CN112899230B - Bladder cancer organoid culture medium and preparation method and application thereof - Google Patents
Bladder cancer organoid culture medium and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of tumor organoid culture, and particularly relates to a bladder cancer organoid culture medium, a preparation method and an application thereof, wherein the culture medium comprises the following components in parts by weight: 8-12mM of cell culture medium, 145ng/mL of fibroblast growth factor 120-. The method has good operability, high repeatability, rapidness, forming within 24 hours and over 90 percent of success rate, successfully reserves immune cells in the tumor, and can provide an ideal research model for the occurrence and development of bladder cancer, the screening of tumor immunity medicines and the in-vitro drug sensitivity test research.
Description
Technical Field
The invention belongs to the field of tumor organoid culture, and particularly relates to a bladder cancer organoid culture medium, a preparation method and application thereof.
Background
Organoids are three-dimensional micro-organs that are highly similar to the source tissues and organs and can be cultured in the laboratory using human stem cells (which differentiate into immature cells of any cell type in the human body and play a key role in tissue function and regeneration), i.e., cells taken from a patient can replicate some characteristics of the patient's tumor, and can be used to construct disease models. Organoids are essentially an avatar of a patient's tumor, can grow to about 1mm in diameter, are similar in shape to a patient's tumor, and have many of the same molecular and genetic characteristics that, over time, produce genetic changes, a phenomenon known as clonal evolution, which is a major contributor to tumor progression and drug resistance, and may be used to test drugs or even replace damaged tissue in a patient.
Tumor organoids have been recently discoveredTo comeCompared with the early tumor microenvironment model, the emerging model for simulating the tumor microenvironment has the following advantages; compared with the early 2D culture, the tumor organoid can more comprehensively simulate the internal characteristics of the tumor on a spatial structure, and the organoid model can more effectively improve the consistency of the reaction with a patient; compared with the recent human tumor xenograft model, the organoid culture period is short, the cost is low, and large-batch reagent can be carried out. The human tumor organoid of bladder cancer is prepared by directly preparing primary cells digested by patient tissues into organoid,the model maintains the functional characteristics of the tissues of the patient, and has short preparation time and low consumption.
Bladder cancer is a common malignant tumor of a urinary system, the incidence rate of the bladder cancer is increased year by year, early bladder cancer symptoms are not obvious, a patient is easy to neglect to cause treatment delay, then distant metastasis to a muscle layer or lymph can occur, the prognosis is poor, part of patients stop treatment because of the failure of bearing side effects brought by medicines, and the life and families of the patients are seriously affected. Therefore, a platform for testing the medicines in advance for different patients needs to be developed, so that personalized treatment is realized. However, the success of the existing bladder cancer organoid culture is very few in reports, the success rate is low, the culture time is long, immune cells in the organoid are deleted, and the tumor immune microenvironment cannot be simulated.
One of the challenges in constructing organoids is how to determine the nutrients, growth factors and tissue culture techniques required to allow a patient's tumor cells to form a tiny tumor organoid in a culture dish. These exact conditions vary greatly from cancer type to cancer type. Organoids cannot be called "miniaturised versions" of real organs, and are still miniature and simple organ models. Current organoids are very difficult to culture even in a standardized, controlled process.
Organoid culture systems include two parts: matrigel and culture medium. The matrigel is a matrigel protein glue rich in laminin and collagen, has the components similar to extracellular matrix of a plurality of tissues, and provides three-dimensional matrix support for in vitro culture of organoids; the culture medium needs to be added with various cytokines and small molecules related to the proliferation and differentiation of stem cells.
Chinese patent CN202010096820.0 describes a method for culturing bladder cancer organoids in vitro, which comprises: manufacturing a gas-liquid interaction culture system; the organoid culture medium comprises: advanced DMEM/F12 medium, Primocin primary cell antibiotic 100X dilution, GlutaMAX cell culture additive 100X dilution, B27 supplement 50X dilution, N-Acetylcysteine (NAC) 1mM, Epidermal Growth Factor (EGF)5ng/mL, Noggin recombinant protein 100ng/mL, R-spondin-1 recombinant protein 250ng/mL, SB202190MAPK inhibitor 10 μ M, A83-01ALK5 inhibitor 500nM, Y-27632ROCK inhibitor 10 μ M, nicotinamide
(Nicotinamide)10mM, fibroblast growth factor (FGF-10)10ng/mL, basic fibroblast growth factor (FGF-basic)5ng/mL, human Heregulin-beta 1 growth factor 10ng/mL and prostaglandin E21 μ M. The success rate of culturing the bladder cancer organoid is improved, the bladder cancer organoid with the reserved immune cells can be obtained by culturing, the operation is simple, the utilization rate of tumor tissues is high, and the method has important significance and value for screening and researching bladder cancer drugs. However, the method of this patent is primarily directed to a Transwell cell, which is relatively expensive.
In addition, other existing organoid preparation techniques have low success rate in preparing bladder cancer organoids, and are hardly subject to balling during the preparation process using agrewell from the industrialized stemchell company. Moreover, because of the problem of bladder cancer species, the cell balling effect is not good, according to the bladder cancer degree in different stages, the high muscle layer infiltration type organoid is more inclined to be grape-shaped in the early balling stage, and the low-level non-basal layer infiltration type organoid is more inclined to be loose at the edge.
Disclosure of Invention
In order to solve the technical problems, the invention provides the special culture medium for the bladder cancer organoid and the rapid preparation method, the method has good operability, high repeatability, rapidness, molding within 24 hours and over 90 percent of success rate, successfully reserves immune cells in tumors, and can provide an ideal research model for the occurrence and development of bladder cancer, tumor immune drug screening and in-vitro drug sensitivity test research; the prepared organoids are uniform in size, so that the method is more beneficial to screening medicines in the later period; the preparation method of the invention leads the cells to be gathered in a short time, thereby quickly forming balls which are biased to embryoid bodies and then growing cavity structures.
The invention provides a bladder cancer organoid culture medium for solving the technical problems, which is characterized in that: the organoid medium comprises the following components in concentrations: 8-12mM of cell culture medium, 145ng/mL of fibroblast growth factor 120-; the other was Advanced DMEM/F12 and minimal solvent, Advanced DMEM/F12 supplemented to the volume required for the culture.
Wherein, the component concentration is the concentration of the final components in the organoid culture medium, the unit of measurement is nM molar concentration, ng/mL is mass concentration,% is volume concentration, the following is the same.
Normolin is a primary cell antibiotic and works as well as primocin. The cell culture medium is Glutamax. It contains dipeptides in the form of stabilized L-glutamine, L-alanyl-L-glutamine, and can prevent degradation of glutamine and accumulation of ammonia during long-term culture.
The fibroblast growth factors comprise FGF10, FGF7 and FGF2, wherein the mass ratio of FGF10 to FGF7 to FGF2 is 8: 2: 1. FGF2, FGF7, FGF10, fibroblast growth factor, are capable of promoting differentiation of bladder cancer organoids. The cells are made to grow structures more easily by the fibroblast growth factor.
The hydrogen ion buffer is Hepes, and can control a constant pH range for a long time.
The antibiotic is penicillin/streptomycin double-resistance.
The epidermal growth factor is EGF.
The recombinant protein is Noggin and Wnt-3a, wherein the mass and dosage ratio of the Noggin to the Wnt-3a is 1: 1.
the secretory protein is R-Spondins, the human leukocyte antigen is B27, and the basement membrane matrix is Matrigel.
The inhibitor is A83-01 and Y-27632, A83-01 is Activin/NODAL/TGF-beta pathway inhibitor, and can inhibit ALK5, ALK4 and ALK 7. Y-27632 is a small molecule specific inhibitor of the Rho-associated serine-threonine protein kinase (ROCK) family, and can prevent apoptosis of stem cells.
Normocin was used at a concentration of 100mg/ml in combination with penicillin/streptomycin to increase the antibacterial spectrum.
The basement membrane matrix is Matrigel, is derived from the basement membrane matrix of EHS mouse sarcoma, contains about 60% laminin, 30% IV collagen and 8% nidogen, and also contains perlecan, TGF-beta, epidermal growth factor, insulin-like growth factor, tissue plasminogen and other growth factors.
Further, the culture medium comprises the following components by mass and using amounts, calculated by the total volume of the culture medium of 500 ml: HEPES 10mM, Glutamax10mM, penicillin/streptomycin double antibody 1%, FGF 10100 ng/mL, FGF7
25ng/mL, FGF212.5 ng/mL, EGF50ng/mL, Noggin 100ng/mL, Wnt3a 100ng/mL, R-Spondin 1500 ng/mL, human Gastrin-110 nM, Nicotinamide 10mM, N-acetylcysteine 1.25mM, Normocin 0.2%, B272%, A83-015 uM, Y2763210.5 uM, Matrigel 37.5%, the balance Advanced DMEM/F12; a trace amount of solvent.
The solvent is used as a buffer solution for preparing the growth factors, the growth factors are solid and need to be dissolved by the buffer solution, but the dosage is relatively trace, and the growth factors do not harm cells. The buffer solution is NAOH, sodium phosphate, Tris, sterile water, complete medium and DMSO. NAOH dissolves Gastrin-1, sodium phosphate dissolves FGF10, Tris dissolves FGF2, sterile water dissolves EGF and Noggin, complete medium dissolves Wnt3a and R-Spondin, DMSO dissolves Nicotinamide, Y27632 and N-acetylcysteine. Dissolving the solvent and the raw materials to be dissolved, taking the required dosage, and then mixing with other raw materials to prepare the compound. The culture medium is prepared by conventional techniques.
The Enzyme-containing medium was prepared using serum-free DMEM or serum-free 1640, 200ul Enzyme H +100ul Enzyme R +25ul Enzyme A being the corresponding Enzyme. Complete medium was 90% DMEM + 10% FBS + 1% double antibody.
The pH value in the culture medium is 7.2-7.4.
The culture medium of the invention reserves the components of the organoid suitable culture medium, and increases the fibroblast growth factor, which is more beneficial to the growth of the bladder cancer organoid. The method can ensure that the bladder cancer organoid can grow quickly, has high success rate, can reserve immune cells in tumors, can prepare the organoid within two days, and then is used for subsequent drug tests, so that the immune cells are reserved, and the method has better effect compared with the condition that the immune cells digested by primary tissues can be apoptotic within two days in vitro.
FGF7, FGF10 and FGF2 which are beneficial to three-dimensional growth and differentiation of organoids are simultaneously added into the culture medium. The fibroblast growth factor family FCFs, which are important regulators of epithelial-mesenchymal interactions in various organogenesis, are FGF10 (keratinocyte growth factor), which is an important regulator of the epiblast, and in the endothelial epithelium, without which embryonic tissues and organs cannot be completed.
FGF7 and FGF10 activate FGFR2-O b, are expressed in epithelial cells, and have higher affinity; FGF2 specifically activates FGFR2-O c, is expressed in mesenchymal cells, and is associated with the cell membrane environment and heparan sulfate. When the extracellular segment of FGFR2 is subjected to point mutation, FGF7 and FGF10 can activate FGFR2-O c, and FGF2 can activate FGFR2-O b, so that the cellular autocrine signals of the ligands are activated.
The special culture medium for the bladder cancer organoid is rapid and efficient, immune cells are reserved, the bladder cancer organoid can stably grow and proliferate in the culture medium by regulating growth factors in the culture medium aiming at the characteristics of proliferation differentiation and physiological functions of bladder cancer tissues of patients with different grades, and meanwhile, the organoids infiltrated by a non-muscular layer and a muscular layer show obvious morphological difference. After about 48 hours of culture, the tumor organoids are subjected to in-situ immunofluorescence staining and observed by a laser confocal microscope, and as shown in fig. 5, the experimental result shows that the organoids prepared by the method of the invention retain immune cells.
The application of the bladder cancer organoid culture medium is the application of the bladder cancer organoid culture, the rapid culture method comprises the steps of manufacturing a hydrophobic inverted culture dish, inverting a single cell suspension on the hydrophobic culture dish in a droplet form, standing on ice and culturing in an incubator to form a hydrophobic module containing cell droplets, culturing the hydrophobic module in a organoid special culture medium with the droplet direction upward, releasing tumor balls obtained by culturing from the hydrophobic module, and continuously culturing in a hole plate provided with the organoid special culture medium to obtain the organoid.
The organoid culture rapid culture method in the application specifically comprises the following steps:
(1) manufacturing a hydrophobic inverted hanging culture dish;
paving a PDMS film on the surface of the glass to prepare a biocompatible hydrophobic module, and baking the hydrophobic module in a baking oven with the baking temperature of 75-85 ℃; adhering the hydrophobic module on the culture dish cover by using a double-sided adhesive tape, wherein the PDMS film faces the bottom of the culture dish to form a hydrophobic inverted culture dish; in the optimized scheme, the hot drying temperature is 80 ℃.
(2) The single cell suspension is inversely hung on a hydrophobic culture dish in a droplet form:
resuspending the single cell suspension dissociated from fresh bladder cancer tissue by matrigel, and inversely hanging the suspension on a hydrophobic culture dish in a droplet form; sucking 0.15-0.25ul of single cell suspension liquid once and placing the single cell suspension liquid on a hydrophobic module to form cell liquid drops until the single cell suspension liquid is completely transferred;
(3) standing on ice: placing on ice, and standing at 0-4 deg.C.
Standing the hydrophobic inverted culture dish containing the cell droplets on ice for 14-16 minutes with the droplet direction facing downwards; then placing the hydrophobic inverted culture dish containing the cell drops into an incubator at 36-38 ℃ for culturing for 1.5-2.5 hours; the carbon dioxide content in the cell culture box is 4-6%; the low-temperature standing is beneficial to the sedimentation of cells in the liquid drops in an inverted state in order to keep the matrigel in a liquid state. The droplets are directed downwards to allow better sedimentation of the cells, forming a multi-layered stack, which is more conducive to the formation of cell spheres.
(4) The hydrophobic module is cultured in organoid special culture medium with the liquid drop direction upwards:
transferring the hydrophobic module containing the cell droplets from the culture dish cover to a low-adhesion 6-hole plate, wherein the droplet direction is upward; adding organoid culture medium until the culture medium exceeds the dripping position of cell sap, and culturing for 9-11 hr in a cell culture box with carbon dioxide content of 4-6% at 36-38 deg.C;
the liquid drop is upward again in order to enable the multilayer cell balls settled at the bottom to be upward and to be in more contact with the culture medium in the culture dish, so that a three-dimensional structure of the culture medium, the cell balls and the matrigel observed from the side is formed, and the three-dimensional growth of the cell balls is facilitated.
(5) The tumor spheres continue to culture after release from the hydrophobic module:
separating the organoid from the hydrophobic film and suspending the organoid in the pore plate for continuous culture, thereby facilitating the subsequent drug test. Releasing the obtained tumor spheres from the hydrophobic module into a low-adhesion 24-well plate for long-term culture; the fluid is changed every 2-3 days according to the growth condition of the organoid.
Compared with the common inverted hanging method, the method of the invention has the advantages that the gel cell balls are placed in the culture medium in the shortest time, and then the gel cell balls grow in a better three-dimensional way, and the nutrition supplement is faster.
Adding 1-3ml of PBS containing 0.8-1.2% of penicillin/streptomycin double-antibody into a hydrophobic culture dish in the step (2); the cell concentration of the single cell suspension is 1.5x107 cells/mL; the mass concentration of matrigel in the resuspended single cell suspension was 37.5%.
The number of cells in the cell droplet in the step (2) is in the range of 2000-4000 cells/cell.
And (5) replacing the organoid special culture medium containing 20% Matrigel every 2-3 days according to the growth condition of the organoid. The Matrigel content was different, as was the other ingredients.
The cells can form tumor organoid spheres in 12 hours by using the culture method of the invention, and the organoid special culture medium containing 20 percent Matrigel is replaced every 2 to 3 days after the cells are transferred to a 24-well plate. When the organoid culture medium is changed to the Matrigel culture medium with low concentration, the Matrigel culture medium in the liquid drop (tumor ball) contains Matrigel with higher concentration, but the Matrigel in the liquid drop is not enough for organoid growth, so that a Matrigel culture medium with gradient decrease is needed to be constructed, and the concentration is not as high as before for better organoid growth. The cultured organoids can be continuously enlarged, have clear edges and have differentiation phenomena. The organoid in the invention grows to more than 200um within 48 hours, and the immunofluorescence staining proves that the organoid contains immune cells, thereby further simulating the tumor microenvironment.
According to the bladder cancer culture medium and the bladder cancer organoid culture method provided by the invention, the organoid containing immune cells is quickly prepared within 24 hours, a bladder cancer tumor microenvironment is successfully simulated, the medicine consumption is low, the time is short, the preparation success rate is over 90 percent, the success rate is high, the organoid retains the immune cells, an ideal research model is provided for the occurrence and development of bladder cancer, tumor immune medicine screening and in-vitro drug sensitivity test research, and the method has great significance for the follow-up research of bladder cancer. The organoid culture method of the invention can also be applied to the rapid preparation and culture of other organoids to form an organoid culture system capable of retaining immune cells.
Drawings
The invention will be described in further detail with reference to the following drawings and detailed description:
FIGS. 1 to 3 are schematic structural views of a hydrophobic inverted hanging culture system according to example 1 of the present invention
FIG. 4 is a Scale bar 100um field map of bladder cancer organoids of the present invention
FIG. 5 is a diagram showing the results of immunofluorescence staining assay of bladder cancer tumor organoids of the present invention
FIG. 6 is a diagram showing the growth state of tumor organoids according to the present invention
FIG. 7 is a H & E staining pattern of tumor organoids and tissues of the present invention (left panel: organoid, right panel: tissue)
FIG. 8 is a graph showing the comparison of the effects of the first experiment of the present invention
FIG. 9 is a diagram showing a process of preparing a medium in the present invention
Wherein, the marks in the figure are specifically:
101. culture dish cover, 102 glass, 103 PDMS film, 104 tumor single cell droplet containing matrigel, 105 tumor ball droplet containing 1% penicillin/streptomycin double antibody PBS, 106 culture dish bottom, 107 bladder cancer organoid special culture medium, 108 tumor ball droplet standing on ice and solidified in culture box for 2 hours, 109 low-adhesion 24-hole plate, 110 bladder cancer organoid formed after 12 hours of culture, 113 immune cell
Detailed Description
The invention will be further described with reference to specific embodiments in which Advanced DMEM/F12, penicillin/streptomycin diabody, Hepes and Normocin are Invitrogen, Glutamax (Thermo-Fisher) is GIBCO, FGF10, EGF, Recombinant Human Noggin, FGF2 and FGF7 are peprotech, Wnt3a is Sigma, RSPO1 is R & D, Human Gastrin 1 is Sigma Aldrich, Nicotinamide (Sigma Aldrich) is N0636, N-acetylcysteine is A9165-5g, B27 (Invitro) is Gibco, A83-01 (Tocrisis) is Tocrisis, Y27632 is STEELL, Matrigel is Corrog, and other enzymes and DMEM media are commercially available:
example 1
A bladder cancer organoid culture medium comprises the following components and dosage: 8mM of cell culture medium, 120ng/mL of fibroblast growth factor, 8mM of hydrogen ion buffer, 0.8% of antibiotic, 45ng/mL of epidermal growth factor, 180ng/mL of recombinant protein, 460ng/mL of secretory protein, 8nM of human Gastrin (human Gastrin 1), 8mM of Nicotinamide or Nicotinamide (Nicotinamide), 1mM of N-acetylcysteine, 0.15% of Normocin, 1.8% of human leukocyte antigen, 14.5uM of inhibitor and 36% of basement membrane matrix; the other was Advanced DMEM/F12 and minimal solvent, Advanced DMEM/F12 supplemented to the volume required for the culture. Trace amount of solvent, which may be NAOH, sodium phosphate, Tris, sterile water, complete medium and DMSO. Complete medium was 90% DMEM + 10% FBS + 1% double antibody medium.
Wherein, the component concentration is the concentration of the final components in the organoid culture medium, the unit of measurement is nM molar concentration, ng/mL is mass concentration,% is volume concentration, the following is the same. This concentration is the final concentration for either 1000ml of medium or 100ml of medium. In the test, the total amount of the culture medium is generally 100ml, and the preparation method of the culture medium is the conventional technology.
The cell culture medium was Glutamax. It contains dipeptides in the form of stabilized L-glutamine, L-alanyl-L-glutamine, and can prevent degradation of glutamine and accumulation of ammonia during long-term culture.
The fibroblast growth factors comprise FGF10, FGF7 and FGF2, wherein the mass ratio of FGF10 to FGF7 to FGF2 is 8: 2: 1.
the hydrogen ion buffer is Hepes. Can control constant pH range for a long time.
The antibiotic is penicillin/streptomycin double-resistant.
The epidermal growth factor is EGF.
The recombinant protein is Noggin and Wnt-3a, wherein the mass dosage ratio of the Noggin to the Wnt-3a is 1: 1.
the secretory protein is R-Spondins, the human leukocyte antigen is B27, and the basement membrane matrix is Matrigel.
The inhibitor is A83-01 and Y-27632, A83-01 is ALK5 inhibitor, and Y-27632 is small molecule specific inhibitor.
Example 2
In other aspects, as in example 1, a bladder cancer organoid culture medium comprises the following components in amounts: 12mM of cell culture medium, 145ng/mL of fibroblast growth factor, 12mM of hydrogen ion buffer, 1.2% of antibiotic, 55ng/mL of epidermal growth factor, 220ng/mL of recombinant protein, 520ng/mL of secretory protein, 12nM of human Gastrin (human Gastrin 1), 12mM of Nicotinamide or Nicotinamide (Nicotinamide), 1.5mM of N-acetylcysteine, 1.22% of Normocin, 2.2% of human leukocyte antigen, 16uM of inhibitor and 38.5% of basement membrane matrix; the other was Advanced DMEM/F12 and minimal solvent, Advanced DMEM/F12 supplemented to the volume required for the culture. Trace amount of solvent, which may be NAOH, sodium phosphate, Tris, sterile water, complete medium and DMSO. In the test, 100ml of the total amount of the medium is generally used. The preparation method of the culture medium is a conventional technology.
Example 3
In other aspects, as in example 1, a bladder cancer organoid culture medium comprises the following components in amounts: 11mM of cell culture medium, 135ng/mL of fibroblast growth factor, 9mM of hydrogen ion buffer, 0.9% of antibiotic, 48ng/mL of epidermal growth factor, 190ng/mL of recombinant protein, 510ng/mL of secretory protein, 11nM of human Gastrin (human Gastrin 1), 11mM of Nicotinamide or Nicotinamide (Nicotinamide), 1.2mM of N-acetylcysteine, 1% of Normocin, 1.9% of human leukocyte antigen, 15.5uM of inhibitor and 38% of basement membrane matrix; the other was Advanced DMEM/F12 and minimal solvent, Advanced DMEM/F12 supplemented to the volume required for the culture. Trace amount of solvent, which may be NAOH, sodium phosphate, Tris, sterile water, complete medium and DMSO. In the test, the total amount of the medium is generally 100 ml. The preparation method of the culture medium is a conventional technology.
Example 4
A bladder cancer organoid culture medium comprises the following components by mass: HEPES 10mM, Glutamax10mM, penicillin/streptomycin diabody 1%, FGF 10100 ng/mL, FGF 725 ng/mL, FGF212.5ng/mL, EGF50ng/mL, Noggin 100ng/mL, Wnt3a 100ng/mL, R-Spondin 1500 ng/mL, human Gastrin-110 nM, Nicotinamide 10mM, N-acetylcysteine 1.25mM, Normocin 0.2%, B272%, A83-015 uM, Y2763210.5uM, Matrigel 37.5%; other details of the procedures for preparing the culture medium with the volume required for supplementing the culture medium with Advanced DMEM/F12, including Advanced DMEM/F12 and the micro solvent, are shown in FIG. 9.
I.e., the components, concentrations or volume percent contents of each ingredient are shown in table 1 below.
TABLE 1
Microbial cell culture contamination and contamination of other cell lines into the culture medium, antibiotics and antifungal agents are used to prevent contamination. Penicillin/streptomycin double-antibody is an antibiotic commonly used in vitro culture for preventing microbial contamination, and is usually prepared into mother liquor with the concentration of 100 times for use. Penicillin mainly inhibits gram-positive bacteria, streptomycin mainly inhibits gram-negative bacteria, and penicillin and streptomycin are complementary to each other and can inhibit most common bacteria. Penicillin acts on cell walls, is sensitive to temperature and pH value, needs to be frozen for storage, and has a pH value of 6.0-6.5; streptomycin only affects the synthesis of bacterial protein, is effective to gram negative and positive bacteria, has no influence on cell (eukaryotic) growth, and has stable pH of 5.0-7.5.
EGF, epidermal growth factor, an active substance in the human body, an active polypeptide consisting of 53 amino groups, achieve the purpose of repairing hyperplastic skin surface cells by stimulating tyrosine phosphorylation of epidermal growth factor receptors. Promoting the proliferation and differentiation of cells, thereby replacing senescent and dead cells with new cells.
Noggin, a recombinant protein, an inhibitor of bone morphogenetic genetic protein (BMP) that plays a key role in development, is secreted in vivo. Wnt3a recombinant and synthetic protein has biological activity, is expressed by CHO, E, Coli system, is one of the important components of Wnt signal path, and plays a key role in cell growth, differentiation and tumorigenesis.
R-Spondins are a family of secreted proteins that are widely expressed in a variety of organisms and all contain furin-like and thrombospondin domains that interact with the Frizzled/LRP6 receptor complex in a manner that stimulates the Wnt/beta-catenin signaling pathway.
Nicotinamide, often abbreviated as NAM, known in Chinese as Nicotinamide or Nicotinamide, also known as Vitamin PP, is a water-soluble Vitamin belonging to Vitamin B3 class, and Nicotinamide is also a commonly used inhibitor of Sirt 1. Sirt1 was selectively inhibited with IC50 ═ 98 nM. Has certain inhibition effect on other Sirt proteins at higher concentration and has no inhibition effect on the deacetylase HDAC family.
N-acetylcysteine, West drug name.
Normocin has antibacterial, antifungal, and mycoplasma effects, and contains two components for killing bacteria and mycoplasma by blocking DNA replication and protein synthesis. The antibacterial spectrum can be increased by combining the penicillin/streptomycin.
Y-27632 can prevent apoptosis of human embryonic stem cells (hES) induced by separation, improve survival rate and cloning efficiency of the separated hES cells without affecting self-renewal or pluripotent properties of hES, and relax smooth muscle and inhibit proliferation of prostate smooth muscle cells.
Matrigel can effectively help the attachment and differentiation of various cells such as epithelial cells.
During the preparation, firstly NAOH is used for dissolving Gastrin-1, sodium phosphate is used for dissolving FGF10, Tris is used for dissolving FGF2, sterile water is used for dissolving EGF and Noggin, complete culture medium is used for dissolving Wnt3a and R-Spondin, and DMSO is used for dissolving Nicotinamide, Y27632 and N-acetylcysteine. Dissolving the solvent and the raw materials to be dissolved, taking the required dosage, and then mixing with other raw materials to prepare the compound. The culture medium is prepared by conventional techniques. The solvent is used as a buffer solution for preparing the growth factors, the growth factors are solid and need to be dissolved by the buffer solution, but the dosage is relatively trace, and the growth factors do not harm cells.
Example 5
The application of the culture medium in the invention is the application of the rapid culture of the bladder cancer organoid, and comprises the steps of preparing a hydrophobic inverted culture dish, inverting a single-cell suspension on the hydrophobic culture dish in a droplet form, standing on ice and culturing in an incubator to form a hydrophobic module containing cell droplets, culturing the hydrophobic module droplets in a organoid special culture medium in an upward direction, releasing tumor balls obtained by culturing from the hydrophobic module, and continuously culturing in a well plate in which the organoid special culture medium is placed to obtain the organoid. The media required starting materials and final concentrations were as described in examples 1-4.
The method specifically comprises the following steps:
(1) manufacturing a hydrophobic inverted hanging culture dish;
spreading a PDMS film on the surface of the glass to prepare a biocompatible hydrophobic module, and thermally drying the hydrophobic module in an oven at the temperature of 80 ℃; and adhering the hydrophobic module to the culture dish cover by using a double-sided adhesive tape, wherein the PDMS film faces the bottom of the culture dish to form a hydrophobic inverted culture dish.
(2) The single cell suspension was hung upside down on a hydrophobic culture dish in the form of droplets:
resuspending the single cell suspension dissociated from fresh bladder cancer tissue by matrigel, and inversely hanging the suspension on a hydrophobic culture dish in a droplet form; sucking 0.2ul of single-cell suspension once and placing the single-cell suspension on a hydrophobic module to form cell droplets until the single-cell suspension is completely transferred; 2ml of 1% penicillin/streptomycin-containing double-resistant PBS was added to the hydrophobic petri dish; the matrigel concentration of the heavy suspension single cell suspension is 37.5 percent, and the cell concentration of the single cell suspension is 1.5x107 cells/mL; the number of cells in the cell droplet ranged from 2000-4000 cells/cell.
The penicillin/streptomycin double antibody added in this step is additionally and newly added into PBS, is not contained in the organoid special culture medium, and is added to keep the air in the culture dish moist and have an antibacterial effect relative to sterile water.
Wherein the fresh bladder cancer tissue dissociated single cell, the operation steps are as follows:
(1) preparing an enzyme-containing culture medium: 4.7ml RPMI 1640 (server-free) +200ul Enzyme H +100ul Enzyme R +25ul Enzyme A, (Miltenyi human promoter association kit # 130-. The enzyme-containing medium was prepared in serum-free DMEM or serum-free 1640.
(2) Clamping tumor blocks in a 15ml centrifuge tube by using forceps with a length of 20cm and teeth, putting the tumor blocks into a confocal culture dish with the diameter of 3.5mm, continuously shearing the tumor blocks by using medical scissors until no meat blocks are visible to the naked eye, adding a proper amount of Enzyme-containing culture medium into the confocal culture dish, continuing shearing, then adding Enzyme mix into the confocal culture dish, and changing the scissors for micro scissors.
(3) And then obliquely shearing a port of a 1ml tip by using medical scissors, increasing the feeding amount of the tip, adding the Enzyme mix into a confocal culture dish, washing for multiple times to completely transfer the tissues in the confocal culture dish into a 15ml centrifuge tube containing the Enzyme mix, and repeatedly and uniformly blowing.
(4) The crushed tumor tissue is placed at 37 ℃ and 5% CO2Cultured in an incubatorThe sample was taken out every 15 minutes, and continuously blown with a 1ml pipette tip with an end cut off obliquely, and shaken by hand. The effect that the tissue meat blocks are smaller and smaller is achieved, and then the tissue meat blocks are placed in the incubator to be continuously digested. The operation is repeated, and the tumor tissue is digested for 60 min. Until the shape of the tissue can not be seen by blowing, the tissue becomes a single cell suspension, and the sealing glue is wound when the tissue is taken out of the incubator and put back.
(5) After one hour of incubation, a 70um sterile filter was placed on a 50ml sterile centrifuge tube, a 1ml tip was cut obliquely across the end, the single cell suspension was aspirated onto the filter, and the single cell suspension was filtered.
(6) The residue on the filter screen was washed above the screen with an additional 15ml of D-PBS (without ca +, without Mg +, serum-free).
(7) And (5) centrifuging at 1500r/7 min.
(8) The supernatant was first aspirated with a 5ml pipette tip, leaving the cells, and a little supernatant, which was carefully removed by changing the 100ul pipette tip.
(9) Cells were re-selected in 1ml organoid medium.
Matrigel resuspension of single cell suspension dissociated from fresh bladder cancer tissue, to the single cell suspension, 37.5% matrigel (basement membrane matrix) was added. The whole process is carried out on ice, and the used gun head needs to be precooled on the ice in advance.
(3) Standing on ice: placing on ice, and standing at 0-4 deg.C.
Standing the hydrophobic inverted culture dish containing the cell droplets on ice for 15 minutes, wherein the direction of the droplets is downward; then placing the hydrophobic inverted culture dish containing the cell drops in an incubator at 37 ℃ for culturing for 2 hours; the carbon dioxide content in the cell culture box is 5 percent;
(4) the hydrophobic module is cultured in the organoid special culture medium with the liquid drop direction upwards:
transferring the hydrophobic module containing the cell droplets from the culture dish cover to a low-adhesion 6-pore plate, wherein the droplet direction is upward; adding the organoid special culture medium until the organoid special culture medium exceeds the dripping level position of cell sap, and culturing for 10 hours in a cell culture box with the carbon dioxide content of 5 percent at 37 ℃;
(5) tumor spheres continued to culture after release from the hydrophobic module:
separating the organoid from the hydrophobic film and suspending the organoid in the pore plate for continuous culture, thereby facilitating the subsequent drug test. The obtained tumor ball. Releasing the obtained tumor balls from the hydrophobic module to a low-adhesion 24-well plate for long-term culture; changing the culture medium for organoid culture with 20% Matrigel every 2-3 days. The Matrigel content was different, as was the other ingredients.
The bladder cancer culture medium and the bladder cancer organoid culture method provided by the invention have the advantages of high success rate and short time, and the organoids retain immune cells, thereby providing an ideal research model for the occurrence and development of bladder cancer, tumor immune drug screening and in-vitro drug sensitivity test research. The organoid containing immune cells can be quickly prepared within 24 hours, the microenvironment of bladder cancer tumor can be successfully simulated, the medicine consumption is low, and the preparation success rate is over 90 percent. Has great significance for the follow-up research of bladder cancer.
Example 6
Other contents are as in example 5, wherein the baking temperature in step (1) is 75 or 85 ℃.
Adding 1 or 3ml of PBS containing 0.8 or 1.2 percent of penicillin/streptomycin double-resistant solution into a hydrophobic culture dish in the step (2); a single aspiration of 0.15 or 0.25ul of single cell suspension was performed on the hydrophobic module to form a cell droplet.
Standing the hydrophobic inverted culture dish containing the cell drops on ice for 14 or 16 minutes, wherein the direction of the drops is downward; then placing the hydrophobic inverted culture dish containing the cell drops in an incubator at 36 or 38 ℃ for culturing for 1.5 or 2.5 hours; the carbon dioxide content in the cell culture chamber was 4 or 6%.
Culturing at 36 or 38 deg.C for 9 or 11 hr in a cell culture box with carbon dioxide content of 4-6% in step (4).
Example 7
Other contents are as in example 5, a method for culturing bladder cancer organoids, comprising the steps of: centrifuging single cell suspension obtained by dissociating fresh bladder cancer tissue, removing supernatant waste liquid, and resuspending inThe required concentration of the special culture medium for the bladder cancer organoid is 1.5x107One per ml. The starting materials and final concentrations required for organoid culture medium are as described in example 4. The rapid culture comprises the following steps:
(1) a PDMS film with the thickness of 1mm is flatly paved on the surface of glass with the length of 22mm, the width of 22mm and the thickness of 3mm to prepare a biocompatible hydrophobic module, and the hydrophobic module is baked in an oven at the temperature of 80 ℃ for 3 hours; adhering a hydrophobic module on a culture dish cover of a 60mm culture dish by using a double-sided adhesive tape with the width of 20mm and the length of 20mm, wherein a PDMS film faces the bottom of the culture dish to form a hydrophobic inverted culture dish;
(2) sucking 0.2ul of single cell suspension once and placing the single cell suspension on a hydrophobic module to form cell suspension micro-droplets, and sucking for multiple times until the single cell suspension is completely transferred; each cell microdroplet contains 2000-4000 primary tumor cells. 2ml of 1% penicillin/streptomycin-containing double-resistant PBS was added to a 60mm hydrophobic petri dish;
(3) the hydrophobic inverted petri dish containing the cell droplets was left to stand on ice for 15 minutes; transferring the hydrophobic inverted culture dish containing the cell drops to a cell culture box with the temperature of 37 ℃ and the carbon dioxide content of 5 percent for culturing for 2 hours, and waiting for the matrigel to be solidified;
(4) transferring the hydrophobic module containing the cell droplets from the culture dish cover to a low-adhesion 6-pore plate, wherein the droplet direction is upward; adding organoid special culture medium until the culture medium exceeds the horizontal position of cell drop, wherein the culture medium is 2-3ml, and culturing for 10 hours in a cell culture box with the carbon dioxide content of 5% at 37 ℃;
(5) releasing the obtained tumor balls from the hydrophobic module to a low-adhesion 24-well plate for long-term culture; the liquid is changed every 2-3 days according to the growth condition of the organoid, and 2ml of liquid is changed every time. The organoids obtained by culture are directly shot in olympus x71, namely, the detection step is to directly take a hole plate and take a picture under a microscope, and fig. 4 is a bright field picture of human muscle-layer invasive bladder cancer organoids and non-muscle-layer invasive bladder cancer organoids in the invention.
And performing immunofluorescence staining on the bladder cancer tumor organoids to identify a result graph, wherein the immunofluorescence staining is a conventional technology, and is shown in figure 5. It can be seen from FIG. 5 that the organoids of the present invention contain immune cells and retain the immune components of bladder cancer.
And (3) shooting the organoids in the same hole by using a bright field IMAGE shot by an Olympus X71 microscope at the same time period every day, then importing the data IMAGE into IMAGE J for size analysis, and judging that the growth state is continuously growing, such as fig. 6, which shows that the organoids in the invention are proliferated and also verifies that the organoids have activity.
H & E staining of tumor organoids and tissues cultured in the present invention was performed by conventional staining techniques, as shown in FIG. 7, left panel is organoids and right panel is tissues. From the H & E staining results of fig. 7, the organoids and tissues are consistent in their morphological structure and pathologically similar.
Test No.)
Let reference to the control group and the experimental group, the experimental group is the content of example 6, and the control group is the content of other contents such as in example 6, but without matrigel.
The same patient sample was taken for culture, and the experimental data of the third day, the data without matrigel in the left panel and the data without matrigel in the right panel in the left panel of fig. 8.
Having cavities illustrates that this is a organoid in morphology. The fact that no cavity exists can be shown from the back that the matrigel has obvious effect on helping the cell to be pelletized.
While the foregoing shows and describes the fundamental principles and principal features of the invention, together with the advantages thereof, the foregoing embodiments and description are illustrative only of the principles of the invention, and various changes and modifications can be made therein without departing from the spirit and scope of the invention, which will fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A bladder cancer organoid culture medium, comprising: the medium comprises the following components in concentration: 8-12mM of cell culture medium, 145ng/mL of fibroblast growth factor 120-; the others are Advanced DMEM/F12 and micro solvent, the Advanced DMEM/F12 is supplemented to the volume required by the culture medium;
the cell culture medium is Glutamax, the hydrogen ion buffer is Hepes, the antibiotic is penicillin/streptomycin double antibody, the epidermal cell growth factor is EGF, the secretory protein is R-Spondins, the human leukocyte antigen is B27, and the substrate membrane matrix is Matrigel;
the fibroblast growth factors are FGF10, FGF7 and FGF2, wherein the mass ratio of FGF10 to FGF7 to FGF2 is 8: 2: 1; the recombinant protein is Noggin and Wnt-3a, wherein the mass and dosage ratio of the Noggin to the Wnt-3a is 1: 1; the inhibitor is A83-01 and Y-27632;
the PH value in the organoid culture medium is 7.2-7.4;
the culture medium comprises the following components by mass: HEPES 10mM, Glutamax10mM, penicillin/streptomycin double antibody 1%, FGF 10100 ng/mL, FGF 725 ng/mL, FGF212.5 ng/mL, EGF50ng/mL, Noggin 100ng/mL, Wnt3a 100ng/mL, R-Spondin 1500 ng/mL, human Gastrin-110 nM, Nicotinamide 10mM, N-acetylcysteine 1.25mM, Normocin 0.2%, B272%, A83-015 uM, Y2763210.5 uM, Matrigel 37.5%, and the balance Advanced DMEM/F12.
2. Use of a bladder cancer organoid medium according to claim 1, wherein: the application is the rapid culture of bladder cancer organoids; the organoid rapid culture method comprises the steps of preparing a hydrophobic culture dish, inversely hanging single cell suspension on the hydrophobic culture dish in a droplet form, standing on ice and culturing in an incubator to form a hydrophobic module containing cell droplets, culturing the hydrophobic module in a organoid special culture medium with the droplet direction upward, releasing cultured tumor balls from the hydrophobic module, and continuously culturing in a well plate in which the organoid special culture medium is placed to obtain the organoid rapid culture method.
3. Use of a bladder cancer organoid medium according to claim 2, wherein: the organoid rapid culture method in the application specifically comprises the following steps:
(1) manufacturing a hydrophobic culture dish; spreading a PDMS film on the surface of the glass, and baking at 75-85 ℃; then, the glass is adhered to the culture dish cover, and the PDMS film faces the bottom of the culture dish;
(2) the single cell suspension was hung upside down on a hydrophobic culture dish in the form of droplets:
resuspending the bladder cancer tissue single cells, and inversely hanging the cells on a hydrophobic culture dish in a droplet form; sucking 0.15-0.25ul of single cell suspension liquid once and placing the single cell suspension liquid on a hydrophobic module to form cell liquid drops until the single cell suspension liquid is completely transferred;
(3) standing on ice:
standing the hydrophobic inverted culture dish in the step (2) on ice for 14-16 minutes with the liquid drop direction facing downwards; then placing the hydrophobic inverted culture dish containing the cell drops in an incubator at 36-38 ℃ for culturing for 1.5-2.5 hours; the carbon dioxide content in the cell culture box is 4-6%;
(4) the hydrophobic module is cultured in organoid special culture medium with the liquid drop direction upwards:
transferring the hydrophobic module containing the cell droplets from the culture dish cover to a low-adhesion 6-hole plate, wherein the droplet direction is upward; adding organoid culture medium until the culture medium exceeds the dripping position of cell sap, and culturing for 9-11 hr in a cell culture box with carbon dioxide content of 4-6% at 36-38 deg.C;
(5) the tumor spheres continue to culture after release from the hydrophobic module:
and releasing the obtained tumor balls from the hydrophobic module into a low-adhesion 24-pore plate for continuous culture, and changing the liquid every 2-3 days according to the growth condition of the organoid to prepare the organoid.
4. Use of a bladder cancer organoid medium according to claim 3, wherein: adding 1-3ml of PBS containing 0.8-1.2% of penicillin/streptomycin double-antibody into a hydrophobic culture dish in the step (2); the cell concentration of the single cell suspension was 1.5x107Per mL; the mass concentration of matrigel in the resuspended single cell suspension was 37.5%.
5. Use of a bladder cancer organoid medium according to claim 3, wherein: the number of cells in the cell droplet in the step (2) is in the range of 2000-4000 cells/cell.
6. Use of a bladder cancer organoid medium according to claim 3, wherein: and (5) replacing the organoid special culture medium containing 20% Matrigel every 2-3 days according to the growth condition of the organoid.
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