阅读说明：本技术 一种胃癌微肿瘤细胞模型的培养方法 (Culture method of gastric cancer micro-tumor cell model ) 是由 尹申意 张函槊 于 2021-09-27 设计创作，主要内容包括：本发明公开了一种胃癌微肿瘤细胞模型的培养方法。本发明提供了一种新的胃癌微肿瘤模型培养技术及配套试剂,该技术的核心是：(1)用温和细胞解离试剂处理胃癌实体瘤组织,最大程度的保证了组织中各种类型细胞的活力；(2)配制特殊的无血清培养基,利用悬浮培养体系使胃癌组织中分离得到的各类型细胞自组装形成具有多种细胞成分的细胞团结构,称之为“胃癌微肿瘤模型”。利用本发明方法得到的胃癌微肿瘤模型可以准确反应患者原病灶的各种特征,是肿瘤精准诊疗领域良好的科研实验模型和临床前实验模型。可以预见,这种培养方法在胃癌的研究和临床诊断治疗领域具有广泛的应用前景。(The invention discloses a culture method of a gastric cancer micro-tumor cell model. The invention provides a novel gastric cancer micro-tumor model culture technology and a matched reagent, and the core of the technology is as follows: (1) the stomach cancer solid tumor tissue is treated by a mild cell dissociation reagent, so that the activity of various cells in the tissue is ensured to the maximum extent; (2) preparing a special serum-free culture medium, and utilizing a suspension culture system to enable various types of cells separated from gastric cancer tissues to self-assemble to form a cell mass structure with various cell components, which is called as a gastric cancer micro-tumor model. The gastric cancer micro-tumor model obtained by the method can accurately reflect various characteristics of the original focus of a patient, and is a scientific research experimental model and a preclinical experimental model in the field of accurate diagnosis and treatment of tumors. It is expected that the culture method has wide application prospect in the fields of research and clinical diagnosis and treatment of gastric cancer.)

1. A culture medium for culturing a gastric cancer micro-tumor model consists of three anti-bacterial and anti-fungal agents, HEPES, GlutaMax, a non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin, human recombinant protein IL-2, human recombinant protein IL-15, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N2 supplement, cholera toxin, B27, ITS-X, Y-27632, gastrin and an Advanced/F12 culture medium;

wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250 ng/mL; the final concentration of the HEPES is 8-12 mM; the final concentration of the GlutaMax is 0.8-1.2% (volume percentage); the final concentration of the non-essential amino acid solution is 0.8-1.2% (volume percentage); the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the human recombinant protein R-spondin is 250-500 ng/mL; the final concentration of the human recombinant protein IL-2 is 10-100 ng/mL; the final concentration of the human recombinant protein IL-15 is 10-100 ng/mL; the final concentration of the CHIR99021 is 1.5-6 mu M; the final concentration of the SB202190 is 5-10 μ M; the final concentration of the A83-01 is 0.25-1.25 mu M; the final concentration of the Primocin is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of nicotine is 5-10 mM; the final concentration of N2 supplement is 1% (volume percent); the final concentration of cholera toxin is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% (volume percentage); the final concentration of ITS-X is 0.8-1.2% (volume percentage content); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the gastrin is 5-20 nM; the balance is Advanced DMEM/F12 medium.

2. A kit for culturing a gastric cancer micro-tumor model, comprising the medium of claim 1 and all or part of: sample dissociation liquid, sample preservation liquid, sample cleaning liquid, cell digestive juice, digestion stop liquid and cell freezing preservation liquid;

the sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I is 150-250U/mL; the final concentration of the collagenase II is 150-250U/mL; the final concentration of the collagenase IV is 150-250U/mL; the balance being PBS;

the sample preservation solution consists of fetal bovine serum, double-antibody P/S, HEPES and HBSS; wherein the final concentration of the fetal calf serum is 1-5% (volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the final concentration of the HEPES is 8-12 mM; the balance is HBSS;

the sample cleaning solution consists of double-antibody P/S and PBS; wherein the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the balance being PBS;

the cell digest consisted of: every 10mL of the cell digestive juice contains 4-6mL of Accutase, EDTA with the final concentration of 5mM, 1.5-2.5mL of TrypLE Express and the balance of PBS;

the digestion stop solution consists of fetal calf serum, double-antibody P/S and a DMEM medium; wherein the final concentration of the fetal calf serum is 8-12% (volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the rest is DMEM culture medium;

the cell freezing medium consists of an Advanced DMEM/F12 culture medium, DMSO and a 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2); the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

3. Use of the medium according to claim 1 or the kit according to claim 2 for culturing a gastric cancer micro-tumor model.

4. A method for culturing a gastric cancer micro-tumor model comprises the following steps:

(a1) dissociating the gastric cancer solid tumor tissue by using the sample dissociation solution as described in claim 2;

(a2) the method comprises the step of performing suspension culture on the cells dissociated in the step (a1) by using the culture medium of claim 1 to form a cell mass, thereby obtaining the gastric cancer micro-tumor model.

5. The method of claim 4, wherein: in the step (a1), the gastric cancer solid tumor tissue is dissociated with the sample dissociation solution according to a method including the following steps: and (3) according to the dosage that 1mL of the sample dissociation solution does not exceed 0.5mg of tissue, carrying out sample dissociation on the sheared gastric cancer solid tumor tissue by using the sample dissociation solution at the temperature of 37 ℃, wherein the dissociation time is 15 minutes to 2 hours.

6. The method according to claim 4 or 5, characterized in that: in the step (a2), the dissociated cells are suspension-cultured (a1) in the medium according to a method comprising the steps of: suspension culture of dissociated cells (a1) in the medium using a cell culture vessel with a low adsorption surface at 37 ℃ with 5% CO₂Culturing is carried out under the conditions.

7. The method according to any one of claims 4-6, wherein: before the step (a1), the method further comprises the following step of performing dissociation pretreatment on the gastric cancer solid tumor tissue: cleaning the surface of the gastric cancer solid tumor tissue sample by using ethanol with the volume percentage of 70-75%; washing the gastric cancer solid tumor tissue sample with the sample washing solution of claim 2 and then with a sterile PBS solution.

8. The method of claim 7, wherein: the gastric cancer solid tumor tissue sample subjected to the pretreatment for dissociation has an in vitro time of not more than 12 hours, and is stored in the sample storage solution according to claim 2 until the pretreatment for dissociation is performed.

9. The method according to any one of claims 4-8, wherein: in the step (a1), the method further includes the following steps after the dissociation treatment of the stomach cancer solid tumor tissue with the sample dissociation solution: terminating the dissociation reaction with the digestion stop solution according to claim 2, and collecting a cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; and re-centrifuging, and then resuspending the cell pellet with the medium of claim 1.

10. A method for obtaining a primary cell of a gastric cancer solid tumor, which is obtained by separating the primary cell of the gastric cancer solid tumor from the gastric cancer micro-tumor model obtained by the method of any one of claims 4 to 9.

Technical Field

The invention relates to the technical field of biology, in particular to a culture method of a gastric cancer micro-tumor cell model.

Background

Gastric cancer is one of the most common health malignancies that severely threaten humans. The existing treatment methods for the gastric cancer mainly comprise surgery, chemotherapy, targeted therapy, immunotherapy and the like. However, the risk of recurrent metastasis of gastric cancer is high, and more than 50% of gastric cancer patients have recurrent metastasis to different degrees within months to years after radical treatment.

Targeted therapies based on next generation gene sequencing technology (NGS) as well as immunotherapy have completely changed the therapeutic modalities of malignancies in the last 10 years. The new drugs and the new diagnosis and treatment technology improve the prognosis of gastric cancer patients to a certain extent, but the targeted drugs and immune drugs for treating gastric cancer are still scarce, the patient income is very limited, and the overall 5-year survival rate is still not satisfactory.

Gastric cancer is a complex disease, has different pathological types, different genetic backgrounds and different life habits, and has very different disease development and treatment prognosis. Scientific research, clinical research and clinical diagnosis in the field of gastric cancer cannot be generalized, and a precise medical way must be taken. Therefore, the development of a tumor model capable of accurately reflecting the original focus characteristics of a patient is very important, and the existing primary tumor cell culture technologies such as 2D culture, 3D culture, reprogramming culture and the like are all faced with the problems of extremely long culture period, low culture success rate, difficulty in removing mixed cells, incapability of reproducing a tumor microenvironment and the like in different degrees.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel gastric cancer micro-tumor model culture technology and a matched reagent, and the core of the technology is as follows: (1) the stomach cancer solid tumor tissue is treated by a mild cell dissociation reagent, so that the activity of various cells in the tissue is ensured to the maximum extent; (2) preparing a special serum-free culture medium, and utilizing a suspension culture system to enable various types of cells separated from gastric cancer tissues to self-assemble to form a cell mass structure with various cell components, which is called as a gastric cancer micro-tumor model.

In a first aspect, the invention claims a medium for culturing a gastric cancer micro-tumor model.

The invention claims a culture medium for culturing a gastric cancer micro-tumor model, which is prepared from three antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin, human recombinant protein IL-2, human recombinant protein IL-15, CHIR99021, SB 190(4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), A83-01(3- (6-Methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazole-1-carbothioamide), Primocin, N-acetyl-L-cysteine (N-acetyl-L-cysteine), nicotine (Nicotinamide), N2 supplement, Cholera Toxin (Cholera Toxin), B27, ITS-X (Insulin, Transferrin, Selenium, ethanomine Solution), Y-27632, Gastrin (Gastrin), and Advanced DMEM/F12 medium.

Wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-200 mu g/mL (such as 100 mu g/mL); the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (volume percent, e.g., 1%); the final concentration of the non-essential amino acid solution is 0.8-1.2% (volume percentage, such as 1%); the final concentration of the human recombinant protein EGF is 10-100ng/mL (such as 50 ng/mL); the final concentration of the human recombinant protein bFGF is 10-50ng/mL (such as 20 ng/mL); the final concentration of human recombinant protein HGF is 5-25ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein FGF-10 is 5-25ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein Wnt-3a is 200-300ng/mL (such as 200 ng/mL); the final concentration of the human recombinant protein Noggin is 100-200ng/mL (such as 100 g/mL); the final concentration of the human recombinant protein R-spondin is 250-500ng/mL (such as 400 ng/mL); the final concentration of the human recombinant protein IL-2 is 10-100ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein IL-15 is 10-100ng/mL (such as 20 ng/mL); the final concentration of CHIR99021 is 1.5-6. mu.M (e.g., 3. mu.M); the final concentration of SB202190 is 5-10 μ M (e.g., 10 μ M); the final concentration of A83-01 is 0.25-1.25 μ M (such as 1 μ M); the final concentration of the Primocin is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine is 0.5-2mM (such as 1 mM); the final concentration of nicotine (Nicotinamide) is 5-10mM (e.g., 10 mM); the final concentration of N2 supplement is 1% (volume percent); the final concentration of the Cholera Toxin (Cholera Toxin) is 0.1-1nM (e.g., 1 nM); the final concentration of B27 is 1.5-2.5% (volume percent, e.g., 2%); the final ITS-X concentration is 0.8-1.2% (volume percentage, such as 1%); the final concentration of Y-27632 is 5-20 μ M (e.g., 10 μ M); the final concentration of said Gastrin (Gastrin) is 5-20nM (e.g., 10 nM); the balance is Advanced DMEM/F12 medium.

Further, the composition of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is as follows: each ml contains 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B. The antimicrobial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is "antibacterial-antibacterial, 100X" (e.g., Gibco #15240062, or other products of the same composition). The "Antibiotic-Antibiotic, 100X" contained 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B per ml, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline as the active ingredientsAn antifungal agent. The GlutaMax is a high-grade cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMax is GlutaMax^TMSupplement "(e.g., Gibco #35050061, or other products of the same composition). The "GlutaMAX^TMThe Supplement "was composed of L-allyl-L-glutamine as a substitute for L-glutamine at a concentration of 200nM in a 0.85% NaCl solution. The solvent of the non-essential amino acid solution is water, and the solutes and the concentrations are as follows: 10mM of glycine; l-alanine 10 mM; 10mM of L-asparagine; 10mM of L-aspartic acid; 10mM of L-glutamic acid; 10mM of L-proline; l-serine 10 mM. The CHIR99021 is a high-specificity glycogen synthase kinase-3 (GSK-3) inhibitor, inhibits GSK-3 alpha and GSK-3 beta, and has IC50 values of 10nM and 6.7nM respectively. The Primocin is an antibacterial agent (such as Invivogen # ant-pm-1 or other products with the same composition) for primary cells, is an antibiotic for protecting the primary cells from being polluted by microorganisms, and has killing effects on gram-positive bacteria, gram-negative bacteria, mycoplasma and fungi. The N-2Supplement is "N-2 Supplement (100X)" (e.g., Gibco #17502001, or other products of the same composition). The "N-2 Supplement (100X)" contained human holotransferrin (H) at a final concentration of 1mMuman Transferrin (Holo)), 500mg/L Recombinant Insulin whole Chain (Insulin Recombinant Full Chain), 0.63mg/L Progesterone (Progesterone), 10mM Putrescine (Putrescine), 0.52mg/L Selenite (Selenite). The B27 is' B-27^TMSupplement (50X), minus vitamin A "(e.g., Gibco #12587010, or other products of the same composition). Said "B-27^TMExample 50X, minus vitamin A "contains Biotin (Biotin), DL-Alpha-tocopheryl Acetate (DL Alpha-tocopheryl Acetate), DL-Alpha-Tocopherol (DL Alpha-tocopheryl), BSA (fat acid fragment V), Catalase (Catalase), Human Recombinant Insulin (Human Recombinant Insulin), Human Transferrin (Human Transferrin), Superoxide Dismutase (Superoxide Dismutase), Corticosterone (Cortisosterone), D-Galactose (D-Galactose), ethanolamine hydrochloric Acid (Ethanolamine HCl), reduced glutathione (reduced)), L-Carnitine HCl (L-Carnitine HCl), Linoleic Acid (Linoleic Acid), Linolenic Acid (Linolenic Acid), Progesterone (Progesterone), Putrescine (Putrescine 2HCl), Sodium Selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). The ITS-X solvent is EBSS solution (Earle's balanced salt solution), and the solutes and the concentrations are as follows: 1g/L of insulin; 0.55g/L of transferrin; 0.00067g/L sodium selenite; ethanolamine 0.2 g/L. Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 220nM and 300nM, respectively)" (e.g. MCE #129830-38-2, or other products of the same composition).

In a specific embodiment of the invention, the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is under the brand code Gibco # 15240062; the brand of HEPES is Gibco # 15630080; the brand name of GlutaMax is Gibco # 35050061; the non-essential amino acid solution is sold under the brand name of Gibco # 11140-050; the brand of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand of the human recombinant protein FGF-10 is Peprotech AF-100-26-100; the brand and commodity number of the human recombinant protein Wnt-3a is R & D5036-WN-500; the brand and cargo number of the human recombinant protein Noggin is Shanghai nearshore # CB 89; the brand of the human recombinant protein R-spondin is Shanghai nearshore # CX 83; the brand and commodity number of the human recombinant protein IL-2 is Peprotech 200-02; the brand code of the human recombinant protein IL-15 is Peprotech 200-015; the brand name of the CHIR99021 is Selleck # S2924; the brand of the SB202190 is Sigma # S7067; the brand name of A83-01 is Tocris # 2939; the brand name of the Primocin is Invivogen # ant-pm-1; the brand and cargo number of the N-acetyl-L-cysteine is Sigma # A9165; the brand of Nicotinamide is Sigma # N0636; the brand goods number of the N-2Supplement is Gibco # 17502001; the brand name of Cholera Toxin is Listlab # 100B; the brand name of B27 is Gibco # 12587010; the ITS-X brand has a goods number of Gibco # 51500056; the brand goods number of the Y-27632 is MCE # 129830-38-2; the brand goods number of Gastrin is NJPeptide # Pep 12307; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

Further, the medium may be present in two forms:

one, wherein the culture medium is composed of the antibacterial antifungal agent tris antibody (penicillin-streptomycin-amphotericin B), the HEPES, the GlutaMax, the non-essential amino acid solution, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the human recombinant protein R-spondin, the human recombinant protein IL-2, the human recombinant protein IL-15, the CHIR99021, the SB202190(4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), the A83-01(3- (6-Methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazole-1H-imidazole) -carbothioamide), said Primocin, said N-acetyl-L-cysteine (N-acetyl-L-cysteine), said nicotine (Nicotinamide), said N2 supplement, said Cholera Toxin (Cholera Toxin), said B27, said ITS-X (Insulin, Transferrin, Selenium, Ethanolamine Solution), said Y-27632, said Gastrin (Gastrin) and said Advanced DMEM/F12 medium.

The media was prepared and sterilized by filtration through a 0.22 μ M needle filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

Secondly, each component in the culture medium exists independently and is prepared according to a formula when in use.

Furthermore, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the human recombinant protein R-spondin, the human recombinant protein IL-2 and the human recombinant protein IL-15 can exist in a stock solution (mother solution) form, wherein the stock solution can be stored for a long time at (-80 ℃), and particularly can be 1000 times of the stock solution (mother solution). SB202190, N-acetyl-L-cysteine, Nicotinamide, Y-27632 and Gastrin can exist in the form of stock solution (mother liquor), and can be stored for a long time at-20 deg.C, specifically 1000 times of stock solution (mother liquor). CHIR99021 and Cholera Toxin can exist in stock solution (mother liquor), and can be stored for a long time at-20 deg.C, specifically 10000 times of stock solution (mother liquor). A83-01 can exist in stock solution (mother liquor) form (long-term storage at-20 deg.C), specifically 100000 times stock solution (mother liquor).

The 1000 Xhuman recombinant protein EGF stock solution consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The stock solution of 1000 Xhuman recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein HGF stock solution consists of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant proteins HGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein FGF-10 stock solution consists of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein Wnt-3a stock solution consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 multiplied human recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein R-spondin stock solution is composed of human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein IL-2 stock solution consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The stock solution of 1000 multiplied human recombinant protein IL-15 consists of human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

In the nine 1000-fold stocks, the BSA can be present (now prepared) in the form of 100-fold stock (mother liquor), and specifically consists of BSA and PBS, wherein the final concentration of BSA (Sigma # A1933) is 0.1g/mL, and the balance is PBS.

In addition, a 10000 × CHIR99021 stock consists of CHIR99021 and DMSO, wherein the final concentration of CHIR99021 is 30mM, and the balance is DMSO.

The 1000 × SB202190 stock consists of SB202190 and DMSO, wherein the final concentration of SB202190 is 10mM, the balance being DMSO.

The 100000 XA 83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25mM, and the balance is DMSO.

The 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is the ultrapure water.

The 1000 XNicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of the Nicotinamide is 5M, and the balance is the ultrapure water.

10000 XCholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin solution, wherein the final concentration of Cholera Toxin is 10 μ M, and the rest is Cholera Toxin solution. The Cholera Toxin dissolving solution comprises the following components: each 10mL of the Cholera Toxin solution contained Tris (1M) pH 7.00.05M, NaCl 0.2M, sodium azide 3mM, EDTA (0.5M) pH 8.01 mM, and the balance ultrapure water.

1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.

1000 XGastrin (Gastrin) stock solution is composed of Gastrin (Gastrin) and ultrapure water, wherein the final concentration of Gastrin is 10 μ M, and the balance is ultrapure water.

In a second aspect, the invention claims a kit for culturing a gastric cancer micro-tumor model.

The kit claimed in the present invention for culturing a gastric cancer micro-tumor model consists of the culture medium as described in the first aspect and all or part of: sample dissociation liquid, sample preservation liquid, sample cleaning liquid, cell digestion liquid, digestion stop liquid and cell freezing and storing liquid.

The sample preservation solution can be used for temporarily preserving a sample after the sample is separated, and can maintain the activity of cells in the sample in a short time after the sample is separated. The sample preservation solution can be preserved for 1 month at 4 ℃ after being prepared.

The sample washing solution can be used for washing and disinfecting a sample. The sample cleaning solution needs to be ready for use.

The sample dissociation liquid can be used for dissociation of a sample. The sample dissociation solution needs to be prepared at present, wherein collagenase I, collagenase II and collagenase IV can be stored for a long time at the temperature of-20 ℃ in a stock solution (mother solution), and specifically, the stock solution (mother solution) can be 10 times. The 10 × collagenase I stock consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; the balance being PBS. A 10 × collagenase II stock solution consists of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance being PBS. The 10 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS. The enzyme activities of collagenase I, collagenase II and collagenase IV are defined below.

The cell digestive juice can be used for digestion and passage of cell masses and can digest gastric cancer tumor masses into single cells. The cell digestive juice is required to be prepared immediately.

The digestion stop solution can be used for stopping the dissociation of the sample or the digestion process of the cells. The prepared digestion stop solution can be stored for one month at 4 ℃.

The sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U/mL (such as 200U/mL); the final concentration of collagenase II is 150-250U/mL (such as 200U/mL); the final concentration of collagenase IV is 150-250U/mL (such as 200U/mL); the balance being PBS.

Wherein the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of a protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I or said collagenase II or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

In a specific embodiment of the present invention, the brand name of collagenase I is Gibco # 17100-017; the brand of collagenase II is Gibco # 17101-015; the brand goods number of the collagenase IV is Gibco # 17104-; the PBS was branded under Gibco # 21-040-CVR.

The sample preservation solution consists of fetal bovine serum, double-antibody P/S, HEPES and HBSS; wherein the final concentration of fetal calf serum is 1-5% (such as 2%,% represents volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the balance being HBSS.

In a specific embodiment of the invention, the brand of fetal bovine serum is Gibco # 16000-; the brand goods number of the double-antibody P/S is Gibco # 15140122; the brand of HEPES is Gibco # 15630080; the HBSS is sold under the brand name Gibco # 14170161.

The sample cleaning solution consists of double-antibody P/S and PBS; wherein the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the balance being PBS.

In a specific embodiment of the invention, the brand of the double antibody P/S is Gibco # 15140122; the PBS was branded under Gibco # 21-040-CVR.

The cell digest consisted of: each 10mL of the cell digest contains 4-6mL (e.g., 5mL) of Accutase, a final concentration of 5mM EDTA, 1.5-2.5mL (e.g., 2mL) of TrypLE Express, and the balance PBS.

Further, the Accutase is StemPro^TMAccutase^TMCell discovery Reagent "(e.g., Gibco # A11105-01, or other products of the same composition). The Accutase is a single-component enzyme, and is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is' TrypLE^TMExpress Enzyme (1X), no phenol red "(e.g., Gibco #12604013, or other products of the same composition). The TrypLE^TMExpress Enzyme (1X), no phenol red "contains 200mg/L KCl and 200mg/L KH₂PO₄8000mg/L NaCl, 2160mg/L Na₂HPO₄·7H₂O, 457.6mg/L EDTA; also contains recombinant protease.

In a specific embodiment of the invention, the brand name of the Accutase is Gibco # A11105-01; the brand name of the 0.5MEDTA is Invitrogen # AM 9261; the brand goods number of the TrypLE Express is Gibco # 12604013; the PBS was branded under Gibco # 21-040-CVR.

The digestion stop solution consists of fetal calf serum, double-antibody P/S and a DMEM medium; wherein the final concentration of fetal calf serum is 8-12% (such as 10%,% represents volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the balance is DMEM medium.

In a specific embodiment of the invention, the brand of fetal bovine serum is Gibco # 16000-; the brand goods number of the double-antibody P/S is Gibco # 15140122; the DMEM medium is sold under the brand name Gibco # 11965-092.

The cell freezing medium consists of an Advanced DMEM/F12 culture medium, DMSO and a 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2) (such as 20:2: 1); the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

In a specific embodiment of the invention, the Advanced DMEM/F12 medium is under the brand code Gibco # 12634010; the brand code of the DMSO is Sigma # D2438; the brand of methylcellulose is Sigma # M7027.

In a third aspect, the invention claims the use of a medium as described in the first aspect above or a kit of parts as described in the second aspect above for culturing a gastric cancer micro-tumor model.

In a fourth aspect, the invention claims a method of culturing a gastric cancer micro-tumor model.

The method for culturing the gastric cancer micro-tumor model claimed by the invention can comprise the following steps:

(a1) dissociating the gastric cancer solid tumor tissue by using the sample dissociation solution described in the second aspect;

(a2) and (3) carrying out suspension culture on the cells dissociated in the step (a1) by using the culture medium of the first aspect to form a cell mass, so as to obtain the gastric cancer micro-tumor model.

Further, in the step (a1), the gastric cancer solid tumor tissue may be dissociated by the sample dissociation solution according to a method including the following steps: and (3) according to the dosage of 1mL of the sample dissociation solution not more than 0.5mg of tissue, carrying out sample dissociation on the minced gastric cancer solid tumor tissue by using the sample dissociation solution at the temperature of 37 ℃, wherein the dissociation time is 15 minutes to 2 hours (such as 1 hour).

Further, in the step (a2), the dissociated cells may be suspension-cultured (a1) with the medium according to a method including the steps of: cell culture vessel using low-adsorption surface (low-adsorption-surface), utilizing the culture mediumSuspension culture (a1) of dissociated cells at 37 ℃ in 5% CO₂Culturing is carried out under the conditions.

Wherein the initial seeding density may be 10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density of individual cells was plated.

Further, the culturing period in the step (a2) is 3 to 5 days.

Further, before the step (a1), the method may further comprise the step of performing dissociation pretreatment on the gastric cancer solid tumor tissue as follows: cleaning the surface of the gastric cancer solid tumor tissue sample for 10-30 seconds by using ethanol with the volume percentage of 70-75%; washing the gastric cancer solid tumor tissue sample 5-10 times (e.g., 5 times) with the sample washing solution of the second aspect, and washing the gastric cancer solid tumor tissue sample 5-10 times (e.g., 5 times) with a sterile PBS solution; then removing impurities, connective tissues, adipose tissues, necrotic tissues and other components which influence the culture of the primary cells in the gastric cancer solid tumor tissue sample.

The step of pretreatment for dissociation of the gastric cancer solid tumor tissue needs to be operated on ice, and the whole operation step needs to be completed within 10 minutes.

Further, the gastric cancer solid tumor tissue sample subjected to the dissociation pretreatment is isolated within 12 hours and stored in the sample storage solution of the second aspect before the dissociation pretreatment.

Further, in the step (a1), the method further includes, after the dissociation treatment of the stomach cancer solid tumor tissue with the sample dissociation solution: terminating the dissociation reaction with an 8-15 (e.g., 10) fold volume of the digestion stop solution described in the second aspect above, and collecting a cell suspension; filtering the cell suspension with a 100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; 800-1000g (e.g., 800g) of the suspension is centrifuged at room temperature for 10-15 minutes (e.g., 10 minutes), and the supernatant is discarded; then resuspend the cells in 3-5mL (e.g., 5mL) sterile PBS; centrifuging at room temperature for 10-15 min (such as 10 min) again at 800-; the cell pellet is then resuspended in the medium as described above in the first aspect.

In a fifth aspect, the invention claims a method for obtaining primary cells of gastric cancer solid tumors.

The method for obtaining the primary cell of the gastric cancer solid tumor claimed by the invention is to separate the primary cell of the gastric cancer solid tumor from the gastric cancer micro-tumor model obtained by the method in the fourth aspect.

The method specifically comprises the following steps:

(b1) digesting the gastric cancer micro-tumor model by using cell digestive juice (such as the cell digestive juice) to obtain single cells;

further, the method also comprises the step of terminating digestion. Digestion was terminated as described previously with a digestion stop solution.

(b2) And (b) selecting CD326 positive cells from the single cells obtained in the step (b1), and obtaining the gastric cancer solid tumor primary cells.

Further, CD326 positive cells can be sorted by CD326 magnetic beads.

In the above aspects, the gastric cancer may be primary gastric cancer, the pathological stage is stage II or stage III, and the weight of the gastric cancer specimen is more than 20 mg.

In the present invention, all of the above PBS's may be 1 XPBS, pH 7.3-7.5. The concrete composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH (Perkin Elmer)₂PO₄ 144mg/L，NaCl 9000mg/L，Na₂HPO₄·7H₂O 795mg/L。

The invention provides a method for extracting and culturing a gastric cancer micro-tumor model from a fresh gastric cancer solid tumor tissue and a matched reagent, and the method has the following advantages:

1. the dosage of the tissue sample is less, and only about 20mg of gastric cancer operation sample is needed;

2. the culture period is short, and only 3-5 days are needed to obtain 10⁵An order of magnitude gastric cancer micro-tumor model;

3. the culture stability is high, and the success rate of in vitro culture of the qualified gastric cancer operation specimen by using the method is up to 75 percent;

4. the cell types are rich, and the gastric cancer micro-tumor model can preserve a plurality of cell types such as tumor cells, interstitial cells, immune cells and the like in the original focus and well reappear the tumor microenvironment;

5. the gastric cancer micro-tumor model can accurately reproduce the pathological subtype of the original focus;

6. the gastric cancer micro-tumor model can accurately reproduce the genetic background of the original focus.

The gastric cancer micro-tumor model obtained by the method can accurately reflect various characteristics of the original focus of a patient, and is a scientific research experimental model and a preclinical experimental model in the field of accurate diagnosis and treatment of tumors. It is expected that the culture method has wide application prospect in the fields of research and clinical diagnosis and treatment of gastric cancer.

Drawings

Fig. 1 is a bright field image of 48 hours before the formation of the gastric cancer micro-tumor model. The scale is 200 μm, 100 times magnification.

FIG. 2 is a comparison graph of HE staining effect of gastric cancer micro-tumor model and corresponding original lesion tissue sample. The scale is 50 μm, 400 times magnification.

FIG. 3 is a comparison graph of multiple immunofluorescence staining effects of gastric cancer micro-tumor models and corresponding primary lesion tissue samples. The scale is 50 μm, 400 times magnification.

FIG. 4 is a bright field diagram and immunofluorescence staining result of isolated and purified gastric cancer primary tumor cells.

FIG. 5 shows the comparison results of copy number variation analysis (CNV) of gastric cancer micro-tumor model and corresponding primary lesion tissue sample obtained by culturing in the four culture media of scheme A, B, C and E.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of reagents for culturing gastric cancer micro-tumor models

1. Sample preservation solution (100mL)

The specific formulation of the specimen preservation solution (100mL) is shown in table 1.

TABLE 1 sample preservation solution (100mL)

After the preparation of the sample preservation solution is completed, the sample preservation solution is subpackaged by 15mL centrifuge tubes, and each tube is 5 mL. Can be stored at 4 deg.C for 1 month after subpackaging.

2. Sample cleaning solution (100mL)

The specific formulation of the sample rinse (100mL) is shown in table 2.

TABLE 2 sample cleaning solution (100mL)

The sample cleaning solution needs to be prepared for use.

3. Sample dissociation liquid (10mL)

The specific formulation of the sample dissociation solution (10mL) is shown in table 3.

TABLE 3 sample dissociation solution (10mL)

Note: the sample dissociation liquid is prepared for use.

In Table 3, the formulation of collagenase stocks is shown in tables 4-6.

TABLE 4 10 Xcollagenase I stock solution (100mL)

After preparing the 10 Xcollagenase I stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 5 10 Xcollagenase II stock solution (100mL)

After preparing the 10 Xcollagenase II stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 6 10 Xcollagenase IV stock solution (100mL)

After preparing the 10 Xcollagenase IV stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

In tables 4, 5 and 6, the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of the protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I or said collagenase II or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

4. Cell digestive juice (10mL)

The specific formulation of the cell digest (10mL) is shown in Table 7.

TABLE 7 cell digest (10mL)

The cell digestive juice is prepared for use.

5. Digestive stop solution (100mL)

The specific formulation of the digestion-stopping solution (100mL) is shown in Table 8.

TABLE 8 digestive stop solution (100mL)

The digestion stop solution can be stored for one month at 4 ℃ after being prepared.

6. Gastric cancer micro-tumor model culture medium (100mL)

The specific formulation of gastric cancer micro-tumor model medium (100mL) is shown in table 9.

TABLE 9 gastric cancer micro-tumor model culture medium (100mL)

After the gastric cancer micro-tumor model medium is prepared, it is sterilized by filtration using a 0.22 μ M needle filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

In Table 9, the preparation of human recombinant protein stocks is shown in tables 11 to 19 (the preparation of BSA stock is shown in Table 10), the preparation of CHIR99021 stock is shown in Table 20, the preparation of SB202190 stock is shown in Table 21, the preparation of A83-01 stock is shown in Table 22, the preparation of N-acetyl-L-cysteine stock is shown in Table 23, the preparation of Nitotinamide stock is shown in Table 24, the preparation of Cholera Toxin stock is shown in tables 25 and 26, the preparation of Y-27632 stock is shown in Table 27, and the preparation of Gastrin (Gastrin) stock is shown in Table 28.

TABLE 10 100 XBSA solution (1mL)

The 100 × BSA solution is ready for use.

TABLE 11 1000 × stock solution of human recombinant protein EGF (5mL)

After 1000 Xhuman recombinant protein EGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 12 stock solution of 1000 × human recombinant protein bFGF (2.5mL)

After 1000 Xhuman recombinant protein bEGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 13 1000 Xhuman recombinant protein HGF stock solution (5mL)

1000 Xthe human recombinant protein HGF stock solution is prepared and subpackaged by a sterile centrifuge tube of 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 14 1000 Xhuman recombinant protein FGF-10 stock solution (5mL)

After 1000 Xhuman recombinant protein FGF-10 stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 15 1000 Xhuman recombinant protein Wnt-3a stock solution (2.5mL)

1000 Xthe human recombinant protein Wnt-3a stock solution is prepared and then subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 16 1000 Xhuman recombinant protein Noggin stock solution (5mL)

1000 times of human recombinant protein Noggin stock solution is prepared and then subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at the temperature of minus 80 ℃.

TABLE 17 stock solutions of 1000 × human recombinant protein R-spondin (4mL)

After 1000 Xhuman recombinant protein R-spondin stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 18 1000 stock solutions of human recombinant protein IL-2 (5mL)

After 1000 Xhuman recombinant protein IL-2 stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 19 stock solution of 1000 × human recombinant protein IL-15 (5mL)

After 1000 Xhuman recombinant protein IL-15 stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 20 10000 XCHIR 99021 stock solution (1.16mL)

10000 XCHIR 99021 stock solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be preserved for a long time at the temperature of 20 ℃ below zero.

TABLE 21 1000 XSB 202190 stock solution (1.51mL)

After preparing the stock solution of 1000 XSB 202190, the stock solution can be stored for a long time at-20 ℃ by subpackaging with a 0.5mL sterile centrifuge tube.

TABLE 22 100000 XA 83-01 stock solution (1.05mL)

After preparing a stock solution of 1000 XA 83-01, the stock solution can be stored for a long time at-20 ℃ by dispensing with a 0.5mL sterile centrifuge tube.

TABLE 23 1000 XN-acetyl-L-cysteine stock solutions (5mL)

After preparing a stock solution of 1000 XN-acetyl-L-cysteine, subpackaging the stock solution by using a sterile centrifuge tube with the volume of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

TABLE 24 1000 XNicotinamide stock solutions (4mL)

1000 XNicotinamide stock solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be stored for a long time at the temperature of minus 20 ℃.

TABLE 25 10000 XCholera Toxin stock solution (1.17mL)

After preparing a stock solution of 1000 XCholera Toxin, subpackaging the stock solution by using a sterile centrifuge tube of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

The specific formulation of Cholera Toxin solution in Table 25 is shown in Table 26.

TABLE 26 Cholera Toxin solution (10mL)

The Cholera Toxin dissolving solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the solution can be preserved for a long time at the temperature of minus 20 ℃.

TABLE 27 1000 XY-27632 stock solution (3.125mL)

After preparing the stock solution of 1000 XY-27632, the stock solution is subpackaged by a sterile centrifuge tube of 0.5mL and can be stored for a long time at the temperature of minus 80 ℃.

TABLE 28 1000 Gastrin stock solution (48mL)

1000 Xgastrin stock solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be preserved for a long time at the temperature of minus 20 ℃.

7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 29.

TABLE 29 cell cryopreservation solution

The cell frozen stock solution is prepared for use at present.

In table 29, the preparation of the 1% methylcellulose solution is shown in table 30.

TABLE 30 1% methylcellulose solution (10mL)

The 1% methyl cellulose solution can be stored for a long time at 4 ℃ after being prepared.

Example 2 acquisition of postoperative specimens for gastric cancer

1. In cooperation with Hospital, samples were obtained in a clinical study format initiated by the investigator, and the cooperative development passed a formal medical ethical review.

2. The attending physician selects patients to be grouped according to clinical indications specified by medical guidelines, and selects appropriate samples for in vitro culture according to intraoperative clinical indications, the selection criteria of the samples are as follows: primary stomach cancer, the pathological stage is stage II or stage III, and the weight of the stomach cancer specimen exceeds 20 mg.

3. All cases are uniformly coded by the sample collection date plus four days after the patient hospitalization number, for example, the sample is provided 1 month and 1 day of 2020, the patient hospitalization number is T001537474, and the sample experiment number is 202001017474. The information related to the privacy of the patient, such as the name, the identification card number and the like of the patient, is hidden. When providing samples, hospitals provide basic clinical information of pathological staging, clinical diagnosis and the like of patients.

The primary physician provides basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc. of the patient. The name, the identification card number and other information of the patient related to the privacy of the patient are hidden and replaced by a uniform experiment number, and the naming principle of the experiment number is eight-digit numerical date of the collected sample plus four digits after the patient is hospitalized.

4. After the tumor tissue is isolated in the operation, a sample collection specialist collects a fresh sample in the sterile environment of an operating room, and the part with rich fresh blood vessels needs to be selected for collecting the sample, so that the part with poor cell activity, such as necrotic tissue, adipose tissue, fibrotic tissue and the like, is avoided. The collected samples were placed in sample stocks (see example 1) pre-chilled at 4 ℃. Temporarily storing the sample storage tube containing the sample on ice, transporting the sample storage tube to a laboratory within 12 hours for next operation, and controlling the temperature to be 2-8 ℃ in the transportation process.

Example 3 pretreatment for dissociation of gastric cancer tissue sample

The following operations required working on ice and the entire procedure required completion within 10 minutes.

Surgical instruments used in the following operations all need to be sterilized by high-temperature steam (120 ℃ for 20 minutes) in advance and then dried for use.

1. The sample was weighed and the surface of the sample was rinsed with medical grade alcohol (75% by volume) for 10 to 30 seconds.

2. The samples were washed 5 times with sample wash and 5 times with sterile PBS solution.

3. The fat tissue, connective tissue and necrotic tissue in the sample are carefully stripped off with the aid of an ophthalmic scissors, an ophthalmic forceps, a scalpel and the like.

Example 4 gastric cancer tissue sample dissociation

The surgical instruments used in the following examples were sterilized with high-temperature steam (120 ℃ C., 20 minutes) and dried before use.

1. Cutting the tissue into 0.5mm pieces with ophthalmic scissors³The left and right small blocks.

2. Tissue was treated with sample dissociation fluid (see example 1), with 1mL sample dissociation fluid for tissue with a sample size of no more than 0.5mg, and 0.1mL sample dissociation fluid for tissue with a sample size of more than 0.5mg per 0.1mg increase in tissue weight. The treatment conditions of the sample dissociation solution were 37 ℃ and the dissociation time was 1 hour. The dissociation of the samples was observed under a microscope every 15 minutes during dissociation until most of the cells were observed to be shed from the tissue.

3. The dissociation reaction was terminated with 10 times the volume of the digestion-terminating solution (see example 1), and after the cell suspension was filtered through a 100 μm sterile cell strainer to remove tissue debris and adherent cells, 800g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

4. The cells were resuspended in 5mL sterile PBS, centrifuged at 800g for 10 minutes at room temperature, and the supernatant discarded.

5. Resuspending the cell pellet with gastric cancer micro-tumor model culture medium (see example 1), counting cells, measuring cell viability by trypan blue staining, and performing cell inoculation culture until the cell viability obtained by separation is more than 70%.

Example 5 gastric cancer micro-tumor model culture

1. The gastric cancer micro-tumor model suspension culture is carried out by using a low-adsorption surface (low-adsorption surface), wherein the used culture medium is a gastric cancer micro-tumor model culture medium in Table 9 of example 1 (wherein the final concentration of the human recombinant protein EGF is 50ng/mL, the final concentration of the human recombinant protein bFGF is 20ng/mL, the final concentration of the human recombinant protein HGF is 20ng/mL, the final concentration of the human recombinant protein FGF-10 is 20ng/mL, the final concentration of the human recombinant protein Wnt-3a is 200ng/mL, the final concentration of the human recombinant protein Noggin is 100g/mL, the final concentration of the human recombinant protein R-spondin is 400ng/mL, the final concentration of the human recombinant protein IL-2 is 20ng/mL, the final concentration of the human recombinant protein IL-15 is 20ng/mL, the final concentration of the CHIR99021 is 3 mu M, the final concentration of the SB202190 is 10 mu M, and the final concentration of the human recombinant protein IL-2 is 20ng/mL The final concentration of A83-01 is 1 μ M; the final concentration of the N-acetyl-L-cysteine is 1 mM; the final concentration of the Nicotinamide is 10 mM; the final concentration of Cholera Toxin is 1 nM; the final concentration of the Y-27632 is 10 mu M; the final concentration of Gastrin is 10nM), as exemplified by a six-well plate, 10 per well⁶Individual cells were plated at density, using 2-3mL of medium per well. Inoculated cells were 37 ℃ and 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

2. The cell state was observed every day until the cells formed clumps of about 100 μm in diameter, after which the medium was changed every 2-3 days to maintain the growth state of gastric cancer micro-tumors.

As shown in FIG. 1, during the first 48 hours of culture, many different types of cells derived from cancer tissues spontaneously aggregate and self-assemble to form a cell mass structure of 100 μm in size, which we callIs a micro-tumor model. The total number of micro-tumor cell masses can reach 10⁵-10⁶. Through a large number of sample tests, the success rate of culturing the micro-tumor models of different gastric cancer operation samples can reach 75%.

Example 6 HE staining comparative identification of gastric cancer micro-tumor model and Primary lesions

The reagent consumables used in the following examples are illustrated:

HE staining kit (beijing solibao biotechnology limited, # G1120);

cation anticreep slide (Beijing China fir Jinqiao Biotech limited);

xylene, methanol, acetone (Beijing chemical reagent company, analytical pure);

neutral resin adhesive (fine chemicals, GmbH, Beijing).

1. Collecting the tissue blocks of the gastric carcinoma primary focus, wherein the size of the soybean grains is 5mm square tissue blocks; the gastric cancer micro-tumor mass obtained in example 5 was collected and centrifuged at 2000rpm for 10min at room temperature to prepare a cell pellet.

2. The original lesion Tissue mass and the micro-tumor cell pellet were dehydrated and fixed separately (standard program of dehydrator, cherry Tissue0Tek VIP 5Jr1 dehydrator).

3. The dehydrated and fixed original lesion tissue blocks and micro-tumor cell precipitation blocks are respectively subjected to paraffin embedding (Leica EG1150H paraffin embedding machine, Leica).

4. The embedded original focus tissue block and the micro-tumor cell precipitation mass wax block are respectively sliced to the thickness of 5 mu m (come Leica RM2245 semi-automatic rotary wheel type slicer).

5. Spreading the slices (come Leica HI1210 spreading machine), attaching on cation anticreep glass sheet, baking (come Leica HI1220 baking machine), and making into paraffin sections for use.

6. HE staining was performed on the primary lesion and the micro-tumor sections with an HE staining kit, respectively: after the gradient ethanol rehydration, the hematoxylin stain was stained for 3 minutes and the slides were washed 3 times with tap water. 100 μ L of the differentiation medium was differentiated for 1 minute, and the slide was washed 2 times with tap water and 1 time with distilled water. After staining with eosin stain for 1 min, gradient ethanol was dehydrated. After the ethanol is dried, 50 mu L of dimethylbenzene is dripped into each glass slide for permeation. After xylene is completely dried, a drop of neutral resin adhesive is dropped, and the piece is sealed by a cover glass.

7. And observing the dyeing effect under a mirror, and taking a picture.

Fig. 2 shows a contrast graph of HE staining effect of the cultured gastric cancer micro-tumor model and the corresponding original lesion tissue sample, and it can be seen that the gastric cancer micro-tumor model can maintain the pathological structural characteristics of the original lesion.

Example 7 multiple immunofluorescence staining identification of gastric cancer micro-tumor model and Primary lesions

The reagents used in the following examples are illustrative:

paraformaldehyde (Beijing chemical reagent company, analytical pure) was dissolved in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;

methanol, dimethyl sulfoxide (Beijing chemical reagent company, analytical pure);

hydrogen peroxide (beijing chemicals, 35%);

mixing methanol, dimethyl sulfoxide and 35% hydrogen peroxide according to a volume ratio of 4:4:1 to prepare a Dan's rinsing solution;

bovine serum albumin (Sigma, # A1933) was dissolved in PBS to prepare a 3% (3g/100mL) BSA solution;

immunofluorescent primary anti-antibodies (Abcam # ab215838, abclonal # A19607, Biolegend # 300434);

immunofluorescent secondary antibodies (CST #4408s, CST #8889 s);

hoechst dye liquor (Beijing Sorleibao Biotech limited, # C0021);

taking the paraffin section obtained in example 6 as a material, multiple immunofluorescent staining was performed according to the following steps, and three antibodies were labeled pan-CK to characterize epithelial-derived tumor cells, Vimentin labeled mesenchymal cells, and CD3 labeled T cells, respectively:

1. paraffin sections were dewaxed and rehydrated, washed once with PBS and treated with pre-chilled methanol solution for 1 hour.

2. The Dane rinse was treated at room temperature for 2 hours, followed by 75%, 50%, 25% (by volume) methanol diluted in PBS for 10 minutes each.

3. 3% BSA solution was blocked for 2 hours at room temperature.

4. Primary antibody mix dilutions (3% BSA solution) primary antibody overnight at 4 ℃.

5. Sections were washed 5 times with PBS solution for 20 minutes each.

6. The secondary antibody mixed dilution (diluted with 3% BSA solution at a ratio of 1: 2000) was used for a secondary antibody at room temperature for 2 hours.

7. Sections were washed 5 times with PBS solution for 20 minutes each.

8. And (5) dyeing by using Hoechst dye liquor for 20 minutes at room temperature.

9. The gel-coated tablets were mounted and stained using a confocal laser microscope and photographed.

Fig. 3 shows the effect diagram of multiple immunofluorescent staining of the gastric cancer micro-tumor model obtained by culturing and the corresponding original lesion tissue sample, and it can be seen that in the gastric cancer micro-tumor model, not only the panCK positive (green fluorescence) tumor cells in the original lesion are retained, but also the Vimentin positive mesenchymal cells (white) and immune cells (T cell blue) are retained, and the cell diversity and microenvironment of the original lesion are reproduced to the greatest extent. The high similarity of the micro-tumor model obtained by the culture of the method and the original focus in terms of cell composition is proved. We performed multiple immunofluorescence staining identification on 10 gastric cancer micro-tumor samples and corresponding original focus tissue blocks, and the statistical results are shown in table 31.

TABLE 31 multiple immunofluorescence staining identification of gastric cancer micro-tumor models and their corresponding primary lesion tissue samples

Example 8 isolation of gastric cancer Primary tumor cells from gastric cancer micro-tumor model

The cell magnetic bead sorting procedure mentioned in the examples below uses the magnetic bead positive selection kit of Meitian whirlwind CD326 (Meitian whirlwind # 130-.

1. The gastric cancer micro-tumor model cultured in suspension in example 5 was collected, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

2. The cell pellet was washed once with sterile PBS solution, centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded.

3. The cell pellet was re-selected with cell digest (see example 1), digested at 37 ℃ for 5-30 minutes, and the digestion of the cell pellet was observed under a microscope every 5 minutes until most of the cell pellet was digested into single cells.

4. Digestion was stopped with 10 volumes of digestion stop solution (see example 1), the cell suspension was centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded.

5. And (3) sorting the single cell suspension obtained by digestion by using a Meitian whirly CD326 magnetic bead positive sorting kit to obtain CD326 positive tumor cells: resuspending the cell pellet with sorting buffer (formula: Table 32) at a cell count of less than 10⁷The system was incubated with 20 μ of LCD326 magnetic beads on ice for 30 minutes. The cell pellet was washed with 2mL of sorting buffer. The column was washed with 5mL of sorting buffer and placed on a magnetic rack. The cell suspension was passed through a cell sorting column, which was washed three times with 3mL of sorting buffer, and CD326 negative cells were eluted. The column was removed from the magnetic frame and washed once with 5mL of sorting buffer to elute CD326 positive cells.

TABLE 32 sorting buffer

Note: it is used as it is.

6. The sorted CD 326-positive cells were centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded. DMEM medium containing 10% serum at 10%⁵And/3.5 cm cell culture dish density for inoculation and adherent culture.

Example 9 passage of gastric cancer Primary tumor cells

1. Adherent cultured gastric cancer primary tumor cells, and the cells are washed by sterile PBS solution after culture medium is removed.

2. 0.05% trypsin digests the cells at room temperature for 30-300s, during which time the cell status is observed until most of the cells are digested into spheres.

3. The digestion reaction was terminated with 10 times volume of DMEM medium containing 10% serum, and 800g of the cell suspension was collected and centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

4. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

5. Cell pellets were resuspended in DMEM medium containing 10% serum and the cells were counted. At 10⁵Seeding and adherent culture were performed at a density of 3.5cm cell culture dishes (medium plus 10% FBS in Table 9).

By the purification procedure of example 8 and subculture of example 9, high-purity gastric cancer primary tumor cells were obtained (fig. 4).

Example 10 cryopreservation of gastric cancer Primary cells

After the gastric cancer primary tumor cells purified and cultured in the embodiment 8 are subjected to passage amplification for 2-3 times, the gastric cancer primary tumor cells can be frozen:

1. adherent cultured gastric cancer primary tumor cells, and the cells are washed by sterile PBS solution after culture medium is removed.

2. 0.05% trypsin digests the cells at room temperature for 30-300s, during which time the cell status is observed until most of the cells are digested into spheres.

3. The digestion reaction was terminated with 10 times volume of DMEM medium containing 10% serum, and 800g of the cell suspension was collected and centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

4. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

5. Cell cryopreservation (see example 1) at 10⁶Resuspending the cell sediment at a density of/mL, freezing 1mL of cell suspension in each tube of a 2mL freezing tube, freezing overnight by using a gradient cooling box, and transferring the cell sediment into liquid nitrogen for long-term storage.

Example 11 recovery of gastric cancer Primary cells

The gastric cancer primary tumor cells preserved in liquid nitrogen can be recovered:

1. sterile water at 37 ℃ was prepared five minutes in advance.

2. The vial was removed from the liquid nitrogen and the cells were rapidly thawed in sterile water at 37 ℃.

3. The cells in the frozen tube were transferred to a 15ml centrifuge tube, supplemented with ten times the volume of DMEM medium containing 10% serum, mixed well, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

4. Cell pellets were resuspended in DMEM medium containing 10% serum and the cells were counted. At 10⁵And/3.5 cm cell culture dish density for inoculation and adherent culture.

Example 12 comparison of gastric cancer micro-tumor Structure formation Capacity in different Primary Medium

The procedures of all primary cultures were identical (see above) and only the media formulations were different. The various primary media tested are shown in table 33. Wherein, the scheme C is the formula adopted in the invention, and the details are shown in the table 9.

TABLE 33 Primary Medium formulation for test (100mL)

Note: in the scheme A, the final concentration of the human recombinant protein EGF is 50 ng/mL; the final concentration of the human recombinant protein bFGF is 20 ng/mL; the final concentration of the human recombinant protein HGF is 20 ng/mL; the final concentration of the human recombinant protein FGF-10 is 20 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200 ng/mL; the final concentration of the human recombinant protein Noggin is 100 ng/mL; the final concentration of SB202190 is 10 μ M; the final concentration of A83-01 is 0.5 mu M; the final concentration of the N-acetyl-L-cysteine is 1 mM; the final concentration of the Nicotinamide is 5 mM; the final concentration of the cortisol is 20 ng/mL; the final concentration of Y-27632 was 10. mu.M. In the scheme B, the final concentration of the human recombinant protein EGF is 50 ng/mL; the final concentration of the human recombinant protein bFGF is 20 ng/mL; the final concentration of the human recombinant protein HGF is 20 ng/mL; the final concentration of the human recombinant protein FGF-10 is 20 ng/mL; the final concentration of the human recombinant protein R-spondin is 400 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200 ng/mL; the final concentration of the human recombinant protein Noggin is 200 ng/mL; the final concentration of SB202190 is 10 μ M; the final concentration of A83-01 is 0.5 mu M; the final concentration of the N-acetyl-L-cysteine is 1 mM; the final concentration of the Nicotinamide is 5 mM; the final concentration of Cholera Toxin is 0.5 nM; the final concentration of the Y-27632 is 10 mu M; the final concentration of Gastrin was 10 nM. In the scheme C, the final concentration of the human recombinant protein EGF is 50 ng/mL; the final concentration of the human recombinant protein bFGF is 20 ng/mL; the final concentration of the human recombinant protein HGF is 20 ng/mL; the final concentration of the human recombinant protein FGF-10 is 20 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200 ng/mL; the final concentration of the human recombinant protein Noggin is 100 g/mL; the final concentration of the human recombinant protein R-spondin is 400 ng/mL; the final concentration of the human recombinant protein IL-2 is 20 ng/mL; the final concentration of the human recombinant protein IL-15 is 20 ng/mL; the final concentration of CHIR99021 is 3. mu.M; the final concentration of SB202190 is 10 μ M; the final concentration of A83-01 is 1 μ M; the final concentration of the N-acetyl-L-cysteine is 1 mM; the final concentration of the Nicotinamide is 10 mM; the final concentration of Cholera Toxin is 1 nM; the final concentration of the Y-27632 is 10 mu M; the final concentration of Gastrin was 10 nM.

Primary medium in Table 34, protocol D (100mL)

Note: the final concentration of the human recombinant protein EGF is 50 ng/mL; the final concentration of the human recombinant protein bFGF is 20 ng/mL; the final concentration of the human recombinant protein MSP is 20 ng/mL; the final concentration of the human recombinant protein IL-2 is 20 ng/mL; the final concentration of the human recombinant protein IL-15 is 20 ng/mL; the final concentration of the human recombinant protein HGF is 20 ng/mL; the Forskolin final concentration is 5 μ M; the final concentration of Y-27632 was 10. mu.M.

Primary medium in Table 35, protocol E (100mL)

Note: the final concentration of the human recombinant protein EGF is 50 ng/mL; the final concentration of the human recombinant protein bFGF is 20 ng/mL; the final concentration of the human recombinant protein HGF is 20 ng/mL; the final concentration of the human recombinant protein Noggin is 100 ng/mL; the final concentration of the human recombinant protein R-spondin is 400 ng/mL; the final concentration of the human recombinant protein IL-2 is 20 ng/mL; the final concentration of the human recombinant protein IL-15 is 20 ng/mL; the final concentration of SB202190 is 10 μ M; the final concentration of the cortisol is 25 ng/mL; the Forskolin final concentration is 5 μ M; the final concentration of A83-01 is 1 μ M; the final concentration of the N-acetyl-L-cysteine is 1 mM; the final concentration of the Nicotinamide is 10 mM; the final concentration of Cholera Toxin is 0.5 nM; the final concentration of Y-27632 was 10. mu.M.

Five primary cell culture medium protocols 20 samples were treated, the sample treatment and culture operations were performed as described in examples 3, 4, and 5, and the formation of a cell mass structure in the culture system was observed after 3 days of culture as shown in Table 36.

TABLE 36 cultivation in different media

It can be seen that the culture of gastric cancer micro-tumor model can be performed by using the A, B, C three culture mediums, while the D and E culture mediums are not suitable for the formation of gastric cancer micro-tumor model. The gastric cancer micro-tumor model formed in the culture medium of the scheme C is larger in size and has little advantage compared with the culture media of the schemes A and B.

Example 13 comparison of genetic background of gastric cancer micro-tumor models formed by different primary cell culture media

The DNA extraction procedure mentioned in the examples below was performed using the tiangen blood/tissue/cell genome extraction kit (DP 304).

The pooling procedures mentioned in the examples below were performed using the NEB DNA sequencing pooling kit (E7645).

The high throughput sequencing referred to in the examples below refers to the Illumina HiSeq X-ten sequencing platform.

The invention obtains a gastric cancer micro-tumor model and a gastric cancer primary focus sample which are obtained by culturing three culture mediums of the scheme A, B, C obtained in the embodiment 12, and respectively carries out DNA extraction, and the whole genome sequencing and copy number variation analysis heteroanalysis method comprises the following steps:

1. 10mg of gastric carcinoma primary lesion tissue mass and about 10 of gastric carcinoma micro-tumor model obtained by culturing the gastric carcinoma primary lesion tissue mass and the A, B, C culture mediums obtained in the scheme of example 12⁵The DNA extraction, the library construction and the whole genome high throughput sequencing (WGS) are respectively carried out on a plurality of cell clusters, and the sequencing depth is 30 x.

2. Copy number variation analysis (CNV) was performed on the two sets of sequencing results of the primary lesion and gastric cancer micro-tumor models, and the copy number variation between the primary gastric cancer tumor tissue and gastric cancer micro-tumor model was compared, as shown in FIG. 5. The results show that the gastric cancer micro-tumor models obtained by the culture of the three culture mediums can well keep the copy number variation characteristics of the primary focus of gastric cancer. There were no significant differences between the three media in terms of maintaining the genetic background of the primary lesion.

Example 14 alignment of gastric cancer micro-tumor model cell formation by different primary cell culture media

The invention obtains a gastric cancer micro-tumor model and a gastric cancer primary focus sample which are obtained by culturing three culture mediums of the scheme A, B, C obtained in the example 12, and respectively carries out multiple immunofluorescence staining to identify the cell composition (the method is shown in the example 7). The results are shown in Table 37.

TABLE 37 comparison of cell components of gastric cancer micro-tumor model cells formed in different media

It can be seen that the culture media A and B can well retain the tumor cells in the original focus, and the formed gastric cancer micro-tumor model is formed by the tumor cells with higher purity. The culture medium C can not only retain the tumor cells in the original focus, but also well preserve fibroblasts and T cells existing in tissues, so that a gastric cancer micro-tumor model is formed by multiple cell types together.

Therefore, the gastric cancer solid tumor micro-tumor model culture medium (table 9) used in the invention can protect various cell types in the primary focus to the maximum extent, promote the spontaneous formation of a multi-cell structure under the suspension condition, and construct an in vitro cell model extremely close to the characteristics of the primary focus.

Example 15 comparison of culture success rates of different dissociation solutions

The procedures of all primary culture methods in this example are identical (see the above description), and only the sample dissociation solution formula is different. The various sample dissociation fluids tested are shown in table 38, wherein protocol D is the formulation employed in the present invention, and is specifically shown in table 3.

TABLE 38 sample dissociation solution for test (10mL)

The sample dissociation liquid is prepared for use.

10 samples with the weight of the gastric cancer solid tumor tissue mass exceeding 100mg are selected, evenly divided into four parts, and the four sample dissociation liquids are respectively used for sample treatment and culture operation according to the methods described in the examples 3, 4 and 5. The formation of the cell mass structure in the culture system after 3 days of culture was observed as shown in tables 39 and 40:

TABLE 39 statistics of gastric cancer micro-tumor formation amount of different sample dissociation fluids

TABLE 40 statistics of sizes of gastric cancer micro-tumor cell masses (in units of μm in major axis) for different sample dissociation solutions

It can be seen that the formula of the sample dissociation solution has certain influence on the forming capability of the gastric cancer micro-tumor model and the size of the micro-tumor cell mass. The sample dissociation liquid (table 3) used in the invention can gently dissociate cells in tissues, maintain the activity of the cells to the maximum extent, and improve the formation efficiency and quality of a micro-tumor model.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.