High-precision drug screening for personalized cancer therapy

Abstract

Methods and devices for high-precision drug screening for personalized cancer therapy include generating a library of mature microorganospheres from a single patient's tissue sample, e.g., from a tumor sample, including patient-derived microorganospheres (PMOS). Further described herein are methods and systems for screening patients using these patient-derived microorganospheres, including personalized therapies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 120,719, filed December 2, 2020, and U.S. Application No. 17 / 118,586, filed December 10, 2020, the entire contents of both of which are incorporated herein by reference.

[0002] The methods, devices, and compositions described herein relate to patient-derived microorganospheres (PMOS), as well as methods and devices for forming and using PMOS. In particular, methods and devices are described herein for identifying one or more formulations that may be effective for treating a particular patient. [Background technology]

[0003] Model cell and tissue systems are useful in biological and medical research. The most common practice is to derive immortalized cell lines from tissues and then culture them in two-dimensional (2D) conditions (e.g., Petri dishes or well plates). However, while 2D cell lines are very useful for basic research, they do not correlate well with individual patient responses to therapy. In particular, 3D 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 3D cell aggregates that have been studied. However, both organoids and spheroids have limitations that limit their effectiveness.

[0004] Multicellular tumor spheroids were first described in the early 1970s and were obtained by culturing cancer cell lines under non-adherent conditions. Spheroids typically form from cancer cell lines as free-floating cell aggregates in ultra-low attachment plates. Spheroids have been shown to maintain more stem cell-associated properties than 2D cell cultures.

[0005] Organoids are in vitro-derived cell aggregates containing stem cell populations that can differentiate into cells of major cell lineages. Organoids typically have a diameter of more than 1 mm and are cultured through subculture. Typically, organoid cultures grow and expand more slowly than 2D cell cultures. Because generating organoids from clinical samples requires a sufficient number of live cells (e.g., hundreds to thousands) for starting, obtaining organoids from low-volume samples, such as biopsies, is often challenging, and even if successful, it takes a considerable amount of time to grow the culture for application, such as drug testing. Furthermore, organoids vary widely in size, shape, and cell number. Organoids may require complex growth factor cocktails and culture conditions to grow and express desired cell types.

[0006] Neither tumor spheroids nor organoids are optimal for rapid and reliable screening, particularly for personalized medicine, e.g., ex vivo testing of drug response. For example, oncology practice continually faces the significant challenge of matching the appropriate treatment regimen with the appropriate patient, as well as balancing relative benefits with risks to achieve the most favorable outcomes. 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 patient response to therapy, there are major limitations to using organoids to guide treatment. As mentioned above, obtaining and growing organoids, especially patient-derived organoids from tumor samples for drug sensitivity testing, takes several months, which reduces their clinical applicability because patients cannot wait that long to receive treatment. Furthermore, the number of organoids required for drug screening using more than a dozen compounds cannot be obtained in a clinically feasible timeframe from core biopsy specimens, which are often the only available form of tissue from patients with metastatic or inoperable cancer.The significant failure rate in obtaining organoids from biopsies also prevents their use as a reliable diagnostic assay.Furthermore, there can be a high degree of variability in organoid size (and potentially response), especially with longer culture times and therefore higher passage numbers.

[0007] Due to their superior correlation with patient outcomes, PDMCs have also been utilized to replace 2D cell lines as a high-throughput screening platform for drug discovery and pharmacological small molecule screening, such as RNAi and CRISPR. However, compared to cell lines, these PDMC models (including spheroids and organoids) typically require much longer growth and manipulation, making them difficult and expensive for high-throughput applications. The long time it takes to grow these models to increase cell numbers also leads to the dominance of the fastest-growing clones in the plastic and the elimination of other clones, making the models more homogeneous and losing their 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. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 5,516,681 [Patent Document 2] U.S. Patent No. 5,559,022 [Patent Document 3] U.S. Patent No. 4,683,195 [Non-patent literature]

[0009] [Non-Patent Document 1] Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 4th edition, Wiley Liss, John Wiley & Sons, 2000. [Non-patent document 2] Basic Cell Culture: A Practical Approach, edited by Davis, Oxford University Press, 2002. [Non-patent document 3] Animal Cell Culture: A Practical Approach, edited by Masters, 2000 [Non-licensed Document 4] Molecular Cloning: A Laboratory Manual (1989), 2nd Edition, edited by Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press, Chapters 16 and 17. [Non-licensed Document 5] DNA Cloning, Volume I and Volume II, edited by Glover, 1985 [Non-licensed Document 6] Oligonucleotide Synthesis, edited by MJ Gait, 1984 [Non-licensed Document 7] Nucleic Acid Hybridization, edited by D. Hames & SJ Higgins, 1984 [Non-licensed Document 8] Transcription and Translation, edited by BD Hames & SJ Higgins, 1984 [Non-licensed Document 9] Culture Of Animal Cells, RI Freshney, Alan R. Liss, Inc., 1987 [Non-licensed Document 10] Immobilized Cells And Enzymes, IRL Press, 1986 [Non-licensed Document 11] Perbal(1984), A Practical Guide To Molecular Cloning [Non-licensed Document 12] Methods In Enzymology (Academic Press, Inc., NY) [Non-licensed Document 13] Gene Transfer Vectors For Mammalian Cells, edited by JH Miller and MP Calos, Cold Spring Harbor Laboratory, 1987 [Non-Patent Document 14] Methods in Enzymology, vol. 154 and vol. 155, edited by Wu et al., Academic Press Inc., NY [Non-Patent Document 15] Immunochemical Methods in Cell and Molecular Biology (eds. Mayer and Walker, Academic Press, London, 1987) [Non-Patent Document 16] Handbook of Experimental Immunology, Volumes I-IV, edited by D. M. Weir and C. C. Blackwell, 1986 [Non-Patent Document 17] R. Mahesparan, Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro. Acta Neuropathol (1999) 97:231-239 Summary of the Invention [Problem to be solved by the invention]

[0010] 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 or biopsies. In particular, it would be useful to provide methods and devices that could enable the generation of large numbers of patient-derived tissue models with predictable and clinically relevant characteristics from a single biopsy, such as an 18-gauge core biopsy that can be completed within 7-10 days of biopsy acquisition. This would enable robust and reliable testing and minimize delays in directing patient-specific therapies. Furthermore, it would be useful to generate patient-derived models that can be rapidly propagated in a highly parallel manner, producing units with smaller and more uniform dimensions, greater control over the number of cells per unit, and better spreadability (e.g., via increased surface area-to-volume ratios) for high-throughput screening applications. [Means for solving the problem]

[0011] Described herein are patient-derived microorganospheres (PMOS), devices and methods for generating PMOS, and devices and methods for using PMOS. Additionally, described herein are methods and systems for screening patients using these patient-derived microorganospheres, including personalized therapies.

[0012] Generally, described herein are methods and devices for forming and expanding patient-derived micro-organospheres containing cells derived from a patient, e.g., cells extracted from a small patient biopsy (e.g., for quick diagnosis to guide treatment), from resected involved tissue, including a resected primary tumor or part of a dysfunctional organ (e.g., for high-throughput screening), and / or from already established PDMCs, including patient-derived xenografts (PDXs) and organoids (e.g., for the purpose of generating micro-organospheres for high-throughput screening).

[0013] These PMOS can be formed from normal primary cells (e.g., normal organ tissue) or tumor tissue. For example, in some variations, these methods and devices can form PMOS from cancerous tumor biopsy tissue, allowing for customized treatments to be selected using the specific tumor tissue being examined. Surprisingly, these methods and devices enable the formation of hundreds, thousands, or even tens of thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000, or more) of PMOS from a single tissue biopsy within hours of the biopsy being removed from the patient. Primary cells dissociated from a patient biopsy may be combined with a fluid matrix material, such as a basement membrane matrix (e.g., MATRIGEL), to form microorganospheres. The resulting plurality of patient-derived microorganospheres can have a defined range of dimensions (e.g., diameters between 10 μm and 700 μm, and any subrange therebetween) and initial number of primary cells (e.g., between 1 and 1000, and particularly lower cell numbers, e.g., between 1 and 200). The number and / or diameter of cells can be controlled, e.g., within + / - 5%, 10%, 15%, 20%, 25%, 30%, etc. These PMOs, 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 to 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 and biologically relevant PMOS, potentially saving significant time in patient treatment, e.g., the development and deployment of cancer treatment plans. The PMOS described herein replicate the tissue environment from which the biopsy was derived, e.g., the three-dimensional (3D) tumor microenvironment, and rapidly form 3D cellular structures representative of that tissue environment. The PMOS described herein are also referred to as "droplets." Each PMOS may include, for example, as part of the fluid matrix material, growth factors and structural proteins (e.g., collagen, laminin, nidogen, etc.) that can mimic the native tissue (e.g., tumor) environment.Virtually any primary cell tissue may be used, including virtually any tumor tissue.

[0014] For example, to date, PMOS have been successfully produced from all tumor types and sites tested (e.g., current success rate of 100%, n=32, including colon, esophageal, skin (melanoma), uterus, bone (sarcoma), kidney, ovary, lung, and breast cancers from primary or metastatic sites, including liver, peritoneum, and diaphragm). Tissue types used for effective generation of microorganospheres may also be metastatic from another site. In some variations, PMOS described herein may be grown from fine needle aspirates (FNAs) or circulating tumor cells (CTCs), e.g., liquid biopsies. Growth and proliferation are typically observed within as little as 3-4 days, and PMOS can be maintained and passaged for several months or may be cryopreserved and / or used immediately for assays (e.g., within the first 7-10 days).

[0015] In particular, methods for forming patient-derived microorganospheres are described herein. Generally, 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 microorganospheres typically less than about 1000 μm in diameter (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) into which the dissociated primary tissue cells are distributed. The number of dissociated cells may be within the predetermined range (e.g., between about 1 and about 500 cells, between about 1 and about 200 cells, between about 1 and about 150 cells, between about 1 and about 100 cells, between about 1 and about 75 cells, between about 1 and about 50 cells, between about 1 and about 30 cells, between about 1 and about 20 cells, between about 1 and about 10 cells, between about 5 and about 15 cells, between about 20 and about 30 cells, between about 30 and about 50 cells, between about 40 and about 60 cells, between about 50 and about 70 cells, between about 60 and about 80 cells, between about 70 and about 90 cells, between about 80 and about 100 cells, between about 90 and about 110 cells, etc., including about 1 cell, about 10 cells, about 20 cells, about 30 cells, about 40 cells, about 50 cells, about 60 cells, about 70 cells, etc.). Any of these methods can be configured to produce microorganospheres of repeatable size (eg, with a narrow size distribution), as described herein.

[0016] Dissociated cells may be freshly biopsied or dissociated in any suitable manner, including mechanical dissociation and / or chemical dissociation (e.g., enzymatic disaggregation using one or more enzymes, e.g., collagenase, trypsin, etc.). Dissociated cells may optionally be 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 labeled (e.g., with one or more markers) that can be used to aid in selection. In some variations, 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. In some examples, PMOS may be sorted by non-cellular objects, e.g., magnetic beads or barcode-like particles. Non-cellular objects that can be used for sorting (e.g., magnetic beads, fluorescently labeled particles, etc.) may be incorporated into PMOS as part of any of the methods described herein. Alternatively, the cells may be used without sorting.

[0017] In some variations, the dissociated cells may be modified by treatment with one or more agents. For example, the cells may be genetically modified. In some variations, the cells may be modified using CRISPR-Cas9 or another gene editing technique. In some variations, 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.

[0018] One or more additional materials may be combined with the dissociated cells and 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 a different biopsy (e.g., primary cells from a different dissociated tissue) and / or cultured cells. The additional cells may be, for example, immune cells, stromal cells, endothelial cells, etc. The additional materials may include culture media (e.g., growth medium, freezing medium, etc.), growth factors, support network molecules (e.g., collagen, glycoproteins, extracellular matrix, etc.), etc. In some variations, the additional materials may include a drug composition. In some variations, the unpolymerized mixture includes only the dissociated tissue sample (e.g., primary cells) and fluid matrix material.

[0019] The method can rapidly generate multiple patient-derived micro-organospheres from a single tissue biopsy, resulting in the generation of greater than about 500 patient-derived micro-organospheres per biopsy (e.g., greater than about 600, greater than about 700, greater than about 800, greater than about 900, greater than about 1000, greater than about 2000, greater than about 2500, greater than about 3000, greater than about 4000, greater than about 5000, greater than about 6000, greater than about 7000, greater than about 8000, greater than about 9000, greater than about 10,000, greater than about 11,000, greater than about 12,000, etc.). The biopsy can be a standard size biopsy, e.g., an 18G (e.g., 14G, 16G, 18G, etc.) core biopsy. For example, the volume of tissue removed by biopsy and used to form a plurality of patient-derived micro-organospheres may be a small cylinder (obtained with a biopsy needle) between about 1 / 32 and 1 / 8 of an inch in diameter and about 3 / 4 to 1 / 4 inch in length, e.g., about 1 / 16 inch in diameter and 1 / 2 inch in length. The biopsy may be obtained by needle biopsy, e.g., core needle biopsy. In some variations, the biopsy may be obtained by fine needle aspiration. Other types of biopsies that can be used include shave biopsy, punch biopsy, incisional biopsy, excisional biopsy, etc. Typically, material from a single patient biopsy may be used to generate a plurality of such patient-derived micro-organospheres (e.g., greater than about 2000, greater than about 5000, greater than about 7500, greater than about 10,000, etc.). A plurality of patient-derived microorganospheres may be formed using an apparatus (as described herein) that can be configured to generate this large number of highly regular (size, cell number, etc.) microorganospheres as described above.In some variations, the methods and apparatus may produce a plurality of microorganospheres at a rapid rate (e.g., greater than about 1 microorganosphere per minute, greater than about 1 microorganosphere per 10 seconds, greater than about 1 microorganosphere per 5 seconds, greater than about 1 microorganosphere per 2 seconds, greater than about 1 microorganosphere per second, greater than about 2 microorganospheres per second, greater than about 3 microorganospheres per second, greater than about 4 microorganospheres per second, greater than about 5 microorganospheres per second, greater than about 10 microorganospheres per second, greater than about 50 microorganospheres per second, greater than about 100 microorganospheres per second, greater than about 125 microorganospheres per second, etc.).

[0020] For example, in some variations, 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 micro-organospheres, at least in part, by controlling one or more flows of the unpolymerized mixture (and / or dissociated tissue and fluid matrix) and a material (e.g., a hydrophobic material, oil, etc.) that is immiscible with the unpolymerized mixture. For example, in some variations, the methods may be performed using a microfluidic device. In some variations, multiple micro-organospheres 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 the same source of dissociated primary tissue and / or fluid matrix.

[0021] The unpolymerized material can be polymerized in a variety of different ways to form patient-derived micro-organospheres. In some variations, the method may include polymerizing the micro-organospheres by a change in temperature (e.g., raising the temperature above a threshold value, e.g., above about 20°C, above about 25°C, above about 30°C, above about 35°C).

[0022] Once polymerized, the patient-derived microorganospheres may be expanded, e.g., in culture, and / or assayed either before or after culture, and / or cryopreserved either before or after culture. Patient-derived microorganospheres may be cultured for any suitable period of time, particularly 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 variations, patient-derived microorganospheres may be cryopreserved or assayed before passage 6, thereby maintaining cellular heterogeneity within the patient-derived microorganospheres and limiting the number of passages to prevent faster-dividing cells from outgrowing slower-dividing cells.

[0023] Generally, because a biopsy from the same patient can provide a large 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 of the patient-derived micro-organospheres may be cryopreserved (e.g., more than half) and a portion may be cultured and / or assayed. As described in more detail herein, the cryopreserved patient-derived micro-organospheres may be stored or used at a later time (e.g., assayed, passaged, etc.).

[0024] Accordingly, methods are described herein, such as methods for forming a plurality of patient-derived microorganospheres. For example, a method for forming a plurality of patient-derived microorganospheres may include combining a dissociated tissue sample with 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 patient-derived microorganospheres having a diameter of between 50 and 500 μm, each having between 1 and 200 dissociated cells distributed therein.

[0025] For example, a method of forming a plurality of patient-derived microorganospheres may include combining a dissociated tissue sample with a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets from a continuous stream of the unpolymerized mixture having a size variation of less than 25%; and polymerizing the droplets by heating to form a plurality of patient-derived microorganospheres, each having between 1 and 200 dissociated cells distributed within each patient-derived microorganosphere.

[0026] In some variations, the methods described herein for forming a plurality of patient-derived micro-organospheres may include combining a dissociated tissue sample with a fluid matrix material to form an unpolymerized mixture; merging 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 dimensional variation of less than 25%; polymerizing the droplets to form a plurality of patient-derived micro-organospheres having a diameter of between 50 and 500 μm and containing between 1 and 200 dissociated cells distributed therein; and separating the plurality of patient-derived micro-organospheres from the immiscible fluid.

[0027] Any of these methods may include modifying the cells within the dissociated tissue sample prior to forming the droplets.

[0028] Forming a plurality of droplets can include forming a plurality of droplets of the unpolymerized mixture of uniform size with a size variation of less than about 25% (e.g., less than about 20% size variation, less than about 15% size variation, less than about 10% size variation, less than about 8% size variation, less than about 5% size variation, etc.). The size variation can also be described as a narrow distribution of size variation. For example, the size distribution can include a patient-derived microorganosphere size distribution (e.g., number of formed microorganospheres relative to microorganosphere diameter) with a small standard deviation (e.g., 15% or less standard deviation, 12% or less standard deviation, 10% or less standard deviation, 8% or less standard deviation, 6% or less standard deviation, 5% or less standard deviation, etc.).

[0029] Any of these methods may further include plating or distributing the patient-derived microorganospheres. For example, in some variations, the methods may include assembling patient-derived microorganospheres from various sources into a container prior to assay. For example, the microorganospheres may be placed in a multiwell plate. Thus, any of these methods may include dispensing the patient-derived microorganospheres into a multiwell plate prior to assaying the patient-derived microorganospheres. One or more (or, in some variations, equal amounts) patient-derived microorganospheres may be included per well. In some variations, applying the patient-derived microorganospheres into the container may include placing the organospheres into multiple chambers separated by at least partially permeable membranes to allow supernatant material to circulate between the chambers. This allows the patient-derived microorganospheres to share the same supernatant.

[0030] In any of these methods, the patient-derived microorganospheres may be assayed. The assay may generally involve exposing or treating individual patient-derived microorganospheres to conditions (e.g., a drug composition) to determine whether the drug composition has an effect (and in some cases, what effect) on the cells of the patient-derived microorganospheres. The assay may involve exposing a subset of the patient-derived microorganospheres (individually or collectively) to one or more concentrations of a drug composition, leaving the patient-derived microorganospheres exposed for a predetermined period of time (minutes, hours, days, etc.), and / or removing the drug composition from the exposure, and then culturing the patient-derived microorganospheres for a predetermined period of time. The patient-derived microorganospheres may then be examined to identify any effects, including, among other things, toxicity to the cells in the patient-derived microorganospheres or changes in the morphology and / or proliferation of the cells in the patient-derived microorganospheres. In some variations, the assay may include labeling live or fixed cells within the patient-derived microorganospheres (e.g., by immunocytochemistry). 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 variations, assaying multiple patient-derived microorganospheres may include sampling one or more of the supernatant, environment, and microenvironment of the patient-derived microorganospheres for secreted factors and other effects. In any of these variations, the patient-derived microorganospheres may be recovered after the assay for further assay, expansion, or storage (e.g., cryopreservation, fixation, etc.) for later examination.

[0031] As stated, virtually any assay can be used. For example, the PMOS described herein can be used to assay genomic, transcriptomic, proteomic, or metagenomic markers (e.g., methylation). Thus, any of the 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 aid in the identification of drugs and / or therapies for treating patients.

[0032] Any suitable tissue sample can be used. In some variations, the tissue sample can include a biopsy sample from a metastatic tumor. For example, the tissue sample can include a clinical tumor sample, which can include both cancer cells and stromal cells. In some variations, the tissue sample includes tumor cells and one or more of mesenchymal cells, endothelial cells, and immune cells.

[0033] Any of the methods described herein may include initially distributing dissociated cells from a tissue biopsy uniformly, or in some variations, non-uniformly, throughout a fluid matrix material at any suitable concentration. For example, in some variations, the methods described herein may include dissociating dissociated tissue cells at a concentration of 1×10 6 within the fluid matrix material. 7 cells / ml (e.g., 9 x 10 6 cells / ml, 7×10 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 combining the dissociated tissue sample with a fluid matrix material so that the dissociated tissue sample is distributed at a density of less than 10 ...

[0034] Generally, droplet formation can involve forming droplets from a continuous stream of unpolymerized mixture. For example, droplet formation can involve applying one or more converging streams of a fluid immiscible with the unpolymerized mixture to the stream of unpolymerized mixture. The streams can be combined in a microfluidic device, such as an apparatus having multiple converging channels through which the unpolymerized mixture and the immiscible fluid interact, to form droplets with precisely controlled volumes. In some variations, the droplets can be formed (e.g., pinched off) in an excess of immiscible material, and the droplets can be simultaneously and / or subsequently polymerized to form patient-derived microorganospheres. For example, a region where the streams converge can be configured to polymerize the unpolymerized mixture after droplet formation, e.g., by heating, and / or a downstream region can be configured to polymerize the unpolymerized mixture after the droplets are formed and surrounded by the immiscible material. In some variations, the immiscible material is heated (or alternatively cooled) to a temperature that promotes polymerization of the unpolymerized material to form patient-derived microorganospheres. For example, polymerization can include heating the droplets to above 35°C.

[0035] Thus, in any of these methods, forming the droplets can include forming the droplets in a fluid that is immiscible with the unpolymerized mixture. Additionally, any of these methods can include separating the immiscible fluid from the patient-derived microorganospheres. For example, any of these methods can include removing the immiscible fluid from the patient-derived microorganospheres. Generally, the immiscible fluid can include a liquid (e.g., oil, polymer, etc.), particularly including a hydrophobic material or another material that is immiscible with the unpolymerized (e.g., aqueous) material.

[0036] The fluid matrix material can be a synthetic or non-synthetic unpolymerized basement membrane material. In some variations, the unpolymerized basement membrane material can comprise a polymeric hydrogel. In some variations, the fluid matrix material can comprise Matrigel. Thus, combining the dissociated tissue sample with the fluid matrix material can include combining the dissociated tissue sample with a basement membrane matrix.

[0037] 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.) of removing the tissue sample from the patient.

[0038] Further described herein are methods for assaying or preserving patient-derived microorganospheres. For example, the methods may include combining a dissociated tissue sample with a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets of the unpolymerized mixture having a dimensional variation of less than 25%; polymerizing the droplets to form a plurality of patient-derived microorganospheres having a diameter of between 50 and 700 μm and containing between 1 and 1000 dissociated cells distributed therein; and assaying or cryopreserving the plurality of patient-derived microorganospheres.

[0039] In some variations, the method may include combining the dissociated tissue sample with 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 patient-derived micro-organospheres having diameters between 50 and 500 μm, each having between 1 and 200 dissociated cells distributed therein; and cryopreserving or assaying the plurality of patient-derived micro-organospheres within 15 days, wherein the micro-organoids are assayed to determine the effect of one or more agents on the cells within the patient-derived micro-organospheres.

[0040] For example, the method includes combining a dissociated tissue sample with a fluid matrix material to form an unpolymerized mixture; combining a stream of the unpolymerized mixture with one or more streams of fluid that are immiscible with the unpolymerized mixture to form a plurality of droplets having a droplet size variation of less than 25%; polymerizing the droplets by warming to form patient-derived micro-organospheres having diameters between 50 and 500 μm, each containing between 1 and 200 dissociated cells distributed therein; and assaying or cryopreserving the patient-derived micro-organospheres prior to passage 6, thereby maintaining the heterogeneity of the cells within the patient-derived micro-organospheres, wherein the assay further includes assaying to determine the effect of one or more agents on the cells within the patient-derived micro-organospheres.

[0041] In any of these methods, the cellular heterogeneity within the patient-derived microorganospheres is maintained by cryopreserving or assaying a plurality of patient-derived microorganospheres prior to passage 6. Any of these methods may further comprise modifying the cells within the dissociated tissue sample prior to forming the droplets.

[0042] Forming the droplets can include forming a plurality of uniformly sized droplets of the unpolymerized mixture having a size variation of less than about 25% (e.g., less than about 20%, less than about 15%, less than about 10%, less than about 7%, less than about 5%, etc.).

[0043] Any of these methods may include culturing the patient-derived microorganospheres for a suitable period of time, as described above (e.g., culturing the patient-derived microorganospheres for between 2 and 14 days prior to assay). For example, these methods may include removing immiscible fluids from the patient-derived microorganospheres prior to culturing. In some variations, culturing the patient-derived microorganospheres includes culturing the patient-derived microorganospheres in suspension.

[0044] In any of the methods described herein, patient-derived microorganospheres may be combined and cultured in bulk. In some variations, for example, when forming a library, patient-derived microorganospheres sorted by cell size and / or number may be combined and cultured. Patient-derived microorganospheres may be cultured in suspension or in layers (e.g., in dishes, chambers, tubes, etc.).

[0045] Generally, assaying patient-derived microorganospheres may include genomic, transcriptomic, epigenomic, and / or metabolic analysis of cells in the patient-derived microorganospheres before and / or after assaying or before and / or after cryopreservation of the patient-derived microorganospheres. Any of these methods may include assaying the patient-derived microorganospheres by exposing them to a drug (e.g., a drug composition).

[0046] In any of these methods, the assay may include visually assaying, either manually and / or automatically, the effect of one or more agents on the cells in the patient-derived microorganospheres. Any of these methods may include labeling or tagging the cells in the patient-derived microorganospheres for visualization. For example, the assay may include fluorescently assaying the effect of one or more agents on the cells.

[0047] The patient-derived microorganospheres described herein are themselves novel and may be characterized as compositions. For example, a composition may comprise a plurality of cryopreserved patient-derived microorganospheres, each having a spherical shape with a diameter between 50 μm and 500 μm, and comprising a polymerized substrate and between about 1 and 1,000 dissociated primary cells that have been passaged less than six times and distributed within the substrate, thereby maintaining cellular heterogeneity within the patient-derived microorganospheres.

[0048] Further described herein is a composition comprising a plurality of cryopreserved patient-derived micro-organospheres, wherein each patient-derived micro-organosphere has a spherical shape with a diameter between 50 μm and 500 μm, the patient-derived micro-organospheres have a dimensional variation of less than 25%, and each patient-derived micro-organosphere comprises a polymerized substrate and between about 1 and 500 dissociated primary cells that have been passaged less than six times distributed within the substrate, thereby maintaining cellular heterogeneity within the patient-derived micro-organospheres.

[0049] The primary cells may be primary tumor cells. For example, the dissociated primary cells may be genetically or biochemically modified. The plurality of cryopreserved patient-derived microorganospheres may have uniform dimensions with a dimensional variation of less than 25%. In some variations, the plurality of cryopreserved patient-derived microorganospheres may include patient-derived microorganospheres from various sources. In any of the microorganospheres, the majority of cells in each microorganosphere may include non-stem cells. In some variations, the primary cells include metastatic tumor cells. The primary cells may include both cancer cells and stromal cells. In some variations, the primary cells include tumor cells and one or more of mesenchymal cells, endothelial cells, and immune cells.

[0050] Primary cells are plated in the polymerized substrate at a density of, for example, 5 × 10 7 cells / ml, 1×10 7 cells / ml, 9×10 6 cells / ml, 7×10 6 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 can be dispensed at a density such as cells / ml.

[0051] Generally, the polymerized substrate may comprise a basement membrane matrix (e.g., Matrigel). In some variations, the polymerized substrate comprises a synthetic material.

[0052] Micro-organoids may have a diameter between 50 μm and 1000 μm, more preferably between 50 μm and 700 μm, more preferably between 50 μm and 500 μm, between 50 μm and 400 μm, between 50 μm and 300 μm, between 50 μm and 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.).

[0053] As noted, the patient-derived microorganospheres described herein can initially comprise any suitable number of primary tissue cells in each patient-derived microorganosphere, e.g., less than about 200 primary cells, more preferably less than about 150 primary cells, more preferably less than about 100 primary cells, more preferably less than about 75 primary cells, less than about 50 cells, less than about 30 cells, less than about 25 cells, less than about 20 cells, less than about 10 cells, less than about 5 cells, etc. In some variations, each patient-derived microorganosphere comprises between about 1 and 500 cells, between about 1 and 400 cells, between about 1 and 300 cells, between about 1 and 200 cells, between about 1 and 150 cells, between about 1 and 100 cells, between about 1 and 75 cells, between about 1 and 50 cells, between about 1 and 30 cells, between about 1 and 25 cells, between about 1 and 20 cells, etc.

[0054] Also described herein are devices for forming patient-derived microorganospheres and methods of operating those devices to form patient-derived microorganospheres. For example, a method of operating a device for forming patient-derived microorganospheres is described herein, comprising the steps of receiving into a first port an unpolymerized mixture comprising a chilled mixture of a dissociated tissue sample and a first fluid matrix material; receiving into a second port a second fluid that is immiscible with the unpolymerized mixture; combining the stream of the unpolymerized mixture with one or more streams of the second fluid to form droplets of the unpolymerized mixture having uniform dimensions that vary by less than 25%; and polymerizing the droplets of the unpolymerized mixture to form a plurality of patient-derived microorganospheres.

[0055] A method of operating an apparatus for forming patient-derived micro-organospheres may include receiving an unpolymerized mixture into a first port, the unpolymerized mixture comprising a cooled mixture of a dissociated tissue sample and a first fluid matrix material; receiving a second fluid that is immiscible with the unpolymerized mixture into a second port; combining a stream of the unpolymerized mixture at a first rate with one or more streams of the second fluid at a second rate to form droplets of the unpolymerized mixture having uniform dimensions that vary by less than 25% and are between 50 μm and 500 μm in diameter; and polymerizing the droplets of the unpolymerized mixture to form a plurality of patient-derived micro-organospheres.

[0056] Any of these methods may include placing a first reservoir containing the unpolymerized mixture in fluid communication with the first port. For example, the method may include combining a dissociated tissue sample with a first fluid matrix material to form an unpolymerized mixture. In some variations, the method includes adding the unpolymerized mixture to a first reservoir in fluid communication with the first port. The methods may include placing a second reservoir containing a second fluid in fluid communication with the 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 variations, receiving the second fluid includes receiving an oil.

[0057] Generally, the methods can include separating a second fluid (e.g., an immiscible fluid) from a plurality of patient-derived microorganospheres. This fluid separation can be manual or automated. For example, the second (immiscible) fluid can be removed by washing, filtration, or any other suitable method.

[0058] 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 variations, the first flow rate is greater than the second flow rate. Either the flow rate or the amount of material (e.g., the unpolymerized mixture), or both the flow rate and the amount of material (e.g., the unpolymerized mixture), can be less than that of the second fluid, such that the unpolymerized mixture can be encapsulated in precisely controlled droplets, as described herein, and then polymerized, for example, within the second fluid.

[0059] In some variations, the combining of the streams includes driving the stream of the unpolymerized mixture across a junction where one or more streams of a second fluid also join. Polymerizing the droplets may include heating the droplets to a temperature above which the unpolymerized material will polymerize (e.g., above about 25°C, above about 30°C, above about 35°C, etc.).

[0060] Any of these methods may include aliquoting a plurality of patient-derived microorganospheres, for example, into a multi-well dish.

[0061] Further described herein are methods for treating patients using these patient-derived microorganospheres and methods for assaying these patient-derived microorganospheres. For example, the methods may include obtaining a patient biopsy from a tumor; forming a plurality of microorganospheres having a diameter of between 50 and 500 μm from the patient biopsy within two weeks of taking the biopsy, the microorganospheres comprising between 1 and 200 dissociated tumor cells distributed throughout a polymerized matrix; and exposing at least some of the patient-derived microorganospheres to a formulation before the dissociated tumor cells have been passaged more than five times, thereby determining that the tumor will respond to the formulation; and measuring the effect of the formulation on the cells within at least some of the microorganospheres and determining, based on the determined effect, whether the drug treats the tumor.

[0062] In some variations, these methods may include obtaining a second patient biopsy after treating the patient with the formulation, forming a second plurality of patient-derived micro-organospheres from the second patient biopsy, exposing at least some of the second plurality of patient-derived micro-organospheres to the formulation, and measuring the effect of the formulation on cells within at least some of the second plurality of micro-organospheres to determine that the tumor remains responsive to the formulation after one or more administrations of the drug.

[0063] Determining that the tumor is responsive to the formulation may include exposing at least some of the patient-derived microorganospheres to a plurality of formulations and recording the measured effect for each of the formulations. In some variations, determining may further include dispensing the patient-derived microorganospheres into a multiwell plate prior to assaying the microorganospheres.

[0064] Any of these methods may include performing a biopsy on the patient to obtain a patient biopsy (or obtain a tissue sample from the patient or a tissue or cell sample derived from the patient), and / or treating the patient with a formulation or assisting a physician in treating the patient (e.g., advising a physician as to which formulations may be effective). Generally, the time between obtaining the biopsy and recording 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.).

[0065] Generally, the described methods and devices are configured to achieve high encapsulation efficiency with minimal sample / cell loss during processing.Therefore, as described herein, a large number of patient-derived microorganospheres can be efficiently formed from a single tumor sample that is resected or biopsied.These methods and systems can overcome the problems associated with other systems, where small clinical samples are completely lost during processing, and larger clinical samples do not reach a sufficient density to establish organoids.

[0066] In particular, these methods and devices may provide a microfluidic system that accepts a dissociated tumor sample (e.g., a single tumor, although in some embodiments multiple tumor samples may be provided) from a patient and controls the viscosity and / or flow rate within the system. Path length and diameter, as well as tortuosity (e.g., curvature), may be controlled to prevent clogging and / or damage to the dissociated cells.

[0067] For example, these methods and devices may control pressure, flow rate (flow), or pressure and flow rate by maintaining constant or near-constant pressure within a flow path of a microfluidic system (which in some embodiments may be configured as a removable cartridge). The cartridge may be disposable or reusable. Thus, fluids (e.g., dissociated cell solutions and unpolymerized matrix material) may be pumped through the microfluidic system at constant or near-constant pressure. In general, the flow of materials, particularly dissociated cells, through a microfluidic system may be laminar. Laminar flow is defined as a flow rate within a predetermined range (e.g., between 0.01 ml / min and 100 ml / min, between 0.01 ml / min and 50 ml / min, between 0.01 ml / min and 20 ml / min, between 0.01 ml / min and 10 ml / min, between 0.01 ml / min and 5 ml / min, between 0.05 ml / min and 100 ml / min, between 0.05 ml / min and 50 ml / min, between 0.05 ml / min and 10 ml / min, between 0.05 ml / min and 5 ml / min, between 0.1 ml / min and 100 ml / min). The blood flow rate can be maintained by maintaining the blood flow rate at a constant rate (for example, between 0.1 ml / min and 50 ml / min, between 0.1 ml / min and 10 ml / min, between 0.1 ml / min and 5 ml / min, between 1 ml / min and 100 ml / min, between 1 ml / min and 50 ml / min, between 1 ml / min and 20 ml / min, between 1 ml / min and 10 ml / min, between 1 ml / min and 5 ml / min, between 5 ml / min and 100 ml / min, between 5 ml / min and 50 ml / min, between 5 ml / min and 10 ml / min, etc.).

[0068] The path length within the microfluidic system from the dissociated cell inlet to the region where droplets of unpolymerized matrix (and dissociated cells) are formed can be, for example, less than 10 cm, less than 9 cm, less than 8 cm, less than 7 cm, less than 6 cm, less than 5 cm, less than 4 cm, less than 3 cm, less than 2 cm, less than 1 cm, etc.

[0069] The diameter of the channel or channels through which dissociated cells pass can be configured to prevent clogging. For example, the diameter of the channels can be greater than 80 μm, greater than 100 μm, greater than 120 μm, greater than 150 μm, greater than 200 μm, greater than 250 μm, greater than 300 μm, greater than 400 μm, greater than 450 μm, greater than 500 μm, etc. (e.g., between 80 and 500 μm, between 100 and 500 μm, between 120 and 500 μm, between 150 and 500 μm, between 200 and 500 μm, etc.).

[0070] Dissociated cells may be added at a port, chamber, container, etc. of the microfluidic system that is fluidly connected to an inlet of the microfluidic system by short or very short tubing (e.g., less than 20 cm, less than 15 cm, less than 10 cm, less than 7.5 cm, less than 5 cm, etc.) to minimize "dead" areas.

[0071] Generally, the structure of the flow channels of a microfluidic system can be configured to avoid clogging. For example, one or more flow channels can have a radius of curvature greater than 1 mm (greater than 2 mm, greater than 3 mm, greater than 5 mm, greater than 10 mm, greater than 15 mm, greater than 20 mm, greater than 25 mm, greater than 30 mm, greater than 40 mm, greater than 50 mm, etc.). All or substantially all of the corners in the pathways may be smooth, curved, or radiused.

[0072] The microfluidic system may include one or more bubble removal chambers (eg, a breathable but fluid impermeable membrane and / or vacuum vents that remove bubbles, etc.).

[0073] Generally, a microfluidic system can control the temperature (and therefore viscosity) of materials within one or more flow channels. As mentioned, the flow channels with dissociated cells and unpolymerized matrix can be cooled to and maintained (within a range of ±1°C or less) at temperatures that are 20°C or less (e.g., 18°C ​​or less, 15°C or less, 12°C or less, 10°C or less, 7°C or less, 5°C or less, 4°C or less, between 1 and 20°C, between 1 and 15°C, between 1 and 12°C, between 1 and 10°C, between 1 and 5°C, between 1 and 4°C, etc.). For example, the system may include integrated Peltier elements to maintain the viscosity of the material during processing. In some embodiments, multiple temperature zones across the microfluidic system may control polymerization of the matrix material only after successful encapsulation.

[0074] For example, described herein is a method for precision drug screening for personalized cancer therapy, the method comprising obtaining a single tumor sample from a patient tumor; dissociating a biopsy tissue sample to form a dissociated tissue sample; conveying the dissociated tissue sample and an unpolymerized fluid matrix material through one or more channels of a microfluidic device, wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the channels to maintain the temperature of the dissociated tissue sample and the unpolymerized fluid matrix material at a temperature of 20° C. or less, and the dissociated tissue sample moves under laminar flow within the one or more channels; combining the dissociated tissue sample and the unpolymerized fluid matrix material within the microfluidic device to form a plurality of droplets of the unpolymerized mixture; and dissociating the droplets of the unpolymerized mixture at a temperature greater than 25° C. to polymerize the fluid matrix material and form patient-derived micro-organospheres, each having a diameter of between 50 and 500 μm and containing between 1 and 200 dissociated cells distributed therein; culturing the patient-derived micro-organospheres for between 2 and 14 days to form mature patient-derived micro-organospheres having structured clusters of cells that recapitulate the structure of the tumor from which the biopsy or resection was derived; and assaying multiple drug therapies in parallel by exposing one or more of the mature patient-derived micro-organospheres to each drug therapy.

[0075] In any of the methods described herein, the patient-derived microorganospheres may be assayed in any suitable assay, including, but not limited to, 3D viability assays, imaging, gene and / or protein expression assays (e.g., DNA, RNA, proteomics, exosomes, etc.). Assays may be performed using cells, patient-derived microorganospheres, extracellular matrix formed as part of the patient-derived microorganospheres, and / or supernatants from cultures of patient-derived microorganospheres, etc.

[0076] Any of the methods described herein, including high-precision drug screening assays, may screen for one or more of chemotherapy, targeted drugs, and immunotherapy. For example, a method of high-precision drug screening for personalized cancer therapy may include screening for drug therapies including one or more of chemotherapy, targeted drugs, and immunotherapy. Chemotherapy or chemotherapeutic agents refer to treatment with cytostatic or cytotoxic agents (i.e., compounds) to reduce or eliminate the growth or proliferation of unwanted cells, such as cancer cells. Thus, as used herein, chemotherapy or chemotherapeutic agents refer to cytotoxic or cytostatic agents used to treat proliferative disorders, such as cancer. Immunotherapy or immunotherapeutic agents, as used herein, typically promote an immune response; for example, the agent may be an immunostimulatory agent or an inhibitor of an immunosuppressant (i.e., an anti-immunosuppressant). Thus, the term includes immunogenic compositions and vaccines. Immunotherapeutic agents may also be checkpoint blockers or checkpoint inhibitors, chimeric antigen receptors (CARs), and adoptive T-cell therapies.

[0077] The microfluidic device may maintain the temperature of all or a portion of the microfluidic device within a cooling target temperature range (e.g., about 20°C or less, e.g., about 18°C ​​or less, about 17°C or less, about 16°C or less, about 15°C or less, about 14°C or less, about 13°C or less, about 12°C or less, about 11°C or less, about 10°C or less, about 9°C or less, about 8°C or less, about 7°C or less, about 6°C or less, about 5°C or less, between about 1°C and about 20°C, between about 2°C and about 15°C, between about 2°C and about 10°C, etc.). The microfluidic device may, for example, have a Peltier device for cooling. The microfluidic device may cool all or a portion of the microfluidic device. For example, the microfluidic device may be configured to cool the injected sample, tubing, and / or the microfluidic device chip, so that the injected material, e.g., a tissue sample, remains cold. Unpolymerized fluid matrix material may also be kept cold. The microfluidic device may include a thermally conductive material (e.g., aluminum, etc.) that can distribute cooling from a cooling source (e.g., a Peltier) to any or all of these regions. The microfluidic device may also include one or more temperature sensors (e.g., thermistors, etc.) that sense the temperature in these regions and may have control feedback that uses the sensor input to regulate the temperature.

[0078] The tissue sample may be a biopsy sample, including a resection sample, and / or a fine needle aspirate and / or a circulating tumor cell tissue sample. The tissue sample may be collected urgently (e.g., within minutes or hours of performing a method, such as a high-resolution drug screening method, involving the formation of patient-derived microorganospheres). For example, the tissue sample may be "fresh" and collected from the patient within about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, etc., of forming patient-derived microorganospheres. The tissue sample may be refrigerated (e.g., between about 0°C and about 20°C, between about 1°C and about 15°C, below about 15°C, below 10°C, etc.).

[0079] The method may include characterizing the response to a drug therapy based on the response to the patient-derived microorganospheres. Forming the patient-derived microorganospheres may further include sorting the patient-derived microorganospheres based on cell number and / or droplet size. For example, forming the patient-derived microorganospheres may include optical sorting or sorting the patient-derived microorganospheres based on cell number and / or droplet size. Sorting may occur before or after polymerization. Sorting may include directing a fluid flow to place a particular PDMO into a particular container (e.g., chamber) based on its sorting characteristics.

[0080] Assays may include assays of more than five different drug therapies (e.g., more than 10, more than 15, more than 20, more than 30, more than 35, more than 40, more than 50, more than 75, more than 100, more than 150, more than 200, more than 250, more than 300, more than 500, more than 1000, etc.). As used herein, drug therapies may include single agents under different conditions (concentrations, carriers, etc.) or different dosing regimens (e.g., repeated administration, duration of administration, etc.), and / or multiple agents in different combinations and / or conditions and / or dosing regimens. For example, different drug therapies may include different concentrations of one or more drugs, different combinations of three or more drugs, different ratios of two or more drugs, different carriers for one or more drugs, and / or different dose times of one or more drugs.

[0081] The formation of more than 1000 patient-derived microorganospheres includes the formation of more than 5000 patient-derived microorganospheres (such as more than 10,000).

[0082] The microfluidic system may maintain the viscosity of the dissociated tissue sample and the unpolymerized fluid matrix material prior to droplet formation.

[0083] The microfluidic system may generally be configured to prevent clogging of one or more channels by the dissociated tissue sample. For example, the microfluidic system may be configured with channel diameters of 100 μm or greater to prevent clogging. The microfluidic system may be configured to maintain an approximately constant pressure and / or flow rate in one or more channels.

[0084] Exposing the droplets of the unpolymerized mixture to a temperature greater than 25°C can include exposing the droplets to a temperature of 30°C or higher (e.g., 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, etc.). In some embodiments, the droplets of the unpolymerized mixture are exposed to a temperature of about 25°C to about 50°C, e.g., about 25°C to about 30°C or higher (e.g., 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or higher, etc.).

[0085] Exposing the droplet of the unpolymerized mixture to a temperature greater than 25° C. can include flowing the droplet into a region of the microfluidic device maintained at a temperature greater than 25° C. In some examples, the droplet can be transported out of the microfluidic device for polymerization in a heated chamber.

[0086] As mentioned, a microfluidic device can maintain a constant flow rate in one or more channels.

[0087] The method described herein can comprise: measuring the effect of each drug therapy on the cells in patient-derived micro-organoid, and based on the effect measured, determining whether the drug therapy treats tumor.The method can comprise: after treating patient with drug therapy, obtain a second patient tumor tissue, form a second plurality of patient-derived micro-organospheres from the second patient tumor tissue, expose at least some of the second plurality of patient-derived micro-organospheres to drug therapy, and measure the effect of drug therapy on the cells in at least some of the second plurality of patient-derived micro-organoids, thereby determining that tumor still responds to drug therapy after one or more administrations of drug therapy.

[0088] Any of these methods can be used for monotherapy testing, including replicate testing, as described above.

[0089] The method may include treating the patient with one medication from multiple medications.

[0090] The time between obtaining the tumor tissue sample and characterizing the response can be less than 21 days (less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, etc.).

[0091] Patient tumor tissue may include biopsy samples from metastatic tumors. Generally, tumor tissue as used herein may be epithelial adenocarcinoma tissue (as seen by all of the examples provided herein).

[0092] Generally, these methods can include forming a plurality of microorganoids, including forming microorganoids with less than 25% variation in size and / or cell number (e.g., less than 22% variation, less than 20% variation, less than 18% variation, less than 15% variation, less than 10% variation, less than 6% variation, etc.). Low variation in size and / or cell number can be particularly beneficial in normalizing responses between different drug therapies. Furthermore, multiple may be used or compared. Assaying multiple drug therapies can include assaying patient-derived microorganospheres with similar size and cell number.

[0093] Generally, patient-derived microorganospheres can replicate the tissue structure of the tissue from which they are derived, e.g., they can form budding clumps of cells and / or hollow structures of cells that replicate the structure of the tumor from which the biopsy or resection is derived.

[0094] For example, a method for precision drug screening for personalized cancer therapy may include obtaining a single tumor sample biopsied or excised from a patient tumor; dissociating the biopsied tissue sample to form a dissociated tissue sample; conveying the dissociated tissue sample and unpolymerized fluid matrix material through one or more channels of a microfluidic device, wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the channels to maintain the temperature of the dissociated tissue sample and the unpolymerized fluid matrix material at a temperature of 20° C. or less, and the dissociated tissue sample moves under laminar flow within the one or more channels; combining the dissociated tissue sample and the unpolymerized fluid matrix material within the microfluidic device to form a plurality of droplets of the unpolymerized mixture; exposing the droplets of the unpolymerized mixture to a temperature above 25° C. to polymerize the fluid matrix material; and The method may include forming a plurality of patient-derived microorganospheres from the dissociated tissue sample by forming patient-derived microorganospheres, each having a diameter of between 50 and 500 μm and containing between 1 and 200 dissociated cells distributed therein, and sorting the patient-derived microorganospheres by size, cell number, or both size and cell number; culturing the patient-derived microorganospheres for between 2 and 14 days to form mature patient-derived microorganospheres having structured clusters of cells that recapitulate the structure of the tumor from which the biopsy or resection was derived; and testing drug therapies by exposing one or more of the mature patient-derived microorganospheres to each drug therapy.

[0095] The novel features of the invention are set forth with particularity in the following claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. [Brief explanation of the drawings]

[0096] [Figure 1]Figure 1A shows patient-derived microorganospheres formed as described herein to contain a single dissociated primary tissue cell per microorganosphere and cultured for 1 day after formation. The cells are derived from colorectal cancer (CRC) tissue. Figure 1B shows patient-derived microorganospheres formed as described herein to contain a single dissociated primary tissue cell per microorganosphere and cultured for 3 days after formation. The cells are derived from colorectal cancer (CRC) tissue. Figure 1C shows patient-derived microorganospheres formed as described herein to contain a single dissociated primary tissue cell per microorganosphere and cultured for 7 days after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 2] Figure 2A shows patient-derived microorganospheres formed as described herein to contain five dissociated primary tissue cells per microorganosphere and cultured for one day after formation. The cells are derived from colorectal cancer (CRC) tissue. Figure 2B shows patient-derived microorganospheres formed as described herein to contain five dissociated primary tissue cells per microorganosphere and cultured for three days after formation. The cells are derived from colorectal cancer (CRC) tissue. Figure 2C shows patient-derived microorganospheres formed as described herein to contain five dissociated primary tissue cells per microorganosphere and cultured for seven days after formation. The cells are derived from colorectal cancer (CRC) tissue. [Figure 3]Figure 3A shows patient-derived microorganospheres formed as described herein to contain 20 dissociated primary tissue cells per microorganosphere and cultured for 1 day after formation. As in Figures 1A-1C and 2A-2C, the cells are derived from colorectal cancer (CRC) tissue. Figure 3B shows patient-derived microorganospheres formed as described herein to contain 20 dissociated primary tissue cells per microorganosphere and cultured for 3 days after formation. As in Figures 1A-1C and 2A-2C, the cells are derived from colorectal cancer (CRC) tissue. Figure 3C shows patient-derived microorganospheres formed as described herein to contain 20 dissociated primary tissue cells per microorganosphere and cultured for 7 days after formation. As in Figures 1A-1C and 2A-2C, the cells are derived from colorectal cancer (CRC) tissue. [Figure 4] Figure 4A. Example of patient-derived microorganospheres formed as described herein to contain 10 dissociated primary tissue cells per microorganosphere. Microorganospheres are shown immediately after formation (low magnification). Figure 4B. Example of patient-derived microorganospheres formed as described herein to contain 10 dissociated primary tissue cells per microorganosphere. Higher magnification view of some of the microorganospheres from Figure 4A taken after 2 days of culture. Figure 4C. Example of patient-derived microorganospheres formed as described herein to contain 10 dissociated primary tissue cells per microorganosphere. Microorganospheres are shown after 3 days of culture. Figure 4D. Example of patient-derived microorganospheres formed as described herein to contain 10 dissociated primary tissue cells per microorganosphere. Microorganospheres are shown after 4 days of culture. Figure 4E. 1 shows an example of patient-derived microorganospheres formed as described herein containing 10 dissociated primary tissue cells per microorganosphere. Microorganospheres are shown after 5 days of culture. [Figure 5]Figure 5A shows an example of micro-organospheres formed as described herein from hepatocytes of a normal mouse liver and cultured for 1 day after formation. The mouse hepatocytes are harvested from a normal (e.g., non-diseased) mouse liver. Figure 5B shows an example of micro-organospheres formed as described herein from hepatocytes of a normal mouse liver and cultured for 10 days after formation. The mouse hepatocytes are harvested from a normal (e.g., non-diseased) mouse liver. [Figure 6] FIG. 1 shows a method for forming patient-derived microorganospheres from primary tissue (eg, biopsy) samples as described herein. [Figure 7] Figure 7A is a schematic diagram of an example of an apparatus for forming patient-derived micro-organospheres described herein, including a microfluidic chip that is part of the assembly. Figure 7B is a perspective view of an example of the microfluidic chip portion of the apparatus shown in Figure 7A. Figure 7C is a schematic diagram of a portion of a microfluidic assembly for an apparatus for forming patient-derived micro-organospheres, as shown in Figure 7A. [Figure 8] FIG. 7B shows an example of an image showing multiple patient-derived microorganospheres formed using a device such as that shown in FIG. 7A, where the patient-derived microorganospheres are shown immediately after polymerization suspended in a channel containing an immiscible fluid (e.g., oil) prior to being dispensed in aliquots from the device. [Figure 9] FIG. 7D shows an image of a portion of a prototype microfluidic assembly for an apparatus for forming patient-derived micro-organospheres, similar to the apparatus shown in FIG. 7C, illustrating the formation of patient-derived micro-organospheres. [Figure 10] 1 shows a plurality of patient-derived micro-organospheres described herein immediately after polymerization, the patient-derived micro-organospheres being suspended in an immiscible fluid. [Figure 11A] FIG. 10 shows another example of multiple patient-derived microorganospheres immediately after formation and suspended in an immiscible fluid (e.g., oil) at low magnification. [Figure 11B]FIG. 10 shows another example of multiple patient-derived microorganospheres immediately after formation and suspended in an immiscible fluid (e.g., oil) at high magnification. [Figure 12A] FIG. 1 shows, at low magnification, multiple patient-derived micro-organospheres after separation from an immiscible fluid within hours of their formation. [Figure 12B] FIG. 1 shows a high magnification of multiple patient-derived micro-organospheres after separation from an immiscible fluid within hours of their formation. [Figure 13] FIG. 10 shows another example of an image showing a plurality of patient-derived micro-organospheres formed as described herein. [Figure 14] 1 is a graph showing the size distribution of diameters from multiple patient-derived micro-organospheres formed from an exemplary biopsy sample. [Figure 15A] 1A-1C show low and high magnification views of an example of multiple patient-derived micro-organospheres formed from a dissociated tissue biopsy sample and a fluid matrix material after polymerization. The images are unstained. [Figure 15B] Figure 1 shows low and high magnification views of an example of multiple patient-derived microorganospheres formed from a dissociated tissue biopsy sample and a fluid matrix material after polymerization, stained with trypan blue to demonstrate that the dissociated cells in the microorganospheres are viable. [Figure 16A] 15A-15B are another example similar to that shown in Figures 15A-15B, showing low and high magnification views, respectively, of an example of multiple patient-derived microorganospheres. The images are unstained. [Figure 16B] 15A-15B show another example similar to that shown in Figures 15A-15B, which show low and high magnification views, respectively, of an example of multiple patient-derived microorganospheres, in which the organospheres were stained with trypan blue (arrows) to demonstrate that the dissociated cells within the microorganospheres remain viable (e.g., are alive) within the microorganospheres. [Figure 17]

[0023] Figure 1 shows an example of a method for assaying multiple patient-derived microorganospheres, in this example formed from patient tumor biopsies, to determine drug response profiles to multiple formulations. The illustrated method takes less than two weeks (e.g., about one week) from biopsy to results. [Figure 18] FIG. 1 is a schematic diagram illustrating an example of a method for treating a patient, including the formation and use of a plurality of patient-derived micro-organospheres as part of a treatment method. [Figure 19] FIG. 1 is a schematic diagram of an example method for treating a patient, including the rapid formation and multiple iterations of assaying multiple patient-derived microorganospheres as part of a treatment method. [Figure 20] FIG. 10 is a schematic diagram illustrating one variation of a portion of an apparatus for forming a plurality of patient-derived micro-organospheres described herein. [Figure 21] FIG. 21 is a schematic diagram illustrating a method of operating an apparatus for forming multiple patient-derived microorganospheres similar to the apparatus shown in FIG. 20. [Figure 22]Figure 22A. An example of validation of the method described herein using multiple patient-derived microorganospheres to identify drug resistance. This figure shows the use of a conventional ("2D") tumor cell assay to predict drug resistance. Figure 22B. An example of validation of the method described herein using multiple patient-derived microorganospheres to identify drug resistance. This figure shows the use of an exemplary patient-derived microorganosphere method described herein to assay drug resistance to predict drug sensitivity. Figure 22C. An example of validation of the method described herein using multiple patient-derived microorganospheres to identify drug resistance. This figure shows that the patient-derived microorganosphere-based method, unlike conventional cultured cells, accurately predicted the tumor's actual response (drug responsiveness). Figure 22D. An example of validation of the method described herein using multiple patient-derived microorganospheres to identify drug resistance. This figure shows that the patient-derived microorganosphere-based method, unlike conventional cultured cells, accurately predicted the tumor's actual response (drug responsiveness). [Figure 23]

[0023] Figure 1 shows another example validating the use of patient-derived microorganospheres described herein to identify drug resistance, showing that the predicted drug response to both oxaliplatin and irinotecan is consistent with the actual tumor response following treatment with those drugs. [Figure 24] FIG. 1 shows an example of a drug screen using the patient-derived microorganospheres described herein, in which a single tumor biopsy can very quickly (e.g., within less than two weeks) generate a large number of nearly identical microorganospheres that can be rapidly tested in parallel against a large number of formulations (e.g., 27 formulations shown). [Figure 25]Figure 25A shows an example of mouse liver microorganospheres formed from mouse liver tissue, with a diameter of 300 μm and one cell per organosphere. The microorganospheres are shown at day 1. Figure 25B shows an example of mouse liver microorganospheres formed from mouse liver tissue, with a diameter of 300 μm and one cell per organosphere. The microorganospheres are shown at day 10. [Figure 26] Figure 26A shows an example of mouse liver microorganospheres formed from partially hepatectomized mouse liver tissue, similar to the mouse liver microorganospheres shown in Figures 25A-B, with a diameter of 300 μm and 25 cells per organosphere. This figure shows microorganospheres at day 1. Figure 26B shows an example of mouse liver microorganospheres formed from partially hepatectomized mouse liver tissue, similar to the mouse liver microorganospheres shown in Figures 25A-B, with a diameter of 300 μm and 25 cells per organosphere. This figure shows microorganospheres at day 10. [Figure 27] Figure 27A. An example of human liver micro-organospheres formed from human liver tissue. Shown are day 1 micro-organospheres seeded at 40 cells / droplet. Figure 27B. An example of human liver micro-organospheres formed from human liver tissue. Shown are day 18 micro-organospheres. In this figure, the micro-organospheres are hepatocyte-like structures. Figure 27C. An example of human liver micro-organospheres formed from human liver tissue. Shown are day 18 micro-organospheres. This figure shows cholangiocyte-like micro-organospheres. [Figure 28]Figure 28A shows an example of a micro-organosphere generated from a patient-derived xenograft tumor line, with a diameter of 300 μm and one cell per organosphere. The figure shows the micro-organospheres at day 1. Figure 28B shows an example of a micro-organosphere generated from a patient-derived xenograft tumor line, with a diameter of 300 μm and one cell per organosphere. The micro-organospheres are shown at day 3. Figure 28C shows an example of a micro-organosphere generated from a patient-derived xenograft tumor line, with a diameter of 300 μm and one cell per organosphere. The micro-organospheres are shown at day 5. Figure 28D shows an example of a micro-organosphere generated from a patient-derived xenograft tumor line, with a diameter of 300 μm and one cell per organosphere. The micro-organospheres are shown at day 7. [Figure 29] Figure 29A shows an example of a micro-organosphere generated from a patient-derived xenograft model, having a diameter of 300 μm and 5 cells per organosphere. The micro-organospheres are shown at day 1. Figure 29B shows an example of a micro-organosphere generated from a patient-derived xenograft model, having a diameter of 300 μm and 5 cells per organosphere. The micro-organospheres are shown at day 3. Figure 29C shows an example of a micro-organosphere generated from a patient-derived xenograft model, having a diameter of 300 μm and 5 cells per organosphere. The micro-organospheres are shown at day 5. Figure 29D shows an example of a micro-organosphere generated from a patient-derived xenograft model, having a diameter of 300 μm and 5 cells per organosphere. The micro-organospheres are shown at day 7. [Figure 30] 1 is a graph comparing the response of conventional organoids and micro-organospheres formed from colorectal cancer patient-derived organoids to oxaliplatin, showing comparable responses of conventional organoids and micro-organospheres. [Figure 31]FIG. 1 is a graph comparing the response of conventional organoids and microorganospheres formed from two colorectal cancer patient-derived xenograft models to SN38 (7-ethyl-10-hydroxy-camptothecin), demonstrating comparable responses. [Figure 32] FIG. 1 is a graph comparing the response of conventional organoids and microorganospheres formed from colorectal cancer patient-derived xenograft models to 5-FU (fluorouracil), demonstrating comparable responses. [Figure 33A] 1 shows an example of a toxicity assay using mouse liver microorganospheres, showing that the dimensions of the tissue in the mouse liver microorganospheres are relatively large in the control group (indicated by the arrow). [Figure 33B] Figure 33B shows an example of a toxicity assay using mouse liver microorganospheres, whereas in Figure 33B, which shows the acetaminophen (10 mM) treated group, the tissue in most microorganospheres is smaller and contains many dead cells. [Figure 34A] 1 shows an example of a toxicity assay using human liver microorganospheres. Typical human liver microorganospheres observed in the control group are shown, including histology (indicated by arrows). [Figure 34B] 1 shows an example of a toxicity assay using human liver microorganospheres, showing microorganospheres from the acetaminophen (10 mM) treatment group, demonstrating abnormal tissue structure (arrows) and debris. DETAILED DESCRIPTION OF THE INVENTION

[0097] Generally, described herein are patient-derived microorganospheres, methods and devices for forming patient-derived microorganospheres, and methods and devices for using patient-derived microorganospheres, e.g., for assaying the response of tissue, including, but not limited to, cancer tissue.

[0098] The patient-derived microorganospheres described herein are typically spheres formed from dissociated primary cells distributed within a substrate. These patient-derived microorganospheres ("PMOS" or "organospheres") can have a diameter of between about 50 μm and about 500 μm (e.g., between about 50 μm and about 400 μm, between about 50 μm and about 300 μm, between about 50 μm and about 250 μm, etc.) and can initially contain between about 1 and 1000 dissociated primary cells (e.g., between about 1 and 750, between about 1 and 500, between about 1 and 400, between about 1 and 300, between about 1 and 200, between about 1 and 150, between about 1 and 100, between about 1 and 75, between about 1 and 50, between about 1 and 40, between about 1 and 30, between about 1 and 20, etc.) distributed within a substrate.

[0099] Surprisingly, despite their small size (often between about 50 and 250 μm) and low cell density (e.g., frequently less than 100 cells per microorganosphere), these microorganospheres can be used immediately or cultured for very short periods (e.g., 14 days or less, 10 days or less, 7 days or less, 5 days or less, etc.) and can maintain many, if not all, of the characteristics of the tissue from which they were extracted, including tumor tissue, while allowing the cells within the microorganospheres to survive. Cell viability within the microorganospheres is remarkably high, and the microorganospheres can be cultured for days (or weeks) through multiple passages, during which time the cells divide, clump, and form structures resembling the parent tissue. Equally surprising, in some variations, cells from dissociated tissue within the microorganospheres form morphological structures within even the smallest microorganospheres. In some applications, the presence of such structures is not necessary for the usefulness of these microorganospheres (e.g., they can be used before substantial structural reorganization occurs), but in some variations they can be particularly useful.

[0100] The methods and devices described herein for forming and using micro-organospheres can be used to generate large numbers (e.g., over 10,000) of patient-derived micro-organospheres from a single biopsy. These micro-organospheres can be used to screen drug compositions and predict which therapies will be effective for the patient from whom the biopsy was taken. This can be useful, for example, in toxicity screening of drugs or other chemical compositions from healthy normal tissue and / or cancerous (e.g., tumor) tissue. In particular, the patient-derived micro-organospheres, methods and devices for forming patient-derived micro-organospheres, and methods and devices for testing patient-derived micro-organospheres can be used in screening to identify one or more drug compositions that can effectively treat a patient (e.g., a cancer patient) prior to administering a drug therapy. This can allow for very rapid screening of cancer patients, for example, before they undergo months of chemotherapy that may otherwise be ineffective for the patient.

[0101] Thus, described herein is a method (and device for performing the method) for high-throughput drug screening using a patient-specific single biopsy (or other suitable tissue / cell source). Described herein are droplet-formed patient-derived microorganospheres that can be formed from patient-derived tumor samples dissociated and suspended in a basement membrane matrix (e.g., Matrigel). The microorganospheres can be patterned onto a microfluidic microwell array, incubated, and loaded with drug compounds. This miniaturized assay maximizes tumor sample utilization, enabling the screening of many more drug compounds from a core biopsy at a much lower cost per sample.

[0102] Patient-derived cancer models (PDMCs), such as cell lines, organoids, and patient-derived xenografts (PDXs), are increasingly being accepted as the "standard" preclinical models for facilitating the identification and development of novel therapeutics. For example, large-scale drug screening of cell lines and organoids derived from cancer patients has been used to identify sensitivity to numerous potential therapeutics. PDXs are also being 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, substantial barriers exist to their effective use. For example, patient-derived organoids (PDOs) are considered the most accurate in depicting patient tumors, as studies have shown that phenotypic and genotypic profiling of organoids frequently shows a high degree of similarity to the patient's original tumor. Unfortunately, at least two limitations hinder the use of PDOs to guide treatment. First, developing and testing drug sensitivity in organoids takes several months, limiting their clinical applicability. Second, the number of organoids obtained from clinically relevant 18-gauge core biopsies is insufficient for high-throughput drug screening. Ideally, assays should be performed within 7-10 days of a single core biopsy. The microorganospheres and methods for generating and using microorganospheres described herein may address these clinical limitations.

[0103] Details of one or more embodiments of the subject matter of this disclosure are described in this document. Modifications to the embodiments described herein, as well as other embodiments, will become apparent to those skilled in the art after consideration of the information provided herein. The information provided in this document, particularly the specific details of the exemplary embodiments described, is provided primarily for clarity of understanding, and no unnecessary limitations should be construed therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0104] Although the terms used herein are believed to be well understood by those of ordinary skill in the art, definitions are provided herein to facilitate explanation of the subject matter of the present disclosure.

[0105] Unless defined otherwise, 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, representative methods, devices, and materials are now described.

[0106] The term "unpolymerized mixture" is used herein in reference to a composition containing a biorelevant 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 for the dissociated tissue (cells) dispersed therein. Once polymerized, the polymerized material can form a hydrogel and / or can contain proteins in addition to cells to form a biocompatible medium. Biocompatible media suitable for use in accordance with the presently disclosed subject matter are typically gels, semi-solids, or liquids, e.g., low-viscosity liquids at room temperature (e.g., 25°C), and can be formed from any biocompatible material that can be used as a three-dimensional matrix 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, collagen, fibrin, chitosan, MATRIGEL™ (BD Biosciences, San Jose, Calif.), polyethylene glycol, dextran, including chemically or photocrosslinkable dextran, and polymers and hydrogels, including electrospun bioblends, synthetic blends, or biological-synthetic blends. In some embodiments, the biocompatible medium is comprised of a hydrogel.

[0107] The term "hydrogel" is used herein to refer to a two-component or multi-component gel that comprises a three-dimensional network of polymer chains, in which water acts as a dispersion medium to fill 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 consideration the parameters used to form the microorganospheres and the effect the selected hydrogel will have on the behavior and activity of biological material (e.g., cells) incorporated into the biological suspension and disposed within the structure. Exemplary hydrogels of the presently disclosed subject matter can be comprised of polymeric materials, including, but not limited to, alginate, collagen (including type I collagen and type VI collagen), elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactides, polyethylene glycols, polycaprolactones, polycolides, polydioxanones, polyacrylates, polyurethanes, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminins, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids, carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof, as well as inorganic materials, e.g., glasses such as bioactive glasses, ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and any combination of the foregoing.

[0108] Further, with regard to the hydrogels used to produce the microorganospheres described herein, in some embodiments, the hydrogel is comprised of a material selected from the group consisting of agarose, alginate, type I collagen, polyoxyethylene-polyoxypropylene block copolymers (e.g., Pluronic® F127 (BASF Corporation, Mount Olive, NJ)), silicone, polysaccharides, polyethylene glycol, and polyurethane. In some embodiments, the hydrogel is comprised of alginate.

[0109] The micro-organospheres described herein may also include biologically relevant materials. The term "biologically relevant material" may describe materials that can be incorporated into a biocompatible medium as defined herein and subsequently interact with and / or affect a biological system. For example, in some implementations, the biologically relevant material is magnetic beads (i.e., beads that are magnetic or contain a material that responds to a magnetic field, such as iron particles) that can be combined as part of the unpolymerized material to produce micro-organospheres that can be used in the present methods and compositions (e.g., to isolate and purify micro-organospheres). As another example, in other implementations, the biologically relevant material may include additional cells in addition to the dissociated tissue sample (e.g., biopsy) material. In the unpolymerized mixture, the dissociated tissue sample and additional biologically relevant material are present as a homogeneous mixture or as a dispersed mixture (e.g., on one half or the other of the micro-organospheres, including only within the core or only within the outer region of the formed micro-organospheres). In some variations, additional biologically relevant material within the unpolymerized material may be in suspension, for example, with the dissociated tissue sample in the suspension, prior to polymerization of the droplets to form microorganospheres.

[0110] In some variations, biologically relevant materials that may be included with dissociated tissue sample (e.g., biopsy) material may contain multiple cell types, including preadipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells and adipose tissue macrophages, as well as small blood vessels or microvascular fragments found within the stromal vascular cell population.

[0111] Generally, with regard to dissociated tissue samples, e.g., biopsy material, included in the micro-organospheres described herein, the tissues can be any suitable tissues from a patient, typically obtained by biopsy. While non-biopsy tissues can be used, the tissues (and resulting dissociated cells) can generally be primary cells obtained from a patient biopsy, e.g., by needle biopsy, as described above. The tissues can be from a healthy tissue biopsy or a cancer (tumor) cell biopsy. Dissociated cells can be incorporated into the micro-organospheres of the presently disclosed subject matter based on the intended use of the micro-organospheres. For example, the relevant tissue (e.g., dissociated biopsy tissue) can typically include cells commonly found within that tissue or organ (or tumor, etc.). In this regard, exemplary relevant cells that may be incorporated into the microorganospheres of the presently disclosed subject matter include neurons, cardiomyocytes, myocytes, bile duct cells, pancreatic acinar cells, islets of Langerhans, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, biliary epithelial cells, and the like. These tissue types 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, liver, 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) tissue may be used. In some variations, the tissue may originate from tumor tissue, including tumors arising in any of these normal tissues.

[0112] Once formed, the microorganospheres may be cryopreserved and / or cultured. Cultured microorganospheres can be maintained in suspension either statically (e.g., in wells, vials, etc.) or in motion (e.g., by rotation or agitation). Microorganospheres can be cultured using known culture techniques. Exemplary techniques can be found, inter alia, 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, edited by Davis, Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, edited by Masters, 2000; and U.S. Pat. Nos. 5,516,681 and 5,559,022.

[0113] In some variations, microorganospheres are formed by the formation of droplets of an unpolymerized mixture (e.g., a cooled mixture in some variations) of a dissociated tissue sample and a fluid matrix material in an immiscible substance, such as a fluid hydrophobic material (e.g., oil). For example, microorganospheres can 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 can be determined by dilution of the dissociated material (e.g., cells) in the unpolymerized material. The dimensions of the microorganospheres can be correlated to the dimensions of the droplets formed. Generally, microorganospheres are spherical structures with stable shapes.

[0114] The practice of the presently disclosed subject matter 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 explained fully in the literature.See, e.g., Molecular Cloning A Laboratory Manual (1989), 2nd ed., Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Vols. I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M.J. Gait, ed., 1984; Nucleic Acid Hybridization, 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., NY; Gene Transfer Vectors For Mammalian Cells, edited by J.H. Miller and M.P. Calos, Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, vols. 154 and 155, edited by Wu et al., Academic Press Inc., NY; Immunochemical Methods In Cell And Molecular Biology (edited by Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, vols. I-IV, edited by D.M. Weir and C.C. Blackwell, 1986.

[0115] As used herein, a drug composition can include any drug, drug diluent, formulation, multi-drug (e.g., multiple active ingredients) composition, drug product, drug form, drug concentrate, combination therapy, etc. In some variations, a formulation refers to a dosage form that includes a mixture of a drug and one or more inactive ingredients.

[0116] The term "passage" as used herein may refer to the average number of doublings of cells within a microorganosphere. While traditional passage numbers refer to the transfer or subculture of cells from one culture vessel to another, cells within a microorganosphere can be stably maintained within the same microorganosphere and continue to grow and divide. Thus, as used herein, passage number typically refers to the average doublings of dissociated cells within a microorganosphere derived from biopsied tissue. Population doublings are the approximate number of doublings a cell population has undergone since isolation (e.g., since the formation of microorganospheres from freshly dissociated biopsied tissue). Generally, the microorganospheres described herein can be cultured for a short period of time (e.g., less than 10 generations, less than 9 generations, less than 8 generations, less than 7 generations, less than 6 generations, less than 5 generations, less than 4 generations, less than 3 generations, etc.) relative to the proliferation, e.g., doubling, of some or all of the cells within the microorganosphere.

[0117] During culture, cells from dissociated biopsy tissue in microorganospheres may aggregate, clump, or aggregate within the microorganospheres. The cell aggregates may be highly organized, forming a consistent morphology, or may be clumps or groups of cells adhered to one another. The organization may reflect the tissue of origin. In some variations, microorganospheres may contain a single cell type (isotypic), but more typically, they may contain two or more cell types (heterotypic).

[0118] As mentioned, the tissue (e.g., dissociated tissue) used to form patient-derived microorganospheres (e.g., biopsy) can be derived from normal or healthy tissue, or from diseased or diseased tissue, e.g., tissue or fluid from a tumor. The tissue used in the microorganospheres can include cells of the immune system, e.g., T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages, and dendritic cells. The cells can be stem cells, progenitor cells, or somatic cells. The tissue can be mammalian cells, e.g., human cells, or cells from animals such as mice, rats, rabbits, etc.

[0119] Generally, the tissues (and resulting cells) can be harvested from a biopsy to form microorganospheres. Thus, tissues can be derived from biopsies, excision specimens, aspirates, drainage, or cell-containing body fluids. Suitable cell-containing body fluids include blood, lymph, sebaceous fluid, urine, cerebrospinal fluid, or peritoneal fluid. For example, in patients with disseminated metastases, ovarian cancer, or colon cancer, cells can be isolated from ascites. Similarly, in patients with cervical cancer, cervical cancer cells can be harvested from the cervix, for example, by radical resection of the transition zone or by cone biopsy. Typically, such microorganospheres contain multiple cell types present in the tissue or body fluid of origin. Cells can be obtained directly from a subject without the intermediate step of subculture, or can first undergo an intermediate culture step to produce a primary culture. Methods for harvesting cells from biological tissues and / or cell-containing body fluids are well known in the art. For example, techniques used to obtain cells from living tissue include those described by R. Mahesparan (Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro. Acta Neuropathol (1999) 97:231-239).

[0120] Generally, cells are first dissociated or separated from one another before forming microorganospheres. Dissociation of cells can be achieved by any conventional means known in the art. Preferably, cells are treated mechanically and / or chemically, for example, by enzymatic treatment. "Mechanical" includes means that disrupt the bonds between associated cells, for example, using scalpels or scissors, or machines such as homogenizers. "Enzymatic" includes means that disrupt the bonds between associated cells, for example, treating cells with one or more enzymes, including any of collagenase, dispase, DNAse, and / or hyaluronidase. One or more enzymes can be used under different reaction conditions, for example, incubation in a 37°C water bath or at room temperature.

[0121] Dissociated tissues may be treated to remove dead and / or dying cells and / or cell debris. Such removal of dead and / or dying cells can be carried out using any conventional means known to those skilled in the art, such as bead and / or antibody methods. For example, it is known that in apoptotic or dead cells, phosphatidylserine redistributes from the inner leaflet to the outer leaflet of the cell membrane. Annexin V-biotin binding, followed by biotin binding to magnetic streptavidin 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, such as filtration.

[0122] The dissociated cells may be suspended in a carrier material prior to combination with the fluid matrix material, and / or the fluid matrix material may be referred to as the carrier material. In some variations, the carrier material may have a viscosity that retards the settling of cells in the cell suspension prior to polymerization and the formation of microorganospheres. The carrier material may have sufficient viscosity to allow dissociated biopsy tissue cells to remain suspended in the suspension until polymerization. The viscosity required to achieve this can be optimized by those skilled in the art by monitoring the settling rate at various viscosities and selecting a viscosity that provides an appropriate settling rate for the anticipated time delay between loading the cell suspension into the microorganosphere-forming device and polymerizing the droplets of unpolymerized material containing the cells to form microorganospheres. In some variations, the unpolymerized material may be fluidized or agitated by the device to keep the cells suspended and / or distributed as desired, even when a low-viscosity material is used.

[0123] As mentioned above, in some variations, the unpolymerized mixture containing the dissociated tissue sample and the fluid matrix material may contain one or more components, such as a biologically relevant substance. For example, biologically relevant substances may include extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides, or antibodies (e.g., to modulate cell survival, proliferation, or differentiation), and / or inhibitors of specific cellular functions. Such biologically relevant substances may be used, for example, to increase cell viability by reducing cell death and / or activating cell proliferation / replication, or to mimic an in vivo environment. The biologically relevant substance may include or mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, saline, amino acids, and hormones. For example, the biologically relevant substance may supplement one or more drugs in the fluid matrix material. In some variations, the fluid matrix material may be a synthetic gel (hydrogel) and supplemented with one or more biologically relevant substances. In some variations, the fluid matrix material is a natural gel. Thus, the gel can 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 contains bioactive polymers that are important for cell viability, proliferation, development, and migration. For example, the matrix material can be a gel containing type 1 collagen, such as type 1 collagen obtained from rat tail. The gel can be a pure type 1 collagen gel, or a gel containing type 1 collagen in addition to another component, such as another extracellular matrix protein. Synthetic gels can refer to gels that do not naturally occur in nature. Examples of synthetic gels include gels derived from polyethylene glycol (PEG), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), and polyethylene oxide (PEO).

[0124] Patient-derived microorganospheres Examples of patient-derived microorganospheres are shown in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E. For example, Figures 1A-1C show microorganospheres with a single cell per microorganosphere. As shown, the microorganospheres are all approximately the same size, e.g., about 300 μm in diameter. Figure 1B shows microorganospheres formed simultaneously after 3 days of culture. The cells expand in size and, in some cases, double and / or proliferate. By 7 days of culture, as shown in Figure 1C, the cells have doubled multiple times and exhibit clumps or clusters of cells.

[0125] Similar results are shown in Figures 2A-2C and 3A-3C, which show microorganospheres formed from 5 cells per microorganosphere or 20 cells per microorganosphere, respectively. Figures 4A-4E show microorganospheres immediately after formation and after 5 days of culture, where approximately identical microorganospheres (e.g., having the same diameter) each contain 10 cells per microorganosphere. Figure 4A shows microorganospheres immediately after formation on day 0, still surrounded by an immiscible fluid, in this case oil. The microorganospheres are removed of the immiscible fluid, washed, and cultured for 5 days. Figure 4B shows microorganospheres after 2 days, Figure 4C shows microorganospheres after 3 days, and Figures 4D and 4E show microorganospheres on days 4 and 5, respectively. 4A-4E show that dissociated tissue (cells) from biopsies within microorganospheres are viable and proliferate at comparable rates within nearly all microorganospheres. As described in more detail, these microorganospheres can be formed in large quantities from even a single average-sized biopsy, resulting in hundreds or thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000 or more) of microorganospheres containing significant numbers of viable cells, allowing multiple rapid assays to be performed in parallel.

[0126] 5A and 5B show examples of microorganospheres formed as described herein from a dissociated biopsy from a mouse liver, e.g., showing mouse hepatocytes distributed within a polymerized fluid matrix material (Matrigel in this example). Each microorganosphere, formed into a sphere, e.g., having a diameter of approximately 300 μm, contains polymerized matrix material 503 with a designated number of hepatocytes 507 dispersed therein. FIG. 5A shows the microorganospheres one day after biopsy, dissociation, and microorganosphere formation. The microorganospheres are then cultured for 10 days, during which time the cells (hepatocytes) continue to survive and proliferate, often doubling multiple times, forming structures 505 as shown in FIG. 5B.

[0127] Microorganospheres are generally prepared by dissociating dissociated, e.g., biopsied tissue (e.g., cells) into a fixed or known cell number and / or concentration (cells / ml or cells / mm 3 ) and may be contained within microorganospheres. As noted above, this matrix material may be one or more of natural polymers, such as alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, elastin, or synthetic polymers, such as one or more of polyethylene glycol (PEG) and polyacrylamide. Both organic and inorganic synthetic polymers may be used.

[0128] In some variations, the number of cells initially contained within a micro-organosphere may be selected from one cell to several hundred. In particular, some assays (e.g., drug toxicity assays) may benefit from containing between about 1 and 75 cells or between about 1 and 50 cells (e.g., smaller cell numbers). The number of cells per micro-organosphere may be set or selected by the user. In some variations described below, the device includes one or more controls for setting the number of cells from the primary tissue to be contained in each micro-organosphere. The number of cells can be selected or set by the user based on how the micro-organospheres are intended to be used. For example, micro-organospheres containing very low numbers of cells (e.g., one cell per micro-organosphere, one to five cells per micro-organosphere, etc.) may be particularly suitable for studying clonal diversity (e.g., tumor heterogeneity). Because each microorganosphere grows from a single cell, it is possible to observe which clones are drug-resistant, and those specific microorganospheres can be investigated (e.g., by genome sequencing) to identify the genomic (mutational) diversity associated with the particular clone. Low to moderate cell numbers per microorganosphere (e.g., between about 3-30 cells, between 5-30 cells, between 5-25 cells, between 5-20 cells, between 10-25 cells, etc.) can be particularly useful for rapid drug testing, including toxicity testing, because these microorganospheres typically grow rapidly. High cell numbers per microorganosphere (e.g., between about 20-100 cells, e.g., between 30-100 cells, 40-100 cells, more than 50 cells, etc.) can be particularly suitable for mimicking tissue composition within each microorganosphere. This is because the microorganospheres can potentially contain different lineages, including epithelial (or cancer, etc.) cells and mesenchymal (or stromal, immune, vascular, etc.) cells.

[0129] Micro-organospheres can be formed in any suitable size consistent with the number of cells to be included. For example, the size can be as small as about 20 μm in diameter up to 500 μm (e.g., an average of 50 μm or 100 μm, e.g., between about 100 and 200 μm). In some variations, the size is about 300 μm, with each micro-organosphere containing between about 10 and 50 cells (e.g., between about 10 and 30 cells). The cell number and size can be varied and / or controlled. In some variations, the cell number and / or micro-organosphere size can be set by one or more controls using the micro-organosphere-forming device. For example, the size of the micro-organospheres and / or the density of cells within the micro-organospheres can be adjusted by adjusting the flow rate and / or the concentration of the dissociated tissue sample (e.g., cells from a biopsy).

[0130] As shown in Figures 1A-5B, the micro-organospheres described herein are capable of containing viable and healthy cells throughout the entire volume of the micro-organosphere, even after culture. The dimensions of the micro-organosphere and / or the number of cells to be contained therein can be selected based on the expected or intended use of the micro-organosphere. For example, in variations using micro-organospheres to examine the relationships between cells in biopsy material, micro-organospheres containing multiple cells can be formed and cultured for extended periods of time (e.g., up to one week or more).

[0131] The patient-derived microorganospheres described herein may be produced by combining a dissociated tissue sample, e.g., a biopsy sample, with a fluid matrix that can be polymerized in a controlled manner to form the microorganospheres. Figure 6 illustrates one method of forming patient-derived microorganospheres. Optionally, the method may include the step of obtaining a sample from the patient, e.g., obtaining a biopsy from the patient's tissue (601). As noted above, such a biopsy may be obtained, for example, using a biopsy needle or biopsy punch. For example, a biopsy can be obtained using a 14-gauge, 16-gauge, 18-gauge, etc. needle inserted into the patient's tissue for its removal. After removal of the tissue from the patient, the tissue may be treated either mechanically and / or chemically to dissociate the material. Dissociated cells may be used immediately to form the patient-derived microorganospheres described herein, or in some variations, all or some of the cells may be modified via genetic modification of the cells (603), e.g., by transfection, electroporation, etc.

[0132] The dissociated tissue sample from the biopsy may be combined with a fluid (e.g., liquid) matrix material to form an unpolymerized mixture (605). The unpolymerized mixture may be kept unpolymerized so that cells from the dissociated tissue may remain suspended within the mixture. In some variations, the cells may remain suspended and unpolymerized, for example, by maintaining cooling below room temperature (e.g., between 1 and 25°C).

[0133] The unpolymerized mixture can then be dispensed as droplets (607), for example, into an immiscible substance, such as oil, in a manner that controls the size of the droplets and, therefore, the size of the patient-derived micro-organospheres formed. For example, droplets of uniform size can be formed by combining a stream of unpolymerized material into one or more (e.g., two converging) streams of immiscible substance (e.g., oil), so that the flow rates and / or pressures of the two streams can determine how the droplets of unpolymerized material form upon crossing the immiscible substance. The droplets can then be polymerized (609) to form patient-derived micro-organospheres (PMOS) in the immiscible substance. In some variations, the immiscible substance can be heated or warmed to a temperature at which the unpolymerized mixture (e.g., the fluid matrix material in the unpolymerized material) polymerizes. Once formed, the patient-derived microorganospheres may be separated from the immiscible fluid, e.g., by washing the PMOS to remove the immiscible fluid (611), and placed in culture medium to allow cells within the patient-derived microorganospheres to grow. The patient-derived microorganospheres may be cultured for any desired period of time, or may be cryopreserved and / or assayed immediately. In some variations, the patient-derived microorganospheres may be cultured for a short period of time (e.g., between 1-3 days, between 1-4 days, between 1-5 days, between 1-6 days, between 1-7 days, between 1-8 days, between 1-9 days, between 1-10 days, between 1-11 days, between 1-14 days, etc.), thereby allowing cells derived from the dissociated biopsy tissue to grow and / or divide (e.g., doubling) for up to five or six passages. After culturing, the cells may be either cryopreserved (615) or assayed (617), or both cryopreserved (615) and assayed (617). Examples of assays that can be used are also described herein.

[0134] In any of the methods and devices described herein, the micro-organospheres may be recovered from the immiscible fluid (e.g., oil) after polymerization. For example, in some variations, the micro-organospheres may be recovered by demulsification and / or demulsification, e.g., by forming emulsified droplets and recovering the micro-organospheres after droplet formation to remove any oil (and other contaminants). This may allow cells to grow within the polymerized droplets (micro-organospheres) without being inhibited by the immiscible fluid.

[0135] While the methods and devices described herein demonstrate forming a plurality of droplets, and thus a plurality of micro-organospheres, by flowing the unpolymerized mixture into one or more streams of an immiscible fluid (e.g., oil or another hydrophobic material), in some variations, the droplets may be formed by other methods that may enable droplet size control as described herein. For example, in some variations, the droplets may be formed by printing (e.g., printing the droplets onto a surface), which may reduce or eliminate the need for an additional emulsification / demulsification recovery step. For example, the droplets may be printed and polymerized onto a surface, such as a flat or shaped surface. In any of these variations, the droplets may be dispensed using pressure, sound, electric charge, etc. In some variations, the droplets may be formed using an automated dispenser (e.g., a pipetting device) adapted to release small amounts of the unpolymerized mixture onto a surface, into air, and / or into a liquid medium (including an immiscible fluid).

[0136] The method for forming patient-derived micro-organospheres may be automated and performed using one or more devices. In particular, the method for forming patient-derived micro-organospheres may be performed by a device that allows for selection and / or control of the size (and thus the density or number of cells) of the patient-derived micro-organospheres. For example, Figure 7A shows an example of an apparatus 700 for forming the described patient-derived micro-organospheres.

[0137] In FIG. 7A , the device typically includes an inlet for injecting an unpolymerized mixture of the dissociated tissue sample and the fluid matrix material (already combined), or may receive the dissociated tissue sample and the fluid matrix material separately, for example, in a holding solution. In some variations, the device includes a holding chamber 706 for holding the unpolymerized mixture, and / or holding chambers (not shown) for holding the dissociated tissue (e.g., biopsy) sample and the fluid matrix material. Some or all of these holding chambers may be pressurized to control and / or accelerate the flow rate out of the chambers and into the device. The device may receive the unpolymerized mixture, or may receive and mix components. In some variations, the device may control the concentration of cells in the unpolymerized mixture and may dilute the mixture (e.g., by adding more fluid matrix material) to achieve a desired density. For example, the device may include a sensor (e.g., an optical reader) for reading the density (e.g., optical density) of cells in the unpolymerized mixture (not shown). The sensor may further be coupled to a controller 724 that automatically or semi-automatically (e.g., by user instruction) controls the dilution of cells in the unpolymerized mixture. The device may further include a port for receiving the unpolymerized mixture. The port may include a valve or be coupled to a valve, which may be controlled by the controller 724 (or a separate controller). The chamber holding the unpolymerized mixture and / or XXX may include:

[0138] The device 700 may include a chamber 708 and / or port for holding and / or receiving an immiscible fluid. In some variations, the immiscible fluid may be held in a pressurized chamber, thereby allowing for control of the flow rate. Either 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.

[0139] 7A, the entire device 700 may be enclosed in a housing 702, or a portion of the device 704 may be enclosed in a housing. In some variations, the housing may include one or more openings or inlets to the device, for example, for the addition of immiscible fluids and / or unpolymerized mixtures.

[0140] As noted, any of these devices 700 may further include one or more sensors 728 for monitoring all or key portions of the manufacturing process. In some variations, the sensors may include optical sensors, mechanical sensors, voltage and / or resistance sensors (or capacitance or inductance sensors), force sensors, etc. These sensors may be used to monitor ongoing operations of the assembly, including the formation of patient-derived micro-organospheres. The device 700 may further include one or more temperature regulators / temperature adjustment devices 718 for controlling the temperature of either the immiscible fluid or the unpolymerized mixture (or fluid matrix material), or both the immiscible fluid and the unpolymerized mixture (and fluid matrix material).

[0141] Any of these devices may further include one or more droplet forming assemblies 720 that can be monitored (e.g., using one or more sensors), as illustrated below in Figures 7C and 9. The droplet micro-organosphere forming assemblies may include (or be coupled to) a dispenser (e.g., a PMOS dispenser) 722, which may dispense, for example, into a multiwell plate 716.

[0142] Generally, the droplet-microorganosphere-forming assembly 720 can include one or more microfluidic chips 730, or structures that shape and control the flow of unpolymerized mixture to form the actual droplets. FIG. 7B shows an example of a microfluidic chip 730 for forming patient-derived microorganospheres. In FIG. 7B, the chip 730 includes a pair of parallel structures for forming microorganospheres. FIG. 7C shows the droplet-forming region of a microfluidic chip forming a PMOS, including an unpolymerized channel outlet 741 and immiscible fluid outlets 743, 743′ that open a “+” junction or intersection region 737 to the channel outlet 741 (in this example, at a right angle). In some variations, the inlet from the immiscible fluid channel may be oblique to the angle (and intersection) with the unpolymerized material. In FIG. 7C, as with all figures in this description that show dimensions, the dimensions are illustrative only and not intended to be limiting unless otherwise specified.

[0143] In Figure 7B, microfluidic chip 730 includes an inlet (entry port) 733 for an immiscible fluid entering the chip (e.g., from the inlet port or reservoir chamber shown in Figure 7A). A second inlet port 735 entering the chip can be configured to receive unpolymerized material and transport it down a semi-serpentine path to a junction region. Similarly, the inlet port for the immiscible fluid can be securely coupled to the outlet or inlet from the immiscible fluid chamber described above.

[0144] An inlet port 735 for unpolymerized material entering the chip may be coupled to a junction region through a delivery channel 741 connecting the inlets 275 (shown in FIG. 7C). Similarly, the inlet 733 for the immiscible fluid may connect to two (or more) connecting channels 743, 743′ leading to a junction region 737. The flow channels leaving the junction region 737 may pass the formed micro-organospheres (in the immiscible fluid) down the flow channel to an outlet 731 that may be connected to a dispenser (not shown), e.g., for dispensing the micro-organospheres into one or more chambers for incubation and / or assay.

[0145] In the example shown in Figures 7B and 7C, the formed droplets, which may polymerize into microorganospheres, can be transported down a long, temperature-controlled microfluidic environment (not shown) before being dispensed from the device. For example, Figure 8 shows an example of a flow channel region 839 (e.g., element 739 in Figure 7B) shown transparently containing multiple microorganospheres 803, each containing a predetermined number of cells 805.

[0146] In FIG. 9 , confluence region 937 is formed as described above, so that channel 911 containing the unpolymerized mixture intersects one or more (e.g., two) channels 909 containing a fluid immiscible with the unpolymerized mixture, e.g., oil. As the unpolymerized mixture is pressurized to flow out of first channel 911 at a first velocity, the immiscible fluid flowing through intersecting channels 909, 909′ forces a defined amount of the unpolymerized mixture to pass through and pinch off to form droplets 903 that pass to outlet channel 939. Thus, in some variations, a minced (e.g., dissociated) clinical (e.g., biopsy or resection) tissue sample, e.g., <1 mm in diameter, may be mixed with a temperature-sensitive gel (e.g., Matrigel, 4°C) to form an unpolymerized mixture. This unpolymerized mixture may be placed in a microfluidic device capable of generating droplets (e.g., water-in-oil droplets) that are uniform in volume and material composition. Dissociated tumor cells may be simultaneously dispensed into the droplets. The gel in the unpolymerized material can solidify upon heating (e.g., at 37°C), forming the resulting patient-derived microorganospheres. In some variations, this method can be used to produce 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, or more than 100,000) uniform droplets (patient-derived microorganospheres) from tissue (e.g., biopsy material). These patient-derived microorganospheres are compatible with conventional 3D cell culture techniques. Figure 10 shows a plurality of patient-derived microorganospheres 1005 formed as described above, suspended in an immiscible substance 1008 (e.g., oil).

[0147] In the exemplary microfluidic chips described above, confluences are shown as T- or X-confluences, where microfluidic flow focusing forms microorganospheres of controllable dimensions. In some variations, droplets may be formed by, for example, robotic micropipetting into immiscible fluids and / or solid or gel substrates rather than by a microfluidic chip. Alternatively, in some variations, droplets of unpolymerized material may be formed with the required size and reproducibility by microcapillary generation. Another example of an alternatively usable technique for forming microorganospheres from unpolymerized material in a specified size range and reproducibility may include colloidal manipulation via external forces such as acoustics, magnetism, inertia, electrowetting, or gravity.

[0148] Figures 11A and 11B show examples of patient-derived microorganospheres in oil formed as described above. The cells within these patient-derived microorganospheres from a single biopsy sample are viable, as seen by the vital dye staining shown in Figures 15A-15B and 16A-16B. For example, Figures 12A-12B show microorganospheres containing tumor cells (similar to the microorganospheres shown in Figures 11A-11B), which may be washed to remove immiscible materials (e.g., oil). The immiscible materials may be removed relatively soon after microorganosphere formation so as not to damage the cells within the microorganospheres.

[0149] In these examples, the gel droplets are recovered from the oil phase and resuspended in PBS, for example, via PFO (perfluorooctanol) and centrifugation. This allows the immiscible fluid to be separated from the microorganospheres. These microorganospheres, including tumor-based microorganospheres, can then be successfully propagated, as shown in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E, as well as Figure 13. This is an important improvement because drug screening must be performed using viable, growing primary tumor cells that retain their characteristics from the patient's tumor in order to predict patient outcomes. The large number and uniformity of these microorganospheres both enables and ensures screening, as described below.

[0150] In any of the microfluidic chips or microfluidic devices described herein, the channels may be coated, for example, the channels of the microfluidic device may be coated with a hydrophobic material.

[0151] Generally, the microorganospheres described herein are highly uniform in diameter and can have, for example, very small size variations in diameter, as illustrated, for example, in Figure 14, which shows an example distribution of droplet diameter sizes.

[0152] As noted, Figures 15A-15B show micro-organospheres formed as described herein, and in Figures 16A-16B, the micro-organospheres are stained with trypan blue (arrows) to demonstrate viability. Micro-organospheres formed as droplets in this manner may contain growth factors and a matrix that mimics the biological environment from which the tissue originated. Patient samples (e.g., biopsies) can be formed into micro-organospheres (containing hundreds, thousands, or tens of thousands of micro-organospheres) within hours of tissue acquisition. Micro-organospheres may have as few as one cell or between 4-6 cells (e.g., cancer cells if a tumor is sampled), or as many as several hundred cells per micro-organosphere. These methods have been shown to work for virtually every type of cancer and non-cancerous tissue tested to date (n=20), including cancer tissues of the colon, esophagus, melanoma, uterus, sarcoma, kidney, liver, ovary, lung, diaphragm, peritoneum, mediastinal lung, and breast. Microorganospheres can be cultured for any desired period of time, typically exhibiting proliferation and growth in as little as 3-4 days. Microorganospheres can be maintained and passaged for several months. As described in more detail below, microorganospheres can be used to screen thousands of drug compositions within as little as 4-6 days of tissue (e.g., biopsy) collection.

[0153] The micro-organospheres described herein may be banked at any time after their formation, for example, by cryopreservation. Tumor micro-organospheres may be harvested from multiple different patients and used individually or collectively to screen numerous formulations to identify toxicity and / or efficacy. Non-tumor cells (healthy tissue) may be biopsied, stored, and / or screened in parallel. Thus, these methods and devices may enable high-throughput screening. In some variations, micro-organospheres may be formed and passaged twice (e.g., two doublings) before cryopreservation. As mentioned, normal, healthy tissue may be used to form micro-organospheres in order to generate hundreds, thousands, or tens of thousands of micro-organospheres that can be used to assay drug efficacy, drug response, biomarkers, proteomic signals, genomic signals, etc.

[0154] These microorganospheres are particularly important because they survive in a biologically relevant manner, enabling them to provide clinically and physiologically relevant data, particularly regarding drug response, as illustrated in Figures 22A-22D and 23A-23D. In particular, the microorganospheres described herein grow cells from tissue extracts / biopsies very well, providing more representative data, particularly compared to organoids or spheroids. Without being bound by theory, this may be because cells may have a more constrained cell density within the microorganospheres, allowing them to communicate unimpededly while sharing signals. Microorganospheres also have a much larger surface-to-volume ratio, making the delivery of growth factors and other signals into the microorganospheres easier (e.g., microorganospheres are less diffusion-limited).

[0155] Assay The patient-derived microorganospheres described herein can be used in a variety of different assays, particularly to determine the effects of pharmaceutical formulations, including toxicity to normal and / or abnormal (e.g., cancer) tissue. For example, drug screening can involve applying microorganospheres to all or a subset of wells in a multiwell (e.g., 96-well) plate. Alternatively, custom plates can be used (e.g., 10,000-microwell arrays formed in 100 x 100 wells). Microorganospheres (e.g., gel droplets) can be applied into, or in some variations, onto, multiple microwell arrays, followed by incubation with culture medium. The microorganospheres can be cultured for 3-5 days. In some variations, on the fifth day, drug compounds based on, for example, a set of FDA-approved anticancer drugs, can be added to the wells (e.g., microreactors) to examine the effects of a drug panel. For example, drugs to be tested may be based on the National Cancer Institute's (Division of Cancer Treatment and Diagnosis) screening of 147 drugs intended to enable cancer research, drug discovery, and combination drug studies. At day 7, microorganospheres may be imaged via standard fluorescence microscopy and ranked based on drug response.

[0156] An example of this assay technique is shown in Figures 17A-17E.

[0157] In this example, the screening assay can be automated. Automation allows for a repeatable and automated workflow that can scale 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) microorganospheres are formed as described above (Figure 17A shows the confluence region where the microorganospheres form). The microorganospheres may then be collected and washed (e.g., to remove immiscible (e.g., oil) materials in which the microorganospheres formed). The microorganospheres may then be plated into one or more microwell plates. As shown in Figure 17C, the microorganospheres may be cultured for one or more generations (e.g., one or more passages), which is shown to occur from day 0 to day 3, day 4, or day 5. The microorganospheres can then be screened, for example, by applying drugs to a subset of replicant wells, as shown in Figure 17D. Then, on day 7, as shown in Figure 17E, the cells in the microorganospheres can be imaged and / or scored automatically or manually to identify drug efficacy (e.g., drug screening and growth profiling).

[0158] The workflow shown in Figures 17A-17E may allow the integrated device to be used to grow, add, and / or review micro-organospheres. In one exemplary device, a freshly biopsied or resected patient tumor sample may be dissociated and seeded into a gel containing reagents for forming micro-organospheres (as described above). Some of the formed micro-organospheres may be cryopreserved. The remainder may be harvested and incubated until seeded into microwell plates for drug testing or screening, as described immediately above. The micro-organospheres may be used to perform growth and viability assays, imaged, and tracked. To identify effective therapeutic agents for a patient's tumor, their response to drug treatment, e.g., IC50, cytotoxicity, and growth curve, may be measured.

[0159] The methods and devices described herein have numerous advantages, including reproducibility. The sample preparation process can be automated with microfluidic sample dispensing, reducing the need for specialized staff for diagnostic testing and manual pipetting, which can be particularly beneficial in clinical settings. Furthermore, it allows for uniformity between signal droplets, increasing assay sensitivity. Additionally, these assays can minimize the time required to generate microorganospheres. Based on preliminary data, these methods can generate libraries of over 100,000 Matrigel-tumor droplets (microorganospheres) in less than approximately 15 minutes. These methods are also highly scalable and can be multiplexed to run multiple patient biopsies in parallel.

[0160] Finally, these methods are adaptable and compatible with other technologies. As research tools, droplet-based microfluidics are generally compatible with a wide range of hydrogel materials, such as agarose, alginate, PEG, and hyaluronic acid. The starting gel composition itself can be easily modified to entrain and promote the growth of microorganospheres. Furthermore, droplet size can be tuned by altering the dimensions of the microfluidic device. Overall, a wide selection of gel material compositions and microreactor dimensions is possible.

[0161] The miniaturized assays described herein, using microorganospheres for example, maximize patient tumor biopsies, enabling the screening of more drug compounds. For example, a 600 μL tumor sample can be distributed into approximately 143,000 individual microreactors, each with a volume of approximately 4 mL. Maximizing tissue sampling allows for multiple experimental replicates, increasing statistical power. These techniques may enable the examination of intratumor heterogeneity, drug perturbation, and identify rare cellular events, such as drug resistance. Microorganospheres are generally compatible with downstream assays, including single-cell RNA transcriptome analysis and epigenetic profiling. Additionally, the maximized tissue (e.g., biopsy) sample efficiency provided by microorganospheres allows for the storage of portions of the microorganospheres (e.g., cryopreservation for biobanking) for future confirmatory analyses, including novel drug assays and / or genetic screening.

[0162] For example, Figures 18 and 19 show exemplary therapeutic methods using the methods and devices described herein, including micro-organospheres. In the context of precision medicine and personalized medicine, these methods and devices can be used as clinical indicators for selecting appropriate drugs to improve clinical outcomes and drug response. In one embodiment, a patient diagnosed with metastatic cancer undergoes biopsy collection for histopathology and screening of multiple micro-organospheres formed from the biopsy as described herein. Screening can be performed from the biopsy to identify the most effective standard of care within 7-10 days, allowing the patient to begin treatment around day 14.

[0163] An example is shown in Figure 18. In this example, a tumor can be identified on day 0 (e.g., by CT scan) (1801), a biopsy taken on day 5 (1805), and hundreds, thousands, or tens of thousands of micro-organospheres can be formed on the same day, cultured for 1-5 days, and screened to identify one or more drug compositions that can be used (1805). This same process (forming and screening micro-organospheres) can be used to guide precision medicine at multiple clinical decision points throughout disease progression. In this example, therapy using the identified drug composition or compositions can begin on day 14 (1809), and the patient can later be monitored over the course of treatment (e.g., a follow-up CT scan at about day 90) to confirm that the tumor is responding to treatment (1811). If there is a response, therapy can be continued (1813) and ongoing progress monitored (1815).

[0164] As noted, the use of microorganospheres for assay purposes can be repeated at multiple time points during the course of treatment, as shown in FIG. 19. For example, when a patient is first diagnosed with a resectable primary tumor (1907), this technique (e.g., generating and screening microorganospheres (1905)) can be used to identify the most effective neoadjuvant therapy (1921). Thus, a biopsy can be taken, and hundreds, thousands, or tens of thousands of microorganospheres can be formed and screened with a panel of potential drug compositions. Once the primary tumor is resected (1923), this technique (1905') can suggest whether and which adjuvant therapy should be selected (1925). If recurrence or metastasis occurs after surgical resection of the primary tumor (1927), the same technique (e.g., generation and screening of microorganospheres from fresh biopsies (1905'', 1905''', 1905''')) may be used to guide standard treatments, including first-line (1929), second-line (1931), and third-line (1933) treatments. If the patient eventually becomes resistant or refractory to all standard treatments, this technique (1905''''') may be performed to identify off-label drugs to treat the resistant tumor (1935). This technique can also be used as a companion diagnostic to identify patients for specific treatments. Finally, this technique may be used to derive and maintain patient-derived microorganospheres toward establishing organosphere-based living cancer banks for screening, genomic profiling, drug discovery, drug testing, and clinical trial design.

[0165] These techniques, and the generation of vast numbers of microorganospheres, can be performed relatively minimally invasively (e.g., by excision or biopsy) to provide reasonable and rapid results from screening, making these methods readily adaptable to standard of care. For example, the volume of cellular material from the tissue (e.g., biopsy) input is fairly small, e.g., dissociated into volumes between 10 μL and 5 mL.

[0166] Generally, the use of the microorganospheres described herein for screening can be automated or manual. Virtually any screening technique can be used, including imaging with one or more of confocal microscopy, fluorescence microscopy, liquid lenses, holography, sonar, bright-field and dark-field imaging, lasers, and planar laser sheets, including high-throughput embodiments of image-based analysis (e.g., using computer vision and / or supervised or unsupervised models, such as CNNs). Downstream screening can include sampling of culture medium and / or genetic or protein screening (e.g., scRNA-seq, ATAC-seq, proteomics, etc.) using cells from the microorganospheres. [Example]

[0167] Figures 20 and 21 show another example of an apparatus for forming a plurality of micro-organospheres described herein. In Figure 20, the apparatus can include a confluence for forming a plurality of micro-organospheres, to which an immiscible substance (e.g., oil) (2002) can be added to a reservoir and / or port (2004) of the apparatus. Similarly, unpolymerized material (2006) (in this example, including dissociated biopsy cells and a fluid matrix material) can be added to a reservoir or port (2008) of the apparatus. In some variations, a second or additional material (e.g., a bioactive agent) can be added through a third set of ports (2010). These components can be combined at a confluence (similar to the previous confluence) to form droplets in the immiscible substance that can be polymerized into micro-organospheres. Figure 20 shows three (or more) parallel confluences with corresponding inputs and outputs.

[0168] Figure 21 illustrates a method of forming micro-organospheres using the apparatus shown in Figure 20. In this variation, the resulting micro-organospheres contain one or more additional bioactive agents that combine to form the micro-organospheres, as well as target (e.g., tumor) biopsy cells. For example, a first channel 2103 can contain unpolymerized material (including dissociated biopsy cells and matrix material), a second channel 2107 contains additional active biological material, and a pair of intersecting channels 2109, 2109' with immiscible materials (e.g., oil) meet at a junction to form size-controlled droplets that polymerize to form micro-organospheres 2107.

[0169] In this example, the additional active biological material can be, for example, a freezing medium (e.g., to aid in banking of the microorganospheres), 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), additional growth factors, and / or pharmaceutical compounds.

[0170] Example 2 Screening results As described above, patient-derived microorganospheres and their use for screening drug compositions can be used to accurately predict a patient's tumor's response to one or more drug therapies. In some cases, the use of microorganospheres can provide accurate results when traditional cultured drug screens do not accurately predict drug response. For example, in Figures 22A-22D, microorganospheres correlated with patient response, but cell lines did not. In Figure 22A, a traditional cell line was tested with the addition of a drug (e.g., oxaliplatin). The drug line showed no effect, predicting that the tumor would be resistant to the drug across the entire dose range tested.

[0171] In comparison, multiple microorganospheres were generated from patient biopsies, as shown in Figure 22B. In this example, the patient-derived microorganospheres showed a significant decrease in cell viability from tumor microorganospheres, predicting drug sensitivity. Indeed, when treated with this drug, the tumor responded to treatment, as shown in Figure 22C (pre-treatment) and Figure 22D (post-treatment).

[0172] Example 3 Correlation of microorganospheres with patient response In a similar set of experiments, microorganospheres were generated from biopsy material (Figure 23A) and used for drug efficacy screening. Figure 23B shows the effect of the first drug (oxaliplatin) on these microorganospheres; the viability of the microorganospheres remained unchanged in the presence of the drug, predicting drug resistance. Similarly, treatment with a second drug, irinotecan, showed a lack of effect on the microorganospheres, as shown in Figure 23C, predicting drug resistance. When patients were treated with both oxaliplatin and irinotecan, there was no response after 6 months of treatment. Thus, microorganospheres strongly correlated with patient response to standard-of-care drugs. In this case, the patient endured 6 months of side effects and toxicity, which could have been avoided by the response predicted from the microorganospheres, which suggested that the tumor would not respond to the drugs (within 7–10 days of biopsy).

[0173] Example 4 Multidrug Screening Figure 24 shows an example of a panel of drugs (e.g., chemotherapeutic agents) that can be generated using multiple patient-derived microorganospheres described herein. In this example, drug screening using patient-derived microorganospheres was performed with multiple replicate additions for each of multiple (27) drugs. A single tumor biopsy was used to generate large quantities of multiple microorganospheres very quickly (e.g., within less than two weeks), and the microorganospheres were tested against a panel of formulations (e.g., 27 formulations shown). This testing could be performed in parallel and automatically quantified (e.g., by optical detection and quantification). In this example, the drug showing the greatest toxicity to this particular tumor was pazopanib.

[0174] Different drug concentrations as well as drug combinations can be investigated in parallel. The methods and devices described herein make such array testing practical, as hundreds, thousands, or tens of thousands of micro-organospheres can be generated from the same tumor biopsy.

[0175] Example 5 Preparation of biopsy samples Materials: Microorganosphere formation equipment as described above, including a droplet microfluidic chip (200 μm); Bio-Rad EvaGreen droplet generation oil (catalog no. 186-4006), 3-5 mL per run; perfluorooctanol (PFO), Sigma; 10% perfluorooctanol (PFO) in Novec HFE 7500; PBS; cell culture medium (i.e., RPMI w / 10% FBS and 1% PenStrep); 70 μm or 100 μm filters; 50 mL conical tubes; and Petri dishes.

[0176] Biopsy sample dissociation: Use a biopsy sample (human / animal) to generate a dissociated sample (i.e., single-cell tissue) from the patient. Coat the microfluidic chip and assemble the microfluidic chip and holder. Connect the microfluidic tubing and fittings to the outputs for microorganospheres and waste oil (e.g., multi-well plate, 15 mL Eppendorf tube, etc.).

[0177] Activate the device to form micro-organospheres. Remove the output (e.g., plate, Eppendorf tube, etc.) containing the droplets from the incubator (after at least 15 minutes). Remove any excess oil from the output. The droplets are buoyant, so the oil will be at the bottom of the vial. Be careful not to remove the droplets from the tube. Add 100 μL of 10% (v / v) PFO to the output. Swirl carefully and wait approximately 1 minute. Do not pipette or disturb the sample. Centrifuge at 300 g for 60 seconds. Remove the supernatant (excess oil / PFO). Do not pipette or disturb the sample. Remove as much PFO as possible, as it can reduce cell viability during the incubation period. Add 1 mL of cell culture medium. Do not pipette or disturb the sample. Centrifuge at 300 g for 60 seconds. Remove the supernatant and excess oil / PFO. Add 1 mL of cell culture medium. Using a 1 mL pipette tip, carefully pipette the sample up and down (approximately 30 times). Be careful not to over-pipette or disturb the droplet sample. Using a 1 mL pipette tip, pass the droplet medium solution through a 70 μm or 100 μm filter (connected to a 50 mL conical tube). Some droplets will stick to the inside of the output (e.g., a 15 mL Eppendorf tube). Rinse each tube with 2-3 mL of PBS and pipette up and down. Filter the rinsed PBS and droplets. Repeat this process twice or until the tube appears 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. Cover the entire surface area of ​​the filter. This washing step removes any excess oil and PFO from the sample, allowing the gel droplets to eventually be recovered in the cell culture medium.

[0178] Once properly drained (approximately 1-2 minutes), carefully remove the filter from the 50 mL conical tube. Turn the filter upside down and wash the underside with fresh cell culture medium, collecting the solution in a fresh Petri dish. A small droplet will come off the filter and fall into the cell culture medium. Using a 1 mL pipette tip and washing with approximately 5 mL of medium is recommended.

[0179] Check the quality of the droplets under a microscope. Most / all of the oil should be removed. If recovery is low, the sample can be filtered again. The density of the recovered microorganospheres can be checked with a hemocytometer.

[0180] Example 6 Kidney Tissue Microorganospheres In another example, microorganospheres 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, and Petri dishes (100 × 15 mm) or tissue culture dishes. Reagents can include EBM-2 medium, collagenase (5 mg / mL stock), Hank's Balanced Salt Solution (HBSS), calcium chloride (10 mM stock solution), phosphate buffer (1x PBS), Matrigel, 0.4% trypan blue solution, and trypsin.

[0181] Renal tissue is stored in cold transport medium and always on ice. 2 mL of enzymatic digestion solution may be placed in a 15 mL conical tube. 600 μL of calcium chloride (final concentration: 3 mM) is added, followed by 200 μL of collagenase (final concentration: 0.5 mg / mL). The kidney sample is transferred to a Petri dish / culture dish. Using a sterile tissue blade or razor blade, excess or non-tumor tissue is removed. 1 mL of enzyme solution is added to the tissue. Using a sterile razor blade, the sample is cut into small pieces (<2 mm 2) and finely chop the tissue. Press the plate down with tweezers or your hands. Return the minced tissue and enzyme solution to the 15 mL tube containing the enzyme solution. Place the tube on a tube rotator or 15 mL tube rotator in a 37°C incubator for 30-60 minutes. Remove the tube from the incubator. Quench the enzyme digestion with at least 6 mL of EBM-2 (at least three times the volume of the enzyme digestion solution). Mix by pipetting. Place a 100 μm or 70 μm cell strainer on a 50 mL conical tube. Transfer the sample through the strainer. Transfer the solution to a new 15 mL conical tube. Centrifuge the sample at 1500 rpm for 5 minutes. Discard the supernatant, leaving the cell pellet. Resuspend the pellet in 1 mL of EBM-2 medium. Add 10 μL of the cell mixture to 10 μL of trypan blue on a piece of parafilm and transfer to a cell counter plate or hemocytometer. Calculate the cell concentration (number / mL). Centrifuge at 1500 RPM for 5 minutes, discard the supernatant, and retain the pellet. The cell pellet is 1.25 x 10 5 Resuspend in 50 μL of Matrigel per cell. Keep on ice. Plate 50 μL domes of Matrigel-cell suspension in the center of wells of a prewarmed 24-well flat-bottom plate. Transfer the plate to a 37°C cell incubator and incubate for at least 20 minutes. Ensure the domes are polymerized. Gently add 500 μL of prewarmed EBM-2 medium through the well wall. Incubate in a 37°C incubator. Continue growing the microorganospheres with a complete medium change every 2 days.

[0182] Example 7 Liver microorganospheres As noted, microorganospheres can be formed from normal (e.g., non-cancerous) and / or diseased tissue. Figures 25A-25B and 26A-26B show an example of microorganospheres formed from mouse liver tissue, which was dislocate and combined with a fluid matrix material to form an unpolymerized mixture, and then droplets of the unpolymerized mixture were polymerized to form microorganospheres. In this example, the microorganospheres have a diameter of approximately 300 μm. In Figures 25A-25B, microorganospheres were formed using a single cell per droplet. In Figures 26A-26B, microorganospheres were formed using 25 cells per droplet. Figure 25A shows microorganospheres one day after formation, and Figure 25B shows microorganospheres after 10 days in culture. Cells within some of the microorganospheres divided and formed clumps that exhibited structure, while others contained slow-dividing or non-dividing cells. Also, in Figures 26A-26B, the microorganospheres initially contained approximately 25 cells per microorganosphere. After 10 days in culture, some of the microorganospheres showed significant cell proliferation and formed structures, while others showed only minimal proliferation. In both cases, the cells within the microorganospheres were found to exhibit characteristics of the primary tissue from which they were derived (e.g., hepatocytes).

[0183] The same method was successfully performed using human liver tissue, as shown in Figures 27A-27C. In this example, microorganospheres were initially formed using approximately 50 cells, as shown in Figure 27A. By day 18 of culture, some of the microorganospheres showed cells forming clumps and structures, while others had smaller structures or the cells had not divided.

[0184] Example 8 Cultured Cell Microorganospheres For example, in addition to primary tissues that are harvested immediately or shortly before forming the microorganospheres, microorganospheres can be formed from cells, including cultured cells, or cells in either 2D or 3D cultures.

[0185] In some variations, microorganospheres may be formed from cell lines grown as part of a patient-derived xenograft (PDX). For example, Figures 28A-28D show microorganospheres formed from cultured PDX240 cells. PDX240 cells are a patient-derived xenograft (PDX) tumor cell line (number 240 based on patient source) grown in immunodeficient mice from human tumors to form in vivo tumors (PDX). The xenograft tissue was extracted, dissociated, and used to form the microorganospheres. In this example, a single cell was contained within each microorganosphere formed. Figure 28A shows a microorganosphere after 1 day of culture, Figure 28B shows a microorganosphere after 3 days of culture, and Figures 28C and 28D show microorganospheres after 5 and 7 days of culture, respectively. As culture time progresses, at least some of the microorganospheres exhibit cells dividing to form structures.

[0186] Figures 29A-29D show a similar experiment in which five PDX240 cells were initially contained in each droplet forming each microorganosphere. With time in culture (e.g., days 1, 3, 5, and 7, as shown in Figures 29A-29D, respectively), the cells were able to divide and form structures.

[0187] Example 9 Comparison of microorganospheres with traditional organoids Organoids were formed from patient-derived xenograft cells (including the PDX240 cells described above and a second PDX cell line, PDX19187) and compared with microorganospheres formed using the same cells. These organoids were formed using conventional techniques in which cells were seeded in a large volume of Matrigel in a well or dish and cultured until proliferation was observed. Microorganospheres were generated from conventional organoids.

[0188] Both conventional ("bulk") organoids and microorganospheres were then treated with the same drug (e.g., oxaliplatin or SN38), and cell viability was measured after 3 days of treatment. The drug response curves shown in Figures 30 and 31 were generated to show similar response curves. For example, in Figure 30, the drug response curves of PDO19187 bulk organoids and microorganospheres showed similar response curves to oxaliplatin concentrations as those of PDX240 bulk organoids and microorganospheres. In Figure 31, the drug response curves of both PDX19187 and PDX240 were the same, and both bulk organoids and microorganospheres showed similar results to SN38. Figure 32 shows the response curves to another anti-cancer drug, 5-FU (fluorouracil), again showing similar drug response curves for both conventional organoids and microorganospheres for both PDZ-19187 and PDX-240.

[0189] Therefore, compared with traditional organoid, the micro-organosphere described herein can be formed more quickly and reliably, and have higher overall survival rate, and can provide drug response comparable to the drug response of the bulk organoid formed using the same cells.However, as described herein, micro-organosphere can be used more quickly and can be formed in much larger numbers.

[0190] Example 10 Drug effects on microorganospheres Generally, the micro-organospheres described herein can be used to perform one or more assays, including toxicity assays. Any suitable assay can be performed, with the results determined by analysis of the tissue (e.g., cells, tissue structures) suspended within the micro-organospheres. The micro-organospheres described herein can be assayed or analyzed optically, chemically, electrically, genetically, or in any other manner known in the art.

[0191] Optical detection (either manual or automated) can be particularly useful and can include optical analysis of the effect of one or more formulations on tissues (including cells, cell clumps, cell structures, etc.) within the micro-organospheres. In some variations, as described above, the formulations can be assayed for cell death (e.g., the number and / or size of tissues within the micro-organospheres being tested). In other variations, the micro-organospheres can be assayed for cell proliferation, including reduction in size, type, and / or growth rate. In some variations, the micro-organospheres can be assayed for changes in the tissue structures formed.

[0192] For example, Figures 33A-33B show the effect of one formulation, in this example, acetaminophen (10 mM), on mouse liver microorganospheres. Figure 33A shows a control group of untreated microorganospheres, showing tissue (arrows) within the microorganospheres that grew in culture. Figure 33B shows a similar set of microorganospheres formed from mouse liver, but these microorganospheres were instead treated with 10 mM acetaminophen. In the control group, the tissue structures within the microorganospheres are relatively large compared to the treated groups. The tissue within most microorganospheres containing acetaminophen is smaller and contains numerous dead cells.

[0193] Figures 34A-B also show toxicity assays using human liver microorganospheres. Figure 34A shows typical human liver microorganospheres observed in the control group, including tissue structures formed therein (indicated by arrows). Figure 34B shows a treatment group in which human liver microorganospheres were treated with acetaminophen (10 mM). Tissue in the treated microorganospheres shows abnormal tissue structures (arrows) and a significant increase in debris compared to the control group.

[0194] Any of these aspects, including optical aspects, may be scored, graded, ranked, or otherwise quantified. For example, in Figures 33A-33B and 34A-34B, the results of these two assays may be quantified to show dimensional differences, number of live / dead cells / tissue, etc. In some variations, scoring may be automated.

[0195] 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 executable by a processor (e.g., a computer, tablet, smartphone, etc.), which when executed by the processor, causes the processor to control the performance of any of the steps, including, but not limited to, displaying, communicating with a user, analyzing, modifying, determining, changing parameters (including timing, frequency, intensity, etc.), etc.

[0196] When a feature or element is referred to herein as being "on" another feature or element, the feature or element may be directly on the other feature or element, or there may be additional intervening features and / or elements. 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 present. Similarly, when a feature or element is referred to as being "connected," "attached," or "coupled" with another feature or element, it is understood that the feature or element may be directly connected, attached, or coupled to the other feature or element, or there may be intervening features or elements present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" with another feature or element, there are no intervening features or elements present. Although described or illustrated with respect to one embodiment, features and elements so described or illustrated may be applicable to other embodiments. Similarly, those skilled in the art will understand that a reference to a structure or feature being located "adjacent" to another feature may have overlapping or underlying portions with the adjacent feature.

[0197] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, steps, operations, elements, and / or components, but are understood not to exclude 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 and may be abbreviated as " / ."

[0198] Spatially relative terms, such as "under," "below," "lower," "over," "upper," etc., may be used for ease of description when describing the relationship of one element or feature to another element or feature as shown in the figures. It is understood that such spatially relative terms are intended to encompass different orientations of the device during use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were turned over, an element described as "under" or "beneath" another element or feature would now be oriented "above" that other element or feature. Thus, the exemplary term "below" can encompass both an upward and downward orientation. The device may be otherwise oriented (rotated 90 degrees or at another orientation), and the spatially relative descriptors used herein can be interpreted accordingly. Similarly, the terms "upward," "downward," "vertically," "horizontally," etc. are used herein for descriptive purposes only, unless otherwise specified.

[0199] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements are not 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 below could be referred to as a second feature / element, and similarly, a second feature / element discussed below could be referred to as a first feature / element, without departing from the teachings of the present invention.

[0200] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" mean that various components may be used together in methods and articles (e.g., compositions, and apparatuses, including devices, and methods). For example, the term "comprising" is understood to imply the inclusion of any specified elements or steps, but not the exclusion of any other elements or steps.

[0201] Generally, any of the devices and methods described herein will be understood to be inclusive, although all or some of the components and / or steps may instead be exclusive and may be expressed as "consisting of" or alternatively "consisting essentially of" various components, steps, subcomponents, or substeps.

[0202] Unless expressly specified otherwise, including in the examples, all numbers used in the description and claims herein may be read as beginning with the word "about" or "approximately," even if not explicitly stated otherwise. The expressions "about" or "approximately" may be used to describe a magnitude and / or location indicating 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 that is + / - 0.1% of the stated value (or value range), + / - 1% of the stated value (or value range), + / - 2% of the stated value (or value range), + / - 5% of the stated value (or value range), + / - 10% of the stated value (or value range), etc. Any numerical value provided herein is also understood to include about or approximately that value unless the context dictates otherwise. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range recited herein is intended to include all subranges subsumed therein. Furthermore, when a value is disclosed, it is understood that "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed, as would be understood by one of ordinary skill in the art. For example, if 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 numeric value) are also disclosed. Furthermore, throughout this application, data is provided in a number of different formats, and it is understood that this data refers to endpoints and starting points, and ranges for any combination of those data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are considered to be disclosed, as are values ​​between 10 and 15. Furthermore, it is understood that each number between two specified numbers is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0203] While various exemplary embodiments have been described above, any of numerous modifications may be made to the various embodiments without departing from the scope of the invention, as set forth in the claims. For example, the order in which the various method steps described are performed may frequently be changed in alternative embodiments, and in other alternative embodiments, one or more method steps may be skipped altogether. Optional features of the various apparatus and system embodiments may be included in some embodiments and not in others. Accordingly, the foregoing description has been provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention, as set forth in the claims.

[0204] The examples and illustrations contained herein show, by way of illustration, not limitation, specific embodiments in which the subject matter may be practiced. As noted, alternative embodiments are available and derivable, such that structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the inventive subject matter may be individually or collectively referred to herein as the "invention" merely for convenience, and without any intention to limit the scope of this application to any single invention or inventive idea if more than one is actually disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement intended to achieve the same purpose may be substituted for the specific embodiment shown. The present disclosure is intended to cover all adaptations or variations of various embodiments. Combinations of the foregoing embodiments, as well as other embodiments not specifically described herein, will be apparent to those of skill in the art upon review of the foregoing specification. [Explanation of symbols]

[0205] 503 Polymeric matrix materials 505 Structure 507 Hepatocytes 700 equipment 706 Holding Chamber 708 Chamber 716 Multiwell Plate 718 Temperature controller / temperature adjustment device 720 Droplet Microorganosphere Formation Assembly 722 Dispenser 724 Control Device 726 Pump 728 Sensors 730 Microfluidic Chips 731 Exit 733 Inlet for immiscible fluids 735 inlet port 737 Areas of junctions or intersections 739 elements 803 Microorganospheres 805 cells 839 Flow Area 903 Droplets 909, 909' flow path 911 Flow path 937 Confluence area 939 Outlet flow path 1005 Patient-derived microorganospheres 1008 Immiscible substances 2002 Immiscible substances 2004 port 2006 Unpolymerized material 2008 port 2010 Third Port Set 2103 First Channel 2107 Second Channel 2109 Intersecting Channel

Claims

1. obtaining a biopsy or resected tissue sample from the patient's tumor; dissociating the tissue sample to form a dissociated tissue sample; conveying the dissociated tissue sample and unpolymerized fluid matrix material through one or more flow channels of a microfluidic device, the microfluidic device controlling the pressure, flow rate, or pressure and flow rate within the one or more flow channels; forming a plurality of unpolymerized fluid mixture droplets from the dissociated tissue sample and the unpolymerized fluid matrix material within the one or more channels of the microfluidic device; polymerizing the fluid matrix material within the plurality of droplets to form a library of spherical droplets within the one or more channels of the microfluidic device, each of the spherical droplets having a diameter between 50 and 500 μm containing between 1 and 500 dissociated cells dispensed therein; forming a library of spherical droplets from the dissociated tissue sample, each of the spherical droplets comprising a polymerized matrix material and one or more three-dimensional cell aggregates; Culturing the library of spherical droplets for 1-14 days to form spherical droplets having three-dimensional cellular aggregates that contain structured clumps of cells that recapitulate the structure of the patient's tumor from which they were taken; and Assaying one or more drug therapies using said library of spherical droplets to determine the effect of said one or more drug therapies on cells within said spherical droplets. A method for high-precision drug screening comprising:

2. 2. The method of claim 1, wherein the assaying step comprises assaying multiple drug therapies in parallel by exposing one or more of the spherical droplets in the library to each drug therapy.

3. 3. The method of claim 1 or 2, further comprising characterizing a response of the patient tumor to the one or more drug therapies based on a response of the spherical droplets in the library to exposure to the one or more drug therapies.

4. 4. The method of claim 3, wherein the time between obtaining the tissue sample and characterizing the response is less than 21 days.

5. The method of claim 1, wherein the step of forming the library of spherical droplets further comprises sorting the spherical droplets based on cell number and / or droplet size.

6. 10. The method of claim 1, wherein the assaying step comprises assaying more than 50 different drug therapies.

7. 13. The method of claim 1, wherein the one or more drug therapies comprise different concentrations of one or more drugs, different combinations of three or more drugs, different ratios of two or more drugs, different carriers for one or more drugs, and / or different administration times of one or more drugs.

8. The method of claim 1 , wherein forming the library of spherical droplets comprises forming more than 1,000 spherical droplets.

9. The microfluidic device comprises: maintaining the viscosity of the dissociated tissue sample and the unpolymerized fluid matrix material prior to forming the plurality of unpolymerized mixture droplets; and / or preventing clogging of the dissociated tissue sample in the one or more channels; and / or having a flow channel diameter of 100 μm or more to prevent clogging; and / or Maintaining an approximately constant pressure in said one or more flow paths The method of claim 1 , wherein the method is configured to:

10. The method of claim 1, wherein the polymerizing step includes exposing the plurality of unpolymerized mixture droplets to a temperature greater than 25°C.

11. 11. The method of claim 10, wherein exposing the plurality of droplets of the unpolymerized mixture to a temperature greater than 25° C. comprises flowing the plurality of droplets of the unpolymerized mixture into a region of the microfluidic device that is maintained at a temperature greater than 25° C.

12. The method of claim 1 , wherein the microfluidic device maintains a constant flow rate in the one or more channels.

13. The method of claim 1 , wherein the patient tissue sample comprises a biopsy sample from a metastatic tumor.

14. The method of claim 1, wherein the spherical droplets in the library of spherical droplets have a dimensional variation of less than 25%.

15. The method of claim 1, wherein the spherical droplets contain budding clumps of cells and / or hollow structures of cells that reproduce the structure of the patient tumor from which they were taken.

16. 10. The method of claim 1, wherein assaying the one or more drug therapies comprises assaying spherical droplets having similar dimensions and cell counts.

17. The method of claim 1, wherein the plurality of unpolymerized mixture droplets have a density of less than 3 x 10 6 cells / ml of unpolymerized liquid matrix material.

18. The method of claim 1, wherein the plurality of unpolymerized mixture droplets have a density of less than 1 x 105 cells / ml of unpolymerized liquid matrix material.

19. The method of claim 1, comprising the step of transporting an immiscible fluid through the one or more flow paths of the microfluidic device, wherein the immiscible fluid is combined with the unpolymerized mixture prior to formation of the plurality of droplets, the plurality of droplets comprising the unpolymerized mixture and the immiscible fluid.

20. The method of claim 19, wherein the immiscible fluid comprises an oil.

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