CN111057680A - Culture medium and three-dimensional culture method for lung tumor cells - Google Patents

Culture medium and three-dimensional culture method for lung tumor cells Download PDF

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CN111057680A
CN111057680A CN201911261165.3A CN201911261165A CN111057680A CN 111057680 A CN111057680 A CN 111057680A CN 201911261165 A CN201911261165 A CN 201911261165A CN 111057680 A CN111057680 A CN 111057680A
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medium
tumor cells
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张文
赵长云
周敏
郑敏
周文涵
杜菲菲
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Chongqing Yuheng Biotechnology Co ltd
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Abstract

The invention provides a culture medium for lung tumor cells, which is particularly suitable for in vitro three-dimensional culture of the lung tumor cells, in particular circulating tumor cells from blood of a lung cancer patient. The invention also provides a method for three-dimensionally culturing circulating tumor cells in vitro to form organoids.

Description

Culture medium and three-dimensional culture method for lung tumor cells
Technical Field
The present invention belongs to the field of cell culture, and relates to a method for in vitro culture of circulating tumor cells and differentiation of the circulating tumor cells into tumor organoid tissues, and a culture medium for the method. In particular, the invention relates to culturing circulating tumor cells from lung tumors in vitro.
Background
Circulating Tumor Cells (CTCs) are formed by a few cells with high invasion and high metastasis capacities in tumor primary foci breaking through a basement membrane and entering a circulatory system, and are closely related to tumor metastasis and recurrence. In recent years, significant correlation between CTCs and survival and therapeutic response of patients has been widely reported in various malignant tumors, and particularly Circulating Tumor Stem Cells (CTSCs) in CTCs are important targets for cancer therapy, so CTCs and CTSCs have wide application prospects as diagnostic and prognostic biomarkers. However, most of the research still stays on CTC counting and molecular typing, and if CTC functional typing, proteomic and transcriptome characteristic analysis, CTC tumorigenic capacity evaluation and tumor recurrence and metastasis mechanism research are required, an ideal CTC in-vitro culture and amplification means must be established.
However, successful in vitro culture of CTCs, particularly ctccs, has presented significant challenges and few reports, one of the reasons being that Circulating Tumor Stem Cells (CTSCs) in CTCs, particularly CTCs, are extremely rare, making capture and expansion very difficult, and enrichment of CTCs isolated to high cell activity and purity remains somewhat difficult with prior art approaches. Another limitation is that the cases of successful in vitro culture of CTCs are mostly performed by 2D suspension culture, but it is difficult to amplify CTSCs having "dry" by this method because the niche (niche) required for stem cell maintenance function is not provided under ordinary 2D culture conditions, and the dry state disappears rapidly. In addition, the 2D culture method cannot simulate the environment in the body, so that the difference exists between the established in vitro model and the in vivo tumor microenvironment, and the 2D culture scheme is not suitable for popularization.
There have been several reports of successful in vitro culture of CTC cell lines in recent years. Yu et al successfully enriched and cultured CTC cell lines from 8ml blood samples of patients with metastatic breast cancer using RPMI-1640 medium containing EGF, bFGF and B27 and ultra-low adsorption dishes (Min Yu et al, Science 11 Jul 2014: Vol.345, Issue 6193, pp.216-220); a similar protocol was used by the Laure Cayresource team to establish colorectal cancer CTC cell lines (LaureCayresource et al, cancer research 75 (5). January 2015). In addition, Zhang et al also successfully established a CTC culture system using DEME/F12 medium supplemented with insulin, hydrocortisone, EGF, FGF2 (Lixin Zhang et al, SciTransl Med.2013Apr 10; 5 (180)).
The prior art has great limitations due to the strict requirements on the number of CTCs in a blood sample and the culture environment, such as the number of CTCs is more than 300 per ml, the culture conditions require culture in a hypoxic incubator, and the like. In addition, the established cell line is cultured under the 2D suspension condition and cannot simulate the microenvironment in an organism, so the current culture scheme is not suitable for popularization.
On the other hand, a portion of CTCs have Stem Cell properties, called Circulating Tumor Stem Cells (CTSCs), which have self-renewal capacity, Stem Cell marker expression capacity, and even immune escape capacity, and are considered to be the root cause of tumor metastasis and recurrence. CTSC in peripheral blood of a tumor patient is separated and captured, and in vitro 3D cell culture is carried out to form an individual specific organoid, so that drug sensitivity test, gene detection and the like can be further carried out, and the method is helpful for guiding treatment. The greatest advantage of the organoids formed by CTSCs is that they originate both from the patient's primary tumor tissue and represent a highly likely metastatic tumor. But the greatest challenge in constructing any type of organoid in vitro is to determine the nutrients, cytokines and culture methods required for formation in the culture dish. These exact culture conditions vary significantly and are difficult to predict for different tumors and tissue types.
Thus, there remains a need in the art for improved means of in vitro culture of CTCs, particularly CTSC cells, and means of organoid formation therefrom, to meet the needs of research, diagnosis, and the like.
Disclosure of Invention
In order to solve the above problems, the inventors of the present invention have conducted extensive experimental studies to obtain a medium particularly suitable for culturing lung tumor cells. The culture medium is also particularly suitable for culturing the circulating tumor cells separated and captured from blood samples of lung cancer patients, and can retain the dryness of the circulating tumor stem cells. In addition, the present invention has been completed by a method for three-dimensionally culturing circulating tumor cells derived from a lung cancer patient in vitro using the medium to form a tumor organoid in which the dryness of the circulating tumor stem cells is retained.
Thus in a first aspect, the present invention provides a culture medium for culturing tumour cells in vitro, said medium comprising or consisting of EGF, FGF2, FGF10, Noggin (Noggin) and R-Spondin and a kinase inhibitor comprising or consisting of a ROCK inhibitor, an ALK inhibitor and a p38 inhibitor.
Preferably, the ROCK inhibitor is a dual inhibitor of ROCK1 and ROCK 2. More preferably the ROCK inhibitor is Y-27632.
Preferably, the ALK inhibitor is a 83-01.
Preferably, the p38 inhibitor is SB 202190.
In a specific embodiment, the medium of the invention comprises EGF at a concentration of 3-7ng/ml, preferably at a concentration of 4-6ng/ml, most preferably at a concentration of 5ng/ml, FGF2 at a concentration of 15-35ng/ml, preferably at a concentration of 20-30ng/ml, most preferably at a concentration of 25ng/ml, noggin at a concentration of 50-150ng/ml, preferably at a concentration of 75-125ng/ml, most preferably at a concentration of 100ng/ml, noggin at a concentration of 50-150ng/ml, preferably at a concentration of 75-125ng/ml, more preferably at a concentration of 90-110ng/ml, most preferably at a concentration of 100ng/ml, and noggin at a concentration of 350-650/ml, preferably at a concentration of 400-600ng/ml, more preferably at a concentration of 450-550ng/ml, most preferably at a concentration of 500ng/ml, R-Spondin1 or alternatively, the medium of the invention comprises inhibitors at a concentration of 6-14. mu.M, preferably at a concentration of 9-11 ng/ml, most preferably at a concentration of 500. mu.5. mu.10. mu.M, most preferably at a concentration of 10. mu.10. mu.5. mu.10. mu.g/ml, most preferably at a concentration of 10. mu.10. sub.10. concentration of inhibitors of 10. preferably at a concentration of 10. sub.10. concentration of 10. sub.10. concentration.
In a specific embodiment, the medium of the invention comprises 3-7ng/ml EGF, 15-35ng/ml FGF2, 50-150ng/ml FGF10, 50-150ng/ml noggin, 350-650ng/ml R-Spondin 1; and 6-14. mu.M of ROCK inhibitor, 350-650nM ALK inhibitor, 6-14. mu.M of p38 inhibitor. More specifically, the medium of the present invention comprises 3-7ng/ml of EGF, 15-35ng/ml of FGF2, 50-150ng/ml of FGF10, 50-150ng/ml of noggin, 350-650ng/ml of R-Spondin 1; and 6-14. mu.M Y-27632, 350-650nM A83-01, 6-14. mu.M SB 202190.
In a specific embodiment, the medium of the invention comprises 5ng/ml EGF, 25ng/ml FGF2, 100ng/ml FGF10, 100ng/ml noggin, 500ng/ml R-Spondin 1; and 10. mu.M ROCK inhibitor, 500nM ALK inhibitor, 10. mu.M p38 inhibitor. More specifically, the medium of the present invention comprises 5ng/ml EGF, 25ng/ml FGF2, 100ng/ml FGF10, 100ng/ml noggin, 500ng/ml R-Spondin 1; and 10. mu.M Y-27632, 500nM A83-01, 10. mu.M SB 202190.
In a further embodiment, the medium of the invention comprises vitamins, preferably nicotinamide. In a specific embodiment, the concentration of nicotinamide is 4-11mM, preferably 5 mM.
In a further embodiment, the medium of the invention comprises amino acids, preferably L-glutamine or N-acetylcysteine, more preferably L-glutamine and N-acetylcysteine. In a particular embodiment, the concentration of N-acetylcysteine is 1-1.5mM, preferably 1.25 mM.
In a further embodiment, the culture medium of the invention comprises a hormone, preferably a prostaglandin, more preferably prostaglandin E2. In a particular embodiment, the concentration of prostaglandin E2 is 0.5 to 1.5. mu.M, preferably 0.75 to 1.25. mu.M, most preferably 1.0. mu.M.
In a particular embodiment, the culture medium of the invention comprises: EGF, FGF2, FGF10, noggin, R-Spondin1, ROCK inhibitors, ALK inhibitors, p38 inhibitors, L-glutamine, N-acetylcysteine, nicotinamide, prostaglandin E2 more specifically, the culture medium of the present invention comprises: EGF, FGF2, FGF10, noggin, R-Spondin1, Y-27632, A83-01, SB202190, L-glutamine, N-acetylcysteine, nicotinamide, prostaglandin E2.
In a particular embodiment, the medium of the invention comprises the above-mentioned components in the amounts as described in table 1.
In another embodiment, the culture medium of the invention optionally comprises an antibiotic, preferably penicillin and/or streptomycin.
The tumour cells of the first aspect of the invention are preferably tumour cells from a patient having a lung tumour, more preferably Circulating Tumour Cells (CTCs). In a specific embodiment, the lung tumor is a malignant tumor.
In a second aspect, the present invention relates to a culture method using the medium of the first aspect.
In a third aspect, the present invention provides a method for three-dimensional culturing lung tumor cells in vitro, comprising the steps of: (1) obtaining tumor cells from a subject having a lung tumor, (2) culturing the tumor cells obtained in step (1) using the culture medium of the first aspect and matrigel.
In specific embodiments, step (1) is performed by enriching circulating tumor cells from a blood sample of the subject.
In a preferred embodiment, the blood sample is a pulmonary venous blood sample. Preferably, the venous blood sample is no less than about 3ml, no less than about 4ml, no less than about 5 ml. Specifically, the venous blood sample is about 4 ml.
In a further embodiment, the circulating tumor cells are enriched from the blood sample in step (1) by density gradient centrifugation. Preferably, the density gradient centrifugation is an immunodensity gradient sorting. More preferably, the density gradient centrifugation is performed using a RossettesepTMThe method is carried out.
In a preferred embodiment, the Matrigel is MatrigelTM。
In a preferred embodiment, matrigel is mixed with the enriched cells and the medium of the first aspect is added after coagulation.
In view of the uniqueness of the combination of a cytokine and a kinase inhibitor of the present invention, in a fourth aspect, the present invention relates to the use of a combination of a cytokine comprising or consisting of EGF, FGF2, FGF10, Noggin (Noggin) and R-Spondin and a kinase inhibitor comprising or consisting of a ROCK inhibitor, an ALK inhibitor and a p38 inhibitor, and a p38 inhibitor, in the culture of prostate tumor cells. In a preferred embodiment of the use of the fourth aspect, the cytokine and the kinase inhibitor are comprised in a culture medium.
In a fifth aspect, the present invention relates to a kit comprising the culture medium of the first aspect.
In a sixth aspect, the present invention relates to the use of the culture medium of the first aspect and the kit of the fourth aspect for culturing cells, preferably lung tumor cells, more preferably circulating tumor cells from a patient having a lung tumor.
The culture medium and the method support the establishment of the lung cancer organoid in vitro from the circulating tumor cells, provide an in vitro model which can better simulate the in vivo tumor microenvironment, and have wide application in the aspects of research, diagnosis, prognosis, medication scheme preparation and the like of the lung cancer.
Brief Description of Drawings
FIG. 1 is a photograph of lung adenocarcinoma cells cultured by the method and medium of example 1 of the present invention taken under an optical microscope after 1 day, 2 days, 3 days, and 7 days of culture.
FIG. 2 is a photograph of lung adenocarcinoma cells cultured by the method and medium of the present invention taken under a light microscope.
FIG. 3 is a photograph of squamous cell lung carcinoma cells cultured by the method and medium of the present invention taken under an optical microscope.
FIG. 4 is a 3D culture immunofluorescence plot, comprising four panels, with the light areas indicating the occurrence of fluorescence. The upper left panel is the EpCAM protein fluorescence labeling diagram, indicating that the cultured cell mass expresses EpCAM; the upper right panel is a CD133 protein fluorescence labeling diagram, and the cultured cell mass expresses CD 133; the lower left panel is hochest33342 staining, marking the cell nucleus; the lower right panel is a fit to the first three photographs.
FIG. 5 compares the expression levels of coherence-associated genes (CD133, CD44, and ABCG2) of lung cancer H2347 cells in normal 2D culture and 3D culture conditions of the present invention.
Fig. 6 shows a comparative example in which a cell population could not be obtained when the amount of growth factor used was too low (below the lower limit of the concentration range defined by the present invention).
Detailed Description
The terms of the present invention have meanings well known to those skilled in the cellular arts, unless otherwise defined herein. Those skilled in the art will appreciate that the numerical ranges defined in the context of the present invention encompass point values, and that the defined point values may encompass approximations due to systematic errors.
"Medium" means a medium used to culture cells in vitro, which provides the cells with the substances required for growth. "defined medium" is a term relative to natural medium derived from tissue extracts and body fluids, characterized by a well-defined chemical composition. The medium eventually used in the culture method of the present invention is also referred to as "organoid medium".
"basal medium" in the present context means a medium commonly used in the art without the addition of the combination of additives, supplements unique to the present invention, which is typically a universal medium, i.e. can be widely used for various types of cells, without customization. In particular in the context herein, the basal medium is preferably a medium for culturing animal cells. The basal medium and buffers used to formulate organoid culture media of the present invention may be media suitable for culturing the cells of interest, and may be known to those skilled in the art. The basal medium used in the organoid medium of the invention may be a medium commonly used for culturing animal cells, in particular tumor cells, in particular for culturing tumor stem cells, especially for culturing lung tumor stem cells. Specifically, the basal Medium may be Eagles's Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), advanced DMEM, RPMI1640 Medium. In a preferred embodiment of the present invention, the basal medium used is a mixture of F12 medium and high-level DMEM medium, abbreviated as "adDMEM/F12 medium". By combining a relatively low but more abundant variety of F12 medium with a higher DMEM medium having a higher concentration of nutrients, a base medium with balanced properties is provided as a basis for further customizing the medium for a particular cell type.
Media for a particular use, such as for culturing a particular type of cell, needs to be formulated by adding specific additives and supplements to the basal media. These additives and supplements may be self-formulated at the time of formulation, particularly for less stable additives such as glutamine, serum, growth factors, hormones; supplements may also be commercially available mixtures. As previously mentioned, circulating tumor stem cells are difficult to culture, and therefore the composition and amount of formulated additives is critical to the present invention. As understood by those skilled in the art, the medium after addition of various additives and supplements may be referred to as a "complete medium" because it contains all the components for culturing a particular cell.
Organoid culture medium of the present invention is a culture medium specifically for culturing cells, in particular circulating tumor cells and circulating tumor stem cells, from patients with lung tumors, comprising a unique combination of cytokines and preferably also a unique combination of kinase inhibitors.
"cytokines" are a generic term for biologically active substances secreted by cells. In the present context, a growth factor is understood as a subset of cytokines, which are capable of promoting cell growth, typically polypeptides. Numerous growth factors have been discovered in the art, including Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), Fibroblast Growth Factor (FGF), Keratinocyte Growth Factor (KGF), Hepatocyte Growth Factor (HGF), Interleukins (IL), and the like. Growth factors may be active in a variety of different tissues and may also act on certain specific types of tissue cells, i.e., be specific. Synergy can be realized among different growth factors or between the growth factors and other substances such as hormones and paracrine factors. Thus, the combination of growth factors added to the medium, and the manner in which the growth factors and other additives are combined, requires judicious choice for the culture of a particular cell type.
The unique cytokine combination of the present invention comprises or consists of the growth factors EGF, FGF2, FGF10, Noggin (Noggin) and R-Spondin 1. In a particular embodiment, the medium of the invention comprises EGF at a concentration of 3-7ng/ml, preferably at a concentration of 4-6ng/ml, most preferably at a concentration of 5 ng/ml. In a particular embodiment, the medium of the invention comprises FGF2 at a concentration of 15-35ng/ml, preferably at a concentration of 20-30ng/ml, most preferably at a concentration of 25 ng/ml. In a particular embodiment, the medium of the invention comprises FGF10 at a concentration of 50-150ng/ml, preferably at a concentration of 75-125ng/ml, most preferably at a concentration of 100 ng/ml. In a particular embodiment, the medium of the invention comprises noggin in a concentration of 50-150ng/ml, preferably in a concentration of 75-125ng/ml, more preferably in a concentration of 90-110ng/ml, most preferably in a concentration of 100 ng/ml. In a specific embodiment, the culture medium of the present invention comprises R-Spondin1 at a concentration of 350-650ng/ml, preferably at a concentration of 400-600ng/ml, more preferably at a concentration of 450-550ng/ml, and most preferably at a concentration of 500 ng/ml.
Another essential group of components in the culture medium of the invention are "kinase inhibitors", which are capable of inhibiting the activity of kinases, typically small molecule compounds. It is crucial and innovative that a combination of three kinase inhibitors, namely the ROCK inhibitor, the ALK inhibitor and the p38 inhibitor, is used simultaneously in the medium of the invention.
A "ROCK inhibitor" or a "Rho kinase inhibitor" is a substance having an inhibitory effect on Ras homolog gene family (Rho) kinases in stem cells. Rho kinase plays an important role in regulating cell growth, division, migration, transformation, invasion, and the like. Rho kinases have two subclasses, called ROCK1 and ROCK2, respectively. The ROCK inhibitor can be a selective inhibitor of ROCK1 or ROCK2, or can be a dual inhibitor of ROCK1 and ROCK 2. In a preferred embodiment of the invention, the ROCK inhibitor is a dual inhibitor of ROCK1 and ROCK2, preferably Y-27632. In a further embodiment, the medium of the invention comprises a ROCK inhibitor at a concentration of 6-14. mu.M, preferably at a concentration of 9-11. mu.M, most preferably at a concentration of 10. mu.M, preferably a dual inhibitor of ROCK1 and ROCK2, most preferably Y-27632.
In a further embodiment, the medium of the invention comprises an ALK inhibitor, preferably A83-01, A83-01, having an inhibitory effect on the TGF- β I receptors ALK5, ALK4 and ALK7, having a reduced transcription induced by these kinases ALK5, ALK4 and ALK7, and having a weak inhibitory effect on the constitutively active ALK-6, ALK-2, ALK-3 and ALK-1. in a further embodiment, the medium of the invention comprises the ALK inhibitor, preferably A83-01, at a concentration of 350-650nM, preferably 400-600nM, more preferably 450-550nM, most preferably 500 nM.
The mitogen-activated protein kinase (MAPK) family comprises four subfamilies of ERK, p38, JNK and ERK5, wherein p38 further comprises p38 α, p38 β, p38 γ and p38 δ having a higher homology to each other the p38 kinase is expressed in a variety of tumor cells, but the effect varies depending on the tumor type. various p38 inhibitors have been found, such as the dual inhibitor SB203580 for protein kinase B and p38, the pan p 8 inhibitor Doramapimod which inhibits p38 α/β/γ/δ, etc. in embodiments of the invention, the p38 inhibitor is preferably SB202190. in further embodiments, the media of the invention comprises the selective inhibitor SB 38 α/38 α, preferably 10 μ M, 38/36190 at a concentration of 6-14 μ M, preferably 9-11 μ M, most preferably 10 μ M.
It will be appreciated by those skilled in the art that the culture medium of the present invention may additionally comprise other additives and supplements, such as vitamins, amino acids, proteins, hormones, salts, etc., in addition to the cytokines and kinase inhibitors that are necessarily included as listed above.
In one embodiment, the media of the invention further comprises nutrients such as vitamins or amino acids, particularly those that require on-site addition prior to use due to instability and/or additional additions due to insufficient levels in the basal media, other supplements to support the culture of the target cells. In a particular embodiment of the invention, the additional amino acid is preferably L-glutamine or N-acetylcysteine, most preferably L-glutamine and N-acetylcysteine. The concentration of N-acetylcysteine used in the medium of the present invention may be 1-1.5mM, preferably 1.25 mM. In a particular embodiment of the invention, said additional vitamin is niacinamide. The concentration of nicotinamide used in the medium of the invention may be 4-11mM, preferably 5 mM.
In one embodiment, the culture medium of the invention further comprises a commercially available supplement which is a mixture of substances, preferably a B27 supplement. The commercially available supplements may be used in the concentrations recommended by their manufacturers.
The culture medium of the present invention may further comprise a hormone, such as a prostaglandin, preferably prostaglandin E2. In a particular embodiment, a prostaglandin, preferably prostaglandin E2, is used in the medium of the invention at a concentration of 0.5-1.5. mu.M, preferably 0.75-1.25. mu.M, most preferably 1.0. mu.M.
The addition of "antibiotics" to the culture medium can avoid the occurrence of contaminations, in particular for controlling the contamination of microorganisms, in particular bacteria. However, it will also be appreciated by those skilled in the art that the use of antibiotics can also have negative effects, such as antibiotics that may render covert contamination undetectable, make mycoplasma infections undetectable, lead to the development of antibiotic-resistant microorganisms, and may have an impact on follow-up studies. Thus, antibiotics may optionally be added in the present invention. Antibiotics that can be used in cell culture are known to those skilled in the art and include, but are not limited to: antibiotics acting on bacteria (including gram-positive and gram-negative bacteria), such as ampicillin, erythromycin, gentamicin, kanamycin, neomycin, penicillin; antibiotics acting on fungi, such as amphotericin B, nystatin; antibiotics acting on mycoplasma, e.g. MRA, cyclopropylAnd (3) the fleroxacin. In a particular embodiment, the antibiotics used in the present invention are penicillin and streptomycin. In a particular embodiment of the invention, the antibiotic used is penicillin and/or streptomycin, e.g. 100 U.ml-1Penicillin and/or 100. mu.g/ml-1Streptomycin of (4). In a further embodiment, penicillin and streptomycin are used simultaneously, e.g. 100 U.ml is used-1Penicillin and 100. mu.g/ml-1Streptomycin of (4).
The buffer used in the present invention may be Good's buffer such as HEPES, Tricine or the like which is commonly used for cell culture. In a particular embodiment of the invention, a HEPES buffer is used, for example at a concentration of 10mM or 20mM, preferably at a concentration of 10 mM.
It will be appreciated by those skilled in the art that when one or more of the additional additives or supplements listed above are provided as a mixture, they are also considered to fall within the scope of the claimed invention as long as they are included in the media ultimately obtained and used for cell culture and in amounts consistent with the numerical ranges defined herein.
"three-dimensional culture" is a culture method as compared with "two-dimensional culture" carried out on a plane, and is characterized by creating a growth environment more similar to that in vivo so as to induce cells to differentiate and form three-dimensional tissues and maintain the three-dimensional tissues, and even simulate the functions of the tissues. Cells in two-dimensional cell culture are often attached to a two-dimensional surface, so that the growth of the cells can only be carried out on the surface, the proliferation can only be expanded along the surface, and the cells often grow in a spindle or oblate shape in an attached manner and cannot be well collected. The three-dimensional cell culture technology is that cells can grow and proliferate in all directions in a three-dimensional space by utilizing a scaffold material with a microscopic 'scaffold' structure or other means, the cell shape is irregular polygon or spherical, and the cells can form a three-dimensional network structure and are accompanied by a large amount of cell-matrix and cell-cell interactions and are gathered into clusters. Compared with two-dimensional cell culture, three-dimensional cell culture can better simulate the growth environment of cells in vivo, and is expected to increase important factors lacking in two-dimensional culture such as interaction between cells and signal transduction.
Three-dimensional culture is typically achieved by providing cultured cells with growth conditions similar to those in vivo and by simulating the physical support of the extracellular matrix, i.e., scaffold materials, which typically include collagen, cellulose, chitosan, gelatin, fibronectin, laminin, and the like, as well as commercially available products such as Corning's MatrigelTMA series of products. Most of them are natural polymers derived from animal or human body, and their network structure, components and biomechanical environment are suitable for cell proliferation, adhesion and metabolism, and these materials have low antigenicity, no toxicity, degradability and can promote cell growth and adhesion, so that they are receiving more and more attention from researchers. In the case of not using a scaffold material, other physical means such as microcarrier, magnetic suspension, etc. may be used to suspend the cells in the culture medium to achieve three-dimensional culture.
On the other hand, due to the complexity of cell-cell interactions, interactions between cells and the matrix, customized conditions are required for three-dimensional cell culture of cells from different sources. Despite the ongoing development of knowledge of cell-cell interactions and interactions between cells and matrices, considerable experimentation is still required to explore ideal culture conditions for some specific cell types. While considering that in vitro three-dimensional culture usually lacks the capillary network in the tissues in vivo, the accessibility of nutrients to cells and the ability to diffuse metabolic waste products are limited due to the three-dimensional stacking formed by the cells, the exploration of culture methods and conditions is also of great importance.
By "Circulating Tumor Cells (CTCs)" is meant herein Tumor cells that originate from a solid Tumor lesion, including primary and metastatic lesions, and are released into the peripheral blood either spontaneously or as a result of a diagnostic procedure. CTCs can be classified according to morphological characteristics: single cell CTCs and cell mass CTCs. A fraction of the CTCs of a single Cell have Stem Cell properties, called Circulating Tumor Stem Cells (CTSCs), which have self-renewal capacity, Stem Cell marker expression capacity, and even immune escape capacity, and are considered to be the root cause of Tumor metastasis and recurrence, and thus are also considered to be important targets for Tumor therapy. Circulating tumor cells are present in the blood and can be used to predict the prognosis of tumor therapy and to monitor the effect of the therapy. In addition, metastasis occurs in the vast majority of patients who are lethal to cancer, and the study of CTCs also helps to elucidate the biological mechanisms of cancer metastasis. Methods for obtaining CTCs from blood are known to those skilled in the art and include, but are not limited to, flow cytometry (FACS), microfluidic devices, centrifugation, density gradient centrifugation, immuno-density gradient centrifugation, magnetic bead adsorption. However, the number of CTCs in blood is very limited and the number of CTSCs is much more rare, so that in recent years much research has been devoted to the in vitro culture of stable CTC and CTSC cell lines.
Many tumor cells are difficult to grow in vitro for complex reasons and are likely to be associated with the specificity of the tumor cells. Tumor cells are essentially dysregulated cells in the body that have nutritional requirements different from normal cells from the same tissue and respond to different nutrients and stimuli different from normal cells. For example, tumor cells may require growth factors that are not the same as those of normal tissue. The variability and heterogeneity of tumor cells themselves also makes this task even harder in exploring the conditions under which they are cultured.
Successful culture of tumor stem cells depends on a variety of factors, including the effects of physicochemical conditions, the "niche sites" provided by three-dimensional culture to maintain normal stem cell activity, cell-cell interactions, and cell-extracellular matrix interactions. In addition, growth factors and other signaling molecules such as hormones can also promote the acquisition of stem cell function. At present, no definite and universal culture method for maintaining the tumor stem cells is established.
Successful culture of tumor cells can be determined by visual observation, for example, whether a cell mass of a certain size is successfully formed. Additionally or alternatively, confirmation may be provided by identifying specific markers expressed by the respective tumor cells. For example, it can be confirmed by immunofluorescence staining using a monoclonal antibody that specifically binds to a tumor surface antigen. Such tumor surface antigens include EpCAM, CD133, and the like. Alternatively, it can be confirmed by measuring the transcription and expression level of a protein expressed specifically in a tumor.
"carcinoid microspheres" as used herein refers to cell masses formed by in vitro culture of CTC cells that exhibit cell structural characteristics similar to those of cancer and have a volume size, e.g., a diameter of about 50 μm or so, and can be passaged. The successful formation of carcinoid microspheres can be identified by observing the cytological structure, and the structure and cell type can be confirmed by preparing pathological sections and using hematoxylin-eosin staining (HE staining). In the context of this document, "carcinoid microspheres" may be used interchangeably with "carcinoid organs" or "cancer organoids". "carcinoid organ" or "cancerous organoid" may also be referred to herein simply as "organoid". In particular, the organoids of the present invention are lung cancer organoids.
The methods herein are suitable for culturing circulating tumor cells, particularly circulating stem cells of a lung tumor, from a subject having a lung tumor. In a specific embodiment, the lung tumor is a benign tumor or a malignant tumor, preferably a malignant tumor. Malignant tumors of the lung can be generally classified into small cell lung cancer and non-small cell lung cancer according to morphology, wherein the non-small cell lung cancer can be further classified into squamous cell carcinoma, lung adenocarcinoma and large cell carcinoma. The methods of the invention are preferably used for the culture of circulating tumor cells and circulating tumor stem cells from patients with squamous and adenocarcinoma lung cancer.
The method of the invention comprises the following steps: (1) obtaining tumor cells from a subject having a lung tumor, (2) culturing the tumor cells obtained in step (1) in matrigel using the organoid culture medium of the present invention.
In one embodiment, step (1) is performed by enriching circulating tumor cells from a blood sample of the subject. In one embodiment, the blood sample is a peripheral blood sample, preferably a pulmonary venous blood sample. In the case of peripheral blood, at least about 8ml of blood is used in step (1). In the case of pulmonary venous blood, at least about 4ml of blood is used in step (1).
The enrichment may be accomplished by methods known in the art, for example, density gradient centrifugation, and in particular, immunodensity gradient sorting. Preferably, the density gradient centrifugation is performed using a RossettesepTMThe method is carried out.
Matrigel is preferred for Matrigel of the present invention. As known to those skilled in the art, Matrigel is a commercially available product.
Temperature, pH, oxygen level, carbon dioxide/carbon acid level, osmotic pressure and surface tension during cell culture all affect the culture, and can be adjusted to appropriate values by adjusting the composition of the culture medium or the culture environment. In general, these parameters can be adjusted to values similar to those of the environment in which the cultured cells are located in vivo. In a particular embodiment, the pH of the medium of the invention is about 7.2 to about 7.4.
Detailed Description
For a more complete understanding and appreciation of the invention, the invention will be described in detail below with reference to examples and the accompanying drawings, which are intended to illustrate the invention and not to limit the scope thereof. The scope of the invention is specifically defined by the appended claims.
Example 1 culturing of circulating tumor cells in Lung adenocarcinoma patients Using organoid Medium
Organoid culture media for culturing tumor circulating cells from lung cancer patients were prepared according to the formulations shown in table 1 according to conventional methods in the art. The specific concentrations in column 3 of Table 1 are used in this example, and the sources of the ingredients are shown in column 2 of Table 1. It will be appreciated by those skilled in the art that while specific manufacturer and lot numbers for the ingredients are set forth in this example, the invention is not limited to these specific commercially available ingredients, but also includes equivalents thereof. The concentration ranges of the individual components in the present invention are given in column 4 of table 1 for reference.
TABLE 1
Figure BDA0002311613210000131
Figure BDA0002311613210000141
The inventors have found during the adjustment of the experimental medium formulation that the combination or content of cytokines, in particular growth factors, is crucial for cell culture. When any one of EGF, FGF2, FGF10 was removed from the culture medium, cell culture became difficult. The amounts of R-Spondin1 and Noggin added also had an effect on the results of cell culture, and when the amounts were too small (i.e., below the lower limit of the column 4 concentration range in Table 1 above), cell growth was affected, resulting in failure to obtain healthy cell clones (FIG. 6).
Cells from lung cancer patients were cultured as follows.
Enrichment of circulating tumor cells:
1) 4ml of pulmonary venous blood was added to 200. mu.l RosseteSepTMAfter incubation of the CD45 depleted mixture (depletioncktail) at room temperature for 20min, an equal amount (4ml) of PBS + 2% FBS was added to the sample and mixed well to dilute the sample.
2) 4ml of density centrifugate (ficoll) is added into a 15ml centrifuge tube, and then the diluted sample obtained in the step 1) is carefully added onto the ficoll, the adherent addition is noticed, and the action is gentle and rapid.
3) Centrifuging at 1200g for 20min at room temperature to separate the plasma layer from the ficoll layer, loosening the brake of the centrifuge, and setting the temperature to 4 ℃.
4) The plasma was removed, about 2ml of the enriched cells were aspirated from the separation layer (between the plasma layer and the ficoll layer), transferred to a new 15ml centrifuge tube, and 4ml of pbs + 2% FBS was added thereto and well mixed.
5) Centrifuge at 200g for 10min at 4 degrees.
And (3) culturing the enriched cells:
6) matrigel was prepared, 200 μ l of the tip pre-cooled and a 24 well plate pre-heated.
7) The supernatant of the centrifuged sample in step 5) was removed, and 1ml of serum-free adDMEM/F12+/+/+ (adDMEM/F12 to which penicillin/streptomycin, 10mM HEPES and 2mM Glutamax had been previously added) was added, followed by centrifugation at 200g for 5min at room temperature.
8) The supernatant was removed and 10. mu.l of liquid was retained.
9) Adding 40 μ l matrigel prepared in step 6), mixing, dripping into the center of 24-well plate, placing 24-well plate in incubator at 37 deg.C and 5% CO2Culturing under the condition.
10) After 5min, the 24-well plate was rapidly inverted and left for another 10 min.
11) 0.5ml of the prepared organoid medium was added.
12) Fresh organoid medium was changed every 3 days for passage for 2-3 weeks.
Circulating tumor cells from lung adenocarcinoma patients were cultured according to the above method, and a cell pellet of about 50 μm in diameter was obtained at 7 days (FIG. 1).
In order to identify the cell composition of the cell mass, the cell mass is subjected to immunofluorescence staining by using an anti-EpCAM monoclonal antibody and an anti-CD 133 monoclonal antibody, and the results are positive (figure 4, upper left and upper right small graphs), which shows that the cells in the cell mass formed by culture have the expression of the two tumor cell specific proteins, and further shows that the tumor organoids can be successfully cultured based on circulating tumor cells in blood by directly taking the blood of a patient as a starting point by using the method disclosed by the invention.
Example 2 culturing of circulating tumor cells in Lung squamous carcinoma patients Using organoid Medium
Using the same medium and method as in example 1, blood samples from patients with squamous cell lung carcinoma were processed, and tumor organoids were successfully obtained by enriching and culturing circulating tumor cells in the blood samples (FIG. 3).
Example 3 drying of cultured cells
In order to determine whether the cell mass obtained by the three-dimensional culture of the present invention retained the sternness of tumor stem cells, the inventors measured the expression levels of sternness-associated genes CD133, CD44 and ABCG2 and compared the expression levels of the associated genes in a mock sample cultured under ordinary two-dimensional (2D) culture conditions using the same medium, which was obtained by adding 10 tumor cell line cells (human non-small cell lung cancer cells H2347 cells) to 4ml of peripheral blood, and the results are shown in fig. 5. As can be seen from fig. 5, the expression levels of CD133 and ABCG2 in the cell mass obtained by the three-dimensional (3D) culture method of the present invention were significantly higher than those of the ordinary 2D cultured cells, wherein the expression level of CD133 of the 3D cultured cells was two hundred times that of the 2D cultured cells and the expression level of ABCG2 was also about 5 times that of the 2D cultured cells, indicating that the 3D cultured cells exhibited stronger dryness.

Claims (18)

1. A culture medium for culturing in vitro lung tumor cells, the culture medium comprising or consisting of a cytokine comprising or consisting of EGF, FGF2, FGF10, Noggin (Noggin), and R-Spondin, and a kinase inhibitor comprising or consisting of a ROCK inhibitor, an ALK inhibitor, and a p38 inhibitor.
2. The culture medium of claim 1, comprising 3-7ng/ml of EGF, 15-35ng/ml of FGF2, 50-150ng/ml of FGF10, 50-150ng/ml of noggin, 350-650ng/ml of R-Spondin 1; and 6-14. mu.M of ROCK inhibitor, 350-650nM ALK inhibitor, 6-14. mu.M of p38 inhibitor.
3. The culture medium of claim 2, comprising 3-7ng/ml EGF, 15-35ng/ml FGF2, 50-150ng/ml FGF10, 50-150ng/ml noggin, 350-650ng/ml R-Spondin 1; and 6-14. mu.M Y-27632, 350-650nM A83-01, 6-14. mu.M SB 202190.
4. The culture medium of any one of claims 1 to 3, further comprising 4-11mM nicotinamide.
5. The culture medium of any one of claims 1 to 4, further comprising 1-1.5mM of L-glutamine and/or 1-1.5mM of N-acetylcysteine.
6. The culture medium of any one of claims 1 to 5, further comprising 0.5-1.5 μ M prostaglandin E2.
7. The culture medium of any one of claims 1 to 6, wherein said pulmonary tumor cells are Circulating Tumor Cells (CTCs) from the blood of the subject.
8. A cell culture method using the medium of any one of claims 1 to 7.
9. A three-dimensional culture method of lung tumor cells comprises the following steps:
(1) obtaining tumor cells from a subject having a lung tumor, and
(2) culturing the tumor cells obtained in step (1) using the medium according to any one of claims 1 to 7 and matrigel.
10. The method of claim 9, step (1) is performed by enriching circulating tumor cells from a blood sample of the subject.
11. The method of claim 10, wherein the blood sample is a pulmonary venous blood sample.
12. The method of claim 11, wherein the blood sample is not less than 3 ml.
13. The method of any one of claims 10 to 12, said enrichment being performed by density gradient centrifugation.
14. The method of any one of claims 9 to 13, wherein the Matrigel is MatrigelTM。
15. The method of any one of claims 9 to 14, wherein matrigel is mixed with the enriched cells in step (2) and the medium is added after coagulation.
16. Use of a combination of a cytokine comprising or consisting of EGF, FGF2, FGF10, Noggin (Noggin), and R-Spondin and a kinase inhibitor comprising or consisting of a ROCK inhibitor, an ALK inhibitor and a p38 inhibitor in the culture of prostate tumor cells.
17. The use of claim 16, wherein the prostate tumor cells are Circulating Tumor Cells (CTCs) from the blood of the subject.
18. The use of claim 16 or 17, wherein the cytokine and kinase inhibitor are comprised in a culture medium.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111424015A (en) * 2019-12-10 2020-07-17 重庆康克唯生物科技有限公司 Culture medium and three-dimensional culture method for prostate tumor cells
CN111548997A (en) * 2020-06-03 2020-08-18 杭州联众医疗科技股份有限公司 Method for in vitro culture of lung cancer tissues
CN112501121A (en) * 2020-11-24 2021-03-16 益善生物技术股份有限公司 Basic culture medium and culture method for circulating tumor cells
CN112592896A (en) * 2020-11-22 2021-04-02 深圳市第二人民医院(深圳市转化医学研究院) Culture solution and culture method for lung adenocarcinoma organoid
WO2021222143A3 (en) * 2020-04-26 2021-12-02 Georgetown University Methods for isolating and culturing tumor cells
CN114214267A (en) * 2021-12-06 2022-03-22 大连大学 Organoid matrigel microspheres and preparation method and application thereof
CN114752566A (en) * 2022-04-21 2022-07-15 中国科学院苏州纳米技术与纳米仿生研究所 Tumor stem cell screening and tumor organoid construction method based on tumor stem cells
CN114874989A (en) * 2022-04-11 2022-08-09 中国科学院苏州纳米技术与纳米仿生研究所 Method for capturing circulating tumor cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834212A (en) * 2017-03-24 2017-06-13 四川大学华西医院 Culture medium for 3D culture of lung tissue
CN108396010A (en) * 2017-02-06 2018-08-14 王琼 A kind of extracorporeal culturing method of colorectal cancer organoid
CN109609441A (en) * 2018-12-29 2019-04-12 创芯国际生物科技(广州)有限公司 A kind of culture medium and organoid cultural method of renal tissue organoid 3D culture
CN110129270A (en) * 2019-05-27 2019-08-16 创芯国际生物科技(广州)有限公司 A kind of Pleural effusions organoid culture medium, cultural method and antibiotics susceptibility test method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108396010A (en) * 2017-02-06 2018-08-14 王琼 A kind of extracorporeal culturing method of colorectal cancer organoid
CN106834212A (en) * 2017-03-24 2017-06-13 四川大学华西医院 Culture medium for 3D culture of lung tissue
CN109609441A (en) * 2018-12-29 2019-04-12 创芯国际生物科技(广州)有限公司 A kind of culture medium and organoid cultural method of renal tissue organoid 3D culture
CN110129270A (en) * 2019-05-27 2019-08-16 创芯国际生物科技(广州)有限公司 A kind of Pleural effusions organoid culture medium, cultural method and antibiotics susceptibility test method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DONG GAO ET AL: "Organoid cultures derived from patients with advanced prostate cancer", 《CELL》 *
JARNO DROST ET AL: "Organoid culture systems for prostate epithelial and cancer tissue", 《NAT PROTOC》 *
Y T LUO ET AL: "The viable circulating tumor cells with cancer stem cells feature, where is the way out?", 《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》 *
周鲁林 等: "3D培养体系富集外周血中的循环肿瘤干细胞的研究", 《中国肿瘤》 *
董研博 等: "类器官研究进展及应用", 《中国药理学与毒理学杂志》 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111424015A (en) * 2019-12-10 2020-07-17 重庆康克唯生物科技有限公司 Culture medium and three-dimensional culture method for prostate tumor cells
WO2021222143A3 (en) * 2020-04-26 2021-12-02 Georgetown University Methods for isolating and culturing tumor cells
EP4143298A4 (en) * 2020-04-26 2024-08-28 Univ Georgetown Methods for isolating and culturing tumor cells
CN111548997A (en) * 2020-06-03 2020-08-18 杭州联众医疗科技股份有限公司 Method for in vitro culture of lung cancer tissues
CN112592896B (en) * 2020-11-22 2024-06-25 深圳市第二人民医院(深圳市转化医学研究院) Culture solution for lung adenocarcinoma organoid and culture method thereof
CN112592896A (en) * 2020-11-22 2021-04-02 深圳市第二人民医院(深圳市转化医学研究院) Culture solution and culture method for lung adenocarcinoma organoid
CN112501121A (en) * 2020-11-24 2021-03-16 益善生物技术股份有限公司 Basic culture medium and culture method for circulating tumor cells
CN112501121B (en) * 2020-11-24 2023-05-23 益善生物技术股份有限公司 Basic culture medium and culture method for circulating tumor cells
CN114214267A (en) * 2021-12-06 2022-03-22 大连大学 Organoid matrigel microspheres and preparation method and application thereof
CN114214267B (en) * 2021-12-06 2024-04-09 大连大学 Organoid matrigel microsphere and preparation method and application thereof
CN114874989A (en) * 2022-04-11 2022-08-09 中国科学院苏州纳米技术与纳米仿生研究所 Method for capturing circulating tumor cells
CN114874989B (en) * 2022-04-11 2024-06-25 中国科学院苏州纳米技术与纳米仿生研究所 Method for capturing circulating tumor cells
CN114752566A (en) * 2022-04-21 2022-07-15 中国科学院苏州纳米技术与纳米仿生研究所 Tumor stem cell screening and tumor organoid construction method based on tumor stem cells
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