Method and device for testing hepatotoxicity using microorganospheres - Patents.com

Abstract

Systems and methods according to the present invention generally relate to micro-organospheres (MOS), as well as methods and devices for forming and using MOS. More specifically, in some embodiments, systems and methods according to the present invention relate to methods and devices for forming and using MOS generated from hepatocytes. MOS generated from hepatocytes is suitable for testing the effects of various drugs on liver toxicity and drug-induced liver damage.

Description

[Technical field]

[0001] Systems and methods according to the present invention generally relate to micro-organospheres (MOS), as well as methods and devices for forming and using MOS. More specifically, in some embodiments, systems and methods according to the present invention relate to methods and devices for forming and using MOS generated from hepatocytes. MOS generated from hepatocytes is suitable for testing the effects of various drugs on liver toxicity and drug-induced liver damage. [Background technology]

[0002] Model cell and tissue systems are useful for biological and medical research. The most common method is to derive immortalized cell lines from tissues and culture them in two-dimensional (2D) conditions (e.g., Petri dishes or well plates). However, 2D cell lines, while very useful for basic research, do not correlate well with individual patient responses to therapy. In particular, three-dimensional cell culture models have proven particularly useful in developmental biology, disease pathology, regenerative medicine, drug toxicity and efficacy testing, and personalized medicine. For example, spheroids and organoids are three-dimensional cell aggregates that have been studied. However, both traditionally formed organoids and spheroids have limitations that reduce their usefulness in certain applications.

[0003] Multicellular tumor spheroids were first described in the early 70s and were obtained by culturing cancer cell lines under non-adherent conditions. Spheroids are typically formed from cancer cell lines as freely floating cell aggregates in ultra-low attachment plates. Spheroids have been shown to maintain more stem cell-associated properties than 2D cell cultures.

[0004] Organoids are in vitro derived cell aggregates that contain a population of stem cells that can differentiate into cells of major cell lineages. Organoids typically have a diameter of more than 1 mm and are cultured through passage. Organoid cultures typically grow and expand slower than 2D cell cultures. Because generating organoids from clinical samples requires starting with a sufficient number (e.g., hundreds to thousands) of viable cells, it is often difficult to derive organoids from small samples such as biopsies, and even if successful, it can take a significant amount of time to expand the culture for applications such as drug testing. Furthermore, there is a large variability in the size, shape, and cell number of organoids. Organoids may require a complex cocktail of growth factors and culture conditions to grow and express the desired cell types.

[0005] Neither tumor spheroids nor organoids are optimal for personalized medicine, such as performing rapid and reliable screening (especially ex vivo testing of drug response). For example, oncology practice is constantly faced with the great challenge of matching the right treatment regimen with the right patient, in addition to balancing the relative benefits and risks to achieve the most favorable outcome. Patient-derived cancer models (PDMCs) may involve the use of organoids (including patient-derived organoids) to facilitate the identification and development of more personalized therapeutic targets. However, although retrospective studies have shown that organoids derived from resected or biopsied patient tumors correlate with the patient's response to therapy, there are significant limitations to using organoids to guide therapy. As mentioned previously, it takes months to derive and expand organoids (especially patient-derived organoids) from tumor samples for drug sensitivity testing, reducing clinical applicability since patients cannot wait that long to receive treatment. In addition, the number of organoids required to perform drug screening with more than a few dozen compounds cannot currently be obtained in a clinically feasible time frame from a core biopsy specimen. Core biopsy specimens are often the only available form of tissue from patients with metastatic or inoperable cancer. Significant failure rates in deriving organoids from biopsies also preclude their use as a reliable diagnostic assay. Furthermore, there can be a high degree of variability in the size (and potential response, especially drug response) of organoids, especially over long culture times and therefore multiple passages.

[0006] Due to their better correlation with patient outcomes, PDMCs have also been utilized to replace 2D cell lines as high-throughput screening platforms for drug discovery, such as RNAi, CRISPR, and pharmacological small molecule screening. However, compared to cell lines, these PDMC models (including spheroids and organoids) are typically much slower to expand and manipulate, making them difficult and expensive for high-throughput applications. As the time required to expand these models and amplify cell numbers increases, the fastest growing clones in the plastic tend to overwhelm and outcompete other clones, thus making the models more homogenous and losing the original tissue composition and clonal diversity. Furthermore, their relatively large and heterogeneous size and limited diffusivity make them challenging for many automated fluorescence and imaging-based readout assays. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need for methods, compositions, and devices for generating patient-derived tissue models (e.g., tumor and / or non-tumor tissue models) from resections, biopsies, and other tissue sources. In particular, it would be useful to provide methods and devices that can enable a large number of patient-derived tissue models with predictable and clinically relevant characteristics from a single biopsy, such as an 18-gauge core biopsy (e.g., can be completed within 7-10 days after the biopsy is obtained). This would allow for robust and reliable testing, minimizing delays in guiding patient-specific therapies. Furthermore, it would also be useful to generate patient-derived models that can be rapidly expanded in a highly parallel manner, generating units with smaller and more uniform size, better controllability over the number of cells per unit, and better diffusivity (e.g., due to increased surface area to volume ratio) for high-throughput screening applications. Additionally, it would be useful to have better hepatocyte models for testing the effects of various agents on hepatotoxicity and drug-induced liver injury. [Means for solving the problem]

[0008] Summary of the Invention Described herein are micro-organospheres (MOS), devices and methods for making MOS, and devices and methods for using MOS. Also described herein are methods and systems for using these MOS to screen patients, including personalized treatment methods.

[0009] Generally, described herein are methods and devices for forming and growing a MOS comprising patient-derived cells extracted from resected patient tissue, including portions of a resected primary tumor or a functional or dysfunctional organ (e.g., for rapid diagnosis to guide therapy) and / or from already established PDMCs, including patient-derived xenografts (PDXs) and organoids (e.g., to generate a MOS for high throughput screening).

[0010] These MOSs can be formed from primary cells that are normal (e.g., normal organ tissue) or from tumor tissue. For example, in some embodiments, these methods and devices form MOSs from cancerous tumor biopsy tissue, allowing for customized treatments that can be selected using the specific tumor tissue examined. Surprisingly, these methods and devices allow for the formation of hundreds, thousands, or tens of thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000, or more) of MOSs from a single tissue biopsy within hours after the biopsy is removed from the patient. Primary cells isolated from a patient biopsy can be combined with a fluid matrix material, such as a substrate basement membrane matrix (e.g., MATRIGEL), to form a MOS. The resulting multiple MOSs can have a predetermined size range (e.g., diameter, e.g., 10 μm to 700 μm, and any subranges therewith), and an initial number of primary cells (e.g., 1 to 1000 cells, and particularly a smaller number of cells, such as 1 to 200 cells). The cell number and / or diameter can be controlled, for example, within + / - 5%, 10%, 15%, 20%, 25%, 30%, etc. These MOSs, when formed as described herein, have very high viability (>75%, >80%, >85%, >90%, >95%) and are stable for use and testing within a very short period of time, including within the first 1-10 days after formation (e.g., within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, etc.). This allows for rapid testing of potentially vast numbers of patient-specific, biologically relevant MOSs, saving significant time in the development and deployment of patient therapies, such as cancer treatment regimens. The MOSs described herein rapidly form three-dimensional (3D) cellular structures that recapitulate and correspond to the biopsied tissue environment, such as the 3D tumor microenvironment. The MOSs described herein may also be referred to as "droplets." Each MOS may include, for example, as part of the fluid matrix material, growth factors and structural proteins (e.g., collagen, laminin, nidogens, etc.) that may mimic the original tissue (e.g., tumor) environment. Each MOS may also include immune cells of the original tissue. Virtually any primary cell tissue may be used, including virtually any tumor or normal tissue.

[0011] For example, to date, MOS have been successfully generated in all tumor types and sites tested (e.g., current success rate 100%, n=32, including colon, esophageal, skin (melanoma), uterine, bone (sarcoma), kidney, ovarian, lung, and breast cancers from primary and metastatic sites including liver, omentum, diaphragm, etc.). Tissue types used to successfully generate MOS may be metastasized from other locations. In some embodiments, MOS described herein may be grown from fine needle aspirates (FNAs) or from circulating tumor cells (CTCs), e.g., from liquid biopsies. Proliferation and growth is typically seen in as little as 3-4 days, and MOS may be maintained and passaged for several months, or they may be cryopreserved and / or used for assays immediately (e.g., within the first 7-10 days).

[0012] In particular, methods of forming a patient-derived MOS are described herein. In some embodiments, these methods include combining dissociated primary tissue cells (including, but not limited to, cancer / abnormal tissue, normal tissue, etc.) with a liquid matrix material to form an unpolymerized material, and then polymerizing the unpolymerized material to form a MOS in which the dissociated primary tissue cells are typically distributed with a diameter of less than about 1000 μm (e.g., less than about 900 μm, less than about 800 μm, less than about 700 μm, less than about 600 μm, and particularly less than about 500 μm). The number of dissociated cells can be within a predetermined range, as described above (e.g., about 1 to about 500 cells, about 1 to 200 cells, about 1 to 150 cells, about 100 cells, about 1 to 75 cells, about 1 to 50 cells, about 35 to about 1 to 30 cells, about 1 to 20 cells, about 1 to 10 cells, about 5 to 15 cells, about 20 to 30 cells, about 30 to 50 cells, about 40 to 60 cells, about 50 to 70 cells, about 60 to 80 cells, about 70 to 90 cells, about 80 to 100 cells, about 90 to 110 cells, etc.). Any of these methods, as described herein, can be adapted to generate MOS of reproducible size (e.g., having a narrow size distribution), as well as MOS that include immune cells.

[0013] Dissociated cells may be freshly biopsied or excised, or may be dissociated in any suitable manner, including mechanical and / or chemical dissociation (e.g., enzymatic digestion by using one or more enzymes such as collagenase, trypsin, etc.). Dissociated cells may be optionally treated, selected, and / or modified. For example, cells may be sorted or selected to identify and / or isolate cells having one or more characteristics (e.g., size, morphology, etc.). Cells may be marked (e.g., with one or more markers) that may be used to aid in selection. In some embodiments, cells may be sorted by known cell sorting techniques, including, but not limited to, microfluidic cell sorting, fluorescence-activated cell sorting, magnetic-activated cell sorting, etc. Alternatively, cells may be used without sorting.

[0014] In some embodiments, the dissociated cells may be modified by treatment with one or more drugs. For example, the cells may be genetically modified. In some embodiments, the cells may be modified using CRISPR-Cas9 or other gene editing techniques. In some embodiments, the cells may be transfected by any suitable method (e.g., electroporation, cell squeezing, nanoparticle injection, magnetofection, chemical transfection, viral transfection, etc.), including transfection with plasmids, RNA, siRNA, etc. Alternatively, the cells may be used without modification.

[0015] One or more additional materials may be combined with the dissociated cells and the fluid (e.g., liquid) matrix material to form an unpolymerized mixture. For example, the unpolymerized mixture may include additional cell or tissue types, including support cells. The additional cells or tissues may be derived from different biopsies (e.g., primary cells from different dissociated tissues) and / or cultured cells. The additional cells may be, for example, immune cells, stromal cells, endothelial cells, etc. The additional materials may include media (e.g., growth media, freezing media, etc.), growth factors, support network molecules (e.g., collagen, glycoproteins, extracellular matrix, etc.), etc. In some embodiments, the additional materials may include drug compositions. In some embodiments, the unpolymerized mixture includes only the dissociated tissue sample (e.g., primary cells) and the fluid matrix material.

[0016] The method can rapidly generate multiple MOSs from a single tissue biopsy, resulting in the generation of more than about 500 (e.g., more than about 600, more than about 700, more than about 800, more than about 900, more than about 1000, more than about 2000, more than about 2500, more than about 3000, more than about 4000, more than about 5000, more than about 6000, more than about 7000, more than about 8000, more than about 9000, more than about 10,000, more than about 11,000, more than about 12,000, etc.) patient-derived MOSs per biopsy. The biopsies can be standard size biopsies, such as 18G (e.g., 14G, 16G, 18G, etc.) core biopsies. For example, the volume of tissue removed by biopsy and used to form the multiple MOSs may be a small cylinder (taken with a biopsy needle) about 1 / 32 to 1 / 8 inch diameter and about 3 / 4 to 1 / 4 inch long, e.g., about 1 / 16 inch diameter by 1 / 2 inch long. The biopsy may be taken by needle biopsy, e.g., by core needle biopsy. In some embodiments, the biopsy may be performed by fine needle aspiration. Other biopsy types that may be used include shave biopsy, punch biopsy, incisional biopsy, excision biopsy, etc. Typically, material from a single patient biopsy may be used to generate multiple (e.g., greater than about 2000, greater than about 5000, greater than about 7500, greater than about 10,000, etc.) MOSs, as described above. The multiple patient MOSs may be formed using an apparatus (described herein) that may be configured to generate this large number of highly regular (size, cell count, etc.) MOSs as described herein. In some embodiments, the methods and apparatus are capable of generating multiple MOSs at high speeds (e.g., greater than about 1 MOS per minute, greater than about 1 MOS per 10 seconds, greater than about 1 MOS per 5 seconds, greater than about 1 MOS per 2 seconds, greater than about 1 MOS per second, greater than about 2 MOS per second, greater than about 3 MOS per second, greater than about 4 MOS per second, greater than about 5 MOS per second, greater than about 10 MOS per second, greater than 50 MOS per second, greater than 100 MOS per second, greater than 125 MOS per second, etc.).

[0017] For example, in some embodiments, the methods may be performed by combining the unpolymerized mixture with a material (e.g., a liquid material) that is immiscible with the unpolymerized material. The methods and devices may control the size and / or cell density of the MOS by at least partially controlling the flow of one or more of the unpolymerized mixture (and / or dissociated tissue and fluid matrix) and the material (e.g., hydrophobic material, oil, etc.) that is immiscible with the unpolymerized mixture. For example, in some embodiments, the methods may be performed using a microfluidic device. In some embodiments, multiple MOSs may be formed in parallel (e.g., two in parallel, three in parallel, four in parallel, etc.). Thus, the same device may include multiple parallel channels that may be coupled to the same source of unpolymerized material, or to the same source of dissociated primary tissue and / or fluid matrix.

[0018] The unpolymerized material can be polymerized to form a MOS in a variety of different ways. In some embodiments, the method can include polymerizing the MOS by changing the temperature (e.g., increasing the temperature above a threshold value, such as above about 20° C., above about 25° C., above about 30° C., above about 35° C., etc.).

[0019] Once polymerized, the MOS may be grown, for example, in culture, and / or assayed either before or after culture, and / or cryopreserved either before or after culture. The MOS may be cultured for any suitable length of time, but in particular for 1-10 days (e.g., 1-9 days, 1-8 days, 1-7 days, 1-6 days, 3-9 days, 3-8 days, 3-7 days, etc.). In some embodiments, the MOS may be cryopreserved or assayed prior to six passages, which may maintain cellular heterogeneity within the MOS and may prevent faster dividing cells from outcompeting slower dividing cells by limiting the number of passages.

[0020] Generally, because the same patient biopsy may provide a high number of cells (e.g., more than 2,000, more than 3,000, more than 4,000, more than 5,000, more than 6,000, more than 7,000, more than 8,000, more than 9,000, more than 10,000, etc.), a portion (e.g., half or more) of the MOS may be cryopreserved, while a portion may be cultured and / or assayed. As described in more detail herein, the cryopreserved MOS may be banked and used at a later time (e.g., assayed and passaged).

[0021] Thus, methods are described herein that include methods of forming a plurality of MOSs. For example, a method of forming a plurality of MOSs can include combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture, forming a plurality of droplets of the unpolymerized mixture, and polymerizing the droplets to form a plurality of MOSs, each having a diameter of 50-500 μm and having 1-200 dissociated cells distributed therein.

[0022] For example, a method of forming a plurality of MOSs may include combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets from a continuous stream of the unpolymerized mixture, the droplets having a size of less than 25% of the embodiments; and polymerizing the droplets by warming to form a plurality of MOSs, each MOS having 1-200 dissociated cells dispersed within each MOS.

[0023] In some embodiments, the methods described herein for forming a plurality of MOSs may include combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; converging a stream of the unpolymerized mixture with one or more streams of a fluid that is immiscible with the unpolymerized mixture to form a plurality of droplets having a size of less than 25% of the embodiment of the droplets; polymerizing the droplets to form a plurality of MOSs having a diameter of 50-500 μm and having 1-200 dissociated cells distributed therein; and separating the plurality of MOSs from the immiscible fluid.

[0024] Any of these methods may involve modifying the cells within the dissociated tissue sample prior to forming the droplets.

[0025] Forming the plurality of droplets may include forming a plurality of uniformly sized droplets of the unpolymerized mixture having a size of less than about 25% of the embodiment (e.g., a size of less than about 20% of the embodiment, a size of less than about 15% of the embodiment, a size of less than about 10% of the embodiment, a size of less than about 8% of the embodiment, a size of less than about 5% of the embodiment, etc.). The size embodiments may also be described as narrow distribution of size embodiments. For example, the size distribution may include a MOS size distribution (e.g., MOS diameter vs. number of MOS formed) having a low standard deviation (e.g., a standard deviation of 15% or less, a standard deviation of 12% or less, a standard deviation of 10% or less, a standard deviation of 8% or less, a standard deviation of 6% or less, a standard deviation of 5% or less, etc.).

[0026] Any of these methods may also include plating or dispensing the MOS. For example, in some embodiments, the methods may include combining MOS from various sources into a receptacle prior to assaying. For example, the MOS may be disposed in a multi-well plate. Thus, any of these methods may include dispensing the MOS into a multi-well plate prior to assaying the MOS. One or more (or in some embodiments, equal amounts) of MOS may be included per well.

[0027] In some embodiments, applying the MOS to the receptacle may include disposing the MOS into multiple chambers separated by at least partially permeable membranes to allow circulation of supernatant material between the chambers, which may allow the MOS to share the same supernatant.

[0028] In any of these methods, the MOS may be assayed. The assay may generally include exposing or treating individual MOS to a condition (e.g., a drug composition or combination of drug compositions, including but not limited to any of the drug compositions described herein) to determine whether (and possibly what effect) the condition has on the cells of the MOS. The assay may include exposing a subset of the MOS (individually or in groups) to one or more concentrations of a drug composition and allowing the MOS to remain exposed for a predetermined period of time (minutes, hours, days, etc.), and / or exposing and removing the drug composition and then culturing the MOS for a predetermined period of time. The MOS may then be examined to identify any effects, including toxicity, particularly to the cells in the MOS, or changes in the morphology and / or growth of the cells in the MOS. In some embodiments, the assaying may include marking live or fixed cells in the MOS (e.g., by immunohistochemistry). The cells may be assayed (e.g., examined) manually or automatically. For example, an automated reader device may be used to examine the cells to determine any toxicity (cell death). In some embodiments, assaying the plurality of MOS may include sampling one or more of the supernatant, the environment, and the microenvironment of the MOS for secreted factors and other effects. In any of these embodiments, the MOS may be recovered after the assay for further assay, expansion, or storage (e.g., cryopreservation, fixation, etc.) for subsequent testing.

[0029] As mentioned above, virtually any assay can be used. For example, genomic, transcriptomic, proteomic, or metagenomic markers (such as methylation) can be assayed using MOS as described herein. Thus, any of these compositions and methods described herein can be used to identify or examine one or more markers and biological / physiological pathways (including, for example, exosomes), which can help identify drugs and / or therapies for the treatment of patients.

[0030] Any suitable tissue sample may be used. In some embodiments, the tissue may be normal, non-cancerous tissue from any part of the body. In some embodiments, the tissue may be tissue from the liver. In some embodiments, the tissue sample may include a biopsy sample from a metastatic tumor. For example, the tissue sample may include a clinical tumor sample, which may include both cancer cells and stromal cells. In some embodiments, the tissue sample includes tumor cells and one or more of mesenchymal cells, endothelial cells, and immune cells.

[0031] Any of the methods described herein may include initially dissociating dissociated cells from a tissue biopsy at any suitable concentration, either uniformly or in some embodiments non-uniformly, throughout the fluid matrix material. For example, in some embodiments, the methods described herein may include combining a dissociated tissue sample and a fluid matrix material, such that the dissociated tissue cells are at least 1×10 in the fluid matrix material. 7 Less than 9 x 10 cells / ml (e.g. 6 <7×10 cells / ml 6 cells / ml, 5×10 6 cells / ml, 3×10 6 cells / ml, 1×10 6 cells / ml, 9×10 5 cells / ml, 7×10 5 cells / ml, 5×10 5 The method may include distributing the cells to a density of 100 μl / ml or less.

[0032] Generally, forming the droplets may include forming the droplets from a continuous stream of the unpolymerized mixture. For example, forming the droplets may include applying one or more converging streams of a fluid that is immiscible with the unpolymerized mixture to the stream of the unpolymerized mixture. The streams may be combined in a microfluidic device, for example, a device having multiple converging channels where the unpolymerized mixture and the immiscible fluid interact to form droplets with precisely controlled volumes. In some embodiments, the droplets may be formed (e.g., pinched off) in an excess of the immiscible material, and the droplets may polymerize simultaneously and / or subsequently to form a MOS. For example, the region where the streams converge may be configured to polymerize the unpolymerized mixture after the droplets are formed, for example, by heating, and / or a downstream region may be configured to polymerize the unpolymerized mixture after the droplets are formed and surrounded by the immiscible material. In some embodiments, the immiscible material is heated (or alternatively cooled) to a temperature that promotes polymerization of the unpolymerized material to form a MOS. For example, the polymerization may include heating the droplets to greater than 35° C.

[0033] Thus, in any of these methods, forming the droplets may include forming the droplets in a fluid that is immiscible with the unpolymerized mixture. Additionally, any of these methods may include separating the immiscible fluid from the MOS. Additionally, any of these methods may include removing the immiscible fluid from the MOS. In general, the immiscible fluid may include liquids (e.g., oils, polymers, etc.), including, among others, hydrophobic materials or other materials that are immiscible with the unpolymerized (e.g., aqueous) material.

[0034] The fluid matrix material may be a synthetic or non-synthetic unpolymerized basement membrane material. In some embodiments, the unpolymerized basement membrane material may comprise a polymeric hydrogel. In some embodiments, the fluid matrix material may comprise MATRIGEL. Thus, combining the dissociated tissue sample and the fluid matrix material may comprise combining the dissociated tissue sample with a basement membrane matrix.

[0035] The tissue sample may be combined with the fluid matrix material within 6 hours or sooner (e.g., within about 5 hours, within about 4 hours, within about 3 hours, within about 2 hours, within about 1 hour, etc.) after the tissue sample is removed from the patient.

[0036] Also described herein are methods of assaying or storing MOS. For example, the methods may include combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture, forming a plurality of droplets of the unpolymerized mixture, the droplets having a size of less than 25% of the embodiments, polymerizing the droplets to form a plurality of MOS having a diameter of 50-700 μm and having 1-1000 dissociated cells distributed therein, and assaying or cryopreserving the plurality of MOS.

[0037] In some embodiments, the method may include combining the dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets of the unpolymerized mixture; polymerizing the droplets to form a plurality of MOSs, each MOS having a diameter of 50-500 μm and having 1-200 dissociated cells distributed therein; and cryopreserving or assaying the plurality of MOSs within 15 days, and assaying the MOSs to determine the effect of one or more agents on cells within the MOS.

[0038] For example, the method may include combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; converging a stream of the unpolymerized mixture with one or more streams of a fluid that is immiscible with the unpolymerized mixture to form a plurality of droplets having a size of less than 25% of the embodiment of the droplets; polymerizing the droplets by warming to form MOSs each having a diameter of 50-500 μm and having 1-200 dissociated cells distributed therein; and assaying or cryopreserving the MOS prior to six passages, thereby maintaining cellular heterogeneity within the MOS, and further comprising assaying to determine the effect of one or more agents on the cells within the MOS.

[0039] In any of these methods, the plurality of MOS may be cryopreserved or assayed prior to the sixth passage, thereby maintaining cellular heterogeneity within the MOS. Any of these methods may further include modifying cells within the dissociated tissue sample prior to forming the droplets.

[0040] Forming the droplets can include forming a plurality of uniformly sized droplets of the unpolymerized mixture having a size of less than about 25% of the embodiments (e.g., less than about 20%, less than about 15%, less than about 10%, less than about 7%, less than about 5%, etc.).

[0041] Any of these methods may include culturing the MOS for an appropriate length of time, as described above (e.g., culturing the MOS for 2-14 days prior to assay). For example, these methods may include removing the immiscible fluid from the MOS prior to culturing. In some embodiments, culturing the MOS includes culturing the MOS in a suspension.

[0042] Generally, assaying the MOS can include genomic, transcriptional, epigenomic, and / or metabolic analysis of cells in the MOS before and / or after assaying or cryopreserving the MOS. Any of these methods can include assaying the MOS by exposing the MOS to a drug (e.g., a drug composition).

[0043] In any of these methods, the assaying may include manually and / or automatically visually assaying the effect of one or more agents on the cells in the MOS. Any of these methods may include marking or labeling the cells in the MOS for visualization. For example, the assaying may include fluorescently assaying the effect of one or more agents on the cells.

[0044] The MOS described herein is novel per se and may be characterized as a composition of matter. For example, the composition of matter may include a plurality of cryopreserved MOS, each MOS having a spherical shape with a diameter of 50 μm to 500 μm, comprising a polymerized substrate and about 1 to 1000 dissociated primary cells distributed within the substrate that have been passaged less than six times, thereby maintaining cellular heterogeneity within the MOS. In some embodiments, the MOS comprises liver cells. In some embodiments, the MOS comprises liver cells. In some embodiments, the MOS comprises primary human hepatocytes (PHHs). In some embodiments, the MOS is formed from PHHs of a single donor. In some embodiments, the MOS is formed from PHHs pooled from multiple donors. In some embodiments, the MOS is formed from adult hepatocytes. In some embodiments, the MOS is formed from adult PHHs. In some embodiments, MOS is prepared by mixing hepatocytes (e.g., PHHs) (e.g., of the type described herein) with a matrix material, thereby forming a mixture, and then crossing the flow of the mixture with a flow of an immiscible fluid, as described herein, thereby forming the MOS. In some embodiments, the MOS is then demulsified according to the methods described herein. Thus, hepatocytes (including PHHs) can be used to form MOS in the same manner as any tissue source described herein, such that after the tissue source has been mechanically and / or enzymatically digested, it is ready to be mixed with a fluid matrix material.

[0045] Also described herein is a composition of matter comprising a plurality of cryopreserved MOS, each MOS having a spherical shape with a diameter of 50 μm to 500 μm, the MOS having a size of less than 25% of the embodiments, each MOS comprising a polymerized substrate, and about 1 to 500 dissociated primary cells that have been passaged less than six times distributed within the substrate, thereby maintaining cellular heterogeneity within the MOS. In some embodiments, the MOS comprises hepatocytes from a tissue of origin.

[0046] The primary cells may be primary tumor cells. For example, dissociated primary cells may be genetically or biochemically modified. The plurality of cryopreserved MOSs may have a uniform size in embodiments with less than 25% variation in size. In some embodiments, the plurality of cryopreserved MOSs may include MOSs from various sources. Any of these MOSs may include a majority of cells in each MOS that are not stem cells. In some embodiments, the primary cells include metastatic tumor cells. The primary cells may include both cancer cells and stromal cells. In some embodiments, the primary cells include tumor cells and one or more of mesenchymal cells, endothelial cells, and immune cells.

[0047] Primary cells are cultured in polymeric substrates at, for example, 5×10 7 cells / ml, 1×10 7 cells / ml, 9×10 6 cells / ml, 7×10 6 10 cells / ml, 5×10 6 cells / ml, 1×10 6 cells / ml, 9×10 5 cells / ml, 7×10 5 cells / ml, 5×10 5 cells / ml, 1×10 5 It may be distributed at a density such as less than cells / ml.

[0048] Generally, the polymeric substrate may comprise a basement membrane matrix (e.g., MATRIGEL). In some embodiments, the polymeric substrate comprises a synthetic material.

[0049] The MOS may have a diameter of 50 μm to 1000 μm, or more preferably 50 μm to 700 μm, or more preferably 50 μm to 500 μm, or 50 μm to 400 μm, or 50 μm to 300 μm, or 50 μm to 250 μm, etc. (e.g., less than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 250 μm, less than about 200 μm, etc.).

[0050] As discussed above, the MOSs described herein may initially include any suitable number of primary tissue cells in each MOS (e.g., less than about 200 primary cells, or more preferably less than about 150 primary cells, or more preferably less than about 100 primary cells, or more preferably less than about 75 primary cells, or less than about 50 cells, or less than about 30 cells, or less than about 25 cells, or less than about 20 cells, or less than about 10 cells, or less than about 5 cells, etc.). In some embodiments, each MOS includes about 1-500 cells, about 1-400 cells, about 1-300 cells, about 1-200 cells, about 1-150 cells, about 1-100 cells, about 1-75 cells, about 30-150 cells, about 1-30 cells, about 1-25 cells, about 1-20 cells, etc.

[0051] Also described herein are apparatus for forming MOSs, and methods of operating these apparatus to form MOSs. For example, a method of operating a MOS forming apparatus is described herein, the method including receiving at a first port an unpolymerized mixture including a cooled mixture of a dissociated tissue sample and a first fluid matrix material, receiving at a second port a second fluid that is immiscible with the unpolymerized mixture, combining the stream of unpolymerized mixture with one or more streams of the second fluid to form droplets of the unpolymerized mixture having uniform sizes that vary by less than 25%, and polymerizing the droplets of the unpolymerized mixture to form a plurality of MOSs.

[0052] A method of operating a MOS formation apparatus includes receiving at a first port an unpolymerized mixture comprising a cooled mixture of a dissociated tissue sample and a first fluid matrix material; receiving at a second port a second fluid that is immiscible with the unpolymerized mixture; combining the flow of the unpolymerized mixture at a first rate with one or more flows of the second fluid at a second rate to form droplets of the unpolymerized mixture having uniform sizes that vary by less than 25%, wherein the droplets are between 50 μm and 500 μm in diameter; and polymerizing the droplets of the unpolymerized mixture to form a plurality of MOSs.

[0053] Any of these methods may include coupling a first reservoir containing the unpolymerized mixture in fluid communication with the first port. For example, the methods may include combining a dissociated tissue sample and a first fluid matrix material to form an unpolymerized mixture. In some embodiments, the methods may include adding the unpolymerized mixture to a first reservoir in fluid communication with the first port. These methods may include coupling a second reservoir containing a second fluid in fluid communication with a second port. Any of these methods may include adding the second fluid to a second reservoir in fluid communication with the second port. In some embodiments, receiving the second fluid includes receiving an oil.

[0054] Generally, these methods can include separating a second fluid (e.g., an immiscible fluid) from a plurality of MOS. This fluid can be separated manually or automatically. For example, the second (immiscible) fluid can be removed by washing, filtration, contact with a hydrophobic membrane, or any other suitable method.

[0055] Combining the flows can include driving a flow of the unpolymerized mixture at a first flow rate across one or more flows of a second fluid moving at a second flow rate. In some embodiments, the first flow rate is greater than the second flow rate. In some embodiments, the second flow rate is greater than the first flow rate. Either or both of the flow rate and / or the amount of material (e.g., the unpolymerized mixture) can be present in an amount less than the second fluid such that the unpolymerized mixture is encapsulated in precisely controlled droplets, as described herein, that can then be polymerized (e.g., within the second fluid).

[0056] In some embodiments, combining the streams includes driving a stream of the unpolymerized mixture across a junction where one or more streams of the second fluid also converge. Polymerizing the droplets can include heating the droplets to a temperature above a temperature at which the unpolymerized material polymerizes (e.g., greater than about 20° C., greater than about 25° C., greater than about 30° C., greater than about 35° C., etc.).

[0057] Any of these methods may include aliquoting multiple MOS, for example into a multi-well dish.

[0058] Also described herein are methods of treating patients with these MOSs and assaying them. For example, the method may include receiving a patient biopsy from a tumor, forming a plurality of MOSs from the patient biopsy within two weeks of taking the biopsy, the MOS having a diameter of 50-500 μm and having 1-200 dissociated tumor cells distributed through a polymeric matrix, exposing at least a portion of the MOS to a drug formulation before the dissociated tumor cells have undergone more than five passages, thereby determining that the tumor will respond to the drug formulation, and measuring the effect of the drug formulation on cells within at least a portion of the MOS to determine whether the drug will treat the tumor based on the determined effect.

[0059] In some embodiments, the methods may include obtaining a second patient biopsy after the patient has been treated with the drug formulation, forming a second plurality of MOSs from the second patient biopsy, exposing at least a portion of the second plurality of MOSs to the drug formulation, and determining that the tumor remains responsive to the drug formulation after one or more administrations of the drug to the patient by measuring an effect of the drug formulation on cells within at least a portion of the second plurality of MOSs.

[0060] Determining that the tumor will respond to a drug formulation can include exposing at least a portion of the MOS to a plurality of drug formulations and reporting the measured effect for each of the drug formulations. In some embodiments, determining further includes distributing the MOS into a multi-well plate prior to assaying the MOS.

[0061] Any of these methods may include biopsying the patient to collect a patient biopsy (or otherwise taking a tissue sample from the patient or a sample of tissue or cells derived from the patient), and / or treating the patient with a drug formulation or assisting a physician in treating the patient (e.g., making a recommendation to the physician as to which drug formulation would be effective). Generally, the time from taking the biopsy to reporting may be less than about 21 days (e.g., less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, etc.).

[0062] Primary tissue-derived hepatocytes are widely used to assess the effects of drug-induced liver injury (DILI), which remains the leading cause of discontinuation of approved drugs. However, there is a lack of a robust high-throughput system to maintain the cell viability and biological function of hepatocytes when cells are cultured in vitro.

[0063] Herein, we provide a new method for culturing primary tissue-derived hepatocytes in vitro based on MOS technology. Hepatocytes generated by this method retain liver-specific functions, including viability and maintenance of biological functions (albumin and urea secretion) for at least 3 weeks. This is impossible to achieve using conventional 2D hepatocyte culture methods.

[0064] Hepatocytes cultured in MOS (HepatoMOS) form the appropriate architecture on day 3 of HepatoMOS culture and maintain the architecture over time.

[0065] Furthermore, HepatoMOS cultures are able to distinguish DILI-positive compounds from non-DILI drugs of concern and capture DILI associated with chronic and repeated dosing regimens. The mass of cells required for a HepatoMOS-based DILI assay is much (5-10x) less than in a 2D culture system, so the throughput of a HepatoMOS-based assay is significantly higher compared to 2D culture systems. Among the main improvements of the HepatoMOS system are the improved 3D environment provided by MOS culture, the optimal density of hepatocytes / droplets, and the efficient growth factor / oxygen diffusion provided by MOS culture.

[0066] HepatoMOS is particularly advantageous for DILI assessment. HepatoMOS distinguishes between DILI compounds of concern and those not associated with DILI effects. The system shows improved sensitivity and can capture the long-term effects of chronic dosing regimens. There is an option to passage / expand HepatoMOS from a single donor for unlimited DILI assays. The turnaround time for HepatoMOS culture-based DILI assays is short compared to other 3D spheroid culture systems.

[0067] MOS generated from hepatocytes (HepatoMOS) can be used to test certain therapies that were previously difficult to test. Unlike the formation of conventional bulk organoids, hepatocytes present in biopsied patient-derived tissues (e.g., from tumors) can be present and persist in the MOS upon their formation, even after extensive processing for MOS formation as described herein. MOS can also be generated directly from patient-derived hepatocytes. Furthermore, when using conventional bulk organoids, some therapies may have difficulty penetrating, reaching, and interacting with patient-derived tissues (e.g., from tumors). In contrast, MOS makes penetrating and testing those therapies much easier.

[0068] Because the MOS formation described herein allows for the incorporation of liver cells (including hepatocytes) from patient-derived tissues, the accuracy of testing the aforementioned drug formulations in the MOS is superior to testing in conventional bulk organoids. In addition, patient-derived hepatocytes may be introduced separately into the already formed MOS, as they are easily infiltrated. Patient-derived tissues (e.g., from tumors) contain a variety of hepatocytes naturally produced by the patient's body. The patient's response to certain drug formulations and therapies may be directly influenced by the hepatocytes present at the target site. The MOS produced as described herein may be advantageous for such drug testing, and the drugs described herein may be tested in MOS containing hepatocytes from the tissue of origin at the time of formation, or in MOS containing hepatocytes introduced after the MOS is formed. The MOS produced using hepatocytes may be used to test the effects of various agents on liver toxicity and drug-induced liver damage.

[0069] It is desirable to utilize MOS generated from hepatocytes (e.g., PHHs) to screen various drugs for their effects on hepatotoxicity and drug-induced liver damage. The size, properties, composition, and improved cell viability of MOS allow for high-throughput screening of various drugs.

[0070] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate various embodiments and aspects of the present invention. [Brief description of the drawings]

[0071] [Figure 1A] 1 shows patient-derived MOS formed as described herein containing a single dissociated primary tissue cell per MOS and cultured for 1 day after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 1B] 1 shows patient-derived MOS formed as described herein containing a single dissociated primary tissue cell per MOS and cultured for 3 days after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 1C] 1 shows patient-derived MOS formed as described herein containing a single dissociated primary tissue cell per MOS and cultured for 7 days after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 2A] 1 shows patient-derived MOS formed as described herein containing 5 dissociated primary tissue cells per MOS and cultured for 1 day after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 2B] 1 shows patient-derived MOS formed as described herein containing 5 dissociated primary tissue cells per MOS and cultured for 3 days after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 2C] 1 shows patient-derived MOS formed as described herein containing 5 dissociated primary tissue cells per MOS and cultured for 7 days after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 3A] 2A shows patient-derived MOS formed as described herein containing 20 dissociated primary tissue cells per MOS and cultured for 1 day after formation. As in Figures 1A and 2A, the cells are derived from colorectal cancer (CRC) tissue. [Figure 3B] 2B, patient-derived MOS formed as described herein to contain 20 dissociated primary tissue cells per MOS and cultured for 3 days after formation. As in FIG. 1B and FIG. 2B, the cells are derived from colorectal cancer (CRC) tissue. [Figure 3C] 1C and 2C, patient-derived MOS were formed as described herein to contain 20 dissociated primary tissue cells per MOS and cultured for 7 days after formation. Similar to Figures 1C and 2C, the cells were derived from colorectal cancer (CRC) tissue. [Figure 4A] 1 shows an example of a patient-derived MOS formed as described herein containing 10 dissociated primary tissue cells per MOS. Shown (at low magnification) are MOS immediately after formation. [Figure 4B]4A shows an example of a patient-derived MOS formed as described herein to contain 10 dissociated primary tissue cells per MOS. 4B shows a higher magnification view of a portion of the MOS of FIG. 4A taken after 2 days in culture. [Figure 4C] 1 shows an example of a patient-derived MOS formed as described herein containing 10 dissociated primary tissue cells per MOS. MOS is shown after 3 days of culture. [Figure 4D] 1 shows an example of a patient-derived MOS formed as described herein containing 10 dissociated primary tissue cells per MOS. MOS is shown after 4 days of culture. [Figure 4E] 1 shows an example of a patient-derived MOS formed as described herein containing 10 dissociated primary tissue cells per MOS. MOS is shown after 5 days of culture. [Figure 5A] Examples of MOS formed as described herein from hepatocytes of normal mouse liver and cultured for 1 day (FIG. 1A) or 10 days (FIG. 1B) after formation are shown. Mouse hepatocytes are taken from normal (e.g., non-diseased) mouse liver. [Figure 5B] Examples of MOS formed as described herein from hepatocytes of normal mouse liver and cultured for 1 day (FIG. 1A) or 10 days (FIG. 1B) after formation are shown. Mouse hepatocytes are taken from normal (e.g., non-diseased) mouse liver. [Figure 6] As described herein, a method for forming a patient-derived MOS from a primary tissue (eg, biopsy) sample is presented. [Figure 7A] 1 shows a schematic diagram of one example of an apparatus for forming a patient-derived MOS as described herein, including a microfluidic chip as part of the assembly. [Figure 7B] FIG. 7B is a perspective view of an example of a microfluidic chip portion of the device as shown in FIG. 7A. [Figure 7C] FIG. 7B shows a schematic diagram of a part of the microfluidic assembly of an apparatus for forming a patient-derived MOS as shown in FIG. 7A. [Figure 8]7A shows an example of an image showing multiple patient-derived MOS formed using a device as shown in Figure 7 A. This shows immediately post-polymerization patient-derived MOS suspended within a channel containing an immiscible fluid (e.g., oil) prior to being aliquoted from the device. [Figure 9] FIG. 7C is an image of a portion of a prototype microfluidic assembly of an apparatus for forming a patient-derived MOS, similar to that shown in FIG. 7C, illustrating the formation of a patient-derived MOS. [Figure 10] 1 shows a plurality of patient-derived MOS as described herein immediately after polymerization, the patient-derived MOS being suspended in an immiscible fluid. [Figure 11A] Another example of multiple patient-derived MOS just after formation suspended in an immiscible fluid (e.g., oil) is shown at low magnification. [Figure 11B] Another example of multiple patient-derived MOS just after formation suspended in an immiscible fluid (eg, oil) is shown at high magnification. [Figure 12A] 1 shows multiple patient-derived MOS after separation from the immiscible fluid within a few hours of their formation at low magnification. [Figure 12B] A high magnification shows multiple patient-derived MOS after separation from the immiscible fluid within hours of their formation. [Figure 13] 11 is another example of an image showing multiple patient-derived MOS formed as described herein. [Figure 14] 13 is a chart showing the size distribution of diameters from multiple patient-derived MOSs generated from exemplary biopsy samples. [Figure 15A] 13 shows a low magnification view of an example of multiple patient-derived MOS formed from a dissociated tissue biopsy and fluid matrix material after polymerization. [Figure 15B] FIG. 1 shows a high magnification view of an example of multiple patient-derived MOS formed from dissociated tissue biopsy and fluid matrix material after polymerization. The MOS was stained with trypan blue to show that the dissociated cells in the MOS are viable. [Figure 16A]Another example showing a low magnification view of an example of multiple patient-derived MOS similar to that shown in Figure 15A. Unstained image. [Figure 16B] Another example shows a high magnification view of one example of multiple patient-derived MOS, similar to that shown in Figure 15B. The MOS was stained with trypan blue (arrows) to show dissociated cells within the MOS, indicating that the cells were viable (e.g., alive) within the MOS. [Figure 17] Figure 17A,B,C,D,E show an example of a method to assay multiple patient-derived MOS formed from patient tumor biopsies to determine drug response profiles to multiple drug formulations. The procedure shown takes less than two weeks (e.g., approximately one week) from biopsy to results. [Figure 18] 1 illustrates generally an example method for treating a patient, including the generation and use of a plurality of patient-derived MOS as part of a treatment procedure. [Figure 19] 1 illustrates generally an example method for treating a patient that involves rapid generation and assay of multiple patient-derived MOS multiple times as part of a treatment protocol. [Figure 20] 1 illustrates a schematic diagram of one embodiment of a portion of an apparatus for forming multiple patient-derived MOSs as described herein. [Figure 21] 21 illustrates a schematic of a method of operating an apparatus for forming multiple patient-derived MOSs similar to that shown in FIG. 20. [Figure 22A-B] 22A and 22B show an example of validation of the method described herein for identifying drug resistance using multiple patient-derived MOS. The use of a traditional ("2D") tumor cell assay method to predict drug resistance is shown. [Fig. 22C-D] Figures 22C and 22D show an example of validation of the method described herein for identifying drug resistance using multiple patient-derived MOS. We show that the patient-derived MOS-based method accurately predicted the actual response (drug responsiveness) of the tumor, unlike conventional cultured cells. [Figure 23]Figures 23A,B,C,D show another example validating the use of the patient-derived MOS described herein to identify drug resistance, showing predicted drug responses to both oxaliplatin and irinotecan that are consistent with actual tumor responses following treatment with these drugs. [Figure 24] An example of drug screening using patient-derived MOS as described herein is shown, where a single tumor biopsy can generate large amounts of nearly identical MOS very quickly (e.g., within 2 weeks) that can be rapidly tested against multiple drug formulations (e.g., 27 are shown) in parallel. [Figure 25A] 1 shows an example of mouse liver MOS formed from mouse liver tissue, with a diameter of 300 pm and one cell per MOS. MOS is shown on day 1. [Figure 25B] 1 shows an example of mouse liver MOS formed from mouse liver tissue, with a diameter of 300 pm and one cell per MOS. MOS is shown at day 10. [Figure 26A] 25A shows an example of mouse liver MOS formed from partial hepatectomy mouse liver tissue, with a diameter of 300 pm and 25 cells per MOS, similar to that shown in FIG. 25A. Day 1 MOS is shown. [Figure 26B] 25B shows an example of mouse liver MOS formed from partial hepatectomy mouse liver tissue, with a diameter of 300 pm and 25 cells per MOS. Day 10 MOS is shown. [Figure 27A] Figure 1 shows an example of human liver MOS formed from human liver tissue. Day 1 MOS seeded with 40 cells / droplet are shown. [Figure 27B] 1 shows an example of human liver MOS formed from human liver tissue. MOS is shown at day 18. MOS is a hepatocyte-like structure. [Figure 27C] 1 shows an example of human liver MOS formed from human liver tissue. MOS at day 18 is shown. Cholangiocellular-like MOS is shown. [Figure 28A]1 shows an example of MOS generated from a patient-derived xenograft tumor line with a diameter of 300 pm and one cell per MOS. MOS is shown on day 1. [Figure 28B] 1 shows an example of MOS generated from a patient-derived xenograft tumor line with a diameter of 300 pm and one cell per MOS. Day 3 MOS is shown. [Figure 28C] 1 shows an example of MOS generated from a patient-derived xenograft tumor line with a diameter of 300 pm and one cell per MOS. MOS is shown at day 5. [Figure 28D] 1 shows an example of MOS generated from a patient-derived xenograft tumor line with a diameter of 300 pm and one cell per MOS. MOS is shown at day 7. [Figure 29A] 1 shows an example of MOS generated from a patient-derived xenograft model with a diameter of 300 pm and 5 cells per MOS. MOS is shown on day 1. [Figure 29B] FIG. 1 shows an example of MOS generated from a patient-derived xenograft model with a diameter of 300 pm and 5 cells per MOS. MOS is shown at day 3. [Figure 29C] 1 shows an example of MOS generated from a patient-derived xenograft model with a diameter of 300 pm and 5 cells per MOS. MOS is shown at day 5. [Figure 29D] 1 shows an example of MOS generated from a patient-derived xenograft model with a diameter of 300 pm and 5 cells per MOS. MOS is shown at day 7. [Diagram 30] Graph comparing the response of MOS formed from conventional organoids and colon cancer patient-derived organoids to oxaliplatin, showing comparable responses for conventional organoids and MOS. [Diagram 31] Graph comparing the response of MOS formed from traditional organoids and two colon cancer patient-derived xenograft models to SN38 (7-ethyl-10-hydroxy-camptothecin), showing comparable responses. [Diagram 32]Graph comparing the response of MOS formed from conventional organoids and colon cancer patient-derived xenograft models to 5-FU (fluorouracil), showing comparable responses. [Figure 33A] An example of a toxicity assay using mouse liver MOS is shown, showing that the size of the tissue in the mouse liver MOS of the control group is relatively large (indicated by the arrow), while the tissue in most MOS is smaller and contains many dead cells. [Figure 33B] An example of a toxicity assay using mouse liver MOS is shown. The acetaminophen (10 mM) treatment group is shown, with tissues in most MOS being smaller and containing many dead cells. [Figure 34A] 1 shows an example of a toxicity assay using human liver MOS. Typical human liver MOS observed in the control group is shown (indicated by arrow), including histology. [Figure 34B] 1 shows an example of a toxicity assay using human liver MOS. MOS in the acetaminophen (10 mM) treated group is shown, showing atypical tissue structure (arrows) and debris. [Diagram 35] Figure 1 shows that HepatoMOS were viable after encapsulation on day 0. 100 and 200 hepatocytes were encapsulated in MOS droplets. Viability assessment was monitored using the live cell dye calcein AM (green channel) and the dead cell dye ethidium homodimer (red channel). The staining results demonstrate that the hepatocytes were viable after encapsulation. [Diagram 36] The viability of HepatoMOS on day 3 is shown. [Figure 37] 1 shows monitoring of HepatoMOS over time. [Figure 38] These results show that HepatoMOS maintained extremely high cell viability over a period of three weeks. [Figure 39] Figure 2 shows that hepatocyte viability was lost in 2D culture conditions. [Diagram 40]We show that HepatoMOS maintain stable levels of urea and albumin secretion, suggesting that HepatoMOS contain functional hepatocytes. [Diagram 41] The results of HepatoMOS-based DILI assessment are presented. [Diagram 42] Figure 1 shows the results of DILI assessment in 2D hepatocyte monolayer cultures. [Figure 43A] Figure 1 shows the establishment and characterization of HepatoMOS from PHH pooled from 10 individual donors. A) Testing the effect of different cell densities in HepatoMOS cultures. [Figure 43B] A) Establishment and characterization of HepatoMOS from pooled PHH from 10 individual donors. B) Evaluation of four media options for successful cultivation of HepatoMOS. [Figure 43C-1] Establishment and characterization of HepatoMOS from pooled PHH from 10 individual donors. C-1) CTG assay measured the viability of HepatoMOS cultures across different cell densities and medium conditions. [Figure 43C-2] Establishment and characterization of HepatoMOS from pooled PHH from 10 individual donors. C-2) CTG assay measured the viability of HepatoMOS cultures across different cell densities and medium conditions. [Fig. 43D] Establishment and characterization of HepatoMOS from PHH pooled from 10 individual donors. D) CDFDA staining confirmed tubule formation in HepatoMOS. [Figure 43E] Establishment and characterization of HepatoMOS from PHH pooled from 10 individual donors. E) ELISA results showed albumin production in HepatoMOS cultures over time. [Figure 43F] Establishment and characterization of HepatoMOS from pooled PHH from 10 individual donors. F) Urea ELISA results showed urea secretion in HepatoMOS cultures over time. [Figure 43G] Establishment and characterization of HepatoMOS from pooled PHH from 10 individual donors. G) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Fig. 43H] Establishment and characterization of HepatoMOS from PHH pooled from 10 individual donors. H) H&E and Ki67 IHC staining of HepatoMOS. [Figure 44A] Live and dead cell staining for HepatoMOS (pooled from 10 individual donors) cultured in various conditions. A) Live and dead dye staining of HepatoMOS with four different cell densities. B) Live and dead dye staining of HepatoMOS in three media conditions. C) Live and dead dye staining of PHH cultured in dome conditions with three matched cell densities. D) Live and dead dye staining of HepatoMOS cultured in the same well with various sizes and densities. [Figure 44B] Live and dead cell staining for HepatoMOS (pooled from 10 individual donors) cultured in various conditions. A) Live and dead dye staining of HepatoMOS with four different cell densities. B) Live and dead dye staining of HepatoMOS in three media conditions. C) Live and dead dye staining of PHH cultured in dome conditions with three matched cell densities. D) Live and dead dye staining of HepatoMOS cultured in the same well with various sizes and densities. [Figure 44C] Live and dead cell staining for HepatoMOS (pooled from 10 individual donors) cultured in various conditions. A) Live and dead dye staining of HepatoMOS with four different cell densities. B) Live and dead dye staining of HepatoMOS in three media conditions. C) Live and dead dye staining of PHH cultured in dome conditions with three matched cell densities. D) Live and dead dye staining of HepatoMOS cultured in the same well with various sizes and densities. [Fig.44D]Live and dead cell staining for HepatoMOS (pooled from 10 individual donors) cultured in various conditions. A) Live and dead dye staining of HepatoMOS with four different cell densities. B) Live and dead dye staining of HepatoMOS in three media conditions. C) Live and dead dye staining of PHH cultured in dome conditions with three matched cell densities. D) Live and dead dye staining of HepatoMOS cultured in the same well with various sizes and densities. [Figure 45A] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of PHH cultured in dome conditions at four matched cell densities. C) Live and dead dye staining of HepatoMOS at day 14 of culture. D) CDFDA staining showed tubule formation in HepatoMOS. E) Albumin production over time in HepatoMOS cultured in different conditions. F) Urea secretion over time in HepatoMOS cultured in different conditions. [Figure 45B] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of PHH cultured in dome conditions at four matched cell densities. C) Live and dead dye staining of HepatoMOS at day 14 of culture. D) CDFDA staining showed tubule formation in HepatoMOS. E) Albumin production over time in HepatoMOS cultured in different conditions. F) Urea secretion over time in HepatoMOS cultured in different conditions. [Figure 45C]Establishment and characterization of HepatoMOS cultures of hepatocytes from a single donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of PHH cultured in dome conditions at four matched cell densities. C) Live and dead dye staining of HepatoMOS at day 14 of culture. D) CDFDA staining showed tubule formation in HepatoMOS. E) Albumin production over time in HepatoMOS cultured in different conditions. F) Urea secretion over time in HepatoMOS cultured in different conditions. [Figure 45D] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of PHH cultured in dome conditions at four matched cell densities. C) Live and dead dye staining of HepatoMOS at day 14 of culture. D) CDFDA staining showed tubule formation in HepatoMOS. E) Albumin production over time in HepatoMOS cultured in different conditions. F) Urea secretion over time in HepatoMOS cultured in different conditions. [Figure 45E] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of PHH cultured in dome conditions at four matched cell densities. C) Live and dead dye staining of HepatoMOS at day 14 of culture. D) CDFDA staining showed tubule formation in HepatoMOS. E) Albumin production over time in HepatoMOS cultured in different conditions. F) Urea secretion over time in HepatoMOS cultured in different conditions. [Fig.45F]Establishment and characterization of HepatoMOS cultures of hepatocytes from a single donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of PHH cultured in dome conditions at four matched cell densities. C) Live and dead dye staining of HepatoMOS at day 14 of culture. D) CDFDA staining showed tubule formation in HepatoMOS. E) Albumin production over time in HepatoMOS cultured in different conditions. F) Urea secretion over time in HepatoMOS cultured in different conditions. [Figure 46A] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single pediatric donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of HepatoMOS cultured at different cell densities. C) Live and dead dye staining of HepatoMOS cultured at different cell densities. D) Live and dead dye staining of hepatocytes cultured in dome conditions. E) CDFDA staining showed the formation of tubules in HepatoMOS. F) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Figure 46B] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single pediatric donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of HepatoMOS cultured at different cell densities. C) Live and dead dye staining of HepatoMOS cultured at different cell densities. D) Live and dead dye staining of hepatocytes cultured in dome conditions. E) CDFDA staining showed the formation of tubules in HepatoMOS. F) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Figure 46C]Establishment and characterization of HepatoMOS cultures of hepatocytes from a single pediatric donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of HepatoMOS cultured at different cell densities. C) Live and dead dye staining of HepatoMOS cultured at different cell densities. D) Live and dead dye staining of hepatocytes cultured in dome conditions. E) CDFDA staining showed the formation of tubules in HepatoMOS. F) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Figure 46D] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single pediatric donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of HepatoMOS cultured at different cell densities. C) Live and dead dye staining of HepatoMOS cultured at different cell densities. D) Live and dead dye staining of hepatocytes cultured in dome conditions. E) CDFDA staining showed the formation of tubules in HepatoMOS. F) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Figure 46E] Establishment and characterization of HepatoMOS cultures of hepatocytes from a single pediatric donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of HepatoMOS cultured at different cell densities. C) Live and dead dye staining of HepatoMOS cultured at different cell densities. D) Live and dead dye staining of hepatocytes cultured in dome conditions. E) CDFDA staining showed the formation of tubules in HepatoMOS. F) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Figure 46F]Establishment and characterization of HepatoMOS cultures of hepatocytes from a single pediatric donor (PHH). A) Representative images of HepatoMOS cultures over time. B) Live and dead dye staining of HepatoMOS cultured at different cell densities. C) Live and dead dye staining of HepatoMOS cultured at different cell densities. D) Live and dead dye staining of hepatocytes cultured in dome conditions. E) CDFDA staining showed the formation of tubules in HepatoMOS. F) IF staining confirmed the expression of key markers in HepatoMOS cultures. [Figure 47] We show that Live / Dead staining detected the effects of DILI with similar specificity and sensitivity as the ATP content-based readout. [Figure 48A] Figure 1 shows that HepatoMOS predicts the impact of DILI. A) DILI response measured by 2D or HepatoMOS approach. B) Comparison of DILI prediction using HepatoMOS or Spheroid approach. C) Table summarizing specificity and sensitivity of HepatoMOS-based DILI prediction. [Figure 48B] Figure 1 shows that HepatoMOS predicts the impact of DILI. A) DILI response measured by 2D or HepatoMOS approach. B) Comparison of DILI prediction using HepatoMOS or Spheroid approach. C) Table summarizing specificity and sensitivity of HepatoMOS-based DILI prediction. [Figure 48C] Figure 1 shows that HepatoMOS predicts the impact of DILI. A) DILI response measured by 2D or HepatoMOS approach. B) Comparison of DILI prediction using HepatoMOS or Spheroid approach. C) Table summarizing specificity and sensitivity of HepatoMOS-based DILI prediction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Generally described herein are MOSs, methods and apparatus for forming them, and methods and apparatus for using them to assay, for example, the response of tissues (including cancerous and non-cancerous tissues).

[0073] The MOS described herein are typically spheres formed from dissociated primary cells distributed within a substrate. These MOS may have a diameter of about 50 μm to about 500 μm (e.g., about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 50 μm to about 250 μm, etc.) and may initially contain about 1 to 1000 dissociated primary cells (e.g., about 1 to 750, about 1 to 500, about 1 to 400, about 1 to 300, about 1 to 200, about 1 to 150, about 1 to 100, about 1 to 75, about 1 to 50, about 1 to 40, about 1 to 30, about 1 to 20, etc.) distributed within the substrate.

[0074] Surprisingly, despite their small size (often about 50-250 μm) and low cell density (e.g., less than 100 cells per MOS), these MOS may be used immediately or cultured for very short periods of time (e.g., less than 14 days, less than 10 days, less than 7 days, less than 5 days, etc.), allowing the cells within the MOS to survive while maintaining many, if not all, characteristics of the tissues, including tumor and non-tumor tissues, from which they were extracted. The viability of cells within the MOS is remarkably high, and the MOS may be cultured for days (or weeks) through multiple passages in which the cells divide, cluster, and form structures similar to the parent tissue. Also, surprisingly, in some embodiments, cells from dissociated tissues within the MOS form morphological structures even within the smallest MOS. In some applications, the presence of such structures is not necessary for the usefulness of these MOS (e.g., they may be used before substantial structural reorganization has occurred), but in some embodiments they may be particularly useful.

[0075] The methods and devices described herein for forming and using MOSs can be used to generate many (e.g., more than 10,000) MOSs from a single biopsy. These MOSs can be used to screen for drug compositions that can predict therapies that can be effectively applied to the patient from whom the biopsy was taken. This can be useful, for example, for toxicity screening of drugs or other chemical compositions from healthy normal tissue and / or from cancerous (e.g., tumor) tissue. In particular, the MOSs, methods, and devices for forming them, as well as methods and devices for testing them, can be used for screening to identify one or more drug compositions or combinations of drug compositions that can effectively treat a patient (e.g., a cancer patient) before they undergo drug therapy. This can allow, for example, very rapid screening of cancer patients before they undergo months of chemotherapy that may not be effective otherwise.

[0076] Thus, described herein are methods (and devices for performing these methods) for high throughput drug screening using a single patient-specific biopsy (or other suitable tissue / cell source). Described herein are droplet-forming MOS that can be formed from patient-derived tumor samples that have been dissociated and suspended in a base matrix (e.g., MATRIGEL). The MOS can be patterned on a microfluidic microwell array, incubated, and administered with drug compounds. This miniaturized assay maximizes the use of tumor samples, allowing many more drug compounds to be screened from a core biopsy at a much lower cost per sample.

[0077] Patient-derived cancer models (PDMCs), such as cell lines, organoids, and patient-derived xenografts (PDXs), are increasingly accepted as the “gold standard” preclinical models to facilitate the identification and development of new therapies. For example, large-scale drug screening of cell lines and organoids derived from cancer patients has been used to identify sensitivity to a number of candidate therapies. PDXs are also used to predict drug response and identify novel drug combinations. Although precision medicine strategies are being developed through the exploration of these various PDMC models, there are substantial barriers to their effective use. For example, patient-derived organoids (PDOs) are considered to be the most accurate in depicting patient tumors, as studies have shown that phenotypic and genotypic profiling of organoids often shows a high degree of similarity to the original patient tumor. Unfortunately, at least two limitations hinder the use of PDOs to guide therapy. First, it takes months to develop and test drug sensitivity in organoids, which reduces clinical applicability. Second, the number of organoids obtained from clinically relevant 18-gauge core biopsies is not sufficient to perform high-throughput drug screening. Ideally, assays should be performed from a single core biopsy within 7-10 days. The MOSs described herein, as well as methods for making and using them, may address these clinical limitations.

[0078] Details of one or more embodiments of the subject matter of the present disclosure are set forth herein. Modifications to the embodiments described herein and other embodiments will be apparent to those skilled in the art upon review of the information provided herein. The information provided herein, particularly the specific details of the exemplary embodiments described, are provided primarily for clarity of understanding, and no unnecessary limitations should be understood therefrom. In case of conflict, the present specification, including definitions, will prevail.

[0079] Although the terms used herein are believed to be well understood by those of skill in the art, definitions are provided herein to facilitate explanation of the subject matter of the present disclosure.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, the preferred methods, devices, and materials are described herein.

[0081] The term "unpolymerized mixture" is used herein to refer to a composition that includes a biologically relevant material, including a dissociated tissue sample and a first fluid matrix material. The fluid matrix material is typically a material that can be polymerized to form a support or support network in which the dissociated tissue (cells) are dispersed. Upon polymerization, the polymerized material can form a hydrogel and can form and / or include proteins in addition to cells that form a biocompatible medium. A suitable biocompatible medium for use in accordance with the subject matter of the present disclosure can typically be formed at room temperature (e.g., 25°C) from any biocompatible material that is a gel, semi-solid, or liquid (e.g., low viscosity liquid) and can be used as a three-dimensional substrate for cells, tissues, proteins, and other biomaterials of interest. Exemplary materials that can be used to form a biocompatible medium in accordance with the presently disclosed subject matter include, but are not limited to, polymers and hydrogels, including collagen, fibrin, chitosan, MATRIGEL™ (BD Biosciences, San Jose, Calif.), polyethylene glycol, dextran (including chemically or photocrosslinkable dextran, etc.), and electrospun bio, synthetic, or biosynthetic blends. In some embodiments, the biocompatible medium comprises a hydrogel.

[0082] The term "hydrogel" is used herein to refer to a two- or multi-component gel that comprises a three-dimensional network of polymer chains in which water acts as the dispersion medium, filling the spaces between the polymer chains. Hydrogels used in accordance with the presently disclosed subject matter are generally selected for a particular application based on the intended use of the structure, taking into account the parameters used to form the MOS, as well as the effect of the selected hydrogel on the behavior and activity of biomaterials (e.g., cells) incorporated in the biosuspension that is placed within the structure. Exemplary hydrogels of the presently disclosed subject matter may be composed of polymeric materials, including, but not limited to, alginates, collagen (including types I and VI), elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactides, polyethylene glycols, polycaprolactones, polycolides, polydioxanones, polyacrylates, polyurethanes, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatins, elastin, fibrin, laminins, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids, carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof, and inorganic materials (e.g., glasses such as bioactive glasses, ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and combinations of all of the foregoing).

[0083] Further, with regard to hydrogels used to generate MOS described herein, in some embodiments, the hydrogel is comprised of a material selected from the group consisting of agarose, alginate, collagen type I, polyoxyethylene-polyoxypropylene block copolymers (e.g., Pluronic® F127 (BASF Corporation, Mount Olive, NJ)), silicones, polysaccharides, polyethylene glycol, and polyurethane. In some embodiments, the hydrogel is comprised of alginate.

[0084] The MOS described herein may also include biologically relevant materials. The phrase "biologically relevant materials" may describe materials that can be included in a biocompatible medium as defined herein and subsequently interact with and / or affect a biological system. For example, in some embodiments, the biologically relevant materials are magnetic beads (i.e., beads that are magnetic themselves or that include a material that responds to a magnetic field, such as iron particles), which can be combined as part of the unpolymerized material to generate MOS that can be used in the methods and compositions (e.g., for the isolation and purification of MOS). As another example, in other embodiments, the biologically relevant materials may include additional cells in addition to the dissociated tissue sample (e.g., biopsy) material. In the unpolymerized mixture, the dissociated tissue sample and the additional biologically relevant materials may be present in a homogenous mixture or as a dispersed mixture (e.g., in just one half or other portion of the MOS, including within the core or outer region of the formed MOS). In some embodiments, the additional biologically relevant materials in the unpolymerized material may be suspended with the dissociated tissue sample in the suspension, for example, before the droplets that form the MOS are polymerized.

[0085] In some embodiments, biologically relevant material that may be contained in dissociated tissue sample (e.g., biopsy) material may include several cell types, including preadipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells, and adipose tissue macrophages, as well as fragments of small blood vessels or microvessels found within the stromal vascular fraction.

[0086] Generally, with regard to dissociated tissue sample (e.g., biopsy) material included in the MOS described herein, these tissues can be any suitable tissue from a patient, typically taken by biopsy. Although non-biopsy tissues may be used, generally these tissues (and the resulting dissociated cells) can be primary cells taken from a patient biopsy as described above, for example, by needle biopsy. The tissues can be derived from healthy tissue biopsies or from cancerous (e.g., tumor) cell biopsies. Dissociated cells can be incorporated into the MOS of the presently disclosed subject matter based on the intended use of the MOS. For example, the relevant tissue (e.g., dissociated biopsy tissue) can typically include cells commonly found in that tissue or organ (or tumor, etc.). In that regard, exemplary associated cells that may be incorporated into the MOS of the presently disclosed subject matter include neurons, cardiomyocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans, bone cells, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, bile duct epithelial cells, and the like. These types of tissues may be dissociated by conventional techniques known in the art. Suitable biopsy tissues may be derived from bone marrow, skin, cartilage, tendon, bone, muscle (including cardiac muscle), vascular, corneal, neural, brain, gastrointestinal, renal, hepatic, pancreas (including islet cells), lung, pituitary, thyroid, adrenal, lymphatic, salivary, ovarian, testicular, cervical, bladder, endometrial, prostate, vulva, and esophageal tissues. Normal or diseased (e.g., cancerous) tissues may be used. In some embodiments, the tissue may originate from tumor tissue, including tumors originating from any of these normal tissues.

[0087] Once MOS is formed, it can be cryopreserved and / or cultured. Cultured MOS can be maintained in suspension, either statically (e.g., in a well, vial, etc.) or in motion (e.g., rolling or agitating). MOS can be cultured using known culture techniques. Exemplary techniques can be found, among others, in Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 4th ed., Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach, Davis, ed., Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, Masters, ed., 2000; and US Pat. Nos. 5,516,681 and 5,559,022.

[0088] In some embodiments, a MOS is formed by forming droplets of an unpolymerized mixture (e.g., in some embodiments, a cooled mixture) of a dissociated tissue sample and a fluid matrix material in an immiscible material, such as a fluid hydrophobic material (e.g., oil). For example, a MOS may be formed by combining a stream of unpolymerized material with one or more streams of immiscible material to form droplets. The density of cells present in the droplets may be determined by the dilution of the dissociated material (e.g., cells) in the unpolymerized material. The size of the MOS may correlate to the size of the droplets formed. Generally, a MOS is a spherical structure having a stable geometric shape.

[0089] The practice of the subject matter of the present disclosure may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art, and such techniques are fully explained in the literature.For example, see Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M.J. Gait, ed., 1984; Nucleic Acid Hybridization, 15 D.Hames & S.J. Higgins, eds., 1984; Transcription and Translation, B.D. Hames & S.J. Higgins, eds., 1984; Culture Of Animal Cells, R.I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells, J.H. Miller and M.P. Calos, eds., Cold Spring Harbor 20 Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV, D.M. Weir and C.C. Blackwell, eds., 1986.

[0090] As used herein, a drug composition may include any drug, drug dilution, drug formulation, composition containing multiple drugs (e.g., multiple active ingredients), drug formulation, drug form, drug concentration, combination therapy, etc. In some embodiments, a drug formulation refers to a formulation containing a mixture of a drug and one or more inactive ingredients. As used herein, the term "passage" may refer to the average number of doublings of cells in a MOS. While traditional passage number refers to the transfer or subculture of cells from one culture vessel to another, cells in a MOS may be stably maintained in the same MOS and continue to grow and divide. Thus, as used herein, passage number typically refers to the average number of doublings by dissociated cells from biopsy tissue in a MOS. The doubling number of a population is the approximate number of doublings a cell population has undergone since isolation (e.g., since the formation of a MOS from freshly dissociated biopsy tissue). Generally, the MOS described herein may be cultured for a short period of time for the growth, e.g., doubling, of some or all of the cells within the MOS (e.g., less than 10 passages, less than 9 passages, less than 8 passages, less than 7 passages, less than 6 passages, less than 5 passages, less than 4 passages, less than 3 passages, etc.).

[0091] During culture, cells from dissociated biopsy tissue in MOS may aggregate, cluster, or aggregate within the MOS. The aggregates of cells may be highly organized and form a defined morphology, or may be clumps of cells clustered or attached together. The tissue may reflect the tissue of origin. In some embodiments, MOS may contain a single cell type (homotypic), but MOS may contain two or more cell types (heterotypic).

[0092] As previously mentioned, the tissue (e.g., biopsy) used to form the MOS (e.g., dissociated tissue) may be derived from normal or healthy biological tissue, or from diseased or diseased biological tissue (e.g., tissue or fluid derived from a tumor). The tissue used in the MOS may include cells of the immune system, such as T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages, and dendritic cells. The cells may be stem cells, progenitor cells, or somatic cells. As described in more detail below, the presence of these immune cells may be used to increase the efficacy and accuracy of drug / biologic testing. The tissue may be mammalian cells, such as human cells, or cells from animals, such as mice, rats, rabbits, etc.

[0093] Generally, tissues (and resulting cells) may be taken, often from a biopsy, to form a MOS. Thus, tissues may be derived from either biopsies, surgical specimens, aspirates, drainage, or cell-containing fluids. Suitable cell-containing body fluids include either blood, lymphatic fluid, sebaceous fluid, urine, cerebrospinal fluid, or ascites. For example, in patients with transcoelomic metastases, ovarian or colon cancer cells may be isolated from ascites. Similarly, in patients with cervical cancer, cervical cancer cells may be removed from the cervix, for example, by extensive excision of the transformation zone or by cone biopsy. Typically, such MOS will contain multiple cell types present in the tissue or fluid of origin. Cells may be obtained directly from a subject without an intermediate step of subculture, or may first undergo an intermediate culture step to produce a primary culture. Methods for recovering cells from biological tissues and / or cell-containing fluids are well known in the art. For example, techniques used to obtain cells from biological tissue include those described by R. Mahesparan (Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro. Acta Neuropathol (1999) 97:231-239).

[0094] Generally, cells are first dissociated or separated from each other before forming MOS. Dissociation of cells can be achieved by any conventional means known in the art. Preferably, cells are mechanically and / or chemically treated, such as by treatment with enzymes. "Mechanically" includes the meaning of disrupting the connections between associated cells, for example, by using scalpels or scissors or by using a machine such as a homogenizer. "Enzymatically" includes the meaning of treating cells with one or more enzymes that disrupt the connections between associated cells, including, for example, any of collagenase, dispase, DNAse, and / or hyaluronidase. One or more enzymes can be used under different reaction conditions, such as incubation in a water bath at 37°C or at room temperature.

[0095] Dissociated tissue may be treated to remove dead and / or dying cells and / or cell debris. Such removal of dead and / or dying cells can be achieved by any conventional means known to those skilled in the art, for example, using beads and / or antibody methods. For example, it is known that phosphatidylserine redistributes from the inside to the outside of the plasma membrane leaflet in apoptotic or dead cells. The use of Annexin V-biotin binding, followed by the binding of biotin to streptavidin magnetic beads, allows the separation of apoptotic cells from live cells. Similarly, removal of cell debris can be achieved by any suitable technique in the art, including, for example, filtration.

[0096] The dissociated cells may be suspended in a carrier material and / or a fluid matrix material (which may be referred to as a carrier material) prior to combination with the fluid matrix material. In some embodiments, the carrier material may be a material with a viscosity level that retards settling of the cells in the cell suspension prior to polymerization and formation of the MOS. The carrier material may have sufficient viscosity to allow the dissociated biopsy tissue cells to remain suspended in suspension until polymerization. The viscosity required to achieve this may be optimized by one skilled in the art by monitoring the settling rate at various viscosities and selecting a viscosity that gives an appropriate settling rate for the expected time delay between loading the cell suspension into an apparatus that forms the MOS by polymerizing droplets of unpolymerized material containing the cells. In some embodiments, the unpolymerized material may be flowed or agitated by the apparatus even when a lower viscosity material is used to keep the cells in suspension and / or distribute them as desired.

[0097] As mentioned above, in some embodiments, the unpolymerized mixture comprising the dissociated tissue sample and the fluid matrix material may include one or more components (e.g., biologically relevant materials). For example, biologically relevant materials may be any of the extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides, or antibodies (e.g., for regulating either cell survival, proliferation, or differentiation), and / or inhibitors of specific cell functions. Such biologically relevant materials may be used, for example, to increase cell viability by reducing cell death and / or activation of cell growth / replication, or to otherwise mimic the in vivo environment. The biologically relevant materials may include or mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids, and hormones. For example, the biologically relevant materials may supplement one or more drugs in the fluid matrix material. In some embodiments, the fluid matrix material is a synthetic gel (hydrogel) and may be supplemented by one or more biologically relevant materials. In some embodiments, the fluid matrix is ​​a natural gel. Thus, the gel may be composed of one or more extracellular matrix components, such as collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose, and chitosan. For example, MATRIGEL includes bioactive polymers important for cell viability, proliferation, development, and migration. For example, the matrix material may be a gel that includes collagen type 1, such as collagen type 1 obtained from rat tail. The gel may be a pure collagen type 1 gel or a gel that contains collagen type 1 in addition to other components, such as other extracellular matrix proteins. Synthetic gels may refer to gels that do not exist in nature. Examples of synthetic gels include gels derived from any of polyethylene glycol (PEG), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), and polyethylene oxide (PEO).

[0098] MOS Examples of MOS are shown in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E. For example, Figures 1A-1C show MOS formed with a single cell per MOS. As shown, the MOS are all approximately the same size (e.g., about 300 μm in diameter). Figure 1B shows MOS formed simultaneously after 3 days in culture. The cells have expanded in size and in some cases have doubled and / or grown. As shown in Figure 1C, by day 7 of culture, the cells have doubled multiple times and show clusters or clumps of cells.

[0099] Similar results are shown in Figures 2A-2C and 3A-3C. MOS formed from 5 cells per MOS or 20 cells per MOS, respectively. In Figures 4A-4E, MOS immediately after formation and MOS cultured for 5 days are shown. The MOS are nearly identical (e.g., have the same diameter) and each contain 10 cells per MOS. In Figure 4A, MOS immediately after formation is shown, day 0, still surrounded by the immiscible fluid (oil in this case). The MOS is removed from the immiscible fluid, washed, and cultured for 5 days. Figure 4B shows MOS after 2 days, Figure 4C shows MOS after 3 days, and Figures 4D and 4E show MOS at days 4 and 5, respectively. Figures 4A-4E show that the dissociated tissue (cells) from the biopsy within the MOS is viable and growing at comparable rates within nearly all MOS. As described in more detail herein, these MOSs can be generated in large quantities from even a single average-sized biopsy, generating hundreds or thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000, or more) of MOSs containing significant numbers of viable cells, allowing multiple rapid assays to be performed in parallel.

[0100] 5A and 5B show examples of MOSs formed as described herein from dissociated biopsies of mouse livers, e.g., MOSs showing mouse hepatocytes distributed within a polymerized fluid matrix material (in this example, MATRIGEL). Each MOS comprises polymerized matrix material 503 formed into spheres, e.g., with a diameter of about 300 μm, in which a set number of hepatocytes 507 are dispersed. In FIG. 5A, MOSs are shown 1 day after biopsy, dissociation, and MOS formation. These MOSs were then cultured for 10 days. During that time, the cells (hepatocytes) continued to survive and grow, often doubling multiple times and forming structures 505, as shown in FIG. 5B.

[0101] The MOS may generally include dissociated (e.g., biopsy) tissue (e.g., cells) at a fixed or known number of cells and / or concentration (cells / ml or cells / mm3) within the MOS. As previously mentioned, the matrix material may be a natural polymer such as one or more of alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, elastin, or a synthetic polymer such as one or more of polyethylene glycol (PEG) and polyacrylamide. Both organic and inorganic synthetic polymers may be used.

[0102] In some embodiments, the number of cells initially included in the MOS may be selected from 1 cell to several hundred cells. Specifically, in some assays (e.g., drug toxicity assays), it may be beneficial to include about 1-75 or about 1-50 (e.g., a smaller number of cells). The number of cells per MOS may be set or selected by the user. In some embodiments, as described below, the device includes one or more controls for setting the number of cells from the primary tissue included in each MOS. The number of cells may be selected or set based on how the user intends to use the MOS. For example, MOSs with very low numbers of cells (e.g., 1 cell per MOS, 1-5 cells per MOS, etc.) may be particularly suitable for studying clonal diversity (e.g., tumor heterogeneity). Since each MOS grows from a single cell, it can be observed which clones are drug resistant, and these specific MOSs may be examined (e.g., by genomic sequencing) to determine the genomic (mutational) diversity associated with the particular clone. Low to moderate numbers of cells per MOS (e.g., about 3-30 cells, 5-30 cells, 5-25 cells, 5-20 cells, 10-25 cells, etc.) may be particularly useful for rapid drug testing (including toxicity testing) since these MOS typically grow rapidly. Larger numbers of cells per MOS (e.g., about 20-100 cells, e.g., 30-100 cells, 40-100 cells, more than 50 cells, etc.) may be particularly suitable to mimic tissue composition in each MOS since MOS may contain different lineages that may include epithelial (or cancer etc.) and mesenchymal (or stromal, immune, vascular etc.) cells.

[0103] The MOS may be formed in any suitable size, which may correspond to the number of cells contained therein. For example, the size may be as small as about 20 μm in diameter and up to 500 μm (e.g., 50 or 100 μm on average, such as about 100-200 μm). In some embodiments, the size is about 300 μm and each MOS contains about 10-50 cells (e.g., about 10-30 cells). The number and size of the cells may be varied and / or controlled. In some embodiments, the number and / or size of the cells in the MOS may be set by one or more controls on the device forming the MOS. For example, the size of the MOS and / or the density of cells within the MOS may be adjusted by adjusting the flow rate and / or concentration of the dissociated tissue sample (e.g., cells from a biopsy).

[0104] As shown in Figures 1A-5B, after culturing the MOS described herein, the MOS allows for viable, healthy cells throughout the entire volume of the MOS. The size of the MOS and / or the number of cells contained in the MOS may be selected based on how the MOS is expected or intended to be used. For example, in embodiments in which the MOS is used to examine relationships between cells of a biopsy, the MOS may be formed to have a plurality of cells and may be cultured for extended periods of time (e.g., up to a week or more).

[0105] The MOS described herein may be made by combining a dissociated tissue sample (e.g., a biopsy sample) with a fluid matrix that can polymerize in a controlled manner to form a MOS. FIG. 6 illustrates one method of forming a MOS. Optionally, the method may include taking a sample from a patient, such as taking a biopsy from patient tissue 601. As previously described, the biopsy may be taken, for example, using a biopsy needle or punch. For example, a biopsy may be taken using a 14-gauge, 16-gauge, 18-gauge, etc. needle that is inserted into the patient tissue to remove the biopsy. After removing the tissue from the patient, the tissue may be processed to mechanically and / or chemically dissociate the material. The dissociated cells may be used immediately to form a MOS, as described, or in some embodiments, all or a portion of the cells may be modified, for example, by genetically modifying the cells 603, such as by transfection, electroporation, etc.

[0106] The dissociated tissue sample from the biopsy may be combined with a fluid (e.g., liquid) matrix material to form an unpolymerized mixture 605. This unpolymerized mixture may be held in an unpolymerized state such that cells from the dissociated tissue may remain suspended within the mixture. In some embodiments, the cells may remain suspect and unpolymerized, for example, by keeping them chilled below room temperature (e.g., 1-25° C.).

[0107] The unpolymerized mixture can then be dispensed as droplets into an immiscible material, such as, for example, oil, in a manner that controls the size of the droplet formation (and thus the size of the MOS 607 formed). For example, uniformly sized droplets can be formed by combining (e.g., the two converge) a stream of unpolymerized material with one or more streams of immiscible material (e.g., oil), such that the flow rates and / or pressures of the two streams can determine how the droplets of unpolymerized material form when they meet the immiscible material. The droplets can be polymerized 609 to form a MOS in the immiscible material. In some embodiments, the immiscible material can be heated or warmed to a temperature that polymerizes the unpolymerized mixture (e.g., the fluid matrix material in the unpolymerized material). Once formed, the MOS can be separated from the immiscible fluid, e.g., the MOS can be washed to remove the immiscible fluid 611, and placed in culture medium to grow cells within the MOS. The MOS can be cultured for any desired time, cryopreserved, and / or assayed immediately. In some embodiments, MOS may be cultured for a short period of time (e.g., 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-8 days, 1-9 days, 1-10 days, 1-11 days, 1-14 days, etc.). This may allow cells derived from the dissociated biopsy tissue to grow and / or divide (e.g., 2 times) for up to 5 or 6 passages. After culture, the cells may be either cryopreserved 615 and / or assayed 617, or both. Examples of assays that may be used are also described herein.

[0108] In any of these methods and devices described herein, the MOS may be recovered from the immiscible fluid (e.g., oil) after polymerization. For example, in some embodiments, the MOS may be recovered by demulsification and / or demulsification, e.g., by forming emulsified droplets and recovering the MOS after the droplets are formed to remove any oil (and other contaminants). This allows cells to grow within the polymerized droplets (MOS) without being inhibited by the immiscible fluid.

[0109] Although the methods and apparatus described herein illustrate methods of forming a plurality of droplets, and thus a plurality of MOSs, by flowing the unpolymerized mixture into one or more streams of an immiscible fluid (e.g., oil or other hydrophobic material), in some embodiments, the droplets may be formed by other methods that may allow for controlling the size of the droplets, as described herein. For example, in some embodiments, the droplets may be formed by printing (e.g., by printing the droplets onto a surface). This may reduce or eliminate the need for an additional recovery step of emulsification / demulsification. For example, the droplets may be printed onto a surface, such as a flat surface or a shaped surface, and polymerized. In any of these embodiments, the droplets may be dispensed using pressure, sound, electric charge, and the like. In some embodiments, the droplets may be formed using an automated dispenser (e.g., a pipetting device) adapted to release a small amount of the unpolymerized mixture onto a surface, into the air, and / or into a liquid medium (including an immiscible fluid).

[0110] The method for forming a MOS may be automated or may be performed using one or more apparatus. In particular, the method for forming a MOS may be performed by an apparatus that allows for selection and / or control of the size (and therefore the density of the cell population) of the MOS. For example, FIG. 7A shows an example of an apparatus 700 for forming a MOS, as described.

[0111] In FIG. 7A, the device typically includes an input for inputting either the unpolymerized mixture (already combined) of the dissociated tissue sample and the fluid matrix material, or may receive the dissociated tissue sample and the fluid matrix material separately (e.g., in a holding solution). In some embodiments, the device includes a holding chamber 706 for holding the unpolymerized mixture, and / or a holding chamber (not shown) for holding the dissociated tissue (e.g., biopsy) sample and for holding the fluid matrix material. Any or all of these holding chambers can be pressurized to control and / or accelerate the flow of fluids out of the chambers and into the device. The device may receive the unpolymerized mixture, or may receive and mix the components. In some embodiments, the device can control the concentration of cells in the unpolymerized mixture and can dilute the mixture (e.g., achieve a desired density by adding additional fluid matrix material). For example, the device may include a sensor (e.g., optical reader) for reading the density (e.g., optical density) of the cells in the unpolymerized mixture (not shown). The sensor may also be coupled to a controller 724. This can automatically or semi-automatically (e.g., by indicating to a user) control the dilution of the cells in the unpolymerized mixture. The apparatus may also include a port for receiving the unpolymerized mixture. The port may include or be coupled to a valve, which may be controlled by controller 724 (or a separate controller).

[0112] The device 700 may include chambers 708 and / or ports for holding and / or receiving immiscible fluids. In some embodiments, the immiscible fluids may be held in pressurized chambers so that the flow rate may be controlled. Any of the pressurized chambers may be controlled by a controller 724, which may use one or more pumps 726 to control the pressure, and therefore the flow, through the device. One or more pressure and / or flow sensors may be included in the system to monitor the flow through the device.

[0113] 7A, the entire apparatus 700 may be housed within a housing 702, or a portion of the apparatus 704 may be housed within a housing. In some embodiments, the housing may include one or more openings or accesses on the device, for example, for adding immiscible fluids and / or unpolymerized mixtures.

[0114] As mentioned above, any of these apparatus 700 may also include one or more sensors 728 for monitoring all or key portions of the manufacturing process. In some embodiments, the sensors may include optical sensors, mechanical sensors, voltage and / or resistance (or capacitance, or inductance) sensors, force sensors, etc. These sensors may be used to monitor ongoing operations of the assembly, including the formation of the MOS. The apparatus 700 may also include one or more heat / temperature regulators 718 for controlling the temperature of either or both of the immiscible fluid and / or the unpolymerized mixture (and / or the fluid matrix material).

[0115] Any of these devices may include one or more droplet forming assemblies 720 that may be monitored (e.g., using one or more sensors), as shown below in Figures 7C and 9. The droplet MOS forming assembly may include (or be coupled to) a dispenser (e.g., a MOS dispenser) 722. The dispenser may dispense, for example, into a multiwell plate 716.

[0116] In general, the droplet MOS formation assembly 720 may include one or more microfluidic chips 730, or structures that form and control the flow of the unpolymerized mixture to form the actual droplets. FIG. 7B shows an example of a microfluidic chip for forming a MOS 730. In FIG. 7B, the chip 730 includes a pair of parallel structures for forming a MOS. FIG. 7C shows the droplet formation region of a microfluidic chip for forming a MOS. This includes an unpolymerized channel outlet 741 that opens (in this example at a right angle) to a channel outlet 741 and immiscible fluid outlet(s) 743, 743′ at a “+” junction or region of intersection 737. In some embodiments, the input from the immiscible fluid channel(s) may be at an angle to the angle (and intersection) with the unpolymerized material. In FIG. 7C, as with all figures in this description that show dimensions, the dimensions shown are merely exemplary and are not intended to be limiting unless they are otherwise specified.

[0117] In Figure 7A, the microfluidic chip 730 includes an inlet (input port) 733 for an immiscible fluid into the chip (e.g., from an inlet port or storage chamber shown in Figure 7A). A second inlet port 735 into the chip can be configured to receive unpolymerized material and transport it in a semi-serpentine path to the junction region. Similarly, the inlet port for the immiscible fluid can be securely coupled to an immiscible fluid chamber or inlet-to-outlet as described above.

[0118] An inlet port 735 for unpolymerized material into the chip may be coupled via a delivery pathway 741 that connects the inlet to the junction region (as shown in FIG. 7C). Similarly, an inlet 733 for an immiscible fluid may connect to two (or more) connecting pathways 743, 743′ to the junction region 737. A channel leaving the junction region 737 may pass the formed MOS (in the immiscible fluid) down the channel to an outlet 731. The outlet 731 may connect to a dispenser (not shown) for dispensing from the MOS to one or more chambers (e.g., for incubation and / or assay).

[0119] In the example shown in Figures 7B and 7C, the droplets formed can polymerize into MOSs and can be transferred to a temperature-controlled microfluidic environment for an extended period before being dispensed from the device (not shown). For example, Figure 8 shows an example of a channel region 839 (e.g., element 739 in Figure 7B) shown in transparency that includes multiple MOSs 803, each containing a predetermined number of cells 805.

[0120] In FIG. 9, the junction region 937 is shaped as described above such that the channel carrying the unpolymerized mixture 911 intersects with one or more (e.g., two) channels 909 carrying a fluid (e.g., oil) that is immiscible with the unpolymerized mixture. When the unpolymerized mixture is pressurized to flow from the first channel 911 at a first velocity, the immiscible fluid flowing in the intersecting channel 909, 909′ allows the unpolymerized mixture to break off and form droplets 903 (sent to the outlet channel 939) after passing a predetermined amount of the unpolymerized mixture. Thus, in some embodiments, a comminuted (e.g., dissociated) clinical (e.g., biopsy or resection) sample of tissue, such as <1 mm in diameter, may be mixed with a temperature-sensitive gel (i.e., MATRIGEL at 4° C.) to form an unpolymerized mixture. This unpolymerized mixture may be placed in a microfluidic device. This may generate droplets (e.g., water-in-oil droplets) that are uniform in volume and material composition. At the same time, dissociated tumor cells may be fractionated into these droplets. The gel in the unpolymerized material may solidify upon heating (e.g., at 37° C.), resulting in the formation of a MOS. In some embodiments, the method may be used to generate more than 10,000 (e.g., more than 20,000, more than 30,000, more than 40,000, more than 50,000, more than 60,000, more than 70,000, more than 80,000, more than 90,000, more than 100,000) uniform droplets (MOS) from tissue (e.g., biopsy material). These MOS are compatible with conventional 3D cell culture techniques. FIG. 10 shows a plurality of MOS 1005 formed as described above and suspended in an immiscible material 1008 (e.g., oil).

[0121] In the exemplary microfluidic chip illustrated above, the junctions are shown as T-junctions or X-junctions where microfluidic flow focusing creates a controllable size of the MOS. In some embodiments, droplets can be formed, for example, in immiscible fluids and / or on solid or gel substrates by robotic micropipetting instead of a microfluidic chip. Alternatively, in some embodiments, droplets of unpolymerized material can be formed with the required dimensions and reproducibility by microcapillary generation. Alternatively, other examples of techniques that can be used to create MOS in a specified size range and reproducibility from unpolymerized material include colloidal manipulation by external forces such as acoustic, magnetic, inertial, electrowetting, or gravity.

[0122] Figures 11A and 11B show examples of MOS in oil formed as described above. As shown in Figures 15A-15B and 16A-16B, the cells in these MOS derived from a single biopsy sample are viable as seen by vital dye staining. For example, Figures 12A-12B show MOS with tumor cells (similar to those shown in Figures 11A-11B) that can be washed to remove immiscible material (e.g., oil). This immiscible material can be removed relatively quickly after the MOS is formed to prevent damage to the cells within the MOS.

[0123] As described in the following examples, the MOS described herein provides a good model for the efficacy of various drug formulations. The effects of pharmaceutical agents (including various drugs and other therapies) can be tested for liver toxicity and the effects of drug-induced liver damage.

[0124] In some embodiments, the gel droplets are recovered from the oil phase and resuspended in, for example, PBS via PFO (perfluorooctanol) and centrifugation. This may separate the immiscible fluid from the MOS. Thus, as shown in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E above, as well as Figure 13, these MOS, including tumor-based MOS, can grow normally. This is an important improvement, since drug screening must be performed on viable and growing primary tumor cells that retain characteristics from the patient's tumor to predict patient outcomes. The large number and uniformity of these MOS makes screening possible and reliable, as described below.

[0125] In any of the microfluidic chips or devices described herein, the channels may be coated, for example, the channels of the microfluidic device may be coated with a hydrophobic material.

[0126] Generally, the MOS described herein can have a very uniform diameter and very small size (e.g., diameter distribution), as shown, for example, in Figure 14, which shows an example distribution of droplet diameter sizes.

[0127] As mentioned above, Figures 15A-15B show MOS formed as described herein. In Figures 16A-16B, these MOS are stained with trypan blue (arrows) to show that they are alive. MOS formed as droplets in this manner may contain growth factors and matrix to mimic the biological environment in which the tissue originated. Patient samples (e.g., biopsy samples) can be formed into MOS (containing hundreds, thousands, or tens of thousands of MOS) within hours of obtaining the tissue. MOS may have as few as one or 4-6 cells (e.g., cancer cells when sampling a tumor) per MOS, or as many as hundreds of cells. These methods have been shown to work for virtually all types of cancer and non-cancerous tissues tested to date (n=32), including colon, esophageal, melanoma, uterine, sarcoma, kidney, liver, ovarian, lung, diaphragm, omentum, diaphragm, and breast cancer tissues. MOS can be cultured for any desired period of time, typically showing proliferation and growth in as little as 3-4 days. They can be maintained and passaged for months, and as described in more detail below, they can be used to screen thousands of drug compositions within just 4-6 days of harvesting the tissue (e.g., a biopsy).

[0128] The MOSs described herein may be banked at any time after they are formed, for example, by cryopreserving them. Tumor MOSs may be taken from many different patients and used individually or collectively to screen multiple drug formulations to determine toxicity and / or efficacy. Non-tumor cells (healthy tissues) may also be biopsied, banded, and / or screened in parallel. Thus, these methods and devices may enable high-throughput screening. In some embodiments, MOSs may be formed, passaged twice (e.g., doubling twice), and cryopreserved. As previously described, these same MOSs may be formed using normal healthy tissue to generate hundreds, thousands, or tens of thousands of MOSs. These may be used to assay drug effects, drug responses, biomarkers, proteomic signals, genomic signals, and the like.

[0129] It is particularly important that these MOS survive in a biologically significant manner, allowing them to provide clinically and physiologically relevant data, especially with respect to drug response, as described in Figures 22A-22D and 23A-23D. In particular, the MOS described herein allows cells of tissue extract / biopsy origin to grow very well and provide more representative data, especially compared to organoids or spheroids. Without being bound to a particular theory, this may be because cells may have a more constrained cell density in the MOS, allowing cells to communicate without inhibiting each other while sharing signals. MOS also have a very large surface area to volume ratio, allowing the transfer of growth factors and other signals to more easily penetrate the MOS (e.g., MOS are less diffusion restricted).

[0130] Assay The MOS described herein can be used in a variety of different assays, particularly to determine the impact (including toxicity) of drug formulations on normal and / or abnormal (e.g., cancerous) tissues. For example, drug screening can include applying MOS to all or some of the wells of a multi-well (e.g., 96-well) plate. Alternatively, custom plates can be used (e.g., a 10,000 microwell array can be formed from 100×100 wells). The MOS (e.g., gel droplets) can be applied into or, in some embodiments, onto the multiple microwell array and incubated with culture medium. The MOS can be cultured for 3-5 days. Then, in some embodiments, on the fifth day, the wells (e.g., microreactors) can be dosed with a drug compound, e.g., based on a set of FDA-approved anti-cancer drugs, to examine the impact of a panel of drugs. For example, the texted drugs can be based on the National Cancer Institute (Division of Cancer Treatment and Diagnosis) screen of 147 drugs intended to enable cancer research, drug discovery, and combination drug testing. On day 7, MOS may be imaged via standard fluorescent microscopy and ranked based on drug response.

[0131] An example of this assay technique is shown in Figures 17A-17E.

[0132] In this example, the screening assay can be automated. This can allow for a repeatable, automated workflow, which can increase the number of drugs screened from a few to hundreds. Figures 17A-17E show an example of this workflow. In Figure 17A, a tumor biopsy is taken and multiple (e.g., >10,000) MOSs are formed as described above (in Figure 17A, the junctional regions forming the MOSs are shown). The MOSs can then be harvested and washed (e.g., to remove immiscible (e.g., oil) materials in which they were formed). The MOSs can then be plated into one or more microwell plates. As shown in Figure 17C, the MOSs can be cultured for one or more generations (e.g., one or more passages). This is shown to occur from day 0 to day 3, day 4, or day 5. The MOSs can then be screened, for example, by applying drugs to a subset of the replicant wells, as shown in Figure 17D. Thereafter, as shown in FIG. 17E, on day 7, cells in the MOS may be imaged and / or automatically or manually scored to identify the effects of drugs (e.g., drug screening and growth profiling).

[0133] The workflow depicted in Figures 17A-17E may allow for the use of an integrated device for the growth, dosing, and / or screening of MOS. In one exemplary device, freshly biopsied or resected patient tumor samples may be dissociated and seeded into a gel with reagents to form MOS (as described above). A portion of the formed MOS may be cryopreserved. The remainder may be harvested and incubated until seeded into microwell plates for drug testing or screening, as previously described. Growth and viability assays may be performed on the MOS, which may be imaged and tracked. Their response to drug treatments, such as IC-50, cytotoxicity, and growth curves, may be measured to identify effective therapies for the patient's tumor.

[0134] The methods and devices described herein have many advantages, including reproducibility. The sample preparation process can be automated by microfluidic sample fractionation, which may reduce the need for specialized personnel for diagnostic testing and manual pipetting. This may be particularly useful in clinical settings. Furthermore, this may allow uniformity between signal droplets, increasing assay sensitivity. In addition, these assays may minimize the time required to generate MOS. Based on preliminary data, these methods may be able to generate a library of over 100,000 MATRIGEL-Tumor Droplets (MOS) in less than about 15 minutes. These methods are also highly scalable and can be multiplexed to perform multiple patient biopsies in parallel.

[0135] Finally, these methods are flexible and compatible with other techniques. As a research tool, droplet-based microfluidics is generally compatible with a wide range of hydrogel materials, such as agarose, alginate, PEG, and hyaluronic acid. Thus, the starting gel composition can be easily modified to accompany and promote the growth of MOS. Furthermore, the droplet size can be adjusted by modifying the size of the microfluidic device. Taken together, they allow a wide selection of gel material compositions and microreactor sizes.

[0136] For example, using MOS, the miniaturized assays described herein may maximize patient tumor biopsies and allow for screening of more drug compounds. For example, a 600uL tumor sample may be fractionated into approximately 143,000 individual microreactors with a volume of approximately 4nL. By maximizing tissue samples, multiple experimental replicates may be examined, increasing power. These techniques may allow for testing of intratumoral heterogeneity, drug perturbations, and may identify rare cellular events such as drug resistance. MOS may generally be compatible with downstream assays including single-cell RNA transcriptome analysis and epigenetic profiling. In addition, by maximizing the efficiency of tissue (e.g., biopsy) samples provided by MOS, a portion of the MOS may be banked (e.g., by cryopreservation for biobanking) for future novel drug assays and / or confirmatory analyses, including genetic screening.

[0137] For example, Figures 18 and 19 show a method of treatment using the methods and devices including the MOS described herein. In the case of precision medicine and personalized medicine, these methods and devices can be used as clinical indicators for appropriate drug selection to improve clinical outcomes and drug response. In one embodiment, a patient diagnosed with metastatic cancer undergoes a biopsy for histopathology and screening of multiple MOS formed from the biopsy as described herein. Within 7-10 days, screening may be performed from the biopsy to identify the most effective standard of care regimen so that the patient can begin treatment around 14 days.

[0138] An example of this is shown in FIG. 18. In this example, a tumor may be identified 1801 (e.g., by CT scan) on day 0, a biopsy may be taken 1805 on day 5, and hundreds, thousands, or tens of thousands of MOSs may be formed on the same day, cultured for 1-5 days, and screened 1805 to identify one or more drug compositions that may be used. This same step (forming and screening MOSs) may be used to guide precision medicine at multiple clinical decision points throughout disease progression. In this example, therapy using the identified one or more drug compositions may be initiated on day 14 (1809), and the patient may be monitored later during the course of treatment (e.g., with a follow-up CT scan at about day 90) to confirm that the tumor is responding to treatment 1811. The therapy may then be continued 1813 and ongoing progression monitored 1815.

[0139] As previously mentioned, the use of MOS for assays can be repeated at multiple time points throughout and during the course of treatment. This is illustrated in FIG. 19. For example, when a patient is first diagnosed 1907 with a resectable primary tumor, this technology (e.g., generating and screening 1905 MOS) can be used to determine the most effective neoadjuvant therapy 1921. Thus, biopsies can be taken and hundreds, thousands, or tens of thousands of MOS can be formed and screened with a panel of candidate drug compositions. Once the primary tumor is resected 1923, this technology 1905' can indicate whether and which adjuvant therapy should be selected 1925. If recurrence or metastasis occurs after surgical removal of the primary tumor 1927, the same technique (e.g., generation and screening of MOS from fresh biopsies 1905", 1905''', 195'''') can be used to guide standard of care regimens including first-line 1929, second-line 1931, and third-line 1933 therapies. If the patient eventually becomes tolerant or resistant to all standard of care regimens, this technique 1905'''''' can be performed to identify off-label drugs to treat the resistant tumor 1935. This technique may also be used as a companion diagnostic to identify patients for specific treatments. Finally, this technique can be used to derive and store patient-derived MOS to establish organosphere-based live cancer banks for screening, genomic profiling, drug discovery, drug testing, and clinical trial design.

[0140] Because these techniques, and the generation of vast numbers of MOS, can be performed relatively minimally invasively (e.g., by resection or biopsy) to provide reasonably rapid results from screening, these methods can be easily adapted to standard of care. For example, the volume of cellular material from the tissue (e.g., biopsy) input is very small, e.g., dissociated into volumes of 10 μL to 5 ml.

[0141] Generally, the use of the MOS described herein for screening may be automated or performed manually. Virtually any screening technique may be used, including imaging by one or more of confocal microscopy, fluorescence microscopy, liquid lens, holography, sonar, bright-field and dark-field imaging, laser, planar laser sheet (including high-throughput embodiments of image-based analysis methods using computer vision and / or supervised or unsupervised models, such as CNN). Downstream screening may include sampling the medium and / or performing gene or protein screening (e.g., scRNA-seq, ATAC-seq, proteomics, etc.) on cells from the MOS. EXAMPLES

[0142] Example 1 20 and 21 show another example of an apparatus for forming multiple MOSs as described herein. In FIG. 20, the apparatus may include multiple MOS forming junctions where immiscible material (e.g., oil) 2002 may be added to a reservoir and / or port 2004 in the device. Similarly, unpolymerized material 2006 (which in this example includes dissociated biopsy cells and a fluid matrix material) may be added to a reservoir or port 2008 in the device. In some embodiments, a second or additional material (e.g., a biologically active agent) may be added via a third set of ports 2010. These components may be combined at a junction (similar to those described above) that forms droplets in the immiscible material that may be polymerized into a MOS. In FIG. 20, three (or more) parallel junctions with corresponding inputs and outputs are shown.

[0143] Figure 21 illustrates a method of forming a MOS using the apparatus shown in Figure 20. In this embodiment, the resulting MOS contains both target (e.g., tumor) biopsy cells and also one or more additional biologically active agents that combine to form the MOS. For example, a first channel 2103 may contain unpolymerized material (including dissociated biopsy cells and matrix material), a second channel 2107 may contain additional active biomaterial, and a pair of intersecting channels 2109, 2109' carrying immiscible materials (e.g., oil) converge at a junction to form size-controlled droplets that are polymerized to form the MOS 2107.

[0144] In this example, the additional active biomaterial may be, for example, freezing medium (e.g., to aid in banking of MOS) and / or co-culture with additional cells (e.g., immune cells, stromal cells, endothelial cells, etc.), additional support network molecules (e.g., ECM, collagen, enzymes, glycoproteins, biomimetic scaffolds, etc.), additional growth factors, and / or drug compounds.

[0145] Example 2: Screening Results As discussed above, MOS and methods of using it to screen drug compositions can be used to accurately predict a patient's tumor response to one or more drug therapies. In some cases, the use of MOS can provide accurate results even when traditional culture drug screens cannot accurately predict drug response. For example, in Figures 22A-22D, MOS, but not cell line, was able to correlate with patient response. In Figure 22A, a traditional cell line administered a drug (e.g., oxaliparitine) was tested. The cell line showed no effect and predicted that the tumor was resistant to the drug at all dose ranges tested.

[0146] For comparison, multiple MOS were generated from patient biopsies, as shown in Figure 22B. In this example, the MOS showed a significant reduction in cell survival from the tumor MOS and predicted drug sensitivity. Indeed, when treated with the drug, the tumor responded to the treatment, as shown in Figure 22C (pre-treatment) and Figure 22D (post-treatment).

[0147] Example 3: Correlation between MOS and patient response In a similar set of experiments, MOS were generated from biopsies (Figure 23A) and the resulting MOS was used to perform drug effect screening. Figure 23B shows the effect of the first drug (oxaliparatidine) on these MOS, showing no change in % viability of MOS in the presence of the drug, predicting drug resistance. Similarly, treatment with a second drug, irinotecan, showed a lack of effect on MOS, as shown in Figure 23C, predicting drug resistance. A patient was treated with both oxaliparatidine and irinotecan and showed no response after 6 months of treatment. Thus, MOS strongly correlated with the patient's response to standard of care drugs. In this case, the patient endured 6 months of side effects and toxicity that could have been avoided by the predicted response from the MOS, demonstrating (within 7-10 days of biopsy) that the tumor was not responsive to these drugs.

[0148] Example 4: Multi-drug screening FIG. 24 shows an example of a panel of drugs (e.g., chemotherapeutic agents) that can be generated using multiple patient-derived MOS as described herein. In this example, drug screening using patient-derived MOS was performed by administering multiple replicates of each of multiple (27) drugs. A single tumor biopsy was used to generate a large number of multiple MOS very quickly (e.g., within less than two weeks) and these MOS were tested against a panel of drug formulations (e.g., 27 formulations are shown). This testing is performed in parallel and can be automatically quantified (e.g., by optical detection and quantification). In this example, the drug with the greatest toxicity to this particular tumor was pazopanib.

[0149] Drug combinations, as well as different drug concentrations, can be examined in parallel. This type of array testing is made practical by the methods and apparatus described herein, since hundreds, thousands, or tens of thousands of MOS can be generated from the same tumor biopsy.

[0150] Example 5: Preparation of biopsy samples Materials: Apparatus containing a droplet microfluidic chip (200um) for forming MOS as described above, Bio-rad droplet generation oil for EvaGreen (catalog #186-4006), 3-5mL per run, perfluorooctanol (PFO), Sigma, 10% perfluorooctanol (PFO) in Novec HFE7500, PBS, cell media (i.e., RPMI with 10% FBS and 1% PenStrep), 70um or 100um filters, 50mL conicals, Petri dishes.

[0151] Dissociation of biopsy samples: Use biopsy samples (human / animal) to generate dissociated samples (i.e., single cell tissue) from patients. Coat the microfluidic chip and assemble the microfluidic chip and holder. Connect the microfluidic tubing and fittings to the MOS and waste oil outputs (multi-wall plate, 15 mL Eppendorf, etc.).

[0152] Run the device to form a MOS. Remove the output (plate, Eppendorf tube, etc.) containing the droplets from the incubator (after at least 15 min). Remove excess oil from the output. The droplets should be buoyant, so the oil should be at the bottom of the vial. Be careful not to remove the droplets from the tube. Add 100uL of 10% (v / v) PFO to the output. Carefully swirl to mix and wait about 1 min. Do not pipette or disturb the sample. Centrifuge at 300g for 60 sec. Remove the supernatant (excess oil / PFO). Do not pipette or disturb the sample. Remove as much PFO as possible, as this chemical can reduce cell viability during incubation. Add 1mL cell medium. Do not pipette or disturb the sample. Centrifuge at 300g for 60 sec. Remove the supernatant and excess oil / PFO. Add 1mL cell medium. Using a 1 mL pipette tip, carefully pipette the sample up and down (approximately 30 times). Be careful not to overpipette or disturb the droplet sample. Using a 1 mL pipette tip, pass the droplet medium solution through a 70um or 100um filter (connected to a 50 mL conical). Some droplets will stick to the inside of the output (e.g., 15 mL Eppendorf). Rinse each tube with 2-3 mL of PBS and pipette up and down. Pass the rinsed PBS and droplets through the filter. Repeat this step twice, or until the tubes are clear and the droplets have been transferred to the filter. Using a 1 mL pipette tip, carefully wash the filter containing the droplets with approximately 5 mL of PBS. Be sure to cover the entire surface of the filter. This washing step removes excess oil and PFO from the sample and allows for the eventual recovery of the gel droplets into cell culture medium.

[0153] Once properly drained (approximately 1-2 min), carefully remove the filter from the 50 mL conical. Flip the filter upside down and wash the backside with fresh cell medium, catching the solution in a fresh Petri dish. This will detach the droplet from the filter and place it in the cell medium. It is recommended to use a 1 mL pipette tip and wash with approximately 5 mL of medium.

[0154] Check the quality of the droplets under a microscope. Most / all of the oil should be removed. If recovery is low, the sample may be refiltered. The density of the recovered MOS may be checked by hemocytometer

[0155] Example 6: Renal tissue MOS In another example, MOS can be formed from biopsied kidney tissue. For example, the equipment used can include a tube rotator or 100 μm and 70 μm cell strainers, 15 mL conical tubes, 50 mL conical tubes, razor blades, tweezers and surgical scissors, Petri dishes (100×15 mm), or tissue culture dishes. Reagents can include the following: EBM-2 medium, collagenase (5 mg / mL stock), Hank's balanced salt solution (HBSS), calcium chloride (10 mM stock solution), phosphate buffer solution (1×PBS), MATRIGEL, 0.4% trypan blue solution, and trypsin.

[0156] Rental tissue should be kept in cold transport media and on ice at all times. 2mL of enzyme digestion solution may be placed in a 15mL conical tube. Add 600uL of calcium chloride (final concentration: 3mM) and 200uL of collagenase (final: 0.5mg / mL). Transfer kidney sample to a Petri / culture dish. Remove all excess or non-tumor tissue with a sterile tissue or razor blade. Add 1mL of enzyme solution to the tissue. Cut the sample into small pieces (<2mm2) with a sterile razor blade. Hold the plate with tweezers or by hand. Place the chopped tissue and enzyme solution back into the 15mL tube containing the enzyme solution. Place the tube in a tube rotator or a 15mL tube rotator in a 37°C incubator for 30-60 minutes. Remove the tube from the incubator. Quench the enzymatic digestion with at least 6mL of EBM-2 (at least 3x the volume of the enzymatic digestion solution). Pipette to mix. Place a 100μm or 70μm cell strainer onto a 50mL conical tube. Transfer the sample through the strainer. Transfer the solution to a new 15mL conical tube. Centrifuge the sample at 1500rpm for 5 minutes. Discard the supernatant, leaving the cell pellet. Resuspend the pellet in 1mL of EBM-2 medium. Add 10μL of cell mixture to 10μL of trypan blue on a piece of parafilm and transfer to a cell counting plate or hemocytometer. Calculate cell concentration (# / mL). Centrifuge at 1500RPM for 5 minutes, discard the supernatant, leaving the pellet. Resuspend the cell pellet in 50uL of MATRIGEL per 1.25x105 cells. Keep on ice. Plate a 50uL dome of MATRIGEL cell suspension in the center of a well in a pre-warmed 24-well flat-bottom plate. Transfer the plate to a 37°C cell incubator and incubate for at least 20 minutes. Ensure the dome is polymerized. Gently add 500uL of pre-warmed EBM-2 medium along the wall of the well. Incubate in a 37°C incubator. To expand MOS, perform a complete medium change every 2 days.

[0157] Example 7: Liver microorganospheres As previously discussed, MOSs can be formed from normal (e.g., non-cancerous) and / or abnormal tissue. For example, FIGS. 25A-25 and 26A-26B show an example of a MOS formed from dislocated mouse liver tissue. The mouse liver tissue was combined with a fluid matrix material to form an unpolymerized mixture, and then droplets of the unpolymerized mixture were polymerized to form the MOS. In this example, the MOS has a diameter of about 300 μm. In FIGS. 25A-25B, the MOS was formed with a single cell per droplet. In FIGS. 26A-26B, the MOS was formed with 25 cells per droplet. In FIG. 25A, the MOS is shown one day after formation, and FIG. 25B shows the MOS after 10 days in culture. The cells in some MOS have divided and formed clusters that exhibit structure. Other MOSs contained cells that divided slowly or did not divide. Similarly, in FIGS. 26A-26B, the MOS initially contains about 25 cells in each MOS. After 10 days in culture, some MOS showed extensive cell growth and formed structures, while other MOS showed only minimal growth. In both cases, the cells within the MOS were found to display properties characteristic of the original tissue from which they were derived (e.g., hepatocytes).

[0158] The same procedure was successfully performed on human liver tissue, as shown in Figures 27A-C. In this example, MOS were initially formed with about 50 cells, as shown in Figure 27A. By day 18 of culture, some MOS had clusters and showed cells forming structures, while others had smaller structures or the cells did not divide.

[0159] Example 8: Cultured Cell Microorganospheres In addition to primary tissues, such as primary tissues removed from a patient immediately prior to or shortly before forming the MOS, a MOS can be formed from cultured cells or cells, including either 2D or 3D cultured cells.

[0160] In some embodiments, MOS may be formed from cell lines grown as part of a patient-derived xenograft (PDX). For example, Figures 28A-D show MOS formed from cultured PDX240 cells. PDX240 cells are a patient-derived xenograft (PDX) tumor cell line (numbered 240 based on patient source), a human tumor grown in immunodeficient mice (PDX) to form tumors in vivo. Xenograft tissue was extracted, dissociated, and used to form MOS as described above. In this example, each MOS contained a single cell at the time of formation. Figure 28A shows MOS after 1 day of culture, Figure 28B shows MOS after 3 days of culture, and Figures 28C and 28D show MOS after 5 and 7 days of culture, respectively. Over time in culture, at least some MOS show cells dividing and forming structures.

[0161] Figures 29A-29D show a similar experiment, where each droplet forming each of the MOS initially contained five PDX240 cells. With time in culture (e.g., from days 1, 3, 5, and 7 as shown in Figures 29A-29, respectively), the cells may divide and form structures.

[0162] Example 9: Comparison of MOS with conventional organoids Organoids were formed from patient-derived xenograft cells (including the PDX240 cells described above and a second PDX cell line, PDX19187) and compared to MOS formed using the same cells. Organoids were formed using conventional techniques, where large amounts of MATRIGEL were seeded into wells or dishes with cells and cultured until growth was observed. MOS were generated from conventional organoids.

[0163] Then, both conventional ("bulk") organoids and MOS were treated with the same drug (e.g., oxaliplatin or SN38), and cell viability was measured after 3 days of treatment. Drug response curves shown in Figure 30 and Figure 31 were generated and show similar response curves. For example, in Figure 30, drug response curves of PDO19187 bulk organoids and MOS showed similar response curves to oxaliplatin concentration, as did PDX240 bulk organoids and MOS. In Figure 31, drug response curves for both PDX19187 and PDX240 also showed similar results for both bulk organoids and MOS to SN38. Figure 32 shows the response curve of another anticancer drug, 5-FU (fluorouracil), again showing similar drug response curves for both PDZ-19187 and PDX-240 conventional organoids and MOS.

[0164] Thus, MOS as described herein can be formed more quickly and reliably, have higher overall viability compared to conventional organoids, and provide drug responses comparable to bulk organoids formed using the same cells.However, as described herein, MOS can be used more quickly and formed in much larger numbers.

[0165] Example 10: Effects of drugs on MOS Generally, the MOS described herein can be used to perform one or more assays, including toxicity assays. Any suitable assay can be performed, and the results determined by analyzing the tissue (e.g., cells, tissue structures) suspended within the MOS. The MOS described herein can be assayed or analyzed optically, chemically, electrically, genetically, or in any other manner known in the art.

[0166] Optical (either manual or automated) detection can be particularly useful and can include optically analyzing the effect of one or more drug formulations on tissues (including cells, clusters of cells, cellular structures, etc.) within the MOS. In some embodiments, as described above, drug formulations can be assayed for cell death (e.g., number and / or size of tissues) within the MOS being tested. In other embodiments, the MOS can be assayed for cell growth, including reduction in size, type, and / or rate of growth. In some embodiments, the MOS can be assayed for changes in the tissue structures formed.

[0167] For example, Figures 33A-B show the effect of one drug formulation, in this example, acetaminophen (10 mM), on mouse liver MOS. Figure 33A is a control group where the MOS was not treated, showing tissue within the MOS (arrows) that grew in culture. Figure 33B shows a similar set of MOS formed from mouse liver that was instead treated with 10 mM acetaminophen. In the control group, the tissue structures within the MOS are relatively large compared to the treated groups. The tissues of most MOS in the acetaminophen group are smaller and contain many dead cells.

[0168] Similarly, Figures 34A-B also show toxicity assays using human liver MOS. Figure 34A shows typical human liver MOS observed in the control group, including tissue structures formed therein (indicated by arrows). Figure 34B shows a treatment group in which human liver MOS was treated with acetaminophen (10 mM). Tissue in the treated MOS showed atypical tissue structures (arrows) and a significant increase in debris compared to the control group.

[0169] Any of these assays, including the optical assays, may be scored, graded, ranked, or otherwise quantified. For example, in Figures 33A-B and 34A-B, the results of these two assays may be quantified to show size differences, number of live / dead cells / tissue, etc. In some embodiments, the scoring may be automated.

[0170] Example 11: Viability of HepatoMOS The morphology of HepatoMOS was assessed using 50, 100, and 200 cells per MOS droplet. Optimal conditions were observed using 100 cells / MOS, as defined by improved morphology, cellular organization, and viability based on bright field (BF) imaging and viability staining. HepatoMOS exhibit sustained viability for at least 24 days.

[0171] FIG. 35 shows that HepatoMOS were viable after day 0 encapsulation. 100 and 200 hepatocytes were encapsulated in MOS droplets. Viability assessment was monitored using the live cell dye calcein AM (green channel) and the dead cell dye ethidium homodimer (red channel). The staining results demonstrate that the hepatocytes were viable after encapsulation.

[0172] Figure 36 shows the viability of HepatoMOS on day 3. The 100 cells / droplet condition showed the best cell viability as indicated by live cell dye (calcein AM) and dead cell dye (ethidium homodimer) (red channel).

[0173] Hepatocytes were packaged into MOS droplets and allowed to mature based on cell density. At 100 cells / droplet, cells began to associate and form organoid structures when in close contact over time based on BF images. The appropriate cell density range is 80-160 cells / droplet (200-300uM diameter). Any medium determined to be suitable for culturing hepatocytes may be used.

[0174] FIG. 37 shows the monitoring of HepatoMOS over time.

[0175] FIG. 38 shows that HepatoMOS maintained very high cell viability over a 3 week period.

[0176] Results from HepatoMOS were improved compared to hepatocytes in 2D culture conditions.

[0177] Figure 39 shows the viability of hepatocytes lost under 2D culture conditions. Figure 39A shows that a large amount of dead hepatocytes were observed on day 7 under 2D culture conditions based on bright field images. Figure 39B shows a representative live / dead image of control wells with bright field showing the low viability of hepatocyte culture under 2D conditions.

[0178] Example 12: Functionality of HepatoMOS HepatoMOS maintained stable levels of urea and albumin secretion, as shown in Figure 40, suggesting that HepatoMOS contained functional hepatocytes. HepatoMOS demonstrated sustained functionality for at least 24 days.

[0179] Examples 11 and 12 show that a method using 100 cells per 300um droplet and 40 droplets per well in a 384-well plate provides sufficient experimental signal for use in toxicological evaluation assays. This significant reduction in cell biomass broadens the application for high throughput screening.

[0180] The MOS generation system described herein enabled screening in 384-well plates using only 4,000 cells per well, compared to traditional 2D methods where typically 30,000 cells are seeded per well.

[0181] Example 13: Evaluation of drug-induced liver injury HepatoMOS and 2D hepatocyte cultures were used to evaluate the drug-induced liver injury (DILI) effects of various agents.

[0182] Frozen human hepatocytes from Lonza were plated at 30,000 cells per well and allowed to establish in plating medium. On day 2, plating medium was removed and replaced with maintenance medium and cultured for 1 day. On day 3, medium was replaced with treatment medium and cells were treated for 72 hours. Prior to harvesting, HepatoMOS were treated with CTG reagent to monitor viability.

[0183] Compounds with known clinical hepatotoxicity were selected to evaluate toxicity parameters in the HepatoMOS 3D model. Published IC50 values ​​are shown for the 3D spheroid and 2D HepG2 models (Table 1). Values ​​above 199 indicate no toxicity in the reported assay. [Table 1]

[0184] Fluorescence-based image analysis was used to observe cytotoxicity and provide dose-dependent responses to known toxicants.

[0185] Figure 41 shows that in a clinical setting, HepatoMOS-based DILI assessment showed more sensitive drug response and improved correlation with DILI impact. In this experiment, 100 cells / droplet were used and cell viability was determined by Celltiter Glo 3D.

[0186] In contrast, as shown in FIG. 42, the 2D hepatocyte culture-based DILI assay did not capture the effects of clinical DILI.

[0187] These results demonstrate that HepatoMOS cultures can distinguish between drugs with no DILI risk and DILI-positive compounds.

[0188] Example 14: Establishment and functional characterization of HepatoMOS cultures To evaluate whether the unique characteristics of MOS provide a favorable 3D environment for maintaining hepatocytes, especially primary human hepatocytes (PHH), a commercial cryopreservation product containing pooled PHH from 10 individual donors, including 5 adult females and 5 adult males, was used to better represent gender and eliminate individual differences. Hepatocytes have been reported to form tight intercellular communication with each other, which is important for the proper function and polarity of PHH. To determine the optimal cell density required for successful HepatoMOS culture, PHH were encapsulated in Matrigel droplets with a diameter of approximately 240 μm and various densities: 10, 50, 100, and 200 cells per droplet. Hepatocyte medium (HCM), a serum-free commercial medium, was initially selected to test HepatoMOS culture. Morphological changes and viability of HepatoMOS were monitored for up to 21 days. Figure 43a shows HepatoMOS culture after 7 days, showing an increase in the number of PHH clusters as the cell density increases. Among the four densities tested, HepatoMOS under the density condition of 100 cells / droplet formed the most uniform and compact microtissue-like structure. Live and dead cell dye staining (calcein AM and EtH) further confirmed that 100 cells / MOS showed the most uniform and viable culture across all four densities tested (Figure 44a). This suggested that an optimal density of approximately 100 cells / droplet was optimal for HepatoMOS cultures. Consistent with this observation, in wells with heterogeneous droplet sizes cultured using the same medium and conditions, we also found that large droplets containing very dense PHHs and droplets containing sparse PHHs showed more dead cells as shown by calcein AM and EtH staining, suggesting an optimal cell density that can provide not only close cell-cell interactions but also efficient nutrient / oxygen penetration. In contrast, in matched dome culture conditions, the live cell dye calcein AM showed positive signals only in the peripheral regions of the domes, whereas most PHHs in the center of the domes stained with the dead cell dye EtH at both days 7 and 14 (Figure 44c), indicating reduced PHH viability within the MATRIGEL domes.We further tested HepatoMOS culture using three other serum-free media options (InSphero, INVITROGRO HI, and William's E) and found that while HepatoMOS showed long-term culture viability in InSphero or HCM media conditions, the majority of HepatoMOS died within 14 days in INVITROGRO HI and William's E media conditions (Figures 43b and 44b). A more rapid increase in CTG signal from day 4 to day 7, and a relatively stable increase in CTG signal from day 7 to day 14, were observed in both InSphero and HCM media conditions (Figure 43c). Therefore, we grew PHHs using 100 cells / droplet and density conditions of HCM or InSphero media for most of the work performed in this study.

[0189] Moreover, CDFDA positive staining is observed in HepatoMOS cultures (Figure 43d), which is a key indicator of tubule formation. To further confirm the preservation of liver-specific functions in HepatoMOS cultures, albumin and urea secretion (the two most widely used indicators of liver-specific functions) were examined in all growth conditions tested. As shown in Figures 43e and 43f, an increase in albumin production from day 4 to day 7 was observed in all HepatoMOS culture conditions, whereas the corresponding 2D culture conditions experienced a rapid decrease in albumin production from day 4 to day 7. Similarly, a steady production of urea was observed in HepatoMOS cultures from day 4 to day 21, whereas the corresponding 2D conditions experienced a rapid decrease in urea production. Notably, the levels of both albumin and urea production were significantly higher than the corresponding 2D PHH culture conditions for up to 21 days. Immunofluorescence staining of albumin and CYP confirmed that the key functions of PHH were preserved in HepatoMOS cultures (Figure 43g).

[0190] An increase in CTG signals and albumin production from day 4 to day 10 was observed in HepatoMOS cultures, and to confirm whether there was an expansion of PHH in HepatoMOS cultures, the histology of HepatoMOS cultures was examined by H&E (hematoxylin and eosin) and IHC (immunohistochemistry) staining. H&E staining results confirmed the presence of polyploidy in HepatoMOS cultures, which is one of the typical characteristic cellular features of PHH observed in vivo. More interestingly, Ki67-positive cells were observed in HepatoMOS cultures on day 7, suggesting that the increase in CTG signals and albumin production may be due to the expansion of PHH in HepatoMOS cultures.

[0191] Furthermore, the reproducibility and robustness of this HepatoMOS culture condition was confirmed in commercially cryopreserved PHH from two individual donors, including one adult and one pediatric donor (Table 2). All individual donor results showed consistent medium, density preferences, and growth patterns as pooled donors (Figures 45 and 46). The above results suggest a clear advantage of using MOS technology to grow PHH. [Table 2]

[0192] Culture of PHHs and generation of HepatoMOS: Pooled and individual donors of PHHs were purchased from BioIVT (www.bioivt.com). The PHHs used to compare the DILI predictability of the HepatoMOS method were provided with the InSphero kit. In this study, four different serum-free media options (InSphero, HCMTM Hepatocyte Culture Medium BulletKit™, BioIVT INVITROGRO HI Medium, and William E) were selected for the initial HepatoMOS culture. PHHs were cultured as 2D monolayers on collagen-coated 96-well plates or encapsulated in MOS to generate HepatoMOS of different cell densities. To compare HepatoMOS with the dome culture method, matched cell densities were used to generate HepatoMOS using domes.

[0193] Quantification of albumin and urea production in HepatoMOS cultures: Supernatant conditioned medium from 2D monolayer cultures or HepatoMOS cultures was collected and analyzed for albumin and urea production. For 2D culture conditions, hepatocytes were plated directly onto collagen-coated 96-well plates at 20,000 cells / well and cultured using Hepatocyte Medium (HCM, Lonza). For MOS conditions, HepatoMOS containing 10, 50, 100, or 200 hepatocytes / MOS were plated in triplicate in 96-well plates at 50MOS / well in HCM medium. HepatoMOS containing 100 cells / MOS were also plated in triplicate at 50MOS / well in medium provided as a component of the 3D InSight human liver microtissue kit from InSphero (hereafter referred to as InSphero medium) as a comparative medium composition. Conditioned medium was collected on days 1, 4, 7, 10, 14, and 21 and stored at -80°C.

[0194] To quantify albumin production, conditioned media were thawed at room temperature (25° C.) and analyzed using a human albumin ELISA kit (Invitrogen). Samples were diluted to bring albumin concentrations within the dynamic range of the assay: 50 cells / MOS (1:50 for all time points), 100 cells / MOS (1:50 for days 1, 4, 21, 1:100 for days 4, 7, 10), 200 cells / MOS (1:50 for days 1, 4, 21, 1:100 for days 4, 7, 10), 100 cells / MOS in InSphero medium (1:50 for all time points), 2D culture (1:50 for all time points). Urea production was assessed using a urea nitrogen BUN colorimetric detection kit (Invitrogen) and samples were diluted in the following ratios: 50, 100, 200 cells / MOS in HCM medium and 100 cells / MOS in InSphero medium (1:5 for days 1 and 4), 50, 100, 200 cells / MOS in HCM medium and 100 cells / MOS in InSphero medium (1:10 for days 7, 10, 14, and 21), 2D culture (1:5 for all time points). For both the albumin ELISA and urea detection kit, the assay procedures described by the manufacturer were followed. Final measurements were captured using a ClarioSTAR plate reader (BMG).

[0195] Example 15: The HepatoMOS platform enables a sensitive and rapid assay for accurate prediction of DILI Given the high fidelity of cell viability, maintenance, and liver-specific function preservation in HepatoMOS cultures, we evaluated their potential application in predicting the impact of DILI during drug development. The sensitivity and specificity of HepatoMOS were compared with two widely used approaches for DILI detection: i) 2D culture and ii) spheroid PHH. As shown in Figure 48a, among the compounds tested, HepatoMOS showed significantly higher sensitivity in predicting the DILI impact of drugs compared to the 2D-based DILI assay. The HepatoMOS-based DILI assay also outperformed the spheroid-based DILI assay. It showed better sensitivity in detecting two severe DILI drugs, tolcapone and troglitazone, as indicated by lower IC50 values ​​and large safety margins (Figures 48b and 48c). None of the three approaches showed any DILI concerns for non-DILI-related compounds.

[0196] In addition to the viability readout detected by the CTG assay, the HepatoMOS-based DILI assay incorporated a combination of live and dead cell staining to determine the acute cytotoxic effects of compounds. As shown in Figure 47, we observed that the live / dead staining detected the effects of DILI with similar specificity and sensitivity as the ATP content-based readout.

[0197] To further validate the predictive potential of the HepatoMOS assay, the assessment of DILI concerns for AMG-510 was evaluated. AMG-520 is a novel KRAS G12C inhibitor approved by the FDA for the treatment of adult NSCLC harboring the Kras G12C mutation. A phase 1 clinical trial study recently reported that this drug was associated with a higher incidence of severe liver side effects when combined with immunotherapy. Both 2D and spheroid-based DILI assays failed to capture DILI concerns for this drug. However, the HepatoMOS-based assay showed that the IC50 of AMG-510 in a batch of 10 donors was less than 10 μM and the IC50 in a single donor was less than 3.7 μM, further below the reported Cmax (13.4 μM) of this drug. This suggested that AMG-510 may cause liver toxicity when administered to patients at excessive doses that were initially considered acceptable due to the mutant-specific nature of the drug.

[0198] Cell viability assay: Cell viability of PHH in 2D monolayer or HepatoMOS cultures was measured by CellTiter-Glo® 3D cell viability assay (Promega). In addition to the previously reported CTG assay, a combination of calcein AM and EtH staining was also applied to measure HepatoMOS viability.

[0199] DILI assessment in 2D PHH and HepatoMOS: For DILI assessment in 2D monolayers of PHH cultures, PHH were seeded in collagen-coated 96-well plates at a cell density of 20,000 viable cells / well. After 24 hours of culture, PHH were exposed to different concentrations of DILI compounds. The cell viability of PHH was then measured using a CTG assay, and the impact of DILI was evaluated by the change in CTG readings. For DILI assessment in HepatoMOS cultures, HepatoMOS were encapsulated at 100 cells / droplet and cultured in HCM or InSphero medium for 4 days. HepatoMOS were then dispensed into 96-well plates and subsequently treated with drugs.

[0200] Discussion of Results: HepatoMOS provides a simple, rapid, high-throughput, automation-compatible platform for the generation and long-term culture of PHHs. HepatoMOS cultures were found to maintain high cell viability and form uniform cell clusters of PHHs. Substantial growth was observed from day 4 to day 14. Importantly, PHHs did not survive or grow under the same conditions in dome cultures. This indicates three key elements provided by the MOS technology, including cell-cell interactions, a surrounding extracellular matrix, and an efficient nutrient / oxygen penetration system, all enabling successful 3D culture of PHHs. HepatoMOS cultures recapitulate authentic functions of PHHs at several different levels: 1) Morphology and Structure: HepatoMOS retains the morphology and key characteristics of PHHs, such as bile canaliculi and multinucleated cells. 2) Liver-specific Functions: Sustainable high-level secretion of albumin and urea in HepatoMOS cultures was detected for up to 21 days. All three of these features make HepatoMOS a unique and robust model that can be used to study liver disease and liver-specific functions in vitro.

[0201] The development of PHHs growing in 3D spheroids or organoids has made great progress in recent years. Unlike previous reports on 3D long-term culture of human liver organoids, the HepatoMOS cultures established here do not require enrichment of EpCAM+ cells for initial expansion and therefore do not require the use of differentiation medium to convert progenitor cells into mature hepatocytes, which greatly simplifies the procedure and reduces the culture time. Growing PHHs as spheroids also poses several challenges. 1) Cell viability: It may be difficult to maintain the viability of PHHs during the process of spheroid formation, 2) Cell function: It may be difficult to maintain liver-specific functions such as metabolism and bile secretion in spheroids. 3) Spheroid size and uniformity: It may be difficult to achieve consistent spheroid size and uniformity, which may affect the reproducibility of results. 4) Long-term maintenance: It may be difficult to maintain spheroids for long periods of time, which may affect the accuracy of results. 5) Scalability: Scalability is a challenge for spheroid culture due to the limited number of cells that can be cultured in a single spheroid. Based on the above results, HepatoMOS offers a novel solution to each of the problems mentioned above, improving the efficiency and reliability of PHH culture in several ways.

[0202] In vitro 2D models for predicting DILI have limited accuracy and reproducibility, making it difficult to accurately predict DILI in humans. 3D culture systems may provide more physiologically relevant information and thus improve the accuracy of DILI prediction. However, current 3D culture systems still face several challenges: 1) preservation of liver-specific functions; 2) lack of standardized protocols; 3) difficulty in maintaining cell viability: Maintaining cell viability over long periods of time in 3D culture can be challenging due to limited oxygen and nutrient availability; 4) Difficulty in drug testing: Accurately assessing the effects of drugs on PHH in 3D culture can be difficult due to the complexity of the system and its limitations as a monitoring and measurement tool. HepatoMOS demonstrated a more sensitive response to drugs where DILI is a concern, and performed similarly to commercially available PHH spheroid kits. HepatoMOS also demonstrated several advantages over other 3D cultures of PHH, including the ability to recapitulate many characteristics of the human liver and liver-specific functions.

[0203] The MOS technology allows for high throughput and automation of organoid culture. Similarly, the HepatoMOS platform also allows for the simultaneous culture of thousands of uniform PHH microtissue-like structures derived from a single donor or pooled donors. The use of the HepatoMOS system for high throughput and automation of PHH culture offers several advantages and has the potential to significantly advance our understanding of liver biology and disease.

[0204] Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware and may be described as a non-transitory computer readable storage medium storing a set of instructions that may be executed by a processor (e.g., a computer, a tablet, a smartphone, etc.) that, when executed by the processor, causes the processor to control / perform any of the steps including, but not limited to, displaying, communicating with a user, analyzing, changing parameters (including timing, frequency, intensity, etc.), making decisions, alerting, etc.

[0205] When a feature or element is referred to herein as being on another feature or element, it may be directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly" on another feature or element, there are no intervening features or elements. It will also be understood that when a feature or element is referred to as being "connected," "attached," or "coupled" to another feature or element, it may be directly connected, attached, or coupled to the other feature or element, or there may be intervening features or elements. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" to another feature or element, there are no intervening features or elements. Although features and elements are described or illustrated with respect to one embodiment, the features and elements so described or illustrated may also apply to other embodiments. Those skilled in the art will also understand that references to structures or features located "adjacent" to another feature may have portions that overlap with the adjacent feature or that underlie the adjacent feature.

[0206] The terms used herein are merely for the purpose of describing particular embodiments and are not intended to be limiting. For example, as used herein, the singular forms "a," "an," and "the" are intended to include the plurals unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items (sometimes abbreviated as " / ").

[0207] Spatially relative terms, such as "below," "lower," "up," "above," and the like, may be used herein for ease of description to describe the relationship of an element or feature shown in the figures to another element(s) or feature(s). It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. For example, if the device in the figures is inverted, an element described as "below" or "beneath" another element or feature would be oriented "above" the other element or feature. Thus, the exemplary term "below" may encompass both an upward and downward orientation. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein will be interpreted accordingly. Similarly, terms such as "upward," "downward," "vertically," "horizontally," and the like are used herein for descriptive purposes only, unless otherwise noted.

[0208] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context dictates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element discussed herein may be referred to as a second feature / element, and similarly, a second feature / element discussed herein may be referred to as a first feature / element, without departing from the teachings of the present invention.

[0209] Throughout this specification, unless the context requires otherwise, the word "comprise" and embodiments such as "comprises" and "comprising" mean that various components can be used together in methods and articles (e.g., compositions and apparatuses, including devices and methods). For example, it will be understood that the term "comprising" implies the inclusion of any stated elements or steps, but not the exclusion of any other elements or steps.

[0210] In general, any of the apparatus and methods described herein should be understood to be inclusive, but alternatively, all or a subset of the components and / or steps may be exclusive and may be expressed as "consisting of" or alternatively "consisting essentially of" the various components, steps, sub-components, or sub-steps.

[0211] As used herein, including in the examples, unless expressly specified otherwise, all numbers may be read as if they are preceded by the word "about" or "approximately", even if the term is not expressly indicated. The phrase "about" or "approximately", when describing a size and / or location, may be used to indicate that the stated value and / or location is within a reasonably expected range of values ​​and / or locations. For example, a numerical value may have a value of + / -0.1% of the stated value (or range of values), + / -1% of the stated value (or range of values), + / -2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / -10% of the stated value (or range of values), etc. Unless the context dictates otherwise, any numerical value given herein should also be understood to include "about" or "approximately" that value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical ranges described herein are intended to include all subranges therein. As would be well understood by one of ordinary skill in the art, when a value is disclosed to be "less than or equal to," it is also understood that "greater than or equal to" and possible ranges between those values ​​are also disclosed. For example, when a value "X" is disclosed, "less than or equal to X" as well as "greater than or equal to X" (e.g., X is a number) are also disclosed. It is also understood that throughout this application, data is provided in several different formats, and this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, when a specific data point "10" and a specific data point 15 are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and equal to 15 are considered to be disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0212] Although various exemplary embodiments have been described above, any of a number of modifications may be made to the various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which the various method steps described are performed may often be changed in alternative embodiments, and in other alternative embodiments, one or more method steps may be omitted entirely. Optional features of the various device and system embodiments may be included in some embodiments but not in other embodiments. Thus, the foregoing description has been provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as described in the claims.

[0213] The examples and figures contained herein show, by way of illustration, not limitation, specific embodiments in which the subject matter may be practiced. As previously mentioned, other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the subject matter of the present invention may be individually or collectively referred to herein by the term "the present invention" merely for convenience, and without any intention to spontaneously limit the scope of the present application to any single invention or inventive concept, if a plurality is actually disclosed. Thus, although specific embodiments have been illustrated and described herein, any embodiment calculated to achieve the same purpose may be substituted for the specific embodiment shown. The present disclosure is intended to cover any adaptations or embodiments of the various embodiments. Combinations of the above embodiments with other embodiments not specifically described herein will be apparent to those skilled in the art upon reviewing the above description.

Claims

1. A method for generating microorganospheres (MOS) from hepatocytes.

2. 2. The method of claim 1, wherein the cell density is 80-160 cells / MOS droplet and the droplet diameter is 200-300 uM.

3. 3. The method of claim 2, wherein the cell density is 100 cells / MOS droplet.

4. MOS obtained by the method of claim 1.

5. MOS, produced from hepatocytes.

6. A method of drug screening using the MOS of claim 4 or 5.

7. 7. The method of claim 6, wherein the method evaluates one or more aspects of the pharmacodynamic profile of a drug.

8. The method of claim 6, wherein the method is applied to high throughput drug screening.

9. The method of claim 6 , wherein the method assesses drug toxicity.

10. 7. The method of claim 6, wherein the method assesses drug-induced liver injury (DILI).

11. The method of claim 6 , wherein the method evaluates the effect of chronic administration of a drug.

12. 6. Use of a MOS according to claim 4 or 5 in a method of drug screening.

13. The MOS or method of any one of claims 1 to 12, wherein the hepatocytes are primary human hepatocytes (PHH).

14. The MOS, method or use according to any one of claims 1 to 13, wherein the hepatocytes are adult hepatocytes.

15. The MOS, method or use according to any one of claims 1 to 14, wherein the hepatocytes are isolated from a donor.

16. 16. The MOS, method or use of claim 15, wherein the donor is a patient in need of treatment.

17. 17. The MOS, method or use of any one of claims 1 to 16, wherein cells within the MOS retain viability for more than 3 weeks in culture.

18. The MOS, method or use of any one of claims 1 to 17, wherein cells within the MOS retain liver-specific functions.

19. The MOS, method or use according to any one of claims 1 to 18, wherein said MOS models liver regeneration.

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