CN114292816B

CN114292816B - Lung cancer organoid culture solution, and culture reagent combination and culture method thereof

Info

Publication number: CN114292816B
Application number: CN202210231127.9A
Authority: CN
Inventors: 肖荣荣; 刘建闯; 李珮文; 孙艳廷; 张晓会; 周宇
Original assignee: Beijing Da Xiang Technology Co ltd
Current assignee: Beijing Da Xiang Technology Co ltd
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2022-05-31
Anticipated expiration: 2042-03-10
Also published as: CN114292816A

Abstract

The application relates to the technical field of biological tissue engineering, and discloses a lung cancer organoid culture solution, a culture reagent combination and a culture method thereof, wherein the lung cancer organoid culture solution comprises a complete culture medium and an autologous condition culture medium; the complete culture medium comprises a conditioned medium, a basal medium, a compound antibiotic and a growth factor; the self-source condition culture medium is obtained by culturing the fibrocyte extracted from the lung cancer tissue by using a tumor-related fibroblast culture medium. The components of the culture solution have synergistic effect, so that the formation and growth of lung cancer organoid cells are accelerated, the cell harvest quantity is increased, and the culture speed is increased; and the organoid cultured in vitro is more bionic, can be suitable for the individual culture of the lung cancer organoid, and can maintain the body structure and pathological characteristics of the primary tissue of each patient.

Description

Lung cancer organoid culture solution, and culture reagent combination and culture method thereof

Technical Field

The application relates to the technical field of biological tissue engineering, for example, a lung cancer organoid culture solution, a culture reagent combination and a culture method thereof.

Background

Lung cancer is one of ten malignant tumors in China. At present, the etiology of lung cancer is not completely understood, and it is thought that it is mainly the result of the combined action of environmental factors and genetic factors. The lung cancer organoid has important significance for research. At present, although some research reports on lung cancer organoid culture media exist, the growth of the lung cancer organoid culture media is slow in the culture process, and the time investment cost is increased; and has low bionic property.

In the process of implementing the embodiment of the present application, it is found that at least the following problems exist in the related art: in the existing culture process of lung cancer organoids, at least 14 days or even more days are needed for each passage to meet the passage requirement. In addition, the obtained cell quantity is very small, the time is longer for harvesting more cells and carrying out downstream tests, so that the construction process of the whole organ is slow, the time cost for researching the lung cancer organoid is high, and more importantly, after the organoid is cultured in vitro for a period of time, the EMT transformation can occur, the interstitial property in vivo is gradually lost, and the difference with the real tumor property in vivo is larger.

Disclosure of Invention

The following presents a simplified summary in order to provide a basic understanding of some aspects of the disclosed embodiments. This summary is not an extensive overview nor is intended to identify key/critical elements or to delineate the scope of such embodiments but rather as a prelude to the more detailed description that is presented later.

The embodiment of the application provides a lung cancer organoid culture solution, a culture reagent combination and a culture method thereof, which are used for accelerating the growth of lung cancer organoid cells and improving the culture speed; and the lung cancer organoid cultured in vitro is more bionic, and the interstitial characteristics of the organ can be kept.

In some embodiments, the lung cancer organoid culture fluid comprises a complete culture medium and an autologous condition culture medium, wherein the volume ratio of the complete culture medium to the autologous condition culture medium is 1-3: 1; wherein the complete medium comprises a conditioned medium, a basal medium, a complex antibiotic and a growth factor; the volume ratio of the conditioned medium to the basic medium is 1-4: 1; the basic culture medium comprises Advanced DMEM/F12, HEPES 5-15 mM and Glutamax 0.5 x-1.5 x; the compound antibiotics comprise 50-500 mu g/mL of Primocin, 0.5-1.5 x of penicillin-streptomycin and 2-10 mu g/mL of metronidazole; the growth factors include: 10-100 ng/mL of EGF, 1010-100 ng/mL of FGF-20.25x-1.5 x of N20.25x, 270.25x-1.5 x of B270, 1-2 mM of n-Acetylcysteine, 5-20 mM of Nicotinamide, 5-20 nM of Gastrin, 20.2-2 mu M of Prostaglandin E and 15 mu M of Y-276325; the conditioned medium comprises two or three protein conditioned media, wherein the protein conditioned media is selected from the group consisting of Wnt-3a conditioned medium, R-spondin1 conditioned medium, and Noggin conditioned medium; the autologous condition culture medium is obtained by culturing autologous tumor fibroblasts extracted from lung cancer tissues by using a tumor-associated fibroblast culture medium; and the self-sourced conditioned medium at least comprises self-sourced growth factors, self-sourced interleukin factors, self-sourced chemokines, self-sourced matrix metalloproteases and self-sourced other factors; the self-derived other factors include SDF1, VEGFA, TGF β, PDGF α and PGE 2.

In some embodiments, the lung cancer organoid culture reagent combination comprises: enzymolysis liquid and the lung cancer organoid culture solution; the enzymolysis liquid comprises a basic culture medium, collagenase type I, collagenase type III and Primocin; wherein the concentration of the collagenase type I is 0.1-2 mg/mL, the concentration of the collagenase type III is 0.1-1 mg/mL, and the concentration of Primocin is 0.2-2 mg/mL; and independently packaging the lung cancer organoid culture solution and the enzymolysis solution.

In some embodiments, the method of culturing a lung cancer organoid comprises: carrying out physical pretreatment on the lung cancer sample to obtain a sample tissue fragment; performing enzymolysis pretreatment on the sample tissue fragments, filtering to obtain filtrate, and centrifuging the filtrate to obtain cell precipitates; resuspending the cell pellet with matrigel to obtain gel mixed with cells; then inoculating the gel into a culture hole, and performing static culture in a cell culture box at 37 ℃ to solidify the gel; adding the lung cancer organoid culture solution into the culture hole for culture to obtain primary lung cancer organoids; and (3) subculturing the primary lung cancer organoid, and culturing by using the lung cancer organoid culture solution in the subculturing process, wherein the culture period of each subculturing is 5-7 days, so as to obtain the corresponding generation of lung cancer organoids.

The lung cancer organoid culture solution, the culture reagent combination and the culture method provided by the embodiment of the application can realize the following technical effects:

the lung cancer organoid culture solution provided by the embodiment of the application comprises a complete culture medium and a self-source condition culture medium, wherein multiple secretion factors in the self-source condition culture medium are mutually synergistic, the secretion factors are mutually synergistic with a condition culture medium, a compound antibiotic and various growth factors in the complete culture medium, and the type and concentration of the compound antibiotic are controlled, so that the cell activity can be kept while pollution is prevented; the formation and growth of lung cancer organoid cells are accelerated, the culture speed is increased, and the subculture process can be carried out for 5-7 days to meet the requirements of subculture; meanwhile, the addition of the self-source condition culture medium enables the lung cancer organoid cells obtained by culture to be more bionic, can maintain the in-vivo tumor characteristics in vitro, cannot generate EMT conversion, and keeps the in-vivo interstitial characteristics. Moreover, the expression of the characteristic protein marker of the lung cancer organoid obtained by multiple passages is consistent with that of the tumor tissue.

The lung cancer organ culture solution provided by the embodiment of the application can be suitable for culturing human lung cancer organoids, is particularly suitable for personalized culture of the lung cancer organoids, and can maintain the body structure and pathological characteristics of primary tissues of each patient.

In the lung cancer organoid culture reagent provided by the embodiment of the application, enzymolysis liquid is subjected to enzymolysis by using collagenase type I and collagenase type III, so that the enzymolysis time can be shortened, the activity of the obtained cells is high, and the pollution of a sample can be effectively reduced; then, the lung cancer organoid culture solution provided by the embodiment of the application is used for culturing lung cancer tumor samples, so that the growth of lung cancer organoid cells can be accelerated, and the culture speed is increased.

The lung cancer organoid culture method provided by the embodiment of the application is simple to operate, the formation and growth time of primary organoids can be effectively shortened by adopting the lung cancer organoid culture solution provided by the embodiment of the application, and the culture time of each generation can be shortened to 5-7 days during subculture. In the process of subculture, the diameter of the organoid can reach 80-110 mu m after 5-7 days of culture, and the subculture period is stably shortened. Moreover, in the process of primary extraction and culture by adopting the lung cancer organoid culture reagent, a large amount of lung cancer organoids can be formed after the lung cancer organoid is cultured for 3 days. Meanwhile, the obtained lung cancer organoid is more bionic and can maintain the in-vivo tumor characteristics in vitro.

The foregoing general description and the following description are exemplary and explanatory only and are not restrictive of the application.

Drawings

One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the accompanying drawings and not in limitation thereof, in which elements having the same reference numeral designations are shown as like elements and not in limitation thereof, and wherein:

FIGS. 1-a to 1-c are respectively a photograph of a autologous tumor fibroblast obtained during the preparation of an autologous conditioned medium according to the examples of the present application;

FIG. 2 is a graph of the number of days cultured in a self-conditioned medium versus cell viability in accordance with an embodiment of the present invention;

FIG. 3 is a histogram of HGF content in a self-conditioned medium of an embodiment of the present application;

FIG. 4 is a bar graph of IL-6 content in a self-conditioned medium according to an embodiment of the present application;

FIG. 5 is a photomicrograph of lung cancer organoids after 7 days of primary culture of a method of lung cancer organoid culture in an embodiment of the present application;

FIG. 6 is a photomicrograph of lung cancer organoids after 7 days of primary culture according to another method of lung cancer organoid culture according to an embodiment of the present application;

FIG. 7 is a graph of trypan blue staining of cells pretreated with enzymatic hydrolysis in another lung cancer organoid culture method according to the present application;

FIG. 8 is a bar graph of the luminescence intensity of ATP content of lung cancer organoids at 7 days of subculture for another lung cancer organoid culture method of the present application;

FIG. 9 is a photograph of a lung cancer organoid at 7 days of subculture according to another lung cancer organoid culture method of the present embodiment;

FIG. 10 is a photoscope image of lung cancer organoids after thawing and conventional culture for 24h after cryopreservation by the cryopreservation method of the embodiments of the present application;

FIG. 11 is a photograph of a lung cancer organoid at 7 days of subculture of the lung cancer organoid culture method of comparative example 1;

FIG. 12 is a histogram of the relative expression of E-cadherin, an epithelial marker of lung cancer organoids according to the example of the present application;

FIG. 13 is a histogram of the relative expression of the mesenchymal marker protein Vimentin of lung cancer organoids according to an embodiment of the present application;

FIG. 14 is a graph of HE staining of lung cancer organoids obtained by the lung cancer organoid culture method of the present application;

FIG. 15 is a graph of HE staining results of lung cancer organoid homologous tissue according to an embodiment of the present application;

in the above drawings, scales of the other drawings except for FIGS. 14 and 15 are all 200 μm.

Detailed Description

So that the manner in which the above recited features and aspects of the present invention can be understood in detail, a more particular description of the embodiments of the invention, briefly summarized above, may be had by reference to the appended drawings, which are included to illustrate, but are not intended to limit the embodiments of the invention. In the following description of the technology, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, one or more embodiments may be practiced without these details. In other instances, well-known structures and devices may be shown in simplified form in order to simplify the drawing.

The terms "first," "second," and the like in the description and in the claims of the embodiments of the application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the present application are described herein. Furthermore, the terms "comprising" and "having," as well as any variations thereof, are intended to cover non-exclusive inclusions.

In the embodiments of the present application, the terms "upper", "lower", "inner", "middle", "outer", "front", "rear", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the embodiments of the present application and embodiments thereof, and are not used to limit the indicated devices, elements or components to a particular orientation or to be constructed and operated in a particular orientation. Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meanings of these terms in the embodiments of the present application can be understood by those skilled in the art as appropriate.

In addition, the terms "disposed," "connected," and "secured" are to be construed broadly. For example, "connected" may be a fixed connection, a detachable connection, or a unitary construction; can be a mechanical connection, or an electrical connection; may be directly connected, or indirectly connected through intervening media, or may be in internal communication between two devices, elements or components. Specific meanings of the above terms in the embodiments of the present application can be understood by those of ordinary skill in the art according to specific situations.

The term "plurality" means two or more unless otherwise specified.

It should be noted that, in the present application, the embodiments and the features of the embodiments may be combined with each other without conflict.

The application embodiment provides a lung cancer organoid culture solution, including complete culture medium and from source condition culture medium, the volume ratio of complete culture medium and from source condition culture medium is 1 ~ 3: 1.

Wherein the complete culture medium comprises a conditioned medium, a basal medium, a complex antibiotic and a growth factor; the volume ratio of the conditioned medium to the basic medium is 1-4: 1, and the basic medium comprises Advanced DMEM/F12, HEPES 5-15 mM and Glutamax 0.5 x-1.5 x; the compound antibiotics comprise 50-500 mu g/mL of Primocin, 0.5-1.5 x of penicillin-streptomycin and 2-10 mu g/mL of metronidazole; the growth factors include: 10-100 ng/mL of EGF, 1010-100 ng/mL of FGF-20.25x-1.5 x, 270.25x-1.5 x of B270, 1-2 mM of n-acetyl cysteine, 5-20 mM of Nicotinamide, 5-20 nM of Gastrin, 20.2-2 mu M of Prostaglandin E and 15 mu M of Y-276325; the conditioned medium comprises two or three protein conditioned media, wherein the protein conditioned media is selected from the group consisting of Wnt-3a conditioned medium, R-spondin1 conditioned medium, and Noggin conditioned medium.

The autologous condition culture medium is obtained by culturing autologous tumor fibroblasts extracted from lung cancer tissues by using a tumor-associated fibroblast culture medium; and the self-sourced conditioned medium at least comprises self-sourced growth factors, self-sourced interleukin factors, self-sourced chemokines, self-sourced matrix metalloproteases and self-sourced other factors; the self-derived other factors include SDF1, VEGFA, TGF β, PDGF α and PGE 2.

Lung cancer organoid culture fluids of the present embodiments include complete media and autologous conditioned media, wherein the autologous conditioned media includes at least endogenous growth factors (e.g., HGF, CTGF, and IGF), endogenous interleukin factors (e.g., IL-6, IL-8, and IL-11), endogenous chemokines (e.g., CCL2, CCL5, CXCL9, and CXCL 10), endogenous matrix metalloproteinases (e.g., MMP1 and MMP 9), and secreted factors such as SDF1, VEGFA, TGF β, PDGF α, and PGE 2) that act synergistically with each other, e.g., matrix Derived Factor-1 (stratum Derived Factor-1, SDF 1) stimulates cancer cell proliferation and can increase tumor angiogenesis by recruiting endothelial precursor cells; HGF in the self-derived growth factor can promote the proliferation, migration and invasion of tumor cells through an HGF/c-Met/STAT3/Twist1 pathway; IL-6 in the self-derived interleukin factor can generate the same effects of promoting the proliferation, migration and invasion of tumor cells through an IL-6/IL-6R/JAK2/STAT3/Twist1 pathway, IL-6 can also up-regulate the expression of c-Met, and further has synergistic effect with HGF in the aspect of enhancing the characteristics of CAF, and the like; meanwhile, the secretion factors and the conditioned medium, the compound antibiotics and the growth factors in the complete culture medium are mutually cooperated, and the type and the concentration of the compound antibiotics are controlled, so that the cell activity can be kept while the pollution is prevented; the formation and growth of lung cancer organoid cells are accelerated, the cell harvest quantity is increased, the culture speed is improved, and the subculture requirement can be met after the cells are cultured for 5-7 days in the subculture process; meanwhile, the addition of the self-source condition culture medium enables the lung cancer organoid cells obtained by culture to be more bionic, can maintain the in-vivo tumor characteristics in vitro, cannot generate EMT conversion, and keeps the in-vivo interstitial characteristics. Moreover, the expression of the characteristic protein marker of the lung cancer organoid obtained by multiple passages is consistent with that of the tumor tissue. The culture solution provided by the embodiment of the application can be suitable for culturing human lung cancer organoids, is particularly suitable for personalized culture of lung cancer organoids, and can maintain the body structure and pathological characteristics of primary tissues of each patient.

In the lung cancer organoid culture solution of the embodiment of the application, each protein conditioned medium in the complete culture medium is obtained by self-preparation, and the self-source conditioned medium can also be obtained by self-preparation in a self-culture mode, so that the cost is greatly reduced. The other components are all commercial products.

In the embodiment of the application, the conditioned medium and the basal medium are mixed according to the volume proportion to obtain the mixed medium, the compound antibiotic and the growth factor are added into the mixed medium and are uniformly mixed to obtain the complete medium, and then the complete medium and the self-source conditioned medium are uniformly mixed according to the volume proportion to obtain the lung cancer organoid culture solution.

In complete media, the concentrations of HEPES and Glutamax in the basal medium are those within the basal medium. Optionally, the basal medium comprises Advanced DMEM/F12, HEPES 8-12 mM and Glutamax 0.75-1.25X. Alternatively, the basal medium comprises Advanced DMEM/F12, HEPES 10 mM and Glutamax 1X.

Optionally, the volume ratio of the conditioned medium to the basal medium is 2-3: 1. Optionally, the volume ratio of conditioned medium to basal medium is 1: 1 or 7: 3 (2.3: 1).

Optionally, the growth factor comprises: 40-80 ng/mL of EGF, 1040-80 ng/mL of FGF-1040, 20.75x-1.25 x of N20.75x, 270.75x-1.25 x of B270, 1.25-1.8 mM of n-acetyl cysteine, 8-15 mM of Nicotinamide, 8-15 nM of Gastrin, 20.5-1.5 mu M of Prostaglandin E and 12 mu M of Y-276328.

Optionally, the growth factor comprises: EGF 50 ng/mL, FGF-1050 ng/mL, N20.75x-1 x, B270.75x-1 x, n-acetyl cysteine 1.25-1.5 mM, Nicotinamide 8-10 mM, Gastrin 8-10 nM, Prostaglandin E20.5-1 μ M, Y-276328-10 μ M.

Optionally, the composite antibiotic comprises Primocin 50-150 μ g/mL, penicillin-streptomycin 0.6-1.4 x and metronidazole 2-6 μ g/mL.

Optionally, the composite antibiotic comprises Primocin 75-125 μ g/mL, penicillin-streptomycin 0.75 x-1.25 x and metronidazole 3-5 μ g/mL.

Alternatively, the antibiotic combinations include Primocin 100. mu.g/mL, penicillin-streptomycin 1X and metronidazole 4. mu.g/mL.

In some embodiments, the conditioned media comprises Wnt-3a conditioned media, R-spondin1 conditioned media, and Noggin conditioned media. The three protein conditioned media can be mixed in any proportion.

Optionally, the volume ratio of the Wnt-3a conditioned medium to the R-spondin1 conditioned medium to the Noggin conditioned medium is 0.1-0.8: 1-2: 1 in sequence.

Optionally, the volume ratio of the Wnt-3a conditioned medium to the R-spondin1 conditioned medium to the Noggin conditioned medium is 0.3 to 0.6 to 1.2 to 1.8 to 1.

Alternatively, the volume ratio of the Wnt-3a conditioned medium, the R-spondin1 conditioned medium and the Noggin conditioned medium was 0.5: 1.5: 1.

Optionally, in the lung cancer organoid culture solution, the conditioned medium is 70% and the basal medium is 30% by volume percentage; wherein the conditioned medium comprises 20 percent of Wnt-3a conditioned medium, 50 percent of R-spondin1 conditioned medium and 30 percent of Noggin conditioned medium by volume percentage.

In some embodiments, the conditioned media include R-spondin1 conditioned media and Noggin conditioned media. The two protein culture conditions can be mixed in any proportion.

Optionally, the volume ratio of R-spondin1 conditioned medium to Noggin conditioned medium is 1: 2: 1.

Optionally, the volume ratio of the R-spondin1 conditioned medium to the Noggin conditioned medium is 1.2-1.8: 1.

Alternatively, the volume ratio of R-spondin1 conditioned medium to Noggin conditioned medium was 1.5: 1.

Optionally, in the lung cancer organoid culture solution, the volume percentage of the conditioned medium is 50% and the volume percentage of the basal medium is 50%; wherein the conditioned medium comprises 60 percent of R-spondin1 conditioned medium and 40 percent of Noggin conditioned medium by volume percentage.

In the examples of the present application, the protein conditioned medium was obtained by the following method:

and S110, sequentially carrying out primary culture, antibiotic screening culture and secondary culture on the cell line producing the target protein, and then entering an incubation culture stage.

In step S110, the cell line to be used is determined according to the type of the target protein, and the cell line is defined as the target cell line in this embodiment for subsequent reference. The target cell line is a target protein overexpression cell strain, and a target gene can be transferred into a CHO cell line in a lentivirus transfection mode to realize overexpression of a specific protein. For example, the target protein is Wnt-3a, and the Wnt-3a overexpression cell strain is used for transferring a Wnt-3a target gene into a CHO cell line by a lentivirus transfection mode to realize overexpression of the specific protein.

Optionally, the primary culturing comprises: the cell line of interest is recovered and cultured using the first complete medium. The first complete medium was prepared using Advanced DMEM/F12+10% FBS (fetal bovine serum) +1% P/S (diabodies (penicillin plus streptomycin)).

Optionally, the antibiotic selection culture comprises: after primary culture is carried out until the confluence degree of cells reaches 90%, passage is carried out to a culture dish, and then primary screening culture is carried out for 2-3 days by using secondary complete culture; after 90% confluence, the cells were passaged to a new dish and then subjected to secondary screening culture for 2-3 days using a second complete culture. Wherein the second complete culture medium is prepared by adding Puromycin (Puromycin) to the first complete culture medium and controlling the concentration to be 5 mug/mL.

Optionally, the secondary culturing comprises: after the secondary screening culture is carried out until the cell confluency reaches 90%, the cells are digested and passaged to a ventilating culture bottle by pancreatin, and the culture is continued by adopting a first complete culture medium (the same as the primary culture), and the culture solution is changed once within 2 days or 3 days. Here, the gas-permeable flask is a T175 gas-permeable flask, and the total culture medium is controlled to be 30 mL per gas-permeable flask, but it may be other volume amounts, and is not limited thereto.

Optionally, the incubating comprises: after secondary culture until the cell confluency reaches 95% -100%, removing the culture medium, washing with PBS, adding a first complete culture medium newly prepared into each bottle, and incubating and culturing. During PBS washing, the volume of PBS is optionally determined, e.g., 10 mL.

S120, in the incubation and culture stage, collecting the culture medium every 24 hours of incubation, centrifuging the culture medium at 4 ℃, transferring the supernatant to a sterile bottle, storing at 4 ℃, and correspondingly obtaining the first conditioned medium to the twelfth conditioned medium according to the collection time sequence; wherein the first to fourth conditioned media are mixed as a first batch of conditioned media; mixing the fifth to eighth conditioned media as a second batch of conditioned media; mixing the ninth-time conditioned medium to the twelfth-time conditioned medium to serve as a third batch of conditioned medium; mixing the first batch of conditioned medium, the second batch of conditioned medium and the third batch of conditioned medium to obtain the protein conditioned medium.

Here, the protein content in the conditioned medium of each batch was also measured, and the protein content was mixed for use after passing the measurement. Meanwhile, the conditioned medium of each batch can be frozen and stored respectively after being filtered, and then thawed and mixed when in use.

In this step S120, optionally, the centrifugation of the medium at 4 ℃ under the conditions comprises: the centrifugal force was 2000 g and the centrifugation time was 5 min.

Alternatively, a 0.22 μm vacuum filter is used for each batch of conditioned media.

Alternatively, the cryopreservation conditions are 80 ℃ at zero (-80 ℃).

Optionally, the first, second and third batches of conditioned medium are mixed in equal volume ratios.

In some embodiments, the autologous conditioned media, the autologous growth factors include HGF, CTGF, and IGF, and the autologous interleukin factors include IL-6, IL-8, and IL-11; autologous chemokines include CCL2, CCL5, CXCL9, and CXCL 10; derived from matrix metalloproteinases such as MMP1 and MMP 9.

In some embodiments, the self-conditioned medium is obtained by culturing:

s210, extracting the autologous tumor-related fibroblasts, carrying out passage and establishing a system to obtain the autologous tumor fibroblasts; here, step S210 specifically includes the following steps: s221, performing physical pretreatment on the lung cancer sample to obtain a sample tissue fragment; s222, performing enzymolysis pretreatment on the sample tissue fragments, and filtering to obtain residual tissue residues after filtering; s223, coating the tissue residues obtained in the step S222 on the bottom of a T25 bottle, allowing an incubator to adhere to the wall for 1-3 hours, and then adding a culture medium (for example, DMEM basic culture medium +5% serum +3% PS) for culture; s224, when the tissue is cultured to the 4 th day, observing whether the tissue block has fiber cells to climb out, and carrying out first liquid change; then, the liquid is changed every 3 days and observed, and after a sufficient amount of fibroblasts are grown, the fibroblasts are harvested and obtained from the original tumor fibroblasts. The CAF (tumor associated fibroblast) with better growth vigor can grow over the bottom of the bottle in about 14 days, and can grow enough fiber cells in about 20 days. Then, the cells are subcultured, expanded and preserved according to the growth condition of the cells.

S220, culturing the autologous tumor fibroblasts by adopting a tumor-associated fibroblast culture medium; wherein the tumor-associated fibroblast cell culture medium comprises: DMEM basal medium +5% serum +3% PS (diabody (penicillin plus streptomycin)). The percentages herein are volume percentages.

Specifically, autologous tumor fibroblasts are seeded in 3D (3 dimensional) and then cultured by adding a tumor-associated fibroblast culture medium.

And S230, after culturing for 3 to 5 days, collecting the culture medium, and centrifuging the collected culture medium to obtain the self-source conditioned culture medium. In this example, the collected medium was centrifuged to remove serum fiber and dead cell debris, and the resulting centrifugate was the autologous conditioned medium.

In this embodiment, the autologous tumor fibroblasts obtained in step S210 may be subjected to subculture, and frozen for seed preservation, in addition to the subsequent preparation of the autologous conditioned medium.

Optionally, the step of subculturing with autologous tumor fibroblasts and cryopreserving the seeds specifically comprises:

s240, subculturing autologous tumor fibroblasts, and performing 1: passage 2. The confluence degree of the cells before passage should reach 80%, and the passage ratio and time can be properly adjusted for the fiber cells with slower or faster growth.

S250, starting to preserve after the third subculture (including or not including the culture of P3), and freezing and storing 1 cell per 0.5X 10cm dish.

The embodiment of the application also provides a lung cancer organoid culture reagent combination, which comprises enzymolysis liquid and the lung cancer organoid culture liquid of any one of the embodiments. Wherein, the enzymolysis liquid comprises a basic culture medium, collagenase type I, collagenase type III and Primocin; wherein the concentration of the collagenase I is 0.1-2 mg/mL, the concentration of the collagenase III is 0.1-1 mg/mL, and the concentration of Primocin is 0.2-2 mg/mL; and independently packaging the lung cancer organoid culture solution and the enzymolysis solution.

The lung cancer organoid culture reagent combination is a kit reagent, enzymolysis is carried out by using enzymolysis liquid, the enzymolysis time can be shortened, the activity of the obtained cells is high, and the pollution to a sample can be effectively reduced; and the lung cancer organoid culture solution is used for culturing a lung cancer tumor sample, so that the growth of lung cancer organoid cells can be accelerated, and the culture speed is increased.

In some embodiments, the basic medium in the enzymatic hydrolysate is a basic medium consistent with lung cancer organoid culture, Advanced DMEM/F12.

In some embodiments, the enzymatic hydrolysate comprises basal medium, collagenase type i, collagenase type iii, and Primocin; wherein, the concentration of the collagenase I is 1-1.5 mg/mL, the concentration of the collagenase III is 0.3-0.7 mg/mL, and the concentration of Primocin is 1-2 mg/mL.

Optionally, an enzymatic hydrolysate comprising basal medium, collagenase type i, collagenase type iii and Primocin; wherein, the concentration of collagenase type I is 1.2 mg/mL, the concentration of collagenase type III is 0.5mg/mL, and the concentration of Primocin is 1.5 mg/mL.

In the culture reagent combination of the embodiment of the application, the ratio of the enzymolysis liquid to the lung cancer organoid culture liquid is not limited and is determined according to the actual requirement of the culture process.

The embodiment of the application also provides a method for culturing the lung cancer organoid, which comprises the following steps:

and S310, carrying out physical pretreatment on the lung cancer sample to obtain a sample tissue fragment.

In step S310, the physical pretreatment includes physical treatment methods such as cleaning and crushing. Optionally, physical pretreatment, including step S311, the lung cancer tumor sample is washed with PBS buffer containing 5% double antibody (penicillin-streptomycin) and 50 ng/mL gentamicin, the sample tissue is transferred to a culture dish, and then minced to obtain a sample tissue fragment. The cleaning frequency is not limited, and the aim of full cleaning can be achieved.

S320, performing enzymolysis pretreatment on the sample tissue fragments obtained in the step S310 to obtain filtered solution; and centrifuging the filtrate to obtain cell precipitate.

In step S320, the enzymolysis pretreatment may adopt conventional enzymolysis solution and enzymolysis operation. Specifically, the enzymolysis pretreatment comprises the following steps: adding the enzymolysis liquid into the sample tissue fragments, incubating on a shaking table at 37 ℃ for a preset incubation time, adding an enzymolysis stopping agent, stopping enzymolysis, and filtering to obtain a filtrate. And (4) completing enzymolysis pretreatment. Wherein, the enzymolysis stopping agent can adopt Advanced DMEM/F12 with the temperature of 4 ℃, and the adding amount is determined according to the adding amount of the enzymolysis liquid.

Optionally, the permeate centrifugation conditions comprise: the centrifugal force was 300 g and the centrifugation time was 5 min.

S330, adopting matrigel to resuspend the cell sediment obtained in the step S320 to obtain gel mixed with cells; then, the gel was seeded into a culture well and cultured in a cell incubator at 37 ℃ in a static manner, so that the gel was solidified.

Alternatively, the gel is seeded in a well plate at an inoculum size of 50 μ L per well. The number of wells of the well plate is not limited, for example, a 24-well plate.

Optionally, the standing culture time is 15-40 min. Optionally, the standing culture time is 15min to 30 min. Alternatively, the incubation time is 30 min.

In step S330, the standard for gel coagulation is that the gel of the vertically placed culture plate cannot flow freely.

S340, adding the lung cancer organoid culture solution of any one of the previous embodiments into the culture hole for culture to obtain primary lung cancer organoids.

And S350, subculturing the primary lung cancer organoids obtained in the step S340, and culturing by using the lung cancer organoid culture solution of any one of the embodiments in the subculturing process, wherein the culture period of each subculturing is 5-7 days, so as to obtain corresponding lung cancer organoids.

The culture method is simple to operate, the lung cancer organoid culture solution can effectively shorten the formation and growth time of primary organoids, and the culture time of each generation can be shortened to 7 days during subculture. In the process of subculture, the diameter of the organoid can reach 80-110 mu m after 5-7 days of culture, and the subculture period is stably shortened. Moreover, the obtained lung cancer organoids are more biomimetic and can maintain the in vivo tumor characteristics in vitro.

In some embodiments, step S320, the enzymatic pretreatment comprises the following steps:

and S321, adding the enzymolysis liquid in the lung cancer organoid culture reagent combination into the sample tissue fragment obtained in the step S310, and incubating for 15-25 min under the conditions of shaking table at 37 ℃ and 200 r/min.

In step S321, the amount of the enzymatic hydrolysate added is related to the size of the sample, and optionally, 5 to 15 mL of the enzymatic hydrolysate is added to every 1g of the sample.

Alternatively, when the weight of the sample tissue fragment is 0.5g, the amount of the enzymatic hydrolysate added is 5 mL. The enzymolysis incubation time can be controlled by controlling the addition amount of the enzymolysis liquid.

S322, adding an enzymolysis stopping agent, stopping enzymolysis, and filtering to obtain filtrate. Wherein, the enzymolysis stopping agent can adopt Advanced DMEM/F12 with the temperature of 4 ℃, and the adding amount is determined according to the adding amount of the enzymolysis liquid.

Optionally, the ratio of the addition volume of the enzymolysis stopping agent to the addition volume of the enzymolysis liquid is 1.5-3: 1. Optionally, the ratio is 2: 1.

Alternatively, in step S321, the amount of the enzymolysis solution is 5mL, and in step S322, the amount of the enzymolysis stopping agent is 10 mL.

Optionally, after the enzymatic hydrolysis terminator is added, the enzymatic hydrolysis is terminated by blowing with a pipette.

Optionally, the filtration is performed using a 100 μm cell sieve.

In the embodiment, after enzymolysis is carried out on the enzymolysis liquid in the lung cancer organoid culture reagent combination, the enzymolysis time can be shortened, the activity of the obtained cells is high, and the pollution of a sample can be effectively reduced; then, the lung cancer organoid culture solution provided by the embodiment of the application is used for culturing lung cancer tumor samples, so that the growth of lung cancer organoid cells can be accelerated, and the culture speed is increased. In the primary extraction culture process, a large number of lung cancer organoids can be formed after 3 days of culture; the diameter of the organoid can reach 80-110 mu m after 5-7 days of culture, and the organoid can be subcultured.

In some embodiments, a method of culturing a lung cancer organoid, further comprises:

s350, embedding the lung cancer organoid obtained in the step S340 by using matrigel, blowing the lung cancer organoid by adopting an Advanced DMEM/F12 culture medium, and centrifuging to obtain a mixed precipitate of the matrigel and the cells.

In step S350, the conditions of the centrifugal processing include: the centrifugal force was 300 g and the centrifugation time was 5 min.

S360, resuspending the mixed precipitate obtained in the step S350 to obtain a resuspension solution; placing the heavy suspension in water bath at 37 ℃ for 8-10 min; then, to the re-suspension was added two volumes of 4 ℃ Advanced DMEM/F12 medium to stop the digestion, and the mixture was centrifuged to obtain a secondary cell pellet.

In this step S360, the resuspension was performed using TrypLE resuspension. The conditions of the centrifugation treatment include: the centrifugal force was 300 g and the centrifugation time was 5 min.

S370, resuspending the secondary cell sediment by adopting a cryopreservation solution, and then performing cryopreservation by using a programmed gradient cooling box; after one day, the cells were transferred to liquid nitrogen for long-term storage. The re-suspension ratio of the secondary cell pellet to the frozen stock solution is 1X 10⁵～5×10⁶The individual cell pellet was resuspended in1 mL of the frozen stock solution. The frozen stock solution adopts a commercial product.

After the organoids subjected to liquid nitrogen cryopreservation are treated in the steps S350 to S370 for 12 months, the organoids can still stably grow, the activity and dryness of the organoids are kept, and the growth state of the organoids after recovery is good.

Specific examples of the embodiments of the present application are given below to explain the effects of the embodiments of the present application.

EXAMPLE 1 protein conditioned Medium

The protein conditioned medium is prepared by the following method:

s101, recovering the target cell line, and culturing by using a first complete culture medium. The first complete medium was prepared using Advanced DMEM/F12+10% FBS (fetal bovine serum) +1% P/S (diabesin). The target cell line is a target protein overexpression cell strain, and a target gene is transferred into a CHO cell line in a lentivirus transfection mode to realize overexpression of a specific protein.

The target proteins include Wnt-3a, R-spondin1 and Noggin.

S102, after primary culture is carried out until the cell confluency reaches 90%, passage is carried out to a culture dish of 10cm, and then primary screening culture is carried out for 2-3 days by using a second complete culture medium; and (4) after the cell confluency reaches 90%, passaging to a new 10cm culture dish, and then performing secondary screening culture for 2-3 days by using a second complete culture. Wherein the second complete culture medium is prepared by adding Puromycin on the basis of the first complete culture medium and controlling the concentration of Puromycin to be 5 mu g/mL.

S103, after secondary screening and culture are carried out until the cell confluency reaches 90%, pancreatin digestion is carried out to transfer to a T175 air-permeable culture bottle, and 30 mL of total culture medium is controlled in each air-permeable culture bottle; adopting a first complete culture medium (synchronous step S101) to continue culturing until the cell confluency reaches 95% -100%, and changing the culture solution once in 2 days or 3 days; then the medium was removed, washed once with 10ml PBS shaking, and the first complete medium freshly prepared was added to each flask for incubation.

S104, in the incubation and culture stage, collecting the culture medium once every 24 hours of incubation, centrifuging the culture medium at 4 ℃ (the centrifugal force is 2000 g, and the centrifugation time is 5 min), transferring the supernatant to a 500mL sterile bottle, storing the sterile bottle at 4 ℃, and correspondingly obtaining the first conditioned medium to the twelfth conditioned medium according to the collection time sequence; wherein the first to fourth conditioned media are mixed as a first batch of conditioned media; mixing the fifth to eighth conditioned media as a second batch of conditioned media; the ninth to twelfth conditioned media were mixed as the third batch of conditioned media. And each batch of conditioned media was applied to a 0.22 μm vacuum filter. The freezing condition is 80 ℃ below zero.

S105, when in use, the first batch of frozen conditioned medium, the second batch of frozen conditioned medium and the third batch of frozen conditioned medium are thawed and mixed in equal volume ratio to serve as the target protein conditioned medium.

Namely, the target cell lines of the Wnt-3a, R-spondin1 and Noggin over-expression cell strains are respectively cultured to obtain corresponding Wnt-3a protein conditioned medium, R-spondin1 protein conditioned medium and Noggin protein conditioned medium.

When in use, the three frozen target protein conditioned media are thawed and then used.

The preparation method of the protein conditioned medium of the embodiment 1 has low cost.

Example 2 self-sourced Condition Medium

The self-source condition culture medium is prepared by the following steps:

s201, performing physical pretreatment on the lung cancer sample to obtain a sample tissue fragment;

s202, carrying out enzymolysis pretreatment on the sample tissue fragments, and filtering to obtain residual tissue residues after filtering;

s203, coating the tissue residues obtained in the step S202 on the bottom of a T25 bottle, allowing an incubator to adhere to the wall for 1-3 hours, and adding a culture medium (for example, DMEM basal medium +5% serum +3% PS) for culture;

s204, culturing until the 4 th day, observing whether the tissue block has fiber cells to climb out, and performing first liquid change; then, the liquid is changed every 3 days and observed, and after a sufficient amount of fibroblasts are grown, the fibroblasts are harvested and obtained from the original tumor fibroblasts. The CAF (tumor associated fibroblast) with better growth vigor can grow over the bottom of the bottle in about 14 days, and can grow enough fiber cells in about 20 days. And then, carrying out passage and expanding culture and conservation according to the growth condition of the cells.

S205, 3D (3 dimensional) planting of the autologous tumor fibroblasts, and then adding a tumor-associated fibroblast culture medium for culture. Wherein the tumor-associated fibroblast cell culture medium comprises: DMEM basal medium +5% serum +3% PS (diabody (penicillin plus streptomycin)). The percentages herein are volume percentages.

S206, respectively culturing for 1 day, 3 days and 5 days, collecting culture mediums, and respectively centrifuging the collected culture mediums to respectively obtain a self-source condition culture medium I, a self-source condition culture medium II and a self-source condition culture medium III. In step S206, the collected culture medium is centrifuged to remove serum fiber and dead cell debris, and the obtained centrifugate is the autologous conditioned culture medium.

In this example, in step S206, the fibroblasts cultured for 1 day, 3 days and 5 days were examined, and the bright field micrographs of the fibroblasts correspond to those shown in FIG. 1-a, FIG. 1-b and FIG. 1-c, respectively, and it was found by comparative analysis that the growth of the fibroblasts reached saturation at3 days of culture and supersaturation at 5 days of culture. Wherein the scale bars in the figures are all 200 μm.

Meanwhile, the fiber cells cultured for 1 day, 3 days and 5 days, respectively, were subjected to cell viability assay, as shown in the cell viability graph of fig. 2. As is clear from FIG. 2, the cells proliferated stably and were in good condition in the first 3 days of culture, and they grew stably from day 3 to day 5.

In example 2, HGF content was measured in autologous conditioned media II and III obtained by culturing for 3 days and 5 days, respectively, to obtain a histogram of HGF content as shown in FIG. 3. As can be seen from FIG. 3, the difference between the HGF contents in culture media II and III was small, and the HGF content in culture medium III was slightly lower than that in culture medium II.

In example 2, IL-6 content was measured in autologous conditioned medium III obtained by 5 days of culture, and a histogram of IL-6 content was obtained as shown in FIG. 4. It can be seen that the conditioned media obtained from this example 1 contains a certain amount of IL-6.

The autologous conditioned medium prepared in this example 2 contains at least autologous growth factors, such as HGF, CTGF and IGF; self-derived interleukin factors, e.g., IL-6, IL-8, and IL-11; autologous chemokines, e.g., CCL2, CCL5, CXCL9, and CXCL 10; self-derived matrix metalloproteinases, e.g., MMP1 and MMP 9; and from other factors, e.g., SDF1, VEGFA, TGF β, PDGF α, and PGE 2.

Example 3 Lung cancer organoid culture fluid

A lung cancer organoid culture solution comprises a complete culture medium and an autologous condition culture medium, wherein the volume ratio of the complete culture medium to the autologous condition culture medium is 3: 1. As the conditioned media, the conditioned media II obtained in example 2 was used.

Complete culture medium includes conditioned medium, basic medium, compound antibiotic and growth factor. Wherein the volume ratio of the conditioned medium to the basal medium is 7: 3, and the conditioned medium comprises 20% of Wnt-3a conditioned medium, 50% of R-spondin1 conditioned medium and 30% of Noggin conditioned medium by volume percentage. Basal media included Advanced DMEM/F12, HEPES 10 mM and Glutamax 1X. Namely, 20% Wnt-3a conditioned medium, 30% R-spondin1 conditioned medium, 20% Noggin conditioned medium and 30% basal medium by volume. The compound antibiotics comprise 100 mu g/mL of Primocin, 1x of penicillin-streptomycin and 4 mu g/mL of metronidazole.

In example 3, the types and contents of growth factors are shown in Table 1 below, so that different complete media can be obtained.

TABLE 1

The lung cancer organoid culture solution of example 3 is prepared by first obtaining a complete culture medium according to the proportional use of each component, and then mixing the complete culture medium and the autologous culture medium according to the volume ratio. Except for the conditioned medium and the self-prepared and obtained self-source conditioned medium, the other components can be all commercial products.

The lung cancer organoid culture solution of the embodiment 3 is suitable for a human-derived lung cancer organoid culture solution.

In example 3, the conditioned media including three proteins were prepared by the method described in example 1.

In this example 3, the following culture media were obtained according to the conditions of the source medium used in example 2 and the above-mentioned complete medium I to complete medium V, respectively.

And mixing the complete culture medium I to the complete culture medium V with the autologous condition culture medium II according to the volume ratio of 3 to 1 to obtain culture solutions I-II to V-II respectively.

And mixing the complete culture medium I to the complete culture medium V with the autologous condition culture medium III according to the volume ratio of 3 to 1 to obtain culture solutions I-III to V-III respectively.

In this example 3, the concentrations of the respective components of the complex antibiotic in complete Medium II were varied on the basis of culture solutions II-II to obtain culture solutions having different antibiotic concentrations, as shown in Table 2 below. The other components and the content of each component are the same as those of the culture solution II-II.

TABLE 2

Example 4

The lung cancer organoid culture solution of this example 4, unlike example 3, was prepared by using a conditioned medium of complete medium II comprising R-spondin1 conditioned medium and Noggin conditioned medium; and the volume ratio of the R-spondin1 conditioned medium to the Noggin conditioned medium was 1.5: 1. Namely, R-spondin1 conditioned medium 30% and Noggin conditioned medium 20%, Advanced DMEM/F1250% by volume. The remainder of the Advanced DMEM/F12 and growth factors were identical.

Likewise, 5 lung cancer organoid cultures were provided, designated as culture broth ' I, culture broth ' II, culture broth ' III, culture broth ' IV and culture broth ' V, based on the same species and content of growth factors as in example 3.

In this example 4, R-spondin 1-conditioned medium and Noggin-conditioned medium were obtained by the preparation methods of step S101 to step S105 in example 1.

Example 5 Lung cancer organoid culture reagent combination

A lung cancer organoid culture reagent composition, comprising an enzymatic hydrolysate and the lung cancer organoid culture solution of any one of embodiment 3 or embodiment 4. Wherein, the enzymolysis liquid comprises a basic culture medium, I type collagenase, III type collagenase and Primocin; wherein the concentration of the collagenase type I is 0.1-2 mg/mL, the concentration of the collagenase type III is 0.1-1 mg/mL, and the concentration of Primocin is 0.2-2 mg/mL. And independently packaging the lung cancer organoid culture solution and the enzymolysis solution.

In this example 5, the basic medium in the enzymatic hydrolysate was Advanced DMEM/F12.

Five enzymatic hydrolysates shown in the following table 3 were obtained according to the difference in the concentrations of collagenase type i, collagenase type iii and Primocin, respectively.

TABLE 3

In example 5, the enzymatic hydrolysate was mixed in the amounts of the respective components.

Example 6

A method for culturing lung cancer organoids comprises the following steps:

s301, a lung cancer tumor sample (with the weight of 0.5 g) is washed by shaking with PBS (phosphate buffer solution) containing 5% double antibody (penicillin-streptomycin) for 5 times, then the sample tissue is transferred to a culture dish (for example, a 60mL culture dish), and then the sample tissue is cut into 2-4 mm tissue fragments by ophthalmic scissors, so as to obtain the sample tissue fragments. And the tissue fragments were transferred to centrifuge tubes.

S302, performing enzymolysis pretreatment on the sample tissue fragments obtained in the step S301, and filtering enzymolysis mixed liquor after enzymolysis to obtain filtered liquor; the filtrate was centrifuged (centrifugal force 300 g, centrifugation time 5 min) to obtain a cell pellet. The enzymolysis pretreatment comprises the following steps: adding 5mL of conventional enzymolysis solution into the sample tissue fragment, incubating for 50 min on a shaker (200 r/min) at 37 ℃, adding 10mL of enzymolysis stopping agent (Advanced DMEM/F12 at 4 ℃), fully blowing with a pipette, stopping enzymolysis, and filtering with a 100 μm cell sieve to obtain filtrate.

Wherein the conventional enzymolysis solution is Advanced DMEM/F12 containing 1.5mg/mL collagenase type IV, 20 mug/mL hyaluronidase and 1% double antibody (penicillin-streptomycin).

S303, adopting matrigel to resuspend the cell sediment obtained in the step S302 to obtain gel mixed with cells; the gel was then inoculated into culture wells of 24-well plates at an inoculum size of 50. mu.L per well, and allowed to stand for 30min to allow the gel to solidify.

S304, adding the lung cancer organoid culture solution of the embodiment 3 into the culture hole for culture to obtain primary lung cancer organoids. In step S304, lung cancer organoid formation is observed after 24h of culture. As shown in FIG. 5, FIG. 5 is a light microscopic image of lung cancer organoids after being subjected to primary culture for 7 days after being subjected to enzymolysis treatment with a conventional enzymolysis solution, and it can be seen that the diameter of the lung cancer organoids is less than 100 μm, and then, the primary culture can be performed for at least 14 days before subculture.

S305, subculturing the primary lung cancer organoid obtained in the step S304, and culturing the lung cancer organoid by using the lung cancer organoid culture solution of the embodiment 3 in the subculturing process, wherein the culture period of each subculturing is 5-7 days, so as to obtain the corresponding generation of lung cancer organoids.

In this embodiment 6, in step S302, the enzymolysis mixture after enzymolysis is filtered, and the obtained filtrate is subjected to subsequent operations. The tissue residue remaining after filtration can be obtained from the original tumor fibroblasts by performing the operations in steps S204 to S206 described in example 2. And subculturing partial autologous tumor fiber cells, and freezing and storing the seeds.

Specifically, the specific steps of freezing and preserving the autologous tumor fibroblasts are as follows:

s401, subculturing the autologous tumor fiber cells, and performing 1: passage 2. The confluence degree of the cells before passage should reach 80%, and the passage ratio and time can be properly adjusted for the fiber cells with slower or faster growth.

S402, starting to preserve the seeds after the third subculture, and freezing and storing 1 seed per 0.5X 10cm dish.

Example 7

The method for culturing a lung cancer organoid of embodiment 7, comprising:

s501, after a lung cancer tumor sample (with the weight of 0.5 g) is washed by shaking with 5% double antibody (penicillin-streptomycin) in PBS buffer for 5 times, the sample tissue is transferred to a culture dish (for example, a 60mL culture dish), and then is cut into 2-4 mm tissue fragments by ophthalmic scissors, so that the sample tissue fragments are obtained. And the tissue fragments were transferred to centrifuge tubes.

S502, performing enzymolysis pretreatment on the sample tissue fragments obtained in the step S501 to obtain filtered solution; the filtrate was centrifuged (centrifugal force 300 g, centrifugation time 5 min) to obtain a cell pellet. The enzymolysis pretreatment comprises the following steps: adding 5mL of enzymolysis liquid into the sample tissue fragments, incubating for 15-25 min on a shaker (200 r/min) at 37 ℃, adding 10mL of enzymolysis stopping agent (Advanced DMEM/F12 at 4 ℃), fully blowing by using a liquid transfer gun, stopping enzymolysis, and filtering by using a 100-micron cell sieve to obtain filtrate.

S503, adopting matrigel to resuspend the cell sediment obtained in the step S502 to obtain gel mixed with cells; the gel was then inoculated into culture wells of 24-well plates at an inoculum size of 50. mu.L per well, and allowed to stand for 30min to allow the gel to solidify.

S504, adding the lung cancer organoid culture solution in the embodiment 3 into the culture hole for culture to obtain primary lung cancer organoids; and after culturing for 7 days, subculturing can be carried out. In step S504, lung cancer organoids can be seen after 3 days of culture, the maximum diameter of the lung cancer organoids reaches more than 100 μm, and the primary culture can be performed for 7 days. In this embodiment, the lung cancer organoids after performing enzymolysis treatment on the enzymolysis solution iii in example 5 in step S502 and performing primary culture for 7 days specifically on the culture solutions ii to ii in example 3 in step S504 are detected, and a light microscope image as shown in fig. 6 is obtained. Therefore, the maximum diameter of the lung cancer organoid cultured for 7 days is more than 100 mu m, and the primary culture can be carried out for 7 days for passage.

And S505, subculturing the primary lung cancer organoids obtained by culturing for 7 days in the step S504, and culturing by using the lung cancer organoid culture solution of the embodiment 3 in the subculturing process, wherein the culture period of each subculturing is 5-7 days, so as to obtain the corresponding generation of lung cancer organoids.

In step S504 of this example 7, the lung cancer organoids after 7 days of culture can be passaged, and the passaging ratio can be 1: 1.5 to 1: 3. the method can stably carry out the subculture of the lung cancer organoid for a long time, and the subculture period is 5-7 days. As shown in fig. 6, which is a picture of lung cancer organoids (denoted as lung cancer organoids ii-iii) obtained by performing enzymolysis pretreatment with the enzymolysis solution iii of example 5 and culturing (primary culture) with the culture solution ii-ii of example 3 for 7 days, it can be seen that the number of organoids with a diameter of 100 μm or more is increased, the minimum diameter of organoids reaches 80 μm or more, and subculture can be performed; and the cell growth density is high.

In this example 7, in step S504, when the culture solution ii-ii, the culture solution iii-ii, and the culture solution iv-ii were subjected to primary culture for 3 days, the lung cancer organoids ii-ii, the lung cancer organoids iii-ii, and the lung cancer organoids iv-ii obtained respectively were subjected to cell mass and cell viability detection by subjecting the lung cancer organoids to enzymatic hydrolysis into single cells, staining with trypan blue, and counting the total cell mass and viable cell count. The results of the measurements are shown in Table 4 below.

TABLE 4

As is clear from Table 4, the lung cancer organoids obtained by culturing in the culture medium II-II had the best activity and the largest amount of cells were harvested.

In this example 7, in step S502, enzymolysis solutions i to v in example 5 are respectively used for enzymolysis pretreatment, and the incubation time corresponding to each enzymolysis solution is shown in table 5 below.

TABLE 5

It can be seen that the enzymolysis solution of example 5 can significantly shorten the degradation incubation time, which is more than half of the incubation time of the conventional enzymolysis solution in example 6.

Meanwhile, trypan blue staining is carried out on cells of the lung cancer sample after enzymolysis, and as shown in fig. 7, the trypan blue staining graph of the cells pretreated by enzymolysis of the enzymolysis liquid III in the embodiment 5 shows that the enzymolysis effect is good, and the cells have good activity.

In this example, in step S504, using culture solutions II-1, II-2, II-3 (i.e., culture solution II-II of example 3), II-4 and II-5 having different antibiotic concentrations in example 3, respectively, lung cancer organoids II-1 to II-5 were obtained, respectively, after culturing for 7 days, and ATP luminescence intensity tests were performed on 5 lung cancer organoids, and the histogram of the test results is shown in FIG. 8. As can be seen from FIG. 8, the total ATP value of the cells of lung cancer organoid II-3 obtained by culturing in culture medium II-3 is high, i.e., the number of cells is higher and the activity is better, and the results of the measurements of the cell amount and the cell viability shown in Table 4 are combined, and the results are found by the overall extraction success rate, so that the contamination can be prevented and the activity can be maintained.

In this embodiment 7, in step S502, the enzymolysis mixture after enzymolysis is filtered, and the obtained filtrate is subjected to subsequent operations. The tissue residue remaining after filtration can be obtained from the original tumor fibroblasts by the same procedure as that described in step S204 to step S206 of example 2. And subculturing partial self-derived tumor fiber cells, and freezing and storing the seeds. For a specific method for cryopreserving seeds, see steps S401 to S402 described in example 6.

Example 8

In this example 8, the primary lung cancer organoids obtained were subcultured based on example 6 or example 7. Specifically, the lung cancer organoid passage treatment comprises the following steps:

s601, using 1 mL of 4 ℃ Advanced DMEM/F12 culture medium to blow off the primary lung cancer organoid embedded by the matrigel in the example 6 or the example 7, recovering the organ in a centrifuge tube, and centrifuging (the centrifugal force is 300 g, and the centrifugal time is 5 min) to obtain a mixed sediment of the matrigel and the cells.

S602, resuspending the obtained precipitate by adopting TrypLE (Gibco) to obtain a resuspension solution; placing the heavy suspension in water bath at 37 ℃ for 8-10 min; digestion was then stopped by adding two volumes of 4 ℃ Advanced DMEM/F12 medium to the resuspension, and pipetting was repeated several times with a pipette gun. And (4) centrifuging (the centrifugal force is 300 g, and the centrifugation time is 5 min) to obtain a secondary cell precipitate.

Wherein, the using amount of TrypLE is based on the amount of the stroma gum embedded in the organoid, and theoretically, 1 mL of TrypLE is used for resuspension of the organoid embedded in every 100 mu L of stroma gum.

Wherein the repeated blowing-beating condition with a pipette after digestion is stopped is directly related to the digestion degree of the organoid. All organoids can be digested into cell clusters of uniform size, typically 2-10 cells, by pipetting 10-20 times with a 1 mL pipette tip and then 10-20 times with a 200. mu.L pipette tip with a 1 mL pipette tip cap.

S603, resuspending the secondary cell sediment by using Advanced DMEM/F12, blowing uniformly, and centrifuging (the centrifugal force is 300 g, and the centrifugal time is 5 min) to obtain a tertiary cell sediment. If the impurities are more, the step can be repeated.

S604, resuspending the cell sediment for three times by using matrigel to obtain gel mixed with cells; the gel was then inoculated into 24-well plates at an inoculum size of 50. mu.L per well and incubated in an incubator at 37 ℃ for 30min to allow the gel to solidify.

S605, the lung cancer organoid culture solution of example 3 was added to the coagulated organoid culture well and cultured. As shown in FIG. 9, it was found that organoids were uniform in size and grew well in a photomicrograph of the organoids after 7 days of culture. The growth diameter is near 100 mu M, and the proportion of large-diameter organoids in the visual field is higher than that of lung cancer organoids cultured for 14 days in the comparative example 1.

Example 9

In example 9, the obtained lung cancer organoids were subjected to cryopreservation treatment in addition to example 6 or example 7. Specifically, the cryopreservation treatment comprises the following steps:

s701, for the lung cancer organoids embedded with matrigel in example 6 or example 7, 1 mL of 4 ℃ Advanced DMEM/F12 medium was used for each well to blow off, and the lung cancer organoids were collected in a centrifuge tube and centrifuged (centrifugal force 300 g, centrifugal time 5 min) to obtain a mixed precipitate of matrigel and cells.

S702, resuspending the organ cell sediment obtained in the step S701 by trypLE (a commercial product, Gibco) to obtain a resuspension solution; placing the heavy suspension in water bath at 37 ℃ for 8-10 min; then, to the re-suspension, two volumes of 4 ℃ Advanced DMEM/F12 medium were added to stop the digestion, and the mixture was centrifuged (centrifugal force 300 g, centrifugation time 5 min) to obtain a secondary cell pellet.

S703, for the secondary cell precipitation, every 1X 10⁵～5×10⁶The individual cell clusters were resuspended in1 mL of cryopreservation solution containing 0.1% -0.5% Y27632 and cryopreserved using a programmed gradient cooling cassette. After one day, the cells were transferred to liquid nitrogen for long-term storage. Here, one day generally means overnight.

After being frozen and stored by liquid nitrogen in the embodiment 9 for 12 months, the lung cancer organoids can still stably grow, and the activity and the dryness of the organoids are kept, and as shown in a light microscope image of the resuscitated lung cancer organoids cultured for 24 hours conventionally shown in fig. 10, the organoids have high activity and good growth state.

The following comparative examples were conducted for analytical comparison with the foregoing examples.

Comparative example 1

Comparative culture solution I of comparative example 1 was prepared by adding no self-source condition medium to culture solutions II to II of example 3. The rest is the same as culture solutions II-II.

In comparative example 1, the lung cancer specimen was cultured using the comparative culture solution I in the same manner as in example 7, wherein a comparative lung cancer organoid was obtained by performing enzymolysis pretreatment with the enzymolysis solution III of example 5 and culturing (primary culture) for 7 days using the comparative culture solution I.

As shown in the comparative lung cancer organoid picture in fig. 11, it can be seen that the size and density of the comparative lung cancer organoids still do not reach the lung cancer organoids in fig. 6 of example 7 and fig. 9 of example 8, and the small organoid ratio is high.

In the present application example, the lung cancer organoids ii to iii in example 7 and the comparative lung cancer organoids in comparative example 1 were subjected to expression detection of the epithelial marker protein ecadherin, respectively, and the detection results are shown in fig. 12, which is a histogram of the relative expression level of the epithelial marker protein ecadherin; where the gray bars correspond to lung cancer organoids II-III of example 7 and the black bars correspond to comparative lung cancer organoids of comparative example 1. Therefore, the expression level of the epithelial marker protein ecadherin is obviously reduced after the culture solution is added with the self-source condition culture medium.

In the present embodiment, the expression detection of the mesenchymal marker protein vimentin was performed on the lung cancer organoids ii-iii in example 7 and the comparative lung cancer organoids in comparative example 1, and the detection results are shown in the histogram of the relative expression level of mesenchymal marker protein vimentin in fig. 13; where the gray bars correspond to lung cancer organoids II-III of example 7 and the black bars correspond to comparative lung cancer organoids of comparative example 1. Therefore, after the autologous condition culture medium is added into the culture solution, the expression level of the mesenchymal marker protein vimentin is obviously increased, which indicates that the autologous condition culture medium is favorable for maintaining the mesenchymal characteristics of the cells.

In the examples of the present application, the primary lung cancer organoids obtained in example 7 (for example, lung cancer organoids ii-iii) were subcultured by the passaging method in example 8, and passaging was performed up to 9 times to obtain the 10 th generation lung cancer organoids. The culture solutions II to II of example 3 were used in both the primary culture in example 7 and the subculture in example 8. The lung cancer organoids obtained after 5 days of subculture each time were identified. The identification method comprises the following steps: lung cancer tumor tissue samples and organoid HE staining formed from the samples were performed.

FIG. 14 shows HE staining results of 10 th generation lung cancer organoid cultured from lung cancer tumor tissue samples, and FIG. 15 shows HE staining results of lung cancer tumor tissue samples (i.e., homologous lung cancer tissues) for primary lung cancer organoid culture in example 7. It can be seen that the 10 th generation lung cancer organoids still showed the same HE staining results as the homologous tumor tissues.

In the examples of the present application, unless otherwise specified, all the percentage concentrations are volume percentage concentrations.

In addition, in the present application example, the culture solution' ii in example 4 is further used to culture the lung cancer tissue, and the detection and analysis result of the lung cancer organoid obtained by the culture is consistent with the detection and analysis result of the lung cancer organoid obtained by the culture solution ii in example 3, which is not described herein again.

The above description and drawings sufficiently illustrate embodiments of the application to enable those skilled in the art to practice them. Other embodiments may include structural and other changes. The examples merely typify possible variations. Individual components and functions are optional unless explicitly required, and the sequence of operations may vary. Portions and features of some embodiments may be included in or substituted for those of others. The embodiments of the present application are not limited to the structures that have been described above and shown in the drawings, and various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Claims

1. The lung cancer organoid culture solution is characterized by comprising a complete culture medium and an autologous condition culture medium, wherein the volume ratio of the complete culture medium to the autologous condition culture medium is 1-3: 1;

wherein the complete medium comprises a conditioned medium, a basal medium, a complex antibiotic and a growth factor; the volume ratio of the conditioned medium to the basic medium is 1-4: 1; the basic culture medium comprises Advanced DMEM/F12, HEPES 5-15 mM and Glutamax 0.5 x-1.5 x; the compound antibiotics comprise 50-500 mug/mL of Primocin, 0.5 x-1.5 x of penicillin-streptomycin and 2-10 mug/mL of metronidazole; the growth factors include: 10-100 ng/mL of EGF, 1010-100 ng/mL of FGF-20.25x-1.5 x, 270.25x-1.5 x of B270, 1-2 mM of n-acetyl cysteine, 5-20 mM of Nicotinamide, 5-20 nM of Gastrin, 20.2-2 mu M of Prostaglandin E and 15 mu M of Y-276325; the conditioned medium comprises a Wnt-3a conditioned medium, an R-spondin1 conditioned medium and a Noggin conditioned medium, the Wnt-3a conditioned medium, the R-spondin1 conditioned medium and the Noggin conditioned medium are obtained by respectively culturing target cell lines of Wnt-3a, R-spondin1 and Noggin overexpression cell strains, and the Wnt-3a conditioned medium, the R-spondin1 conditioned medium and the Noggin conditioned medium are respectively 20%, 50% and 30% in percentage by volume;

2. The lung cancer organoid culture fluid of claim 1, wherein the growth factor comprises: EGF 50 ng/mL, FGF-1050 ng/mL, N20.75x-1 x, B270.75x-1 x, n-acetyl cysteine 1.25-1.5 mM, Nicotinamide 8-10 mM, Gastrin 8-10 nM, Prostaglandin E20.5-1 μ M, Y-276328-10 μ M.

3. The lung cancer organoid culture solution according to claim 1,

the compound antibiotics comprise 100 mu g/mL of Primocin, 1x of penicillin-streptomycin and 4 mu g/mL of metronidazole.

4. The lung cancer organoid culture fluid of claim 1, 2 or 3, said self-derived conditioned medium obtained by the following culture method:

extracting the autologous tumor-associated fibroblasts, carrying out passage and establishing a system to obtain the autologous tumor fibroblasts;

culturing the autologous tumor fibroblasts by adopting a tumor-associated fibroblast culture medium; wherein the tumor-associated fibroblast cell culture medium comprises: DMEM basal medium +5% serum +3% PS;

after 3 to 5 days of culture, the medium is collected and the collected medium is centrifuged to obtain a self-conditioned medium.

5. A lung cancer organoid culture reagent combination, comprising: an enzymatic hydrolysate and a lung cancer organoid culture solution according to any one of claims 1 to 4; the enzymolysis liquid comprises a basic culture medium, collagenase type I, collagenase type III and Primocin; wherein the concentration of the collagenase type I is 0.1-2 mg/mL, the concentration of the collagenase type III is 0.1-1 mg/mL, and the concentration of Primocin is 0.2-2 mg/mL; and independently packaging the lung cancer organoid culture solution and the enzymolysis solution.

6. The lung cancer organoid culture reagent combination according to claim 5, wherein the enzymatic hydrolysate comprises basal medium, collagenase type I, collagenase type III and Primocin; wherein the concentration of the collagenase type I is 1.5mg/mL, the concentration of the collagenase type III is 0.5mg/mL, and the concentration of Primocin is 1.5 mg/mL.

7. A method of culturing a lung cancer organoid, comprising:

carrying out physical pretreatment on the lung cancer sample to obtain a sample tissue fragment;

performing enzymolysis pretreatment on the sample tissue fragments, filtering to obtain filtrate, and centrifuging the filtrate to obtain cell precipitates;

resuspending the cell pellet with matrigel to obtain gel mixed with cells; then inoculating the gel into a culture hole, and performing static culture in a cell culture box at 37 ℃ to solidify the gel;

adding the lung cancer organoid culture solution of any one of claims 1 to 4 into the culture well for culture to obtain primary lung cancer organoids;

subculturing the primary lung cancer organoid, wherein the lung cancer organoid culture solution according to any one of claims 1 to 4 is adopted for culturing in the subculturing process, and the culture period of each subculturing is 3-5 days, so that the corresponding generation of lung cancer organoid is obtained.

8. The culture method according to claim 7, wherein the enzymatic pretreatment comprises:

adding the enzymolysis liquid in the lung cancer organoid culture reagent combination of claim 5 or 6 into the sample tissue fragment, and incubating for 20-30 min under the conditions of shaking at 37 ℃ and 200 r/min;

adding enzymolysis stopping agent, stopping enzymolysis, and filtering to obtain filtrate.

9. The culture method according to claim 7 or 8, further comprising:

embedding the obtained lung cancer organoid by using matrigel, blowing the lung cancer organoid by adopting an Advanced DMEM/F12 culture medium, and centrifuging to obtain mixed sediment of the matrigel and cells;

resuspending the mixed pellet with a cell dissociation reagent to obtain a resuspension solution; placing the heavy suspension in a water bath at 37 ℃ for 8-10 min; then adding two times of volume of Advanced DMEM/F12 culture medium at 4 ℃ into the resuspension, mixing uniformly, and centrifuging to obtain secondary cell sediment;

resuspending the secondary cell sediment by adopting a freezing medium, and then freezing by using a programmed gradient cooling box; transferring to liquid nitrogen for long-term storage after one day; wherein the re-suspension ratio of the secondary cell sediment to the freezing medium is 1 × 10⁵～5×10⁶The individual cell pellet was resuspended in1 mL of the frozen stock solution.