Device and method for multidimensional cell culture

Abstract

SOLUTION: The present invention discloses a device and method for multidimensional cell culture, and more particularly three-dimension (3D) and four-dimension (4D) device and method. The device and method of the present invention comprises growing cells as spheroids / tissueoids on non-woven fabric scaffold to create 3D tissue-like structures. The fourth dimension is provided by the ability of the system to generate the 3D tissueoids in a much less time span and their ability to grow for an extended period of time, even for greater than one year. The present invention also provides methods of use for analysis of cell-drug sensitivity of the device. Further, the invention provides a device for growth and drug sensitivity characterization of cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention is in the field of molecular cell biology and provides cell culture devices and methods for performing multidimensional cell analysis, more particularly, three-dimensional (3D) and four-dimensional (4D) based devices and methods. Also provided are methods for making such devices. The cell culture methods and devices are useful for drug discovery and development, clinical trials, treatment decisions, and focused Patient Genome / Cancer Genome Treatment-Outcome data. [Background technology]

[0002] Human physiology, pathology and tissue-based analyses have, until recent years, been performed with two-dimensional (2D) cell culture methods, which have played a crucial role in driving various developments in the research areas of developmental biology, tissue morphogenesis, disease mechanisms, drug discovery, tissue engineering, regenerative medicine and organ printing. Based on this method, significant discoveries have been made and utilized, benefiting the world population. However, research forces have undergone a tremendous change of perspective and paradigm shift, revealing numerous differences and inadequacies associated with 2D cultures, especially the inability of 2D cultures to emulate in vivo conditions or to have physiological relevance. The differences that exist between in vivo and in vitro scenarios are a well-recognized challenge, especially in the field of cancer diagnostics and medicine. 2D cell-based assays suffer from drawbacks such as flawed design, problems with 3D space, difficult accessibility, and generally do not represent a valid 3D in vivo environment. To bridge this gap, rapid progress has been made in the last few years and genomic tools have been tried, but they have not been efficient enough to address the complexities present in patients and animal models. The discrepancies between data generated in 2D cell-based assays / functional assays are often 2-20 fold in dose intensity for drug dose-finding studies depending on fast cut-offs of candidate molecules. Thus, animal models have their flaws and dynamics, and are therefore not representative of real-world judgment of a molecule's potential or efficacy.

[0003] Scientists have been trying for the last few years to create environments for cells in vitro or artificially, where these cells can grow in three dimensions (3D) and interact with their surroundings. In the biomedical field, three-dimensional (3D) cell cultures are currently gaining status as the new normal in the cell culture space. In the current practice, 3D cultures are grown in cell culture bioreactors or small plate-based systems / capsules, where the cells can proliferate and become spheroids or 3D cell colonies [Goodman et al, Microsc. Microanal. 22 (Suppl 3), 2016]. Cell culture of mammalian and human cells in 3D to create tissue-like organs is a revolutionary analysis in cell culture technology and is finding applications in various fields with promising future growth.

[0004] The key to successfully growing homotypic or heterotypic 3D tissue culture models is to mimic the physiological, organizational and functional properties of the respective tissue. Homotypic systems include pure cell lines, while heterotypic systems include real tumor biopsies containing cells of mixed lineages, for example. Further development of 3D cell culture methods and various applications has led to significant inroads in various medical, pharmaceutical and biotechnology-based applications. Numerous studies are ongoing in the fields of cancer, stem cell research, drug discovery and regenerative medicine, to name a few [Report ID: GVR-1-68038-091-0, Published Date: Jun, 2018]. Hospitals, pharmaceutical companies, research institutes and laboratories are adopting 3D cell culture methods and their derivatives for better results, and the penetration rate is expected to rise rapidly in the next decade. The establishment of 3D cell culture methods is based on the use of either scaffold-based platforms, scaffold-free platforms, gels, bioreactors and / or microchips. Various scaffold-based platforms such as macroporous, microporous, nanoporous or solid scaffolds have been described in the literature. However, these systems are not completely efficient in that they are laborious to produce and use, are very time-consuming, are not stable over time, have low throughput, may have biocompatibility issues with tissue samples, may pose sample uptake challenges, etc. (Archana Swami et al., 3D Tumor Models: History, Advances and Future Perspectives; Future Oncology, May 2014).

[0005] The present invention addresses existing problems in the prior art, thereby providing multi-dimensional systems and methods that achieve convincing functional results, thereby closely mimicking the internal micro- or macro-scale functions of engineered tissues. Summary of the Invention

[0006] The present invention provides high throughput devices and methods for performing multidimensional cell culture, more specifically, three-dimensional (3D) and four-dimensional (4D) devices and methods. The devices and methods of the present invention include growing cells as spheroids and / or tissueoids on a nonwoven base matrix system to generate 3D tissue-like structures. The present invention also provides methods and devices for analyzing cellular drug sensitivity. Furthermore, the present invention provides devices for characterizing and analyzing the functions of cellular proliferation and drug sensitivity characteristic of various cell lines and biopsy samples.

[0007] The devices and methods of the present invention address the challenges faced when applying existing 2D / 3D systems and offer a wide range of industrial applications in cancer drug development, clinical trials, regenerative medicine, and personalized medicine assays, among others.

[0008] An embodiment of the present invention provides an apparatus for growing cells, the apparatus comprising a plurality of sterile culture chambers, each containing a sterile non-woven base matrix system for containing and supporting an inoculum selected from the group of spheroids in hanging drop cultures, bulk cell cultures, and primary cultures of biopsies or explants, each sterile culture chamber having a bottom and side for holding culture medium, the base matrix system, and cells for growing cells in three dimensions (3D).

[0009] In a general embodiment of the device, the base matrix-based fabric comprises a non-woven matrix of polymer or copolymer fibers consisting of at least one selected from the group consisting of polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), polyamide (PA), polyethylene (PE), PBT (polybutylene terephthalate), glass fiber, acrylic resin and cotton. Typically, the base matrix-based fabric has a thickness of about 10 to 50 gm / m 2 and has a thickness of at least about 0.05 mm and less than about 5 mm.

[0010] In a general embodiment, the cells are of mammalian origin, primarily human cells, although visualization of cell growth in the device allows for growth of eukaryotic cells such as birds and reptiles, as well as eukaryotic microbial cells such as yeast. In additional embodiments, the device is utilized with cells of origin selected from plants, fungal species, and bacterial species.

[0011] In another aspect of the present invention, the device is useful for generating artificial biological tissues from cells of different origins in a relatively short time for conducting further screening studies, and the artificial biological tissues are visible in less than 72 hours, or less than 48 hours, or even less than 24 hours.

[0012] In another aspect of the invention, the device is useful for creating extracellular and intracellular architecture of artificial biological tissues containing at least one component of an extracellular matrix, whereby the extracellular matrix includes the production and further growth of collagen or vascular tubules, and the intracellular matrix includes at least one intracellular microscopic structure such as tubulin and / or actin.

[0013] Another aspect of the invention herein provides a method for making a device for growing cells three-dimensionally, the method comprising the steps of providing a cell sample of origin selected from a patient biopsy, a biopsy explant, a cell culture in a tissue culture plate, and a spheroid of cells in a hanging drop culture, to obtain a resulting multicellular inoculum or a plurality of multicellular inocula; transferring the inoculum to a corresponding plurality of culture vessels, each of which contains a nonwoven base matrix system and a growth medium; and incubating the vessels to obtain a three-dimensional spheroid of cells in the device. In a particularly preferred embodiment of the method, each cell sample contains less than about 1,000 cells, less than about 500 cells, or even less than about 250 cells, or even less than about 25 cells. Thus, a single sample, such as a biopsy sample or a hanging drop culture, provides multiple aliquots of inoculum to a plurality of culture vessels. The cells in the device have been demonstrated to remain viable and functional for at least about 30 days, or at least about 60 days, or at least about 90 days, or at least about 250 days, or at least about 380 days, and even substantially longer.

[0014] Another aspect of the invention herein provides a method for use in analyzing cellular drug response and sensitivity by a device for three-dimensionally growing spheroids of cells on a nonwoven support base matrix system, the method comprising contacting at least one test chamber of the spheroid with at least one concentration of a drug, and comparing the growth and viability of cells in the spheroid with the growth in an otherwise identical control chamber in which the drug is absent, whereby the spheroid is cultured from a patient, or from a disease cell line, or from a disease model animal. In certain embodiments, the at least one concentration is a plurality of concentrations of a drug in a corresponding plurality of test chambers, and / or the drug is a plurality of drugs in a plurality of test chambers. Overall, the test chamber and the control chamber contain spheroids / artificial living tissues cultured from biopsy tissues of tumor patients, or cultured cell lines. An additional control chamber contains spheroids / artificial living tissues containing non-tumorous normal cells of the patient. In certain embodiments, for patients with tumors, the drug is an anti-cancer chemical agent or an anti-cancer antibody or binding protein. For patients with tumors, some embodiments of the method include at least one chamber containing a combination of two or more drugs. In alternative or additional embodiments, at least one chamber contains a drug selected from antibacterial, anti-inflammatory, antiviral, antihelminthic and antipsychotic. Method embodiments include continuing to grow spheroids / artificial tissues for at least about 30 days, or at least about 60 days, or at least about 90 days, or at least about 250 days, or at least about 380 days, or even for one year or more, and continuing to analyze cell function and response.

[0015] Thus, an aspect of the present invention provides an apparatus for characterizing the proliferation and drug sensitivity of cells of a subject having cancer, the apparatus comprising a plurality of sterile culture chambers, each of which comprises a sterile non-woven polyethylene terephthalate (PET) fabric base matrix system for containing and supporting an inoculum of cells of the subject, selected from the group of spheroids of hanging drop culture, bulk cell culture, and primary culture of biopsy, the plurality of cultures of test originating from cancer tissue of the subject, and the control culture or control biopsy originating from normal tissue of the subject, each of which has a bottom and a side for holding culture medium, base matrix system, and cells, for characterizing the proliferation and viability of cells in three dimensions (3D) under a set of variable medium components. In certain embodiments, the apparatus further comprises cells cultured in the sterile culture chamber. For example, the sterile culture chamber is a well of a multi-well culture dish, such as a 24-well culture dish or a 96-well culture plate.

[0016] An embodiment of the invention provided herein is a set of one or more artificial biological tissue cell cultures produced by the methods herein.

[0017] Another aspect of the invention herein provides a device for cell culture and artificial tissue production, the device includes at least one or more sterile culture chambers, each of which includes a sterile non-woven polyethylene terephthalate (PET) fabric base matrix system for containing and supporting an inoculum of cells, the cells being selected from the group of spheroids of hanging drop culture, bulk cell culture, and primary culture of biopsy, each of which has a bottom and a side for holding culture medium, base matrix system, and cells, each of which has a port for adding fresh culture medium and a drain for draining spent medium. In general, the origin of the cells is avian or mammalian. For example, the origin of the cells is muscle, epithelium, or other tissue. [Brief description of the drawings]

[0018] [Figure 1]1A-1C are a set of photographs of microscopic images of three-dimensional tissues of nonwoven fabrics, either empty (FIG. 1A) or in the presence of artificial biological tissue of MCF-7 (breast cancer cell line) as a base matrix system (FIG. 1B and FIG. 1C), demonstrating that the system provided herein forms tissue-like structures. The base matrix system is a fabric mat made using spunbond technology. The images (FIG. 1B and FIG. 1C) show the growth of artificial biological tissue on the AXTEX-4D base matrix system in a 3D manner. To create the 3D cultures, spheroids are formed using the hanging drop method, and then grown on the base matrix system using the methods and systems provided herein, resulting in the formation of artificial biological tissue. FIG. 1A shows a photograph of the AXTEX-4D base matrix system in its original form, without any cells or artificial biological tissue growing on AXTEX-4D, as observed by scanning electron microscope (SEM). FIG. 1B shows an artificial tissue derived from breast cancer cell line MCF-7 grown on AXTEX-4D-based matrix system, as observed by compound microscope (magnification 100x). FIG. 1C shows an image of an artificial tissue derived from breast cancer cell line MCF-7 grown on AXTEX-4D-based matrix system, as observed by SEM (magnification 350x). [Diagram 2] Figure 2 is a set of photographs of HT-29 cell line grown on base matrix system to form 3D artificial biological tissue observed by phase contrast microscopy. Observe the growth of artificial biological tissue on base matrix system with different density of fabric mat (19g / m2, 20g / m2, 30g / m2, 35g / m2). [Figure 3A] Figures 3A-B are a set of photographs of spheroids and / or artificial biological tissues showing that cells interact with each other and as a result grow three-dimensionally on the AXTEX-4D system. Figure 3A shows images of spheroids derived from MCF-7 and HUVEC cell lines (upper panel) or spheroids cultured on the AXTEX-4D base matrix to obtain artificial biological tissues (lower panel). The photographs are observed by phase contrast microscopy (10x magnification). [Figure 3B] Figures 3A-3B are a set of photographs of spheroids and / or artificial tissues showing the interaction of cells with each other resulting in cells growing in a 3-dimensional manner on the AXTEX-4D system. Figure 3B is a set of images showing the spatiotemporal organization of artificial tissues grown on the AXTEX-4D system. Along with the extracellular matrix, the connectivity and organization between cells was observed, including the 3D tissue-like organization, connectivity and interactions between cells of a biopsy specimen taken from a colon cancer and grown directly on the base matrix system and grown as an artificial tissue on the AXTEX-4D system. The photographs were taken using SEM. (1500x, 7000x magnification). [Figure 4] Figure 4 shows artificial tissues of transformed cell lines HEK-293 and CHO-K1 as observed by scanning electron microscopy. Upper panel: Three-dimensional organization of artificial tissues of human embryonic kidney cell line (HEK-293) grown on AXTEX-4D base matrix system (magnifications 1000x, 1500x). Lower panel: Three-dimensional organization of artificial tissues of Chinese hamster ovary cell line (CHO-K1) grown on AXTEX-4D base matrix system (magnifications 1000x, 1500x). [Diagram 5] Figure 5 shows scanning electron microscopy images of biopsy explants taken from lung cancer and so grown on a base matrix system. The cells of the biopsy were cultured on the AXTEX-4D base matrix and, upon growth, produced an artificial living tissue as a result, without any prior treatment of the cells. (Magnifications 1000x and 1500x) [Figure 6] Figure 6 shows confocal microscope images of the artificial tissue on the AXTEX-4D system, where PC3 cell line was taken as an example and stained with Calcein AM to show the cell growth / proliferation and viability of the artificial tissue observed on different days, namely, day 3, day 25, day 108 and day 250. Calcein AM, a cell permeable dye, was used to determine the cell viability. [Figure 7]7A-7F show the formation of the extracellular matrix of a 2D monolayer and of a 3D artificial tissue on the AXTEX-4D system, using the MCF-7 cell line as an example. On day 7, staining was performed with anti-collagen type I antibody (green) at a dilution of 1:50 and DAPI (blue - nuclear stain) and observed by fluorescence microscopy. It was observed that an ECM was formed in the artificial tissue formed on the AXTEX-4D system, but it was more continuous compared to that of cells cultured in a 2D monolayer, as can be seen in the images (7A-7F). (10x magnification). For the 2D cultures, the figures represent 7A - nuclei stained with Hoechst, 7B - collagen stained with anti-collagen antibody, and 7C - composite image. For the artificial tissue analysis, the figures represent 7D - nuclei stained, 7E - collagen stained, and 7F - composite image. Figures 7G-H are images of whole blocks of engineered tissue generated from MCF-7 cell line and grown on AXTEX-4D system. On day 7, they were stained with anti-collagen type I antibody (green) at 1:50 dilution and DAPI (blue - nuclear stain) and observed by fluorescence microscopy (10x magnification). [Figure 8] Figure 8 compares the formation of intracellular matrix of a 2D monolayer and a 3D artificial tissue on the AXTEX-4D system, using the MCF-7 cell line as an example. The artificial tissue containing cytoskeletal components is shown, stained with anti-phalloidin antibody (red) and DAPI (blue - nuclear stain) at a dilution of 1:1000, grown on the base matrix system. The cytoskeletal organization of the artificial tissue was observed to be more continuous compared to that of cells cultured in a 2D monolayer, as can be seen in the above image (Figure 8). (10x magnification) [Figure 9]9A-9E show engineered tissues generated from MCF-7 cells at various cell numbers ranging between 250 cells and 25 cells using phase contrast microscopy. The engineered tissues were grown on the AXTEX-4D system. 9A, 9B, 9C, 9D, and 9E are images of phase contrast microscopy studies (10x magnification), and 9F is an image of up to 25 cells grown on the AXTEX-4D system using scanning electron microscopy (1500x magnification). Inocula ranging in cell numbers up to 5000 cells were evaluated, and the data shown here depict good growth of approximately 250 cells to 25 cells on the AXTEX-4D system. [Figure 10] 10A-10B are a set of photographs showing the morphological characteristics of MCF-7 cells grown either as 2D monolayer cultures or as artificial tissues grown on the AXTEX-4D system. FIG. 10A shows the morphology of MCF-7 cells treated with or without doxorubicin after 3 days of growth in 2D cultures. Dose-dependent growth inhibition was observed in the treated groups compared to that of cells cultured in the presence of DMSO (vehicle control). Vacuoles were observed even with 1 μM doxorubicin in 2D cultures, indicating the sensitivity of the cells to doxorubicin treatment, which eventually led to cell death. The inset of the image in the top panel is shown as a magnification in the image in the bottom panel. FIG. 10B is a set of photographs showing the sensitivity of MCF-7 artificial tissues to doxorubicin after 3 days of incubation. The growth of the artificial tissue generated from the MCF-7 cell line was not inhibited by doxorubicin at a concentration of 2.5 μM, and was comparable to the growth observed for the artificial tissue grown without drug in the presence of the vehicle control (DMSO). At a concentration of 5 μM, the growth of the artificial tissue was observed to be partially inhibited. At higher concentrations (5 μM), the artificial tissue was observed to shrink, but the artificial tissue remained attached without disintegrating from the AXTEX-4D base matrix system. [Figure 11A]11A-11B are a set of bar graphs illustrating the sensitivity of MCF-7 cells grown either as monolayers in 96-well plates or as artificial tissues on the AXTEX-4D system to suggested concentrations of doxorubicin in the presence or absence of bevacizumab antibody. FIG. 11A shows the analysis of drug sensitivity using three different concentrations of doxorubicin in both 2D monolayer cultures and 3D artificial tissue systems for 48 hours. The viability of cells in monolayer cultures (2D) and artificial tissues (3D), respectively, was assessed by analyzing viability using PrestoBlue. Data are expressed in relative fluorescence units (RFU) and normalized to vehicle control as 100% viability. Resistance to drug activity was observed (up to 80% viability) in artificial tissues grown on the AXTEX-4D system, even at a doxorubicin concentration of 1 μM. Cells cultured in 2D monolayers showed 35% viability at the same concentration (1 μM doxorubicin). [Figure 11B] 11A-11B are a set of bar graphs illustrating the sensitivity of MCF-7 cells grown either as monolayers in 96-well plates or as artificial tissues on the AXTEX-4D system to suggested concentrations of doxorubicin in the presence or absence of bevacizumab antibody. FIG. 11B shows the combined effect of doxorubicin and bevacizumab on cell proliferation of VEGF-165-induced MCF-7 cells grown as artificial tissues. MCF-7 artificial tissues were cultured in wells of 96-well plates. Cells were serum starved for approximately 5 hours and then treated with either 100 ng / ml VEGF-165 alone or a combination of 1 μM doxorubicin and 25 μg / ml bevacizumab for 6 days. Cell viability was analyzed using PrestoBlue. Engineered tissue grown on the AXTEX-4D system demonstrated that the combined effect of both drugs was more effective (about 57%) in inhibiting cell proliferation compared to single drug treatment (as shown in FIG. 11A). [Figure 12]Figure 12 shows phase contrast images of HT-29 bioprosthetic tissue before (Figure 12A) and after (Figure 12B) treatment with a combination of cytokines TNF-α (20 ng / ml) and IFN-γ (0.5 ng / ml) for 16-18 hours. After treatment with cytokines, the intact bioprosthetic tissue was stripped to demonstrate the effect of cytotoxicity away from the AXTEX-4D-based matrix system. [Figure 13] FIG. 13 is a set of photographs showing the duration of time (days as shown in each panel) during which the artificial living tissues remained in culture. Phase contrast microscopy was used to observe the longevity of artificial living tissues derived from HepG2 and PC3 cells. When different fields were photographed on different days, an increase in cell number was observed with increasing density. As of the filing date of this application, the artificial living tissues of PC3 are viable in culture (day 364). In FIG. 13A-FIG. 13B, the viability of the artificial living tissues of HepG2 was observed until day 82. In FIG. 13C-FIG. 13D, the viability of the artificial living tissues of PC3 was observed until day 364. [Figure 14]Figure 14 shows a set of photographs of monocultures, cocultures and tricultures of three cell lines, which were grown as 2D monolayer cultures or as artificial tissues on the base matrix system AXTEX-4D by adding cell suspensions of a transformed fibroblast cell line (NIH-3T3), endothelial cells (HUVEC) and a breast cancer cell line (MCF-7). The cocultures of each combination were analyzed by taking either the breast cancer cell line (MCF-7) and endothelial cells (HUVEC) or the endothelial cells (HUVEC) and fibroblast cells (NIH-3T3) in a 1:1 ratio, respectively. For the tricultures, the NIH-3T3, HUVEC and MCF-7 cell lines were added in a 2:1:1 ratio. Spheroids were formed in all combinations and cultured on the 2D monolayer as well as on the base matrix system AXTEX-4D. It was observed that spheroids attached on the AXTEX-4D base matrix system within 24 hours, and all combinations were further grown as artificial biological tissues. Figure 14 (upper panel) shows monolayer cultures of spheroids made of HUVEC, HUVEC:MCF-7, HUVEC:3T3 and HUVEC:MCF-7:3T3 grown in 2D format. Figure 14 (lower panel) shows artificial biological tissues of HUVEC, HUVEC:MCF-7, HUVEC:3T3 and HUVEC:MCF-7:3T3 grown on the AXTEX-4D base matrix system. [Figure 15] Figure 15 is a set of photographs showing the HEK-293, NIH-3T3 and PC3 artificial tissues grown on AXTEX-4D system. Figure 15 shows the minimum time required for artificial tissues to attach and begin to grow on AXTEX-4D system. The HEK-293 and NIH-3T3 cell line artificial tissues took less than 24 hours to attach and grow on AXTEX-4D system, while the PC3 artificial tissue took about 48 hours to attach and grow on AXTEX-4D system. [Figure 16]FIG. 16A-FIG. 16B are a set of photographs showing the application of the AXTEX-4D system as a cell factory. When the adherent CHO-DG44 stable cell line expressing tocilizumab is grown as an artificial tissue on the AXTEX-4D base matrix system, secretion of the monoclonal antibody tocilizumab is observed in the culture supernatant as analyzed by SDS-PAGE. FIG. 16A shows growing CHO-DG44 cells expressing tocilizumab as an artificial tissue on AXTEX-4D. FIG. 16B shows the expression analysis of tocilizumab by non-reducing SDS-PAGE. Briefly, culture supernatants were taken from cells grown as 2D cultures on Petri plates and as artificial tissue grown on the AXTEX-4D system at different days of culture. Growing artificial biotissues on the AXTEX-4D base matrix system allowed for increased longevity (6 days in monolayer and 26 days as artificial biotissue, when samples were taken for analysis), improved productivity, and increased cell numbers in a more compact space. Analysis by SDS-PAGE showed the expression of a monoclonal antibody (tocilizumab) in the adherent CHODG44 cell line in 2D and 3D formats, where each lane of a 10% SDS-PAGE was loaded with a different sample: lane 1: pre-stained protein marker, lane 2: positive control (1 μg), lane 3: sample in the supernatant of a 2D culture on day 6, lane 4: sample in the supernatant of artificial biotissue on day 6, lane 5: sample in the supernatant of artificial biotissue on day 12, lane 6: sample in the supernatant of artificial biotissue on day 18, lane 7: sample in the supernatant of artificial biotissue on day 26. Equal cell numbers were seeded on the 2D and 3D formats. After 6 days, the 2D cultures were terminated due to culture confluency, while the engineered tissues were maintained until day 26. SDS-PAGE revealed that expression of tocilizumab antibody was observed at day 6 in the 2D monolayer cultures, but not in the engineered tissues grown on AXTEX-4D. However, expression of tocilizumab in the 3D cultures was observed to increase as a function of incubation time from days 12 to 26. [Figure 17] FIG. 17A-FIG. 17B are a set of photographs showing endothelial cells HUVECs grown as artificial tissue on AXTEX-4D base matrix system treated with (FIG. 17B) and without (FIG. 17A) VEGF-165 for 72 hours and observed by phase contrast microscopy. FIG. 17A shows that spheroids attach to the base matrix system with minimal cell proliferation. FIG. 17B shows that cells proliferate along the formation of tube-like structures, demonstrating that angiogenesis was observed in the AXTEX-4D system. FIG. 17C (enlarged view of artificial tissue treated with VEGF) is a magnified view depicting a closer look at the tube-like structures. [Figure 18] FIG. 18 is a diagram of an embodiment of the invention that is a device with 3D and 4D elements. [Figure 19] FIG. 19 is a diagram illustrating various applications and relative advantages of the devices provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following description, with reference to the accompanying drawings, is provided to aid in a comprehensive understanding of exemplary embodiments of the present invention. Although the following description includes various specific details to aid in the understanding thereof, these should be considered as merely exemplary.

[0020] Although the present invention is susceptible to various modifications and alternative forms, the following detailed description of specific embodiments thereof is provided. It should be understood that the present invention is not intended to be limited to the specific forms disclosed, but rather, the present invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention. Also, descriptions of well-known functions and constructions are omitted for clarity and consistency.

[0021] The terms and words used in the following description and claims are not limited to their bibliographical meanings, but are merely used by the inventors to enable a clear and consistent understanding of the present invention. Therefore, it should be clear to those skilled in the art that the following description and embodiments of the present invention are provided for illustrative purposes only, and are not provided for the purpose of limiting the present invention as defined by the appended claims and equivalents.

[0022] Features described and / or illustrated with respect to one embodiment may be used in the same or similar manner in one or more other embodiments and / or may be used in combination with or instead of features of the other embodiments.

[0023] As used herein, the term "comprises / comprising" is to be interpreted as specifying the presence of stated features, integers, steps or components, but not excluding the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0024] Thus, the present invention relates to a multidimensional cell culture system, in particular a 3D / 4D system for studying cells or molecules. It also provides a device that contains a novel 3D / 4D tissue culture model. Furthermore, the present invention provides a method for producing said multidimensional cell culture system.

[0025] The aim of this study is to provide a cell culture system for analyzing multidimensional tissue models, more specifically 3D / 4D tissue culture and artificial tissue generation systems for analyzing cells and molecules, and their applications.

[0026] It is yet another object of the present invention to provide methods for making the above-described systems and culture devices.

[0027] Yet another object of the present invention is to provide a high throughput device for growing cells, the device comprising a plurality of sterile culture chambers, each containing a sterile non-woven base matrix for receiving and supporting an inoculum selected from the group of spheroids of hanging drop culture or spheroids of direct suspension of cells derived from tissue, bulk cell cultures derived from cell lines, and primary cultures of biopsies or explants, each sterile culture chamber having a bottom and side for holding culture medium, base matrix, and cells for growing cells in three dimensions (3D).The device provided herein contains a non-woven mat of polymeric fibers consisting of at least one selected from the group of PET, PP, PBT, glass fiber, and cotton.

[0028] In another embodiment, a device is provided, wherein the base matrix fabric is about 10 gm / m 2 ~50gm / m 2 Density in the range of, e.g., 19-25 gm / m 2 and has a thickness of at least about 0.05 mm and less than about 5 mm, for example 0.12 mm. The fiber has a thickness of 0.5 to 10 dtex, for example 2.5 to 3.0 dtex, and a porosity in the range of 20 to 80 microns.

[0029] In another embodiment, the device of the invention is used to grow cells selected from mammalian species, in embodiments the mammalian cells are human cells, such as established cell lines or fresh biopsy samples from a patient, or other mammalian cells, such as Chinese Hamster Ovary-derived cells (CHO and CHO-derived cells).

[0030] Artificial tissues that have been successfully grown using this technology include MCF7, an adenocarcinoma breast cancer cell line, HepG2, an epithelial liver cancer cell line, PC3, an adenocarcinoma prostate cancer cell line, and A375, an epithelial cell line, a cutaneous melanoma cancer cell line.

[0031] Non-malignant cell lines also grew well; these are CHO cells (Chinese Hamster Ovary), HEK-293 (human embryonic kidney cells), and NIH-3T3 (fibroblasts), as shown in the examples herein.

[0032] Additionally, primary tissues that have been successfully used as a source of cells include breast cancer tissue, colon cancer tissue, stomach cancer tissue, lung cancer tissue, and thyroid cancer tissue.

[0033] In the overall embodiment of the device, the artificial biological tissue grown by the method herein produces extracellular structure, namely collagen.By analyzing the expression of F-actin, it was observed that the artificial biological tissue grown on this base matrix AXTEX-4D system produces 3D-like reconstruction of skeletal elements.The device herein provides an artificial biological tissue that continues cell growth for a long period of time, which indicates a favorable growth condition.

[0034] Embodiments of the present invention provide methods for expanding cell samples utilizing samples containing less than about 1,000 cells, less than about 500 cells, less than about 250 cells, and even less than about 25 cells as an inoculum. The cells are derived from stable cell lines or from biological tissue, such as a tumor biopsy, cultured ex vivo using the devices and methods herein.

[0035] In another aspect, the present invention provides a method for use in analyzing cellular drug sensitivity using an apparatus for three-dimensionally growing artificial biological tissues cultured from patient cells on a nonwoven support base matrix, the method comprising contacting at least one test chamber of the artificial biological tissue with at least one concentration of a drug and comparing the proliferation and viability of cells in the artificial biological tissue with that of an otherwise identical control chamber in which the drug is absent.

[0036] In an embodiment, the at least one concentration is a plurality of concentrations of a drug in a corresponding plurality of chambers and / or the drug is a plurality of drugs in a plurality of chambers, The drug is selected from an anti-cancer chemical agent or an anti-cancer antibody or binding protein.

[0037] In another preferred embodiment, the test and control chambers contain artificial living tissue cultured from biopsy tissue of tumor patients.

[0038] In yet another embodiment, an additional control chamber is provided containing an artificial living tissue containing non-neoplastic, physiologically normal cells of the patient.

[0039] In another embodiment, at least one of the chambers contains a drug or a combination of two or more drugs.

[0040] In a further embodiment, at least one laboratory contains a drug selected from antibacterial, anti-inflammatory, antiviral, anti-helminthic and antipsychotic.

[0041] In another aspect of the present invention, a device is provided for characterizing the proliferation and drug sensitivity of cells of a tumor-bearing inoculum. The device includes a plurality of sterile culture chambers, each containing a sterile non-woven polyethylene terephthalate (PET) fabric base matrix for containing and supporting an inoculum of cells. The inoculum is selected from the group of hanging drop culture spheroids, bulk cell cultures, and biopsy primary cultures or explants, whereby the test cultures originate from the cancerous tissue of the inoculum and the control cultures or control biopsies originate from the normal tissue of the inoculum. Each sterile culture chamber has a bottom and a side for holding culture medium, a base matrix, and cells to characterize the proliferation and viability of cells in three dimensions (3D) under a set of variable medium components. In a further embodiment, the cultured cells are present in the chambers of a multi-well culture dish, for example a 24-well culture dish or a 96-well culture plate.

[0042] The present invention also provides a cell culture and ex vivo tissue production apparatus, the apparatus comprising at least one or more sterile culture chambers, each containing cells and a sterile non-woven polyethylene terephthalate (PET) fabric base matrix for containing and supporting an inoculum of cells, the cells being selected from the group of spheroids in hanging drop cultures, bulk cell cultures, and primary cultures of biopsies, each sterile culture chamber having a bottom and sides for holding culture medium, base matrix, and cells, each sterile culture chamber having a port for adding fresh culture medium and a drain for draining spent medium.

[0043] The present invention provides a device having artificial tissue whose cells are avian or mammalian in origin.

[0044] In embodiments, the origin of the cells may be selected from muscle, epithelium or other tissue.

[0045] The invention also provides for the use of the product resulting from the device as a therapeutic artificial skin or muscle.

[0046] In another aspect, the cells of the artificial biological tissue produced by the method and device provided herein of the present invention have a longer lifespan, i.e., a longer period of cell viability, compared to those reported in the prior art. In this example using different cell lines, as shown in Figure 6, a viability of up to 250 days was observed, and as shown in Figure 13, the cells were shown to continue to grow for up to 364 days. The artificial biological tissue was observed to remain viable with sufficient morphology and function for a period of more than 12 months, etc. Various examples were carried out using various cell lines and primary cells, and the lifespan of the artificial biological tissue was reproducibly confirmed to be substantially longer than those previously reported.

[0047] In another aspect, the method and device provide a 3D culture assay that can be initiated in less time than previously reported (less than 72 hours). Zanoni M et al. reported that 2×10 cells per well can be cultured in 3D. 3 , 4×10 3 and 6×10 3 Cells have been used to form spheroids using the hanging drop method, but it was reported that these spheroids require a period of 7 days.

[0048] [Table 1]

[0049] In the device and method of the present invention, spheroids were generated in about 24 hours or less. Moreover, such spheroids could be attached onto a base matrix in less than about 24 hours. In another preferred embodiment of the present invention, the artificial biological tissue is shown to grow on a base matrix in less than 24 hours.

[0050] Yet another advantage is that the device and method can be used with different cell lines. Even cell types that are not normally very compact in nature, such as PC3 cell line (prostate cancer) and HT29 cell line (colon cancer), have been observed to exhibit good binding to the base matrix. The 3D / 4D device and method of the present invention have been observed to grow artificial living tissues that have been observed to have similar functions and structures to the original tissue across all cell lines tested herein.

[0051] In yet another aspect, the devices and methods of the present invention provide results that are comparable to genomic and proteomic profiling of primary tumor cells and replace studies performed on monolayer cultures, which have produced inconsistent and sometimes irreproducible results.

[0052] In the present invention, the nature and use of the multi-dimensional physical and analytical readout of the device / base matrix AXTEX-4D system are as follows: The artificial tissues were grown as single pure cultures or as combinations of multiple cell types; in attached or suspended states and combinations thereof, to support, maintain, or mimic the in vivo microenvironment of a particular organ, tumor, or immune system interaction with cancer or infection, similar to that observed in in vivo networks; multiple combination therapies using one or more combinations of both chemical and biological drugs were designed, tested, and evaluated; genetic changes due to the effects of administering drugs / combinations at various doses / forms were determined as a function of time by accelerated studies; the artificial tissues were tested for physical stimuli that promote attachment, growth, and accelerated growth to rapidly produce results for drug effects, efficacy studies, gene mutations, etc. For example, the 3D / 4D models provided herein are also used to study various drug concentrations that can be used as effective doses for treatment regimens.

[0053] By reducing the time required to obtain results, the model can deliver more valid analyses, benefiting patients in clinical outcomes.

[0054] In another embodiment, the system of the present invention is used to establish drug combination therapy using both chemical and biological drugs. It is envisioned that the system will help select patients for clinical trials of oncology-related studies to determine multiple treatment plans, studying multiple concentrations simultaneously, i.e., simultaneously, to generate data for patients with disease, and correlating the data received after treatment effects with tissue grown in vitro. Thus, by generating a result database focused on the patient's genomic / cancer genomic treatment, and using the ex vivo AXTEX-4D-based matrix system provided herein for patient screening, a valid future treatment plan can be created that identifies the appropriate and effective therapeutic agent for each patient.

[0055] In yet another embodiment, the present invention provides methods and apparatus for characterizing tissue-like structures capable of supporting various tumor cell lines in 3D and in the fourth dimension (4D) as a function of time over extended periods of time, and their applications in drug discovery and other clinical analyses, diagnostics, and the like.

[0056] A device incorporating the 3D tissue system and its configuration is presented in Figure 18, which depicts a 3D tumor-based matrix system at the center of a cubic design. In one embodiment, the device of the invention is provided as a 4D base matrix system used to study tissue under in situ conditions, allowing efficient observation and evaluation of input information and device results as a function of time.

[0057] The time dimension involved in carrying out cell culture in vitro provides a way to diagnose disease more quickly compared to existing methods. Furthermore, the lifespan of the artificial biological tissue produced by the method described herewith is also increased, thereby increasing the test window for different analyses. The AXTEX-4D-based matrix system envisioned in the present invention, used for a longer period of time, is depicted photographically in FIG. 6.

[0058] In another important aspect of the present invention, the device is useful for generating artificial biological tissues from cells of different origins in a relatively short time for conducting further screening studies, where the artificial biological tissues are visible in less than 72 hours, or less than 48 hours, or even less than 24 hours.

[0059] An important observation and therefore an important aspect of the study is that there is evidence that tubule-like structures grow, and thus, in some cases, the present invention provides an angiogenesis model for anti-angiogenic drug / assay studies and other uses.

[0060] In certain embodiments, the systems of the invention serve as a source of material in determining proteomic and genomic profiles.

[0061] In yet another embodiment, the device is used as a biotransformation reactor, for example to produce high value proteins such as antibodies over a time course of several months.

[0062] In yet another embodiment, a method is provided for determining an effective treatment or therapeutic regimen for treating a disease, such as cancer.

[0063] In yet another embodiment, the system of the present invention may be used as a cell factory / bioreactor to grow large scale cultures to produce therapeutic / antigen / vaccine candidates, etc.

[0064] Overall, the systems of the present invention closely represent tissue-like structure and tissue-like function, grow more rapidly, are robust, viable and sustainable for longer periods of time. EXAMPLES

[0065] The present invention will be described in more detail below with reference to examples and comparative examples, but it should be noted that the present invention is not intended to be limited to these examples.

[0066] Example 1: Compounds and Reagents Compounds and reagents used to analyze drug sensitivity using the device and method provided as AXTEX-4D as base matrix system. Doxorubicin, cisplatin, colchicine, paclitaxel and DMSO were purchased from Sigma. These drugs were tested for sensitivity / resistance of cells of different cancer cell lines grown as 2D (monolayer) and 3D (on AXTEX-4D base matrix system as artificial living tissue) formats and data were compared. Exemplary data are shown in Figure 10 and Figure 11.

[0067] Example 2: Cell line and tumor analysis Various human cancer cell lines (MCF-7, HepG2, PC3, HT29, etc.) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) A375, and CHO-K1 cell line was received from NCCS (Pune, India). HUVEC were obtained from Lonza. MCF-7 and HepG2 were cultured in EMEM (Sigma-Aldrich, St. Louis, MO, USA). PC3 and CHOK-1 were cultured in F12K (Sigma-Aldrich, St. Louis, MO, USA), and HT-29, A-375, NIH-3T3, HEK-293 cells were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA). HUVEC cells were cultured in EBM-2 basal medium and EGM-2 Single Quots supplement. Figures 1, 2, 3A and 4 are representative photographs showing cell lines growing on 3D base matrix systems to form artificial biological tissues. All adherent cell lines were cultured in 10% FBS (Gibco) supplemented with 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured under static conditions at 37°C with 8% CO2 humidification.

[0068] The morphological appearance of the engineered biotissues for each cell line was analyzed as phase contrast and SEM images are shown in (Figure 1, Figure 2, Figure 3A, Figure 4).

[0069] Generation of artificial tissue using primary tumor biopsies: Tumor biopsies of colon, gastric, lung and thyroid carcinomas were taken from the pathology specimens and shipped for culture and analysis ex vivo in the cell culture laboratory. The tissues were cultured in 1x PBS (Ca ++ and Mg ++The cells were then rinsed with 0.1% NaCl (no HCl), sliced ​​into smaller pieces using a scalpel, and further treated with a plunger to separate and isolate the cells. The cells were then cultured in DMEM medium containing 2 mM glutamine, 2x antibiotic solution (penicillin and streptomycin, HiMedia) and 20% FBS. Tumor tissue specimens were harvested and grown on the AXTEX-4D system as suspension cultures or explants. Growth of tumor tissue on the AXTEX-4D base matrix system is shown in Figure 3B and Figure 5 as an example.

[0070] Formation of spheroids and artificial biological tissues. Unless otherwise indicated, spheroids were formed using the hanging drop method. This further led to the formation of artificial biological tissues. Figure 3A (upper panel) shows the growth of spheroids generated by the hanging drop method, and Figures 1, 2, 3A (lower panel), and 4 are a set of representative photographs of artificial biological tissues growing on AXTEX-4D base matrix. The formation process of spheroids of various cell lines is described below.

[0071] Briefly, the day before the hanging drops were made, cells were seeded at approximately 80% confluency. After trypsinization, the cells were resuspended in an appropriate amount of the respective medium, and the hanging drop formation process was started only if the cell viability was greater than 90%. Each cell suspension was diluted with 10 3 ~10 4 Spheroids were generated containing cell numbers ranging from 100 to 1500 cells. Droplets were pipetted onto the inner surface of the lid of a sterile culture dish, and the bottom of the dish was filled with PBS. After 24-48 h, the inner lid was inverted to resuspend the droplets in fresh medium. Spheroids were analyzed by phase contrast microscopy. Representative photographs show the development of spheroids using the hanging drop method in 3A (upper panel), and artificial living tissues grown on 3D base matrix systems (Figure 1B, Figure 1C, Figure 2, Figure 3A (lower panel) and Figure 4).

[0072] Scanning electron microscopy. The 3D morphology of cells attached to the base matrix AXTEX-4D system was evaluated by SEM analysis (EVO-18 Research, Zeiss) (Figure 1C and Figure 4). Samples (fixative: 2.5% glutaraldehyde and 2% paraformaldehyde in PBS, pH 7.4) were fixed on stubs and dried in vacuum at 0.1 mbar pressure for 10 min, followed by addition of argon gas. Samples were coated with gold particles using a sputter coater. The coated samples were then analyzed by scanning electron microscopy.

[0073] Example 3: Creation of 3D cell culture system: In the examples herein, a commercially available spunbond PET material (A in FIG. 1) was used, which is flat-bonded and consists of extruded round continuous filaments. The fabric used is a nonwoven mat of endless polymeric fibers. The density of the fabric is about 19-35 gm / m 2 The porosity is approximately 65 microns. To create artificial tissue, spheroids are prepared using the hanging drop method and grown in culture on a base matrix (Figure 1B, Figure 1C, and Figure 3A (lower panel)).

[0074] The attachment and growth of the artificial tissue was continuously monitored as a function of time using phase contrast microscopy. It was observed that the entire process was completed in less than 24 hours, or less than 48 hours, or less than 72 hours, including about 24 hours to prepare the spheroids, about 24 hours to attach the spheroids on the base matrix system, and several hours to grow and generate the spheroids as artificial tissue. After this, the AXTEX-4D system (cells grown in 3D culture on the base matrix system) was ready to perform screening studies and other analyses as demonstrated in other examples. In this artificial tissue base matrix system, spheroids of various primary cells and tissues, pathological and non-pathological cancer cells or patient tumor biopsies, transfected or non-transfected cell lines were observed to grow with morphology similar to in vivo tissues.

[0075] The bioartificial tissues were generated from tumor biopsies by taking either suspension cultures and / or explants. The inocula of explants that formed the bioartificial tissues were observed to grow in 8% CO2 incubators for less than 24, 48, or 72 hours. After this, the platform was ready to perform screening studies and other analyses.

[0076] Example 4: Type of construction material for the base matrix system, as well as thickness parameters: (19, 20, 30, 35) gm / m 2 Different types of spun-woven materials such as PET fabrics with various densities (19, 20, 30 and 35 gm / m) were used as the 3D base matrix system. A representative example, Figure 2, shows that HT-29 artificial living tissue was 2 The results show that the tissue grows efficiently on the same fabric with a thickness ranging from 0.1 to 0.2 mm. Other materials tested as base matrix systems for growing artificial tissue are Bestbond PP / PS / PA-40g / m by FNT, 22 , FNT Cisellina PET 250g / m 2 , FNT Newjet Viscose 80g / m 2 , FNT Polibond PP 45g / m 2 , Hydroweb BicoPET / PP 150g / m 2 , JM 011 / 120PET / 120g / m 2 , Mogul Buffalo bico PET / coPET, round, 80g / m 2 , Mogul Buffalo bico PET / coPET, trilobal 80g / m 2 , Mogul Mopet PET Flat Bond 19g / m 2 , Mogul Mopet PET Flat Bond 75g / m 2 , Resintex Master PE, Acrylic resin 220g / m 2 , AS10, AS03 and ASO3A.

[0077] Example 5: Cell-to-extracellular matrix interactions: Cell-to-extracellular matrix interactions play a key role in tumor growth and invasion and serve as a key component of the tumor microenvironment. Collagen, present as an ECM component, is involved in cancer and fibrosis. In the presence of other components such as hyaluronic acid, fibronectin, laminin and matrix metalloproteinases, collagen influences the activity of cancer cells. It was observed that engineered tissues generated from MCF-7 cell line and grown on AXTEX-4D system produced collagen (Figure 7). The ECM was formed more continuously in the case of engineered tissues grown on AXTEX-4D compared to 2D monolayer cultures.

[0078] Example 6: Analysis of drug sensitivity of 2D and 3D cell cultures MCF-7 cell lines were grown as 2D monolayer cultures, as well as engineered tissues grown on 96-well plates, where spheroids were cultured on top of a membrane and incubated for 1-3 days. Approximately 5 × 10 spheroids were cultured in each well of either 96-well plates pre-coated with 1.5% tissue culture agarose or not pre-coated with tissue culture agarose. 3 Cell numbers of cells were added. After attachment of spheroids, the medium was replaced with fresh medium in the presence or absence of drugs. For 2D cultures, 5 × 10 cells were added to each well of a 96-well plate. 3 Cells were seeded and allowed to attach for 48-72 hours before drug treatment was initiated.

[0079] As shown in Figure 10, MCF-7 cells grown in 2D cultures or on the AXTEX-4D base matrix system were treated with doxorubicin (1-5 μM), and cells grown in a 3D format showed greater resistance to growth arrest or killing compared to cells cultured as a 2D monolayer (Figure 10). Figure 10A shows the effect of different concentrations (1-5 μM) of doxorubicin on cell viability when grown in a 2D monolayer, and Figure 10B shows the effect of different concentrations (1-5 μM) of doxorubicin on cell viability when grown as a 3D artificial tissue on the AXTEX-4D base matrix system. As shown in Figure 11A, cells and artificial tissue were treated with different doses (1-5 μM) of doxorubicin, and some resistance was observed in artificial tissues with 1 μM doxorubicin compared to the vehicle control.

[0080] FIG. 11B shows the combined effect of both doxorubicin and bevacizumab on the growth of MCF-7 artificial tissue. MCF-7 artificial tissue grown on AXTEX-4D base matrix system was first serum-starved for 5 hours and then treated with 100ng / ml VEGF-165 (R&D Systems, Cat No. 293-VE-010, splice variant or isoform of vascular endothelial growth factor-165) alone or in the presence of 1μM doxorubicin and 25μg / ml bevacizumab (sourced from Roche, 100mg / 4ml) for 6 days at 37℃ and 8% CO2. Viability was assessed by using PrestoBlue. Drug sensitivity was analyzed by a fluorescence-based test (excitation 485nm / emission 595nm) using PrestoBlue.

[0081] Artificial tissue grown on the AXTEX-4D-based matrix system demonstrated that 1 μM doxorubicin in the presence of 25 μg / ml bevacizumab prevented cell proliferation with greater efficacy (approximately 57%).

[0082] Example 7. Evaluation of artificial tissue grown on AXTEX-4D base matrix system by fluorescence microscopy: As a process for performing immunofluorescence analysis, the samples were fixed with 4% PFA for 15 min and washed with PBS three times for 5 min each. The samples were permeabilized with 0.1% Triton-x. On the 7th day, staining was performed with anti-collagen type I antibody (green) and DAPI (blue - nuclear stain) at a dilution of 1:50 and observed based on fluorescence microscopy. In the artificial tissue formed on the AXTEX-4D system, it was observed that the ECM formed was more continuous compared to that of cells cultured in a 2D monolayer, as can be seen in the images (Figure 7A-Figure 7F). (10x magnification). The samples were analyzed using an ApoTome microscope.

[0083] Example 8. Confocal analysis The 3D growth of artificial tissue was visualized using confocal microscopy.To carry out confocal analysis, samples were fixed, stained and analyzed using Leica TCS SP8.In this example, spheroids of MCF-7 cells were prepared (as described herein), added onto the membrane and incubated to obtain the growth of artificial tissue.

[0084] Cells were stained using phalloidin for F-actin and Hoechst dye for nuclear staining. Cells were fixed with fixative and blocked in PBS with 1% BSA for 30 min at room temperature. Later, specimens were washed in PBS and stained for actin followed by counterstaining with Hoechst to visualize nuclei. Phalloidin staining was performed using a 1:1000 dilution at 25°C for 40 min. Nuclear staining was performed using a 1:1000 dilution of Hoechst in PBS for 15 min at 25°C (Figure 8). Pictures were taken at 10x magnification in 3D and 40x magnification in 2D using a Leica confocal microscope (Leica SP8).

[0085] To analyze collagen expression in the bioartificial tissues, cells were fixed and blocked as described previously. Bioartificial tissues of MCF-7 cell line were stained with anti-collagen I antibody at 1:50 dilution for 16 hours. Nuclear staining was performed using DAPI. Pictures were taken at the same magnification as described previously.

[0086] To analyze the proliferation longevity of the PC3 cell line derived artificial tissue, the artificial tissue was stained with calcein AM for 30 min according to the manufacturer's protocol. As shown in Figure 6, the artificial tissue generated from the PC3 cell line grown on the base matrix AXTEX-4D was viable and capable of cell proliferation for up to 250 days.

[0087] To analyze the proliferation and viability of the PC3 bioartificial tissues, calcein AM (Thermo Fisher) staining was performed at different time points (days 3, 25, 108 and 250). The PC3 bioartificial tissues were stained with 1 μM calcein AM for 30 min and kept at 37 °C and 8% CO2. The bioartificial tissues were then analyzed by confocal microscopy and the results showed an increase in cell number and viability as shown by calcein AM staining (Figure 6).

[0088] The confocal analysis data showed a fibrous organization of the engineered biotissue of the MCF-7 cell line, with the continuity of the cells clearly visible, in contrast to the pictures seen from cells grown in Petri dishes in a 2D format where the cells have defined edges and margins and are discontinuous (Figure 8).

[0089] Example 9: Low cell numbers in initial samples as inoculum for growing artificial tissue on AXTEX-4D The data showed that as few as 25 cells successfully grew and formed artificial biological tissues on the base matrix system. The analysis was performed by making a cell suspension by dilution method such that 20 μl of medium contained the exact cell number ranging from 25 to 250 cells. A drop was pipetted onto the inner surface of the lid filled with PBS at the bottom. After 24 hours, the inner lid was inverted and the drop was resuspended in fresh medium. The spheroids were analyzed by phase contrast microscopy, added on top of the base matrix placed on tissue culture plates (24 or 96 well plates) and incubated in a humidified incubator at 37 °C and 8% CO2. Using different cell numbers for inoculation, artificial biological tissues were attached and grown and investigated under phase contrast and scanning electron microscopy (Figure 9).

[0090] As shown in the figures, different cell numbers of MCF-7 cell line are used as starting material for growing spheroids and 3D artificial biological tissues, ranging from about 250 cells to less than 25 cells. Spheroids were grown on the 3D base matrix system, as shown in Figure 9A, Figure 9B, Figure 9C, Figure 9D and Figure 9E using phase contrast microscopy (10x magnification) and in Figure 9F using SEM analysis (1500x magnification). The pictures show the growth of artificial biological tissues and clearly showed that only 25 cells were required to create artificial biological tissues on the base matrix system provided herein.

[0091] Example 10: Time course of viability of cell cultures on base matrix systems and culture duration The growth and viability of artificial tissues grown on the base matrix AXTEX-4D was observed to extend for over a year for PC3 (approximately 364 days, FIG. 13, lower panel), for HepG2 approximately 3 months (up to 82 days, FIG. 13, upper panel), and for MCF-7 artificial tissues close to 130 days. 2 We analyzed and observed this phenomenon using three different cell lines (HepG2, MCF-7, and PC3) on 100-nm-long platelets (100-nm-long platelets).

[0092] Furthermore, the viability of the PC3 artificial tissue was analyzed by FACS analysis using LIVE / DEAD staining on day 100 of its growth, and among the gated population (i.e., up to 75%), 47% of the cells were live cells and 18.68% of the cells were dead cells, indicating that even after 100 days of culture, the viability of the PC3 artificial tissue was almost 60%.

[0093] The clear advantage of long-term growth and viability is given by the data herein, which show that it is possible to mimic the fibrous situation ex vivo for extended periods of time, thereby performing different assays over extended periods of time as a way to obtain drug susceptibility data in order to design optimal therapeutic regimens for patients.

[0094] Example 11: Several different cell lines were grown on the base matrix system: The artificial biological tissue generation method and system described in this application is a universal base matrix system that has been shown in the examples herein to be usable for culturing various types of cell cultures in a 3D format.Using the process described above in Example 2, the following cell lines have been successfully grown on the cloth-based matrix system (Figure 1B, Figure 1C, Figure 2, Figure 3A, Figure 4), including MCF7: breast cancer cell line, adenocarcinoma, HepG2: liver cancer, epithelial cells, PC3: prostate cancer cell line, adenocarcinoma, A375: skin melanoma, epithelial cell line, HT-29: colorectal, adenocarcinoma and non-malignant cell line CHO-K1 cells (stably expressing surface proteins), HEK-293, NIH3T3 fibroblasts.In addition, this system has been tested for the growth of artificial biological tissues derived from primary tumor cells such as colon, stomach, lung, and thyroid (represented in Figure 3B and Figure 5).

[0095] Example 12: Co-cultures and tri-cultures of different cell mixtures on a base matrix system Artificial tissues were generated by co-culturing two or more cell lines from mixed cell populations. Each combination of co-cultures was analyzed by taking either breast cancer cell line (MCF-7) and endothelial cells (HUVEC) or endothelial cells (HUVEC) and fibroblasts (NIH-3T3) in a 1:1 ratio, respectively. These were grown in a 2D monolayer format and on an AXTEX-4D-based matrix system, as shown in Figure 14. Artificial tissues were found to grow very efficiently.

[0096] Artificial tissues were generated from mixed cell populations by co-culturing three cell lines. A cell suspension of MCF-7 cell line was mixed with NIH-3T3 and HUVEC cell lines in a 1:2:1 ratio. These mixed cell populations were grown on AXTEX-4D and the data, shown in Figure 14, indicate that the populations grew very efficiently.

[0097] Producing and analyzing co-cultures and tri-cultures using artificial tissue-based matrix systems is envisioned as useful in studying cell-cell interactions, drug discovery and development, and patient treatment regimens, particularly in cancer immunotherapy and infectious disease-based matrix systems (Figure 14).

[0098] Example 13: Primary patient cell and tissue samples grown on AXTEX-4D based matrix system Primary cell lines and samples of tissue biopsies from tumor patients were grown on the base matrix system as artificial living tissues. Tumor tissue specimens were harvested as suspension cultures or explants and grown on the AXTEX-4D base matrix system. Growth of tumor tissue on the base matrix system is shown as an example in Figure 3B and Figure 5, demonstrating that the AXTEX-4D base matrix system can be used effectively and universally to generate artificial living tissues from primary tissue samples / biopsies.

[0099] Example 14: Reducing the time it takes to grow artificial tissues on a base matrix system and begin assays Observations herein report that cells of cell lines / primary cells attach to the AXTEX-4D base matrix system and begin to grow as artificial tissue in a time interval of 24 hours or less. The artificial tissue was observed to be optimal for analyzing drug sensitivity and resistance appropriate for assaying therapeutic drug dosing regimens. For certain cell lines, cells took somewhat longer, but overall, for human cell lines, it took 72 hours or less to attach to the base matrix system and begin to grow as artificial tissue. This rapid culturing of artificial tissue addresses the key factor of time, a long-needed factor in patient-drug related research, making it a 4-dimensional system. (Figure 15).

[0100] Example 15: Cell Factory The AXTEX-4D base matrix system sustains the growth of artificial biological tissue for a longer period of time. It is envisioned that the 3D system and method and format provided herein will be very useful for the mass production of cells, vaccines, therapeutic proteins, antibodies, and secreted proteins. The system is easy to handle and does not require special tubing, and antibody production was observed to increase as a function of time, as shown in Figure 16.

[0101] By growing CHO-DG44 cells stably expressing the anti-IL-6R antibody tocilizumab on the AXTEX-4D-based matrix system, we were able to increase the life span of the antibody-expressing cells, improve productivity, and increase the number of cells in a more compact space (Figure 16), confirming the use of the AXTEX-4D-based matrix system as a cell factory for the biological production of biologics and vaccines.

[0102] Example 16: Angiogenic base matrix system Endothelial cell dysfunction is involved in diabetes, lung disease, inflammatory diseases, cardiovascular diseases, and immune diseases. Angiogenesis is a key process in tissue development, wound healing, and tumor progression. The method utilizing the 3D format provided useful insights for studying angiogenesis or screening the tumor microenvironment for inhibitors of antiangiogenic drugs.

[0103] The artificial tissues generated from HUVEC cells in a loosely compact form were grown on the AXTEX-4D system in the presence of VEGF-165, which is a potent mediator of angiogenesis. Figure 17 shows that the artificial tissues grow together with the formation of tubular structures after 72 hours of treatment with 50ng / ml VEGF.

[0104] The 3D methods and systems provided herein have provided useful insights into angiogenesis and the formation of the tumor microenvironment, and the need to screen potent anti-angiogenic drugs in a system that closely resembles that of an in vivo tumor.

Claims

**Claim 1**: An apparatus for rapidly growing artificial biological tissues, the apparatus comprising: at least one sterile culture chamber, each chamber containing a non-electrospun sterile non-woven polyethylene terephthalate (PET) fabric-based matrix system for accommodating and supporting an inoculation material selected from the group consisting of spheroids of hanging drop culture, cell suspensions, and explants of biopsies, and each chamber having a bottom surface and side surfaces for holding a culture medium, the base matrix system, and the artificial biological tissue for growing the artificial biological tissue in three dimensions (3D). **Claim 2**: The apparatus according to claim 1, wherein the artificial biological tissue is visible in less than 72 hours, or less than 48 hours, or even less than 24 hours. **Claim 3**: The apparatus according to claim 1 or 2, wherein the apparatus has an application as a bioreactor for growing a large amount of culture to produce a therapeutic protein, a vaccine, or an antigen. **Claim 4**: The apparatus according to claim 3, wherein the sterile culture chamber has an inlet port for adding fresh culture medium and an outlet port for discharging used medium. **Claim 5**: The apparatus according to claim 3, comprising the artificial biological tissue grown for at least 10 days, at least 20 days, at least 30 days, at least 90 days, or at least 250 days, or at least 380 days. **Claim 6**: A method for manufacturing an artificial tissue using the apparatus according to claim 1, the method comprising: (I) providing the apparatus according to claim 1; and (II) growing cells of avian or mammalian origin in the base matrix system. **Claim 7**: The method according to claim 6, wherein the artificial tissue is a therapeutic artificial skin or muscle for transplantation. **Claim 8**: The method according to claim 6, wherein the artificial tissue is available for clinical or edible use. **Claim 9**: A method for studying angiogenesis and angiogenesis inhibitors using the apparatus according to claim 1, the method comprising: (I) providing the apparatus according to claim 1; (II) growing an artificial biological tissue for angiogenesis in the base matrix system; (III) exposing the artificial biological tissue for angiogenesis to an angiogenesis inhibitor; and (IV) evaluating the angiogenesis inhibitor by measuring changes in angiogenesis. ​

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