CN112760288B - Special culture medium and culture method for lung cancer organoid - Google Patents

Special culture medium and culture method for lung cancer organoid Download PDF

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CN112760288B
CN112760288B CN202110094775.XA CN202110094775A CN112760288B CN 112760288 B CN112760288 B CN 112760288B CN 202110094775 A CN202110094775 A CN 202110094775A CN 112760288 B CN112760288 B CN 112760288B
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lung cancer
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cell
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CN112760288A (en
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于言
朱宇
李刚
黄敏
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Accurate International Biotechnology Guangzhou Co ltd
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Abstract

The invention provides a special culture medium for lung cancer organoid and a culture method, wherein the culture medium comprises a basic culture medium and a specific additive factor; the specific addition factors comprise: GSK 429286A; y27632; EGF; BMP 4; human FGF 4; Insulin-Transferrin-Selenium (Insulin-Transferrin-Selenium); b27; all-trans retinoic acid (all-trans retinic acid); KGF; DAPT; GlutaMAX; R-Spondin-1; CHIR 99021; 8-Br-cAMP; bovine Serum Albumin (BSA); phosphodiesterase (PDEs) inhibitors. The culture medium can realize the rapid growth of the lung cancer organoid, can be stably cultured for a long time, and can well keep the heterogeneity of tumor tissues of patients in vitro, and the formed tumor organoid cells have regular sphere shapes and uniform sizes.

Description

Special culture medium and culture method for lung cancer organoid
Technical Field
The invention belongs to the technical field of organoid culture, and particularly relates to a special culture medium for lung cancer organoids and a culture method.
Background
Lung cancer is one of the malignant tumors threatening human health and life safety, and about 100 to 300 million people die of lung cancer every year worldwide. Lung cancer also becomes the first malignant tumor threatening human life in China, the number of deaths per year is about 60 thousands, and the number of deaths per year tends to increase year by year. Although researchers have made breakthrough progress in lung cancer pathogenesis and treatment by using various lung cancer cell lines with the development of molecular biology in recent years, due to the difference of genetic background, the molecular mechanisms and treatments of different patients have individuation characteristics. Currently, individualized treatment of lung cancer is based on three layers: clinics, molecular and cytology; the cytological factors are determined by detecting the drug sensitivity result of the tumor cells, but because the difference between the drug sensitivity of bacteria and the drug sensitivity of tumor cell lines and the clinical application value is large, a new in-vitro substitute drug sensitivity model is urgently needed to be established to select a reasonable comprehensive treatment scheme according to the clinical condition, so that patients can be better treated or cured.
The primary tumor cells cultured by the autologous tumor tissues of tumor patients can show the original characteristics of tumors, and are an important means for in vitro research of the tumors. Organoids are a 3-dimensional cell culture that contains some key features of the tissue from which they are derived; the organoid culture of lung cancer tissue refers to the culture of tumor tissue taken out from the body of a patient with lung cancer, and is suitable for the research of cell morphology, function, differentiation, drug therapy and the like due to the short culture in vitro time and similar genetic characters and in vivo cells. However, the current research technology for establishing the lung cancer organoid culture method in China is blank, and the main problem is that the development of a special culture system for the lung cancer organoid is difficult. If the culture technology of lung cancer organoids can be successfully researched, an ideal in-vitro model is provided for drug research, drug sensitivity detection and drug screening of lung cancer patients, and a great deal of gospel is undoubtedly brought to lung cancer patients in China.
Disclosure of Invention
In view of the above, the present invention aims to provide a special culture medium for lung cancer organoid and a culture method thereof to solve the above problems. The technical scheme of the invention is as follows:
in a first aspect, the invention provides a special culture medium for lung cancer organoid, which comprises a basic culture medium and a specific additive factor; the specific addition factors comprise: GSK 429286A; y27632; epidermal Growth Factor (EGF); bone morphogenetic protein 4(BMP 4); human fibroblast growth factor 4(Human FGF 4); Insulin-Transferrin-Selenium (Insulin-Transferrin-Selenium); b27; all-trans retinoic acid (all-trans retinic acid); keratinocyte Growth Factor (KGF); (3, 5-difluorophenylacetyl) -L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT); GlutaMAX; R-Spondin-1; CHIR 99021; 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP); bovine Serum Albumin (BSA); phosphodiesterase (PDEs) inhibitors.
Further, the specific addition factor comprises the following components in final concentration: GSK429286A, 2-20 μ M; y27632, 2-20 μ M; EGF, 10-500 ng/ml; BMP4, 10-500 ng/ml; human FGF4, 50-1000 ng/ml; 0.5-2x of Insulin-Transferrin-Selenium; b27, 0.5-3 x; all-trans retinoic acid, 0.1-1 μ M; KGF, 10-500 ng/ml; DAPT, 2-20 mM; GlutaMAX, 0.5-2 x; R-Spondin-1, 100-1000 ng/ml; CHIR99021, 1-20. mu.M; 8-Br-cAMP, 10-200 nM; 0.1% -1% of BSA; PDEs inhibitor, 10-200nM, the aforementioned percentages being percentages by mass.
Preferably, the specific addition factor comprises the following components in final concentrations: GSK429286A, 5-15 μ M; y27632, 5-12.5 μ M; EGF, 50-200 ng/ml; BMP4, 50-200 ng/ml; human FGF4, 200 and 600 ng/ml; 0.75-1.5x of Insulin-Transferrin-Selenium; b27, 0.75-1.5 x; all-trans retinoic acid, 0.4-0.6 μ M; KGF, 50-200 ng/ml; DAPT, 5-15 mM; GlutaMAX, 0.75-1.25 x; R-Spondin-1, 300-800 ng/ml; CHIR99021, 5-10. mu.M; 8-Br-cAMP, 50-150 nM; 0.2-0.6% of BSA; 3-isobutyl-1-methylxanthine, 50-150nM, the aforementioned percentages being percentages by mass.
Preferably, the basal medium is DMEM/F12(1:1) medium.
Further, the preparation method of the culture medium comprises the following steps: adding the specific additive factor into the basic culture medium, and uniformly mixing to obtain the product.
In a second aspect, the present invention provides a lung cancer organoid culture method, comprising the steps of:
1) pretreating the separated lung cancer tissue, digesting twice, and collecting cell precipitate;
2) cracking cell precipitate, mixing with matrigel gel, inoculating, coagulating, adding the culture medium, and culturing at 37 deg.C under 5% CO2Culturing at the concentration for 6-14 days to obtain the final product.
Further, the process control of the first digestion in the step 1) comprises: the method comprises the steps of adopting a 'digestive juice 1', wherein the 'digestive juice 1' comprises 200-.
Further, the process control of the second digestion in the step 1) comprises: and (2) digesting the cell sediment subjected to primary digestion for 15-20 min at 37 ℃ and 220rpm by using a 'digestive juice 2' which comprises 2-20mg/ml of dispase II and 0.2-2mg/ml of DNase, adding HBSS to terminate digestion, and centrifuging to obtain the cell sediment subjected to secondary digestion.
Further, the step 1) further comprises: cell pellets obtained by the two digestions were resuspended in HBSS and passed through a 100 μm cell strainer, after which the cell pellets were collected by centrifugation.
In a third aspect, the invention provides a lung cancer organoid, which is obtained by the above culture method.
The invention has the beneficial effects that:
the culture medium can realize the rapid growth of the lung cancer organoid, can be stably cultured for a long time, and the formed tumor organoid has good activity, tight connection among cell balls and large diameter, and can well keep the heterogeneity of tumor tissues of patients in vitro.
Secondly, the culture method of the invention has simple operation, less influence of personnel operation and high stability.
Drawings
FIG. 1 is a morphological diagram of tumor cells cultured for 0 day in example 1 of the present invention.
FIG. 2 is a morphological diagram of tumor cells cultured for 3 days in example 1 of the present invention.
FIG. 3 is a morphological diagram of tumor cells cultured for 6 days in example 1 of the present invention.
FIG. 4 is a morphological diagram of tumor cells cultured for 12 days in example 1 of the present invention.
FIG. 5 is a structural morphology chart of lung cancer organoids obtained by 14 days of culture in example 2 of the present invention.
FIG. 6 is a structural morphology chart of lung cancer organoids obtained by 14 days of culture in example 3 of the present invention.
FIG. 7 is a structural morphology chart of lung cancer organoids obtained by 14 days of culture in example 4 of the present invention.
FIG. 8 is a morphological diagram of tumor cells cultured for 30 days in example 5 of the present invention.
FIG. 9 is a morphological diagram of tumor cells cultured for 90 days in example 5 of the present invention.
FIG. 10 is a morphological diagram of tumor cells cultured for 120 days in example 5 of the present invention.
FIG. 11 is a morphological diagram of tumor cells cultured for 180 days in example 5 of the present invention.
FIG. 12 is a morphological diagram of tumor cells cultured for 5 days in comparative example 1 of the present invention.
FIG. 13 is a morphological diagram of tumor cells cultured for 10 days in comparative example 1 of the present invention.
FIG. 14 is a morphological diagram of tumor cells cultured for 14 days in comparative example 2 of the present invention.
FIG. 15 is a morphological diagram of tumor cells cultured for 14 days in comparative example 3 of the present invention.
Detailed Description
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The embodiment provides a special culture medium and a culture method for lung cancer organoids, which comprise a basic culture medium and a specific additive factor; the specific addition factor comprises the following components in final concentration: GSK429286A, 5 μ M; y27632, 12.5. mu.M; EGF, 50 ng/ml; BMP4, 150 ng/ml; human FGF4, 300 ng/ml; Insulin-Transferrin-Selenium, 1.5 x; b27, 0.75 x; all-trans retinoic acid, 0.5 μ M; KGF, 200 ng/ml; DAPT, 5 mM; GlutaMAX, 1.25 x; R-Spondin-1, 300 ng/ml; CHIR99021, 5 μ M; 8-Br-cAMP, 150 nM; BSA, 0.5%; 3-isobutyl-1-methylxanthine, 50 nM; the foregoing percentages are by mass.
The lung cancer organoid culture method comprises the following steps:
(1) after being washed by normal saline, the taken lung cancer tissues are sheared on ice, 10ml of digestive juice 1 is added for resuspension, wherein the digestive juice 1 comprises 500ng/ml hydrocortisone and 500U/ml Collagenase enzyme type IV, the mixture is transferred to 37 ℃ and is digested for 2 hours by a shaker at 100rpm, and 5ml of HBSS is added after the digestion is finished to stop the digestion. Centrifuge at 1200rpm for 5 minutes and remove supernatant.
(2) Adding the cell sediment subjected to primary digestion into 5ml of digestive juice 2, wherein the components of the digestive juice 2 comprise 10mg/ml of dispase II and 0.5mg/ml of DNase, transferring to 37 ℃, performing shake digestion for 15 minutes at 220rpm, and adding 5ml of HBSS to terminate the digestion after the digestion is completed. Centrifuge at 1200rpm for 5 minutes and remove supernatant.
(3) The twice digested cell pellet was resuspended in 5ml of HBSS, the cell suspension was filtered through a 100. mu.M cell strainer, centrifuged at 1200rpm for 5 minutes, and the cell pellet was collected.
(4) Adding 5ml erythrocyte lysate into the cell precipitate to resuspend the precipitate, terminating the lysis with 5ml HBSS, centrifuging, removing supernatant, counting cells, mixing matrigel, dropping 20000 cells per drop in 24-well plate, placing at 37 deg.C and 5% CO2Adding the special culture medium into the incubator after matrigel gel is solidified, and adding 5% CO at 37 DEG C2Culturing at the concentration for 12 days to obtain the final product. FIGS. 1 to 4 provide the state diagrams of the change process from the cell development to the organoid during the culture process, and it can be seen that the culture medium of the present embodiment can realize the rapid growth of the lung cancer organoid, and has good activity, regular cell sphere shape of the formed tumor organoid, tight connection between cell spheres, and uniform and controllable size.
Example 2
The embodiment provides a special culture medium and a culture method for lung cancer organoids, which comprise a basic culture medium and a specific additive factor; the specific addition factor comprises the following components in final concentration: GSK429286A, 15 μ M; y27632, 6. mu.M; EGF, 200 ng/ml; BMP4, 50 ng/ml; human FGF4, 550 ng/ml; Insulin-Transferrin-Selenium, 0.8 x; b27, 1.5 x; all-trans retinoic acid, 0.6 μ M; KGF, 100 ng/ml; DAPT, 15 mM; GlutaMAX, 0.75 x; R-Spondin-1, 600 ng/ml; CHIR99021, 8 μ M; 8-Br-cAMP, 80 nM; BSA, 0.3%; 3-isobutyl-1-methylxanthine, 150 nM; the foregoing percentages are by mass. The lung cancer organoid culture method was the same as in example 1. FIG. 5 provides a topographical map of lung cancer organoids obtained after 14 days of culture, with the resulting tumor organoid cell spheroids regular and of larger diameter.
Example 3
The embodiment provides a special culture medium and a culture method for lung cancer organoids, which comprise a basic culture medium and a specific additive factor; the specific addition factor comprises the following components in final concentration: GSK429286A, 2.5 μ M; y27632, 20. mu.M; EGF, 400 ng/ml; BMP4, 30 ng/ml; human FGF4, 300 ng/ml; Insulin-Transferrin-Selenium, 2 x; b27, 0.5 x; all-trans retinoic acid, 1 μ M; KGF, 10 ng/ml; DAPT, 20 mM; GlutaMAX, 0.5 x; R-Spondin-1, 1000 ng/ml; CHIR99021, 1 μ M; 8-Br-cAMP, 150 nM; BSA, 0.1%; 3-isobutyl-1-methylxanthine, 150 nM; the foregoing percentages are by mass. The lung cancer organoid culture method was the same as in example 1. FIG. 6 provides a morphological map of lung cancer organoids obtained after 14 days of culture, and the formed tumor organoids have regular cell spheres, tight cell sphere connection and uniform and controllable size.
Example 4
The embodiment provides a special culture medium and a culture method for lung cancer organoids, which comprise a basic culture medium and a specific additive factor; the specific addition factor comprises the following components in final concentration: GSK429286A, 20 μ M; y27632, 2.5. mu.M; EGF, 20 ng/ml; BMP4, 400 ng/ml; human FGF4, 800 ng/ml; Insulin-Transferrin-Selenium, 1 x; b27, 2 x; all-trans retinoic acid, 0.2 μ M; KGF, 400 ng/ml; DAPT, 2.5 mM; GlutaMAX, 2 x; R-Spondin-1, 100 ng/ml; CHIR99021, 5 μ M; 8-Br-cAMP, 20 nM; BSA, 0.8%; 3-isobutyl-1-methylxanthine, 20 nM; the foregoing percentages are by mass. The lung cancer organoid culture method was the same as in example 1. FIG. 7 provides a morphological map of lung cancer organoids obtained after 14 days of culture, and the formed tumor organoids have regular cell spheres, tight cell sphere connection and uniform and controllable size.
Example 5
This example provides a method for culturing lung cancer organoids, using the culture medium of example 1, comprising the steps of:
(1) after being washed by normal saline, the taken lung cancer tissues are sheared on ice, 10ml of digestive juice 1 is added for resuspension, wherein the digestive juice 1 comprises 500ng/ml hydrocortisone and 500U/ml Collagenase enzyme type IV, the mixture is transferred to 37 ℃ and is digested for 2 hours by a shaker at 100rpm, and 5ml of HBSS is added after the digestion is finished to stop the digestion. Centrifuge at 1200rpm for 5 minutes and remove supernatant.
(2) Adding the cell sediment subjected to primary digestion into 5ml of digestive juice 2, wherein the components of the digestive juice 2 comprise 10mg/ml of dispase II and 0.5mg/ml of DNase, transferring to 37 ℃, performing shake digestion for 15 minutes at 220rpm, and adding 5ml of HBSS to terminate the digestion after the digestion is completed. Centrifuge at 1200rpm for 5 minutes and remove supernatant.
(3) The twice digested cell pellet was resuspended in 5ml of HBSS, the cell suspension was filtered through a 100. mu.M cell strainer, centrifuged at 1200rpm for 5 minutes, and the cell pellet was collected.
(4) Adding 5ml erythrocyte lysate into the cell precipitate to resuspend the precipitate, terminating the lysis with 5ml HBSS, centrifuging, removing supernatant, counting cells, mixing matrigel, dropping 20000 cells per drop in 24-well plate, placing at 37 deg.C and 5% CO2Adding the special culture medium into the incubator after matrigel gel is solidified, and adding 5% CO at 37 DEG C2Cultured at the concentration for 6 months. FIGS. 8-11 provide graphs of cell culture status for 30, 90, 120, 180 days of lung cancer organoids during the aforementioned culture process, and it is evident that long-term stable culture of lung cancer organoids for up to six months can be maintained using the culture medium and culture method of the present invention.
Comparative example 1
This comparative example provides a method for culturing lung cancer organoids using the conventional common medium (DMEM + 10% FBS) in the same manner as in example 1. FIG. 12 provides a state diagram of cells cultured for 5 days and 10 days, wherein the cells of lung cancer tissue grow slowly and are partially adherent during the culture process, and organoids with spherical structure and multi-cellular components can not be formed.
Comparative example 2
The culture medium provided in this comparative example was supplemented with GSK429286A factor, otherwise as in example 1.
The organoid state obtained by 14 days of culture using the above medium according to the method of example 1 is shown in fig. 13, with the results that: after the GSK429286A factor was subtracted, the cell mass was loosely connected and less active.
Comparative example 3
The culture medium provided in this comparative example was supplemented with CHIR99021 factor, and the procedure was as in example 1.
The organoid state obtained by 14 days of culture using the above medium according to the method of example 1 is shown in fig. 14, with the results that: after the factor CHIR99021 is subtracted, the number of organoids formed is reduced, the growth speed of cell mass is slower, and the diameter is smaller.
In conclusion, the culture medium can realize the rapid growth of the lung cancer organoid, can be stably cultured for a long time, forms the spherical cell rules of the tumor organoid, has uniform size, and can well keep the heterogeneity of the tumor tissues of patients in vitro.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (6)

1. A special culture medium for lung cancer organoid is characterized in that: consists of a basic culture medium and a specific additive factor; the specific additive factor consists of the following components in final concentration: GSK429286A, 2-20 μ M; y27632, 2-20 μ M; 10-500ng/ml of epidermal growth factor; bone morphogenetic protein 4, 10-500 ng/ml; human fibroblast growth factor 4, 50-1000 ng/ml; insulin-transferrin-selenium, 0.5-2 x; b27, 0.5-3 x; all-trans retinoic acid, 0.1-1 μ M; keratinocyte growth factor, 10-500 ng/ml; (3, 5-difluorophenylacetyl) -L-alanyl-L-2-phenylglycine tert-butyl ester, 2-20 mM; GlutaMAX, 0.5-2 x; R-Spondin-1, 100-1000 ng/ml; CHIR99021, 1-20. mu.M; 8-bromo-cyclic adenosine monophosphate, 10-200 nM; bovine serum albumin, 0.1% -1%; 10-200nM phosphodiesterase inhibitor, the aforementioned percentage is mass percentage; the basic culture medium is a DMEM/F121: 1 culture medium.
2. The special culture medium for the lung cancer organoid according to claim 1, wherein: the specific additive factor consists of the following components in final concentration: GSK429286A, 5-15 μ M; y27632, 5-12.5 μ M; epidermal growth factor, 50-200 ng/ml; bone morphogenetic protein 4, 50-200 ng/ml; human fibroblast growth factor 4, 200 and 600 ng/ml; insulin-transferrin-selenium, 0.75-1.5 x; b27, 0.75-1.5 x; all-trans retinoic acid, 0.4-0.6 μ M; keratinocyte growth factor, 50-200 ng/ml; (3, 5-difluorophenylacetyl) -L-alanyl-L-2-phenylglycine tert-butyl ester, 5-15 mM; GlutaMAX, 0.75-1.25 x; R-Spondin-1, 300-800 ng/ml; CHIR99021, 5-10. mu.M; 8-bromo-cyclic adenosine monophosphate, 50-150 nM; bovine serum albumin, 0.2-0.6%; 3-isobutyl-1-methylxanthine, 50-150nM, the aforementioned percentages being percentages by mass.
3. A lung cancer organoid culture method is characterized in that: the method comprises the following steps:
1) pretreating the separated lung cancer tissue, digesting twice, and collecting cell precipitate;
2) lysing the cell pellet, mixing with matrigel gel and inoculating, adding the culture medium of claim 1 or 2 after the gel has solidified, and adding 5% CO at 37 deg.C2Culturing at the concentration for 6-14 days to obtain the final product.
4. The lung cancer organoid culture method of claim 3, wherein: the process control of the first digestion in the step 1) comprises the following steps: adopting a ' digestive juice 1 ', wherein the ' digestive juice 1 ' comprises 200-1000ng/ml hydrocortisone and 200-1000U/ml collagenase IV, digesting the lung cancer tissue for 2-3 h at 37 ℃ and 220rpm, adding Hank's balanced salt solution to stop digestion, and centrifuging to obtain a primary digested cell precipitate.
5. The lung cancer organoid culture method of claim 3, wherein: the process control of the second digestion in the step 1) comprises the following steps: and (2) digesting the cell sediment subjected to primary digestion for 15-20 min at 37 ℃ and 220rpm by using a ' digestive juice 2 ', wherein the components of the ' digestive juice 2 ' comprise 2-20mg/ml of dispase II and 0.2-2mg/ml of DNase, adding Hank's balanced salt solution to terminate digestion, and centrifuging to obtain the cell sediment subjected to secondary digestion.
6. The lung cancer organoid culture method of claim 3, wherein: the step 1) further comprises: cell pellets obtained from the two digestions were resuspended in Hank's balanced salt solution and passed through a 100 μm cell strainer, after which the cell pellets were collected by centrifugation.
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