Cancer modeling platform and methods of use thereof

Abstract

To provide an in vitro cell construct that is useful as a tumor model comprising live patient-derived tumor cells and method of use thereof.SOLUTION: An in vitro cell construct is provided comprising (a) a core comprising a live tumor cell and (b) a shell surrounding the core, the shell comprising live benign cells. A method for producing and using the construct is also provided. A device is further provided which is useful for evaluating tumor cells in vitro and which comprises: (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber; (b) a live tumor cell construct in the chamber; (c) a growth medium in the chamber and channel; (d) a pump operatively coupled to the chamber and the channel and configured to circulate medium from the chamber through the channel and back to the chamber; and (e) a microporous membrane in the channel positioned to allow medium to flow therethrough.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Application Information This application is a continuation of U.S. Provisional Patent Application No. 62 / 370,320, filed August 3, 2016, and and claims the benefit of U.S. Provisional Patent Application No. 62 / 522,313, filed June 20, 2017. , the disclosures of each of which are incorporated herein by reference.

[0002] Statement of government support This invention was made with funding from the National Institutes of Health under grant number 1U54TR001362-01. This work was made with government support under grants 5UL1TR001420-03 and 5UL1TR001420-03. The government has made no grant in respect of this invention. have certain rights in the

[0003] The present application describes in vitro cell constructs (also known as cellular constructs) that can be used as tumor models. "organoids") and methods for making and using same. [Background technology]

[0004] Precision medicine holds great promise for improving the care of cancer patients. Identification will be performed on tumor biopsies to scope drug selection based on key mutations of interest. Although this narrows the scope of treatment options, selecting the absolute best drug for a particular patient remains difficult. Drug selection can be screened using tumor models made from the patient's own cells. This may complement genetic screening to determine the most effective drugs. Cancer models provide personalized predictions of the effects of drugs on tumor growth and their metastatic potential. This can provide statistical data. Summary of the Invention

[0005] The first aspect of the present invention comprises (a) a core of viable tumor cells and (b) a matrix surrounding the core (e.g., and a shell that encases living benign cells (e.g., tissue cells, benign or differentiated tumor cells, etc.) useful as a tumor model. (or "organoids"). The viable tumor cells and / or viable benign cells are from a subject, e.g., a biopsy (e.g., a tumor biopsy and / or a tissue biopsy) taken from the subject etc.

[0006] A second aspect of the present invention provides a method for the preparation of a tumor model comprising administering to a subject a tumor cell line comprising the steps of: In some embodiments, the organoid is an in vitro cellular construct (or "organoid"). The viable tumor cells are derived from a biopsy (eg, a tumor biopsy) taken from the subject.

[0007] A further aspect of the invention is a method for producing a liquid crystal display comprising: (a) a chamber; and a channel in fluid communication with the chamber. (b) a microfluidic device with a chamber containing a living tumor cell construct (e.g., (e.g., organoids), (c) growth medium in the chambers and channels, and (d) the operatively coupled to the bar and the channel, and extending from the chamber through the channel and back to the chamber (e) a microporous membrane (e.g., a TR ANSWELL® microporous membrane) and positioned to allow medium to flow through it. These assays are useful for evaluating tumor cells in vitro (e.g., tumor cell metastasis). To assess in vitro markers of tumor cell migration and / or invasion, to assess the proliferation of tumor cell-containing constructs in vitro and / or response to a compound of interest, e.g., a drug or prodrug, etc. It is a useful device for evaluating

[0008] Another aspect of the invention is useful for evaluating tumor cells in vitro (e.g., To assess tumor cell metastasis in vitro, tumor cell migration and / or invasion were used. To assess the in vitro effect of CTCT, tumor cell-containing constructs were grown in vitro. To evaluate and / or identify compounds of interest, e.g., drugs or prodrugs, etc. The device is useful for assessing the response to The device comprises two or more chambers each having at least one organoid. The organoid or organoids may be generated from cells obtained from a single subject. In some embodiments, the device contains live tumor cell organelles in the first chamber. The organoids are placed in a second chamber and contain liver organoids. and liver organoids may be formed from cells obtained from the same subject, thereby allowing for individual A differentiated analysis can be provided.

[0009] The above and other objects and aspects of the present invention are described in more detail below. It should be understood that the present invention may be embodied in various forms, and the embodiments described herein may be modified in various ways. Rather, such embodiments should not be construed as being limited to the embodiments described herein. This disclosure is thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. This is provided to. [Brief description of the drawings]

[0010] [Figure 1] A conceptual diagram showing how modified 3D in vitro models can be used to better inform precision medicine treatment regimens for individual patients. The solid arrows represent the current state of the art precision medicine pipeline, where treatment is determined for a patient based on their tumor genetic profile. However, in practice, even after the identification of key mutations of interest, oncologists are often forced to choose between several possible drugs, resulting in a best guess or guess as to what the optimal treatment will be. The dashed arrows show that the introduction of organoids made with patient cells can complement genetic screening of biopsied tumor cells and ultimately predict the optimal therapy for the patient. [Figure 2A] FIG. 1 is a schematic diagram showing the assembly of the microfluidic device layers: (from top to bottom) tubing, polystyrene (PS) slide with fluidic inlets and outlets, adhesive film with chambers for organoids and corresponding fluidic inlets and outlets, glass slide, and photomask layer. [Figure 2B] Schematic diagram showing the in situ patterning technique. The microfluidic chamber (i) is filled with a hydrogel mixture containing HA hydrogel, photoinitiator and tumor cells (ii) and then exposed to UV illumination through a photomask (iii). The exposed hydrogel is crosslinked (iv) and then the non-crosslinked gel is washed away using a clean buffer (v). Finally, the washed-out buffer is replaced with culture medium for incubation (vi). [Figure 2C] FIG. 1 is a schematic diagram showing the entire measurement apparatus, featuring a low-volume, closed-loop fluidic circuit for each organoid, with flow driven by a computer-controlled peristaltic pump. [Figure 2D] 13A-13D are maximum projection confocal and segmented images of tumor constructs on the device after LIVE / DEAD assay on days 7 and 14, showing percent viability of 93.3% and 86.4%, respectively. [Figure 3A-3F]Panels af(i) are maximum projection confocal images of tumor constructs after LIVE / DEAD assay, and panels af(ii) are segmented images using Imaris software. Medium grey represents dead cells, light grey represents live cells. Panel a) is an image of a control at week 1 with >90% viability. Panel b) is an image of a control at week 2. Panel c) shows constructs that were under the influence of carboplatin and pemetrexed drugs for 7 days. Panel d) shows constructs that were under the influence of carboplatin and pemetrexed drugs for 7 days. Panel e) shows constructs that were under the influence of cisplatin and pemetrexed drugs for 7 days. Panel f) shows constructs that were under the influence of cisplatin and pemetrexed drugs for 7 days. [Figure 3G] Graph showing viability (live / dead cells) measurements from segmented images of control and drug-treated organoids. Drug exposure began on day 7. Significance: *p<0.1, **p<0.05, ***p<0.01. [Figure 4A.4B] Figure 1 shows the biomarker-based therapeutic drug screening in patient-derived tumor constructs. In panel A), genetic analysis of tumor biopsy samples identified two mutations, PBRM1 and BAP1. The BAP1 mutation was identified as a potential target by the EZH2 inhibitor DZNep. In panel B), MTS mitochondrial metabolism assay showed that control organoids were the most proliferative compared to organoids treated with DZNep drug, with a decrease in mitochondrial metabolism occurring in proportion to the decrease in cell number. Conversely, HCT116 organoids do not appear to respond negatively to DZNep. [Fig. 4C-4F]FIG. 1 shows biomarker-based therapeutic drug screening in patient-derived tumor constructs. In panel C), further analysis was performed using Annexin V and Ki67 biomarker images, showing that all drug treatments had a higher ratio of Annexin V to Ki67, indicating increased apoptotic cell mass. Representative images of Annexin V and Ki67 fluorescent biomarkers are shown for DZNep treatments. Red staining (medium grey in black and white images) is Annexin V, and green staining (light grey in black and white images) is Ki67. Scale bar indicates 100 um. In panel D), further analysis was performed using Annexin V and Ki67 biomarker images, showing that all drug treatments had a higher ratio of Annexin V to Ki67, indicating increased apoptotic cell mass. Representative images of Annexin V and Ki67 fluorescent biomarkers are shown for DZNep treatments. The red staining (medium gray in the black and white image) is Annexin V, and the green staining (light gray in the black and white image) is Ki67. The scale bar indicates 100 um. In panel E), hematoxylin and eosin stained organoid sections show that as the DZNep concentration increases in mesothelioma organoids, they lose their normal morphology and nucleus-less ghost cells appear. In panel F), contrary to the mesothelioma organoids in E, HCT116 cells do not show dose-dependent changes like the mesothelioma organoids in E, suggesting that DZNep induces cell death more effectively in mesothelioma organoids than in HCT116 organoids. [Figure 5A] Figure 1 shows viable organoids derived from difficult to culture tumors. Panel A) Low-grade appendix (LGA). [Figure 5B] Figure 1 shows viable organoids derived from difficult to culture tumors.Panel B) Well-differentiated papillary peritoneal mesothelioma. [Figure 5C] Figure 1 shows viable organoids derived from difficult to culture tumors. Panel C) As already suspected in clinical practice, LGA organoids do not respond to therapy, whereas high-grade appendiceal tumor organoids do. [Figure 5D] Figure 1 shows viable organoids derived from difficult to culture tumors. Panel D) Generation of viable tumor organoids from a rare sarcoma. [Figure 6] Figure 6A is a diagram showing the 2D drug testing protocol. Figure 6B is a graph showing percent relative cell viability values ​​of HCT116, HT29, CACO2, and SW480 colorectal cancer cell lines in 2D microenvironments after treatment with various concentrations of anticancer drugs. A=regorafenib (μM) B=sorafenib (μM) C=trametinib (nM) D=5-FU (mM) E=dabrafenib (μM). [Figure 7] Figure 7A is a diagram showing the 3D drug testing protocol. Figure 7B is a graph showing percent relative cell viability values ​​of HCT116, HT29, CACO2, and SW480 colorectal cancer cell lines in 3D microenvironments after treatment with various concentrations of anticancer drugs. A=regorafenib (μM) B=sorafenib (μM) C=trametinib (nM) D=5-FU (mM) E=dabrafenib (μM). [Figure 8] Graph comparing the results of 2D and 3D drug screening with regorafenib, sorafenib, and 5-FU on HCT116 and CACO2 colorectal cell lines. Significant differences are again displayed as determined by p-values ​​(<0.10=* <0.05=** <0.01=***). [Figure 9A] FIG. 1 shows the circular organoid drug testing protocol. [Figure 9B] Microscopic images detailing targeted cell death of HCT116 by regorafenib, and universal cell death of both HCT116 and CACO2 by 5-FU at day 7. Green (light grey in black and white image) fluorescent dye (calcein AM) indicates live cells, whereas red (medium grey in black and white image) fluorescent dye (ethidium homodimer 1) indicates dead cells (scale bar = 200 μm). [Figure 9C] Graph showing the ratios of regorafenib and 5-FU at each concentration versus the control ratio, which is the ratio of no treatment. [Figure 10]FIG. 1 depicts the internal and external components of a microfluidic device that circulates media through the device. [Figure 11] 1 is an image showing LIVE / DEAD results showing that liver organoids are required to metabolize capecitabine into the pharmacologically active form of 5-FU and induce increased cell death in cardiac and pulmonary organoids. [Figure 12] Images showing LIVE / DEAD results in a miniaturized system, showing that in the presence of the liver, capecitabine is metabolized to the toxic drug 5-FU, causing toxicity to the heart and lungs. Without the liver, this metabolism does not occur and heart and lung organoid viability is not compromised. [Figure 13] Graph of initial biomarker analysis in standard size and miniaturized systems. [Figure 14] Diagram of a hybrid microreactor fabrication strategy incorporating tape fluidics and adhesive film and / or double-sided tape (DST), poly(methyl methacrylate) (PMMA), and polydimethylsiloxane (PDMS) components. [Figure 15A] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 15B] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 15C] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 15D] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 16] FIG. 1 shows an example six chamber device according to an embodiment of the present invention. [Figure 17] 1 is a schematic diagram of a device structure according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will now be described in more detail hereinafter with reference to the accompanying drawings, which show embodiments of the invention. However, the present invention may be embodied in various forms and the embodiments set forth herein are merely illustrative. The present invention should not be construed as being limited to the embodiments described herein. This disclosure is complete and complete, and will fully convey the scope of the present invention to those skilled in the art. This is provided so that

[0012] The terminology used herein is for the purpose of describing particular embodiments only. It is not intended to limit the present invention. Unless expressly stated otherwise, the singular forms "a," "an," and "the" include the plural forms as well. The terms "include" and "comprising" as used herein are intended to include In the case of The presence of a combination of one or more other features, integers, steps, operations, elements, It is further understood that the presence or addition of components and / or groups or combinations thereof is not precluded. is understood to be.

[0013] Unless otherwise defined, all terms used herein, including technical and scientific The terms used herein (including the term "term") have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries have their meanings and are included in the present specification. shall be interpreted as having a consistent meaning in the context of the specification and claims. Unless expressly defined as such herein, ideal or It is further understood that the invention should not be construed in a formal sense. Some structures may not be described in detail for the sake of brevity and / or clarity. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference. In case of conflict in terminology, the present specification shall control. Distribute.

[0014] Also, as used herein, "and / or" refers to the multiple instances of the associated listed items. Any or all possible combinations of one or more of the above, as well as When "or" is used, it refers to and includes the absence of combinations.

[0015] Unless the context dictates otherwise, the various features of the invention described herein may be combined in any It is specifically contemplated that some embodiments of the present invention may be used in combination. In some embodiments, any feature or combination of features described herein may be omitted. For example, the present specification may state that a complex comprises components A, B, and C. When indicating that, any of A, B, or C, or any combination thereof, may be omitted and excluded. It is specifically intended to obtain

[0016] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) The claimed invention is directed to the recited materials or steps "as well as to basic and novel features." Therefore, this should be interpreted as including "things that do not substantially affect As used in the specification, the term "consisting essentially of" is understood to be equivalent to "comprising." should not be interpreted.

[0017] As used herein, the term "about" refers to a measurable value, such as an amount or concentration. Without limitation, ±10%, ±5%, ±1%, ±0.5%, or even ±0% of a particular value. .1%, etc., refers to a variation of up to ±20% of a particular value, as well as to a particular value. Examples For example, "about X" means X, where X is a measurable value, as well as ±20%, ±10%, This is meant to include variations of ±5%, ±1%, ±0.5%, or even ±0.1%. Any ranges of measurable values ​​provided in the specification may be combined with any other ranges and / or subranges thereof. It may include each value in the range.

[0018] As used herein, "cells" generally refer to animal cells, particularly mammalian and primate cells. Examples of cells include human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, or goat. The cells may be derived from liver, intestine, pancreas, lymph node, smooth muscle At least one of these is expressed in specific cell or tissue types, such as skeletal muscle, central nervous system, peripheral nervous system, skin, and immune system. Preferably, the cells are somewhat differentiated. As discussed further below, some cells may be In some cases, the cells may be cancer cells, in which case some of the cells may also be mutated, as discussed further below. Thus, optionally but preferably, a detectable compound is added (either naturally or recombinantly). It is manifested by surgery.

[0019] As used herein, a "subject" is generally a human subject, but in aspects of the present invention , other animal subjects, particularly veterinary mammalian subjects (e.g., dogs, cats, horses, goats, sheep, etc.) The subjects may be male or female and may be infants, juveniles, adolescents, adults, or both. The subjects may be of any age, including adults and the elderly.

[0020] "Organoid" is used herein to refer to a "cell construct" and a "three-dimensional tissue construct." Used interchangeably, a membrane protein is a membrane protein that is fabricated in a three-dimensional or multilayer structure (as opposed to a monolayer), typically a support. Carrier media refers to a composition of living cells in a carrier medium. Suitable carrier media include cross-linked Further examples of hydrogels include hydrogels such as No. 6,399,413, filed on Oct. 23, 2015, the disclosure of which is incorporated herein by reference. Organoids include, but are not limited to, those that have been used as models or Depending on the particular tissue or organ to be mimicked, one differentiated cell type or two or more differentiated cell types may be used. Some organoids can be of the cytoplasmic type, as discussed further below. When the organoids contain cancer cells, the organoids are The cells may contain cellular components and / or extracellular matrix (or tissue derived therefrom). Proteins or polymers), hyaluronic acid, gelatin, collagen, alginic acid, etc. The present invention can include cell-free tissue mimics, such as those that include combinations of In some embodiments, the cells are mixed with an extracellular matrix, or a crosslinked matrix. In some embodiments, the cells are combined to form organoids or constructs, while in other embodiments, the cells are combined to form spheroids or constructs. Alternatively, cell aggregates such as organoids may be preformed and then mixed with extracellular matrix. In some embodiments, the organoids are selected from the group consisting of tumor-bearing and / or non-tumor-bearing organoids. It is a tumor organoid.

[0021] In some embodiments, the organoids comprise cells that are human-derived cells, and some In an embodiment, the organoid comprises cells of human origin. The identity is a single or multiple copies of a biomarker that is identical to a biomarker produced by cells in vivo. and / or expressing a number (e.g., 1, 2, 3, 4, or more) of biomarkers. For example, liver cells in vivo can produce albumin, including liver cells. The organoids of the present invention, including those described herein, may express albumin. In some embodiments, the organoids may express albumin. The peptides are produced and / or expressed in the same amounts or amounts as the corresponding cells in vivo. The samples may express the biomarker at levels ±20%, ±10%, or ±5% of the mean level. Examples of biomarkers include albumin, urea, and glutathione S-transferase (G ST) (e.g., α-GST), chemokines (e.g., IL-8, IL-1β, etc.), pro Stacyclin, SB100B, neuron-specific enolase (NSE), myelin base Sex protein (MBP), hormones (e.g., testosterone, estradiol, progesterone, gesterone, inhibin A / B, lactate dehydrogenase (LDH), and / or tumor necrosis factor (TNF) These include, but are not limited to, tumor necrosis factor (TNF).

[0022] In some embodiments, the organoids of the present invention have a diameter of about 200 μm to about 350 μm. m, for example, about 200, 250, 300, or 350 μm. The total number of cells in each culture was approximately 1,500 or 2,000 to approximately 3,000 or 3,500. The organoids of the present invention can be formed, for example, in any three-dimensional or multi-layered shape. In some embodiments, the organoids of the present invention are In some embodiments, the organoids of the present invention are in the form of spheroids. They may be allowed to self-assemble in a liquid or medium (eg, a cross-linked hydrogel).

[0023] As used herein, "growth medium" refers to a medium for maintaining the cells used to practice the present invention. The medium may be any natural or artificial growth medium (typically an aqueous liquid) that supports the growth of the plant. For cultures containing essential or minimal essential medium (MEM), or a variant thereof, such as Eagle's minimum essential medium, Minimal essential medium (EMEM) and Dulbecco's modified Eagle's medium (DMEM), as well as blood , serum, plasma, lymph, and others, including synthetic mimetics thereof. In some embodiments, the growth medium does not contain a pH color indicator (e.g., phenol Red).

[0024] As used herein, a "candidate compound" or "compound of interest" refers to a compound that is isolated from a primary site. To determine the inhibitory activity of the compound in preventing the spread of cancer cells to the 2 sites or in reducing the survival rate of cancer cells. The compound may be any compound for which antitumor and / or anti-tumor activity is to be determined. To this end, marimastat (N-[2,2-dimethyl-1-(methylcarbamoyl)propyl ]-2-[hydroxy-(hydroxycarbamoyl)methyl]-4-methyl-pentane However, any compound can be used, and generally These include proteins, peptides, nucleic acids, and small organic compounds (aliphatic, aromatic, and Candidate compounds can be selected from a wide range of organic compounds, including aryl, aryl, phenyl ... Randomly generated by technology and / or intelligently designed based on a specific target The method may be performed by any suitable technique, including techniques for generating AM Stock et al.Targets for anti-metastatic drug development, Curr.Pharm.Des.19(28):5 127-34 (2013). In some embodiments, the candidate compound is These are prodrugs that can be converted into active drugs during the performance of the tests and / or methods of the invention. In some embodiments, the prodrug is converted to an active drug by the liver organoids. do.

[0025] As used herein, a "detectable compound" refers to a fluorescent protein (e.g., a red fluorescent photoproteins, green fluorescent proteins, etc.); enzymes, fluorescent or radioactive groups, or other labels the antigen protein or peptide to which the antibody bound to the target protein specifically binds; or any other The detectable compound may be a suitable detectable compound that is naturally occurring in cancer cells. These include those that occur (e.g., cytoplasmic mammary glands that are expressed at higher levels in cancer cells than in non-cancerous cells) marker protein), or inserted into cancer cells by genetic engineering / recombinant DNA technology The nucleic acid may be heterologous (i.e., heterologous).

[0026] The cells used to practice the present invention may be animal cells (e.g., avian, reptile, amphibian, etc.). In some embodiments, the cells are mammalian cells (e.g., canine, feline, Preferably, the cells are from a mouse, rat, monkey, ape, or human. The cells may be differentiated or undifferentiated. In some embodiments, the cells may be tissue cells (e.g., hepatocytes, etc.) liver cells, pancreatic cells, cardiac muscle cells, skeletal muscle cells, etc.

[0027] The choice of cells will depend on the particular organoid being generated. For example, for liver organoids, In the case of peripheral or central nervous organoids, peripheral nerve cells, Central nerve cells, glial cells, or a combination thereof may be used. In bone organoids, osteoblasts In the lung organoids, lung airway epithelial cells, osteoclasts, or a combination thereof may be used. In lymph node organoids, follicular dendritic lymphocytes, fibroblastic reticular lymphocytes, , leukocytes, B cells, T cells, or a combination thereof may be used. In the organoids, smooth muscle cells, skeletal muscle cells, or a combination thereof may be used. In the present invention, skin keratinocytes, skin melanocytes, or a combination thereof may be used. , undifferentiated cells which may be differentiated when first incorporated into the composition, or which subsequently differentiate Additional cells may be added to any of the above compositions, and As noted above, primary or "first" organoids may be supplemented with the following cancer cells:

[0028] The cancer cells used in the present invention include melanoma, carcinoma, sarcoma, blastoma, glioma, The cancer cells may be of any type, including but not limited to, cells of the cancer types, and the like. In some embodiments, the cancer cells used in the present invention are derived from cancer cells cultured according to the methods taught herein. The cancer cells express N-cadherin and / or show epithelial-mesenchymal transition in the intestine ( small intestine, large intestine, colon), lungs, breasts, prostate, skin, bones, brain, liver, pancreas, uterus, cervix, Cancer cells derived from any primary tissue, including, but not limited to, testicular and ovarian cancer cells. It is also possible.

[0029] In some embodiments, the cells are derived from, for example, a subject or patient undergoing cancer treatment; and / or subjects or patients with cancer and / or subjects with immunodeficiency. In some embodiments, the cells can be obtained from a subject, e.g., from a biopsy. The tumor cells are tumor cells obtained from the tumor, and organoids made from such cells are used to This allows screening for potentially effective drugs and / or treatments. Examples of tumor organoids obtained from this study include mesothelioma, colorectal, appendix, lung, melanoma, and In some embodiments, the organoids include, but are not limited to, sarcoma organoids. The cells include benign cells obtained from tissue biopsies. The cells are from the brain, liver, intestine, pancreas, lymph nodes, and brain. It is less prevalent in certain cell or tissue types, such as smooth muscle, skeletal muscle, central nervous system, peripheral nervous system, skin, and immune system. Cells obtained from a biopsy (e.g., tumor and / or benign ) can be used to form and / or generate the organoids of the present invention, The organoids are cultured in accordance with the methods and / or devices of the present invention for about 1, 2, 3, 4 days after biopsy. , 5, 6, 7, or 8 days. In one embodiment, the probe may be a fluorescent compound (e.g., a dye, a protein, etc.), but is not limited to the fluorescent compound. The cells may be labeled with an extractable compound.

[0030] In some embodiments, the organoids of the present invention are generated from cells derived from immortalized cell lines. The organoids of the present invention do not contain cells derived from an immortalized cell line and / or do not contain cells derived from an immortalized cell line. Cells and / or including, but not limited to, primary cells and / or stem cells (e.g., artificially and / or contain highly functional cells such as pluripotent stem cells and / or differentiated iPS-derived cells Alternatively, it may be prepared using

[0031] The aspects and features of the invention described herein will be readily apparent to those skilled in the art to which the invention pertains. It can be carried out in accordance with certain known materials, methods and techniques, or modifications thereof. A reduct, Skardal A, Devarasetty M, et al. ionist metastasis-on-a-chip platform for in vitro tumor progression modeling and drug screening.Biotechnology and Bioeng ineering 113(9), 2020~32(2016);Skardal A, Devarasetty M, et al. A hydrogel bioink to olkit for mimicking native tissue bioche mical and mechanical properties in biopr inted tissue constructs.Acta Biomaterial ia 25, 24~34(2015); Bhise N, Manoharan V, et al.A liver-on-a-chip platform with biop rinted hepatic spheroids.Biofabrication 8(1);014010(2016).

[0032] As described above, the present invention provides a method for the preparation of a tumor cell comprising: (a) a core of viable tumor cells; and (b) a tumor cell-containing matrix surrounding the core. and a shell that contains living benign cells (e.g., tissue cells, good In vitro cell constructs useful as tumor models, consisting of cells that are either resistant or differentiated The present invention provides a method for the preparation of a human organoid.

[0033] In some embodiments, the viable tumor cells comprise malignant cells.

[0034] In some embodiments, the live tumor cells are colorectal cancer cells (e.g., HCT11 6 cells, HT29 cells, SW480 cells, Caco2 cells, etc.

[0035] In some embodiments, the tumor cells and benign cells are mammalian cells (e.g., human, mice, rats, monkeys, etc.).

[0036] In some embodiments, the viable benign cells comprise epithelial cells.

[0037] In some embodiments, the live benign cells comprise Caco-2 cells.

[0038] In some embodiments, the tumor cells contain a detectable compound (e.g., a fluorescent compound). nothing.

[0039] In some embodiments, the core and / or the shell comprise a hydrogel.

[0040] In some embodiments, the construct is incubated for 1 or 2 days, or for 1 to 2 weeks, or The cells are allowed to grow in the medium for a longer period of time.

[0041] In some embodiments, the method is useful as a tumor model comprising live tumor cells derived from a subject. In some embodiments, the present invention provides an in vitro cellular construct (or "organoid"). In some embodiments, live tumor cells are obtained and / or derived from a subject, e.g., from a tumor in a subject. Viable tumor cells may be harvested and / or derived from a tumor biopsy. In terms of morphology, live tumor cells form the core of the organoids, and the organoids are composed of live, well-formed tumor cells. Contains a shell of sex cells.

[0042] In some embodiments, organoids are provided that comprise living benign cells from a subject. Benign cells may be obtained and / or derived from a subject, such as, for example, from a subject's tissue. In an embodiment of the present invention, the viable benign cells may be taken and / or derived from a tissue biopsy; and Organoids containing live benign cells may be tissue specific. Live tumor cells may be isolated from organoids containing live benign cells. Separate organoids containing the endothelial cells (e.g., formed and grown separately in different chambers of the device) from the endothelial cells. In some embodiments, the samples may be collected and / or present from a single subject. and / or using cells derived therefrom (e.g., using one or more biopsies from the subject). ), at least one organoid comprising viable tumor cells obtained from a tumor biopsy from the subject; and at least one other organoid comprising viable liver cells derived from the subject. At least two (e.g., 2, 3, 4, 5, 6, 7, 8, or more) different In some embodiments, the ganoids are harvested and / or derived from a single subject. using cells from the subject (e.g., using one or more biopsies from the subject), At least one organoid containing viable tumor cells obtained from a tumor biopsy, and a liver tumor At least one other olfactory vector containing viable benign cells from the subject, of the same tissue type as the cells. At least two (e.g., 2, 3, 4, 5, 6, 7, 8, or more) The cells form organoids of size 100 or more.

[0043] The organoids of the present invention can be used to treat a patient, optionally prior to the initiation of therapy and / or treatment. These studies can provide patient-specific model systems that can be used to determine treatment options. In embodiments, including but not limited to, genetic biomarker assessment and / or genetic profiling Optionally, the target substance is screened for antitumor activity in addition to additional screening methods such as ELISA. In some embodiments, the organophosphate phosphatase of the present invention can be used to screen for compounds. Noids and / or methods for cellular biomarker recognition, quantification of biomarker expression and / or allows for real-time testing of chemotherapy drug efficacy; and / or may be provided.

[0044] A method for screening compounds of interest for antitumor activity in vitro (a ) providing at least one construct comprising a live tumor cell as described herein; (b) contacting the compound with the construct in vitro, and then (c) determining the desired compound. exhibits antitumor activity of the compound (e.g., at least one similar compared to the tumor cells of the construct and / or present in the shell surrounding the construct, and / or or increase in viable tumor cells (compared to viable benign cells present in a separate organoid) proliferation, decreased proliferation of viable tumor cells (e.g., lack of proliferation of tumor cells, death of tumor cells, tumor and determining whether or not the tumor cell is invading the shell. In some embodiments, tumor cells are obtained from a patient suffering from a tumor. In some embodiments, the compound of interest is a prodrug. If the compound is determined to reduce tumor cell proliferation in vitro, the method of the invention can further comprise the step of administering a therapeutically effective amount of the compound to the patient.

[0045] In some embodiments, the methods of the invention include obtaining a tumor biopsy from a subject (e.g., a patient). and using the cells to prepare the organoids of the present invention. Tumor biopsies can be sequenced in part or in whole to identify mutations. Any mutations identified may contribute to the formation of one or more compounds of interest for the treatment of a subject (e.g., antitumor activity). The methods of the invention can indicate and / or suggest a gene sequence or sequences. screening one or more of the compounds of interest identified in and / or combinations thereof, The method can include contacting the organoids with organoids generated using the cells of the present invention. The method of the invention involves determining whether one or more of the compounds of interest reduces tumor cell proliferation in vitro. If it is determined that the compound or compounds of interest reduces the tumor size, one or more of the compounds of interest are administered to a subject from whom a tumor biopsy was taken. The method may further include administering a therapeutically effective amount to

[0046] In some embodiments, the methods of the present invention involve the use of viable tumor cells obtained from a tumor biopsy from a subject. Preparing tumor cell organoids from the cells, and from the same subject or from a different source. and preparing liver organoids from the obtained live liver cells. In this embodiment, viable tumor cells and viable liver cells are obtained from the same subject. The liver organoids and liver organoids are placed in fluid communication with each other (e.g., using a device, e.g. (with the device described herein) and live tumor cell organoids and The target compound can be contacted with the organoids and / or the liver. The proliferation of live tumor cells in tumor cell organoids can be assessed. In embodiments of the present invention, the compound of interest is metabolized by the liver organoids to produce an active drug. and the activity of the active drug can be determined (e.g., in living tumor cells and / or liver If cell proliferation is affected by the prodrug and / or active drug, this activity is determined. The one or more additional organoids are prodrugs that can be used to treat or inhibit the growth of the organoids of the present invention. In some embodiments, one or more additional compounds may be used in the method of The organoids can be obtained from the same subject, from the same tissue or from different tissues of the subject. It may be of origin.

[0047] In some embodiments, the methods and / or devices of the invention involve two or more (e.g. 1, 2, 3, 4, 5, 6, 7 or more compounds of interest In some embodiments, two of the compounds of interest may be provided and / or enabled. At least one of the or more is a prodrug.

[0048] In some embodiments, the methods and / or devices of the invention use the cells. The organoids of the present invention used in the methods and / or devices of the present invention were prepared by Also, within about one or two weeks (e.g., within about one or two weeks) after obtaining a biopsy (e.g., a tumor biopsy) from the subject. Within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 days), results (e.g., drug screening The present invention may provide and / or enable the provision of clinical diagnostics, including clinical testing, clinical trials, and / or clinical analysis (e.g., clinical trial results and / or treatment analysis).

[0049] Also, (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber. (b) a fluidic device and a living tumor cell construct (e.g., an organoid) in the chamber. (c) growth medium in the chambers and channels; and (d) the chambers and channels. and operatively couples to circulate medium from the chamber, through the channel, and back to the chamber. (e) a microporous membrane (e.g., TRANSWELL ( A tumor cell culture medium is positioned to flow through the tumor cell culture medium. Described herein are devices useful for evaluating cells in vitro. In some embodiments, the device is adapted to assess tumor cell metastasis in vitro by: To assess tumor cell migration and / or invasion in vitro, To assess the in vitro proliferation of constructs containing In some embodiments, the device is useful for evaluating the response of a construct to a target cell. size in vitro, tumor cell count in vitro, and / or tumor cell death It may be useful to assess in vitro

[0050] In some embodiments, a device useful for evaluating tumor cells in vitro The method comprises the steps of: (a) forming a culture medium comprising: a culture chamber having at least one organoid in each chamber; The device is designed to assess tumor cell metastasis in vitro. To assess tumor cell migration and / or invasion in vitro, tumor cell and / or a compound of interest for evaluating in vitro proliferation of a construct comprising In some embodiments, the device may be useful for assessing the response to The size of the constructs was measured in vitro, the number of tumor cells in vitro, and / or the tumor This may be useful for evaluating tumor cell death in vitro. Alternatively, multiple organoids may be generated from cells obtained from a single subject. In embodiments, the device includes a first chamber containing live tumor cell organoids, and and in the second chamber, liver organoids. Visceral organoids may be formed from cells obtained from the same subject, thereby allowing for individualized Analysis can be provided.

[0051] In some embodiments, the device comprises a primary chamber containing live tumor cell organoids. at least one secondary chamber containing different organoids; primary and secondary chambers A small number of plates are connected to the members of the casing to allow fluid communication (e.g., flow of growth medium) between them. at least one primary conduit; and optionally, a primary chamber, each secondary chamber, and and a growth medium in the primary conduit. In some embodiments, two or more ( For example, 2, 3, 4, 5, 6, 7, 8 or more secondary chambers, In some embodiments, the organoids include at least one of the following: One secondary chamber contains liver organoids, optionally from this same subject. The obtained cells are used to generate live tumor cell organoids and liver organoids. Examples of such devices include International Application PCT / US2016, the entire contents of which are incorporated herein by reference. Examples of suitable fluoropolymers include, but are not limited to, those described in / 054611.

[0052] An example of a device with two chambers is shown in Figure 15. A device with five chambers is shown in Figure 15. Another example of a device is how the medium flows through the chambers of the device at different stages. FIG. 17 shows a method for labeling a detectable compound according to an embodiment of the present invention. Schematic diagram of a device structure that can be used to track infected cells (e.g., tumor cells).

[0053] In some embodiments, the tumor cell construct comprises a hydrogel.

[0054] In some embodiments, the tumor cell construct consists of a single cell type.

[0055] In some embodiments, the tumor cell construct is a human tumor cell construct that is capable of expressing a tumor cell lineage ... 6 cells, HT29 cells, SW480 cells, Caco2 cells, etc.

[0056] In some embodiments, at least a portion of the microfluidic device is transparent, The device is operatively associated with a microporous membrane and is adapted to detect (e.g., image) tumor cells on the microporous membrane. The system further comprises a detector (e.g., a camera) configured to detect the presence of the detected object.

[0057] In some embodiments, the device used in the methods of the invention is a fraction of the tissue volume. The fluid may be configured to deliver physiological or supra-physiological fluid to, for example, One or more of the chambers of the device have an average volume ranging from about 2 μL to about 10 μL. In some embodiments, one or more of the chambers of the device may include can have an average volume of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 μL. In some embodiments, the devices (e.g., 1, 2, 3, 4, 5, 6, or more For example, in an apparatus having a chamber of 100 or more, the Less than about 100μL, such as 0, 65, 60, 55, 50, 45, 40μL or less The amount of liquid used may be less than about 100 μL and / or may have a volume of less than about 100 μL. The volume of the device used refers to the volume that fills the chambers and channels of the device. In some embodiments, the device uses a volume of liquid less than about 50 μL, and / or Or it may have a volume of less than about 50 μL.

[0058] The organoids of the present invention are viable for at least 1, 2, 3, 4 weeks or more. In some embodiments, in the device and / or in the method of the invention The organoids used in this study are viable for at least 1, 2, 3, 4 weeks or longer. In some embodiments, the organoids of the present invention may be viable. Based on the average number of cells present in the organoids, 1, 2, 3, 4 weeks or more At least about 75% or more (e.g., about 80%, 85%, 90%, 95% or more) The above) may include viable cells.

[0059] As described herein, cells and / or cell samples are used in the methods of the invention. In the method of the present invention, viable organoids can be formed. In some embodiments, the methods of the invention can be used to generate ganoids, e.g. Approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% Engraftment rates of at least 50% or more, such as 98% or more, can be achieved. For example, a 90% engraftment rate indicates viable organoids or multiple organoids (e.g. 90 times the number of times a set of organoids (i.e., a set of organoids) is obtained and / or produced by the method of the present invention. %. In other words, when the method of the present invention is used to produce a 90% engraftment rate, the Nine of the cell samples (e.g., tumor cell samples) produced viable organoids. Or a plurality of organoids are obtained. The method can be used in other methods and / or diagnostics of the present invention. In embodiments, the cells and / or cell samples are, for example, mesothelioma biopsies and / or G The biological sample may be derived from a tumor, such as a tumor biosample.

[0060] Methods for screening tumor cells for metastatic activity in vitro include (a) (b) providing a device as described above; and (b) circulating a medium through the device. (c) quantitatively or qualitatively detecting the tumor cells captured on the microporous membrane. In this case, the more tumor cells captured, the greater the number of tumor cells captured ( For example, compared to other tumor cells under similar conditions and / or to non-metastatic cells under similar conditions. (compared to control) indicates increased metastatic activity of tumor cells.

[0061] Methods for screening tumor cells for metastatic activity in vitro include (a) As described above, a device (e.g., a primary chamber containing live tumor cell organoids) can be used. providing a device comprising a first chamber, a second chamber, and at least one secondary chamber; (b) circulating a medium through the device; and (c) one of the secondary chambers. or in a second chamber (e.g., optionally in a third chamber) Quantitatively or qualitatively detecting tumor cells (on and / or in the ganoids) In this case, the secondary chambers may be one or more of the secondary chambers. The more tumor cells that are involved (e.g., other tumor cells under similar conditions, and / or This indicates that the tumor cells have increased metastatic activity (compared to non-metastatic cells under similar conditions). Screen compounds of interest in vitro for antimetastatic and / or antitumor activity The method of scanning comprises the steps of: (a) providing a device as described herein above; (b) circulating a medium through the device; and (c) delivering a compound to the tumor cells (e.g., (d) administering the compound to the culture medium; and then (e) capturing the compound on a microporous membrane. and quantitatively or qualitatively detecting the tumor cells. In this case, the fewer the number of captured tumor cells (e.g., under similar conditions but with The anti-metastatic activity of the compound of interest is increased compared to other tumor cells that have not been treated with the compound. In some embodiments, the tumor cells are obtained from a patient suffering from a tumor. In some embodiments, the methods of the present invention include determining whether a compound inhibits metastasis of tumor cells in vitro. o, reducing the size of the construct in vitro and reducing tumor cell numbers in vivo. determined to reduce tumor cell death in vitro and / or induce tumor cell death in vitro. If so, the method further comprises administering a therapeutically effective amount of the compound to the patient.

[0062] Screening compounds of interest in vitro for antimetastatic and / or antitumor activity The method of cleaning comprises the steps of: (a) providing a device as described herein above; (b) circulating a medium through the device; and (c) delivering a compound to the tumor cells (e.g., (d) administering the compound to the primary chamber; and / or a method for quantitatively or qualitatively detecting tumor cells present in the second chamber. This can be done by the following steps: The greater the number of tumor cells treated with the compound of interest (e.g., under similar conditions, but without the compound of interest), the greater the the anti-metastatic and / or anti-tumor activity of the compound of interest (compared to the cells) In some embodiments, tumor cells are obtained from a patient suffering from a tumor. In some embodiments, the methods of the invention include determining whether a compound reduces metastasis of tumor cells in vitro. Reduce the size of the construct in vitro and reduce the number of tumor cells in vitro and / or induce tumor cell death in vitro The method further comprises the step of administering a therapeutically effective amount of the compound to the patient.

[0063] In some embodiments, the methods of the present invention relate to anti-metastatic and / or anti-tumor activity. screening the compound of interest in vitro; The method includes the steps of: (a) providing a device as described herein above; and (b) administering to the liver The first chamber containing the liver organoids was then transferred to the second chamber containing the live tumor cell organoids. (c) circulating a growth medium through the chamber; and (b) culturing the liver organoids and / or viable cells. The tumor cell organoids are then treated with a compound of interest (e.g., by adding the compound to the growth medium). (d) administering the test compound to a living tumor cell organelle in the absence of administration of the test compound. A reduction in the presence of tumor cells in the second chamber compared to the number of tumor cells present in the nodal and determining whether the tumor is benign or not (e.g., a change in number or density). One or more additional organoids, optionally comprising cells, such as, but not limited to, However, organoids such as brain, colon, lung, endothelium, heart, and epithelium can be cultured in additional chambers of the device. may be present in one or more of the chambers, which are in fluid communication with each other.

[0064] In some embodiments, the methods of the present invention relate to anti-metastatic and / or anti-tumor activity. screening the compound of interest in vitro; The method includes (a) administering to a subject a device (e.g., a living tumor cell organelle) as described herein above. The primary chamber containing the organoids, and the different organoids (e.g., organoids containing benign cells) providing a device having at least one secondary chamber containing a ganoid; (b) circulating a medium through the device; and (c) delivering a compound to the tumor cells (e.g., (e.g., by adding the compound to the culture medium); and (d) administering a test compound. Live tumor cells in the organoids and / or tumors present in the secondary chamber without Compared to tumor cell numbers, the primary chamber (e.g., associated with live tumor cells organoids) ) and / or a reduction in the presence of tumor cells in the second chamber (e.g., a change in number or density and determining the identity of the different organophosphate ions in the one or more secondary chambers. Examples of ids may include benign and / or tumor cells, such as brain, colon, lung, endothelium, heart, Organoids include, but are not limited to, organoids such as epithelium, liver, etc.

[0065] The methods of the present invention include, but are not limited to, detection of fluorescent compounds (e.g., dyes, proteins, etc.). The present invention also includes a labeled cell (e.g., a tumor cell) that contains an extractable compound. and other aspects are further described in the Examples below. EXAMPLES

[0066] [Example 1] The first objective of this project is to develop 3D models of colorectal cancer tumors surrounded by normal host tissue. The objective of the study was to evaluate the differentiation of colorectal tumor cores and The tumor is composed of an outer ring of colonic epithelium and a reductionist, but physiologically relevant, environment. This construct was used to model tumors. The penetration of hyaluronic acid and gelatin-based drugs into the epithelial layer and the efficacy of the drugs were measured. The constructs were made with cells suspended in hydrogels of four colorectal cancer cell types. One was used to generate tumor cores, which allowed the generation of a model with four levels of malignancy. CT116 cells were the most malignant, followed by HT29, SW480, and Caco2 cells. Caco2 cells were the most similar to healthy epithelial cells. Tumor cores were fluorescently labeled and fluorescence imaging was used to study tumor progression over 7–14 days. Tumor cores were monitored throughout the study. After this period, the tumor cores were shown to be dense. However, little invasion was observed. The circular constructs were also treated with the chemotherapeutic drug regorafenib. After treatment, a decrease in the amount of viable cells was confirmed, and LIVE / DEAD staining revealed Gorafenib selectively targets the inner ring of the construct over less tumorigenic Caco2 cells It was shown that:

[0067] The second objective is to develop a system to quantify the number of cells metastasizing from 3D colorectal cancer tumor constructs. The objective of this study was to develop a microfluidic perfusion device to detect and diagnose circulating metastatic cells. The polydimethylsiloxane was captured on a Transwell membrane sealed in a channel within the device. The devices were fabricated using polydiphenyl ether (PDMS) soft lithography. Tumor constructs consisting of a single tumor cell type suspended in rogel are placed into appropriate chambers; The device was sealed and fixed. Culture medium was circulated through the device and incubated every 2-3 days for approximately 2 weeks. After two weeks, the cells were observed on the Transwell membrane, and the device The results showed that the ELISA kit can assist in the quantification of tumor cells that enter the circulation from tumor constructs. Further tests were performed to demonstrate that tumor cells with different levels of malignancy have different migration rates. Check whether it can be shown quantitatively.

[0068] In summary, two such models, the 3D concentric ring tumor model and the microfluidic The circulating tumor cell quantification device is designed to measure tumor cell proliferation, the effect of drugs on tumor cell responses, and This provides a novel method to test the quantification of circulating cells.

[0069] [Example 2] Microengineer 3D tumor organoids directly from fresh tumor biopsies to create patient-specific model systems that allow for treatment optimization prior to the initiation of therapy. Here, we demonstrate the efficacy of this platform using mesothelioma tumor biopsies removed from patients. Demonstration of initial implementation. Live tumor-on-a-chip microfluidic device. The ability to generate and maintain biologically viable 3D tumor constructs is then demonstrated. -chip chemotherapy screening identifies drugs cisplatin and pemetrexed Finally, we will demonstrate that the gene expression profile of the serotonin receptor agonist (SAR) mimics the patient response to the serotonin receptor agonist (SAR). The efficacy of identified candidate compounds is demonstrated in vitro.

[0070] method Fabrication of thin-film microfluidic devices The microfluidic device fabrication method is based on methods already published elsewhere. Using a GraphTec-CE6000-40 plotter, A channel was formed in the bottom of the patterned layer, and the bottom surface of the patterned layer was attached to a clean microscope slide. The device features drilled holes to act as ports for fluid delivery to the channels. A polystyrene slide (Tedpella, Inc.) was attached to the top surface. After construction, PTFE tubing was connected to each port and solidified using UV-curable polyester resin. It was determined.

[0071] Patient History The patient was a 50-year-old man with debilitating cachexia, ECOG2 functional status and abdominal distension. The patient was a male. Diagnostic laparoscopy was performed for histological diagnosis, which revealed an epithelioid malignant peritoneal tumor. A diagnosis of rheumatoid arthritis was confirmed. Precision medicine analysis of his tumor identified available targeted drugs or clinical treatments. Two genomic alterations (BAP1 splice site 1729+1G>A and and PBRM1 N258fs×6) were revealed. Computed tomography (CT) showed voluminous malignant ascites without signs of disease outside the peritoneal cavity. He has been investigating hyperthermic intraperitoneal chemotherapy (HIPEC) combined with cytoreductive surgery (CRS). Due to the excessive tumor volume, the chances of achieving complete CRS at that time were slim. Therefore, he was recommended to undergo modern systemic chemotherapy with the aim of reducing the tumor volume. Six cycles of cisplatin-gemcitabine were administered, followed by cisplatin-associated ototoxicity. Because of concerns about pulmonary embolism, he was treated with a single cycle of carboplatin-gemcitabine. He had a good clinical response to cisplatin-based chemotherapy and underwent repeated staging. Both cross-sectional CT and laboratory tests performed prior to carboplatin-based chemotherapy showed that The malignant ascites disappeared almost completely. He was then taken to the operating room and diagnosed with CRS / Cisplatin. After the procedure, the patient underwent chin-based HIPEC, and the postoperative course was uneventful. During the diagnostic examination and before HIPEC, the area of ​​omental disease was excised with scissors and placed on ice. The tumor organoids were then immediately transferred to the laboratory for construction.

[0072] Obtaining tumor biospecimens and cell processing Tumors were delivered to the laboratory within 1 hour of removal for cell processing. 3 cycles for 5 min in phosphate buffered saline (PBS) containing 5% penicillin-streptomycin The tumors were washed and then cultured in Dulbecco's modified sucrose buffer containing 2% penicillin-streptomycin. The tumors were then washed for 2 cycles of 5 min in Eagle's medium (DMEM). 2% penicillin-streptomycin and 10% colloids were incubated on a shaker plate at 37 °C for 1 h. The cells were incubated for 18 hours in DMEM containing 10X Col lagenaze / hyaluronidase in DMEM, STEMCELL The digested tumor was then The cells were filtered through a 0.1 μm cell filter and centrifuged to produce a pellet. Remove non-cellular material and place in 1 mL of BD PharmLyse containing 9 mL of deionized water. The pellet was resuspended for 5 min (BD PharmLyse, San Diego, CA Fill the conical beaker to 50 mL with deionized water and centrifuge to lyse the cells. The lysis buffer was aspirated and the cell pellet was ready for use.

[0073] Preparation of extracellular matrix hydrogels and basic organoid formation ECM-mimicking HA / gelatin-based hydrogel (HyStem-HP, ESI-BIO, Alameda, CA) was prepared as described above. 15、18、27 Briefly, thiol The thiolated HA component (Glycosil) and the thiolated gelatin component (Gelin-S) were added at 0 0.05% w / v of the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl Dissolved in sterile water containing ethylpropiophenone (Sigma, St. Louis, MO). A 1% w / v solution was prepared using polyethylene glycol diacrylate (PEGDA ) Crosslinker (Extralink, ESI-BIO) was dissolved in the photoinitiator solution at 2% w A solution of 100 ml / v was then prepared. Glycosil, Gelin-S, and Extra link was mixed in a volume ratio of 2:2:1. Mix thoroughly by using a vortex to resuspend the cells (20 million cells). / mL), followed by 1 second of UV exposure (365 nm, 18 Wcm -2 ) crosslinking and and initiate organoid formation.

[0074] Tumor-on-a-chip biofabrication Using photopatterning techniques similar to our previous work, we fabricated microfluidic devices Tumor constructs were biologically generated in the 15 Here, the same Using cutting plotter technology, we cut photomasks with openings of 1 to 3 mm into aluminum. Fabricate foil and apply a matching patterned adhesive film to the bottom surface of the microfluidic device. The hydrogel precursor solution was then applied to the patient's tissue at a density of 20 million cells / mL. The mixture was introduced into each device channel through an inlet and mixed with cells from a mesothelioma biopsy. The ganoid constructs were exposed to UV light through an attached photomask for 1 s ( 365nm, 18Wcm -2 ) was clarified. A stepwise cross-linking reaction was rapidly initiated, thereby encapsulating the cells in the 3D columns. The unexposed precursor / cell mixture was washed from the device with phosphate-buffered saline to remove each channel. Each channel of the device with a cell construct was then left with a 3D patient-derived mesothelioma construct in the well. , connected to separate reservoirs containing DMEM. MP2 Precision mic ro-peristaltic pump(Elemental Scientific Silastic tubing connected to a septum tube (Omaha, NE) was used to pass the channel. The flow was started and maintained at 10 μL / min.

[0075] Histological evaluation 5 μm thick organoid sections were prepared from paraffin-embedded constructs and then stained. The tissue was deparaffinized to detect the biomarker cytokeratin 5 CK5 / 6 (CK5 / 6), calretinin, and thrombomodulin were visualized. Blocking was achieved by incubation in the presence of Protein Block for 15 min. Primary antibody CK5 / 6 (Abcam, ab17133, produced in mouse) and and calretinin (Abcam, ab702, produced in rabbits) or CK5 / 6 and Thrombomodulin (Abcam, ab109189, produced in rabbit) was purchased from Dako A Dilute 1:200 with antibody diluent and apply to sections on slides. The mixture was incubated at room temperature for 1 hour. Alexa Fluo r 488 or Alexa Fluor 594 secondary antibodies were used with Dako Antibodies. Dilute 1:200 with y Diluent and apply to all samples. Allow to stand at room temperature for 1 hour. (Anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594, Life Technologies, Carlsbad, CA, A-110 70) The sections were then incubated in Dapi for 5 min before being covered with a coverslip. Subsequent biomarker studies showed that annexin V and Ki67 staining (Abca m, Cambridge, MA, ab14196 and ab16667, respectively) Protocol. Fluorescent images were acquired using a Leica DM400B compound microscope. and overlaid for analysis.

[0076] Cisplatin, Carboplatin, and Pemetrexed Drug Trials At the end of the first week, the two constructs were stained for LIVE / DEAD as described below. To ensure that the cells were viable before adding the drugs, two combinations of drugs were considered: This cancer model was tested. Two different concentrations of cisplatin plus pemetrexed and carboplatin plus pemetrexed The DMEM reservoir was replaced with a volume of 2 mL of this drug mixture.

[0077] LIVE / DEAD staining (LIVE / DEAD Viability / for mammalian cells) Cytotoxicity Kit, Life Technologies, Grand The study (Hamburg Island, NY) was performed on day 7, at the end of the drug exposure period. The medium was washed out of the device channels with clean PBS, and then 2 μM calcein A was added. PBS and DMEM (1:1) containing 2 μM ethidium homodimer 1 1 mL of the mixture was introduced. The construct was incubated for 60 min, after which the channel was cleaned. The cells were washed again with pure PBS. Olympus Fluo View™ FV1000 In situ imaging was performed using confocal microscopy. A 5 μm z-stack was acquired for each construct using the same filters, then -Barley.

[0078] Imaris MeasurementPro (Bitplane, Concord, Confocal images were processed using the Sigma-Aldrich ... (Sigma-Aldrich) software, which allows the identification of the individual color channels. Images were analyzed for cell size, shape, and fluorescence intensity, and the location of cells was labeled. For the synthesis, an area of ​​450 μm × 900 μm of each image was considered. Identified in the green channel (LIVE) compared to the total number of cells identified in the DEAD channel Cell viability was calculated by quantification of the total number of cells that were cultured.

[0079] Biomarker-guided experimental drug testing Patients from whom tumor biopsies were obtained were enrolled at Wake Forest Baptist Medical Center ( Comprehensive Cancer Center at Wake Forest Baptist Medical Center High-precision Cancer Center Participants in the clinical program will receive biopsies to identify potentially actionable mutations in tumors. The anonymized data showed a positive mutation in the BAP1 gene. , testing enhancer of zeste homolog 2 (EZH2) inhibitors as experimental targeted therapies. showed favorable conditions for

[0080] Tumor constructs were once again biologically generated and then incubated for 3 days prior to drug screening. The patient's organoids were then cultured in cell culture medium at 0.1 μM, 1 μM, and drug 3, a histone methyltransferase EZH2 inhibitor, at a concentration of 10 μM. -3-deazaneplanocin A (DZNep) The cells were exposed to a 96-hour incubation period. Biologically generated organoids were generated using HCT116 cells, a colorectal cancer cell line. The MTS assay and immunohistochemistry showed that the IL-11 markers were expressed in the IL-11 markers. The drug effects were quantified using the MTS assay (CellTiter 96 One So). Mitochondrial synthesis was performed using mitochondrial filtration reagent (Promega, Madison, WI). Chondriac metabolism was quantified to determine relative cell number. The Molecular Devices SpectrumM Absorbance values ​​were measured on an ax M5 (Molecular Devices).

[0081] Organoid sections were sliced ​​at 5 μm thickness and analyzed using the biomarkers Annexin V and Ki67. Immunohistochemistry was performed using the ELISA kit. The tissue was embedded in paraffin, sectioned, and then stained with IHC. Sections were taken from previously unstained organoids that were deparaffinized for color. Primary antibody annexin V was diluted with Dako Antibody Diluent at a concentration of 1:200. The antibody was diluted and applied to the slides and left at room temperature for 60 min (Annexin V, Abcam, In parallel, a separate sample was incubated with the primary antibody Ki67 was diluted 1:200 with Dako Antibody Diluent. The secondary antibody, Alexa488, was applied at 100 nm (Ki67, Abcam, ab16667). All samples were diluted 1:200 with ko Antibody Diluent. and left at room temperature for 1 hour (Alexa 488, Life Technology ies, Carlsbad, CA, A-11070). Leica DM400B compound microscope Fluorescence images were taken using a microscope and annexin V and Ki67 were detected adjacent to GFP. Images of the sections were taken and overlaid for analysis. Annexin V images are colored red. The images of Ki67 were colored green (light gray in the black and white images).

[0082] result Tumor Organoid Strategy for Precision Medicine Our goal is to test anticancer drugs for efficacy and then provide information for treatment optimization. The goal was to develop a platform of patient-specific tumors that could be used to treat cancer. The concept of personalized precision medicine works as follows: A tumor biopsy is taken from a patient. In some or all of the biological samples, mutations that may represent druggable targets are identified. To identify such biomarkers, some or all of the genes are sequenced. FDA-approved drugs are available or in clinical trials targeting one of the following: If ongoing, the medication or therapy the patient receives for that study should be adjusted accordingly. Under optimal conditions, this strategy is comparable to standard chemotherapy. However, in practice, multiple mutations are frequently identified and The lack of a definitive optimal treatment has led to a range of possible drug options. Additional means of testing compounds, as well as potential experimental drugs, are needed. Our method: A patient was identified using the same biological sample material sent for genetic profiling. The aim of this method is to incorporate tumor organoids derived from living tissue (Figure 1, dashed arrow). A portion of the sample was used to study the cell-supporting extracellular matrix hydrogel biomaterial. A large number of microscale 3D tumor organoids are biologically generated. This was then used in a drug screen designed to test agents identified in the screening. Using these organoids, researchers can determine which drugs will be most effective for a patient. do.

[0083] Patient-specific tumor-on-a-chip biofabrication Panels A-C of Figure 2 show the thin-film microfluidic device for biological fabrication of tumor organoids. The process of in situ incorporation of and general device operation is shown. The device capable of housing tumor organoids comprises fluidic channels and The chamber is made of an adhesive film that blocks the chamber. This is a polystyrene slab with an inlet and outlet. The image is sandwiched between the substrate and a glass slide with a photomask attached (Figure 2, Panel A). Tumor organoids were generated using a hydrogel precursor solution containing cells derived from the patient's tumor. The tumor organoids were then patterned and introduced into each of the six channels, and the photodetector was used to measure the tumor size. The organoids were then photopolymerized through the opening in the mask (Figure 2, Panel B). After fabricating the biological medium, the deionized water was pumped through a micro peristaltic pump and tubing to the medium reservoir. The vice was then connected and the flow of organoid medium was then initiated (Figure 2, panel C).

[0084] First, each tumor construct was left in the presence of circulating DMEM for 7 or 14 days. To validate the platform's ability to maintain viable patient-derived tumor models, The inventors performed LIVE / DEAD analysis on organoids on days 7 and 14. (Figure 2, Panel D). The results were greater than 90% on day 7 and 85% on day 14. This shows a higher cell viability, which is consistent with the inventors' device structure and its support. The hydrogel matrix can support patient-derived cells for the expansion of experimental methods. It has been demonstrated that this is possible.

[0085] Tumor-on-a-chip provides viable patient-derived models that correlate with patient drug response and supporting chemotherapy drug trials On day 7, a subset of tumor constructs were treated with two different doses of carboplatin / pepidine. one of the chemotherapy drug mixtures methotrexed and cisplatin / pemetrexed, After 7 days of treatment, LIVE / DEAD analysis was performed on all organelles. The results were analyzed for cytoplasmic endothelial cells (Figure 3, panels a(i)–f(i) and a(ii)–f(ii)). Under control (drug-free) conditions, the tumor constructs were observed to maintain high viability. After a total of 14 days in the device, there was a statistically significant decrease of up to 87%. Organoids exposed to the mixture showed a significant decrease in viability. In methotrexate-treated samples, a circulating concentration of 0.1 μM resulted in a survival rate of 52.1%. The inventors confirmed that at 10 μM, the effect was reduced to 39.8%. Although the concentrations were two orders of magnitude higher than those of the control, the observed changes in viability were of low significance ( p<0.1), indicating a potentially moderate drug effect. In organoids treated with Xed, the 0.1 μM concentration reduced the cellular cellular fate by 39.0% and the 10 μM concentration reduced the cellular fate by 39.0%. The survival rate was reduced by 11.8% in the control group. These results were significantly higher than those in the control group (p<0.05). and control measurements (p<0.05 and p<0.001, respectively). Therefore, cisplatin / pemetrexed was shown to be effective in our device. , and was significantly more effective than carboplatin / pemetrexed. These results are shown in Figure 3. Summarised in Panel G.

[0086] Such data have two central outcomes. First, the data are For this study, we will study patient tumor biopsies longitudinally in bioengineered organoid systems. This demonstrates that it is possible to sustain cells derived from the This invention is the first to report this ability. Second, the data support the idea that such It is feasible to perform drug screening on ganoids in vitro, and cell viability The results show that the drug-dependent reduction in the rate of This represents a promising advantage for therapy design, as it allows the establishment of in principle For example, in such assays, our results show that cisplatin / pemetrexed We propose EGFR-1 as a superior treatment option over carboplatin / pemetrexed. The nature of treatment does not allow for a systematic comparison of the efficacy of the two drugs in patients. However, crucially, patients were treated with cisplatin / pemetrexed and received favorable responses. This indicates that the efficacy of patient-specific drugs is consistent in our in vitro system. This suggests that it is precisely reproducible.

[0087] Experimental drug screening by genetic biomarker identification Precision medicine testing identified two known mutations in tumor biopsies: BAP1 splice site 1729+1G>A and PBRM1 N258fs×6. BAP 1 (BRCA1-associated protein 1) is a deubiquitinase 28 On the other hand, PBRM 1 is a tumor suppressor gene associated with several cancers 29 At the time of testing, mesothelioma or In any other tumor type, no mutations were associated with FDA-approved therapies.

[0088] However, in further investigation, the inventors conducted animal models and in vitro studies. Inhibiting EZH2, an enzyme involved in DNA methylation and transcriptional repression, And therefore 30 , and confirmed that they have been successful in targeting mutations in BAP1 (Figure 4, Panel A). Therefore, the inventors used the EZH2 inhibitor DZNep to As a control population, we performed screening experiments using extensively studied colorectal cancer cell lines. HCT116 was used to generate organoids. Importantly, HCT116 cells DZNep 31 Treatment with induced cell cycle arrest, but not necessarily at significant levels. It has been found that HCT116 cells do not undergo cell death, and therefore the inventors believe that It was considered an appropriate control for the study.

[0089] Our results showed that there was no significant decrease in HCT116 proliferation after drug treatment, but mesothelial The results show that there was a significant reduction in the proliferation of HCT116 and mesothelial tumors (Figure 4, Panel B). Tumor controls were similar, but DZNep-treated mesotheliomas increased with increasing drug concentration. As the DZNep concentration increased, the cells Furthermore, when comparing HCT116 and mesothelioma, the proliferation was reduced at all therapeutic drug concentrations. For both cell types, mesothelioma cell proliferation was significantly reduced. Annexin V and Ki67 immunohistochemistry was performed across all conditions. In Syn V, mesothelioma appears to increase with increasing DZNep concentration, which may be due to apoptosis. The increase in apoptotic cells was not observed in all HCT116 conditions (Figure 4, Panel C). There was minimal change in proliferation after 1 h of incubation, indicating that DZNep did not significantly affect proliferation. (Figure 4, Panel D). Hematoxylin and eosin staining of each organoid. In the representative images of mesothelioma, ghost cells were observed in the tumor treated with DZNep. and ghost cells without nuclei (Figure 4, Panel E). This response was consistent with mesothelioma or HC It was not seen in the T116 controls and was absent in any of the HCT116 treatments (Figure 4 , Panel F). Only mesothelioma responded, demonstrating that DZNep enabled measurable proliferation in patient samples. Furthermore, the ability of the IL-16 receptor agonist to induce IL-16 expression was significantly reduced, and HCT116 served as an appropriate non-responsive positive control. As shown in et al.

[0090] Consideration Precision oncology, which sequences tumor DNA to identify actionable genetic mutations, is It is poised to become standard clinical practice in cancer treatment decision-making. Any correlation between previously reported gene mutations and potential drugs could not be confirmed before treatment was started. Furthermore, only 11% of patients who trial precision medicines adhere to precision medicine-guided treatments. In addition, there are many different biological behaviors of cancer based on histology, grade, and tumor volume. There is degeneration.

[0091] The study uses patient-derived xenografts (PDXs) to monitor patients' tumor progression and drug therapy. Such models have been used to test the therapeutic response of mice to biopsy specimens that are infiltrated with mouse-derived cells. There is no need for immunodeficient mice to receive tumor samples or to place tumor samples in. The cells are also They have adapted to new environments and have shown genetic drift since the initial samples, which also makes them ideal One further limitation in the widespread adoption of PDX technology is The reason is that only the most aggressive tumor biopsies were able to "engraft" when introduced into immunodeficient mice. The generally accepted success rate range is 25-33%. The poor engraftment rate of PDX has led to uncertainty about the applicability and predictive value of PDX technology for the majority of cancer patients. The studies described here and the early stages of ongoing parallel trials have limited application. The main criticism of our method is that we were unable to improve the rate of PDX engraftment. It is noteworthy that this was the first time that the inventors' platform As shown in Figure 2, the inventors were able to culture In addition, the inventors confirmed high cell viability on days 7 and 14 of culture. When performing similar studies with leiomyoma biosamples and other GI tumor biosamples, the inventors observed engraftment rates of greater than 90%. A 10% non-engraftment rate was observed in patients with low-grade mucinous tumor of the appendix. It is important to note that the results may reflect acellular samples obtained from individuals. This is based on the finding that all high-grade samples to date have been successfully grown into organoids. This makes sense, and using 3D, ECM microenvironment-supported organoid technology This will allow for greater adoption for a wider range of patients than is possible with PDX technology. This shows us that it may be possible to create a model that can However, this further evidence of engraftment efficiency requires significant further confirmatory testing.

[0092] In this series of experiments, beyond engraftment and survival assessments, the inventors aimed to: 1) assess the relationship between patients and tumors; 2) correlation with organoids and 3) biomarker testing of experimental drugs First, the inventors demonstrate the feasibility of several proof-of-concept scenarios. Using the ID platform, we have developed a common chemotherapy drug for clinical use in this type of cancer. Two of the regimens were screened. In particular, the inventors screened cisplatin and pembrolizumab. Organoid response to methotrexed versus the combination of carboplatin and pemetrexed The answer was tested by injecting the cells into the medium circulating through a microfluidic system (Figure 3). Cisplatin-pemetrexed treatment was more effective when patients were exposed to the drug for 7 days. The results were clear, showing that the ratio of live to dead cells was a function of the relative concentration. 10 μM cisplatin and pemetrexed compared with 10 μM carboplatin and pemetrexed The results showed that the platform was able to detect and treat This result is in itself a significant step in enabling successful implementation of screening tests. But more importantly, such Patients from whom organoids were derived also underwent chemotherapy at the end of their cisplatin-based chemotherapy. CT imaging demonstrated that the large amount of ascites had almost completely disappeared. In fact, this patient responded dramatically to cisplatin-based chemotherapy. If the tumor did not respond to cisplatin-based therapy, the patient was not a valid candidate. This is important, as such correlative results between patients and our organoid technology These results provide important data for the continued development of this method to validate it for clinical application. It is a set.

[0093] Second, the inventors confirmed the genetic test results using some of the patient's tumor biosamples. This report showed that two mutations specific to this tumor were identified. Rice site 1729+1G>A and PBRM1 N258fs×6. BAP1 is a deubiquitinase 28 On the other hand, PBRM1 is involved in several cancers. Tumor suppressor genes associated with 29 At the time of such testing, mesothelioma or any other None of the mutations were associated with FDA-approved therapy in any tumor type, and no appropriate ongoing clinical trials were performed. However, the inventors have also used animal models and in vitro studies. In a study, inhibition of EZH2 30 , and targeting BAP1 mutations. Thus, the inventors have confirmed that such a gene expression pattern, as shown in the genetic analysis, A study to determine whether biomarkers could indeed be used as actionable targets As a scenario, we conducted a drug screening experiment using the EZH2 inhibitor DZNep. (Figure 4). In fact, in the treatment with this compound, the inventors generally They found a decline in mitochondrial metabolism, a metric that is proportional to cell number. They found a tendency for increased expression of annexin V, indicative of apoptosis, whereas proliferating cells Furthermore, H&E staining revealed that the cell morphology and damage It has been shown that the cell nuclei without nuclei are damaged by the compound in a dose-dependent manner. Organoids generated by the same method from the HCT116 colorectal cancer cell line, which has been Instead, DZNep had little effect during It appeared that there was no

[0094] We have developed a parallelized microfluidic platform with a circulatory system to study the development of such 3D tumors. By introducing a tumor model, the inventors were able to evaluate multiple drug types and In addition to having the ability to assess disease progression and dose, the inventors also aim to detect tumors that occur prior to metastasis to distant sites. This provides advantageous conditions for visualizing and tracking the dynamics of tumor progression, migration, and intravasation into the circulatory system. Also obtain the item.

[0095] [Example 3] With engraftment rates of over 90% in vitro (vs. less than 10% in 2D cell culture), Multiple sets of patient tumor-derived organoids have been generated, representing the most aggressive tumors. Previously reported engraftment rates in patient-derived xenograft (PDX) models where only somatic samples were grown2 This compares favorably with 5-33%. This poor engraftment rate leaves many cancer patients with poor prognosis. The application of PDX technology to diagnostics has been limited. They will continue to be used for drug screening to determine whether the tumor organoids are different, just like the patients. To date, the results have demonstrated that the selective response to anticancer drugs is maintained. and low-grade appendix metastatic sites to the omentum, ovaries and liver, peritoneal mesothelioma, and extremity A diverse set of primary cells, including sarcomas, and patient-specific organoids from their metastatic sites 1) Tumor organoid drug response and drug response in patients from which organoids are derived Correlation with response; 2) Design based on identification of druggable mutations in patient tumors and 3) the ability to extract viable cells from difficult-to-culture tumor populations. Several examples are described herein that demonstrate the construction of viable organoids.

[0096] The inventors have isolated viable tumor oocytes from rare and difficult-to-isolate and culture cancer types. We have demonstrated the ability to generate ganoid models. For example, we have demonstrated the ability to generate ganoid models of low-grade appendiceal (LGA) ) tumors (Figure 5, Panel A), and well-differentiated papillary mesotheliomas, traditionally referred to as "benign" mesotheliomas. We generated viable patient-specific tumor organoids from mesothelioma (Figure 5, Panel B). Both have a slow growth pattern and unpredictable transformation potential. It was essentially impossible to establish cell cultures from cells with low proliferation indices. In particular, compared to high-grade organoids and 2D cultures, which respond better to treatment, the invention Our LGA organoids did not respond to chemotherapeutic agents (Figure 5, Panel C). The data indicate that LGA is indeed a chemotherapy-resistant tumor and therefore This is the first useful and confirmed study to show that treatment of LGA patients is not beneficial and results in unnecessary toxicity. Furthermore, the inventors have recently treated a sarcoma in the extremities and We have generated a set of patient-specific sarcoma organoids that also demonstrate high survival rates in the current platform ( (Figure 5, Panel D). Such sarcoma organoids have been used in current drug screening studies. Currently employed.

[0097] [Example 4] The restriction of normal cell types to malignant tumor cells is due to altered cell-cell interactions and physical (Lodish H, Berk A, Zipursky SL, Anticancer drugs travel to tumors via the circulatory system and penetrate extravascular tissues. The drug must be able to successfully target cancer cells inside the organ (T In the circular organoid model, different cell types They create simple 3D micro-organs, or organoids, that contain the invasive cancer types they encase, and then By targeting internal invasive cell lines with specific anti-cancer drugs, In addition, the experiments described herein attempt to recapitulate the in vivo process that This allows the external and internal cells to be placed in a 3D microenvironment similar to that found in vivo. When both types are present, the anticancer drug may target the internal, more invasive cell type as opposed to the external cell type. We then determined whether the 3D drug screening assays could successfully target the cell lines of (Efficacy was determined by screening.)

[0098] To investigate the effect of the extracellular microenvironment on the efficacy of anticancer drugs, anti-cancer drugs (regorafenib, sorafenib, trametinib, 5-fluorouracil, and dapagliflozin) Brafenib) in four colorectal cancer cell lines (HCT116, HT29, SW480, and and CACO2), which were then embedded in a hyaluronic acid-based hydrogel. Or directly seeded into the wells of a well plate. Embedding in hyaluronic acid-based hydrogel The colorectal cancer cells were cultured to mimic the 3D microenvironment of cancer cells in vitro. The cells seeded directly into the tube were cancer cells grown in a conventional 2D microenvironment.

[0099] 2D tumor construct drug screening A 2D drug screen was performed using colorectal cancer cell lines present in monolayer cultures. Conventional drugs lack the cell-ECM interactions typically seen in malignant tumors in vivo. The effectiveness of anticancer drugs was determined in screening practice.

[0100] Cancer cells were seeded directly into the wells and were given 4 to 5 hours to re-attach to the bottom of the wells. Then, anticancer drug solution was administered instead of the medium (Figure 6, Panel A), and MTS uptake was performed 2 days later. The MTS absorbance results of the anticancer drugs were compared to those of the control without the drug. The MTS values ​​are compared with those of the control (Figure 6, Panel B). At 100 mM concentration of 5-FU, cell viability was significantly improved by nivolumab and sorafenib. All four colorectal cancer cell lines tested had less than 20% of the viability of controls However, screening with dabrafenib and trametinib did not reveal high concentrations of other The three anticancer drugs produced different results. Dabrafenib at a concentration of 100 μM was Relative cell numbers ranged from approximately 36% for CT116 cells to approximately 93% for SW480 cells. The results showed that trametinib at a concentration of 100 nM yielded a survival value of approximately 39% for CACO2 cells. This resulted in relative cell viability values ​​ranging from 0.1% to approximately 58% for SW480 cells. When treated with 5-FU at 100 mg / mL, the relative cell viability was approximately 39% for SW480 cells. and less than 10%–22% for HCT116, HT29, and CACO2 cells. These values ​​were significantly lower than the relative cell viability values ​​of the lowest concentrations of other anticancer drugs. It was quite low.

[0101] 3D tumor construct drug screening We performed 3D drug screening on colorectal cancer cell lines and further investigated the effect of hyaluronic acid vectors. The cells were embedded in a hydrogel construct of hyaluronic acid to investigate cell-ECM interactions in the presence of hyaluronic acid. , and observed how it affected the effectiveness of anticancer drugs.

[0102] Polydimethylsiloxane, or PDMS, was used to coat the bottom of the wells. The cells were embedded in a uronic acid-based hydrogel and allowed to acclimate to the 3D environment for 5 days. On the fifth day, different anticancer drug solutions were administered (Figure 7, Panel A). was performed and the results are shown relative to the control values ​​(Figure 7, Panel B). For regorafenib and sorafenib, cell viability was significantly improved in the colorectal cancer cell lines used. All four of the 10 0 μM regorafenib was administered to CACO2 cells. Percent relative cell viability The survival rate increased to approximately 48%. When screened with 100 mM 5-FU, From 48% when used in SW480 cells to 81% when administered to HCT116 cells However, only the 10 mM concentration of 5-FU was able to inhibit the cell viability of the four cells. For all cell lines, the percentage of cells ranged between 9% in SW480 cells and 50% in HT29 cells. At a concentration of 100 μM, dabrafenib inhibited HCT116 and S Showed high efficacy in targeting the W480 cell line, with relative cell viability of 38% and 43% This is in contrast to the HT29 and CACO2 cell lines, which showed 93% and At a concentration of 100 μM, trametinib showed a higher cell viability value of 4. For the three colorectal cancer cell lines, results for relative cell viability fell within a narrow range. The values ​​were 54% in SW480 cells and 74% in HT29 cells.

[0103] Comparing 2D and 3D Drug Screening Perform significance statistics for 2D and 3D drug screening to determine whether In one case, they looked to see if colorectal cancer cells responded differently to anticancer drugs. The significant difference in cellular response to drugs in such drug screens was due to the difference in the cell and hyaluronan. This suggests that the cause is cell-ECM interactions between the The response of HCT116 and CACO2 to nib and 5-fluorouracil was Due to the morphological differences and similarities between cell types such as , using such cells and drugs.

[0104] Even when the same concentration of anticancer drug solution was administered to the same cell line, the results showed no significant difference in 2D vs. 3D in vitro o It is clear that cells responded differently to drug treatment when placed in a microenvironment. HCT 116 and CACO2 with regorafenib, sorafenib, and 5-fluorouracil Between the results of 2D and 3D drug screening using A significant difference in cell viability was observed between the two groups (Figure 8). was significantly different between 2D and 3D screening with regorafenib. different (both <0.05), and one significantly different for sorafenib (<0.10); Two were significantly different for 5-FU (both <0.01). In CACO2 cells, both The results showed that regorafenib was significantly different between 2D and 3D screening. significantly different (both <0.01), and three significantly different for sorafenib (two < 0.01 and one <0.05), three were significantly different for 5-FU (two < 0.01 and one <0.10).

[0105] In HCT116 cells, a 10 μM solution of sorafenib and a 10 mM solution of 5-FU were Except, when performing 3D drug screening, the relative cell viability values ​​were significantly higher than those of 2D drug screening. In CACO2 cells, the agonist activity of regorafenib and sorafenib at 10 μM was higher than that of serogroup A. Except for the 10 mM solution of 5-FU and the 10 mM solution of 5-FU, 3D drug screening showed a relative The cell viability values ​​were also higher than in the 2D drug screen. Comparing the results of cleaning (Figure 8), regorafenib inhibited H The effect of sorafenin on the cell viability of CT116 cells was more significant. The effect of α-amycin on cell viability was more significant in CACO2 cells than in HCT116 cells. (HCT116 was already significantly affected in both screens.) ), and 5-FU significantly reduced cell viability in both HCT116 and CACO2 cells. During the 3D drug screening, the 100 μM concentration of the Gorafenib and sorafenib significantly increased the expression of HCT116 cells compared to CACO2 cells by 4 Lower percent relative cell viability values ​​of 21% versus 8% and 15% versus 25% 100 mM 5-FU was used for drug screening of 10 mM 5-FU. The results showed that HCs in the 3D microenvironment yielded viability values ​​much higher than those during incubation. Results for 5-FU at a concentration of 10 mM for both T116 and CACO2 cells When noted, these relative cell viabilities decreased to similar values ​​of 18% and 22%. It is clear that it was dropped.

[0106] Circular organoid drug screening The circular organoid study demonstrated that different cells grow in a 3D hyaluronic acid-based hydrogel microenvironment. Cell-ECM interactions or cell-cell interactions between cell types may be key in targeting specific cancer cells. In 3D drug screening, we will investigate whether this affects the efficacy and specificity of anti-cancer drugs. and tested as previously observed.

[0107] Polydimethylsiloxane, or PDMS, was again used to coat the bottom of the wells. HCT116 cells were embedded in hyaluronic acid hydrogels and constructs were fabricated on PDMS surfaces. CACO2 cells similarly embedded in hyaluronic acid gel were used to investigate the function of the HCT116 inner ring structure. An outer ring was created surrounding the construct (Figure 9, Panel A). Regorafenib was administered at a concentration of 100 μM. When administered to HCT116 cells, the expression of IL-1 was significantly lower in the 3D microenvironment than in CACO2 cells. The results showed that 5-FU significantly improved the survival rate of 3D cells compared with control mice (21% vs. 48%). showed similar effects on the reduction of relative cell viability in The circular constructs were administered at 2 μM, 10 μM, and 100 μM concentrations for comparison. The efficacy of such drugs was observed and quantified in two cell lines.

[0108] On day 7, LIVE / DEAD staining was performed on the circular constructs and images were taken on an Axiovert microscope. The circular constructs were imaged using the imaging software MATLAB (Figure 9, Panel B). % Dead:% Alive (or % Red (medium gray in black and white images) vs. % Green (bright in black and white images) The ratio of regorafenib (in gray) to 50 (in gray) was derived. The values ​​were then graphed to show the relative abundance of regorafenib and 50 (in gray). The ratios of the 100%-FU group to the 100%-FU group, as well as the control ratios, which are the ratios to the untreated group, are shown in Fig. 9, Panel C). At 100 μM, both regorafenib and 5-FU showed 0.98% The mortality rate was 100%. This means that approximately half of the HCT116 and CACO2 cells survived. This means that half of the cells were targeted by the anticancer drug. When analyzing the relative cell viability when treated with regorafenib and 5-FU at concentrations of % Kill:% Viability was significantly higher for the 5-FU solution than for the regorafenib solution. The % killing:% survival at 1 μM is 0.48 in contrast to regorafenib. In contrast, the % killing:% survival ratio at 10 μM was 0.95 for 5-FU and 0.5 for 10 μM for 5-FU. The mean mean IL-16 concentration was 0.78 for 5-FU, in contrast to 0.08 for 5-FU. % death with regorafenib and 5-FU: % survival rate was only 0.03 % death with control The survival rate was substantially higher than the % survival rate values.

[0109] Looking at the live-dead image in panel B, the red (middle gray in the black and white image) cells Most of the HC in the inner ring (right side of the image) appears in the inner ring (left side of the image) rather than the outer ring (left side of the image). T116 cells appear to be more sensitive to regorafenib than CACO2 cells. In contrast, red (middle gray in black and white images) stained with ethidium homodimer 1 dye. Because dead cells appear in both the inner (HCT116) and outer (CACO2) rings, -FU appears to target both cell types to the same extent. A ten-fold increase in the intensity results in an observable increase in target cell death by the drug.

[0110] A microfluidic device to quantify metastasis A PDMS-based microfluidic device (Figure 10) was developed to culture cancer cells in 3D hyaluronan. When anticancer drug solutions were administered to the cancer cells, which were embedded in the phosphoric acid-based construct and flowed through a simplified circulatory system, The researchers then determined how the ratio of cell transfer to cytoplasmic DNA changed when the cells were transferred to the microfluidic device. The anticancer drug was administered to the "primary" tumor construct and the "secondary" tumor construct captured on the transwell membrane. in cancer cells, we aimed to determine how it affected cancer cell viability.

[0111] After 7 days of incubation, the debate in capturing cells that had entered the medium circulation on the transwell membrane was Two microfluidic devices were set up with the intention of observing the feasibility of the device. 3D constructs embedded with HCT116 cells were placed in the "construction chambers" of both devices. Untreated medium was added to the fluid reservoir of one device (control), and A 10 μM solution of sorafenib was added to the fluid reservoir of the Zei Using a ss Axiovert 200M 487-1 microscope, constructs of control devices were Images of the control device and transwell membrane were taken between days 1 and 7. Increase in cells captured on transwell membrane as shown by the green membrane dye DiO After disassembly of the device, the transwell membrane was examined under a confocal microscope on the 7th day. The device also allowed the capture of cells that had migrated out of the 3D construct and entered the circulation. The flow rate of the device was recorded as 0.20 mL / min.

[0112] The device circulating 10 μM sorafenib drug medium solution suppressed the internal fungal infection on the third day. After this observation, the device showed signs of contamination and was dismantled and discarded. Any data that could have been collected was lost. The fungal contamination was identified as the source of the fungal contamination.

[0113] Consideration Such studies may suggest that the 3D extracellular microenvironment plays an important role in determining the efficacy of anti-cancer drugs. Four colorectal cancer cell lines were identified that expressed hyaluronan-based hydrogels. After embedding and encapsulating the cells in PBS and administering five anticancer drugs, significant differences in cell viability were observed. The circular organoids were able to target different cancer cells when embedded in a 3D hydrogel environment. The circular organoids provide a method for measuring cell migration, allowing for qualitative comparison of drugs that affect cell function. The developed microfluidic device was shown to be capable of trapping metastatic cancer cells. The study demonstrated the ability to perform continuous microscopic observation of 3D constructs and captured cells, and to perform personalized chemistry. We propose its use as a model and platform for therapeutic drug screening. .

[0114] material and method Origin of cancer cell lines The HCT-116, HT-29, SW-480, and CACO-2 colorectal Cancer cell lines were obtained from Wake Forest University School of Medicine. ity School of Medicine) Cell and Viral V The 15 cm cell culture plate was sourced from the Cell Core Laboratory. The cells were cultured in DMEM high glucose solution (HyClone, Utah, USA), 1 0% fetal bovine serum, 1% penicillin / streptomycin, and 1% L-glutamate Standard DMEM-10 (Dulbecco's Modified Eagle Medium) containing methylaminobutyric acid was used as the cell growth medium. Used.

[0115] 2D Drug Screening Sterilization and cell passaging: Aspirate the medium from the cell culture plate and add 10 mL of PBS (25 °C). ) was pipetted into each plate. The cell culture plate was manipulated to separate the cells in the plate. The entire cells were covered with PBS, which was then aspirated off the plate. 5 mL of psulin (37 °C) was pipetted into each cell culture plate. The culture plate was placed in a 37°C incubator for approximately 8 minutes. After this, 5 mL of DMEM- 10 mL of medium was added to each plate. Then, 10 mL of cells and medium components were added to four clean plates. The mixture was placed in a 15 mL centrifuge tube.

[0116] Counting and loading of cells on the hemocytometer: Use a 1000 μL single channel pipette. Resuspend the cells in a 15 mL centrifuge tube with water and transfer the cells from each cell line tube to a new 15 mL centrifuge tube. Then, 400 μL of DMEM-10 medium and 500 μL of Gibco Trypan Blue stain (0.4%) (Thermo-Fischer Science Pipet 100 µL of ... The second tube was diluted 1:10 to facilitate cell counting and diluted with trypan blue. Dead cells were excluded from the cell count by adding Lou's stain. The diluted sample was dropped into a 2-20 µL single-channel pipette and placed in a pre-sterilized hemocytometer. The viable cells were counted and calculations were performed to determine the cellular abundance of each cancer cell line. The total number of cells was determined.

[0117] Cell seeding: The first 15 mL contains 10 mL of DMEM-10, cells, and trypsin. The tube was placed in the centrifuge. The centrifuge settings were 1500 RPM and maximum acceleration for 5 minutes. The medium and trypsin mixture was then aspirated from the tube to remove the cell pellet. The cells were left intact. 5 mL of DMEM-10 was then pipetted into each tube. Drop-wise addition of 5 mL of cell culture medium solution resuspended the cells in the process. To confirm the results, 25,000 cells of each of the four colorectal cancer cell lines were cultured on polystyrene plates. It was confirmed that the cells were directly seeded into each well of a clear-bottom 96-well plate. Then, 100 1 μL of DMEM-10 medium was pipetted directly into the wells. Incubate at 37°C and 5% CO2 for 5 hours, then aspirate the medium and add the anticancer drug solution. The cells were allowed to re-adhere to the bottom of the wells until

[0118] Addition of anticancer drugs for screening: The anti-colorectal The drugs are 5-FU, regorafenib, sorafenib, trametinib, and dabrafenib. Nibs (Sigma-Aldrich, Missouri, USA) were included. The drug stock solution was prepared using DMSO. The low concentration drug stock solution was prepared using DMSO. The solution was prepared by mixing with DMEM-10 medium. Drug solutions of 0 mM and 100 mM were prepared, containing regorafenib, sorafenib, and For dabrafenib, drug solutions of 1 μM, 10 μM, and 100 μM were prepared. For trametinib, drug solutions of 1 nM, 10 nM, and 100 nM were prepared. The medium was carefully aspirated from the wells and 200 μL of the various drug-medium solutions were added to the wells containing the cells. For each anticancer drug concentration, three duplicates each with a different cell type were placed in a well. A well plate was prepared, the average value was calculated, and statistics were performed on the obtained data. The plates were incubated at 37°C and 5% CO2 for 24 hours.

[0119] Cell viability assay: After 48 hours of exposure to the anticancer drug solution, cell viability was determined. CellTiter 96® AQueous One Solution C Membrane proliferation (MTS) assay (Promega, Wisconsin) The cell viability was examined using a ELISA kit (Nippon Immunosorbent Inc., USA). The anticancer drug-medium solution was aspirated from the wells. Then, 200 μL of MTS reagent was carefully pipetted into the wells. The incubation time for the cells was approximately 45 min. Cell viability assays were performed and the EnVision Multilabel Plates were The absorbance was measured using a spectrophotometer (PerkinElmer, California, USA). was quantified.

[0120] 3D Cancer Organoids for Drug Screening Well Coating and Cell Isolation with PDMS: Polydimethylsiloxane, i.e. PDMS (DOW Corning, North Carolina, USA) was mixed at 1: Coat the bottom of a 96-well plate using a hardener ratio of 10:1 and then plate The plates were placed in an oven at 80°C for at least 1 hour. For all four colorectal cancer cell types, , each containing 100,000 cells (10,000,000 / μL) 3D hyaluronic acid To place 10 μL of base hydrogel construct into a well of a 96-well plate, use the method described above. The cells were subcultured in the same manner as in (A). Depending on the cell number, fresh cell-medium solution was The desired number of cells was isolated in a new 15 mL centrifuge tube and subjected to centrifugation.

[0121] Hydrogel component of single cell type constructs: hyaluronic acid-based hydrogel (ESI BIO , California, USA) were used as Heprasil, Gelin-S, and Ext Using a three-component mixture of ralink PEGDA 2-ARM acrylate crosslinker The components were mixed in a volume ratio of 2:2:1. Prior to mixing, 0. 1% Irgacure photoinitiator (Sigma-Aldrich, Missouri, USA) SA) was added to each of the three components, and the three components were placed in a 37°C incubator for approximately 3 The mixture was left for 10 minutes to solubilize.

[0122] Formation of organoid constructs: Immediately after solubilization, aspirate the medium from the 15 mL centrifuge tube (centrifuge After centrifugation, the cells were isolated as a pellet. The hydrogel components were directly added to the centrifuge tube containing the cells. The cells were carefully resuspended in the mixture. 10 μL of the gel-cell solution was added to the PDM Continue this for all wells containing cells. This was repeated for each cell type. After this time point, Blue Wave 200 spots were The gel was exposed to UV light from a light source (Dymax, Connecticut, USA). The hydrogel can be allowed to solidify or the gel can be allowed to form spontaneously for about 30 minutes. DMEM-10 medium is then placed into the wells and the constructs are incubated for 5 days. The mixture was incubated for 1 h.

[0123] Anticancer drug screening and cell viability: The same five anticancer drugs were added on day 5 and then incubated for 7 days. A cell viability assay was performed on the eyes.

[0124] Circular organoids for comparing the effectiveness of anti-cancer drugs Hydrogel components of circular organoid constructs: Hyaluronan was used to generate circular organoids. Polyurethane-based hydrogels include Heprasil, Gelin-S, and PEGDA 2 -ARM acrylate crosslinker (Extralink), and alkyne-PEG-alkyne The four components of 2-ARM crosslinker (ESI BIO, California, USA) The components were mixed in a volume ratio of 4:4:1:1. Then, 0.1% Irgacure photoinitiator was added to each of the four components, and the three components were mixed. The mixture was solubilized in a 37°C incubator for about 30 minutes. O2 cells were isolated by the passaging method described above and coated the bottom of a 96-well plate using PDMS. I did.

[0125] Formation of circular organoid constructs: Place the cytoplasmic organoids in the 15 mL centrifuge tube containing the HCT116 cell pellet. The four components of the hydrogel were added and the cells were resuspended in this mixture. Place 1 μL of HCT116 in the center of the well and cover the 96-well plate with aluminum foil for approximately 3 The gel was left for 5 minutes. During this time, the PEGDA 2-ARM acrylate crosslinked, whereas the UV-sensitive 2-ARM alkyne-PEG-alkyne remained uncrosslinked. do.

[0126] While waiting for the PEGDA to crosslink, add the 15 mL centrifuge tube containing the isolated CACO2 cells Add the four hydrogel components and resuspend the cells in the gel. Cover the 96-well plate Remove the aluminum foil and inspect the constructs to see if they have solidified yet. In this case, 25 μL of CACO2-embedded hydrogel was taken and placed around the HCT116 construct. Carefully place the construct on the plate. Using the tip of the pipette, physically remove the CACO2 gel from around the construct on all four sides. Using UV light from a Blue Wave 200 spotlight, both The gels are cross-linked together to form a fused circular construct. DMEM-10 medium is added to the gel. Fill the well.

[0127] Anticancer drug screening: On day 5, 1 μM, 10 μM, and 100 μM 5-FU Drug solutions were added to the circular constructs at 1 μM, 10 μM, and 100 μM of regorafenib Drug solutions were added to separate circular constructs. Three replicates were made for each drug concentration, and A no-subject control was also maintained.

[0128] Live-Dead staining and microscopy: On day 7, the circular constructs were stained with Live-Dead staining. d staining and observed under an Axiovert 200M 487-1 microscope (Carl Zeiss The constructs were imaged on a 350 nm microscope (Microscopy, Sigma-Aldrich AG, Oberkochen, Germany). Calcein A M and ethidium homodimer 1 staining (Thermo-Fischer Scientific ific, Massachusetts, USA) at an equivalent concentration of 1.0 μl / mL The circular constructs were exposed to culture medium. The constructs were then cultured in PB before and after exposure to Live-Dead staining solution. The mixture was washed twice with S. MATLAB (Math Works, Massachusetts) The percent red to percent green ratio was calculated using Regola (USA) software. Drugs for HCT116-CACO2 cyclic constructs in the presence of phenytoin and 5-FU The efficacy of was determined. The % red to % green ratio represents the % dead:% live cell ratio.

[0129] Microfluidic Devices Build: Graphtec Studio software to develop novel microfluidic A device model was created. The device model is a Graphtec ce6000-60 Pro. Aluminum foil was attached to one side of the plate using a thermal annealer (Graphtec, Tokyo, Japan). The mold was then etched onto a double-sided tape with a tungsten ion beam attached to it, and the mold was then physically attached to a cell culture plate. 20 mL of PDMS (1:10 curing agent ratio) was placed in the plate, and then the PDMS The PDMS mold was placed in an oven at 80 °C overnight to solidify. The next day, the PDMS mold was immediately transferred to a plate. was disconnected from

[0130] 8.0μm Isopore Transwell Membrane Filter s (Merck Millipore, Massachusetts, USA) was cleaved. Specifically adapted to the size of the device compartment with a transwell membrane for cell capture Plasma Cleaner PDC-32G (Harrick Plasma a Inc., New York, USA) to plasma bond the PDMS layer. A transwell membrane was placed between two PDMS layers by using a lab filter. The PDMS layer of the device was disinfected using a 70% ethanol solution in the oven.

[0131] Execution: HCT116 cells were passaged and stained with DiO membrane dye (Thermo-Fischer S Scientific, Massachusetts, USA) at 5 μL / mL The cells were treated with DMEM-10 medium for about 20 minutes. 10 μL of hyaluronic acid-based hydrogel construct containing cells (10,000,000 / μL) The PDMS layers were pressed together and secured in place with plastic clamps. was used on either side of the device and the PDMS layer was secured in place with four screws. Three milliliters of the medium was added to the fluid reservoir. The constructs and transwell membranes were imaged using a microscope, and on day 7, The transwell membrane was examined using a confocal microscope (Leica Camera, Wetzlar The images were taken at the National Atomic Energy Agency (NAEA) in Germany.

[0132] [Example 5] Screening of five drugs incorporated into organoids Experimental objective: To evaluate the dependence of compound organoid toxicity on liver organoid metabolic function. As an example, hepatic metabolic activity can severely affect the outcome and efficacy of some drugs. For example, 5FU, a common first-line cancer treatment, can affect not only tumors but also It is not a cytotoxic agent, but is cytotoxic to many cells in the body. Therefore, 5FU is usually used as a prodrug. It is administered in the form of a late-stage drug, capecitabine, which is essentially inactive until it passes through the liver. It is metabolized to its active form.

[0133] Experimental Design and Results: Five organoids were cultured, including liver, heart, lung, testis, and brain organoids. The creation of one tissue platform was initiated and maintained for 7 days before the start of drug testing. At this point, the liver module was removed from one half of the platform. Capecitabine (20 μM ) was administered to all platforms, followed by 7 days after exposure (14 of all studies). On the day after the experiment, the survival rate was evaluated by live / dead staining or heart rate behavior.

[0134] The results showed that in the presence of the liver, capecitabine is metabolized to the toxic drug 5-FU, which is then transported to the heart. They demonstrated that ethanolamine produces toxicity in the liver and lungs (Figure 11). Without the liver, this metabolism cannot occur. Surprisingly, brain organoids were not impaired in viability. , both groups were damaged (Figure 11). Metabolizing 5-FU into the pharmacologically active form of 5-FU, which increases cell death in cardiac and pulmonary organoids We show that liver organoids are required to induce gliomas-induced gliomas-induced hyperplasia.

[0135] On days 1, 3, 5, 7, 9, 11, 13, and 15, urea, albumin, and α-GST were Soluble biomarkers including IL-8, IL-1β, and IL-1β were quantified from aliquots of culture medium. An aliquot of the medium was sent for mass spectrometry to determine the binding of capecitabine to 5-FU. Metabolism was examined.

[0136] [Example 6] Organoids incorporated in a miniaturized microfluidic platform Screening for the top 5 drugs We created a microfluidic platform with a reduced footprint and fluid volume. We evaluated whether it would be possible to perform multiple drug tests on the same platform. By using a circulating marker, the signal-to-noise ratio of the relevant biomarkers can be increased. do.

[0137] Experimental Design and Results: Adhesive Film-Based Microfluidic Platform The system was constructed as follows. Similar to the full-scale platform described in Example 4, We have begun generating five tissue constructs, including liver, heart, lung, testis, and brain organoids. The liver modules were then maintained for 7 days prior to the start of drug testing. At this point, the liver modules were Capecitabine (20uM) was administered to all platforms, Then, 7 days after exposure (14th day of the entire study), live / dead staining or cardiac Survival was assessed by beat behavior.

[0138] In a miniaturized system, the presence of the liver inhibited capecitabine from inhibiting the toxic drug 5-FU. Without the liver, this metabolism would not occur. Importantly, this platform does not compromise cardiac and pulmonary organoid viability. In this form, the survival rate of brain organoids was high (Figure 12). , metabolizing capecitabine into the pharmacologically active form of 5-FU in cardiac and pulmonary organoids. We show that liver organoids are required to induce increased cell death in the brain. Luganoids are viable, and without wishing to be bound by any particular theory, Perhaps scaling down the system volume would allow for better medium preparation and better differentiation of brain organelles. This suggests that this system, along with other organoid types, supports cell viability. We demonstrate that it is possible to maintain cerebral organoids in vitro.

[0139] On days 7 and 15, urea, albumin, α-GST, IL-8, and IL-1 Soluble biomarkers, including β, were quantified from an aliquot of the medium (Figure 13). The cot was sent for mass spectrometry to verify the metabolism of capecitabine to 5-FU.

[0140] Miniaturized System Design This system has a smaller amount of media exposure compared to other systems, such as standard size systems. Reduce or eliminate the surface area of ​​PDMS that is confined to drug compounds, toxins, and soluble proteins. The opportunity for proteins and secreted compounds to be adsorbed or absorbed onto or into the device walls. Device fabrication strategies include tape microfluidics, laser cutting PMMA, and several PDMS mouldings, which allow the device to come into contact with the medium. This significantly minimizes the amount of PDMS surface area that is required. Figure 14 provides an overview of this fabrication method.

[0141] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. As defined by the following claims, together with equivalents thereto, The publications, patent applications, patents, patent disclosures, and references herein are incorporated by reference in their entirety. All sequences and / or SNP accession numbers and other references are included in the reference statement. The following sections are incorporated by reference into this specification for their teachings regarding chapters and / or paragraphs: It shall be so.

Claims

1. A hydrogel, the hydrogel being a crosslinked hydrogel comprising thiolated hyaluronic acid; Viable tumor cells, Live immune cells Including, the live tumor cells and the live immune cells are from a single subject; the live tumor cells and the live immune cells are from the same subject; An in vitro cell construct useful as a tumor model, wherein the living tumor cells and the living immune cells are mixed in the hydrogel.

2. The construct of claim 1 , wherein the live tumor cells and / or the live immune cells are derived from a tumor biopsy from the single subject.

3. The construct of claim 1 , wherein the live tumor cells comprise colorectal cancer cells, mesothelioma cancer cells, appendix cancer cells, melanoma cancer cells, glioma cancer cells, lung cancer cells, ovarian cancer cells, breast cancer cells, prostate cancer cells, liver cancer cells, and / or sarcoma cancer cells.

4. The construct of claim 1 , wherein the live immune cell is selected from a dendritic cell, a lymphocyte, a leukocyte, a B cell, a T cell, and any combination thereof.

5. The construct of claim 1 , wherein the live immune cells are derived from a lymph node.

6. 2. The construct of claim 1, wherein the construct grown in culture for one week contains at least 75% viable cells based on the average number of cells in the construct cultured for one week.

7. The construct of claim 1, wherein the construct has a diameter of about 200 μm to about 350 μm.

8. The construct of claim 1 , wherein the hydrogel further comprises collagen.

9. 2. The construct of claim 1, wherein the live tumor cells and the live immune cells are present in the construct in a combined amount of about 20 million cells per mL of hydrogel.

10. The construct of claim 1 , wherein the live tumor cells and / or the live immune cells comprise a detectable compound.

11. The construct of claim 1 , wherein the construct comprises a total of about 1,500 to about 3,500 cells.

12. 2. The construct of claim 1, wherein the hydrogel further comprises thiolated gelatin and at least two polyethylene glycol (PEG) crosslinkers, at least one of the at least two PEG crosslinkers comprising an alkyne.

13. 1. A method for screening a compound of interest for antitumor activity in vitro, comprising: contacting said compound with the construct of claim 1 in vitro; and then determining the proliferation of said viable tumor cells, wherein a decrease in proliferation of said viable tumor cells indicates anti-tumor activity of said compound of interest; The method includes:

14. A device useful for in vitro screening of tumor cells for anti-tumor activity, or for evaluating in vitro proliferation and / or migration of tumor cells and / or immune cells, or for in vitro screening of compounds of interest for anti-metastatic and / or anti-tumor activity, comprising: a microfluidic device having a chamber and a channel in fluid communication with the chamber; A construct according to claim 1 in said chamber; a growth medium in the chamber and the channel; a pump operatively associated with the chamber and the channel, the pump configured to circulate the medium from the chamber, through the channel, and back to the chamber; The device that contains

15. 15. The device of claim 14, further comprising a microporous membrane in the channel positioned for the medium to flow therethrough.

16. 1. A method for screening cancer cells in vitro for anti-metastatic activity, comprising: Providing a device according to claim 14, said device comprising a primary chamber containing said construct and at least one secondary chamber containing at least one organoid comprising cancer cells; circulating the medium through the device; detecting the amount of cancer cells present in the at least one secondary chamber, wherein a greater number of cancer cells present in the at least one secondary chamber indicates anti-metastatic activity; The method includes:

17. 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: Providing a device according to claim 15; circulating the medium through the device; administering said compound to said construct; and then detecting cancer cells trapped in said microporous membrane, a lower number of trapped cancer cells indicating a higher anti-metastatic and / or anti-tumor activity of said compound of interest; The method includes:

18. 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: Providing a device according to claim 14, said device comprising a primary chamber containing said construct and at least one secondary chamber containing additional organoids comprising cells different from said tumor cells; circulating a medium through the device; administering said compound to said construct; determining a reduction in the number of cancer cells present in said construct and / or said at least one secondary chamber compared to the number of cancer cells present in said construct and / or said at least one secondary chamber when said compound of interest is not administered; The method includes:

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