TW202237858A - Precision drug screening for personalized cancer therapy - Google Patents

Abstract

Precision drug screening methods and apparatuses for personalized cancer therapies include the formation of a library of mature Micro-Organospheres, including Patient-Derived Micro-Organospheres (PMOSs), from a single patient tissue sample, such as from a tumor sample, are described. Also described herein are methods and systems for screening a patient using these Patient-Derived Micro-Organospheres, including personalized therapies.

Description

Precision Drug Screening for Personalized Cancer Therapy

The methods, devices and compositions of matter described herein relate to patient-derived micro organospheres (PMOSs), and methods and devices for forming PMOSs, and methods and devices for using PMOSs. In particular, described herein are methods and apparatus for identifying one or more pharmaceutical formulations that are effective in treating a particular patient.

Model cell and tissue systems are suitable for biological and medical research. The most common practice is to derive immortalized cell lines from tissues and culture them in two-dimensional (2D) conditions (eg, in Petri dishes or well plates). However, while well suited for basic research, 2D cell lines are not well correlated with individual patient responses to therapy. In particular, three-dimensional cell culture models prove particularly useful in developmental biology, disease pathology, regenerative medicine, drug toxicity and efficacy testing, and personalized medicine. For example, spheroids and organoids are three-dimensional cell aggregates that have been studied. However, both organoids and spheroids have limitations that reduce their efficacy.

Multicellular tumor spheroids were first described in the early 1970s and were obtained by culturing cancer cell lines under non-adherent conditions. Spheroids are typically formed from cancer cell lines as free-floating cell aggregates in ultra-low attachment dishes. Spheroids have been shown to maintain more stem cell-related properties compared to 2D cell cultures.

Organoids are in vitro derived aggregates of cells that include populations of stem cells that can differentiate into cells of major cell lineages. Organoids typically have a diameter of more than one millimeter in diameter and are cultured through passage. Organoid cultures are generally slower to grow and expand than 2D cell cultures. To generate organoids from clinical samples, sufficient numbers of viable cells (e.g., hundreds to thousands) are required to start with, so deriving organoids from smaller volume samples such as biopsies is often challenging and—even if successful—expanded in culture For applications such as drug testing would be time consuming. In addition, organoid size, shape, and cell number exhibit substantial variability. Organoids may require complex mixtures of growth factors and culture conditions in order to grow and express desired cell types.

Neither tumor spheroids nor organoids are optimal for rapid and reliable screening, especially for personalized medicine, such as performing in vitro tests of drug response. For example, in addition to balancing relative benefits and risks to achieve the most favorable outcome, the practice of oncology is constantly faced with the enormous challenge of matching appropriate treatment regimens to appropriate patients. Patient-derived cancer models (PDMC) can include the use of organoids, including patient-derived organoids, to facilitate the identification and development of more individualized therapeutic targets. However, while 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 organoid-guided therapy. As mentioned above, deriving and expanding organoids (and patient-derived organoids in particular) from tumor samples for drug susceptibility testing can take months, which reduces clinical applicability because patients cannot wait It took so long to receive treatment. Additionally, the number of organoids needed to perform drug screens with more than a few dozen compounds, typically in patients with metastatic or inoperable cancer, is currently not available in a clinically feasible time frame from core biopsy samples The only available form of organization. The high failure rate of biopsy-derived organoids also prevents their use as reliable diagnostic assays. Furthermore, there can be a high degree of variability in the size (and potentially response) of organoids, especially over longer periods of time in culture, and thus many passages.

Due to their better correlation with patient outcomes, PDMCs have also been adopted instead of 2D cell lines as high-throughput screening platforms for drug discovery, such as RNAi, CRISPR, and pharmaceutical small molecule screens. However, such PDMC models, including spheroids and organoids, are generally much slower to expand and manipulate than cell lines, making high-throughput applications challenging and costly. The longer time required to expand these models to expand cell numbers also tends to allow the fastest growing strain in the plastic to dominate and outperform the others, thus making the model more homogeneous and losing the original tissue composition and strain diversity. Furthermore, its relatively large and heterogeneous size and limited diffusivity make it challenging for many automated fluorescence- and imaging-based readouts.

Accordingly, there is a need for methods, compositions and devices for generating patient-derived tissue models (eg, tumor models and/or non-tumor tissue models) from resections or biopsies. In particular, it would be useful to provide methods and apparatus that enable a large number of patient-derived tissue models to have predictable and clinically relevant properties of a single biopsy, such as an 18-scale core biopsy, which This can be done, for example, within 7 to 10 days of obtaining a biopsy. This will allow robust and reliable testing and minimize delays in leading to patient-specific therapy. In addition, it would also be useful to generate patient-derived models that can be rapidly scaled up in a highly parallel fashion, resulting in better models with smaller and more uniform size, better controllability of the number of cells per unit, and high-throughput screening applications. Cells with better diffusivity (eg, by increasing the surface area to volume ratio).

Described herein are patient-derived micro-organoid spheroids (PMOS), devices and methods for making PMOS, and devices and methods for using PMOS. Also described herein are methods and systems for screening patients, including personalized therapy approaches, using these patient-derived mini-organoid spheroids.

In general, described herein are methods and apparatus for forming and growing patient-derived miniature organoid spheroids containing patient-derived cells, e.g., extracted from smaller patient biopsies (e.g., for rapid diagnostics) to guide therapy); extraction from resected patient tissue, including resected primary tumors or fractions of dysfunctional organs (e.g., for high-throughput screening); and/or extraction from established PDMCs, including Patient-derived xenografts (PDX) and organoids (e.g., to generate miniature organoid spheroids for high-throughput screening).

These PMOSs can be formed from primary cells that are normal (eg, normal organ tissue) or from tumor tissue. For example, in some variations, these methods and devices can form PMOSs from cancerous tumor biopsy tissue, thereby enabling the selection of customized treatments using the specific tumor tissue tested. Surprisingly, such methods and devices permit the formation of hundreds, thousands, or even tens of thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000, or more) PMOS. Dissociated primary cells from patient biopsies can be combined with a fluid matrix material, such as a substrate basement membrane matrix (eg, Matrigel), to form miniature organoid spheroids. The resulting plurality of patient-derived micro-organoid spheroids can have a predefined size range (such as diameter, e.g., 10 µm to 700 µm and any subrange therein), and an initial number of primary cells (e.g., 1 to 1000 , and especially lower numbers of cells, such as 1 to 200). Cell number and/or diameter can be controlled within, eg, +/- 5%, 10%, 15%, 20%, 25%, 30%, etc. These PMOSs had exceptionally high survival rates (>75%, >80%, >85%, >90%, >95%) when formed as described herein and were used and tested in extremely short periods of time. Stable, 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 days, within 10 days, etc.). This allows rapid testing of potentially large numbers of patient-specific and biologically relevant PMOSs, which can save critical time in generating and launching patient therapies, such as cancer treatment plans. The PMOS described herein rapidly forms 3D cellular structures that replicate and correspond to the tissue environment in which they are biopsied, such as a three-dimensional (3D) tumor microenvironment. The PMOS described herein may also be referred to as a "droplet." Each PMOS can include, for example, as part of a fluid matrix material, growth factors and structural proteins (eg, collagen, laminin, nestin, etc.) that can mimic the original tissue (eg, tumor) environment. Almost any primary cell tissue can be used, including almost any tumor tissue.

For example, all tumor types and sites tested to date have successfully produced PMOS (eg, 100% success rate to date, n=32, including from original sites including liver, omentum, and vibrating membrane) or metastatic tumors. colon cancer, esophageal cancer, skin cancer (melanoma), uterine cancer, bone cancer (sarcoma), kidney cancer, ovarian cancer, lung cancer and breast cancer). Tissue types used to successfully generate miniature organoid spheroids can be transferred from other locations. In some variations, the PMOS described herein can be grown from fine needle aspirate (FNA) or from circulating tumor cells (CTC), eg, from liquid biopsy. Proliferation and growth are typically seen in as few as 3-4 days, and PMOSs can be maintained and subcultured for months, or they can be cryopreserved and/or used immediately for analysis (eg, within the first 7-10 days).

In particular, methods for forming patient-derived mini-organoid spheroids are described herein. Generally, these methods involve combining dissociated primary tissue cells (including but not limited to cancer/abnormal tissue, normal tissue, etc.) with a liquid matrix material to form an unpolymerized material, and then polymerizing the unpolymerized material to form a typical diameter Miniature organoid spheroids of less than about 1000 µm (e.g., less than about 900 µm, less than about 800 µm, less than about 700 µm, less than about 600 µm, and especially, less than about 500 µm) having dissociated primary tissue cells distributed therein. The number of dissociated cells can be within a predetermined range, as mentioned above (e.g., between about 1 to about 500 cells, about 1 to 200 cells, about 1 to 150 cells, about 1 to 100 cells, about 1 to 75 cells, about 1 to 50 cells, about 1 to 30 cells, about 1 to 20 cells, about 1 to 10 cells, about 5 to 15 cells, about 20 to 30 cells, about 30 to 50 cells, about 40 to 60 cells, about 50 to 70 cells, about 60 to 80 cells, about 70 to 90 cells, about 80 to 100 cells, about 90 to 110 cells, etc., 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 structurally engineered as described herein to produce miniature organoid spheroids of reproducible size (eg, with a narrow size distribution).

Dissociated cells can be freshly biopsied and can be dissociated in any suitable manner, including mechanical and/or chemical dissociation (eg, by enzymatic disaggregation using one or more enzymes, such as collagenase, trypsin, etc.). Dissociated cells can optionally be treated, selected and/or modified. For example, cells can be sorted or selected to identify and/or isolate cells with one or more characteristics (eg, size, morphology, etc.). Cells that can be labeled (eg, with one or more markers) can be used to aid in selection. In some variations, known cell sorting techniques can be used, including but not limited to microfluidic cell sorting, fluorescence activated cell sorting, magnetic activated cell sorting, and the like. In some examples, PMOS can be sorted by acellular objects such as magnetic beads or barcode-like particles. Acellular objects useful for sorting (eg, magnetic beads, fluorescently labeled particles, etc.) can be incorporated into the PMOS as part of any of the methods described herein. Alternatively, cells can be used without sorting.

In some variations, dissociated cells can be modified by treatment with one or more reagents. For example, cells can be genetically modified. In some variations, cells can be modified using CRISPR-Cas9 or other gene editing techniques. In some variations, cells can be transfected by any suitable method (e.g., electroporation, cell extrusion, nanoparticle injection, magnetofection, chemical transfection, viral transfection, etc.), including with plastids, RNA, siRNA, etc. transfection. Alternatively, cells can be used without modification.

One or more additional materials can be combined with dissociated cells and fluid (eg, liquid) matrix material to form an unpolymerized mixture. For example, the unpolymerized mixture can include additional cell or tissue types, including supporting cells. Additional cells or tissues may be derived from different biopsies (eg, primary cells from different dissociated tissues) and/or cultured cells. Additional cells can be, for example, immune cells, stromal cells, endothelial cells, and the like. Additional materials may include media (eg, growth media, freezing media, etc.), growth factors, support network molecules (eg, collagen, glycoproteins, extracellular matrix, etc.), or the like. In some variations, the additional material can include a pharmaceutical composition. In some variations, the unpolymerized mixture includes only a dissociated tissue sample (eg, primary cells) and a fluid matrix material.

The method can rapidly form a plurality of patient-derived micro-organoid spheroids from a single tissue biopsy, such that greater than about 500 patient-derived micro-organoid spheroids (e.g., greater than about 600, greater than about 700) are formed from each biopsy , 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 may be a standard size biopsy, such as an 18G (eg, 14G, 16G, 18G, etc.) core biopsy. For example, the volume of tissue removed by biopsy and used to form the plurality of patient-derived micro-organoid spheroids can be as small as about 1/32 to 1/8 inch in diameter and about ¾ inch to ¼ inch long. Cylinder (obtained with a biopsy needle), such as a cylinder approximately 1/16 inch diameter by ½ inch long. The biopsy may be obtained by needle biopsy, for example by core needle biopsy. In some variations, biopsies may be obtained by fine needle aspiration. Other types of biopsies that may be used include shaving biopsies, punch biopsies, excisional biopsies, excisional biopsies, and the like. Typically material from a single patient biopsy can be used to generate a plurality (e.g., greater than about 2000, greater than about 5000, greater than about 7500, greater than about 10,000, etc.) of patient-derived mini-organoid spheroids as described above. A plurality of patient-derived mini-organoid spheroids can be formed using equipment (as described herein) that can be structurally designed to produce such large numbers of highly regular (size, cell number, etc.) mini-organoid spheroids as described herein . In some variations, these methods and devices can be used at rapid rates (e.g., greater than about 1 micro-organoid spheroid per minute, greater than about 1 micro-organoid spheroid per 10 seconds, greater than about 1 micro-organoid per 5 seconds Spheroids, greater than about 1 micro-organoid spheroid per 2 seconds, greater than about 1 micro-organoid spheroid per second, greater than about 2 micro-organoid spheroids per second, greater than about 3 micro-organoid spheroids per second, greater than about 4 micro-organoid spheroids, greater than about 5 micro-organoid spheroids per second, greater than about 10 micro-organoid spheroids per second, greater than 50 micro-organoid spheroids per second, greater than 100 micro-organoid spheroids per second, More than 125 micro-organoid spheroids per second, etc.) generate multiple micro-organoid spheroids.

For example, in some variations, the methods may be performed by combing the unpolymerized mixture with a material that is immiscible with the unpolymerized material (eg, a liquid material). Methods and apparatus may be controlled by at least partially controlling one or more of an unpolymerized mixture (and/or dissociated tissue and fluid matrix) and a material immiscible with the unpolymerized mixture (e.g., hydrophobic material, oil, etc.) Flow rate to control the size and/or cell density of the micro-organoid spheroids. For example, in some variations, microfluidic devices may be used to perform such methods. In some variations, multiple micro-organoid spheroids can be formed in parallel (eg, 2 in parallel, 3 in parallel, 4 in parallel, etc.). The same device may thus include multiple parallel channels, which may be coupled to the same source of unpolymerized material, or the same source of dissociated primary tissue and/or source of fluid matrix.

Unpolymerized materials can be polymerized to form patient-derived micro-organoid spheroids in a number of different ways. In some variations, the method may include changing the temperature (e.g., raising the temperature above a threshold value, such as, for example, above about 20°C, above about 25°C, above about 30°C, above about 35°C, etc.) to aggregate micro-organoid spheroids.

Once aggregated, the patient-derived micro-organoid spheroids can be allowed to grow, for example, by culture and/or can be analyzed before or after culture and/or can be cryopreserved before or after culture. Patient-derived mini-organoid spheroids can be cultured for any suitable length of time, but in particular can be cultured for 1 day to 10 days (e.g., 1 day to 9 days, 1 day to 8 days, 1 day to 7 days, 1 day to 6 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, etc.). In some variations, patient-derived micro-organoid spheroids can be cryopreserved or analyzed prior to six passages, which preserves cellular heterogeneity within patient-derived micro-organoid spheroids; limiting the number of passages prevents Faster dividing cells outnumber slower dividing cells.

In general, because the same patient biopsy can provide a higher number (eg, greater than 2,000, greater than 3,000, greater than 4,000, greater than 5,000, greater than 6,000, greater than 7,000, greater than 8,000, greater than 9,000, Greater than 10,000, etc.) cells, therefore some of the patient-derived micro-organoid spheroids can be cryopreserved (eg, more than half), while some are cultured and/or analyzed. As will be described in more detail herein, cryopreserved patient-derived mini-organoid spheroids can be stored and used later (eg, analysis, passage, etc.).

Accordingly, methods are described herein, including methods for forming a plurality of patient-derived micro-organoid spheroids. For example, a method of forming a plurality of patient-derived micro-organoid spheroids can include: combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets of the unpolymerized mixture; and making the small Droplets aggregate to form patient-derived micro-organoid spheroids, each between 50 µm and 500 µm in diameter, distributed with 1 to 200 dissociated cells.

A method of, for example, forming a plurality of patient-derived micro-organoid spheroids may comprise combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets from the continuous flow of the unpolymerized mixture, wherein the droplets having a size change of less than 25%; and polymerizing the droplets by increasing the temperature to form a plurality of patient-derived micro-organoid spheroids, each having 1 to 200 dissociated cells distributed within each patient-derived micro-organoid spheroid.

In some variations, a method for forming a plurality of patient-derived micro-organoid spheroids as described herein may comprise: combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; Convergence of a stream of a polymerized mixture with one or more streams of a fluid immiscible with an unpolymerized mixture to form a plurality of droplets that vary in size by less than 25%; causing droplets to coalesce to form diameters between 50 µm and patient-derived micro-organoid spheroids between 500 µm with 1 to 200 dissociated cells distributed therein; and separating the patient-derived micro-organoid spheroids from immiscible fluids.

Any of these methods can include modifying the cells within the dissociated tissue sample prior to forming the droplets.

Forming a plurality of droplets can comprise forming a droplet having a size change of less than about 25% (e.g., a size change of less than about 20%, a size change of less than about 15%, a size change of less than about 10%, a size change of less than about 8%) , less than about 5% size variation, etc.) of uniformly sized droplets of a plurality of unpolymerized mixtures. Size changes can also be described as a narrower distribution of size changes. For example, the size distribution can include samples with a low 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.) patient-derived micro-organoid spheroid size distribution (eg, micro-organoid diameter and number of micro-organoid spheroids formed).

Any of these methods may also include plating or distributing patient-derived micro-organoid spheroids. For example, in some variations, methods can include combining patient-derived micro-organoid spheroids from different sources into a container prior to analysis. For example, miniature organoid spheroids can be placed in porous dishes. Accordingly, any of these methods may comprise dispensing the patient-derived micro-organoid spheroids into a multi-well disc prior to analyzing the patient-derived micro-organoid spheroids. Each well can include one or more (or, in some variations, equivalent amounts) of patient-derived mini-organoid spheroids. In some variations, coating the patient-derived micro-organoid spheroids into the container may include placing the organoid spheroids into a plurality of chambers separated by at least partially permeable membranes to allow the supernatant material to Circulation between chambers. This allows patient-derived micro-organoid spheroids to share the same supernatant.

In any of these methods, patient-derived micro-organoid spheroids can be analyzed. Assays can generally involve exposing or treating individual patient-derived mini-organoid spheroids to conditions (e.g., pharmaceutical compositions) to determine whether the pharmaceutical composition has an effect on the cells of the patient-derived mini-organoid spheroids (and in some cases, what effect does it have). Assays may include exposing a subset of patient-derived micro-organoid spheroids (individually or in groups) to one or more concentrations of the pharmaceutical composition, and allowing the patient-derived micro-organoid spheroids to remain exposed for a predetermined period of time (minutes, minutes, Hours, days, etc.) and/or exposure and removal of the pharmaceutical composition, followed by culturing the patient-derived micro-organoid spheroids for a predetermined period of time. The patient-derived micro-organoid spheroids can then be assayed to identify any effects including, inter alia, toxicity to cells in the patient-derived micro-organoid spheroids, or changes in the morphology and/or growth of cells in the patient-derived micro-organoid spheroids . In some variations, the analysis can include labeling (eg, by immunohistochemistry) living or fixed cells within the patient-derived mini-organoid spheroids. Cells can be analyzed (eg, inspected) manually or automatically. For example, cells can be assayed using an automated reader device to determine any toxicity (cell death). In some variations, analyzing the plurality of patient-derived micro-organoid spheroids can include sampling one or more of the supernatant, the environment, and the microenvironment of the patient-derived micro-organoid spheroids for analysis of secreted factors and other effects. In any of these variations, the patient-derived micro-organoid spheroids can be recovered after analysis for further analysis, expanded, or stored (eg, cryopreserved, frozen, etc.) for subsequent examination.

As mentioned, almost any analysis can be used. For example, gene body, transcriptome, proteomic or inter-genome markers such as methylation can be analyzed using the PMOS described herein. Accordingly, any of the compositions and methods described herein can be used to identify or test one or more markers and biological/physiological pathways, including, for example, exosomes, which can aid in the identification of Medications and/or therapies used to treat the patient.

Any suitable tissue sample can be used. In some variations, the tissue sample may comprise a biopsy sample from a metastatic tumor. For example, a tissue sample can include a clinical tumor sample; a clinical tumor sample 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.

Any of the methods described herein may comprise initially dissociated cells from a tissue biopsy distributed uniformly (or in some variations non-uniformly) throughout the fluid matrix material at any suitable concentration. For example, in some variations, the methods described herein can include combining a dissociated tissue sample and a fluid matrix material such that the dissociated tissue cells are distributed within the fluid matrix material to less than ¹ x 107 cells/ mL (e.g., less than 9×10 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 cells/ml, etc.).

In general, forming droplets may comprise forming droplets from a continuous stream of unpolymerized mixture. For example, forming droplets can include applying a converging stream of one or more fluids immiscible with the unpolymerized mixture to the unpolymerized mixture stream. These streams can be combined in a microfluidic device, such as a device with a plurality of converging channels in which unpolymerized mixtures and immiscible fluids interact to form droplets with precisely controlled volumes. In some variations, droplets are formed (eg, pinched off) in an excess of immiscible material, and the droplets can be simultaneously and/or subsequently polymerized to form patient-derived miniature organoid spheroids. For example, the region where the streams converge can be configured to polymerize the unpolymerized mixture after the droplets have been formed, for example by heating, and/or the region downstream can be configured to polymerize the unpolymerized mixture after the droplets have been formed. Mixed and surrounded by immiscible materials. In some variations, the immiscible material is heated (or alternatively cooled) to a temperature that promotes polymerization of the unpolymerized material, thereby forming patient-derived micro-organoid spheroids. For example, polymerizing may involve heating the droplets to above 35°C.

Thus, in any of these methods, forming the droplets may comprise 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 micro-organoid spheroids. For example, any of these methods can include removing immiscible fluid from the patient-derived micro-organoid spheroids. In general, immiscible fluids may include liquids (eg, oils, polymers, etc.), including hydrophobic materials or other materials that are immiscible with unpolymerized (eg, aqueous) materials, among others.

The fluid matrix material may be a synthetic or non-synthetic unpolymerized basement membrane material. In some variations, the unpolymerized base material may comprise a polymerized hydrogel. In some variations, the fluid matrix material may comprise Matrigel. Thus, combining the dissociated tissue sample and the fluid matrix material may comprise combining the dissociated tissue sample with the basement membrane matrix.

The tissue sample can be mixed with the fluid within six hours or earlier (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. Matrix material combined.

Also described herein are methods of analyzing or preserving patient-derived micro-organoid spheroids. For example, the method may include: combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets of the unpolymerized mixture and the size of the droplets vary by less than 25%; polymerizing the droplets to form Multiple patient-derived micro-organoid spheroids between 50 µm and 700 µm in diameter with 1 to 1000 dissociated cells distributed therein; and analysis or cryopreservation of multiple patient-derived micro-organoid spheroids.

In some variations, the method may include: combining the dissociated tissue sample and the fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets of the unpolymerized mixture; Multiple patient-derived micro-organoid spheroids between 500 µm with 1 to 200 dissociated cells distributed; The effect of one or more reagents on cells within the patient-derived mini-organoid spheroids is determined.

For example, the method may comprise: combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; Converge to form multiple droplets with a droplet size variation of less than 25%; the droplets are aggregated by heating to form patient-derived micro-organoid spheroids with individual diameters between 50 µm and 500 µm, distributed with 1 to 200 dissociated cells; and analyzing or cryopreserving the patient-derived micro-organoid spheroids prior to six passages, thereby maintaining the heterogeneity of cells within the patient-derived micro-organoid spheroids, further wherein the analysis includes facilitating the determination of one or more Analysis of the effect of reagents on cells within patient-derived micro-organoid spheroids.

In either of these methods, the plurality of patient-derived micro-organoid spheroids can be cryopreserved or analyzed prior to six passages, thereby maintaining the heterogeneity of the cells within the patient-derived micro-organoid spheroids. Any of these methods can further include modifying the cells within the dissociated tissue sample prior to forming the droplets.

Forming the droplets can include forming a plurality of uniformly sized droplets 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.). Polymerized mixture droplets.

Any of these methods may include culturing the patient-derived mini-organoid spheroids for an appropriate length of time, as mentioned above (eg, culturing the patient-derived mini-organoid spheroids for 2 to 14 days prior to analysis). For example, such methods can include removing immiscible fluids from patient-derived micro-organoid spheroids prior to culturing. In some variations, culturing the patient-derived micro-organoid spheroids comprises culturing the patient-derived micro-organoid spheroids in suspension.

In any of the methods described herein, patient-derived mini-organoid spheroids can be cultured together in large quantities. In some variations, for example when forming a bank, patient-derived mini-organoid spheroids sorted by size and/or number of cells can be cultured together. Patient-derived micro-organoid spheroids can be cultured in suspension or in layers (eg, in dishes, chambers, tubes, etc.).

In general, analyzing patient-derived micro-organoid spheroids may comprise genomic, transcriptomic, epigenomic and/or metabolic testing prior to and/or after analysis or cryopreservation of patient-derived micro-organoid spheroids. Analysis of cells in patient-derived micro-organoid spheroids. Any of these methods can comprise analyzing the patient-derived micro-organoid spheroids by exposing the patient-derived micro-organoid spheroids to a drug (eg, a pharmaceutical composition).

In any of these methods, analyzing can comprise manually and/or automatically visually analyzing the effect of one or more reagents on the cells in the patient-derived mini-organoid spheroids. Any of these methods can include labeling or labeling the cells in the patient-derived mini-organoid spheroids for visualization. For example, analysis can include fluorometric analysis of the effect of one or more reagents on cells.

The patient-derived micro-organoid spheroids described herein are novel per se and can be characterized as compositions of matter. For example, the composition of matter may comprise a plurality of cryopreserved patient-derived micro-organoid spheroids, wherein each patient-derived micro-organoid spheroid has a spherical shape with a diameter between 50 µm and 500 µm and comprises a polymeric base material, and about 1 to 1000 dissociated primary cells of less than six passages distributed within the base material, thereby maintaining the heterogeneity of the cells within the patient-derived micro-organoid spheroids.

Also described herein are compositions of matter comprising a plurality of cryopreserved patient-derived micro-organoid spheroids, wherein each patient-derived micro-organoid spheroid has a spherical shape with a diameter between 50 µm and 500 µm, wherein the patient-derived micro-organoid The organoid spheroids have a size variation of less than 25%, and wherein each patient-derived micro-organoid spheroid comprises a polymeric substrate material and about 1 to 500 dissociated primary cells of less than six passages distributed within the substrate material, The heterogeneity of the cells within the patient-derived mini-organoid spheroids is thereby maintained.

Primary cells may be primary tumor cells. For example, dissociated primary cells may have been genetically or biochemically modified. The plurality of cryopreserved patient-derived micro-organoid spheroids can be of uniform size with less than 25% variation in size. In some variations, the plurality of cryopreserved patient-derived micro-organoid spheroids may comprise patient-derived micro-organoid spheroids from different sources. In any of these mini-organoid spheroids, the majority of cells in each mini-organoid spheroid may comprise cells that are not stem cells. In some variations, the primary cells comprise metastatic tumor cells. Primary cells can include both cancer cells and stromal cells. In some variations, the primary cells comprise tumor cells and one or more of: mesenchymal cells, endothelial cells, and immune cells.

Primary cells can be less than, for example, ⁵ x 107 cells/ml, 1 x 107 cells/ml, ⁹ x 106 cells/ml, ⁷ x 106 cells/ml, ⁵ x ¹⁰⁶ cells/ml, The density distribution of 1×10 ⁶ cells/ml, 9×10 ⁵ cells/ml, 7×10 ⁵ cells/ml, 5×10 ⁵ cells/ml, 1×10 ⁵ cells/ml, etc. in aggregation in the base material.

Generally, the polymeric base material can comprise a basement membrane matrix (eg, Matrigel). In some variations, the polymeric base material comprises a synthetic material.

The micro-organoids may have a diameter between 50 µm and 1000 µm, or more preferably between 50 µm and 700 µm, or more preferably between 50 µm and 500 µm, or between 50 µm and 400 µm, or 50 Between µm and 300 µm, or between 50 µm and 250 µm, etc. (eg, 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.).

As mentioned, the patient-derived micro-organoid spheroids described herein can include any suitable number of primary tissue cells initially in each patient-derived micro-organoid spheroid, such as less than about 200 primary cells, or more Preferably less than about 150 primary cells, or more preferably less than about 100 primary cells, or more preferably less than about 75 primary cells, or less than about 50 cells, or less than about 30 cells, or less than about 25 cells, or less than about 20 cells, or less than about 10 cells, or less than about 5 cells, etc.). In some variations, each patient-derived micro-organoid spheroid comprises about 1 to 500 cells, about 1 to 400 cells, about 1 to 300 cells, about 1 to 200 cells, about 1 to 150 cells, About 1 to 100 cells, about 1 to 75 cells, about 1 to 50 cells, about 1 to 30 cells, about 1 to 25 cells, about 1 to 20 cells, etc.

Also described herein are devices for forming patient-derived micro-organoid spheroids, and methods of operating such devices to form patient-derived micro-organoid spheroids. For example, described herein is a method of operating a patient-derived micro-organoid spheroid forming device comprising: receiving in a first port an unpolymerized mixture comprising a dissociated tissue sample and a cooled mixture of a first fluid matrix material; Receiving a second fluid immiscible with the unpolymerized mixture in the second orifice; combining the stream of the unpolymerized mixture with one or more streams of the second fluid to form an unpolymerized mixture of uniform size and variation less than 25% droplets; and polymerizing the unpolymerized mixture droplets to form a plurality of patient-derived micro-organoid spheroids.

A method of operating a patient-derived micro-organoid spheroid forming device may include: receiving an unpolymerized mixture comprising a dissociated tissue sample and a cooled mixture of a first fluid matrix material in a first port; receiving an unpolymerized mixture in a second port; A second fluid in which the polymerized mixture is immiscible; combining the stream of the unpolymerized mixture at the first rate with one or more streams of the second fluid at the second rate to form unpolymerized streams of uniform size and less than 25% variation polymerizing the mixture droplets, wherein the diameter of the droplets is between 50 µm and 500 µm; and polymerizing the unpolymerized mixture droplets to form a plurality of patient-derived micro-organoid spheroids.

Any of these methods can include coupling a first reservoir containing the unpolymerized mixture in fluid communication with the first orifice. For example, a method can include combining a dissociated tissue sample and 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 orifice. Such methods may include coupling a second reservoir containing a second fluid in fluid communication with a second orifice. Any of these methods can include adding the second fluid to a second reservoir in fluid communication with the second orifice. In some variations, receiving the second fluid includes receiving oil.

Generally, such methods can include separating a second fluid (eg, an immiscible fluid) from the plurality of patient-derived micro-organoid spheroids. This fluid can be separated manually or automatically. For example, the second (immiscible) fluid may be removed by washing, filtering or any other suitable method.

Combining the streams may comprise driving the unpolymerized mixture stream at the first flow rate through one or more streams of the second fluid moving at the second flow rate. In some variations, the first flow rate is greater than the second flow rate. Either or both of the flow rate and/or amount of material (e.g., unpolymerized mixture) may be present in a smaller amount than the second fluid so that the unpolymerized mixture is encapsulated in precisely controlled droplets , as described herein, the droplets may then polymerize, for example, within a second fluid.

In some variations, combining the streams includes driving the stream of the unpolymerized mixture through a junction into which one or more streams of the second fluid also converge. Polymerizing the droplets can comprise heating the droplets to a temperature above which the unpolymerized material polymerizes (eg, above about 25°C, above about 30°C, above about 35°C, etc.).

Any of these methods may comprise aliquoting a plurality of patient-derived mini-organoid spheroids. For example, aliquot into multi-well Petri dishes.

Also described herein are methods of treating patients using these patient-derived mini-organoid spheroids and methods of analyzing the same. For example, the method may comprise: receiving a patient biopsy from the tumor; determining that the tumor will respond to the drug formulation within 2 weeks of obtaining the biopsy by: forming a tumor from the patient biopsy between 50 µm and 500 Multiple micro-organoid spheroids between µm with 1 to 200 dissociated tumor cells distributed throughout the polymeric substrate material, and patient-derived micro-organoids before the dissociated tumor cells have been passaged for more than five passages exposing at least some of the spheroids to the drug formulation; and measuring the effect of the drug formulation on cells within at least some of the mini-organoid spheroids to determine whether the drug will treat the tumor based on the determined effect.

In some variations, the methods may include determining that the tumor remains responsive to the drug formulation after one or more administrations of the drug by receiving a second patient biopsy after the patient has been treated with the drug formulation and Forming a second plurality of patient-derived micro-organoid spheroids from a second patient biopsy, exposing at least some of the second plurality of patient-derived micro-organoid spheroids to the drug formulation, and measuring the effect of the drug formulation on the second The role of cells within at least some of the plurality of miniature organoid spheroids.

Determining that a tumor will respond to a drug formulation can include exposing at least some of the patient-derived mini-organoid spheroids to a plurality of drug formulations, and reporting a measured effect of each of the drug formulations. In some variations, determining further comprises dispensing the micro-organoid spheroids into the porous disc prior to analyzing the patient-derived micro-organoid spheroids.

Any of these methods may include taking a biopsy of the patient to collect the patient biopsy (or otherwise obtain a tissue sample from the patient or a sample of patient-derived tissue or cells), and/or treat with the drug formulation patient, or assisting a physician in treating a patient (eg, advising the physician which drug formulation will be effective). Generally, the time between receipt of a biopsy and reporting can 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 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, etc.).

In general, the methods and apparatus described are structurally designed to achieve high encapsulation efficiency and minimal loss of sample/cells during processing. Thus, as described herein, large numbers of patient-derived mini-organoid spheroids can be efficiently formed from a single resected or biopsied tumor sample. These methods and systems can overcome problems associated with other systems in which smaller clinical samples are completely lost in processing and larger clinical samples cannot reach sufficient densities for organoid establishment.

In particular, such methods and apparatus can provide microfluidic systems that receive a dissociated sample of a tumor from a patient (e.g., a single tumor, although in some examples multiple tumor samples can be provided), and control the viscosity and/or flow rate. Path length and diameter and twist rate (eg, curve) can be controlled to prevent clogging and/or damage to dissociated cells.

For example, the methods and apparatus can control pressure, flow rate by maintaining a constant or near constant pressure within the channels of a microfluidic system (which in some instances can be configured with a removable filter cartridge) (flow rate) or pressure and flow rate. Filter cartridges can be single use or reusable. Fluids (eg, solutions of dissociated cells and unpolymerized matrix material) can thus be pumped through the microfluidic system with constant or nearly constant pressure. In general, the flow of materials through a microfluidic system can be laminar, especially the flow of dissociated cells. By maintaining the flow rate within a predetermined range (for example, 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 Between 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 between min and 10 ml/min, between 0.05 ml/min and 5 ml/min, between 0.1 ml/min and 100 ml/min, between 0.1 ml/min and 50 ml/min, between 0.1 ml/min and Between 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.) to maintain laminar flow.

The path length within the microfluidic system from the input of the dissociated cells to the region where the unpolymerized matrix is formed (and the dissociated cells) may be less than, for example, 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.

The diameter of the channel or channels through which dissociated cells pass can be engineered to prevent clogging. For example, the diameter of the channel 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 µm and 500 µm, between 100 µm and 500 µm, between 120 µm and 500 µm, between 150 µm and 500 µm, between 200 µm and 500 µm, etc.).

Dissociated cells can be passed through short or very short conduits (e.g., less than 20 cm, less than 15 cm, less than 10 cm, less than 7.5 cm, less than 5 cm, etc.) are added to the microfluidic system to minimize the "dead" area.

In general, the geometry of the channels of a microfluidic system can be engineered to avoid clogging. For example, one or more channels may have a diameter 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.) radius of curvature. All or substantially all corners within the path may be smooth, curved or rounded.

A microfluidic system can include one or more air bubble removal chambers (eg, gas-permeable but fluid-impermeable membranes and/or vacuum ports, etc. for removing air bubbles).

In general, microfluidic systems can control the temperature (and thus viscosity) of materials within one or more channels. As mentioned, channels carrying dissociated cells and unpolymerized matrix can be cooled and maintained (within +/- 1°C or preferably) to 20°C or lower (e.g., 18°C or lower, 15°C or Lower, 12°C or lower, 10°C or lower, 7°C or lower, 5°C or lower, 4°C or lower, between 1°C and 20°C, between 1°C and 15°C, 1 °C to 12 °C, 1 °C to 10 °C, 1 °C to 5 °C, 1 °C to 4 °C, etc.). For example, the system may include integrated Peltier elements to maintain the viscosity of the material during processing. In some examples, multiple temperature zones throughout the microfluidic system can control polymerization of the matrix material only after encapsulation has been successful.

For example, described herein is a method for precision drug screening for personalized cancer therapy comprising: receiving a single tissue sample from a patient's tumor; dissociated the biopsy tissue sample to form a dissociated tissue sample; by Formation of more than 1000 patient-derived micro-organoid spheroids from dissociated tissue samples: driving dissociated tissue samples and unpolymerized fluid matrix material through one or more channels of a microfluidic device, where the microfluidic device controls the pressure within the channel , flow rate or pressure and flow rate and maintaining the temperature of the dissociated tissue sample and the unpolymerized fluid matrix material at a temperature of 20° C. or lower, wherein the dissociated tissue sample passes through one or more channels under laminar flow, combining the dissociated tissue sample and unpolymerized fluid matrix material within the microfluidic device to form a plurality of unpolymerized mixture droplets; exposing the unpolymerized mixture droplets to a temperature above 25° C. to polymerize the fluid matrix material and form Patient-derived micro-organoid spheroids, wherein the patient-derived micro-organoid spheroids each have a diameter between 50 µm and 500 µm and have 1 to 200 dissociated cells distributed therein; the patient-derived micro-organoid spheroids are cultured for 2 to 14 to form mature patient-derived mini-organoid spheroids with structured cell clusters replicating the structure of their biopsied or resected tumors; and by deriving the mature patient-derived One or more of the miniature organoid spheroids were exposed to each drug therapy and multiple drug therapies were analyzed in parallel.

In any of the methods described herein, patient-derived micro-organoid spheroids can be analyzed by any suitable assay, including but not limited to: 3D viability analysis, imaging, analysis of gene and/or protein expression (e.g. , DNA, RNA, proteomics, exosomes, etc.). Cells, patient-derived micro-organoid spheroids, extracellular matrix forming part of the patient-derived micro-organoid spheroids, and/or supernatants from culturing the patient-derived micro-organoid spheroids, etc. can be assayed.

Any of the methods described herein, including precision drug screening assays, can screen for one or more of: chemotherapy, targeting agents, and immunotherapy. For example, a method for precision drug screening for personalized cancer therapy can include screening drug therapies that include one or more of chemotherapy, targeting agents, and immunotherapy. Chemotherapy or chemotherapeutic agents refer to treatment with cytostatic or cytotoxic agents (ie, 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 in the treatment of proliferative disorders such as cancer. As used herein, an immunotherapy or immunotherapeutic agent generally promotes an immune response, eg, the agent may be an immunostimulator or an inhibitor of an immunosuppressant (ie, an anti-immunosuppressant). The term thus includes immunogenic compositions as well as vaccines. Immunotherapeutics may also include checkpoint blockers or inhibitors, chimeric antigen receptors (CARs), and T cell therapy.

The microfluidic device can maintain the temperature of all or a portion of the microfluidic device within a cooled 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 °C or lower, about 15 °C or lower, about 14 °C or lower, about 13 °C or lower, about 12 °C or lower, about 11 °C or lower, about 10 °C or lower, about 9 °C or lower, about 8°C or lower, about 7°C or lower, about 6°C or lower, about 5°C or lower, between about 1°C and about 20°C, between about 2°C and about 15°C between, between about 2°C and about 10°C, etc.). Microfluidic devices may include, for example, Peltier devices for cooling. The microfluidic device can cool all or a portion of the microfluidic device. For example, the microfluidic device can be structurally designed to cool the input sample, the catheter, and/or the microfluidic device wafer so that the input material (eg, tissue sample) remains cool. The unpolymerized fluid matrix material can also be kept cool. The microfluidic device can include a thermally conductive material (eg, aluminum, etc.) that can distribute coolant from a cooling source (eg, Peltier) to any or all of these regions. Microfluidic devices may include one or more thermal sensors (eg, thermistors, etc.) for sensing temperature in these regions, and may include control feedback to adjust temperature using sensor input.

The tissue sample can be a biopsy sample (including resected), and/or can be a fine needle aspirate, and/or a tissue sample of circulating tumor cells. Tissue samples can be obtained acutely (eg, within minutes or hours of performing a method, eg, a precision drug screening method involving the formation of patient-derived micro-organoid spheroids). For example, the tissue sample can be "fresh" and can be within about 12 hours, within about 8 hours, 4 hours, within about 3 hours, within about 2 hours, within about 1 hour of formation of the patient-derived micro-organoid spheroids Nether is obtained from the patient. Tissue samples can be stored at low temperatures (eg, between about 0°C and about 20°C, between about 1°C and 15°C, below about 15°C, below 10°C, etc.).

The method can include characterizing the response to a drug therapy based on the response to the patient-derived miniature organoid spheroids. Forming the patient-derived micro-organoid spheroids may further comprise sorting the patient-derived micro-organoid spheroids based on cell number and/or droplet size. For example, forming the patient-derived micro-organoid spheroids can include optically sorting the patient-derived micro-organoid spheroids or based on cell number and/or droplet size. Sorting can be performed before or after polymerization. Sorting can include directing fluid flow to deposit specific PDMOs into specific bins (eg, chambers) based on their sorting characteristics.

Analysis may include analysis of more than 5 different drug therapies (more than 10, more than 15, more than 20, more than 30, more than 35, more than 40, more than 50, more than 75 species, more than 100 species, more than 150 species, more than 200 species, more than 250 species, more than 300 species, more than 500 species, more than 1000 species, etc.). As used herein, drug therapy may include a single drug and/or different combinations and/or conditions in different conditions (concentration, vehicle, etc.), or in different dosing regimens (e.g., repeated doses, duration of administration, etc.) and/or multiple drugs in the dosing regimen. For example, different drug regimens 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 of one or more drugs, and/or a or different administration times of multiple drugs.

Forming more than 1000 patient-derived micro-organoid spheroids includes forming more than 5000 patient-derived micro-organoid spheroids (greater than 10,000 etc.), as mentioned above.

The microfluidic system can maintain the viscosity of the dissociated tissue sample and the unpolymerized fluid matrix material prior to the formation of droplets.

Microfluidic systems can typically be structurally designed to prevent clogging of dissociated tissue samples within one or more channels. For example, microfluidic systems can be engineered to prevent clogging by having channel diameters of 100 μm or greater. Microfluidic systems can be engineered to maintain an approximately constant pressure and/or flow rate within one or more channels.

Exposing the unpolymerized mixture droplets to a temperature above 25°C may comprise exposing the droplets to 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.).

Exposing the unpolymerized mixture droplet to a temperature above 25°C may comprise flowing the droplet to a region of the microfluidic device maintained at a temperature above 25°C. In some examples, droplets can be output from a microfluidic device to polymerize in an elevated temperature chamber.

As mentioned, a microfluidic device can maintain a constant flow rate within one or more channels.

The methods described herein can include measuring the effect of each drug therapy on cells within a patient-derived mini-organoid spheroid to determine whether the drug therapy will treat the tumor based on the measured effect. The method can include determining that the tumor remains responsive to the drug therapy after one or more administrations of the drug therapy by receiving a second patient tumor tissue after the patient has been treated with the drug therapy and forming a second patient tumor tissue from the second patient tumor tissue. The plurality of patient-derived micro-organoid spheroids, exposing at least some of the second plurality of patient-derived micro-organoid spheroids to a drug therapy, and measuring the effect of the drug therapy on at least some of the second plurality of patient-derived mini-organoids The role of some inner cells.

Any of these methods can be used to test monotherapy, including repeated testing, as described above.

The method may comprise treating the patient with a drug therapy from a plurality of drug therapies.

The time between receiving 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.).

Patient tumor tissue may comprise a biopsy sample from a metastatic tumor. In general, tumor tissue as used herein may be epithelial adenocarcinoma tissue (as seen in all examples provided herein).

In general, such methods can include forming a plurality of mini-organoids, including forming mini-organoids with less than 25% variation in size and/or cell number variation (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.) miniature organoids. Lower variation in size and/or cell number can be especially helpful in normalizing responses between different drug therapies. Also, multiple times can be used or compared. The step of analyzing the plurality of drug therapies may comprise analyzing patient-derived mini-organoid spheroids of similar size and cell number.

In general, patient-derived micro-organoid spheroids can be recoated with tissue structures from the tissue from which they were derived, e.g., they can form sprouting clusters of cells and/or hollow structures of cells that replicate their The structure of the tumor after biopsy or resection.

For example, a method for precision drug screening for personalized cancer therapy, the method may comprise: receiving a single biopsy or resected tissue sample from a patient's tumor; dissociating the biopsy tissue sample to form a dissociated tissue sample ; forming a plurality of patient-derived micro-organoid spheroids from a dissociated tissue sample by driving the dissociated tissue sample and unpolymerized fluid matrix material through one or more channels of a microfluidic device, wherein the microfluidic device controls the channel pressure, flow rate or pressure and flow rate and maintain the temperature of the dissociated tissue sample and the unpolymerized fluid matrix material at a temperature of 20° C. or lower, wherein the dissociated tissue sample passes through one or more chambers under laminar flow channels for combining dissociated tissue samples and unpolymerized fluid matrix material within the microfluidic device to form a plurality of unpolymerized mixture droplets; exposing the unpolymerized mixture droplets to temperatures greater than 25°C to polymerize the fluid matrix material and form patient-derived micro-organoid spheroids, wherein the patient-derived micro-organoid spheroids each have a diameter between 50 µm and 500 µm, with 1 to 200 dissociated cells distributed therein, wherein by size, number of cells The patient-derived mini-organoid spheroids were sorted for both size and number of cells, and the patient-derived mini-organoid spheroids were cultured for 2 to 14 days to form mature patient-derived mini-organoid spheroids. Organoid spheroids have structured cell clusters that replicate the architecture of their biopsied or resected tumors; and drug therapies are tested by exposing each drug therapy to one or more of the mature patient-derived miniature organoid spheroids .

incorporated by reference

All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. .

In general, described herein are patient-derived miniature organoid spheroids, methods and apparatus for forming them, and methods and apparatus for using them, for example, to analyze the response of tissues, including but not limited to cancerous tissues.

The patient-derived micro-organoid spheroids described herein are generally spheres formed from dissociated primary cells distributed within a substrate material. These patient-derived miniature organoid spheroids ("PMOS" or "organoid spheroids") can have a diameter between about 50 µm and about 500 µm (e.g., between about 50 µm and about 400 µm, between about 50 µm and between about 300 µm, between about 50 µm and about 250 µm, etc.), and may initially contain about 1 to 1000 dissociated primary cells (e.g., about 1 to 750, about 1 to 500 about 1 to 400, about 1 to 300, about 1 to 200, about 1 to 150, about 1 to 100, about 1 to 75, about 1 to 50, about 1 to 40, about 1 to 30, about 1 to 20, etc.).

Surprisingly, despite their small size (typically between about 50 µm and 250 µm) and low cell density (e.g., typically less than 100 cells per micro-organoid spheroid), these micro-organoid spheroids are ready-to-use Or cultured for a very short period of time (e.g., 14 days or less, 10 days or less, 7 days or less, 5 days or less, etc.) Including many, if not all, features of the extracted tumor tissue). The survival rate of the cells inside the micro-organoid spheroids is significantly higher, and the micro-organoid spheroids can be subcultured for several days (or weeks) through multiple subcultures, where the cells will divide, cluster and form structures similar to the parent tissue. Also surprisingly, in some variations, cells from dissociated tissue within micro-organoid spheroids form morphological structures inside even the smallest micro-organoid spheroids; The utility of organ spheroids (eg, they can be used before substantial structural reorganization occurs) is not essential, but in some variations they may be particularly useful.

The methods and apparatus described herein for forming and using miniature organoid spheroids can be used to generate many (eg, greater than 10,000) patient-derived miniature organoid spheroids from a single biopsy. Pharmaceutical compositions can be screened using these miniature organoid spheroids, which can predict which therapies can be effectively applied to patients who undergo biopsy. This can be applied, for example, to toxic screening of drugs or other chemical compositions from healthy normal tissue and/or from cancerous (eg, tumor) tissue. In particular, patient-derived micro-organoid spheroids, methods and devices for forming them, and devices for testing them can be used in screening to identify patients (e.g., cancer patients) who can be effectively treated prior to drug therapy one or more pharmaceutical compositions. This may allow, for example, very rapid screening of cancer patients before they would otherwise be subjected to chemotherapy for months which may not be equally effective.

Thus, described herein are high-throughput drug screening methods (and devices for performing such methods) using individual patient-specific biopsies (or other suitable tissue/cell sources). Described herein are droplet-forming patient-derived mini-organoid spheroids that can be formed from patient-derived tumor samples that have been dissociated and suspended in a base matrix (eg, Matrigel). Miniature organoid spheroids can be patterned onto microfluidic well arrays for incubation and administration of pharmaceutical compounds. This miniaturized assay maximizes the use of tumor samples and enables screening of more drug compounds from the core biopsy at a significantly lower cost per sample.

Patient-derived cancer models (PDMC) such as cell lines, organoids and patient-derived xenografts (PDX) are increasingly accepted as "standard" preclinical models to facilitate the identification and development of new therapies. For example, large-scale drug screens of cell lines and organoids derived from cancer patients have been used to identify sensitivities to a large number of potential therapies. PDX is also used to predict drug response and identify novel drug combinations. Despite the development of precision medicine strategies by studying these different PDMC models, there are substantial barriers to their effective use. For example, patient-derived organoids (PDOs) are considered the most accurate in delineating patient tumors because studies have shown that the phenotype and genotype profiles of organoids often exhibit a high degree of similarity to the original patient tumor. Unfortunately, at least two limitations prevent the use of PDO-guided therapy. First, developing and testing drug sensitivity in organoids can take months, which reduces clinical applicability. Second, the number of organoids obtained from clinically relevant 18-size core biopsies is insufficient to perform high-throughput drug screening. Ideally, the analysis should be performed from a single core biopsy within 7 to 10 days. The miniature organoid spheroids and methods of making and using them described herein can address these clinical limitations.

The details of one or more embodiments of the presently disclosed subject matter are set forth in this document. Modifications to the embodiments are described in this document, and other embodiments will be apparent to those of ordinary skill in the art after studying the information provided in this document. The information provided in this document, and particularly specific details of the described exemplary embodiments, are provided primarily for clarity of understanding and should not be construed as unnecessary limitations. In case of conflict, the specification, including definitions, of this document will control.

While the terms used herein are believed to be well understood by those of ordinary skill in the art, definitions are set forth herein in order to facilitate the explanation of the presently disclosed subject matter.

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

The term "unpolymerized mixture" is used herein to refer to a composition comprising biologically relevant material, including a dissociated tissue sample and a first fluid matrix material. A fluid matrix material is typically a material that can be polymerized to form a carrier or carrier network in which dissociated tissue (cells) are dispersed. Once polymerized, the polymeric material may form a hydrogel and may be formed from or and/or may include, in addition to cells, proteins that form a biocompatible medium. Suitable biocompatible media for use in accordance with the presently disclosed subject matter may generally be formed from any biocompatible material that is a gel, semi-solid, or liquid, such as a low-viscosity liquid at room temperature (e.g., 25° C.), And it can be used as a three-dimensional substrate for cells, tissues, proteins and other related biological materials. Exemplary materials that may be used to form biocompatible media in accordance with the presently disclosed subject matter include, but are not limited to, those comprising collagen, fibrin, polyglucosamine, MATRIGEL™ (BD Biosciences, San Jose, Calif.), polyethylene glycol Polymers and hydrogels of alcohols, polydextrose (including chemically or photocrosslinkable polydextrose) and the like, and electrospun biological, synthetic or biosynthetic blends. In some embodiments, the biocompatible medium is comprised of a hydrogel.

The term "hydrogel" is used herein to refer to a gel of two or more components comprising a three-dimensional network of polymer chains, wherein water serves as the dispersion medium and fills 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, considering the parameters to be used to form miniature organoid spheroids and the effect that the selected hydrogel will have on incorporation into the microspheres to be placed. Effects on the behavior and activity of biological material (eg, cells) in a biological suspension in a structure. Exemplary hydrogels of the presently disclosed subject matter can be composed of polymeric materials including, but not limited to: alginate, collagen (including collagen types I and VI), elastin, keratin, fibronectin, protein Glycans, glycoproteins, polylactides, polyethylene glycols, polycaprolactones, polyglycolides, polydioxanones, polyacrylates, polyurethanes, polyethylenes, peptide sequences , proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylate, polyacetate, polyester, polyamide, polycarbonate, polyanhydride, polyamine Acid carbohydrates, polysaccharides and modified polysaccharides, their derivatives and copolymers, and inorganic materials such as glass (bioactive glass), ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone and All combinations of the foregoing.

Additionally with respect to the hydrogels used to generate the micro-organoid spheroids described herein, in some embodiments, the hydrogels are composed of materials selected from the group consisting of: agarose, alginate, collagen Type I, polyoxyethylene-polyoxypropylene block copolymers (eg, Pluronic® F127 (BASF Corporation, Mount Olive, N.J.)), silicones, polysaccharides, polyethylene glycols, and polyurethanes. In some embodiments, the hydrogel is composed of alginate.

The miniature organoid spheroids described herein can also include biologically relevant materials. The phrase "biologically relevant material" may describe a material capable of being included in a biocompatible medium as defined herein and subsequently interacting with and/or affecting a biological system. For example, in some implementations, the biologically relevant material is a magnetic bead (that is, a bead that is itself magnetic or contains a material that responds to a magnetic field, such as an iron particle), which can be incorporated into an Portions of the material are polymerized to produce micro-organoid spheroids that can be used in methods and compositions (eg, for the isolation and purification of micro-organoid spheroids). As another example, in other implementations, the biologically relevant material may include additional cells in addition to the dissociated tissue sample (eg, biopsy) material. In unpolymerized mixtures, the dissociated tissue sample and additional biologically relevant material are in a homogeneous mixture or as a distributed mixture (e.g., on just one half or other portion of a microorganoid spheroid, including just in the core or just in the outer region of the formed miniature organoid spheroids). In some variations, for example, additional biologically relevant material within the unpolymerized material may be suspended in suspension with the dissociated tissue sample prior to polymerization to form droplets of micro-organoid spheroids.

In some variations, biologically relevant materials, which can include dissociated tissue sample (e.g., biopsy) material, can 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 vessels or microvascular fragments found within the stromal vascular fraction.

In general, with respect to dissociated tissue sample (eg, biopsy) material included in the miniature organoid spheroids described herein, such tissue may be any suitable tissue from a patient, typically obtained from a biopsy. Although non-biological tissues can be used, generally such tissues (and resulting dissociated cells) will be primary cells obtained from patient biopsies (eg, by needle biopsy) as described above. The tissue can be from a healthy tissue biopsy or from a cancer (eg, tumor) cell biopsy. Dissociated cells can be incorporated into the mini-organoid spheroids of the presently disclosed subject matter based on the intended use of that mini-organoid spheroid. For example, a tissue of interest (eg, dissociated biopsy tissue) can generally include cells normally found in that tissue or organ (or tumor, etc.). In this regard, exemplary related cells that can be incorporated into the miniature organoid spheroids of the presently disclosed subject matter include neurons, cardiomyocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans , bone cells, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, biliary epithelial cells, and the like. These types of tissues can be dissociated by conventional techniques known in the art. Suitable biopsy tissues can be derived from: bone marrow, skin cartilage, tendon, bone, muscle (including cardiac muscle), blood vessel, cornea, nerve, brain, stomach, kidney, liver, pancreas (including islet cells), lung, pituitary, thyroid , adrenal gland, lymph, saliva, ovary, testis, cervix, bladder, endometrium, prostate, vulva and esophagus tissue. Normal or diseased (eg, cancer) tissue can be used. In some variations, the tissue may arise from tumor tissue, including tumors originating in any of these normal tissues.

Once formed, the miniature organoid spheroids can be cryopreserved and/or cultured. Cultured micro-organoid spheroids can be maintained in suspension, static (eg, in wells, bottles, etc.) or in motion (eg, rolling or agitating). Micro-organoid spheroids can be grown using known culture techniques. Exemplary techniques can be found in, among other places: Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 4th Edition, Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach, Davis, eds. , Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, Masters, Ed., 2000; and US Patent Nos. 5,516,681 and 5,559,022.

In some variations, by forming an unpolymerized mixture of a dissociated tissue sample and a fluid matrix material in an immiscible material such as a fluid hydrophobic material (e.g., oil) (e.g., in some variations, cooling mixture) droplets to form miniature organoid spheroids. For example, micro-organoid spheroids 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 a droplet can be determined by diluting the dissociated material (eg, cells) in the unpolymerized material. The size of the miniature organoid spheroids can be related to the size of the droplets formed. In general, micro-organoid spheroids are spherical structures with stable geometries.

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, which are found in within the skill range of this technology. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual (1989), 2nd Edition, Eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent No. 4,683,195; DNA Cloning, Volumes I and II, Glover , ed., 1985; Oligonucleotide Synthesis, M.J.Gait, ed., 1984; Nucleic Acid Hybridization, D.Hames & S.J.Higgins, ed., 1984; Transcription and Translation, B.D.Hames & S.J.Higgins, ed., 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; see Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells, J.H.Miller and M.P.Calos, eds., Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, Volumes 154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I to IV, D.M. Weir and C.C. Blackwell, eds., 1986.

As used herein, a pharmaceutical composition may include any drug, drug dilution, drug formulation, composition comprising multiple drugs (e.g., multiple active ingredients), drug formulation, drug form, drug concentration, combination therapy, and the like By. In some variations, a drug formulation refers to a formulation comprising a drug and a mixture of one or more inactive ingredients.

As used herein, the term "passaging" can refer to the average number of doublings of cells within a miniature organoid spheroid. While traditional passage numbers refer to the transfer or subculture of cells from one culture vessel to another, cells within micro-organoid spheroids are stably retained within the same micro-organoid spheroid and continue to grow and divide. Accordingly, passage number as used herein generally refers to the average number of doublings performed by dissociated cells from biopsy tissue within mini-organoid spheroids. The population doubling number is the approximate number of doublings a population of cells has undergone since isolation (eg, from freshly dissociated biopsy tissue to form miniature organoid spheroids). In general, the mini-organoid spheroids described herein can be cultured for relatively short periods of time (eg, less than 10 passages, less than 9 Second passage, less than 8 passages, less than 7 passages, less than 6 passages, less than 5 passages, less than 4 passages, less than 3 passages, etc.).

Cells from dissociated biopsy tissue within the micro-organoid spheroid can aggregate, cluster, or collect within the micro-organoid spheroid during culture. Aggregates of cells may be highly organized and may form a defined morphology or may be clumps of cells that have clustered or stuck together. Organization can mirror the source organization. Although in some variations mini-organoid spheroids may contain a single cell type (homotype), more typically such mini-organoid spheroids may contain more than one cell type (heterotype).

As mentioned, the (e.g., biopsy) tissue used to form the patient-derived mini-organoid spheroids (e.g., dissociated tissue) may be derived from normal or healthy biological tissue, or from biological tissue suffering from a disease or affliction, such as Tumor-derived tissue or fluid. The tissues used in the micro-organoid spheroids may include cells of the immune system, such as T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages, and dendritic cells. Cells can be stem cells, progenitor cells or somatic cells. Tissues may be mammalian cells, such as human cells, or cells from animals such as mice, rats, rabbits, and the like.

In general, such tissues (and resulting cells) can be harvested typically from biopsies to form miniature organoid spheroids. Thus, tissue may be derived from any of a biopsy, surgical sample, aspiration, drain, or cell-containing fluid. Suitable cell-containing fluids include any of blood, lymph, sebum fluid, urine, cerebrospinal fluid, or peritoneal fluid. For example, in patients with implanted metastases, ovarian or colorectal cancer cells can be isolated from peritoneal fluid. Similarly, in patients with cervical cancer, cervical cancer cells can be harvested from the cervix, eg, by mass excision of the transition region or by cone biopsy. Typically, such miniature organoid spheroids will contain multiple cell types residing in the tissue or fluid of origin. Cells can be obtained directly from an individual without an intermediate step of subculturing, or they can first be subjected to an intermediate step of culturing to generate a primary culture. Methods for harvesting cells from fluids containing biological tissues and/or cells are well known in the art. For example, techniques for obtaining cells from biological tissues include those described by R. Mahesparan (Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro. Acta Neuropathol (1999) 97:231-239).

Generally, cells are first dissociated or separated from each other prior to formation of miniature organoid spheroids. Dissociation of cells can be accomplished by any conventional means known in the art. Preferably, the cells are treated mechanically and/or chemically, such as by treatment with enzymes. By "mechanically" we include the meaning of disrupting the connections between the cells concerned, for example, using a scraper or scissors, or by using a machine such as a homogenizer. By "enzymatically" we include the meaning of treating cells with one or more enzymes that disrupt the junctions between the cells involved, including, for example, any of collagenase, dispase, DNase, and/or hyaluronidase. one. One or more enzymes can be used under different reaction conditions, such as incubation at 37°C in a water bath or at room temperature.

Dissociated tissue can be treated to remove dead and/or dying cells and/or cellular debris. Removal of such dead and/or dying cells can be accomplished by any conventional means known to those skilled in the art (eg, using beads and/or antibody methods). For example, phosphatidylserine is known to redistribute from the inner plasma membrane leaflet to the outer plasma membrane leaflet in apoptotic or dying cells. Apoptotic cells were isolated from viable cells using phospholipid binding protein V-biotin conjugation followed by conjugation of the biotin to streptavidin magnetic beads. Similarly, removal of cellular debris can be achieved by any suitable technique in the art, including, for example, filtration.

The dissociated cells may be suspended in a carrier material prior to combining with the fluid matrix material, and/or the fluid matrix material may be referred to as a carrier material. In some variations, the carrier material may be a material having a viscosity grade that retards the settling of cells in the cell suspension prior to polymerization and formation of micro-organoid spheroids. The carrier material may have sufficient viscosity to keep the dissociated biopsy cells suspended in suspension until polymerized. The viscosity required to achieve this can be optimized by the skilled person by monitoring the sedimentation rate at different viscosities and selecting a viscosity that yields a suitable viscosity for loading the cell suspension into the device and for forming miniature organoid spheroids (by including An appropriate settling rate in terms of the expected time delay between the aggregation of droplets of unpolymerized material of cells to form micro-organoid spheroids). In some variations, even with lower viscosity materials, the unpolymerized material can still be flowed or agitated by the device in order to keep cells in suspension and/or maintain cell distribution as desired.

As mentioned above, in some variations, the unpolymerized mixture (including the dissociated tissue sample and the fluid matrix material) may include one or more components, such as biologically relevant materials. For example, biologically relevant materials that can be included can include any of the following: extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides, or antibodies (e.g., to regulate cell survival, proliferation or differentiation); and/or inhibitors of specific cellular functions. Such biologically relevant materials can be used, for example, to increase cell survival or otherwise mimic an in vivo environment by reducing cell death and/or reducing activation of cell growth/replication. Biologically relevant materials may include or may mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids, and hormones. For example, biologically relevant materials may be supplemented with one or more reagents in a fluid matrix material. In some variations, the fluid matrix material is a synthetic gel (hydrogel) and can be supplemented by one or more biologically relevant materials. In some variations, the fluid matrix is a natural gel. Thus, the gel may be composed of one or more extracellular matrix components, such as any of the following: collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate , agarose and polyglucosamine. For example, Matrigel comprises bioactive polymers important for cell survival, proliferation, development and migration. For example, the matrix material may be a gel comprising collagen type 1, such as collagen type 1 obtained from a rat tail. The gel may be a pure collagen type 1 gel or may be a gel containing collagen type 1 among other components such as other extracellular matrix proteins. Synthetic gels may refer to gels that do not occur naturally in nature. Examples of synthetic gels include gels derived from any of the following: polyethylene glycol (PEG), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyethylene oxide (PEO). Patient-derived micro-organoid spheroids

Examples of patient-derived micro-organoid spheroids are shown in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E. By way of example, Figures 1A-1C illustrate the formation of micro-organoid spheroids, each micro-organoid spheroid having a single cell. As shown, the miniature organoid spheroids are all approximately the same size, eg approximately 300 µm diameter. Figure IB shows miniature organoid spheroids formed at the same time after 3 days of culture. Cells have expanded in size, and in some cases, doubled and/or grown. By seven days of culture, the cells had doubled multiple times, exhibiting clusters or clumps of cells, as shown in Figure 1C.

Similar results are shown in Figures 2A-2C and 3A-3C, which show micro-organoid spheroids formed from five cells per micro-organoid spheroid or 20 cells per micro-organoid spheroid, respectively. In FIGS. 4A-4E , mini-organoid spheroids are shown immediately after formation and cultured for five days, where nearly identical mini-organoid spheroids (e.g., with the same diameter) each included 10 cells per mini-organoid spheroid . In Figure 4A, miniature organoid spheroids are shown immediately after formation, still surrounded by immiscible fluid (in this case, oil) at day 0. The mini-organoid spheroids were removed from the immiscible fluid and washed, and cultured for five days. Figure 4B shows micro-organoid spheroids after 2 days, Figure 4C shows micro-organoid spheroids after 3 days, and Figure 4D and Figure 4E show micro-organoid spheroids at 4 days and 5 days, respectively. Figures 4A-4E demonstrate that dissociated tissue (cells) from biopsies within micro-organoid spheroids are viable and grow at comparable rates in almost all micro-organoid spheroids. As will be described in more detail herein, such miniature organoid spheroids can be formed in large numbers from a single average-sized biopsy, and hundreds or thousands (e.g., 500, 750, 1000, 2000, 5000 1, 10,000 or more) micro-organoid spheroids containing large numbers of living cells, allowing multiple rapid assays to be performed in parallel.

5A and 5B illustrate examples of miniature organoid spheroids formed from dissociated biopsies of mouse livers as described herein, such as showing mice distributed within a polymeric fluid matrix material (in this example, Matrigel). Hepatocyte. Each micro-organoid spheroid includes a polymeric matrix material 503 formed into a sphere having a diameter of, for example, about 300 µm, with a set number of hepatocytes 507 dispersed therein. In FIG. 5A , micro-organoid spheroids are shown one day after biopsy, dissociation, and formation of micro-organoid spheroids. These mini-organoids were then cultured for 10 days, during which time the cells (hepatocytes) remained viable and grew, in many cases doubling multiple times, to form structures 505, as shown in Figure 5B.

Mini-organoid spheroids can typically include dissociated (e.g., biopsy) tissue (e.g., cells) at a fixed or known number and/or concentration (cells/ml or cells/mm ³ ) within a mini-organoid . As mentioned above, this matrix material can be a natural polymer, such as one or more of the following: alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, elastin; or a synthetic polymer, such as polyethylene One or more of glycol (PEG) and polyacrylamide. Both organic and inorganic synthetic polymers can be used.

In some variations, the number of cells initially included in the miniature organoid spheroids can be selected from 1 cell up to several hundred. In particular, in some assays (eg, drug toxicity assays), it may be beneficial to include about 1 to 75 or about 1 to 50 (eg, lower numbers of cells). The number of cells per micro-organoid spheroid can be set or selected by the user. In some variations, as described below, the device will include one or more controls to set the number of cells from primary tissue to include in each micro-organoid spheroid. The number of cells can be selected or set based on how the user intends to use the micro-organoid spheroids. For example, mini-organoid spheroids with very low cell numbers (e.g., 1 cell per mini-organoid spheroid, 1 to 5 cells per mini-organoid spheroid, etc.) may be particularly useful for studying strain diversity ( For example, tumor heterogeneity). Since each mini-organoid is grown from a single cell, we can observe which strains are drug-resistant and can test (e.g., by genome sequencing) these specific mini-organoids to determine whether they are compatible with a particular strain. Associated gene body (mutation) diversity. A low to moderate number of cells (e.g., about 3 to 30 cells, 5 to 30 cells, 5 to 25 cells, 5 to 20 cells, 10 to 25 cells, etc.) per miniature organoid spheroid can It is especially suitable for rapid drug testing, including toxicity testing, since these miniature organoid spheroids usually grow rapidly. Larger numbers of cells per mini-organoid spheroid (e.g., about 20 to 100 cells, e.g., 30 to 100 cells, 40 to 100 cells, greater than 50 cells, etc.) Tissue composition is simulated in organoid spheroids, since miniature organoids can contain cells of different lineages, potentially including epithelial (or carcinoma, etc.) and mesenchymal (or stromal, immune, vascular, etc.) cells.

Miniature organoid spheroids can be formed of any suitable size that can be matched to 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 (eg, on average 50 µm or 100 µm, eg, between about 100 µm and 200 µm, etc.). In some variations, the size is about 300 μm, with about 10 to 50 cells (eg, about 10 to 30 cells) included in each miniature organoid spheroid. The number and size of cells can be varied and/or controlled. In some variations, the number of cells and/or the size of the micro-organoid spheroids can be set by one or more controls on the apparatus for forming the micro-organoid spheroids. For example, the size of a micro-organoid spheroid and/or the density of cells within a micro-organoid spheroid can be adjusted by adjusting the flow rate and/or concentration of a dissociated tissue sample (eg, cells from a biopsy).

As shown in Figures 1A-5B, even after culturing the mini-organoid spheroids described herein, viable and healthy cells were allowed to pass through the entire volume of the mini-organoid spheroids. The size of the micro-organoid spheroid and/or the number of cells included in the micro-organoid spheroid can be selected based on how the micro-organoid spheroid is intended or intended to be used. For example, in variations of micro-organoid spheroids to be used to examine relationships between cells of biopsy material, micro-organoid spheroids can be formed with multiple cells and can be cultured for extended periods of time (e.g., up to a week or more long time).

The patient-derived micro-organoid spheroids described herein can be prepared by combining a dissociated tissue sample (eg, a biopsy sample) with a fluid matrix that can polymerize in a controlled manner to form micro-organoid spheroids. Figure 6 illustrates a method for forming patient-derived micro-organoid spheroids. Optionally, the method may include obtaining a sample from the patient, such as obtaining a biopsy 601 from patient tissue. As mentioned above, a biopsy can be obtained, for example, using a biopsy needle or punch. For example, a biopsy may be obtained with a 14 gauge, 16 gauge, 18 gauge, etc. needle that is inserted into patient tissue to remove the biopsy. After the tissue is removed from the patient, the tissue may be treated mechanically and/or chemically to dissociate the material. The dissociated cells can be used immediately to form patient-derived mini-organoid spheroids, as described; in some variations, all or some of the cells can be modified, such as by genetically modifying the cells 603 (e.g., by transfection, electroporation, etc.) .

The dissociated tissue sample from the biopsy material may be combined with a fluid (eg, liquid) matrix material to form an unpolymerized mixture 605 . This unpolymerized mixture can be maintained in an unpolymerized state so that cells from the dissociated tissue can remain suspended within the mixture. In some variations, cells can be kept in suspension and unaggregated, for example, by cooling them, eg, below room temperature (eg, between 1°C and 25°C).

The unpolymerized mixture can then be dispensed as droplets into, for example, an immiscible material such as oil in a manner that controls the size of the formed droplets, and thus forms the size 607 of patient-derived micro-organoid spheroids. For example, uniformly sized droplets can be formed by combining a stream of unpolymerized material into one or more (e.g., two converging) streams of immiscible material (e.g., oil) such that both The flow rate and/or pressure of the stream can determine how droplets of unpolymerized material are formed when they intersect with immiscible materials. Droplets 609 can be polymerized to form patient-derived micro-organoid spheroids (PMOS) in an immiscible material. In some variations, the immiscible materials may be heated or raised to a temperature that polymerizes the unpolymerized mixture (eg, the fluid matrix material in the unpolymerized material). Once formed, patient-derived micro-organoid spheroids can be isolated from the immiscible fluid, e.g., PMOS can be washed to remove immiscible fluid 611, and placed in culture medium to allow patient-derived micro-organoid Cell growth within organ spheres. Patient-derived micro-organoid spheroids can be cultured for any desired time, or can be cryopreserved and/or analyzed immediately. In some variations, patient-derived mini-organoid spheroids can be cultured for brief periods of time (e.g., 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days). days, 1 to 9 days, 1 to 10 days, 1 to 11 days, 1 to 14 days, etc.). This can allow cells derived from the dissociated biopsy to grow and/or divide (eg, double) up to five or six passages. After culturing, the cells may be either or both cryopreserved 615 and/or analyzed 617 . Examples of assays that can be used are also described herein.

In any of the methods and apparatus described herein, the micro-organoid spheroids can be recovered from an immiscible fluid (eg, oil) after polymerization. For example, in some variations, micro-organoid spheroids can be recovered by demulsification and/or de-emulsification, such as by forming emulsified droplets after forming droplets to remove any oil (and other contaminants) and Recover the miniature organoid spheroids. This can allow cells to grow within aggregated droplets (miniature organoid spheroids) without being inhibited by immiscible fluids.

Although the methods and apparatus described herein illustrate the formation of a plurality of droplets and thus a plurality of microscopic The method of organoid spheroids, but in some variations, droplets can be formed by other methods that allow control of droplet size as described herein. For example, in some variations, droplets may be formed by printing (eg, by printing droplets onto a surface). This can reduce or eliminate the need for an additional recovery step of emulsification/de-emulsification. For example, droplets can be printed onto a surface, such as a flat or shaped surface, and polymerized. In any of these variations, pressure, sound waves, electrical charges, etc. may be used to dispense the droplets. In some variations, automated dispensers (e.g., pipetting devices) adapted to release small amounts of unpolymerized mixture onto surfaces, into the air, and/or into liquid media (including immiscible fluids) can be used to form small drop.

Methods for forming patient-derived micro-organoid spheroids can be automated or performed using one or more devices. In particular, the method of forming patient-derived micro-organoid spheroids can be performed by a device that allows selection and/or control of the size (and thus the density of the number of cells) of the patient-derived micro-organoid spheroids. For example, FIG. 7A illustrates one example of an apparatus 700 for forming patient-derived micro-organoid spheroids as described.

In FIG. 7A, the device generally includes an input for inputting an unpolymerized mixture of a dissociated tissue sample and a fluid matrix material (combined) or may receive a dissociated tissue sample (e.g., in a holding solution) and a fluid separately. matrix material. In some variations, the apparatus includes a holding chamber 706 for holding the unpolymerized mixture, and/or a holding chamber (not shown) for holding a dissociated tissue (eg, biopsy) sample and holding a fluid matrix material. Any or all of these containment chambers may be pressurized to control and/or accelerate fluid flow from the chamber and into the device. The equipment can receive the unpolymerized mixture or its receivable components and mix it. In some variations, the device can control the concentration of cells in the unpolymerized mixture and can dilute the mixture (eg, by adding additional fluid matrix material) to achieve a desired density. For example, an apparatus may include a sensor (eg, an optical reader) (not shown) for reading the density (eg, optical density) of cells in the unpolymerized mixture. The sensors can also be coupled to a controller 724, which can automatically or semi-automatically (eg, by indicating to the user) control the dilution of cells in the unaggregated mixture. The apparatus may also include an orifice for receiving the unpolymerized mixture. The orifices may include or be coupled to valves and the valves may be controlled by controller 724 (or a respective controller).

Apparatus 700 may include a chamber 708 and/or an orifice for containing and/or receiving immiscible fluids. In some variations, immiscible fluids can be contained in a pressurized chamber such that the flow rate can be controlled. 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 thus the flow of fluid through the device. One or more pressure and/or flow sensors may be included in the system to monitor fluid passing through the device.

In FIG. 7A, the entire device 700 may be enclosed in a housing 702 or a portion 704 of the device may be enclosed in a housing. In some variations, the housing may include, for example, one or more openings or inlet portions on the device for adding immiscible fluid and/or unpolymerized mixture.

As mentioned, any of these apparatuses 700 may also include one or more sensors 728 for monitoring all or critical portions of the manufacturing process. In some variations, the sensors may include optical sensors, mechanical sensors, voltage and/or resistance (or capacitance, or inductance) sensors, force sensors, and the like. These sensors can be used to monitor the ongoing operation of the assembly, including the formation of patient-derived micro-organoid spheroids. Apparatus 700 may also include one or more heat/temperature regulators 718 for controlling the temperature of either or both of the immiscible fluid and/or the unpolymerized mixture (and/or the fluid matrix material).

Any of these apparatuses can also include one or more droplet forming assemblies 720 that can be monitored (eg, using one or more sensors) as will be explained below in FIGS. 7C and 9 . The droplet micro-organoid spheroid formation assembly can include or be coupled to a dispenser (eg, a PMOS dispenser) 722 . Distributor nozzle 712 may dispense into perforated disc 716, for example.

In general, droplet micro-organoid spheroid formation assembly 720 may include one or more microfluidic chips 730 or structures that form and control the flow of unpolymerized mixture and form actual droplets. FIG. 7B illustrates an example of a microfluidic chip 730 used to form patient-derived micro-organoid spheroids. In FIG. 7B, wafer 730 includes a pair of parallel structures for forming miniature organoid spheroids. Figure 7C illustrates the droplet formation region of a microfluidic chip used to form a PMOS, including an unpolymerized channel outlet 741, which directs a "+" junction or intersection region 737 to the channel outlet 741 and immiscible fluid outlets 743, 743' open (in this example, at right angles). In some variations, the input from the immiscible fluid channel may be at an angle relative to the angle (and point of intersection) with the unpolymerized material. In FIG. 7C , in all figures in this specification where dimensions are shown, the dimensions shown are illustrative only and are not intended to be limiting unless otherwise indicated.

In FIG. 7A, a microfluidic wafer 730 includes an inlet (input port) 733 for immiscible fluids to enter the wafer (eg, from an inlet port or storage chamber shown in FIG. 7A). The second inlet port 735 into the wafer can be structured to receive unpolymerized material and deliver it along a semi-tortuous path to the bonding area. Similarly, the immiscible fluid inlet port can be securely coupled to the outlet of the immiscible fluid chamber or the inlet described above.

The inlet port 735 for unpolymerized material into the wafer can be coupled to the bonding region via a delivery path 741 connecting the inlet 275 (as shown in FIG. 7C ). Similarly, an inlet 733 for an immiscible fluid may connect two (or more) connection paths 743 , 743 ′ to the junction area 737 . A channel exiting the junction region 737 allows the formed micro-organoid spheroids (in an immiscible fluid) to pass along the channel to an outlet 731 which can be connected to a channel for dispensing from the micro-organoid spheroids to one or Dispensers (not shown) in multiple chambers, eg, for culture and/or analysis.

In the example shown in FIGS. 7B and 7C , the formed droplets that once aggregated can become miniature organoid spheroids can travel along a longer temperature-controlled microfluidic environment before being dispensed from the device (not shown). transmission. For example, FIG. 8 illustrates an example of a channel region 839 (eg, element 739 in FIG. 7B ), shown transparently containing a plurality of micro-organoid spheroids 803 each containing a predetermined number of cells 805 .

In FIG. 8 , junction region 937 is shaped as described above such that channel 911 carrying the unpolymerized mixture is in contact with one or more (eg, two) channels carrying a fluid immiscible with the unpolymerized mixture, such as oil. ) channel 909 crossed. As the unpolymerized mixture is pressurized to flow out of the first channel 911 at a first rate, the immiscible fluid flowing in the intersecting channels 909, 909' permits a predetermined amount of the unpolymerized mixture to pass before it is pinched off to form a pass to the outlet. Droplet 903 in channel 939. Thus, in some variations, minced (e.g., dissociated) clinical (e.g., biopsy or resected) tissue samples (such as <1 mm in diameter) can be combined with a temperature-sensitive gel (i.e., Matrigel, at 4°C) to form an unpolymerized mixture. This unpolymerized mixture can be placed in a microfluidic device that can generate droplets (eg, water-in-oil droplets) of uniform volume and material composition. At the same time, dissociated tumor cells can be divided into these droplets. The gel in the unpolymerized material can solidify upon heating (eg, at 37° C.) and the resulting patient-derived micro-organoid spheroids can form. In some variations, this method can be used to self-organize (e.g., biopsy material) to generate more than 10,000 (e.g., more than 20,000, more than 30,000, more than 40,000, more than 50,000, more than 60,000, more than 70,000, More than 80,000, more than 90,000, more than 100,000) uniform droplets (patient-derived micro organoid spheroids). These patient-derived micro-organoid spheroids are compatible with traditional 3D cell culture techniques. Figure 10 illustrates a plurality of patient-derived micro-organoid spheroids 1005 suspended in an immiscible material 1008 (eg, oil) as described above.

In the exemplary microfluidic chips described above, the junctions are shown as T or X junctions, where the flow of microfluidics is focused to form micro organoid spheroids of controllable size. In some variations, droplets rather than microfluidic chips may be formed by robotic micropipetting, eg, into immiscible fluids and/or onto solid or gel substrates. Alternatively, in some variations, droplets of unpolymerized material can be formed with the requisite size and reproducibility by microcapillary generation. Other examples of alternative techniques for forming micro-organoid spheroids in a specified size range and reproducibility from unpolymerized materials may include colloidal manipulation, for example, via external forces such as acoustics, magnetism, inertia, electrowetting, or gravity.

11A and 11B show examples of patient-derived mini-organoid spheroids in oil formed as described above. Cells within these patient-derived mini-organoid spheroids derived from a single biopsy sample were viable, as seen by key dye staining, as demonstrated in Figures 15A-15B and Figures 16A-16B. For example, FIGS. 12A-12B illustrate miniature organoid spheroids with tumor cells (similar to those shown in FIGS. 11A-11B ) that can be washed to remove immiscible material (e.g., Oil). This immiscible material can be removed relatively quickly after formation of the micro-organoid spheroids in order to prevent damage to the cells within the micro-organoid spheroids.

In these examples, gel droplets were recovered from the oil phase and resuspended via PFO (perfluorooctyl alcohol) and centrifugation eg into PBS. It can separate immiscible fluids from miniature organoid spheroids. Accordingly, these mini-organoid spheroids, including tumor-based mini-organoid spheroids, can be grown successfully, as in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E above and shown in Figure 13. This is an important improvement because drug screening must be performed on viable and growing primary tumor cells, which retain their properties from the patient's tumor, to predict patient outcome. The higher number and uniformity of these mini-organoid spheroids made screening possible and reliable, as will be described below.

In any of the microfluidic chips or devices described herein, the channels can be coated. For example, channels of microfluidic devices can be coated with hydrophobic materials.

In general, the micro-organoid spheroids described herein are highly uniform in diameter and can have extremely small dimensions, eg, diameter, variance. This is illustrated, for example, in Figure 14, which shows the distribution of one example of droplet diameter sizes.

As mentioned, Figures 15A-15B show mini-organoid spheroids formed as described herein; in Figures 16A-16B, these mini-organoid spheroids have been stained with trypan blue (arrows) showing their survival. Microorganoid spheroids formed into droplets in this way can contain growth factors and a matrix to mimic the biological environment in which the tissue emerges. Patient samples (eg, biopsy samples) can be formed into micro-organoid spheroids (including hundreds, thousands, or tens of thousands of micro-organoid spheroids) within hours of tissue harvesting. Micro-organoid spheroids can have as few as 1 or 4 to 6 cells (eg, cancer cells when sampling a tumor) or as many as several hundred cells per micro-organoid spheroid. These methods have been shown to work on nearly all types of cancerous and non-cancerous tissue tested so far (n=20), including colon, esophagus, melanoma, uterus, sarcoma, kidney, liver, ovary, lung, diaphragm, Omentum, mediastinal lung and breast cancer tissues. Miniature organoid spheroids can be cultured for any desired period of time and typically exhibit proliferation and growth in as little as 3 to 4 days. They can be maintained and subcultured for several months. As will be described in more detail below, they can be used to screen thousands of pharmaceutical compositions in as little as 4 to 6 days from harvesting tissue (eg, biopsy).

The micro-organoid spheroids described herein can be stored at any time after formation, for example by cryopreserving them. Tumor mini-organoid spheroids can be collected from many different patients and used individually or collectively to screen various drug formulations for toxicity and/or efficacy. Non-tumor cells (healthy tissue) can be biopsied, stockpiled and/or screened in parallel. Accordingly, such methods and devices may allow high throughput screening. In some variations, miniature organoid spheroids can be formed and passaged twice (eg, doubled twice) and cryopreserved. As mentioned, normal healthy tissue can be used to form these same miniature organoid spheroids to generate hundreds, thousands or tens of thousands of spheroids that can be used to analyze drug effects, drug responses, biomarkers, proteosome signatures, gene body signatures. and other miniature organoid spheroids.

Importantly, these miniature organoid spheroids survived in a biologically significant manner, making them clinically and physiologically relevant, especially in terms of drug response, as shown in Figures 22A to 22D and Figures 23A to 23D describe. In particular, the miniature organoid spheroids described herein allow tissue extraction/biopsy-derived cells to grow exceptionally well and provide more representative data, especially compared to organoids or spheroids. Without being bound by a particular theory, this may be because the cells may have a more restricted cell density in the miniature organoid spheroids, thereby allowing the cells to communicate with each other while sharing signals without inhibition. Micro-organoid spheroids also have an extremely large surface area-to-volume ratio, which more easily permits the transmission of growth factors and other signals for infiltration into the micro-organoid spheroids (eg, the micro-organoid spheroids are less limited by diffusion). analyze

The patient-derived mini-organoid spheroids described herein can be used in a variety of different assays, and are particularly useful for determining drug formulation effects, including toxicity, on normal and/or abnormal (eg, cancer) tissue. For example, drug screening can include coating mini-organoid spheroids into all or some wells of a multi-well (eg, 96-well) plate. Alternatively, a custom plate can be used (eg, a 10,000 microwell array that can be formed from 100 x 100 wells). Miniature organoid spheroids (eg, gel droplets) can be coated into or, in some variations, onto multiple microwell arrays and incubated with culture medium. Miniature organoid spheroids can be cultured for a time course of 3 to 5 days. In some variations, at day 5, the wells (eg, microreactors) can then be dosed with drug compounds (eg, based on a panel of FDA-approved anticancer drugs) to examine the effect of drug pools. For example, the drugs tested can be based on the National Cancer Institute (Division of Cancer Treatment and Diagnosis (11)) screen of 147 agents intended to enable cancer research, drug discovery, and combination drug research composition. At day 7, the mini-organoid spheroids can be imaged via standard fluorescence microscopy and graded based on drug response.

An example of this analytical technique is shown in Figures 17A-17E.

In this example, screening analysis can be automated. This enables a reproducible and automated workflow that can increase the number of drugs screened from a few to hundreds. Figures 17A-17E illustrate an example of this workflow. In FIG. 17A , tumor biopsies were taken and a plurality (eg, >10,000) of mini-organoid spheroids formed as described above (in FIG. 17A , junctional regions forming mini-organoid spheroids are illustrated). Thereafter, the micro-organoid spheroids can be recovered and washed (eg, to remove immiscible (eg, oil) material forming them therein). The micro-organoid spheroids can then be plated into one or more microwell plates. As shown in Figure 17C, mini-organoid spheroids can be cultured for one or more passages (eg, one or more passages). It is shown to appear on day 0 to day 3, day 4 or day 5. The mini-organoid spheroids can then be screened, for example, by coating a drug to a subset of replica wells, as shown in Figure 17D. Thereafter, as shown in FIG. 17E , at day 7, the cells in the mini-organoid spheroids can be imaged and/or scored automatically or manually to identify drug effects (eg, drug screening and growth profiling).

The workflow shown in Figures 17A-17E can enable an integrated device for growing, dosing, and/or examining micro-organoid spheroids. In one exemplary setup, fresh biopsied or resected patient tumor samples can be isolated and seeded into gels with reagents to form miniature organoid spheroids (as described above). Parts of the resulting miniature organoid spheroids can be cryopreserved. The remainder can be recovered and grown until inoculated into microwell plates for drug testing or screening as just described. Growth and viability assays can be performed on imageable and trackable miniature organoid spheroids. Their responses to drug treatments, such as IC-50, cytotoxicity, and growth curves, can be measured to identify effective therapies for a patient's tumor.

The methods and apparatus described herein have various advantages, including reproducibility. The sample fabrication process can be automated through microfluidic sample segmentation, which can reduce the need for specialists in diagnostic testing and manual pipetting. This may be especially helpful in clinical settings. In addition, it enables uniformity of signal droplet to increase assay sensitivity. In addition, these assays minimize the time required to generate miniature organoid spheroids. Based on preliminary data, these methods may be capable of generating a library of over 100,000 matrigel tumor droplets (miniature organoid spheroids) in less than about 15 minutes. These methods are also highly scalable and can be multiplexed to run multiple patient biopsies in parallel.

Ultimately, these methods are flexible and compatible with other techniques. As a research tool, droplet-like microfluidics are generally compatible with a wide range of hydrogel materials such as agarose, alginate, PEG, and hyaluronic acid. Thus, the starting gel composition can be readily modified to accompany and facilitate the growth of miniature organoid spheroids. Furthermore, droplet size can be tuned by modifying the size of these microfluidic devices. At the same time, these allow a large choice of gel material composition and microreactor size.

The miniaturized assays described herein (eg, using miniature organoid spheroids) can maximize patient tumor biopsy, enabling screening of more drug compounds. For example, a 600 uL tumor sample can be divided into about 143,000 individual microreactors with a volume of about 4 nL. By maximizing the tissue sample, multiple experimental replicates can be examined, increasing statistical power. These techniques may allow detection of intra-tumor heterogeneity, drug perturbation, and identification of rare cellular events such as drug resistance. Miniature organoid spheroids can generally be compatible with subsequent analyzes including single cell RNA transcriptome analysis and epigenetic profiling. Additionally, by maximizing the efficiency of tissue (e.g., biopsy) samples provided by the micro-organoid spheroids, a portion of the micro-organoid spheroids can be stockpiled (e.g., biobanked by cryopreservation) for future novel drug analysis and and/or for confirmatory analysis, including genetic screening.

For example, Figures 18 and 19 illustrate examples of therapeutic methods using the methods and devices described herein, including miniature organoid spheroids. For precision and personalized medicine, these methods and devices can be used as clinical indicators for appropriate drug selection to improve clinical outcomes and drug responses. As an example, a histopathological biopsy of a patient diagnosed with metastatic cancer will be taken and used to screen a plurality of mini-organoid spheroids formed from the biopsy as described herein. Within 7 to 10 days, screening can be performed from the biopsy to identify the most effective standard-of-care therapy, so patients can begin treatment in approximately 14 days.

This example is illustrated in FIG. 18 . In this example, a tumor 1801 can be identified on day 0 (e.g., by CT scan), and a biopsy 1805 can be taken on day 5, and on the same day, hundreds, thousands, or tens of thousands of miniature species can be formed Organ spheroids were cultured for 1 to 5 days and screened 1805 to identify one or more pharmaceutical compositions that could be used. This same step (formation of miniature organoid spheroids and screening) can be used to guide precision medicine at multiple clinical decision points throughout the disease process. In this example, therapy 1809 with the identified one or more pharmaceutical compositions can be initiated on day 14, and the patient can be monitored later during the course of treatment (e.g., follow-up CT scans at about day 90) to confirm the tumor 1811 responded to treatment. If so, therapy can be continued 1813 and ongoing progress monitored 1815.

As mentioned, the analysis using mini-organoid spheroids can be repeated at multiple points throughout the treatment course. This technique is illustrated in FIG. 19 . For example, this technique (eg, generation and screening 1905 of mini-organoid spheroids) can be used to determine the most effective neoadjuvant therapy 1921 when a patient is first diagnosed 1907 with a resectable primary tumor. Thus, biopsies can be obtained and hundreds, thousands or tens of thousands of miniature organoid spheroids can be formed and screened with a panel of potential drug compositions. Once the primary tumor has been resected 1923, this technique 1905' can indicate whether and what adjuvant therapy should be chosen 1925. If recurrence or metastasis occurs after surgical removal of the primary tumor 1927, the same technique can be used (e.g., miniature organoid spheroids generated and screened from fresh biopsies 1905'', 1905''', 195'''') To guide the standard of care therapy, including 1-line therapy 1929, 2-line therapy 1931 and 3-line therapy 1933. If the patient eventually becomes tolerant or resistant to all standard-of-care therapies, this technique can be performed 1905''''' to identify off-label drugs 1935 for treatment of drug-resistant tumors. This technology can also be used as a companion diagnostic to identify patients for specific treatments. Ultimately, this technique can be used to derive and preserve patient-derived micro-organoid spheroids to establish living organoid-spheroid-based cancer libraries for screening, genome profiling, novel drug discovery, drug testing, and clinical trial design.

Because these techniques can be accomplished in a relatively less invasive manner (e.g., by excision or biopsy) and generate large numbers of miniature organoid spheroids to provide reasonably rapid results for self-screening, these methods are readily adaptable to standard of care . For example, cellular material from tissue (eg, biopsy) input is very small in volume and can be dissociated into volumes of, eg, 10 µL to 5 ml.

In general, screening using the miniature organoid spheroids described herein can be performed automatically or manually. Virtually any screening technique can be used, including imaging by one or more of the following: confocal microscopy, fluorescence microscopy, liquid lensing, holography, sonar, brightfield and darkfield imaging, laser, planar laser Shots, including high-throughput embodiments of image-based analysis methods (eg, using computer vision and/or supervised or unsupervised modalities, such as CNNs). Subsequent screening may include sampling the culture medium and/or performing genetic or protein screening (eg, scRNA-seq, ATAC-seq, proteomics, etc.) on the cells from the mini-organoid spheroids. example

20 and 21 illustrate another example of an apparatus for forming a plurality of miniature organoid spheroids as described herein. In FIG. 20, the device can include a plurality of micro-organoid spheroids forming a junction where an immiscible material (eg, oil) 2002 can be added to a reservoir and/or orifice 2004 in the device. Similarly, unpolymerized material 2006 (including, in this example, dissociated biopsy cells and fluid matrix material) can be added to a reservoir or orifice 2008 in the device. In some variations, a second or additional material (eg, a bioactive agent) can be added via the third set of orifices 2010 . These components can combine at junctions 2012 (similar to those described above), forming droplets in immiscible materials that can be polymerized into miniature organoid spheroids. In FIG. 20, three (or more) parallel junctions with corresponding inputs 2004, 2008, 2010 and output 2014 are shown.

FIG. 21 illustrates a method of forming miniature organoid spheroids using the apparatus as shown in FIG. 20 . In this variation, the resulting mini-organoid spheroids include both target (eg, tumor) biopsy cells combined to form the mini-organoid spheroids and one or more additional bioactive agents. For example, the first channel 2103 may include unpolymerized material (including dissociated biopsy cells and matrix material), the second channel 2107 includes additional active biological material, and carries a pair of immiscible materials (such as oil). Intersecting channels 2109 , 2109 ′ converge at the junction to form size-controlled droplets that aggregate to form micro-organoid spheroids 2107 .

In this example, the additional active biomaterial can be, for example, a freezing medium (e.g., to aid in the storage of micro-organoid spheroids) and/or have additional cells (e.g., immune cells, stromal cells, endothelial cells, etc.), additional supportive mesh Co-cultures of molecules (eg, ECM, collagen, enzymes, glycoproteins, biomimetic scaffolds, etc.), additional growth factors, and/or pharmaceutical compounds. Example 2 : Filter results

As mentioned above, patient-derived micro-organoid spheroids and methods of screening pharmaceutical compositions using the same can be used to accurately predict the response of a patient's tumor to one or more drug therapies. In some cases, the use of miniature organoid spheroids can provide precise results where traditional cultured drug screens do not precisely predict drug response. For example, in FIGS. 22A-22D , miniature organoid spheroids rather than cell lines can be correlated with patient response. In Figure 22A, conventional cell lines dosed with drug (eg, oxaliplatin) were examined; the drug line showed no effect, and tumors were predicted to be resistant to the drug at all dose ranges tested.

For comparison, a plurality of miniature organoid spheroids were generated from patient biopsies, as shown in Figure 22B. In this example, patient-derived micro-organoid spheroids exhibited significantly reduced cell survival in tumor micro-organoid spheroids, predictive of drug sensitivity. Indeed, when treated with the drug, the tumors responded to the treatment, as shown in Figure 22C (before treatment) and Figure 22D (after treatment). Example 3 : Correlation Between Miniature Organoid Spheroids and Patient Response

In a similar set of experiments, mini-organoid spheroids were generated from biopsy material ( FIG. 23A ), and drug effect screening was performed using the resulting mini-organoid spheroids. Figure 23B shows the effect of the first drug (oxaliplatin) on these mini-organoid spheroids, showing no change in the percent survival of the mini-organoid spheroids in the presence of the drug, predicting drug resistance. Similarly, treatment with a second drug (irinotecan) showed no effect on mini-organoid spheroids, predicting drug resistance, as shown in Figure 23C. The patient was treated with both oxaliplatin and irinotecan and showed no response after 6 months of treatment. Thus, miniature organoid spheroids were largely correlated with patient response to standard-of-care medications. In this case, a patient suffering six months of side effects and toxicity could have been avoided by the predicted response of the mini-organoid spheroids, indicating (within 7 to 10 days of self-biological examination) that the tumor would not respond to the drugs . Example 4 : Multiple Drug Screening

Figure 24 shows an example of a panel of drugs (eg, chemotherapeutic agents) that can be produced using patient-derived plurality of mini-organoid spheroids as described herein. In this example, drug screening using patient-derived mini-organoid spheroids was performed by administering multiple replicates of each of multiple (27) drugs. A single tumor biopsy was used to generate large numbers of multiple mini-organoid spheroids extremely rapidly (eg, in less than two weeks) and test these mini-organoids against the panel of drug formulations (eg, 27 formulations demonstrated) sphere. This test is performed in parallel and can be quantified automatically (eg, by optical detection and quantification). In this example, the drug exhibiting the greatest toxicity for this particular tumor was Pazopanib.

Combinations of drugs and different drug concentrations can be tested in parallel. Since hundreds, thousands, or tens of thousands of mini-organoid spheroids can be generated from the same tumor biopsy, array testing of this sort is made feasible by the methods and devices described herein. Example 5 : Biopsy sample preparation

Materials: An apparatus for forming miniature organoid spheroids, as described above, including droplet microfluidic wafers (200 µm); Bayer-rad Droplet Generation Oil from EvaGreen (Catalog #186 -4006), 3 to 5 mL per run, perfluorooctyl alcohol (PFO), Sigma, Novec HFE 7500 with 10% perfluorooctyl alcohol (PFO), PBS, cell culture medium (i.e., RPMI w/10% FBS with 1% Penicillin-Streptomycin), 70 µm or 100 µm filter, 50 mL conical Petri dish.

Biopsy Dissociation: A biopsy (human/animal) is used to generate a dissociated sample (ie, single cell tissue) from a patient. The microfluidic wafer is coated, and the microfluidic wafer and holder are assembled. Connect microfluidic tubing and fit to micro-organoid spheroids and output of waste oil (eg, multi-well disc, 15 mL Eppendorf, etc.).

Run the device to form miniature organoid spheroids. The output (eg, tray, Eppendorf tube, etc.) containing the droplets is removed from the incubator (after at least 15 minutes). Remove any excess oil from the output. The droplets should be floating, so the oil should be at the bottom of the bottle. Be careful not to remove the droplet from the tube. Add 100 µL of 10% (v/v) PFO to the output. Vortex carefully and wait about 1 min. Do not pipette or disturb the sample. Centrifuge at 300 g for 60 seconds. The supernatant (excess oil/PFO) was removed. Do not pipette or disturb the sample. Remove as much PFO as possible, as this chemical can reduce cell viability during culture. 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 any excess oil/PFO. Add 1 mL of cell culture medium. Carefully pipette the sample up and down (approximately 30 times) with a 1 mL pipette tip. Be careful not to overshoot the pipette or disturb the droplet sample. Using a 1 mL pipette tip, pass a small drop of media solution through a 70 µm or 100 µm filter (attached to a 50 mL conical tube). Some droplets will stick to the inside of the output (eg, 15 mL Eppendorf). Rinse each tube with 2 to 3 mL of PBS and pipette up and down. Pass the rinsed PBS and droplet through the filter. Repeat this step twice, or until the tube appears clear and the droplets have transferred to the filter. Using a 1 mL pipette tip, carefully wash the filter containing the droplet with approximately 5 mL of PBS. Attempt to cover the entire surface area of the filter. This washing step removes any excess oil and PFO from the sample and allows for eventual recovery of the gel droplets into the cell culture medium.

Once properly drained (approximately 1 to 2 minutes), the filter was carefully removed from the 50 mL conical tube. The filter was turned upside down and the backside was washed with fresh cell culture medium, and the solution was retained in a fresh petri dish. This operation separates the droplets from the filter and places them in the cell culture medium. It is recommended to use a 1 mL pipette tip and wash with about 5 mL of medium.

The quality of the droplets was checked under a microscope. Most/all oil should be removed. If recovery is poor, the sample can be refiltered. The density of the recovered micro-organoid spheroids can be checked by a hemocytometer. Example 6 : Kidney Tissue Miniature Organoid Spheroids

In another example, miniature organoid spheroids can be formed from biopsy kidney tissue. Examples of tools used may include: tube rotators or 100 μm and 70 μm cell strainers, 15 mL conical tubes, 50 mL conical tubes, razor blades, forceps and surgical scissors, petri dishes (100× 15 mm) or tissue culture dishes. Reagents may include: EBM-2 medium, collagenase (5 mg/mL stock solution), Hank's Balanced Salt Solution (HBSS), calcium chloride (10 mM stock solution), phosphate buffered saline ( 1×PBS), matrigel, 0.4% trypan blue solution and trypsin.

Kidney tissue was stored in cryogenic transfer medium and kept on ice at all times. 2 mL of enzymatic digestion solution can be placed in a 15 mL conical tube. Add 600 μL of calcium chloride (final concentration: 3 mM) and add 200 μL of collagenase (final: 0.5 mg/mL). Transfer kidney samples to petri/petri dishes. Remove all excess or non-neoplastic tissue with sterile tissue or a razor blade. Add 1 mL of Enzyme Solution to the tissue. Samples were minced into small pieces with a sterile razor blade (<2 mm ² ). Hold the disc with tweezers or by hand. Transfer the minced tissue and enzymatic solution back to the 15 mL tube with enzymatic solution. Place the tube in a tube rotator or a 15 mL tube rotator in a 37 °C incubator between 30 and 60 min. Remove the tube from the incubator. Quench the enzyme digestion solution with at least 6 mL of EBM-2 (at least 3 times the volume of the enzyme digestion solution). Pipette to mix. Place a 100 µm or 70 µm cell strainer onto a 50 mL conical tube. Transfer the sample through the filter. Transfer the solution to a new 15 mL conical tube. The samples were centrifuged 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 the parafilm and transfer to a cytometer or hemocytometer. Calculate the cell concentration (#/mL). Centrifuge at 1500 RPM for 5 minutes and discard the supernatant, leaving the pellet. Resuspend the cell pellet in 50 uL Matrigel per 1.25 x 105 cells. Perform on ice. Plate 50 μL of the dome with Matrigel cell suspension in the center of each well in a pre-warmed 24-well flat bottom plate. The plate was transferred to a 37°C cell incubator and incubated for at least 20 minutes. Confirm that the dome is polymerized. Gently add 500 μL of pre-warmed EBM-2 medium along the wall of the well. Cultivate in a 37°C incubator. Complete media changes were performed every 2 days to expand the micro-organoid spheroids. Example 7 : Liver Micro-Organoid Spheroids

As mentioned, miniature organoid spheroids can be formed from normal (eg, non-cancerous) and/or abnormal tissue. For example, Figures 25A-25B and Figures 26A-26B illustrate an example of a miniature organoid spheroid formed from self-exposed mouse liver tissue that was combined with a fluid matrix material to form an unpolymerized mixture , followed by polymerizing the unpolymerized mixture droplets to form the miniature organoid spheroids. In this example, the micro-organoid spheroids were approximately 300 µm in diameter. In FIGS. 25A-25B , a single cell per droplet was used to form miniature organoid spheroids. In FIGS. 26A-26B , 25 cells per droplet were used to form miniature organoid spheroids. In FIG. 25A , miniature organoid spheroids are shown one day after formation; FIG. 25B shows miniature organoid spheroids after ten days of culture. The cells in some micro-organoid spheroids had divided, forming clusters that exhibited structures; other micro-organoid spheroids included cells that divided slowly or did not divide. Similarly, in FIGS. 26A-26B , the mini-organoid spheroids initially included about 25 cells in each mini-organoid spheroid. After ten days in culture, some micro-organoid spheroids exhibited substantial cell growth forming structures, while others exhibited only minimal growth. In both cases, cells within micro-organoid spheroids have been found to exhibit characteristics characteristic of their original tissue of origin (eg, hepatocytes).

The same procedure was successfully performed on human liver tissue, as shown in Figures 27A-27C. In this example, approximately fifty cells were initially used to form miniature organoid spheroids, as shown in Figure 27A. By day 18 in culture, some micro-organoid spheroids exhibited cells that clustered and formed structures, while others had smaller structures or cells that did not divide. Example 8 : Cultured cell micro-organoid spheroids

Micro-organoid spheroids can be formed from one or more cultured cells, including 2D-cultured cells or 3D-cultured cells, in addition to, for example, primary tissue removed from a patient immediately or shortly prior to formation of the micro-organoid spheroids.

In some variations, mini-organoid spheroids can be formed from cell lines grown as part of patient-derived xenografts (PDX). For example, Figures 28A-28D illustrate miniature organoid spheroids formed from cultured PDX240 cells. PDX240 cells are a patient-derived xenograft (PDX) tumor cell line (number 240 based on patient origin), which are human tumors grown in immunodeficient mice (PDX) to form tumors in vivo. Xenograft tissue was extracted, dissociated, and used to form miniature organoid spheroids as described above. In this example, a single cell was included in each miniature organoid spheroid formed. Figure 28A shows mini-organoid spheroids after one day of culture, while Figure 28B shows mini-organoid spheroids after three days of culture, and Figure 28C and Figure 28D show mini-organoid spheroids after five and seven days of culture, respectively. Over time in culture, at least some miniature organoid spheroids exhibit cell division and formation of structures.

Figures 29A-29D show similar experiments in which five PDX240 cells were initially included in each droplet forming each mini-organoid spheroid. Over time in culture (eg, Day 1, Day 3, Day 5, and Day 7, as shown in Figures 29A-29, respectively), cells can divide and form structures. Example 9 : Comparison of Miniature Organoid Spheroids and 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 to miniature organoid spheroids formed using the same cells. Organoids are formed using well-known techniques, in which masses of Matrigel in wells or dishes are seeded with cells and cultured until growth is confirmed. Generate miniature organoid spheroids from conventional organoids.

Both traditional ("bulky") organoids and mini-organoid spheroids were then treated with the same drug (eg, oxaliplatin or SN38) and cell viability was measured after 3 days of treatment. The drug response curves shown in Figure 30 and Figure 31 were generated and exhibit similar response curves. For example, in Figure 30, the drug response curves of PDO19187 bulk organoids and mini-organoid spheroids showed similar response curves to oxaliplatin concentrations, as did PDX240 bulk organoids and mini-organoid spheroids. In FIG. 31 , the drug response curves of both PDX19187 and PDX240 also showed similar results against SN38 for both bulk organoids and mini-organoid spheroids. Figure 32 shows the response curve of another anticancer drug 5-FU (fluorouracil), again showing the similar drug response curves of PDZ-19187 and PDX-240 traditional organoids and micro-organoid spheroids.

Thus, the miniature organoid spheroids described herein, which can be formed more quickly and reliably and can have a higher overall survival rate compared to traditional organoids, can provide drug responses comparable to those of bulk organoids formed using the same cells. considerable drug reaction. However, as described herein, miniature organoid spheroids can be used relatively quickly and can be formed in significantly larger numbers. Example 10 : Drug Effects of Miniature Organoid Spheroids

In general, the micro-organoid spheroids described herein can be used to perform one or more assays, including toxicity assays. Any suitable analysis can be performed, as determined by analysis of tissue (eg, cells, tissue structures) suspended within the micro-organoid spheroids. The micro-organoid spheroids described herein can be analyzed or analyzed optically, chemically, electrically, genetically, or in any other manner known in the art.

Optical (manual or automated) detection may be particularly useful and may include optically analyzing the effect of one or more drug formulations on tissue (including cells, cell clusters, cellular structures, etc.) within the micro-organoid spheroid. In some variations, as mentioned above, the drug formulation can be assayed for cell death (eg, the number and/or size of tissue within the tested mini-organoid spheroids). In other variations, miniature organoid spheroids can be assayed for cell growth, including reduction in size, type, and/or growth rate. In some variations, micro-organoid spheroids can be analyzed for changes in the formed tissue architecture.

For example, Figures 33A-33B illustrate the effect of a drug formulation (in this example) acetaminophen (10 mM) on mouse liver mini-organoid spheroids. Figure 33A is a control group in which the micro-organoid spheroids were untreated, showing the organization within the micro-organoid spheroids (arrows) grown in culture. Figure 33B shows a similar set of mini-organoids formed from mouse livers treated with 10 mM acetaminophen replacement. In the control group, the organized structures within the micro-organoid spheroids were relatively larger compared with the treated group. The tissue in most micro-organoid spheroids of acetaminophen was small and contained many dead cells.

Similarly, Figures 34A-34B also show toxicity assays using human liver micro-organoid spheroids. Figure 34A shows typical human liver micro-organoid spheroids observed in a control group including tissue structures formed therein (indicated by arrows). Figure 34B shows the treatment group in which human liver micro-organoid spheroids were treated with acetaminophen (10 mM). Tissue in treated micro-organoid spheroids exhibited significantly increased atypical tissue architecture (arrows) and fragmentation compared to controls.

Any of these inspections, including optical inspections, can be scored, graded, rated or otherwise quantified. For example, in Figures 33A-33B and 34A-34B, the results of these two assays can be quantified to indicate size differences, numbers of live/dead cells/tissues and the like. In some variations, scoring may be automatic.

Any of the methods described herein (including user interfaces) can be implemented as software, hardware, or firmware, and can be described as storing data that can be read by a processor (e.g., computer, tablet, smartphone, etc.) ) a non-transitory computer-readable storage medium for a set of instructions that, when executed by a processor, cause the processor to control the execution of any of the steps, including but not limited to: displaying, communicating with a user, analyzing, Modify parameters (including timing, frequency, intensity, etc.), decisions, alerts, or the like.

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

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 as well, unless the context clearly dictates otherwise. It should be further understood that the term "comprises and/or comprising" when used in this specification specifies the presence of stated features, steps, operations, elements and/or components, but does not exclude one or more other features , the existence or addition of 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 "/".

For ease of description, spatially relative terms such as "under", "below", "lower", "above", "upper" and the like may be used herein to describe The relationship between one element or feature and another element or feature illustrated in the drawings. It will be understood that spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "beneath" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upward," "downward," "vertical," "horizontal," and the like are used herein for purposes of explanation only, unless specifically indicated otherwise.

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

Throughout this specification and claims that follow, unless the context requires otherwise, the word "comprise" and variations thereof, such as "comprises/comprising", mean that various elements can be collectively applied to methods and In articles of manufacture (eg, compositions and devices including devices and methods). For example, the term "comprising" should be understood as implying the inclusion of any stated elements or steps, not the exclusion of any other elements or steps.

In general, any of the apparatus and methods described herein should be understood to be inclusive, but all or a subset of the components and/or steps may alternatively be exclusive and may be expressed as "by" Each component, step, sub-component or sub-step "consists of" or alternatively "consists essentially of" each component, step, sub-component or sub-step.

As used in the specification and claims herein (including as used in the examples), unless expressly stated otherwise, all numbers can be read as if preceded by the word "about" or "approximately," even if that term is not expressly stated presented. When describing degrees and/or locations, the phrases "about" or "approximately" may be used to indicate that the described value and/or location is within a reasonable expectation of the value and/or location. For example, a value may have a stated value (or range of values) of +/-0.1%, a stated value (or range of values) of +/-1%, a stated value (or range of values) of +/-2%, +/-5% of the specified value (or value range), +/-10% of the specified value (or value range), etc. Unless the context dictates otherwise, any numerical value given herein should also be understood to include about or approximating that value. For example, if the value "10" is revealed, "about 10" is also revealed. Any numerical range recited herein is intended to include all subranges subsumed therein. It is also understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as properly understood by those skilled in the art. For example, if the value "X" is disclosed, then "less than or equal to X" and "greater than or equal to X" are also disclosed (eg, where X is a numerical value). It should also be understood that throughout this application, data is provided in a number of different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood to be deemed to disclose greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15 . It is also understood that every element between the two specified elements is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

While various illustrative embodiments are described above, any of a number of changes may be made to the various embodiments without departing from the scope of the invention as described in the claims. For example, in alternative embodiments, the order of performing differently described method steps may generally be varied, and in other alternative embodiments, one or more method steps may be skipped entirely. In some embodiments, and not in others, optional features of different device and system embodiments may be included. Accordingly, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

By way of example and not limitation, the examples and descriptions included herein show specific embodiments of the subject matter that can be practiced. As mentioned, other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of the present invention. Such embodiments of the present subject matter may be referred to herein individually or collectively by the term "invention," which is for convenience only and is not intended to limit the scope of this application to any single invention or inventive concept. Concepts (where more than one concept is actually disclosed). Thus, while specific embodiments have been illustrated and described herein, any configuration calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above-described embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

275: Entrance 503: polymer matrix material 507: Liver cells 505: structure 601: Step 603: Step 605: Step 607: Step 609: Step 611: Step 613: Step 615: Step 617: Step 700: Equipment 702: Shell 704: part of the device 706: holding chamber 708: chamber 712: Distributor nozzle 716: porous disc 718: Heat/Temperature Regulator 720: Droplet forming assembly 722: Allocator 724: Controller 726: pump 728: sensor 730: microfluidic chip 731: export 733:Entrance 735: Second entrance orifice / entrance orifice 737:joint area/intersection area 739: Element 741: Channel exit/delivery path 743: Immiscible fluid outlet/connection path 743': Immiscible fluid outlet/connection path 803: Micro Organoid Spheroids 805: cells 839: Channel area 903: droplet 909: Channel/Cross Channel 909': cross aisle 911: first channel/channel 937: joint area 939: exit channel 1005:Patient-derived micro-organoid spheroids 1008: immiscible materials 1801: step 1803: step 1805: step 1807: step 1809: step 1811: step 1813: step 1815: step 1905: Technology 1905' : Technology 1905'': Technology 1905''': Technology 1905'''': Technology 1905''''': Technology 1907: Technology 1921: Technology 1923: Technology 1925: Technology 1927: Technology 1929: Technology 1931: Technology 1933: Technology 1935: Technology 2002: Immiscible materials 2004: Reservoirs and/or orifices; inputs 2006: Unpolymerized materials 2008: Reservoir or orifice; input 2010: Third set of orifices; input 2012: Junction 2014: Output 2103: first channel 2107: Second Passage/Miniature Organoid Spheroids 2109:Cross passage 2109': Cross Passage

The novel features of the invention are set forth in detail in the claims that follow. A better understanding of the features and advantages of the invention will be gained by reference to the following detailed description and accompanying drawings which illustrate illustrative embodiments utilizing the principles of the invention:

Figures 1A-1C illustrate patient-derived micro-organoid spheroids formed as described herein, each micro-organoid spheroid comprising a dissociated primary tissue cell, cultured for one day after formation (Figure 1A), Cultured for three days after formation (Fig. IB) and seven days after formation (Fig. 1C). Cells were derived from colorectal cancer (CRC) tissue.

Figures 2A-2C illustrate patient-derived micro-organoid spheroids formed as described herein, each micro-organoid spheroid comprising five dissociated primary tissue cells, cultured for one day after formation (Figure 2A) , three days after formation (Fig. 2B) and seven days after formation (Fig. 2C). Cells were derived from colorectal cancer (CRC) tissue.

Figures 3A-3C illustrate patient-derived micro-organoid spheroids formed as described herein, each micro-organoid spheroid comprising twenty dissociated primary tissue cells, cultured for one day after formation (Figure 3A ), three days after formation (Fig. 3B) and seven days after formation (Fig. 3C). As shown in Fig. 1A to Fig. 1C and Fig. 2A to Fig. 2C, the cells are derived from colorectal cancer (CRC) tissue.

4A-4E illustrate examples of patient-derived micro-organoid spheroids formed as described herein, each micro-organoid spheroid comprising ten dissociated primary tissue cells. Figure 4A shows miniature organoid spheroids (at low magnification) shortly after formation. Figure 4B shows a higher magnification view of some of the miniature organoid spheroids of Figure 4A taken after two days in culture. Figure 4C shows miniature organoid spheroids after three days in culture. Figure 4D shows miniature organoid spheroids after four days in culture. Figure 4E shows miniature organoid spheroids after five days in culture.

Figures 5A-5B illustrate examples of miniature organoid spheroids formed as described herein from normal mouse liver hepatocytes cultured one day after formation (Figure 5A) and ten days after formation ( Figure 5B). Mouse hepatocytes are obtained from normal (eg, non-diseased) mouse livers.

6 illustrates a method of forming patient-derived mini-organoid spheroids from primary tissue (eg, biopsy) samples as described herein.

Figure 7A schematically illustrates one example of an apparatus for forming patient-derived micro-organoid spheroids as described herein, including a microfluidic chip as part of the assembly. 7B is a perspective view of an example microfluidic wafer portion of a device such as that shown in FIG. 7A. Figure 7C schematically illustrates a portion of a microfluidic assembly of an apparatus for forming patient-derived micro-organoid spheroids, such as the one shown in Figure 7A.

8 shows an example of an image depicting a plurality of patient-derived micro-organoid spheroids formed using an apparatus such as that shown in FIG. Previously suspended in a channel containing an immiscible fluid (eg, oil).

Figure 9 is an image of a portion of a prototype microfluidic assembly of an apparatus for forming patient-derived micro-organoid spheroids, similar to those shown in Figure 7C, illustrating the formation of patient-derived micro-organoid spheroids.

Figure 10 illustrates a plurality of patient-derived micro-organoid spheroids as described herein shortly after polymerization; the patient-derived micro-organoid spheroids are suspended in an immiscible fluid.

11A-11B illustrate a plurality of patient-derived microclasses shortly after formation and suspended in an immiscible fluid (e.g., oil) at low magnification (FIG. 11A) and higher magnification (FIG. 11B). Another example of an organ spheroid.

Figures 12A-12B show multiple patients after isolation from immiscible fluids within hours of formation of patient-derived miniature organoid spheroids at low magnification (Figure 12A) and higher magnification (Figure 12B) Derived miniature organoid spheroids.

13 is another example of an image showing a plurality of patient-derived micro-organoid spheroids formed as described herein.

14 is a graph illustrating the size distribution of diameters of a plurality of patient-derived micro-organoid spheroids formed from exemplary biopsy samples.

15A-15B illustrate low and higher magnification views, respectively, of an example of a plurality of patient-derived micro-organoid spheroids formed from dissociated tissue biopsy samples and fluid matrix material after polymerization. Figure 15A is an unstained image, while in Figure 15B the organoid spheroids have been stained with trypan blue to show that the dissociated cells in the miniature organoid spheroids are viable.

FIGS. 16A-16B are another example, similar to those shown in FIGS. 15A-15B , showing low and higher magnification views, respectively, of one example of a plurality of patient-derived micro-organoid spheroids. Figure 16A is an unstained image, while in Figure 16B the organoid spheroids have been stained with trypan blue (arrow) to demonstrate dissociated cells in the mini-organoid spheroids, which indicates that the cells remain viable (e.g., viable) within the mini-organoid spheroids. of).

Figures 17A-17E illustrate one example of a method for analyzing a plurality of patient-derived mini-organoid spheroids to determine drug response profiles to multiple drug formulations in this example formed from patient tumor biopsies. The illustrated procedure takes less than two weeks (eg, approximately one week) from biopsy to result.

Figure 18 schematically illustrates an example of a method for treating a patient, including forming and using a plurality of patient-derived micro-organoid spheroids as part of a treatment procedure.

Figure 19 schematically illustrates an example of a method for treating a patient, including multiple iterations of rapid formation and analysis of a plurality of patient-derived micro-organoid spheroids as part of a treatment procedure.

Figure 20 schematically illustrates one variation of a portion of an apparatus for forming a plurality of patient-derived micro-organoid spheroids as described herein.

FIG. 21 schematically illustrates a method of operating an apparatus similar to those shown in FIG. 20 for forming a plurality of patient-derived micro-organoid spheroids.

Figures 22A-22D illustrate one example of validation of a method for identifying drug resistance using a plurality of patient-derived mini-organoid spheroids as described herein. Figure 22A illustrates the use of traditional ("2D") tumor cell analysis methods to predict drug resistance. Figure 22B illustrates the use of one example of the patient-derived micro-organoid spheroid method as described herein to analyze drug resistance and thereby predict drug sensitivity. Figures 22C and 22D demonstrate that the patient-derived micro-organoid spheroid-based approach accurately predicts the actual tumor response (drug responsiveness) as opposed to traditional cultured cells.

Figures 23A-23D illustrate another example of validating the use of patient-derived mini-organoid spheroids as described herein to identify drug resistance, showing resistance to both oxaliplatin and irinotecan Predicted drug responses were consistent with actual tumor responses following treatment with these drugs.

Figure 24 illustrates an example of drug screening using patient-derived mini-organoid spheroids as described herein, where a single tumor biopsy can very rapidly (e.g., in less than two weeks) generate large numbers of nearly identical Rapid testing of miniature organoid spheroids and in parallel against a large number of drug formulations (eg, 27 displayed).

Figures 25A-25B illustrate examples of mouse liver micro-organoid spheroids formed from mouse liver tissue with a diameter of 300 µm and 1 cell per organoid spheroid. Figure 25A shows mini-organoid spheroids at day 1, and Figure 25B shows mini-organoid spheroids at day 10.

Figures 26A-26B illustrate examples of mouse liver micro-organoid spheroids formed from partially hepatectomized mouse liver tissue, similar to those shown in Figures 25A-25B , having a diameter of 300 µm, and each Each organoid spheroid has 25 cells. Figure 26A shows mini-organoid spheroids at day 1, and Figure 26B shows mini-organoid spheroids at day 10.

27A-27C illustrate examples of human liver micro-organoid spheroids formed from human liver tissue. Figure 27A shows miniature organoid spheroids at day 1, seeded with 40 cells/droplet. Figure 27B and Figure 27C show miniature organoid spheroids at day 18. In Figure 27B, the micro-organ spheroids are hepatocyte-like structures, while Figure 27C shows biliary epithelial-like micro-organoid spheroids.

Figures 28A-28D show examples of mini-organoid spheroids generated from patient-derived xenograft tumor lines with a diameter of 300 µm and 1 cell per mini-organoid spheroid, shown in Figure 28A at day 1 Figure 28B shows mini-organoid spheroids at day 3, Figure 28C shows mini-organoid spheroids at day 5, and Figure 28D shows mini-organoid spheroids at day 7.

Figures 29A-29D show examples of miniature organoid spheroids generated from a patient-derived xenograft model with a diameter of 300 µm and 5 cells per organoid spheroid. Figure 29A shows mini-organoid spheroids on day 1, Figure 29B shows mini-organoid spheroids on day 3, Figure 29C shows mini-organoid spheroids on day 5, and Figure 29D shows mini-organoid spheroids on day 7 organ sphere.

Figure 30 is a graph comparing the response to oxaliplatin of conventional organoids and mini-organoid spheroids formed from organoids derived from colorectal cancer patients, showing comparable responses of traditional organoids and mini-organoid spheroids.

Figure 31 is a graph comparing the response to SN38 (7-ethyl-10-hydroxy-camptothecin) of conventional organoids and mini-organoid spheroids formed from xenograft models derived from two colorectal cancer patients, It exhibits considerable response.

Figure 32 is a graph comparing the responses to 5-FU (fluorouracil) of conventional organoids and miniature organoid spheroids formed from colorectal cancer patient-derived xenograft models showing comparable responses.

Figures 33A and 33B show examples of toxicity assays using mouse liver mini-organoid spheroids. Figure 33A shows that the size of the tissue in the mouse liver mini-organoid spheroids was relatively larger (as indicated by the arrows) in the control group. In contrast, in Figure 33A showing the acetaminophen (10 mM) treated group, most of the tissue within the mini-organoid spheroids was smaller and contained many dead cells.

Figures 34A and 34B show examples of toxicity assays using human liver mini-organoid spheroids. Figure 34A shows typical human liver micro-organoid spheroids (indicated by arrows) observed in controls including tissue structures. Figure 34B shows miniature organoid spheroids in the acetaminophen (10 mM) treatment group showing atypical tissue architecture (arrows) and debris.

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Claims (28)

A method for precision drug screening for personalized cancer therapy, the method comprising: Receive tissue samples from patient tumors; dissociating the tissue sample to form a dissociated tissue sample; A library of patient-derived mini-organoid spheroids was formed from the dissociated tissue sample by: Driving the dissociated tissue sample and unpolymerized fluid matrix material through one or more channels of a microfluidic device, wherein the microfluidic device controls pressure, flow rate, or pressure and flow rate within the one or more channels and will The temperature of all or a portion of the microfluidic device is maintained at 20° C. or lower such that the dissociated tissue sample and the unpolymerized fluid matrix pass through the one or more channels in laminar flow, combining the dissociated tissue sample and the unpolymerized fluid matrix material within the microfluidic device to form a plurality of unpolymerized mixture droplets; exposing the plurality of unpolymerized mixture droplets to a temperature greater than 25° C. to polymerize the fluid matrix material and form the patient-derived micro-organoid spheroids, wherein each of the patient-derived micro-organoid spheroids has a diameter between 50 μm and Between 500 µm in diameter, with 1 to 200 dissociated cells distributed in it; culturing the bank of patient-derived mini-organoid spheroids for 2 to 14 days to form mature patient-derived mini-organoid spheroids having a structure that replicates the structure of the patient's tumor from which they were sampled Cell clusters; and One or more drug therapies are analyzed using the patient-derived mini-organoid spheroid library. The method of claim 1, wherein analyzing comprises analyzing a plurality of drug therapies in parallel by exposing one or more of the mature patient-derived micro-organoid spheroids to each drug therapy. The method of claim 1, further comprising characterizing the response of the one or more drug therapies to the patient's tumor based on the response of the patient-derived micro-organoid spheroids to exposure to the one or more drug therapies. The method of claim 3, wherein the time between receiving the tissue sample and characterizing the response is less than 21 days. The method of claim 1, wherein forming the patient-derived micro-organoid spheroid library further comprises sorting the patient-derived micro-organoid spheroids based on cell number and/or droplet size. The method of claim 1, wherein forming the patient-derived micro-organoid spheroid library comprises optically sorting the patient-derived micro-organoid spheroids or sorting the patient-derived micro-organoid spheroids based on cell number and/or droplet size organ sphere. The method of claim 1, wherein the analyzing comprises analyzing more than 50 different drug therapies. The method of claim 7, wherein the one or more drug therapies include different concentrations of one or more drugs, different combinations of three or more drugs, different ratios of two or more drugs, different doses of one or more drugs Different timing of administration of the vehicle and/or one or more drugs. The method of claim 1, wherein forming the patient-derived micro-organoid spheroid library comprises forming more than 100 to 600 patient-derived micro-organoid spheroids. The method of claim 1, wherein forming the patient-derived micro-organoid spheroid library comprises forming more than 1,000 patient-derived micro-organoid spheroids. The method of claim 1, wherein forming the patient-derived micro-organoid spheroid library comprises forming more than 10,000 patient-derived micro-organoid spheroids. The method of claim 1, wherein the microfluidic device maintains the viscosity of the dissociated tissue sample and the unpolymerized fluid matrix material before forming the plurality of unpolymerized mixture droplets. The method of claim 1, wherein the microfluidic device is structurally designed to avoid clogging of the dissociated tissue sample in the one or more channels. The method of claim 13, wherein the microfluidic device is structurally designed to avoid clogging by having a channel diameter of 100 μm or greater. The method of claim 1, wherein the microfluidic device is structurally designed to maintain an approximately constant pressure in the one or more channels. The method of claim 1, wherein exposing the plurality of unpolymerized mixture droplets to a temperature higher than 25° C. comprises exposing the plurality of unpolymerized mixture droplets to a temperature of 30° C. or higher. The method of claim 1, wherein exposing the plurality of unpolymerized mixture droplets to a temperature higher than 25° C. comprises flowing the plurality of unpolymerized mixture droplets to a temperature maintained above 25° C. in the microfluidic device Area. The method of claim 1, wherein the microfluidic device maintains a constant flow rate in the one or more channels. The method of claim 1, wherein the total length of the path taken by the dissociated tissue sample before being combined with the unpolymerized fluid matrix material in the microfluidic device is less than 10 cm. The method of claim 1, further comprising measuring the effect of the one or more drug therapies on the cells in the patient-derived mini-organoid spheroid. The method of claim 1, further comprising determining that after one or more administrations of one of the one or more drug therapies, the patient's tumor still responds to the drug therapy by: receiving the second patient's tumor tissue following treatment with the drug therapy and forming a second plurality of patient-derived micro-organoid spheroids from the second patient's tumor tissue, at least one of the second plurality of patient-derived micro-organoid spheroids Some are exposed to the drug therapy, and the effect of the drug therapy is measured on cells within the at least some of the second plurality of patient-derived mini-organoid spheroids. The method of claim 1, further comprising treating the patient with one of the one or more drug therapies. The method of claim 1, wherein the patient tissue sample comprises a biopsy sample from a metastatic tumor. The method of claim 1, wherein the patient-derived micro-organoid spheroids in the patient-derived micro-organoid spheroid bank have a size variation of less than 25%. The method of claim 1, wherein the patient-derived micro-organoid spheroids form budding clusters of cells and/or hollow structures of cells that replicate the structure of the patient's tumor from which they were sampled. The method of claim 1, wherein analyzing the one or more drug therapies comprises analyzing patient-derived micro-organoid spheroids with similar size and cell number. A method for precision drug screening for personalized cancer therapy, the method comprising: Receive a biopsy or resected tissue sample from the patient's tumor; dissociating the tissue sample to form a dissociated tissue sample; A bank of more than 100 patient-derived micro-organoid spheroids was formed from the dissociated tissue sample by: Driving the dissociated tissue sample and unpolymerized fluid matrix material through one or more channels of the microfluidic device, wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate in the one or more channels and maintains a temperature of 20° C. or lower, so that the dissociated tissue sample and the unpolymerized fluid matrix pass through the one or more channels in laminar flow, combining the dissociated tissue sample and the unpolymerized fluid matrix material within the microfluidic device to form a plurality of unpolymerized mixture droplets; exposing the plurality of unpolymerized mixture droplets to a temperature above 25° C. to polymerize the fluid matrix material and form the patient-derived micro-organoid spheres, wherein the patient-derived micro-organoid spheres each have a diameter of 50 μm and 500 μm. diameter between µm, in which 1 to 200 dissociated cells are distributed, wherein the patient-derived miniature organoid spheroids are sorted by size, number of cells or both; The patient-derived mini-organoid bank is cultured for 2 to 14 days to form mature patient-derived mini-organoids with reproducible biopsy or resected tumors of the patient Structured cell clusters of the structure; and Multiple drug therapies were analyzed using the patient-derived mini-organoid spheroid library. A method for precision drug screening for personalized cancer therapy, the method comprising: Receive tissue samples from patient tumors; dissociating the tissue sample to form a dissociated tissue sample; A bank of more than 1000 patient-derived micro-organoid spheroids was formed from the dissociated tissue sample by: Driving the dissociated tissue sample and unpolymerized fluid matrix material through one or more channels of the microfluidic device, wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate in the one or more channels and maintains a temperature of 20° C. or lower, so that the dissociated tissue sample and the unpolymerized fluid matrix pass through the one or more channels in laminar flow, combining the dissociated tissue sample and the unpolymerized fluid matrix material within the microfluidic device to form a plurality of unpolymerized mixture droplets; exposing the plurality of unpolymerized mixture droplets to a temperature above 25° C. to polymerize the fluid matrix material and form the patient-derived micro-organoid spheres, wherein the patient-derived micro-organoid spheres each have a diameter of 50 μm and 500 μm. diameter between µm, in which 1 to 200 dissociated cells are distributed, wherein the patient-derived miniature organoid spheroids are sorted by size, number of cells or both; culturing the patient-derived micro-organoid spheroids for 2 to 14 days to form mature patient-derived micro-organoid spheroids having a structure that replicates the structure of the patient's tumor from which they were sampled cell clusters; and The plurality of drug therapies are tested in parallel by exposing each of the drug therapies to one or more of the mature patient-derived mini-organoid spheroids.

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