CN116410929A - Lung cancer organoid culture solution, culture reagent combination and culture method - Google Patents
Lung cancer organoid culture solution, culture reagent combination and culture method Download PDFInfo
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Abstract
The invention relates to a lung cancer organoid culture solution, a culture reagent combination and a culture method, wherein the lung cancer organoid culture solution comprises a basal medium, a compound antibiotic and a growth factor, the basal medium comprises Advanced DMEM/F12, HEPES10mM and Glutamax 1.0x, the compound antibiotic comprises Primocin 200 mug/mL, penicillin-streptomycin 1x and ornidazole 5 mug/mL, and the culture reagent combination further comprises an enzymolysis solution, wherein the enzymolysis solution comprises a basal medium, type I collagenase, type III collagenase, type I DNase and Primocin. By the method for culturing the lung cancer organoids, the growth of lung cancer organoid cells can be accelerated, the culture speed is improved, and the lung cancer organoids cultured in vitro can retain tumor-related immune cells, so that the lung cancer organoids are more similar to the growth conditions in vivo.
Description
Technical Field
The invention relates to the technical field of biological tissue engineering, in particular to lung cancer organoid culture solution, a culture reagent combination and a culture method.
Background
Lung cancer is one of ten major malignant tumors in our country. The etiology of lung cancer is not completely clear today, and is currently thought to be mainly the result of the combined actions of environmental factors and genetic factors. The lung cancer organoids have important significance for research, and although some research reports on lung cancer organoid culture mediums exist at present, the culture process of the lung cancer organoids is slow in growth, time input cost is increased, and the bionics is low.
In the existing lung cancer organoid culture process, at least 14 days or even more days are needed for each passage, and the passage requirement is met. In addition, more importantly, after the organoid is cultured in vitro for a period of time, a great deal of loss of tumor microenvironment cell components such as T cells and B cells in the organoid occurs, and the difference between the tumor microenvironment cell components and the in-vivo actual tumor characteristics is larger.
The large heterogeneity of Tumor Microenvironment (TME) cell types has a critical impact on therapeutic response. Recently, the broad prospect of tumor-infiltrating immune cell therapies has created great pressure to reproduce the diverse human cancer models of TMEs. However, there is a lack of 2D or 3D models to reproduce in vivo interactions of tumor and immune cells in TMEs. Immune cells from blood or patient tumors have been recombined in traditional monolayer, spheroid or primary organoid cultures with a heterogeneously established cancer cell line. However, this in vitro tumor immune model does not maintain the complex diversity and physical structure of TMEs well, and in particular does not allow for the overall co-culture of primary tumor epithelial cells with their own infiltrating immune population. The use of custom microfluidic devices derived from human tumor suspension containing immune cells was responsive to immunotherapy, but not tumor immunospecific. In view of the above, it is particularly important to reproduce the structural complexity of the tumor organoids TME.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects in the prior art and providing lung cancer organoid culture solution, a culture reagent combination and a culture method.
The invention is realized by the following technical scheme:
a lung cancer organoid culture broth comprising basal medium comprising Advanced DMEM/F12, HEPES10mM and Glutamax 1.0x, a complex antibiotic comprising Primocin 200 μg/mL, penicillin-streptomycin 1x and ornidazole 5 μg/mL, and a growth factor comprising IL2 3000U/mL, IL4500U/mL, IL10 500U/mL, IL15 250U/mL, g-CSF 500U/mL, M-CSF300U/mL, CD28mAb 5ug/mL, OX-40mAb 10ug/mL, PD-1mAb 10ug/M and human a/B serum (v/v%) 5%.
According to the above technical scheme, preferably, the composition comprises the following components: 1mM N-Acetylcysteine (Sigma), 10mM Nicotinamide (Sigma), 50ng/mL recombinant human EGF (Peprotech), 100ng/mL FGF-10,25ng/mL HGF (Peprotech), 500ng/mL R-Spondin (Peprotech), 10. Mu.M Forskolin (BioGems, CA, USA), 5. Mu.M SB431542 (Cayman Chemical, michigan, USA) and 10nM gamin (BioGems), 10. Mu. M Y-27632 (Tocris Bioscience), 25ng/mL hNoggin (Peprotech) and 100ng/mL Wnt-3a (Peprotech).
Meanwhile, the patent also discloses a lung cancer organoid culture reagent combination, which comprises the lung cancer organoid culture solution and further comprises an enzymolysis solution, wherein the enzymolysis solution comprises a basic culture medium, and the enzymolysis solution further comprises type I collagenase, type III collagenase, type I DNase and Primocin.
According to the above technical scheme, preferably, the concentration of the type I collagenase is 2mg/mL, the concentration of the type III collagenase is 1mg/mL, the concentration of the type I DNase is 0.1mg/mL, and the concentration of the Primocin is 1mg/mL.
In addition, the patent discloses a lung cancer organoid culture method based on the lung cancer organoid culture reagent combination, comprising the following steps:
s1, preprocessing a lung cancer sample to obtain sample tissue fragments;
s2, carrying out enzymolysis pretreatment on the sample tissue fragments, filtering to obtain filtrate, and centrifuging the filtrate to obtain cell sediment;
s3, adopting matrigel to resuspend the cell sediment to obtain gel mixed with cells;
s4, inoculating the gel into a culture hole, and performing stationary culture in a cell incubator at 37 ℃;
s5, adding the lung cancer organoid culture solution into the culture holes for culture to obtain primary lung cancer organoids;
s6, carrying out subculture on the primary lung cancer organoid by adopting the lung cancer organoid culture solution to obtain the corresponding generation lung cancer organoid.
According to the above technical solution, preferably, step S2 includes: adding the enzymolysis liquid into the sample tissue fragments; after incubating for a preset incubation time under the conditions of a shaking table at 37 ℃ and 200r/min, adding an enzymolysis stopping agent; filtering after stopping enzymolysis to obtain the filtrate; and centrifuging the filtered solution to obtain a cell precipitate.
According to the above technical solution, preferably, the preset incubation time in step S2 is 15-25min, and the ratio of the added volume of the enzymolysis suspension to the added volume of the enzymolysis solution is 2:1.
According to the above technical scheme, preferably, the stationary culture time in step S4 is 15-40min.
The beneficial effects of the invention are as follows:
the lung cancer organoid culture solution disclosed by the invention has the advantages that various secretion factors of the lung cancer organoid culture solution are in synergistic interaction with the compound antibiotics and the growth factors, and the compound antibiotics are matched with the control of the type and the concentration of the compound antibiotics, so that the pollution is prevented, and the cell activity is maintained; the formation and growth of lung cancer organoid cells are accelerated, the culture speed is improved, the requirements of passage can be met after the culture for 7 days in the process of passage culture, and meanwhile, the immune component maintenance factors are added, so that the lung cancer organoid cells obtained by culture are more bionic, the tumor characteristics in vivo can be maintained in vitro, and the tumor microenvironment can be well maintained;
meanwhile, in the lung cancer organoid culture reagent combination disclosed by the invention, the enzymolysis liquid is subjected to enzymolysis by using the type I collagenase and the type III collagenase, so that the enzymolysis time can be shortened, the obtained cell activity is high, the sample pollution can be effectively reduced, then the lung cancer organoid culture liquid is used for culturing a lung cancer tumor sample, the growth of lung cancer organoid cells can be accelerated, and the culture speed is increased;
in addition, the invention also discloses a method for culturing lung cancer organoids, which can effectively reduce the formation and growth time of primary organoids, can shorten the culture time to 5-7 days in subculture, can reach 80-110 mu m in diameter of organoids in 5-7 days in culture, can stably shorten the passage period, can realize the formation of a large number of lung cancer organoids after 3 days in the primary extraction culture process by adopting a lung cancer organoid culture reagent, and can simultaneously obtain more bionic lung cancer organoids and maintain the tumor characteristics in vivo in vitro.
Drawings
FIG. 1 is a photomicrograph of lung cancer organoids after 7 days of primary culture in example 4 of the present invention.
FIG. 2 is a photomicrograph of lung cancer organoids 7 days after primary culture in a comparative example of the invention.
FIG. 3 is a comparison of the luminous intensity of ATP content of lung cancer organoids at 7 days of subculture in example 4 of the present invention with that of organoids in the comparative example.
FIG. 4 shows the expression of Macrophages (Macrophages), B lymphocytes (CD 19), T lymphocytes (CD 3), CD8+ T lymphocytes, and PD1+ T lymphocytes in the organoids of comparative example 1 after 14 days of organoid culture.
FIG. 5 shows the expression of Macrophages (Macrophages), B lymphocytes (CD 19), T lymphocytes (CD 3), CD8+ T lymphocytes, and PD1+ T lymphocytes in organoids 21 days after organoid culture (+IL 2) in example 4.
Detailed Description
The present invention will be described in further detail below with reference to the drawings and preferred embodiments, so that those skilled in the art can better understand the technical solutions of the present invention. All other embodiments, based on the embodiments of the invention, which would be apparent to one of ordinary skill in the art without making any inventive effort are intended to be within the scope of the invention.
Example 1 the invention includes basal medium comprising Advanced DMEM/F12, HEPES10mM and Glutamax 1.0x, complex antibiotics comprising Primocin 200 μg/mL, penicillin-streptomycin 1x and ornidazole 5 μg/mL, and growth factors comprising IL2 3000U/mL, IL4500U/mL, IL 10U/mL, IL15 250U/mL, g-CSF 500U/mL, M-CSF300U/mL, CD28mAb 5ug/mL, OX-40mAb 10ug/mL, PD-1mAb 10ug/M and human A/B serum (v/v%) 5%.
According to the above embodiment, it is preferable to include the following components: 1mM N-Acetylcysteine (Sigma), 10mM Nicotinamide (Sigma), 50ng/mL recombinant human EGF (Peprotech), 100ng/mL FGF-10,25ng/mL HGF (Peprotech), 500ng/mL R-Spondin (Peprotech), 10. Mu.M Forskolin (BioGems, CA, USA), 5. Mu.M SB431542 (Cayman Chemical, michigan, USA) and 10nM gamin (BioGems), 10. Mu. M Y-27632 (Tocris Bioscience), 25ng/mL hNoggin (Peprotech) and 100ng/mL Wnt-3a (Peprotech).
The embodiment 2 also discloses a lung cancer organoid culture reagent combination, which comprises the lung cancer organoid culture solution and an enzymolysis solution, wherein the enzymolysis solution comprises the basic culture medium, and the enzymolysis solution further comprises type I collagenase, type III collagenase, type I DNase and Primocin.
According to the above embodiment, preferably, the concentration of the type I collagenase is 2mg/mL, the concentration of the type III collagenase is 1mg/mL, the concentration of the type I DNase is 0.1mg/mL, and the concentration of Primocin is 1mg/mL.
Example 3 this patent discloses a lung cancer organoid culture method based on the above lung cancer organoid culture reagent combination comprising the steps of:
s1, preprocessing a lung cancer sample to obtain sample tissue fragments. Specifically, after a lung cancer tumor sample is washed by a PBS buffer solution containing 5% of double antibody (penicillin-streptomycin) and 50ng/mL of gentamicin (the washing times are not limited, and the purpose of sufficient washing is achieved), sample tissues are transferred into a culture dish, and then sheared to obtain sample tissue fragments.
S2, carrying out enzymolysis pretreatment on the sample tissue fragments, filtering to obtain filtrate, and centrifuging the filtrate to obtain cell sediment. Specifically:
s2-1, adding the enzymolysis liquid into the sample tissue fragments, wherein the addition amount of the enzymolysis liquid is related to the size of the sample, preferably 5-15mL of the enzymolysis liquid is added to each 1g of sample, in this case, when the weight of the sample tissue fragments is 0.5g, the addition amount of the enzymolysis liquid is 5mL, and the addition amount of the enzymolysis stopping agent is 10mL. The addition amount of the enzymolysis liquid is controlled, so that the enzymolysis incubation time can be controlled;
s2-2, after incubating for a preset incubation time under the conditions of a shaking table at 37 ℃ and 200r/min, adding an enzymolysis stopping agent, wherein the enzymolysis stopping agent can be Advanced DMEM/F12 at 4 ℃, the ratio of the adding volume of the enzymolysis stopping agent to the adding volume of the enzymolysis liquid is 2:1, and after adding the enzymolysis stopping agent, fully blowing by using a pipetting gun to stop enzymolysis;
s2-3, filtering by adopting a 100 mu m cell sieve after stopping enzymolysis to obtain filtrate;
s2-4, centrifuging the filtered solution under the condition that the centrifugal force is 300g and the centrifugal time is 5min to obtain cell sediment.
S3, adopting matrigel to resuspend the cell sediment to obtain gel mixed with cells, wherein the standard of gel solidification is that the gel of the vertically placed culture plate cannot flow freely. Specifically:
s3-1, after blowing off by adopting Advanced DMEM/F12 culture medium, carrying out centrifugal treatment (preferably, the centrifugal force is 300g, and the centrifugal time is 5 min), so as to obtain mixed sediment of matrigel and cells;
s3-2, re-suspending the mixed precipitate to obtain a heavy suspension;
s3-3, placing the heavy suspension in a water bath at 37 ℃ for 8-10min, then adding twice volume of Advanced DMEM/F12 culture medium at 4 ℃ into the heavy suspension to stop digestion, and centrifuging (preferably, the centrifugal force is 300g, and the centrifugal time is 5 min) to obtain secondary cell sediment;
s3-4, re-suspending the secondary cell sediment by adopting a freezing solution, freezing and preserving by using a programmed gradient cooling box, and transferring the secondary cell sediment into liquid nitrogen for long-term preservation after one day. Wherein the secondary cell pellet and the freezing solution have a weight ratio of 1×10 5 -5×10 6 Each cell pellet was resuspended in 1mL of frozen stock. The organoids subjected to liquid nitrogen freezing storage after the treatment of the steps can still grow stably after being recovered for 12 months, the activity and dryness of the organoids are maintained, and the organoids have good growth state after recovery.
S4, inoculating the gel into a culture hole, and performing stationary culture in a cell culture box at 37 ℃. Wherein the gel is inoculated into the well plate in an inoculum size of 100. Mu.L per well, wherein the number of wells of the well plate is not limited, and a 12-well plate may be preferred in this case. In addition, the stationary culture time is 15 to 40 minutes, and in this case, it is preferable that the stationary culture time is 30 minutes.
S5, adding the lung cancer organoid culture solution into the culture holes for culture to obtain primary lung cancer organoids;
s6, adopting the lung cancer organoid culture solution to subculture the primary lung cancer organoid, wherein the culture period of each subculture is 5-7 days, and obtaining the corresponding lung cancer organoid.
Furthermore, to characterize the technical effects of the present application, the obtained primary lung cancer organoids were subjected to detection of immune component maintenance conditions while being subjected to passaging treatment by example 4 and comparative example.
Example 4:
s101, after a lung cancer tumor sample (weight of 0.5 g) is subjected to shaking cleaning for 5 times by adopting PBS buffer solution containing 5% of double antibody (penicillin-streptomycin), transferring sample tissues into a culture dish (for example, a 60mL culture dish), and then shearing the sample tissues into 2-4 mm tissue fragments by using ophthalmic scissors to obtain the sample tissue fragments. And transferring the tissue fragments into a centrifuge tube.
S102, carrying out enzymolysis pretreatment on the sample tissue fragments obtained in the step S101 to obtain filtrate; the filtrate was centrifuged (centrifugal force: 300g, centrifugal time: 5 min) to obtain a cell pellet. Wherein, the enzymolysis pretreatment comprises the following steps: 5mL of enzymolysis liquid is added into the sample tissue fragments, after incubation is carried out for 15-25min on a shaking table (200 r/min) at 37 ℃,10 mL of enzymolysis stopping agent (Advanced DMEM/F12 at 4 ℃) is added, the enzymolysis is stopped by fully blowing with a pipetting gun, and a 100 mu m cell sieve is adopted for filtering, so that filtered liquid is obtained.
S103, adopting matrigel to resuspend the cell sediment obtained in the step S102 to obtain gel mixed with cells; the gel was then inoculated into the culture wells of a 24-well plate at an inoculum size of 50. Mu.L per well, and allowed to stand still for 30 minutes to solidify.
S104, adding the lung cancer organoid culture solution into the culture holes for culture to obtain primary lung cancer organoids; and after 7 days of culture, passaging can be performed. In the step S104, lung cancer organoids are formed after 3 days of culture, the maximum diameter of the lung cancer organoids reaches more than 100 mu m, and the primary culture can be carried out for 7 days. In this embodiment, the enzymolysis treatment is performed by using the enzymolysis solution iii in the step S102, and the detection is performed on the lung cancer organoids after the primary culture is performed for 7 days by using the culture solution ii-ii in the step S104, so as to obtain the optical lens diagram shown in fig. 1. As can be seen, the lung cancer organoids cultured for 7 days reach a maximum diameter of more than 100 μm, and can be subjected to primary culture for 7 days for passage.
S105, subculturing the primary lung cancer organoids obtained in the step S104 for 7 days, and culturing by adopting the lung cancer organoids culture solution in the subculture process, wherein the culture period of each subculture is 5-7 days, so as to obtain the corresponding lung cancer organoids.
S106, digesting the primary lung cancer organoids obtained in the step S104 after 7 days of culture,
the luminescence method cell viability detection kit determines the cell viability of the 3D cultured cells by quantifying ATP.
Comparative example:
s101, after a lung cancer tumor sample (weight of 0.5 g) is subjected to shaking cleaning for 5 times by adopting PBS buffer solution containing 5% of double antibody (penicillin-streptomycin), transferring sample tissues into a culture dish (for example, a 60mL culture dish), and then shearing the sample tissues into 2-4 mm tissue fragments by using ophthalmic scissors to obtain the sample tissue fragments. And transferring the tissue fragments into a centrifuge tube.
S102, carrying out enzymolysis pretreatment on the sample tissue fragments obtained in the step S101 to obtain filtrate; the filtrate was centrifuged (centrifugal force: 300g, centrifugal time: 5 min) to obtain a cell pellet. Wherein, the enzymolysis pretreatment comprises the following steps: 5mL of enzymolysis liquid is added into the sample tissue fragments, after incubation is carried out for 15-25min on a shaking table (200 r/min) at 37 ℃,10 mL of enzymolysis stopping agent (Advanced DMEM/F12 at 4 ℃) is added, the enzymolysis is stopped by fully blowing with a pipetting gun, and a 100 mu m cell sieve is adopted for filtering, so that filtered liquid is obtained.
S103, adopting matrigel to resuspend the cell sediment obtained in the step S102 to obtain gel mixed with cells; the gel was then inoculated into the culture wells of a 24-well plate at an inoculum size of 50. Mu.L per well, and allowed to stand still for 30 minutes to solidify.
S104, adding a common lung cancer organoid culture solution (without immune component maintenance factors) into the culture holes for culture to obtain primary lung cancer organoids; and after 7 days of culture, passaging can be performed. In the step S104, lung cancer organoids are formed after 3 days of culture, the maximum diameter of the lung cancer organoids reaches more than 100 mu m, and the primary culture can be carried out for 7 days. In this embodiment, the enzymolysis treatment is performed by using the enzymolysis solution iii in step S102, and the lung cancer organoids after the primary culture is performed for 7 days by using the culture solution ii-ii in step S104 are detected, so as to obtain the optical lens diagram shown in fig. 2. As can be seen, the lung cancer organoids cultured for 7 days reach a maximum diameter of more than 100 μm, and can be subjected to primary culture for 7 days for passage.
S105, subculturing the primary lung cancer organoids obtained in the step S104 for 7 days, and culturing by adopting the lung cancer organoids culture solution in the subculture process, wherein the culture period of each subculture is 5-7 days, so as to obtain the corresponding lung cancer organoids.
S106, digesting the primary lung cancer organoids obtained in the step S104 after 7 days of culture,
the luminescence method cell viability detection kit determines the cell viability of the 3D cultured cells by quantifying ATP.
Meanwhile, the primary lung cancer organoids obtained in example 4 were tested for immune component maintenance, specifically:
collecting cells: when the organoids grow for 7 days, the organoids with a diameter of more than 100 mu m increase, the minimum diameter of the organoids reaches more than 80 mu m, and cells are collected after digestion by digestive enzymes, and the supernatant and the digested cells are combined;
washing the cells: washing the cells with pre-chilled PBS for 2 times;
grouping: each experiment was divided into a non-staining group, a single-staining CD3 group, a single-staining CD8 group, and a double-staining CD3 and CD8 group;
dyeing: diluting 10×binding buffer to 1×buffer with PBS, sucking residual PBS from centrifuge tube, adding 100 μl of 1×binding buffer per tube, blowing cells with a pipette to fully resuspend cells, and adding dye under dark condition;
and (3) detecting: after incubation for 15 min at room temperature in the dark, 1 Xbinding buffer 300. Mu.L was added and mixed well and the cell suspension was transferred to a 5mL flow tube in the dark and detected on-line in a flow cytometer for 1 h.
Data analysis: and (3) drawing a bicolor scatter diagram through software analysis, and carrying out data analysis.
In addition, on the basis of example 4, the primary lung cancer organoids obtained were subjected to passaging, in particular:
(1) For the primary lung cancer organoids embedded with matrigel in example 4, 1mL of Advanced DMEM/F12 medium at 4deg.C was used for blowing off each well, recovered in a centrifuge tube, and centrifuged (centrifugal force 300g, centrifugation time 5 min) to obtain matrigel and cell mixed precipitate.
(2) Resuspension of the obtained pellet with TrypLE (Gibco) to obtain a resuspension; placing the heavy suspension in a water bath at 37 ℃ for 8-10 min; digestion was then stopped by adding twice the volume of 4℃advanced DMEM/F12 medium to the resuspension and repeatedly blown several times with a pipette. And (3) centrifuging (the centrifugal force is 300g, the centrifugal time is 5 min) to obtain secondary cell sediment. Wherein, the TrypLE is used in an amount based on the amount of matrigel embedding the organoid, and 1mL TrypLE is theoretically used for resuspension per 100 mu L matrigel embedding the organoid. In addition, repeated blows using a pipette after stopping digestion are directly related to the extent of organoid digestion. All organoids can be digested into uniform size cell clusters, typically containing 2-10 cells, using a 1mL pipette gun to blow 10-20 times, then a 1mL pipette gun cap to blow again with 200 μl pipette tips 10-20 times.
(3) The secondary cell pellet was resuspended with Advanced DMEM/F12, blown uniformly, and centrifuged (centrifugal force 300g, centrifugation time 5 min) to obtain a tertiary cell pellet. If the impurities are more, the step can be repeated.
(4) Re-suspending the cell sediment for three times by adopting matrigel to obtain gel mixed with cells; the gel was then seeded into 24-well plates at an inoculum size of 50 μl per well and incubated in an incubator at 37 ℃ for 30min to allow the gel to solidify.
(5) And adding lung cancer organoid culture solution into the coagulated organoid culture hole for culture. As shown in FIG. 1, the photomicrograph of the organoid after 7 days of culture showed that the organoid was uniform in size and good in growth. The growth diameter was approximately 100. Mu.M.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A lung cancer organoid culture broth comprising basal medium comprising Advanced DMEM/F12, HEPES10mM and Glutamax 1.0x, a complex antibiotic comprising Primocin 200 μg/mL, penicillin-streptomycin 1x and ornidazole 5 μg/mL, and a growth factor comprising IL2 3000U/mL, IL4500U/mL, IL10 500U/mL, IL15 250U/mL, g-CSF 500U/mL, M-CSF300U/mL, CD28mAb 5ug/mL, OX-40mAb 10ug/mL, PD-1mAb 10ug/M and human a/B serum (v/v%) 5%.
2. The lung cancer organoid culture fluid of claim 1, comprising the following components: 1mM N-Acetylcysteine (Sigma), 10mM Nicotinamide (Sigma), 50ng/mL recombinant human EGF (Peprotech), 100ng/mL FGF-10,25ng/mL HGF (Peprotech), 500ng/mL R-Spondin (Peprotech), 10. Mu.M Forskolin (BioGems, CA, USA), 5. Mu.M SB431542 (Cayman Chemical, michigan, USA) and 10nM gamin (BioGems), 10. Mu. M Y-27632 (Tocris Bioscience), 25ng/mL hNoggin (Peprotech) and 100ng/mL Wnt-3a (Peprotech).
3. A lung cancer organoid culture reagent combination comprising the lung cancer organoid culture solution of claim 1 or 2, further comprising an enzymatic hydrolysate comprising the basal medium, the enzymatic hydrolysate further comprising type I collagenase, type iii collagenase, type I dnase, and Primocin.
4. A lung cancer organoid culture reagent combination according to claim 3, wherein the concentration of type I collagenase is 2mg/mL, the concentration of type iii collagenase is 1mg/mL, the concentration of type I dnase is 0.1mg/mL, and the concentration of Primocin is 1mg/mL.
5. A lung cancer organoid culture method based on the lung cancer organoid culture reagent combination of claim 3, comprising the steps of:
s1, preprocessing a lung cancer sample to obtain sample tissue fragments;
s2, carrying out enzymolysis pretreatment on the sample tissue fragments, filtering to obtain filtrate, and centrifuging the filtrate to obtain cell sediment;
s3, adopting matrigel to resuspend the cell sediment to obtain gel mixed with cells;
s4, inoculating the gel into a culture hole, and performing stationary culture in a cell incubator at 37 ℃;
s5, adding the lung cancer organoid culture solution into the culture holes for culture to obtain primary lung cancer organoids;
s6, carrying out subculture on the primary lung cancer organoid by adopting the lung cancer organoid culture solution to obtain the corresponding generation lung cancer organoid.
6. The method of claim 5, wherein step S2 comprises: adding the enzymolysis liquid into the sample tissue fragments; after incubating for a preset incubation time under the conditions of a shaking table at 37 ℃ and 200r/min, adding an enzymolysis stopping agent; filtering after stopping enzymolysis to obtain the filtrate; and centrifuging the filtered solution to obtain a cell precipitate.
7. The method according to claim 6, wherein the predetermined incubation time in step S2 is 15-25min, and the ratio of the added volume of the enzyme inhibitor to the added volume of the enzyme solution is 2:1.
8. The method according to any one of claims 5 to 7, wherein the stationary culture time in step S4 is 15 to 40min.
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