RU2778891C2 - Ex-vivo cultivation system and methods for its application - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: present invention relates to the field of cell biology and discloses a tissue cultivation system, a method for tissue cultivation, as well as a method for determining the effectiveness of a drug, a method for selecting a drug for the treatment of a disease and a method for treating a malignant tumor in a subject in need, using the above method for tissue cultivation. The specified tissue cultivation system contains a culture medium and one high-precision cut of tissue placed on an insert for tissue cultures in the well of a tablet for tissue cultures. Moreover, the specified high-precision tissue cut is maintained in an atmosphere with a high oxygen content containing at least 60% and less than 95% of oxygen. And the specified culture medium is suitable for maintaining the viability of the specified tissue cut. Moreover, the specified culture is mixed rotationally, contributing to the periodic immersion of the specified tissue cut into the specified culture medium.

EFFECT: present invention makes it possible to increase the efficiency of cultivating a high-precision cut of tissue while preserving the viability of cells.

24 cl, 11 dwg, 6 tbl, 2 ex

Description

TECHNICAL FIELD OF THE INVENTION

The present invention, in some embodiments, relates to an ex vivo culture system and methods of using the same.

The usual choice of anti-cancer therapeutic regimens is primarily based on the origin of the malignant tissue, the stage and grade of the tumor, and the general health of the patient. While these regimens are effective in some patients, they are toxic, causing many side effects that can be life-threatening.

The availability of targeted drug therapies and the ability to identify patient-specific tumor markers have indicated individualized treatment options but also require the widespread use of genetic profiling [see, for example, Dancey et al. (2012) Cell 148, 409-420 and Garraway et al. (2013) J. Clin. oncol. off. J. Am. soc. Clin. oncol. 31, 1806-1814]. However, extrapolating genetic data to specific treatment regimens is difficult and predicting patient response is unreliable. For example, in the case where a patient's tumor has more than one targetable mutation, it is very difficult to predict which of the potential drugs will be most effective. The same is true when there is more than one drug for a particular mutation. Thus, the choice of treatment method is highly dependent on trial and error.

Various technologies are available to directly investigate the response of a patient's malignant cells to a particular treatment, such as tumor-derived cell lines derived from patient tumors and patient-derived xenograft (PDX) models [see, for example, Crystal et al. (2014) Science 346, 1480-1486; Clever et al. (2016) Cell 165, 1586-1597 and Hidalgo et al. (2014) Cancer Discov. 4, 998-1013]. However, these models are expensive and time consuming, and most importantly, they do not preserve the original tumor microenvironment, which can have a marked effect on drug sensitivity [Straussman et al. (2012) Nature 487, 500-504].

Ex vivo organ culture (EVOC) systems used in cancer biology are cultures of highly precise sections of a patient's tumor. EVOC has been used for a variety of applications, including the study of drug toxicity, virus uptake, tumor susceptibility to radiation, or specific anti-cancer drugs [see, for example, Vaira et al. (2010) Proc. Natl. Acad. sci. U.S.A. 107, 8352-8356; Vickers et al. (2004) Chem. Biol. Interact. 150, 87-96; de Kanter et al. (2002) Curr. drug metab. 3, 39-59; Stoff-Khalili et al. (2005) Breast Cancer Res. BCR 7, R1141-1152; Merz et al. (2013) Neuro-Oncol. 15, 670-681; Gerlach et al. (2014) Br. J. Cancer 110, 479-488; Meijer et al. (2013)Sr. J. Cancer 109, 2685-2695; Grosso et al. (2013) Cell Tissue Res. 352, 671-684; Vaira et al. (2010) PNAS 107, 8352-8356; Roife et al. (2016) Clin. Cancer Res. June 3, 1-10; Maund et al. (2014) Lab. Invest. 94, 208-221; Vickers et al. (2004) Toxicol Sci. 82(2):534-44; Zimmermann et al. (2009) Cytotechnology 61(3): 145-152); Parajuli et al. (2009) In Vitro Cell. dev. Biol-Animal 45:442-450; Koch et al. (2014) Cell Communication and Signaling 12:73; Graaf et al. Nature Protocols (2010): 5: 1540-1551; Majumder et al. Nat. commun. 6, 6169 (2015); US Patent Application Nos: US 2014/0228246, US 2010/0203575 and US 2014/0302491 and International Patent Publication No. WO2002/044344.

Brief summary of the present invention

According to an aspect of some embodiments of the present invention, there is provided a culture system comprising a culture medium and a precision tissue section placed on a tissue culture insert, wherein the precision tissue section is maintained in a high oxygen atmosphere containing at least 50% oxygen, and wherein the culture is agitated. rotation, contributing to the periodic immersion of the tissue section in the culture medium.

In some embodiments of the present invention, the culture is in a tissue culture plate.

According to some embodiments of the present invention, the culture system contains a drug.

According to an aspect of some embodiments of the present invention, there is provided a tissue culture method comprising: culturing a high fidelity tissue section on a tissue culture insert in a culture medium in a high oxygen atmosphere containing at least 50% oxygen; and agitating the culture by rotating to cause the tissue section to be intermittently immersed in the culture medium.

According to some embodiments of the present invention, the method further comprises adding a drug to the culture medium.

According to some variants of implementation of the present invention, a method for determining the effectiveness of a medicinal product or a batch of medicinal products is provided, which includes:

(i) culturing tissue in accordance with the method of the present invention;

(ii) adding the drug or batch of drugs to the culture medium; as well as

(iii) determining the effect of the drug or batch of drugs on tissue, where tissue sensitivity to the drug is indicative of drug efficacy.

According to some embodiments of the present invention, the tissue is a pathological tissue.

In some embodiments, the present invention provides a method for selecting a drug for the treatment of a disease in a subject in need thereof, the method comprising:

(i) culturing the pathological tissue obtained from the subject in accordance with the method of the present invention;

(ii) adding the drug to the culture medium; as well as

(iii) determining the effect of the drug on the tissue, wherein tissue sensitivity to the drug is indicative of the efficacy of the drug in treating a disease in the subject.

In some embodiments, the present invention provides a method of treating a disease in a subject in need thereof, the method comprising:

(a) the choice of drug in accordance with the method of the present invention; as well as

(b) administering to the subject a therapeutically effective amount of a drug that is effective in treating a disease in the subject, thereby treating the disease in the subject.

According to some embodiments of the present invention, the drug is a combination of drugs.

In some embodiments of the present invention, the tissue or precision tissue section is freshly isolated.

According to some variants of implementation of the present invention, the tissue or a high-precision section of tissue is stored at a temperature of 4°C.

In some embodiments of the present invention, tissue or a high-precision tissue section is cryopreserved.

According to some embodiments of the present invention, the determination is influenced by a morphology score, a viability score, a proliferation score, and/or a cell death score.

According to some variants of implementation of the present invention, the determination is carried out by assessing the morphology.

According to some variants of implementation of the present invention, the determination is carried out within 3-5 days of cultivation.

According to some embodiments of the present invention, the disease is a malignant tumor.

According to some embodiments of the present invention, the cancer is selected from the group consisting of cancer of the ovary, colon and rectum, lung, pancreas, stomach, gastrointestinal tract, and breast.

According to some embodiments of the present invention, the pathological tissue is cancerous tissue.

According to some embodiments of the present invention, tissue is selected from the group consisting of ovary, colon and rectum, lung, pancreas, stomach, esophagus, and breast.

According to some embodiments of the present invention, tissue is selected from the group consisting of ovary, colon and rectum, lung, pancreas, stomach, gastrointestinal tract, breast, liver, cartilage, and bone.

According to some variants of implementation of the present invention, the addition is carried out 12-24 hours after the start of cultivation.

According to some embodiments of the present invention, the drug is an anti-inflammatory drug or an anti-cancer drug.

In some embodiments, the drug is selected from the group consisting of 5-fluorouracil (5FU), oxaliplatin, irinotecan, cisplatin, 4-hydroperoxycyclophosphamide, docetaxel, doxorubicin, navelbine, gemcitabine, gefitinib, tamoxifen, olaparib, trametinib, everolimus, and palbociclib .

According to some embodiments of the present invention, the drug concentration is higher than the IC50 drug dose in the cell line.

According to some embodiments of the present invention, the drug concentration is obtained from a dosing experiment in which a dose-dependent response is observed.

According to some variants of implementation of the present invention, the cultivation is carried out for at least 4 days.

According to some variants of implementation of the present invention, the cultivation is carried out for at least 5 days.

According to some variants of implementation of the present invention, the cultivation is carried out up to 7 days.

According to some embodiments of the present invention, culturing is carried out in a tissue cult plate.

According to some embodiments of the present invention, the high precision cut is 200-300 microns.

In some embodiments of the present invention, the slice is placed in the middle of a cell culture insert.

According to some embodiments of the present invention, the slice is a single slice.

In some embodiments of the present invention, the slice is in direct contact with the tissue culture insert.

In some embodiments of the present invention, the tissue culture insert is a titanium mesh insert.

In some embodiments of the present invention, the tissue is human tissue.

According to some embodiments of the present invention, the high oxygen atmosphere contains at least 70% oxygen.

According to some embodiments of the present invention, the high oxygen atmosphere contains less than 95% oxygen.

According to some embodiments of the present invention, the method is carried out in combination with genetic profiling.

Unless otherwise indicated, all technical and/or scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. While methods and materials similar or equivalent to those described herein may be used in the practice or study of embodiments of the present invention, exemplary methods and/or materials are described below. In the event of a conflict, the description of the patent, including the definitions, will take precedence. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

Brief description of several types of graphic materials

Some embodiments of the present invention are described herein by way of example only, with reference to the accompanying drawings. With specific reference to the drawings, it is emphasized in detail that the details shown are by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the description taken with the drawings makes it obvious to those skilled in the art how embodiments of the present invention may be implemented.

On graphics:

In FIG. 1 shows representative histological micrographs showing the morphology of xenograft sections derived from human ovarian cancer after 5 days of ex vivo culture. Precise sections of harvested tissue were incubated on a titanium insert (left column) in a 6-well plate or directly in a 6-well plate (right column); in 21% O ₂ (lower row) or in 80% O ₂ (upper row) for 5 days; and stained with H&E. The scale bar represents 50 µm. For each condition, the tissues were examined in triplicate.

In FIG. 2 shows representative histological micrographs showing the morphology of xenograft sections derived from human breast cancer after 5 days of ex vivo culture in various media. Precise sections of harvested tissue were incubated on a titanium insert in 80% O ₂ in DMEM/F12 (left panel) or M199 (right panel) for 5 days; and stained with H&E. The scale bar represents 50 µm. For each condition, the tissues were examined in duplicate.

In FIG. 3 shows the ability of the developed ex vivo culture method to predict the responses of xenografts derived from human breast carcinoma to anticancer drug treatment. Shown are graphs generated by Jackson Laboratories demonstrating the in vivo response of two xenografts derived from human breast carcinoma to treatment with 4-hydroperoxycyclophosphamide, showing that TM00096 breast carcinoma is resistant to treatment while TM00098 breast carcinoma is sensitive to treatment. . The Y-axis indicates the size of the tumor in mm ³ .

Representative histological micrographs show the morphology of sections of breast carcinomas TM00096 (left column) and TM00098 (right column) after 5 days of ex vivo cultivation on a titanium insert in 80% O ₂ treated for 4 days with the indicated concentrations of 4-hydroperoxycyclophosphamide. The scale bar represents 50 µm. For each condition, the tissues were examined in duplicate.

In FIG. 4 shows the ability of the developed ex vivo culture method to predict the response of xenografts derived from human colorectal carcinoma (CRC) to anti-cancer drug treatment. Graphs generated by Jackson Laboratories showing the in vivo response of three CRC-derived xenografts to oxaliplatin treatment are shown, indicating that TM00134 CRC is resistant to treatment, while TM00170 tumor is partially susceptible and TM00164 CRC is very sensitive. to treatment. The Y-axis indicates the size of the tumor in mm ³ .

Representative histological micrographs show the morphology of sections of CRC TM00134 (left panel), TM00170 (middle panel) and TM00164 (right panel) after 5 days of ex vivo cultivation on a titanium insert in 80% O ₂ treated for 4 days with oxaliplatin at a concentration of 0.2 µM. DMSO treatment served as control. The scale bar represents 50 µm. For each condition, the tissues were examined in triplicate.

In FIG. 5 shows that tumor tissue sections cultured according to the developed ex vivo culture method have different sensitivities to various anticancer drugs. Tumor tissue of human colorectal cancer (CRC) was taken from a metastatic lesion in the peritoneum, removed during surgery to remove the tumor and intraperitoneal chemotherapy. Representative histological micrographs are shown showing the morphology of tumor sections after 5 days of ex vivo culture on a titanium insert in 80% O ₂ treated for 4 days with the indicated anticancer drugs 5-fluorouracil (5-FU), irinotecan or oxaliplatin. DMSO treatment served as control. Of note, the tissue was dose-dependently sensitive to oxaliplatin and irinotecan, but resistant to 5-fluorouracil. The scale bar represents 50 µm. For each condition, the tissues were examined in triplicate.

In FIG. 6 shows that sections of tumor tissue obtained from different individuals and cultured according to the developed ex vivo culture method have different sensitivity to the same anticancer drug. Tumor tissue of human colorectal cancer (CRC) was taken from a metastatic lesion in the peritoneum, removed during surgery to remove the tumor and intraperitoneal chemotherapy. Representative histological micrographs are shown showing the morphology of tumor sections after 5 days of ex vivo culture on a titanium insert in 80% O ₂ treated for 4 days with the indicated anti-cancer drugs: 5-fluorouracil (5-FU) or oxaliplatin. DMSO treatment served as control. PR indicates a partial response and NR indicates no response. It should be noted that while the tumor obtained in patient #8 was characterized by a strong partial response to oxaliplatin and no response to 5-fluorouracil, another patient, patient #6, partially responded to both drugs. The scale bar represents 50 µm. For each condition, the tissues were examined in duplicate.

In FIG. 7 shows the ability of the developed ex vivo culture method to predict sensitivity to target therapy as predicted by molecular profiling. Shown are representative histological micrographs demonstrating the morphology of sections of triple-negative breast cancer tumors from two patients after 5 days of ex vivo culture on a titanium insert in 80% O ₂ treated for 4 days with AZD4547 (an FGFR inhibitor). DMSO treatment served as control. Of note, Patient #1, whose tumor causes FGFR1 amplification, is susceptible to AZD4547 (an FGFR inhibitor), while Patient #2, who does not have this mutation, was resistant to treatment. The scale bar represents 50 µm. For each condition, the tissues were examined in duplicate.

In FIG. 8 shows that Ki67 proliferation staining can be used to assess tissue sensitivity to various treatments. Representative histological micrographs are shown showing morphology (H&E stain) and proliferation (Ki67 stain) in one patient after 5 days ex vivo culture on a titanium insert in 80% O ₂ treated for 4 days with the indicated drugs. The graph on the right, obtained by Jackson Laboratories, demonstrates the in vivo response of this CRC-derived xenograft to treatment with oxaliplatin and 5-fluorouracil. The Y-axis indicates the size of the tumor in mm ³ . This patient was partially sensitive to 5-fluorouracil but highly sensitive to oxaliplatin both in vivo and ex vivo. DMSO treatment served as control. The scale bar represents 50 µm. For each condition, the tissues were examined in duplicate.

In FIG. 9 shows the processing of core biopsies for EVOC. In this example, three core biopsies are placed side by side in liquid agarose in a metal tray. More agarose is added and the tray is cooled. Biopsies are excised from agarose and the block is fixed to the plunger with a contact adhesive, embedded in more agarose (using the same method as with tissue pieces), and then cut with a microtome. Biopsy sections are exposed and processed similarly to tissue sections. The scale bar represents 100 µm.

In FIG. 10 shows histological micrographs demonstrating that tissue can be cut and stored in the medium for up to 48 hours prior to the start of the experiment without any adverse effects on the tissue or any change as a result. Colon cancer tissue is shown that was treated with control DMSO or 5-fluorouracil (5-FU) for 4 days either immediately after sectioning or 48 hours after cooling. BrdU was added 18 hours before the end of the experiment; and the tissue was stained with an anti-BrdU antibody to identify dividing cells. Of note, 5-FU-treated tissue is still somewhat viable, but begins to respond to treatment with increased cell death and less BrdU under both conditions. The scale bar represents 50 µm.

In FIG. 11 shows representative histological images showing these tissues after 5 days of ex vivo culture; and a table summarizing the examined tissues that maintained tissue structure and viability for at least 5 days of culture. Precise sections of the harvested tissue were incubated on a titanium insert in DMEM/F12 in 80% O ₂ for 5 days and stained with H&E. All experiments were performed in duplicate on two different cases of the specified malignant tumor.

Description of specific embodiments of the present invention

The present invention, in some embodiments, relates to an ex vivo culture system and methods of using the same.

Before a detailed explanation of at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the details set forth in the following description or exemplified in the examples section. The present invention is capable of other embodiments or may be practiced or carried out in various ways.

Personalized treatment options for diseases such as cancer typically use genetic profiling and study of the response of a patient's cells to a particular treatment using expensive and time-consuming models, such as cell line models derived from patient tumors and patient-derived xenograft (PDX). ). In recent years, ex vivo organ culture (EVOC) systems have been proposed for use in various fields, including the study of drug toxicity, virus uptake, tumor susceptibility to radiation, or specific anti-cancer drugs.

Although the present invention has been put into practice, the present inventors have developed an EVOC system that can maintain the structure and viability of a high-precision tissue section for 7 days in culture, and thus can predict the response of pathological tissues to various drugs.

As illustrated below and in the example section that follows, the newly developed EVOC system is based on culturing a single tissue section directly placed on a culture insert (in this case, in the middle of a reusable titanium mesh) that allows the slice to be periodically immersed in the medium, thereby facilitating the diffusion of nutrients and gases across the shear, in a highly oxygenated atmosphere containing oxygen (80%), using a standard medium supplemented with only fetal calf serum (FCS) and antimicrobial agents (example 1, table 1, fig. 1- 2 and 9). EVOC systems of various malignant tissues, including ovarian, colon, rectum, lung, pancreas, and breast, cultured according to this method showed high viability after 4-6 days of culture (Example 1, Table 1, Fig. eleven).

Further, the authors of the present invention show that the developed EVOC system can be used to predict the response of tumors to anticancer drugs (example 2). In particular, the present inventors demonstrate that the sensitivity of ex vivo malignant tissue sections obtained from various patient-derived xenograft (PDX) models to anticancer treatment is consistent with the response of PDX models to in vivo treatment (Example 2, Table 2, FIG. 3-4); and that the sensitivity to targeted therapy of ex vivo triple-negative breast cancer tissue sections obtained from patients is consistent with molecular profiling data (Example 2, Figure 7). In addition, the authors of the present invention show that EVOC systems of human tumors from different patients exhibit different sensitivity to different anticancer treatments (example 2, tables 3-4, Fig. 5-6 and 8). It is important to note that the tissue can be stored in the medium for at least 48 hours prior to the start of the experiment without any adverse effects on tissue or drug sensitivity (Example 2, Figure 10).

Overall, the new EVOC system preserves tissue microenvironment, architecture, viability, and genetic heterogeneity. This system allows you to quickly, reliably and cost-effectively study the response of human tissues (for example, malignant tissues). Therefore, the present teachings further contemplate the application of this developed EVOC system to preclinical and basic research, as well as to the evaluation of the effectiveness of a drug for the treatment of diseases in general and for personalized therapy in particular.

Thus, according to a first aspect of the present invention, there is provided a culture system comprising a culture medium and a high-precision tissue section placed on a tissue culture insert, said high-precision tissue section being maintained in a high oxygen atmosphere containing at least 50% oxygen. , and moreover, the specified culture is stirred by rotation, contributing to the intermittent immersion of the specified tissue section in the specified culture medium.

In accordance with another aspect of the present invention, there is provided a method for culturing tissue, comprising: culturing a highly accurate tissue section on a tissue culture insert in a culture medium in a high oxygen atmosphere containing at least 50% oxygen; and agitation of the culture by rotation, facilitating the periodic immersion of the specified section of tissue in the specified culture medium

As used herein, the term "culture system" refers to at least a high-precision tissue section, insert, and medium in an ex vivo environment.

In specific embodiments, the culture system retains the structure and viability of the high-precision tissue section for at least 2-10, 2-7, 2-5, 4-7, 5-7, or 4-5 days in culture. In a specific embodiment, the high-precision tissue section remains viable for at least 5 days, 6 days, 7 days, or even 10 days. In a specific embodiment, the high-precision tissue section remains viable for at least 5 days.

In specific embodiments, at least 60%, at least 70%, at least 80% of the cells in a high-precision tissue section remain viable for 4-5 days in culture, as determined, for example, by morphological analysis of the optimal viability area.

As used herein, "optimum viability area" refers to the microscopic field of tissue (e.g., at 20x magnification) that contains the highest number of viable cells per unit area as judged by a pathologist compared to an immediate pre-EVOC sample of the same species. .

Thus, according to specific embodiments, culture is carried out for 2-10, 2-7, 2-5, 4-7, 5-7, or 4-5 days.

According to a specific embodiment, the cultivation is carried out for at least 4 days.

According to a specific embodiment, the cultivation is carried out for at least 5 days.

According to a specific embodiment, cultivation is carried out for up to 7 days.

As used herein, the term "tissue" refers to a portion of a solid organ (i.e., not blood) of an organism that has some vascularity that includes more than one cell type and maintains at least some of the macrostructure of the in vivo tissue from which it was derived. deleted

Examples include, without limitation, ovarian tissue, colon and rectum tissue, lung tissue, pancreatic tissue, breast tissue, brain tissue, retina, skin tissue, bone, heart tissue, and kidney tissue. In specific embodiments, tissue is selected from the group consisting of ovary, colon and rectum, lung, pancreas, stomach, gastrointestinal tract, and breast. In specific embodiments, the tissue is selected from the group consisting of ovary, colon and rectum, lung, pancreas, stomach, gastrointestinal tract, breast, liver, cartilage, and bone. In specific embodiments, the tissue is metastatic cancer tissue derived from sites such as, but not limited to, liver, bone, lung, and peritoneum.

In specific embodiments, the tissue is not liver tissue.

In specific embodiments, the tissue is not prostate tissue.

In specific embodiments, the tissue is mammalian tissue.

In a particular embodiment, the tissue is human tissue.

In another specific embodiment, the tissue is mouse or rat tissue.

In specific embodiments, the tissue is healthy tissue.

In other specific embodiments, the tissue is pathological tissue. The method may use a plurality of screened high-precision tissue sections (e.g., each on a separate insert), each of which may be from pathological tissue(s), healthy tissue(s), or a combination of both (e.g., when healthy tissue serves as a control, if taken from the same tissue as the pathological tissue).

In specific embodiments, the tissue is pathological tissue.

Used in this document, the term "pathological tissue" refers to the tissue that causes the disease. Therefore, removal of such tissue is expected to result in a treatment, as further defined below. Specific examples of diseases treatable according to some embodiments of the present invention are described in detail below. In specific embodiments, the pathological tissue is inflamed tissue, fibrous tissue, or malignant tissue. In a specific embodiment, the pathological tissue is cancerous tissue.

In specific embodiments, tissue is obtained surgically or by biopsy, laparoscopy, endoscopy, or as a xenograft, or any combination thereof.

Tissue may be cut and cultured immediately after tissue extraction (i.e. primary tissue) or after implantation in an animal model [i.e. patient-derived xenograft (PDX)], each possibility is a separate embodiment of the present invention.

The tissue or tissue section according to some embodiments of the present invention may be freshly isolated or stored, eg, at 4°C, or cryopreserved (ie, frozen), eg, in liquid nitrogen.

In specific embodiments, the tissue or tissue section is freshly isolated (i.e., not more than 24 hours after being removed from the subject and has not been subjected to preservation processes), as further disclosed below.

In specific embodiments, tissue is cryopreserved after tissue is removed and before sectioning.

In specific embodiments, tissue is thawed prior to sectioning.

In specific embodiments, the tissue section is cryopreserved after sectioning.

In specific embodiments, the tissue section is thawed prior to culturing.

In specific embodiments, the tissue is maintained at 4°C, for example, in an environment after tissue is removed and until sectioned.

In specific embodiments, the tissue section is maintained at a temperature of 4° C., for example, in a post-sectioning medium and prior to culture.

In specific embodiments, storage at 4°C is for up to 120 hours, up to 96 hours, up to 72 hours, or up to 48 hours.

In specific embodiments, preservation at 4° C. is carried out for 24-48 hours.

Thus, in specific embodiments, the method further comprises obtaining a tissue from a subject or from an animal model containing the tissue.

As used herein, "patient-derived xenograft" (PDX) refers to tissue obtained by implanting primary tissue into an animal of a different species relative to a primary tissue donor. In specific embodiments, the PDX is a tissue created by implantation of primary human tissue (eg, malignant tissue) from an immunocompromised mouse.

After the tissue is removed from the tissue, sections are obtained to obtain high-precision sections.

As used herein, the phrase "high-precision tissue section" refers to a viable section obtained from isolated hard tissue with a reproducible, well-defined thickness (eg, ±5% thickness variation between sections).

Typically, a tissue section is a mini-model of tissue that contains tissue cells in their natural environment and retains three-dimensional connectivity such as intercellular and cell-matrix interaction of intact tissue without selecting a specific cell type among the various cell types that make up the tissue or organ. . High-precision cutting reduces sources of error due to differences in slice thickness and damage to slice surfaces that contribute to uneven exchange of gases and nutrients in tissue sections; improves reproducibility and allows evaluation of adjacent sections for histology and comparison in pairs under different experimental conditions.

The slice segment can be cut in different directions (eg, anteroposterior, dorsal-ventral, or nasal-temporal) and thickness. The size/thickness of the tissue section depends on the tissue source and the method used to obtain the section. In a particular embodiment, the thickness of the high-precision section allows the tissue structure to be maintained in culture.

In particular embodiments, the high-precision slice thickness allows full access of oxygen and nutrients to the inner layers of the cells so that the inner layers of the cells are exposed to sufficient concentrations of oxygen and nutrients.

In certain embodiments, the high precision slice thickness allows full oxygen and nutrient access to the inner cell layers so that the inner cell layers are exposed to the same concentrations of oxygen and nutrients as the outer cell layers.

In specific embodiments, the high precision cut is 50 to 1200 µm, 100 to 1000 µm, 100 to 500 µm, 100 to 300 µm, or 200 to 300 µm.

According to a specific embodiment, the high precision cut is 200-300 µm.

Methods for obtaining tissue sections are known in the art and are described for examples in the Examples section that follows and in Roife et al. (2016) Clin. Cancer Res. June 3, 1-10; Vickers et al. (2004) Toxicol Sci. 82(2):534-44; Zimmermann et al. (2009) Cytotechnology 61(3): 145-152); Koch et al. (2014) Cell Communication and Signaling 12:73 and Graaf et al. Nature Protocols (2010) 5: 1540-1551, the contents of each of which are incorporated herein by reference in their entirety. Such methods include, without limitation, sectioning using a vibratome, immersion in agarose followed by cutting with a microtome, or sectioning using a matrix.

As a non-limiting example, tissue is isolated and immediately placed in a physiological dissection medium (eg, ice-cold PBS), which can be supplemented with antibiotics.

In specific embodiments, the warm ischemic time is less than 2 hours, less than 1.5 hours, or less than 1 hour.

In specific embodiments, the cold ischemic time is less than 96 hours, less than 72 hours, less than 48 hours, less than 24 hours, less than 12 hours, less than 5 hours, or less than 2 hours.

Prior to sectioning, the tissue is attached to a tissue cutter, for example, with a contact adhesive, followed by dipping, for example, in a low-melting agarose gel. The tissue is then cut into high-precision sections. Numerous suitable tissue cutting devices are commercially available, such as, but not limited to, Compresstome™ VF-300 (Precision Instruments Inc. NC, USA), Brendel-Vitron tissue cutter (Tucson, AZ), Krumdieck precision tissue cutter (model no. MD4000-01; Alabama R&D) and a vibrating blade microtome Leica VT1200S (Leica, Wetzlar, Germany). In specific embodiments, the slicer is filled with an ice-cold medium such as Williams Medium E or Krebs-Henseleit (KHV) buffer. One of skill in the art would be aware of the necessary media and conditions for dissection and for tissue preservation and tissue section prior to culturing for each type of tissue.

The tissue section is then placed on a tissue culture insert in a tissue culture vessel filled with culture medium. One slice or multiple slices can be placed in one tissue culture insert. In specific embodiments, one slice is placed per tissue culture insert.

In specific embodiments, the culture vessel is filled with culture medium up to the bottom of the tissue section (eg, 4 ml of medium in a 6-well plate containing an insert).

The culture may be in a glass, plastic, or metal vessel, which may provide an aseptic environment for tissue culture. In specific embodiments, the culture vessel includes plates, flasks, bottles, and vials. Culture vessels such as COSTAR®, NUNC® and FALCON® are commercially available from various manufacturers.

In specific embodiments, the culture vessel is a tissue culture plate such as a 6-well plate, a 24-well plate, a 48-well plate, and a 96-well plate.

In a specific embodiment, the culture vessel is a 6-well tissue culture plate.

In specific embodiments, the culture vessel is not pre-coated with proteins extracted from the selected tissue (eg, when the tissue is cancerous tissue, then the culture vessel is not pre-coated with proteins extracted from the tumor of the appropriate stage and grade). Non-limiting examples of such proteins include ECM proteins such as collagen, fibronectin, laminin, vibronectin, cadherin, filamin A, vimentin, osteopontin, decorin, tenascin X, basement membrane proteins, cytoskeletal proteins, and matrix proteins; and growth factors.

The culture medium used in the present invention may be an aqueous-based medium that includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids and/or proteins such as cytokines, growth factors and hormones, all of which are essential for cell proliferation and are capable of maintaining tissue structure and viability. For example, the culture medium may be a synthetic tissue culture medium such as DMEM/F12 (available from Biological Industries, for example), M199 (available from Biological Industries, for example), RPMI (available from Biological Industries, for example). from Gibco-Invitrogen Corporation products), M199 (can be obtained, for example, from Sigma-Aldrich), Ko-DMEM (can be obtained, for example, from Gibco-Invitrogen Corporation products), supplemented with the necessary additives, as further described below. Preferably, all ingredients included in the culture medium of the present invention are substantially pure relative to the degree of tissue culture.

One of skill in the art will know how to select a growth medium for each tissue type under consideration.

According to specific embodiments of the present invention, the culture medium contains serum, for example, fetal calf serum (FCS, can be obtained, for example, from Gibco-Invitrogen Corporation products).

In specific embodiments, the culture medium contains less than 10% serum.

In specific embodiments, the culture medium contains less than 2% serum derived from the same species as the cultured tissue.

In specific embodiments, the culture medium does not contain serum derived from the same species as the cultured tissue.

In specific embodiments, the culture medium contains less than 2% serum that is autologous to the cultured tissue (ie, from the same subject).

In specific embodiments, the culture medium does not contain serum that is autologous to the cultured tissue (ie, from the same subject).

In specific embodiments, the culture medium contains less than 2% human serum.

According to some embodiments of the present invention, the culture medium does not contain human serum.

According to some embodiments of the present invention, the culture medium does not contain any animal contaminants, i.e. animal cells, fluid or pathogens (e.g. viruses that infect animal cells), i.e. does not contain xeno.

According to some embodiments of the present invention, the culture medium may further contain antibiotics (eg, penicillin, streptomycin, gentamicin), antifungals (eg, amphotericin B), L-glutamine, or NEAAs (non-essential amino acids).

In a specific embodiment, the medium contains serum and antibiotics.

In a specific embodiment, the medium contains DMEM/F12, 5% FCS, glutamine, penicillin, streptomycin, gentamicin, and amphotericin B

It should be noted that the culture medium may be refreshed periodically to maintain sufficient supplementation and to remove metabolic products that can damage tissues. In specific embodiments, the culture medium is refreshed every 12-72 hours, every 24-72 hours, every 24-48 hours, or every 12-48 hours.

In specific embodiments, the culture medium is refreshed every 12-48 hours.

In a specific embodiment, the culture medium is refreshed once every 12-24 hours and then every approximately 48 hours.

As used herein, the phrase "tissue culture insert" refers to a porous membrane suspended in a tissue culture vessel and is compatible with subsequent ex vivo culture of a tissue section. The pore size is capable of supporting the tissue section while being permeable to the culture medium, allowing the passage of nutrients and metabolic waste to and from the section, respectively. In specific embodiments, the tissue section is placed on a tissue culture insert, thereby allowing access to the culture medium to both the apical and basal surface of the tissue section.

In specific embodiments, the pore size is 0.1 µm to 20 µm, 0.1 µm to 15 µm, 0.1 µm to 10 µm, 0.1 µm to 5 µm, 0.4 µm to 20 µm, 0.4 µm to 10 µm, or 0.4 µm to 5 µm.

In specific embodiments, the pore size is 0.4 mm to 4 mm, 0.4 mm to 1 mm, 1 mm to 4 mm, 1 mm to 3 mm, or 1 mm to 2 mm.

In specific embodiments, the tissue culture insert is sterile.

In specific embodiments, the tissue culture insert is disposable.

In specific embodiments, the cell culture insert is reusable and autoclavable.

The cell culture insert can be synthetic or natural, inorganic or polymeric, such as titanium esters, alumina, polytetrafluoroethylene (PTFE), Teflon, stainless steel, polycarbonate, nitrocellulose, and cellulose. In specific embodiments, the cell culture insert is a titanium insert. Cell culture inserts that can be used with specific embodiments of the present invention are commercially available from, for example, Alabama R&D, Millipore Corporation, Costar, Corning Incorporated, Nunc, Vitron Inc. and SEFAR and include, but are not limited to, MA0036 Well, BIOCOATTM, Transwell®, Millicell®, Falcon®-Cyclopore, Nunc® Anapore plate inserts, titanium shield, and Teflon shield.

In specific embodiments, the tissue culture insert is a titanium mesh insert such as, but not limited to, Titanium MA0036 Well plate inserts (Alabama R&D).

In specific embodiments, the tissue culture insert is not coated with organic material such as collagen, fibronectin, or polyethylene glycol (PEG).

In specific embodiments, the tissue culture insert is not coated with proteins isolated from the appropriate tissue (e.g., when the tissue is cancerous tissue, then the tissue culture insert is not pre-coated with proteins isolated from the tumor of the appropriate stage and grade).

In specific embodiments, the tissue section is in direct contact with the tissue culture insert.

In specific embodiments, the tissue section is not separated from the culture medium by heterologous organic material such as collagen, fibronectin, or a synthetic polymer (which is not part of the culture vessel), such as polyethylene glycol (PEG).

In specific embodiments, the tissue section is in direct contact with the culture medium.

In specific embodiments, the tissue section is placed in the middle of the cell culture insert. Thus, for example, when a titanium mesh is used, such as Titanium MA0036 Well inserts, the tissue section is placed in a concavity located in the middle of the insert.

The tissue section is then cultured (or maintained) at physiological temperature, eg 37° C., in a highly oxygenated humidified atmosphere containing at least 50% oxygen and eg 5% CO ₂ .

In specific embodiments, the high oxygen atmosphere contains at least 60%, at least 70%, or at least 80% oxygen.

In particular embodiments, the high oxygen atmosphere contains at least 70% oxygen.

In other specific embodiments, the high oxygen atmosphere contains less than 95% oxygen.

In a specific embodiment, the high oxygen atmosphere contains approximately 80% oxygen.

According to a specific embodiment, during the culture process, the culture is agitated by rotation, facilitating periodic immersion of the tissue section in the culture medium.

As used herein, the phrases "rotational agitation to promote intermittent dipping of the tissue section into the culture medium" or "rotational agitation to promote intermittent immersion of the tissue section into the culture medium" refers to agitation that allows the tissue section to be periodically immersed in the medium, which facilitates diffusion nutrients and gas throughout the medium and through the tissue section.

In specific embodiments, the mixing is orbital mixing.

According to specific embodiments, the mixing is an angular mixing, for example, at an angle of 30°-45°.

In specific embodiments, mixing is accomplished with a tilt rotator (such as the MD2500 incubation block commercially available from Alabama Research and Development).

In specific embodiments, the agitation frequency is 50-200 rpm, 50-150 rpm, or 50-100 rpm.

In a particular embodiment, the agitation frequency is approximately 70 rpm.

The tissue culture and methods of the present invention can be adapted for many uses, including but not limited to:

1. Preclinical and basic research, including:

- study of mechanisms related to health and disease;

- screening of pathological tissue (for example, tumor tissue) for the presence of specific markers;

- screening and/or development of new drugs, such as anti-cancer drugs or new combinations of drugs;

- determination of the effectiveness of a batch of medicines;

- study of the mechanisms of sensitivity and resistance of human tissues. Thus, for example, the treatment of the same tumor with several drugs and combinations of drugs can be used for a detailed mechanistic study, which cannot be easily formed in human patients;

- study of the tumor microbiome: bacteria, viruses or fungi that may be present in malignant tumors.

2. Study of the effect of new drugs or drug combinations on tumor sections to prioritize drugs or drug combinations for further drug development, as well as mechanistic studies, in vivo preclinical trials and clinical trials.

3. Predicting a patient's response to drugs (eg, anti-cancer drugs) and drug combinations, and therefore using this prediction to tailor a specific treatment regimen for the patient (ie, a personalized drug).

4. Since the tissue section retains its structure and has high viability after 5 days of culture, this system allows modeling both chronic and acute toxicity studies, including the metabolic activity of the tissue.

5. A culture showing a positive ex vivo response may be used as a criterion for inclusion of a patient in a clinical trial. Thus, this stage may pre-select patients with a high response rate and therefore may increase the likelihood of successful clinical trials. In such cases, ex vivo tissue culture may later become part of the drug acceptance criteria.

Thus, according to specific embodiments, the culture system contains a drug.

In other specific embodiments, the method of the present invention described above includes the addition of a drug or combination of drugs, as further described herein below.

As used herein, the phrase "drug" refers to an agent that has an antipathological effect, including small molecules and biological drugs (eg, nucleic acid agents, polypeptides, antibodies, aptamers, etc.). Typically, the drug is exogenous to the high-precision tissue section or to the human body or tissue from which the high-precision tissue section is obtained. The drug may be a drug approved by regulatory authorities for the treatment of a pathology, or a drug under development. In specific embodiments, the drug is a batch of drug.

In specific embodiments, the drug is an anti-inflammatory drug.

Non-limiting examples of anti-inflammatory drugs that can be used with specific embodiments of the present invention include the following: alkofenac; alkomethasone dipropionate; algestone acetonide; alpha-amylase; amcinafal; amcinafide; amfenac sodium; amiprilose hydrochloride; anakinra; anirolac; anitrazafen; range; balsalazide disodium; bendazac; benoxaprofen; benzydamine hydrochloride; bromelains; broperamol; budesonide; carprofen; cycloprofen; cintazone; klirofen; clobetasol propionate; clobetasone butyrate; clopirac; Cloticasone Propionate; cormetasone acetate; cortodoxone; deflazacort; dosonide; deoxymethasone; dexamethasone dipropionate; diclofenac potassium; diclofenac sodium; diflorazone diacetate; diflumidone sodium; diflunisal; difluprednate; diphthalone; dimethyl sulfoxide; drocinonide; endrison; enlimomab; enolicam sodium; epirizol; etodolac; etofenamate; felbinac; fenamol; fenbufen; fenclofenac; fenclorac; fendozal; fenpipalone; fentiazak; flazalon; fluazacort; flufenamic acid; flumizol; flunisolide acetate; flunixin; flunixin meglumine; fluorocortine butyl; fluorometholone acetate; fluquazone; flurbiprofen; fluretofen; fluticasone propionate; fluraprofen; furobufen; halcinonide; halobetasol propionate; halopredone acetate; ibufenac; ibuprofen; aluminum ibuprofen; ibuprofen piconol; ilonidap; indomethacin; indomethacin sodium; indoprofen; indoxol; intrazole; isoflupredone acetate; isoxepak; isoxicam; ketoprofen; lofemizole hydrochloride; lomoxicam; loteprednol etabonate; meclofenamate sodium; meclofenamic acid; meclorison dibutyrate; mefenamic acid; mesalamine; meseklazone; methylprednisolone suleptanate; momiflumate; nabumetone; naproxen; naproxen sodium; naproxol; nimazon; olsalazine sodium; orgotein; orpanoxin; oxaprozin; oxyphenbutazone; paraniline hydrochloride; pentosan sodium polysulphate; fenbutazone sodium glycerate; pirfenidone; piroxicam; piroxicam cinnamate; piroxicam olamine; pyrprofen; intended; prifelon; prodolic acid; proquazone; proxazole; proxazole citrate; rimexolone; romazarite; salcolex; salnacedin; salsalate; sanguinary chloride; seclazone; sermetacin; sudoxam; sulindac; Suprofen; talmethacin; talniflumat; talosalate; tebufelon; tenidap; tenidap sodium; tenoxicam; Tesikami; tesimide; tetridamine; thiopinacus; thixocortol pivalate; tolmetin; tolmetin sodium; triclonide; triflumidate; zidomethacin; Zomepirac sodium.

In other specific embodiments, the drug is an anticancer drug.

As used herein, the phrase "anticancer drug" refers to an agent that has an antitumor effect, including chemotherapy, small molecules, biologics, hormonal therapy, antibodies, and targeted therapy.

Anticancer drugs that can be used with specific embodiments of the present invention include, without limitation, the following: acivicin; aclarubicin; acodazole hydrochloride; acronin; adriamycin; adoselezin; aldesleukin; altretamine; ambomycin; amethantron acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brekinar sodium; bropyrimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; desaguanine; deaguanine mesylate; diaziquon; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromat; epipropidin; epirubicin hydrochloride; erbulozol; esorubicin hydrochloride; estramustine; estramutin sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozol hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidon; fostriecine sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ylmofosine; interferon alpha-2a; interferon alfa-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-1a; interferon beta-1b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; mazoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaryl; mercaptopurine; methotrexate; sodium methotrexate; methoprine; meturedepa; mitindomide; mitocarcin; mitocromine; mitogillin; mitomalcin; mitomycin; mitosper; mitotane, mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxysuran; paclitaxel; pegaspargas; peliomycin; pentamustine; peplomycin sulfate; perphosphamide; pipobroman; piposulfan; pyroxantrone hydrochloride; plicamycin; plomestan; sodium porfimer; porphyromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprin; safingol; safingol hydrochloride; semustine; symtrazen; sodium sparphosate; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; steptonigrin; streptozocin; sulofenur; talisomycin; taxol; sodium tecogalan; tegafur; teloxantrone hydrochloride; temporfin; teniposide; teroxirone; testolactone; thiamiprin; thioguanine; thiotepa; thiazofurin; tirapazamine; tapotecan hydrochloride; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulosole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; winepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozol; zeniplatin; zinostatin; zorubicin hydrochloride. Additional antineoplastic agents include those described in Chapter 52 of Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner) and Introduction thereto, 1202-1263, Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill Inc. (Health Professions Division).

Non-limiting examples of approved anti-cancer drugs include the following: abarelix, aldesleukin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, AZD4547, AZD2281, bortenomybezamybose, bevacizumab, bevacizumab, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin, actinomycin D, darbepoetin alfa, darbepoetin alfa, daunorubicin liposomal, daunorubicin liposomal, daunorubicin, decitabinoxan , dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, Elliott B solution, epirubicin, epoetin alfa, erlotinib, estramustine, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil 5-FU, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, ozogamicin acetate, histrelin acetate, hydroxyurea, ibritumomab thiuk setan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, CCNU, meclorethamine, nitrogen mustard, megestrol acetate, melphalan, L-PAM, mercaptopurine 6-MP, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, nandrolone fenpropionate, nelarabine, nofetumomab, oprelvekin, oprelvekin, oxaliplatin, paclitaxel, palbociclib palifermin, pamidronate, pegademase, pegaspargasa, pegfilgrastim, pemetrexed disodium, pimanbrostim, pentostatin, mithramycin, sodium porfimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib maleate, tamoxifen, temozolomide, teniposide VM-26, testolactone, thioguanine 6-TG, thiotepa, thiotepa, topotecan, toremifene, tositumomab, trametinib, trastuzumab, tretinoin ATRA, uracil mustard, valrubicin, vinblastine, vinorelbine, zoledronate, and zoledronic acid.

In specific embodiments, the anticancer drug is selected from the group consisting of the following: gefitinib, lapatinib, afatinib, BGJ398, CH5183284, linsitinib, PHA665752, crizotinib, sunitinib, pazopanib, imatinib, ruxolitinib, dasatinib, BEZ235, pictilisib, everolimus, MK-2206 , trametinib/AZD6244, vemurafinib/dabrafenib, CCT196969/CCT241161, baracertib, VX-680, nutlin3, palbociclib, BI 2536, bardoxolone, vorinostat, navitoclax (AVT263), bortezomib, vismodegib, olaparib (AZD2281), simvastatin, 5-fluorocil, irinotecan, epirubicin, cisplatin and oxaliplatin.

In specific embodiments, the anticancer drug is selected from the group consisting of 4-hydroperoxycyclophosphamide, 5-fluorouracil (5FU), oxaliplatin, irinotecan, docetaxel, cisplatin, trametinib, palbociclib, and AZD4547.

In specific embodiments, the anticancer drug is selected from the group consisting of 5-fluorouracil (5FU), oxaliplatin, irinotecan, cisplatin, 4-hydroperoxycyclophosphamide, decetaxel, doxorubicin, navelbine, gemcitanim, gefitinib, tamoxifen, olaparib, trametinib, everolimus, and palbociclib.

It is understood that the culture system and methods described herein can be used to investigate the differential effect of drug combinations.

Thus, in specific embodiments, the drug is a combination of drugs.

Specific drug concentrations in the culture system and methods of the present invention can be obtained by culturing different concentrations of drug on several malignant tissues (of the same type or different types) and assessing the range over which a dose-dependent response is achieved. Typically, this dose-dependent range reflects the spectrum according to which the cultured malignant tissue should respond or not respond, which gives a predictive result.

In specific embodiments, the drug concentration in the culture system and methods of the present invention is at least the same as the IC50 dose of the drug in the cell line (eg, a cell line of the same tissue type and/or species).

In specific embodiments, the drug concentration in the culture system and methods of the present invention is higher than the IC50 dose of the drug in the cell line (eg, a cell line of the same tissue type and/or species).

According to specific embodiments, the drug concentration in the culture system and methods of the present invention is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50 times, at least 100 times the IC50 dose of the drug in a cell line (eg, in a cell line of the same tissue type and/or species).

In specific embodiments, the drug concentration in the culture system and methods of the present invention is at least 10 times the IC50 dose of the drug in the cell line (eg, cell line of the same tissue type and/or species).

Specific concentrations of anticancer drugs that can be used with some embodiments of the present invention are shown in tables 5-6 below.

In specific embodiments, multiple tissue sections from a single tissue are prepared and cultured in multiple culture vessels (eg, plate wells) to allow testing of a range of drugs and drug combinations.

In specific embodiments, the methods of the present invention include:

(i) determining the effect of multiple (eg, at least 2) drugs in accordance with the method of the present invention; as well as

(ii) determining the effect of drug combinations shown to be effective according to step (i), wherein increased tissue sensitivity to the drug combination is indicative of the effectiveness of the drug combination.

As used herein, the term "hypersensitivity" refers to an increase in the effect induced by a combination of drugs over the effect induced by each of the drugs in the combination, which may be an additive or synergistic effect. In specific embodiments, the effect is a synergistic effect.

In specific embodiments, the magnification is at least 1.5x, at least 2x, at least 3x, at least 5x, at least 10x, or at least 20x, compared with the effect caused by each of the drugs in combination.

In other specific embodiments, the increase is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 100% of the effect produced by each of the drugs in combination.

As mentioned, in particular embodiments, the tissue or tissue sections may be stored, i. at 4°C, or be cryopreserved and thawed at will, allowing sequential testing of multiple drugs and drug combinations.

Thus, for example, according to specific embodiments, the methods of the present invention include:

(i) determining the effect of at least two drugs in accordance with the method of the present invention;

(ii) culturing an additional tissue section obtained from the pathological tissue obtained from the subject (eg, after cryopreservation and thawing) in accordance with the method of the present invention;

(iii) adding combinations of drugs determined to be effective in treating the disease in the subject according to step (i); as well as

(iv) determining the effect of the drug combination on the tissue section, wherein increased sensitivity of the tissue section to the drug combination is indicative of the efficacy of the drug combination in treating a disease in the subject.

The drug or combination of drugs can be added to the culture at different times. In specific embodiments, the drug is added to the culture 2-48 hours, 2-36, 2-24, 12-48, 12-36, or 12-24 hours after the start of culture.

In a specific embodiment, the drug or combination of drugs is added to the culture 12-24 hours after the start of the culture.

In a specific embodiment, the drug or combination of drugs is added to the culture 12-36 hours after the start of the culture.

In a specific embodiment, the drug or combination of drugs is added to the culture 12-48 hours after the start of the culture.

Cultivation in the presence of a drug or combination of drugs may be carried out during the entire period of cultivation from the time of the first addition of the drug, or may be limited in time. Alternatively or additionally, the drug or combination of drugs may be added to the culture multiple times, such as when the culture medium is updated.

The choice of drug concentration and incubation time with the drug or drug combination is within the ability of those skilled in the art.

In specific embodiments, the drug concentration and incubation time with the drug or drug combination results in a detectable effect on tissue, as further described below.

The selection of the concentration of drug used in an ex vivo study that will result in a detectable effect on tissue is within the ability of those skilled in the art. Preferably, the concentration used should be in the linear range of the selected parameter.

The amount of study drug can be at least 1, at least 2, at least 3, at least 5, at least 6, 1-10, 2-10, 3-10, 5-10, 1-5 , 2-5 and 3-5 different concentrations in the same assay.

The number of sample replicates for each of the tested drug concentrations can be 2, 3, 4, 5, or 6 replicates.

After culturing, the effects of the drug or combination of drugs on tissues can be determined to thereby determine the efficacy of the drug.

Thus, according to another aspect of the present invention, there is a method for determining the potency of a medicinal product or batch of medicinal products, the method comprising:

(i) culturing the high precision tissue section on a tissue culture insert in a culture medium in a high oxygen atmosphere containing at least 50% oxygen; and agitation of the culture by means of a rotation causing said tissue section to be intermittently immersed in said culture medium;

(ii) adding the drug or batch of drugs to said culture medium; as well as

(iii) determining the effect of said drug or said batch of drugs on said tissue, wherein the sensitivity of said tissue to said drug is indicative of the efficacy of said drug.

In specific embodiments, the detection step is performed after a predetermined culture time. The culturing time may vary, and determining the culturing time that will result in a detectable effect is within the ability of those skilled in the art.

In specific embodiments, the determination is carried out within 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, or 4-5 days of culture.

According to a specific embodiment, the determination is carried out within 3-5 days of cultivation.

According to another specific embodiment, the determination is carried out within 4-5 days of cultivation.

Methods for determining effects caused by a drug or drug combination are known in the art and include, for example:

- Viability assessment using, for example, the MTT assay based on the selective ability of live cells to reduce the yellow salt of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (Sigma, Aldrich St Louis, MO , USA) to a purple-blue insoluble formazan precipitate; WST analysis or ATP uptake analysis;

- Evaluation of proliferation using, for example, BrDu assay [BrdU ELISA colorimetric kit for cell proliferation (Roche, Mannheim, Germany] or Ki67 staining;

- Cell death assessment using e.g. TUNEL assay [Roche, Mannheim, Germany], annexin V assay [ApoAlert® Annexin V apoptosis kit (Clontech Laboratories, Inc., CA, USA)], LDH assay, activated caspase 3 assay, activated caspase 8 assay and nitric oxide synthase assay;

- Assessing aging using, for example, the aging-associated p-galactosidase assay (Dimri GP, Lee X, et al. 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92: 9363-9367) and telomerase shortening assay;

- Evaluation of cellular metabolism using, for example, glucose uptake analysis;

- Various methods for detecting RNA and protein (which determine the level of expression and/or activity); as well as

- Assessment of morphology using, for example, staining with hematoxylin and eosin (H&E).

In specific embodiments, the determination is influenced by a morphology score, a viability score, a proliferation score, and/or a cell death score.

In specific embodiments, the determination is made through morphology evaluation.

Morphology assessment using H&E staining can provide detailed information such as cell content, size and density, viable cell/dead cell ratio, diseased (e.g. tumor) cell/healthy cell ratio, immune cell infiltration, fibrosis, size and nuclear density, integrity, apoptotic bodies and mitotic figures. In specific embodiments, the effect of the drug on tissue is determined by morphology assessment, such as by a pathologist.

Typically, the results of each of the assays are expressed in numerical form, with a high score correlated with drug sensitivity and a low score correlated with drug resistance.

As used herein, the phrase "drug sensitivity" or "drug combination sensitivity" refers to the ability of a drug or combination of drugs to induce cellular changes such as changes in cell viability, proliferation rate, differentiation, cell death, necrosis, apoptosis, aging, the rate of transcription and/or translation of specific genes, and/or changes in protein states such as phosphorylation, dephosphorylation, translocation, and any combination thereof. In specific embodiments, the cellular changes are reflected in decreased cell viability, decreased proliferation rate, increased cell death and/or aberrant morphology compared to those in the absence of the drug.

In a particular embodiment, the cellular changes are reflected in a decrease in cell viability.

In a specific embodiment, cellular changes are reflected by aberrant morphology.

In specific embodiments, the change is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold in compared with the same in the absence of the drug.

In other specific embodiments, the change is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 100% compared to those in the absence of drug.

In specific embodiments, a certain drug potency is indicative of the potency of a drug lot, i.e. drug susceptibility indicates that the lot is potent.

As used herein, the term "potency" refers to a measure of the biological activity of a drug based on an attribute of the drug that is associated with the relevant biological properties (i.e., drug susceptibility).

According to specific embodiments of the present invention, the method comprises comparing tissue sensitivity to a drug with tissue sensitivity to a reference standard lot of drugs to determine the relative potency of the lot.

As used herein, the term "relative potency" refers to a qualitative measure of the potency of a batch of drugs relative to a standard reference (RS) of a drug of known potency.

In specific embodiments, the potency of a batch of drugs is determined relative to the known potency of a reference standard (RS).

As used herein, the expression "reference standard" or "RS" refers to a standardized drug that is used as the basis of measurement for a drug. The RS provides a calibrated level of biological effect against which new drug formulations can be compared.

In a particular embodiment, RS is characterized by optimal activity and quality of the active ingredient that is effective in treating a disease (eg, inflammatory disease, cancer).

The calculation of activity and relative activity is known in the present technical field. In specific embodiments, relative activity is calculated using software suitable for biological assays such as parallel line analysis software such as PLA (Stegmann Systems GmbH) and Gen5 data analysis software (BioTek).

The implementation of the method and/or system of embodiments of the present invention may involve the implementation or execution of selected tasks manually, automatically, or a combination of both. In addition, according to the actual tooling and hardware of the method and/or system embodiments of the present invention, several selected tasks may be implemented in hardware, software, or firmware, or a combination thereof, using an operating system.

In specific embodiments, a certain efficacy of a drug or combination of drugs is indicative of the suitability of the drug for the treatment of a disease.

Since the present inventors show that the developed EVOC system can predict tumor responses to anti-cancer drugs and combination therapy (Example 2 in the next section of examples), the present invention also contemplates using the culture systems and methods described herein to predict the response of a particular subject. to a treatment regimen, thereby selecting an appropriate treatment regimen for that particular subject and treating the subject in accordance with that selection.

Thus, according to an aspect of the present invention, there is provided a method for selecting a drug for the treatment of a disease in a subject in need thereof, the method comprising:

(i) culturing a highly accurate section of pathological tissue obtained from the subject on a tissue culture insert in a culture medium in a high oxygen atmosphere containing at least 50% oxygen; and agitation of the culture by means of a rotation causing said tissue section to be intermittently immersed in said culture medium;

(ii) adding a drug to said culture medium; as well as

(iii) determining the effect of said drug on said tissue, wherein the sensitivity of said tissue to said drug is indicative of the efficacy of said drug in treating said disease in said subject.

According to another aspect of the present invention, there is provided a method for treating a disease in a subject in need thereof, the method comprising:

(a) selecting a drug or combination of drugs in accordance with the method described herein; as well as

(b) administering to said subject a therapeutically effective amount of a drug or combination of drugs shown to be effective in treating said disease in said subject, thereby treating the disease in the subject.

As used herein, the phrase "subject" refers to a mammalian subject (eg, a human) who has been diagnosed with a disease or is at risk of developing a disease. Use in animals is also under consideration. The subject may be of any gender and any age, including neonates, infants, juveniles, adolescents, adults, and the elderly.

The term "treatment" refers to the inhibition or cessation of the development of a pathology (disease, disorder or condition) and/or the reduction, remission or regression of the pathology. Those skilled in the art will appreciate that various methodologies and assays may be used to assess the progression of pathology, and similarly, various methodologies and assays may be applied to assess reduction, remission, or regression of pathology.

Determining a therapeutically effective amount of a drug or combination of drugs is within the skill of the art. The dosage may vary depending on the drug selected, the dosage form used and the route of administration used. The precise composition, route of administration, and dosage can be selected by the individual physician based on the condition of the patient. (See, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

In specific embodiments, the disease is an inflammatory disease, which may be a chronic inflammatory disease or an acute inflammatory disease.

Inflammatory Disorders Associated with Hypersensitivity Non-limiting examples of hypersensitivity include: Type II hypersensitivity, including but not limited to rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15(3) :791), spondylitis, ankylosing spondylitis (Krenn V. et al., Histol Histopathol 2000 Jul; 15(3):791), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998 ; 17 (1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar; 6 (2): 156); Chan FROM. et al., Immunol Rev 1999 Jun; 169:107), glandular disease, glandular autoimmune disease, autoimmune pancreatic disease, type I diabetes mellitus (Zimmet P. Diabetes Res Clin Pract 1996 Oct; 34 Suppl:S125), thyroid disease, autoimmune thyroid disease, Graves' disease ( Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29(2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165(12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug; 57 (8): 1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug; 57 (8): 1759); autoimmune reproductive diseases, ovarian diseases, autoimmune ovarian diseases (Garza KM. et al., J Reprod Immunol 1998 Feb; 37 (2): 87), autoimmune antispermal infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar ;43(3):134), recurrent fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH et al ., J Neuroimmunol 2001 Jan 1; 112 (1-2): 1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18 (1-2): 83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May; 7 (3): 191), Guillain-Barré syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr; 319 (4): 234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr; 319 (4): 204), paraneoplastic neuro logical diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic rigidity syndrome in men, cerebellar atrophy, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Hidey's syndrome, Gilles de la Tourette's syndrome, polyendocrinopathy, autoimmune polyendocrinopathy (J.Antoine). and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156(1):23); neuropathies, disimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999; 50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13; 841:482), cardiovascular disease, cardiovascular autoimmune disease, atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), granulomatosis, granulomatosis Wegener, arteritis, Takayasu arteritis and Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112(15-16):660); autoimmune disease against factor VIII (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26 (2): 157); vasculitis, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151(3):178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171); heart failure, anti-β-adrenergic agonist-like antibodies in heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17; 83(12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr- Jun 14(2):114); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan; 28(3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory bowel diseases (Garcia Herola A. et al., Gastroenterol Hepatol 2000 Jan; 23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), autoimmune muscle diseases, myositis, autoimmune myositis, Sjögren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123(1):92); autoimmune smooth muscle disease (Zauli D. et al., Biomed Pharmacother 1999 Jun; 53(5-6):234), liver disease, autoimmune liver disease, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug; 33(2): 326) and primary biliary cirrhosis (Strassburg SR. et al., Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6): 595);

Type IV or T cell mediated hypersensitivity including, but not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci USA 1994 Jan 18;91(2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998; 7(9):591), glandular diseases, autoimmune glandular diseases, pancreatic diseases, pancreatic autoimmune diseases, type 1 diabetes mellitus (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S. et al., Mol Cell Endocrinol 1993 Mar; 92(1):77); ovarian disease (Garza KM. et al., J Reprod Immunol 1998 Feb; 37(2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec; 50(6):893), polyglandular syndrome , autoimmune polyglandular syndrome, autoimmune polyglandular syndrome type I (Hara T. et al., Blood. 1991 Mar 1; 77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M et al., J Neurol Neurosurg Psychiatry 1994 May;57(5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990 Dec;20(12):2563), muscle stiffness syndrome (Hiemstra HS et al., Proc Natl Acad Sci USA 2001 Mar 27; 98 (7): 3988), cardiovascular disease, cardiac autoimmunity in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15; 98 (8 ):1709), autoimmune thrombocytopenic purpura (Semple JW. et al., Blood 1996 May 15; 87(10):4245), autoimmunity against helper T-lymphocytes (Caporossi AP. et al., Viral Immunol 1998; 11 (1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 Mar; 74 (3): 139), liver disease, autoimmune liver disease, hepatitis, chronic active hepatitis (Franco A. et al. , Clin Immunol Immunopathol 1990 Mar; 54(3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91(5):551), kidney disease, autoimmune kidney disease, nephritis , interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2): 140), connective tissue diseases, ear diseases, connective tissue autoimmune diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug; 157 (1):249), inner ear disease (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29; 830:266), skin diseases, skin diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigus, and pemphigus foliaceus ;

Delayed type hypersensitivity including, but not limited to, contact dermatitis and drug dermatitis.

Autoimmune diseases

Include, without limitation, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, muscle diseases, kidney diseases, reproductive diseases, connective tissue diseases, and systemic diseases.

Examples of autoimmune cardiovascular diseases include, without limitation, atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112 (15-16 ):660), autoimmune disease against factor VIII (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000; 26 (2): 157), necrotizing vasculitis of small vessels, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171), antibody-induced heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17; 83(12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 199 9 Apr-Jun; 14(2):114; Sample JW. et al., Blood 1996 May 15; 87(10):4245), autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan; 28(3-4):285; Sallah S. et al., Ann Hematol 1997 Mar; 74(3): 139), cardiac autoimmunity in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15; 98 (8): 1709) and autoimmunity against helper T-lymphocytes (Caporossi AP. et al., Viral Immunol 1998 ; 11(1):9).

Examples of autoimmune rheumatoid diseases include, without limitation, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15(3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3(3):189).

Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, type I diabetes mellitus, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune antispermal infertility, autoimmune prostatitis, and autoimmune type I polyglandular syndrome. Diseases include, without limitation, autoimmune diseases of the pancreas, type I diabetes mellitus (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct; 34 Suppl:S125) , autoimmune thyroid disease, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun; 29(2):339; Sakata S. et al., Mol Cell Endocrinol 1993 Mar; 92(1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12): 7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug; 57 (8): 1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug; 57 (8): 1759), autoimmune ovarian disease (Garza KM. et al., J Reprod Immunol 1998 Feb; 37 (2): 87), autoimmune antisperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43(3):134), autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50(6):893), and type I autoimmune polyglandular syndrome (Hara T et al., Blood. 1991 Mar 1; 77(5):1127).

Examples of autoimmune gastrointestinal diseases include, without limitation, chronic inflammatory bowel disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan; 23(1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), colitis, ileitis and Crohn's disease.

Examples of autoimmune skin diseases include, without limitation, autoimmune bullous skin diseases such as, but not limited to, pemphigus vulgaris, bullous pemphigus, and pemphigus foliaceus.

Examples of autoimmune liver diseases include, without limitation, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar; 54 (3): 382), primary biliary cirrhosis (Jones DE. Clin Sci ( Colch) 1996 Nov;91(5):551; Strassburg CP et al., Eur J Gastroenterol Hepatol 1999 Jun;11(6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33(2) :326).

Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH et al., J Neuroimmunol 2001 Jan 1; 112 (1-2): 1), Alzheimer's disease (Oron L. et al., J Neural Transm. Suppl. 1997; 49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18(1-2):83; Oshima M. et al., Eur J Immunol 1990 Dec; 20(12) :2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May; 7 (3): 191); Guillain-Barré syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr; 319 (4): 234), myasthenia gravis, myasthenic Lambert-Eaton syndrome (Takamori M. Am J Med Sci. 2000 Apr; 319 (4) :204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiffness syndrome (Hiemstra HS. et al., Proc Natl Acad Sci units S A 2001 Mar 27; 98(7):3988); non-paraneoplastic stiffness syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sideghem's chorea, Gilles de la Tourette's syndrome and autoimmune polyendocrinopathy (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1): 23); disimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999; 50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13; 841:482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May; 57 ( 5):544) and neurodegenerative diseases.

Examples of autoimmune muscle diseases include, without limitation, myositis, autoimmune myositis, and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123(1):92) and autoimmune smooth muscle disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53(5-6):234).

Examples of autoimmune kidney diseases include, without limitation, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1(2): 140).

Examples of autoimmune reproductive-related diseases include, without limitation, repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl 2:S 107-9).

Examples of autoimmune connective tissue diseases include, without limitation, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug; 157(1):249) and autoimmune diseases of the inner ear (Gloddek B. et al. , Ann N Y Acad Sci 1997 Dec 29; 830:266).

Examples of autoimmune systemic diseases include, without limitation, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17(l-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol Mar. 1999;6(2):156); Chan FROM. et al., Immunol Rev 1999 Jun; 169:107).

Infectious diseases

Examples of infectious diseases include, without limitation, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasmal diseases, and prion diseases.

In specific embodiments, the disease is a malignant tumor.

The terms "cancer" and "malignant" describe a physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, the terms "cancer" and "malignant" refer to any solid tumor, cancer metastasis, and/or solid precancer.

Examples of a malignant tumor include, without limitation, carcinoma, blastoma, sarcoma, and lymphoma. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung carcinoma), glioma, melanoma, peritoneal cancer, hepatocellular cancer, gastric cancer. , stomach and esophagus or stomach (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon and rectum cancer, endometrial or uterine carcinoma, salivary gland carcinoma, soft tissue sarcoma, kidney cancer, prostate cancer, malignant vaginal cancer, thyroid cancer, hepatic carcinoma, Kaposi's sarcoma, carcinoid carcinoma, and various types of head and neck cancer.

Precancerous conditions are well characterized and known in the art (see, for example, Berman JJ. and Henson DE., 2003. Classifying the precancers: a metadata approach. BMC Med Inform Decis Mak. 3:8). Examples of precancerous diseases include, but are not limited to, acquired small precancerous conditions, acquired large lesions with nuclear atypia, precancerous lesions arising from hereditary hyperplastic syndromes that progress to malignancy, and acquired diffuse hyperplasias and diffuse metaplasias. Non-limiting examples of small precancerous conditions include HGSIL (high grade squamous intraepithelial lesion of the cervix), AIN (anal intraepithelial neoplasia), vocal cord dysplasia, aberrant crypts (colon), PIN (prostate intraepithelial neoplasia).

Non-limiting examples of acquired large lesions with nuclear atypia are tubular adenoma, AILD (angioimmunoblastic lymphadenopathy with dysproteinemia), atypical meningioma, gastric polyp, large plaque parapsoriasis, myelodysplasia, papillary transitional cell carcinoma in situ, refractory anemia with blast excess, and Schneiderian papilloma. Non-limiting examples of precursor lesions occurring with hereditary hyperplastic syndromes that progress to malignancy include atypical mole syndrome, C-cell adenomatosis, and MEA. Non-limiting examples of acquired diffuse hyperplasia and diffuse metaplasia include Paget's disease of the bone and ulcerative colitis.

In specific embodiments, the cancer is selected from the group consisting of ovarian cancer, colon and rectal cancer, lung cancer, pancreatic cancer, gastric cancer, gastroesophageal cancer, and breast cancer.

In specific embodiments, the cancer is a metastatic cancer.

Since various complementary technologies are available for predicting a patient's response to a treatment regimen, the methods of the present invention can be combined with other methods known in the art, such as, but not limited to, genetic profiling, tumor-derived patient models of tumor-derived cell lines, and derived from patient xenografts (PDX), as also described in the examples section below.

In specific embodiments, the method is carried out in combination with genetic profiling.

As used herein, the term "genetic profiling" refers to the molecular signature of a set of genes in biological samples using any suitable profiling technology such as, but not limited to, DNA sequencing, RNA sequencing, and microarray techniques.

As used herein, the term "about" refers to ±10%.

The terms "comprises", "comprising", "includes", "including", "having" and their related words in root and meaning mean "including, without limitation".

The term "consisting of" means "including and limited".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not significantly change the main and new characteristics of the claimed composition, method or structures.

As used herein, the singular forms include plural references unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" can include a variety of compounds, including mixtures thereof.

Throughout the present application, various embodiments of the present invention may be presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered as specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and and so on, as well as individual numbers in that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

Whenever a numerical range is specified herein, it is intended to include any cited number (fractional or integer) within the specified range. The phrases "range/ranges between" the first specified number and the second specified number and "range/ranges from" the first specified number "to" the second specified number are used interchangeably herein and are intended to include the first and second specified numbers and all fractional and integer numbers. between them.

As used herein, the term “method” refers to methods, means, techniques, and procedures for accomplishing a given task, including, without limitation, those methods, means, techniques, and procedures known or readily developed from known methods, means, techniques. and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

When reference is made to specific sequence listings, it is to be understood that such reference also covers sequences that substantially correspond to its complementary sequence, including minor sequence variations resulting, for example, from sequence errors, cloning errors, or other changes, resulting in a base substitution, base deletion, or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5000 nucleotides, alternatively less than 1 in 10000 nucleotides.

It is understood that certain features of the present invention, which are described in the context of separate embodiments for clarity, may also be provided in combination in one embodiment. Conversely, various features of the present invention, which for brevity are described in the context of one embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment operates without those elements.

The various embodiments and aspects of the present invention described above and claimed in the claims section below find experimental support in the following examples.

EXAMPLES

The following examples, together with the above descriptions, illustrate some embodiments of the present invention in a non-limiting manner.

Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological, and recombinant DNA methods. Such techniques are described in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal., "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methodologies set forth in US Pat. No. 4,666,828; 4683202; 4801531; 5192659 and 5272057; "Cell Biology: A Laboratory Handbook," Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are described in detail in the patent and scientific literature, see, for example, US patent No. 3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; "Oligonucleotide Synthesis" Gait, M.J., ed. (1984); "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., eds. (1985); "Transcription and Translation" Hames, B.D., and Higgins S.J., eds. (1984); "Animal Cell Culture" Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal., B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if they were set forth herein in their entirety. Other general references are provided in this document. It is believed that the procedures in this document are well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Tissue Sections - Healthy and malignant tissues (liver, lung, colon, breast, pancreas, or metastatic malignant tissue originating from the lung, colon, breast, or pancreas) were obtained from PDX mouse models (Nod Scid Gamma Mice Jackson Laboratories USA and/or Harlan Laboratories); human biopsies and surgically removed tissues; and human-derived xenografts (ovarian cancer, pancreatic PDX). After collection, fresh tissue was immediately placed in ice-cold phosphate buffered saline (PBS, Biological Industries, Kibbutz Beit Haemek, Israel) or ice-cold culture medium (RPMI Biological Industries). A high precision tissue cutter (CompresstomeTM VF-300: Precision Instruments Inc. NC, USA) was cleaned with 70% ethanol, rinsed twice with sterile water, and a new blade (Double Edge Blades 32017759 Belgar, Jerusalem, Israel) was attached and positioned in the desired location.

The fabric was left on ice until it was transferred to a pre-cleaned fabric cutter; the tissue was then removed from PBS and carefully attached to the plunger with a contact adhesive and stabilized in 3% low gel agarose, heated to 50°C and then cooled to solidify before use (agarose, low gel A0701 Sigma-Aldrich, 3% in 0.9 g of NaCl). The tissue plunger was inserted into the designated location in the tissue cutter and the bath filled with ice-cold William's E medium supplemented with penicillin and streptomycin. To maintain the environment in the bath at low temperatures, a hollow cooling coil was designed, which is located in the bath. Ice water was then pumped through the coil using a standard submersible pump. The tissue was cut into sections up to ~250 µM thick. Approximately 30-40 sections were prepared from a 1 cm ³ piece of tissue. Two or three sections were immediately placed in disposable plastic histological cassettes and fixed in 4% PFA.

cultivation procedure. Tissue sections were placed individually in 6-well plates (Corning CC-3516 Getter, Petach-Tikva, Israel) directly, over titanium inserts (MA0036 Well Inserts, Alabama R&D, Alabama, USA) or on PEG or PEG placed on a titanium insert . Each well contained 4.5 ml of DMEM/F12 or M199 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), gentamicin (G1397 50 µg/ml Sigma-Aldrich), 5% fetal calf serum (FCS , 10270106 Gibco Sigma-Aldrich, Rehovot, Israel), amphotericin B (A-4888 2.5 µg/ml Sigma-Aldrich) and glutamine (L-glutamine 03-020-1 B 100 µl/ml Biological Industries). The plates were incubated in a humidified incubator at 37°C with 5% CO ₂ and 21% or 80% O ₂ (using oxygen chamber: BioSpherix C274, NY, USA; oxygen controller: Biospherix ProOx C21, NY, USA, and oxygen 98 % Maxima Air Innovations, Ashdod, Israel).

The entire incubator was placed in an orbital shaker with agitation (TOU-120N MRC, Holon, Israel) at 70 rpm. After incubation overnight, the medium was replaced with fresh medium. In these wells, fresh medium contained the anticancer drug [4-hydroperoxycyclophosphamide (sc-206885 ENCO, Petah Tikva, Israel), 5-fluorouracil (5FU, A10042 Adooq Biotag, Kfar Yona, Israel), oxaliplatin (09512 Sigma-Aldrich), irinotecan (A10479-200 Adooq Biotag, Kfar Yona, Israel), docetaxel (01885 Sigma-Aldrich), cisplatin (Sigma-Aldrich,), trametinib (A11029 Adooq Biotag, Kfar Yona, Israel), CDK4/6 inhibitor palbociclib (PZ0199 Sigma-Aldrich) or FGFR inhibitor AZD4547 (A11075-5 Adooq Biotag) at the indicated concentrations. The medium was replaced once every 48 hours with fresh drug/DMSO for 4-6 days.

Functional analyses. Eighteen to twenty-four hours before the end of the culture, BrdU was added to the medium (final concentration: 10 μM/ml) for subsequent staining with an anti-BrdU antibody (BRDU+anti-BRDU B5002 Sigma-Aldrich and 94-MS-1058-P Eldan, Petach- Tikva, Israel) and/or Ki67 (94-RM-9106-S Eldan, Petah Tikva, Israel). At the end of the culture, tissues were placed between two glass coverslips, placed in a disposable plastic histology cassette, and fixed overnight in 4% paraformaldehyde (PFA). Sections were then paraffinized, blocked and cut into 5 μM sections for hematoxylin and eosin (H&E) staining and other IHC staining such as Ki67 staining.

Example 1

Conditions for ex vivo organ culture to maintain the structure and functions of sections of healthy and tumor tissues

To obtain tissue structure and function for at least 5 days in ex vivo organ culture (EVOC), several conditions were investigated. Conditions included:

1. Culture of tissue sections on different surfaces such as titanium inserts versus direct immersion of the sections in culture medium;

2. Tissue incubation under standard tissue culture conditions with 21% O ₂ and 5% CO ₂ versus a high oxygen atmosphere containing 80% O ₂ ;

3. Culturing 1 slice per well versus multiple slices per well;

4. Using different media such as DMEM/F12 and M199;

5. Cultivation on PEG with or without titanium inserts. The results are summarized in Table 1 below.

To this end, tissue incubation in a high oxygen atmosphere containing 80% O ₂ significantly increased tissue viability compared to standard tissue culture conditions with 21% O ₂ (a representative culture of xenografts derived from human ovarian cancer is shown in FIG. one). In addition, tissue survives better when supported on titanium inserts that allow interaction between media and air compared to directly inserting the tissue into the tissue culture well so that the section is completely immersed in the media (representative culture of xenografts derived from ovarian cancer human, shown in Fig. 1). In addition, DMEM/F12 was a better medium than M199 (a representative xenograft culture derived from human ovarian cancer is shown in Figure 2).

All tissues for EVOC presented in Table 1 above were obtained from surgery. However, the assay developed can be adapted to small core biopsies taken from tumor tissue (Fig. 9). Specifically, tissue is immersed in agarose in two steps: first, liquid agarose is placed in a small metal tray, core biopsies are placed on top of the agarose, and agarose is added on top of the biopsies. The tray is cooled and the biopsy agarose is excised in the block. In the second step, the agarose block is attached to the plunger with a contact key and surrounded by agarose in a manner similar to that used for tissue pieces.

In general, EVOCs were obtained from a variety of sources representing multiple tissues that maintained tissue structure and viability for at least 5 days of culture (FIGS. 1, 2 and 11). It is important to note that all tissues were examined by a pathologist and were considered fully viable with the same morphology as normal tissue pathology from the patient.

Overall, culturing a single tissue section directly in the middle of the titanium insert in DMEM/F12 medium in a high oxygen atmosphere containing 80% O ₂ and agitating the culture with orbital shaking, which promotes intermittent dipping of the tissue section into the culture medium, maintained structure and viability. tissue for at least 5 days of cultivation. This newly developed EVOC system was used in the following examples.

Example 2

Developed EVOC can be used to predict the response of tumors to anticancer drugs

Designed EVOC can predict the in vivo response of patient-derived xenografts to treatment: Several mouse-derived xenograft (PDX) models with known in vivo responses to anticancer drugs were purchased from Jackson Laboratories. Tumors were excised from mice, cultured as described in Materials and Methods and Example 1 above, and sensitivity of tissue sections to anticancer drugs was examined. A total of seven PDX models originating from 3 types of tumors (breast cancer, lung cancer, and colon and rectal cancer) were studied, and in all of the models studied, the developed EVOC system was able to correctly predict the susceptibility of tumors to drug treatment (Table 2 below). and Fig. 3-4). In addition, a clear dose-dependent response was observed in the EVOC system.

Thus, as can be seen in FIG. 3-4 and in Table 2 below, tumors that were resistant to in vivo treatment were also resistant to ex vivo treatment, even when very high doses were used. In contrast, when using a treatment to which tumors were sensitive in vivo, cell death was detected ex vivo at low drug concentrations, and drug sensitivity increased with increasing drug concentration.

The developed EVOC demonstrates that human tumors have differential sensitivity to various anti-cancer therapies. Patient tumor tissues excised from the patient's tumors during surgical excision were directly cultured as described in the materials and methods above and in Example 1, and the sensitivity of the tissue sections to anticancer drugs was examined. The most common anti-cancer regimens have been used for each tumor type, ie, for example, for human colon and rectal cancer, standard treatment is 5-fluorouracil, oxaliplatin, and/or irinotecan. In FIG. 5 shows an example of a tumor that was found to be resistant to high doses of 5-fluorouracil, but relatively sensitive to irinotecan and oxaliplatin. Determination of cell proliferation, such as Ki67 staining, can also be used to evaluate tissue section viability and sensitivity to anticancer drugs. Thus, in FIG. 8 shows an example of a malignant tumor of the colon and rectum that was partially sensitive to 5-fluorouracil, but very sensitive to oxaliplatin treatment by Ki67 staining.

Overall, the developed EVOC system has clearly shown that a patient's tumors can be sensitive to one anti-cancer drug but resistant to another. Moreover, EVOCs from different patients responded differently to the same anti-cancer treatment (FIG. 6 and Tables 3-4 below). In addition, a dose-dependent response was observed for each anti-cancer treatment (eg, Tables 3-4).

Developed EVOC can predict sensitivity to targeted therapy as predicted by molecular profiling: As a tool for personalized medicine, a patient's tumor tissue can be examined for possible beneficial genetic mutations, for example, by exome sequencing. One of the tumors was obtained from a patient with triple negative breast cancer, which was found to carry somatic genetic changes that could potentially be targeted, including FGFR1 amplification and CDK6 amplification. As can be seen in FIG. 7, although the tumor derived from this patient (Patient 1) was ex vivo resistant to the CDK4/6 inhibitor palbociclib, it showed high sensitivity to the AZD4547 FGFR inhibitor. Most importantly, breast cancer derived from another patient who did not have FGFR1 amplification (Patient 2) was resistant to FGFR inhibition in the developed EVOC system.

Tissue may be stored for 48 hours prior to culture: Patient tumor tissues excised from patient tumors during surgical excision were directly cultured as described in Materials and Methods above and in Example 1, or cultured after 48 hours of storage in medium at temperature 4°C; and investigated the sensitivity of tissue sections to anticancer agents. In FIG. 10 shows an example of colon cancer tissue found to be partially sensitive to 5-fluorouracil in a similar manner under both conditions (i.e., direct culture or after 48 hours of storage at 4°C).

In general, tissue can be stored in the medium for at least 48 hours prior to the start of the experiment without any adverse effects on tissue or drug sensitivity.

Overall, the newly developed EVOC makes the process of studying the response of human malignant tissue fast, economical, and versatile for many malignant tissues. All malignant tissues examined, including those of the ovary, colon and rectum, lung, pancreas, and breast, showed high viability after 4-7 days in culture.

Although the present invention has been described in connection with its specific implementation options, it is obvious that many alternatives, modifications and variations will be obvious to experts in the present field of technology. Accordingly, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents, and patent applications referred to in this specification are incorporated herein in their entirety by reference in the specification to the same extent as if each individual publication, patent, or patent application were specifically and individually designated for inclusion in this document. through a link. In addition, the citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be regarded as mandatory restrictions.

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Claims (32)

1. A culture system comprising a culture medium and one high-precision tissue section placed on a tissue culture insert in a well of a tissue culture plate, said high-precision tissue section being maintained in a high oxygen atmosphere containing at least 60% and less than 95% oxygen. % oxygen, and the specified culture medium is suitable for maintaining the viability of the specified tissue section and wherein the specified culture is mixed rotationally, contributing to the periodic immersion of the specified tissue section in the specified culture medium. 2. A culture system according to claim 1 containing a drug. 3. The culture system according to any one of paragraphs. 1, 2, wherein said tissue culture insert is a titanium mesh insert or a stainless steel insert. 4. The culture system according to any one of paragraphs. 1, 2, wherein said high oxygen atmosphere contains at least 70% oxygen. 5. The culture system according to any one of paragraphs. 1, 2, in which the specified culture medium contains DMEM/F12. 6. A tissue culture method comprising culturing a single precision tissue section on a tissue culture insert in a well of a tissue culture plate in a culture medium suitable for maintaining the viability of said tissue section in a high oxygen atmosphere containing at least 60% or less than 95% oxygen; and agitating the culture by rotation causing said tissue section to be intermittently immersed in said culture medium. 7. The method of claim 6, further comprising adding the drug to said culture medium. 8. A method for determining the effectiveness of a medicinal product or a batch of medicinal products, which includes: (i) tissue culture according to the method of claim 6; (ii) adding the drug or batch of drugs to said culture medium; as well as (iii) determining the effect of said drug or said batch of drugs on said tissue, wherein the sensitivity of said tissue to said drug is indicative of the efficacy of said drug. 9. The method according to any one of paragraphs. 6-8, wherein said tissue is a pathological tissue. 10. A method for selecting a drug for the treatment of a disease in a subject in need thereof, the method comprising: (i) culturing the pathological tissue obtained from the subject in accordance with the method of claim 6; (iii) adding a drug to said culture medium; as well as (iii) determining the effect of said drug on said tissue, wherein the sensitivity of said tissue to said drug is indicative of the efficacy of said drug in treating said disease in said subject. 11. A method of treating a malignant tumor in a subject in need thereof, the method comprising: (a) selecting a drug according to the method of claim 10, wherein said pathological tissue is malignant tissue; as well as (b) administering to said subject a therapeutically effective amount of a drug exhibiting efficacy in treating cancer in said subject, thereby subjecting said subject to treatment of cancer. 12. The method according to any one of paragraphs. 8 and 10, 11, wherein said determination is affected for 3-5 days of cultivation. 13. The method according to claim 10, wherein said disease is a malignant tumor. 14. The method according to any one of paragraphs. 11 and 13, wherein said cancer is selected from the group consisting of cancer of the ovary, colon, rectum, lung, pancreas, stomach, gastrointestinal tract, and breast. 15. The method of claim 9, wherein said pathological tissue is malignant tissue. 16. The method according to any one of paragraphs. 6-8 and 10, 11, wherein said tissue is selected from the group consisting of ovary, colon and rectum, lung, pancreas, stomach, esophagus, breast, liver, cartilage and bone. 17. The method according to any one of paragraphs. 7, 8 and 10, 11, wherein said drug is selected from the group consisting of 5-fluorouracil (5FU), oxaliplatin, irinotecan, cisplatin, 4-hydroperoxycyclophosphamide, docetaxel, doxorubicin, navelbine, gemcitabine, gefitinib, tamoxifen, olaparib, trametinib, everolimus and palbociclib. 18. The method according to any one of paragraphs. 7, 8 and 10, 11, wherein the indicated drug concentration is higher than the indicated IC50 dose of the drug in the cell line. 19. The method according to any one of paragraphs. 7, 8 and 10, 11, wherein said cultivation is carried out for at least 4 days and/or up to 7 days. 20. The method according to any one of paragraphs. 6-8 and 10, 11 wherein said slice is placed in the middle of said cell culture insert. 21. The method according to any one of paragraphs. 6-8 and 10, 11 wherein said slice is in direct contact with said tissue culture insert. 22. The method according to any one of paragraphs. 6-8 and 10, 11, wherein said tissue culture insert is a titanium mesh insert or a stainless steel insert. 23. The method according to any one of paragraphs. 6-8 and 10, 11, wherein the oxygen-rich atmosphere contains at least 70% oxygen. 24. The method according to any one of paragraphs. 6-8 and 10, 11, in which said culture medium contains DMEM/F12.

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