different cells in different positions of the structure are different, so that phenomena observed in the two-dimensional cells may not reflect actual reflection of tumors in a patient to different treatments. In addition, in vivo tumors are composed of various cells, are polyclonal tissues and have tumor heterogeneity, but tumor cell lines are of monoclonal origin and cannot reduce the complexity of tumor tissues, so that the prediction value of different curative effects is very limited.
The application of the mouse transplanted tumor model in tumor research is very wide, compared with a cell line, the transplanted tumor model can well retain the structure, the cell composition, the molecular biological characteristics and the complexity of the original tumor, but the model has higher cost, long culture period and lower success rate of culture, so the model is not suitable for guiding clinical personalized medicine and high-flux medicine sensitivity screening. Organoids are 3D cultures of cells with dry potential to form organs/tissues similar to the original organs/tissues. Tumor organoids (PDOs) are multicellular clusters obtained by three-position culture of tumor tissue that retain the characteristics of the original tumor. The research proves that the PDO of various tumor types including colorectal cancer concentrates the characteristics of the original tumor in the aspects of genome, transcriptome, immunohistochemical staining characteristics and the like, namely, the PDO and the transcriptome have high coincidence degree on the multiple aspects of genetics. There are also many studies in terms of functionality that demonstrate that PDO has highly consistent drug-sensitive properties with the original tumor tissue. In addition, PDO has a fast proliferation rate, a short culture period, a high culture success rate, and high implementation and operability, which makes PDO a hotspot model in the fields of accurate treatment of cancer and research and development of new drugs after cell lines and humanized mouse models.
Although the traditional organoid model has a plurality of advantages, the traditional organoid model does not contain tumor microenvironment components including immune cells, so that the application of the traditional organoid model is limited to a certain extent, and research related to tumor immunotherapy cannot be carried out.
Disclosure of Invention
Aiming at the technical problems, the invention provides a colorectal cancer tissue organoid culture method capable of realizing immune cell retention, so that immune cells in tumor tissues are retained in organoids, thereby further widening the application range of the organoids and serving the researches related to tumor immune microenvironment and tumor immune treatment. In particular, the method comprises the steps of,
the first aspect of the invention provides a colon cancer organoid construction method retaining immune cells, comprising the steps of:
a1 Cleaning and mechanical shredding of fresh colon cancer tissue;
a2 Placing the minced tissue blocks in a centrifuge tube, centrifuging to remove the supernatant and re-suspending the tissue with a special culture medium to obtain a tissue block suspension;
a3 Adding special matrix into the bottom of the transwell chamber, and placing the matrix in a cell culture box for solidification;
a4 Mixing the tissue block suspension with the unfrozen matrigel, and inoculating the mixture to the solidified matrix upper layer at the bottom of the transwell chamber;
a5 After the matrigel is solidified, adding special culture medium into the lower layer of the transwell chamber.
In certain embodiments, the solution used for the washing in step A1) is penicillin/streptomycin-containing PBS; preferably, the penicillin/streptomycin concentration is 2%.
In certain embodiments, the minced tissue mass in step A2) has a diameter of 0.5-2mm; preferably, the tissue mass has a diameter of 0.6-1.2mm.
In certain embodiments, the centrifugation in step A2) is performed at a rotational speed of 500g/min and a centrifugation time of 5min, wherein both the ramp-up and ramp-down are 9.
In certain embodiments, the dedicated medium components in steps A2) and A5) comprise: DMED/F12 culture medium, R-spondin1 protein, epidermal growth factor, prostaglandin E-2 and interleukin-2.
In certain embodiments, the special purpose medium in steps A2) and A5) comprises DMED/F12 medium, R-spondin1 protein, noggin, epidermal growth factor, HEPES, glutamax, normocin, gentamicin/amphoteritin, N2, B27, n-Acetylcysteine, SB202190, gastin, prostaglandin E-2, interleukin-2.
In certain embodiments, the transwell chamber in step A3) has a diameter of 12mm, a pet film, and a pore size of 04.Um.
In certain embodiments, the specialized substrate in step A3) comprises: matrigel, DMEM medium, PBS; preferably, the Matrigel content is 70% -90%, the DMEM medium content is 5% -15%, and the PBS content is 5% -15%.
In certain embodiments, the setting time of the specialized substrate in step A3) is 20-40min; further preferably, the setting time of the dedicated matrix is 25-35min.
In certain embodiments, the Matrigel in step A4) is preferably Matrigel.
The second aspect of the invention provides the use of colon cancer organoids obtained according to the construction method of the first aspect of the invention in colorectal cancer mouse model construction, colorectal mechanism research, drug screening, therapy assessment.
Compared with the prior art, the invention has the beneficial effects that:
the colorectal cancer organoid construction method for retaining immune cells is different from the existing colorectal cancer organoid culture method, in the method, firstly, the upper layer of the organoid is exposed to air, and the lower layer is immersed in a culture medium, so that the nutrition supply of the organoid is ensured, and meanwhile, the oxygen content of a culture system is increased, and the method can simulate the growth environment of intestinal cancer in vivo more than the traditional organoid culture method. In addition, the special matrix added at the bottom of the transwell chamber plays a role in mechanically fixing the original immune cells in the tumor tissue, so that the immune cells are not easy to be dissociated in the culture medium, and the retaining capacity of the immune cells of the organoid is promoted. Therefore, compared with the current popular method, the method disclosed by the invention can retain immune cells to a greater extent, so that the immune microenvironment of the tumor is retained, the simulation degree of the organoid model on the original tumor is further improved, and the organoid model has application potential in the aspects of tumor immune microenvironment research and personalized immune accurate treatment of patients.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 shows a schematic structure of the method of the present invention. Wherein A represents a colorectal cancer tissue mass; b represents matrigel; c represents a special matrix; d represents a transwell chamber; e represents a colorectal cancer dedicated medium; f represents a cell culture dish.
FIG. 2 shows a physical diagram of the method of the present invention. A, B, C, D, E, F in the figure corresponds to A, B, C, D, E, F in fig. 1, respectively.
FIG. 3 shows representative growth states of organoids cultured using the methods of the invention at various time points during the culture process. Wherein, FIG. 3A shows the growth state of the organoids cultured using the method of the present invention on days 0 to 10 of the culture. Wherein the same organoids were photographed every two days and their growth status was recorded. From top to bottom 3 rows represent 3 independent experiments, respectively. FIG. 3B shows organoids cultured for 1 to 21 days using the methods of the invention.
FIG. 4 shows representative growth states of organoids cultured using the methods of the invention at various time points after passage. From top to bottom 3 rows represent 3 independent experiments, respectively.
FIG. 5 shows immunofluorescent staining of immune cells in colorectal cancer organoids. Wherein FIG. 5A is a organoid cultured using the methods of the invention and FIG. 5B is a organoid cultured using conventional organoid culture methods. The organoids cultured using both culture methods were each immunofluorescent stained for immune cell-related markers on day 14 of culture, with red fluorescence representing CD45 positive cells and green fluorescence representing CD8 positive cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
EXAMPLE 1 preparation of colon cancer organoids retaining immune cells
1.1 preparation of tissue pieces
Fresh intestinal cancer tissues are collected, and the total volume of the tissues is more than 1 cubic centimeter to ensure the success rate of culture and the reservation of tumor immunity microenvironment. Conditions allow to obtain as much material as possible at different locations of the tumor, so as to preserve the tumor heterogeneity to the maximum and to restore the true tumor microenvironment to the maximum. Tissues were washed with pre-chilled PBS containing 2% penicillin/streptomycin for 5min X5 times. The sterile petri dish was placed on an ice box in a sterile operating table and the tissue was transferred into the petri dish. The macroscopic necrotic tissue is removed by a sterile blade, and the rest part is cut into tissue blocks with the diameter of 0.8-1.2mm by the sterile blade, so that the volume can keep a relatively complete tumor immunity microenvironment and prevent the anoxic necrosis inside the organoids in the culture process caused by the overlarge volume. The cut tissue pieces were collected into sterile 15ml centrifuge tubes, washed 5 times with pre-chilled PBS containing 2% penicillin/streptomycin, 500 g.times.5 min, and then centrifuged at 200g for 5min to pellet all tissue pieces.
1.2transwell cell pretreatment
Matrigel (Matrigel), DMEM high sugar medium, sterilized PBS were uniformly mixed in 8:1:1 to prepare a dedicated matrix. A quantity of the special matrix is sucked by a precooled 1ml gun head and added to the bottom of the transwell cell, so that the whole bottom of the cell is covered by the special matrix. Gently shake the chamber to make the thickness of the bottom-dedicated substrate uniform. The transwell chamber was placed in an incubator at 37℃for 30min until the bottom matrix was sufficiently coagulated.
1.3 preparation of organoids
The supernatant centrifuged in step 1 was discarded and the matrigel was aspirated with a pre-chilled 1ml gun head to resuspend the tissue mass. The pretreated transwell chamber was removed from the incubator and gently rocked to confirm that the bottom matrix had sufficiently coagulated. The mixture of matrigel and tissue blocks is sucked by a precooled 1ml gun head, the mixture is added into the upper layer of the matrigel layer at the bottom of the transwell chamber, and the chamber is gently swayed to ensure that the tissue blocks are uniformly distributed. The transwell chamber was placed in an incubator at 37℃for 30min. And taking out the small chamber from the incubator after 30min, and slightly shaking for detection to ensure that the matrigel is fully solidified. Adding proper amount of intestinal cancer organoid special culture medium (comprising DMED/F12 culture medium, R-spondin1 protein, epidermal cell growth factor, prostaglandin E-2, and preferred components comprise DMED/F12 culture medium, R-spondin1 protein, noggin, epidermal cell growth factor, HEPES, glutamax, normocin, gentamicin/amphoteritin, N2, B27, n-Acetylcysteine, SB202190, gastrin, prostaglandin E-2) and 300u/ml IL-2 into the lower layer of the transwell chamber, and maintaining the liquid level at the junction of the tissue mass layer and the special matrix layer.
FIG. 1 is a schematic diagram of the method of the present invention. Wherein A represents a colorectal cancer tissue mass; b represents matrigel; c represents the special matrix described above; d represents a transwell chamber; e represents a colorectal cancer dedicated medium; f represents a cell culture dish. FIG. 2 is a schematic representation of the method of the present invention. A, B, C, D, E, F in the figure corresponds to A, B, C, D, E, F in fig. 1, respectively.
Example 2 validation of preparation of colon cancer organoids retaining immune cells
Experiment group 1: intestinal cancer organoids were cultured as described in example 1, organoid growth was observed daily and photographed, medium was changed every 3 days, and cultured for 10 days.
Control group 1: the intestinal cancer tissue from the same source as that of experiment group 1 is cultured according to the traditional organoid culture mode. Fresh intestinal cancer tissue was taken and washed with pre-chilled PBS containing 1% penicillin/streptomycin for 5min X5 times. A sterile petri dish was placed on an ice box in a sterile operating table, the tissue was transferred into it and minced as much as possible with a sterile surgical blade. The tissue fragments were transferred to a 37℃pre-heated digest (DMEM medium+1% FBS+collagenase IV+collagenase II) with a disposable sterile pipette and digested for 20-40min in a 37℃water bath shaker. After digestion, the mixture was blown about 20 times with a pipette and filtered through a 100um filter. Supernatant was removed by centrifugation at 800g 1min, and resuspended in pre-chilled PBS. Wash 5 times with pre-chilled PBS 100g containing 1% penicillin/streptomycin for 5min each, then pellet all cell clusters by centrifugation at 200 g. The cell mass is resuspended by sucking a proper amount of matrigel with a precooling 1ml gun head, and inoculated into a preheated cell culture plate, wherein the quantity of the cell mass is about 200, and the matrigel is 50 mu L per hole. The inoculated culture plate is placed in a cell culture box at 37 ℃ for standing for 5-8min, and 500 mu L of organoid culture medium is added into each hole. Then placing the cells in a cell culture box for culture, observing the growth condition of the organoids every day, photographing, changing the culture medium every 3 days, and culturing for 10 days.
The intestinal cancer specimens used in the experimental group 1 and the control group 1 are derived from the same patient, the tissue amount used in the process of extracting the organoids in the two groups is the same, and the composition, the culture condition and the culture process of all the other culture mediums are the same except that the extraction methods of the organoids in the two groups are different and 300u/ml IL-2 is added into the culture medium in the control group 1.
Table 1 below shows the organoid growth in different organoid culture systems.
TABLE 1 organoid growth under different culture systems
Group of | Percentage of organoid |
Experiment group | |
1 | 386 |
Control group | |
1 | 374% |
FIG. 3 is a representative growth state of a organoid cultured using the methods of the invention at various time points during the culturing process. Wherein panel a shows the growth status of organoids cultured using the methods of the invention from day 0 to day 10 of culture. Wherein the same organoids were photographed every two days and their growth status was recorded. From top to bottom 3 rows represent 3 independent experiments, respectively. As shown in fig. 3, colorectal cancer tumor organoids can maintain a normal growth state under the culture conditions of the method of the present invention, and the organoids are increasing in volume and exhibit a typical colorectal cancer tumor organoid morphology with sprouting. Organoids are highly refractive under light lenses, representing good activity. Panel B represents organoids cultured for 1 to 21 days according to the method of the invention. Under this condition, the tumor organoid can maintain normal growth in a relatively long culture period, and the stability of the culture system is reflected.
From the results in Table 1 in combination with the organoid morphology of FIG. 3, it can be shown that the system of the present invention maintains stable growth of human intestinal organoids compared to conventional organoid culture systems. The culture system of the invention can successfully culture intestinal cancer organoids.
EXAMPLE 3 passage of immune cell-retaining organoids
Example 2 demonstrates that the culture method of the present invention can achieve normal culture of primary intestinal cancer organoids, but requires organoid passaging if long-term stable culture of organoids is desired. Therefore, the example verifies that the culture method of the invention can realize normal passage of organoids.
The organoids of example 1 were passaged after 14 days of culture. At passage, the medium under the transwell chamber was aspirated. Sucking 1ml of precooled PBS with precooled gun head, adding the upper layer of a transwell chamber, blowing off the mixture of the matrigel organoid and the bottom matrix, transferring into a 15ml sterile centrifuge tube, blowing off and beating until organoid and matrigel are completely blown off and mixed uniformly, centrifuging at 500g for 5min at 4 ℃, and sucking off the supernatant and the upper matrigel deposited at the bottom as much as possible. The above washing steps were repeated 3 times until the matrigel was washed. Adding 5ml of precooled PBS to resuspend the organoid, lightly blowing with a gun head until the organoid volume is blown away to about 1/2 of that before passage, taking part of organoid suspension, observing under a microscope to confirm that the organoid is blown away to a proper size, and centrifuging 500g at 4 ℃ for 5min to precipitate the organoid. The transwell chamber pretreatment step and organoid preparation step of example 1 were repeated and passaged at a rate of 2 cells per chamber. The organoids after passage are continuously cultured according to the organoid culture flow, the growth condition of the organoids is observed every day, the organoids are photographed, and the culture medium is replaced every 3 days. After 4 days of culture, the organoid morphology is shown in FIG. 4. FIG. 4 is a representative growth state of organoids cultured using the methods of the invention at various time points after passage. From top to bottom 3 rows represent 3 independent experiments, respectively. As shown in FIG. 4, colorectal cancer organoids cultured using the methods of the present invention can still grow normally after passage. Part of the smaller cell mass was generated by blowing during passage. The organoids form a typical budding structure and have good refraction, which indicates that the organoids are in good state after passage, and the method does not influence the normal passage of organoids. The example shows that the organoid after passage can keep the normal growth state, which shows that the organoid culture method for retaining immune cells can realize the normal passage of the organoid of intestinal cancer, so that the long-term application of the model is possible.
EXAMPLE 4 comparison of the different culture methods to preserve the immunocyte Capacity
Experiment group 1: intestinal cancer organoids were cultured as described in experimental group 1 of example 2, organoid growth was observed daily and photographed, medium was changed every 3 days, and cultured for 14 days.
Control group 1: intestinal cancer organoids were cultured as described in control group 1 of example 2, organoid growth was observed daily and photographed, medium was changed every 3 days, and cultured for 14 days.
The intestinal cancer tissues used in the experimental group 1 and the control group 1 were derived from the same patient. Organoids of the experimental and control groups after 14 days of culture were aspirated, and washed twice with 500 μl PBS per well. 500. Mu.L of 4% paraformaldehyde solution was added to each well, the mixture was placed in a 37℃cell incubator for 30min, the organoids after fixation were removed, and the paraformaldehyde solution was aspirated and washed twice with 500. Mu.L of PBS and 500. Mu.L of 70% ethanol were added to each well. Matrigel was scraped completely from the culture plate with forceps and transferred to an embedding cassette. The embedding box is put into a dehydrator to dehydrate tissues. The dehydrated tissue mass was paraffin embedded and sectioned. Paraffin sections were immunofluorescent stained with red fluorescence 568 to label CD45 positive cells and green fluorescence 488 to label CD8 positive cells. The stained sections were observed under a microscope and photographed. The fluorescent images obtained in the observation experiment group 1 and the control group 1 are shown in fig. 5. FIG. 5 is an immunofluorescent staining of immune cells in colorectal cancer organoids. Wherein FIG. 5A is a organoid cultured using the methods of the invention and FIG. 5B is a organoid cultured using conventional organoid culture methods. The organoids cultured using both culture methods were each immunofluorescent stained for immune cell-related markers on day 14 of culture, with red fluorescence representing CD45 positive cells and green fluorescence representing CD8 positive cells. The figure shows that immune cells are still reserved in the organoids after 14 days of culture by using the method of the invention, and the expression of immune cell related markers is not found in the organoids cultured by the traditional culture method, which proves that the method of the invention has the capability of reserving immune cells within a certain time range and has certain advantages in teaching the traditional culture method. This example demonstrates that the culture method of the present invention has a greater immune cell retention capacity than the conventional culture method.
While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and the description is provided for clarity only, and those skilled in the art will recognize that the embodiments of the disclosure may be combined appropriately to form other embodiments that will be understood by those skilled in the art.
Claims (10)
1. A method for constructing colon cancer organoids retaining immune cells, comprising the steps of: a1 Cleaning and mechanical shredding of fresh colon cancer tissue;
a2 Placing the minced tissue blocks in a centrifuge tube, centrifuging to remove the supernatant and re-suspending the tissue with a special culture medium to obtain a tissue block suspension;
a3 Adding special matrix into the bottom of the transwell chamber, and placing the matrix in a cell culture box for solidification;
a4 Mixing the tissue block suspension with the unfrozen matrigel, and inoculating the mixture to the solidified matrix upper layer at the bottom of the transwell chamber;
a5 After the matrigel is solidified, adding special culture medium into the lower layer of the transwell chamber.
2. The method according to claim 1, wherein the solution used for washing in step A1) is penicillin/streptomycin-containing PBS; preferably, the penicillin/streptomycin concentration is 2%.
3. The method of claim 1, wherein the minced tissue mass of step A2) has a diameter of 0.5-2mm; preferably, the tissue mass has a diameter of 0.6-1.2mm.
4. The method according to claim 1, wherein the rotational speed of the centrifugation in the step A2) is 500g/min, the centrifugation time is 5min, and the rising speed and the falling speed are 9.
5. The method of claim 1, wherein the dedicated medium components in steps A2) and A5) comprise: DMED/F12 culture medium, R-spondin1 protein, epidermal growth factor, prostaglandin E-2 and interleukin-2; preferably, the special culture medium in the steps A2) and A5) comprises DMED/F12 culture medium, R-spondin1 protein, noggin, epidermal growth factor, HEPES, glutamax, normocin, gentamicin/amphoteritin, N2, B27, n-Acetylcysteine, SB202190, gastrin, prostaglandin E-2, interleukin-2.
6. The method according to claim 1, wherein the transwell cells in step A3) have a diameter of 12mm, a pet film, and a pore size of 0.4um.
7. The method of claim 1, wherein the specialized substrate in step A3) comprises: matrigel, DMEM medium, PBS; preferably, the Matrigel content is 70% -90%, the DMEM medium content is 5% -15%, and the PBS content is 5% -15%.
8. The method of claim 1, wherein the setting time of the specialized substrate in step A3) is 20-40min; further preferably, the setting time of the dedicated matrix is 25-35min.
9. The method of claim 1, wherein the Matrigel in step A4) is preferably Matrigel.
10. Use of a colon cancer organoid obtained by the construction method according to any of claims 1-9 in colorectal cancer mouse model construction, colorectal mechanism studies, drug screening, therapy assessment.
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