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  • C12M23/00—Constructional details, e.g. recesses, hinges
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  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
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  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]

Definitions

• the methods, apparatuses and compositions of matter described herein relate to Patient-Derived Micro-Organospheres (PMOSs), and methods and apparatuses for forming PMOSs, and methods and apparatuses for using PMOSs.
• PMOSs Patient-Derived Micro-Organospheres
• described herein are methods and apparatuses for identifying one or more drug formulations that may be effective to treat a particular patient.
• Model cell and tissue systems are useful for biological and medical research.
• the most common practice is to derive immortalized cell lines from tissue and culture them in two- dimensional (2D) conditions (e.g., in Petri dish or well plate).
• 2D cell lines do not correlate well with individual patient response to therapy.
• three-dimensional cell culture models are proving particularly helpful in developmental biology, disease pathology, regenerative medicine, drug toxicity and efficacy testing, and personalized medicine.
• spheroids and organoids are three-dimensional cell aggregates that have been studied. Both organoids and spheroids have limitations that reduce their efficacy, however.
• Multicellular tumor spheroids were first described in the early 70s and obtained by culture of cancer cell lines under non-adherent conditions. Spheroids are typically formed from cancer cell lines as freely floating cell aggregates in ultra-low attachment plates. Spheroids have been shown to maintain more stem cell associated properties than 2D cell culture.
• Organoids are in-vitro derived cell aggregates that include a population of stem cells that can differentiate into cell of major cell lineages. Organoids typically have a diameter of more than one mm diameter, and are cultured through passages. It is typically slower to grow and expand organoid culture than 2D cell culture. To generate organoids from clinical samples, it requires sufficient number of viable cells (e.g., hundreds to thousands) to start with, so it is often challenging to derive organoids from low volume samples such as biopsy, and— even if successful— it will take considerable time to expand the culture for applications such as drug testing. In addition, there is a large amount of variability in organoid size, shape and cell number. Organoids may require complex cocktails of growth factors and culture conditions in order to grow and express desired cell types.
• PDMC Patient-Derived Models of Cancer
• organoids including patient-derived organoids
• organoids and particularly patient- derived organoids
• the significant failure rate for deriving organoids from biopsies also prevents its use as a reliable diagnostic assay.
• PDMC small molecule screens
• these PDMC models are typically much slower to expand and manipulate, making it challenging and costly for high-throughput applications.
• the longer time required to expand these models to amplify the cell numbers also tend to allow the fastest growing clone in plastics to dominate and outcompete other clones, hence making the model more homogeneous and losing the original tissue compositions and clonal diversity.
• patient derived tissue models e.g., tumor models and/or non-tumor tissue models
• PMOSs Patient-Derived Micro-Organospheres
• apparatuses and methods of making PMOSs apparatuses and methods of using PMOSs.
• methods and systems for screening a patient using these Patient-Derived Micro- Organospheres including personalized therapy methods.
• a patient for example, extracted from a small patient biopsy, (e.g., for quick diagnostics to guide therapy), from resected patient tissue, including resected primary tumor or part of a dysfunctional organ (e.g., for high- throughput screening), and/or from already established PDMCs, including patient-derived xenografts (PDX) and organoids (e.g., to generate Micro-Organospheres for high-throughput screening).
• PDX patient-derived xenografts
• organoids e.g., to generate Micro-Organospheres for high-throughput screening.
• PMOSs may be formed from primary cells that are normal (e.g., normal organ tissue) or from tumor tissue.
• these methods and apparatuses may form PMOSs from cancerous tumor biopsy tissue, enabling tailored treatments that can selected using the particular tumor tissue examined.
• these methods and apparatuses permit the formation of hundreds, thousands or even tens of thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000 or more) of PMOSs from a single tissue biopsy, within a few hours of the biopsy being removed from the patient.
• Dissociated primary cells from the patient biopsy may be combined with a fluid matrix material, such as a substrate basement membrane matrix (e.g., MATRIGEL), to form the micro-organosphere.
• a fluid matrix material such as a substrate basement membrane matrix (e.g., MATRIGEL)
• the resulting plurality of Patient-Derived Micro- Organospheres may have a predefined range of sizes (such as diameters, e.g., between 10 pm and 700 pm and any sub-range therewithin), and initial number of primary cells (e.g., between 1 and 1000, and in particular lower numbers of cells, such as between 1-200).
• the number of cells and/or the diameter may be controlled within, e.g., +/-5%, 10%, 15%, 20%, 25%, 30%, etc.
• PMOSs when formed as described herein, have an exceptionally high survival rate (>75%, >80%, >85%, >90%, >95%) and are stable for use and testing within a very short period of time, including within the first 1-10 days after being formed (e.g., within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, etc.).
• This allows for rapid tests on a potentially huge number of patient- specific and biologically relevant PMOSs which may save critical time in developing and deploying a patient therapy, such as a cancer treatment plan.
• the PMOSs described herein rapidly form 3D cellular structures that replicate and correspond to the tissue environment from which they were biopsied, such as a three-dimensional (3D) tumor microenvironment.
• the PMOSs described herein may also be referred to as“droplets”.
• Each PMOSs may include, e.g., as part of the fluid matrix material, growth factors and structural proteins (e.g., collagen, laminin, nidogen, etc.) that may mimic the original tissue (e.g., tumor) environment.
• Virtually any primary cell tissue may be used, including virtually any tumor tissue.
• the tissue types used to successfully generate Micro-Organopheres may be metastasized from other locations.
• the PMOSs described herein can be grown from fine needle aspirate (FNA) or from circulating tumor cells (CTCs), e.g., from a liquid biopsy. Proliferation and growth is typically seen in as few as 3-4 days, and the PMOSs can be maintained and passaged for months, or they may be cryopreserved and/or used for assays immediately (e.g., within the first 7-10 days).
• FNA fine needle aspirate
• CTCs circulating tumor cells
• Described herein are methods of forming Patient-Derived Micro- Organospheres.
• these methods include combining dissociated primary tissue cells (including, but not limited to cancer/abnormal tissue, normal tissue, etc.) with a liquid matrix material to form an unpolymerized material, and then polymerizing the unpolymerized material to form micro-Organospheres that are typically less than about 1000 pm (e.g., less than about 900 pm, less than about 800 pm, less than about 700 pm, less than about 600 pm, and in particular, less than about 500 pm) in diameter in which the dissociated primary tissue cells are distributed.
• dissociated primary tissue cells including, but not limited to cancer/abnormal tissue, normal tissue, etc.
• a liquid matrix material to form an unpolymerized material
• micro-Organospheres that are typically less than about 1000 pm (e.g., less than about 900 pm, less than about 800 pm, less than about 700 pm, less than about 600 pm, and in particular, less than about 500
• the number of dissociated cells may be within a predetermined range, as mentioned above (e.g., between about 1 and about 500 cells, between about 1-200 cells, between about 1- 150 cells, between about 100 cells, between about 1-75 cells, between about 1-50 cells, between about 1-30 cells, between about 1-20 cells, between about 1-10 cells, between about 5-15 cells, between about 20-30 cells, between about 30-50 cells, between about 40-60 cells, between about 50-70 cell, between about 60-80 cells, between about 70-90 cells, between about 80-100 cells, between about 90-110 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 may be configured as described herein to produce Micro-Organospheres of repeatable size (e.g., having a narrow distribution of sizes).
• the dissociated cells may be freshly biopsied and may be dissociated in any appropriate manner, including mechanical and/or chemical dissociation (e.g., enzymatic disaggregation by using one or more enzymes, such as collagenase, trypsin, etc.).
• the dissociated cells may optionally be treated, selected and/or modified.
• the cells may be sorted or selected to identify and/or isolate cells having one or more characteristics (e.g., size, morphology, etc.).
• the cells may be marked (e.g., with one or more markers) that may be used to aid in selection.
• the cells may be sorted by a known cell-sorting technology, including but not limited to microfluidic cell sorting, fluorescent activated cell sorting, magnetic activated cell sorting, etc. Alternatively, the cells may be used without sorting.
• the dissociated cells may be modified by treatment with one or more agents.
• the cells may be genetically modified.
• the cells may be modified using CRISPR-Cas9 or other genetic editing techniques.
• the cells may be transfected by any appropriate method (e.g., electroporation, cell squeezing, nanoparticle injection, magnetofection, chemical transfection, viral transfection, etc.), including transfection with of plasmids, RNA, siRNA, etc. Alternatively, the cells may be used without modification.
• the unpolymerized mixture may include additional cell or tissue types, including support cells.
• the additional cells or tissue may originate from different biopsy (e.g., primary cells from a different dissociated tissue) and/or cultured cells.
• the additional cells may be, for example immune cells, stromal cells, endothelial cells, etc.
• the additional materials may include medium (e.g., growth medium, freezing medium, etc.), growth factors, support network molecules (e.g., collagen, glycoproteins, extracellular matrix, etc.), or the like.
• the additional materials may include a drug composition.
• the unpolymerized mixture includes only the dissociated tissue sample (e.g., primary cells) and the fluid matrix material.
• the methods may rapidly form a plurality of Patient-Derived Micro-Organospheres from a single tissue biopsy, so that greater than about 500 Patient-Derived Micro-Organospheres are formed from per biopsy (e.g., greater than about 600, greater than about 700, greater than about 800, greater than about 900, greater than about 1000, greater than about 2000, greater than about 2500, greater than about 3000, greater than about 4000, greater than about 5000, greater than about 6000, greater than about 7000, greater than about 8000, greater than about 9000, greater than about 10,000, greater than about 11,000, greater than about 12,000, etc.).
• per biopsy e.g., greater than about 600, greater than about 700, greater than about 800, greater than about 900, greater than about 1000, greater than about 2000, greater than about 2500, greater than about 3000, greater than about 4000, greater than about 5000, greater than about 6000, greater than about 7000, greater than about 8000, greater than about 9000, greater than about 10,000, greater
• the biopsy may be a standard size biopsy, such as an 18G (e.g., 14G, 16G, 18G, etc.) core biopsy.
• the volume of tissue removed by biopsy and used to form the plurality of Patient- Derived Micro-Organospheres may be a small cylinder (taken with a biopsy needle) of between about 1/32 and 1/8 of an inch diameter and about 3 ⁇ 4 inch to 1 ⁇ 4 inch long, such as a cylinder of about 1/16 inch diameter by 1 ⁇ 2 inch long.
• the biopsy may be taken by needle biopsy, e.g., by core needle biopsy. In some variations the biopsy may be taken by fine needle aspiration.
• Other biopsy types that may be used include shave biopsy, punch biopsy, incisional biopsy, excisional biopsy, and the like.
• the material from a single patient biopsy may be used to generate the plurality (e.g., greater than about 2000, greater than about 5000, greater than about 7500, greater than about 10,000, etc.) of Patient-Derived Micro-Organospheres as described above.
• the plurality of Patient-Derived Micro-Organospheres may be formed using an apparatus (as described herein) that may be configured to generate this large number of highly regular (size, cell number, etc.) Micro-Organospheres as described herein.
• these methods and apparatuses may generate the plurality or Micro-Organospheres at a rapid rate (e.g., greater than about 1 Micro-Organosphere per minute, greater than about 1 Micro-Organosphere per 10 seconds, greater than about 1 Micro-Organosphere per 5 seconds, greater than about 1 Micro- Organosphere per 2 seconds, greater than about 1 Micro-Organosphere per second, greater than about 2 Micro-Organospheres per second, greater than about 3 Micro-Organospheres per second, greater than about 4 Micro-Organospheres per second, greater than about 5 Micro- Organospheres per second, greater than about 10 Micro-Organospheres per second, greater than 50 Micro-Organospheres per second, greater than 100 Micro-Organospheres
• these methods may be performed by combing the unpolymerized mixture with a material (e.g., liquid material) that is immiscible with the unpolymerized material.
• the method and apparatus may control the size and/or cell density of the Micro-Organospheres by, at least in part, controlling the flow of one or more of the unpolymerized mixture (and/or the dissociated tissue and fluid matrix) and the material that is immiscible with the unpolymerized mixture (e.g., a hydrophobic material, oil, etc.).
• these methods may be performed using a microfluidics apparatus.
• multiple Micro-Organospheres may be formed in parallel (e.g., 2 in parallel, 3 in parallel, 4 in parallel, etc.).
• the same apparatus may therefore 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 a source of fluid matrix.
• the unpolymerized material may be polymerized in order to form the Patient-Derived Micro-Organospheres in a variety of different ways.
• the methods may include polymerizing the Micro-Organospheres by changing the temperature (e.g., raising the temperature above a threshold value, such as, for example greater than about 20 degrees C, greater than about 25 degrees C, greater than about 30 degrees C, greater than about 35 degrees C, etc.).
• the Patient-Derived Micro-Organospheres may be allowed to grow, e.g., by culturing and/or may be assayed either before or after culturing and/or may be cryopreserved either before or after culturing.
• the Patient-Derived Micro-Organospheres may be cultured for any appropriate length of time, but in particular, may be cultured for between 1 day and 10 days (e.g., between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, etc.).
• the Patient-Derived Micro-Organospheres may be cryopreserved or assayed before six passages, which may preserve the heterogeneity of the cells within the Patient-Derived Micro-Organospheres; limiting the number of passages may prevent the faster-dividing cells from outpacing more slowly dividing cells.
• some of the Patient-Derived Micro-Organospheres may be cryopreserved (e.g., over half) while some are cultured and/or assayed.
• cryopreserved Patient- Derived Micro-Organospheres may be banked and used (e.g., assayed, passaged, etc.) later.
• a method of forming a plurality of Patient- Derived Micro-Organospheres 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 polymerizing the droplets to form a plurality of Patient-Derived Micro-Organospheres each having a diameter of between 50 and 500 pm with between 1 and 200 dissociated cells distributed therein.
• a method e.g., of forming a plurality of Patient-Derived Micro-Organospheres, may include combining a dissociated tissue sample and a fluid matrix material to form an
• a method as described herein for forming a plurality of Patient- Derived Micro-Organospheres may include: combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets having less than a 25% variation in size of the droplets by converging a stream of the unpolymerized mixture with one or more streams of a fluid that is immiscible with the unpolymerized mixture;
• Any of these methods may include modifying the cells within the dissociated tissue sample prior to forming the droplets.
• Forming the plurality of droplets may comprise forming a plurality of droplets of the unpolymerized mixture of uniform size with less than about 25% variation in size (e.g., less than about 20% variation in size, less than about 15% variation in size, less than about 10% variation in size, less than about 8% variation in size, less than about 5% variation in size, etc.).
• the variations in size may also be described as a narrow distribution of size variation.
• the distribution of sizes may include a Patient-Derived Micro-Organospheres size distribution (e.g., Micro-Organosphere diameter vs.
• the number of formed Micro-Organospheres having a low standard deviation (e.g., a standard deviation of 15% or less, a standard deviation of 12% or less, a standard deviation of 10% or less, a standard deviation of 8% or less, a standard deviation of 6% or less, a standard deviation of 5% or less, etc.).
• a standard deviation of 15% or less e.g., a standard deviation of 15% or less, a standard deviation of 12% or less, a standard deviation of 10% or less, a standard deviation of 8% or less, a standard deviation of 6% or less, a standard deviation of 5% or less, etc.
• any of these methods may also include plating or distributing the Patient-Derived Micro-Organospheres.
• the method may include combining Patient-Derived Micro-Organospheres from various sources into a receptacle prior to assaying.
• the Micro-Organospheres may be placed into a multi-well plate.
• any of these methods may include dispensing the Patient-Derived Micro-Organospheres into a multi- well plate prior to assaying the Patient-Derived Micro-Organosphere.
• One or more (or in some variations in equal amounts of) Patient-Derived Micro-Organospheres may be included per well.
• applying the Patient-Derived Micro-Organospheres into a receptacle may include placing the Organopsheres into a plurality of chambers that are separated by an at least partially permeable membrane to permit circulation of supernatant material between the chambers. This may allow the Patient-Derived Micro-Organospheres to share the same supernatant.
• the Patient-Derived Micro-Organospheres may be assayed.
• An assay may generally include exposing or treating individual Patient-Derived Micro- Organospheres to conditions (e.g., drug compositions) to determine if the drug composition has an effect on the cells of the Patient-Derived Micro-Organospheres (and in some cases, what effect it has).
• Assays may include exposing a subset of the Patient-Derived Micro-Organospheres (individually or in groups) to one or more concentrations of a drug composition, and allowing the Patient-Derived Micro-Organospheres to remain exposed for a predetermined time period (minutes, hours, days, etc.) and/or exposing and removing the drug composition, then culturing the Patient-Derived Micro-Organospheres for a predetermined time period.
• the Patient-Derived Micro-Organospheres may be examined to identify any effects, including in particular toxicity on the cells in the Patient-Derived Micro-Organospheres, or a change in morphology and/or growth of the cells in the Patient-Derived Micro-Organospheres.
• assaying may include marking (e.g., by immunohistochemistry) live or fixed cells within the Patient-Derived Micro-Organospheres. Cells may be assayed (e.g., examined) manually or automatically. For example, cells may be examined to determine any toxicity (cell death) using an automated reader apparatus.
• assaying the plurality of Patient- Derived Micro-Organospheres may include sampling one or more of a supernatant, an environment, and a microenvironment of the Patient-Derived Micro-Organosphere for secreted factors and other effects.
• the Patient-Derived Micro-Organospheres may be recovered following the assay for further assaying, expansion or preservation (e.g., cryopreserving, fixation, etc.) for subsequent examination.
• transcriptomic, proteomics, or meta-genomic markers may be assayed using the PMOSs described herein.
• any of these compositions and methods described herein may be used to identify or examine one or more markers and biological/physiological pathways, including, for example, exosomes, which may assist in identifying drugs and/or therapies for patient treatment.
• the tissue sample may include comprises a biopsy sample from a metastatic tumor.
• a tissue sample may comprise a clinical tumor sample; the clinical tumor sample may comprise both cancer cells and stroma cells.
• the tissue sample comprises tumor cells and one or more of: mesenchymal cells, endothelial cells, and immune cells.
• Any of the methods described herein may include initially distributing the dissociated cells from the tissue biopsy uniformly, or in some variations non-uniformly, throughout the fluid matrix material, in any appropriate concentration.
• the methods described herein may include combining the dissociated tissue sample and the fluid matrix material so that the dissociated tissue cell are distributed within the fluid matrix material to a density of less than lxlO 7 cells/ml (e.g., less than 9 x 10 6 cells/ml, 7 x 10 6 cells/ml, 5 x 10 6 cells/ml, 3xl0 6 cells/ml, 1 x 10 6 cells/ml, 9 x 10 5 cells/ml, 7 x 10 5 cells/ml, 5 x 10 5 cells/ml, etc.).
• lxlO 7 cells/ml e.g., less than 9 x 10 6 cells/ml, 7 x 10 6 cells/ml, 5 x 10 6 cells/ml, 3xl0 6 cells/ml, 1 x 10 6 cells/ml, 9 x 10 5 cells/ml, 7 x 10 5 cells/ml, 5 x 10 5 cells/ml, etc.
• forming the droplet may comprise forming the droplet from a continuous stream of the unpolymerized mixture.
• forming the droplet may comprise applying one or more convergent streams of a fluid that is immiscible with the unpolymerized mixture to the stream of unpolymerized mixture.
• the streams may be combined in a microfluidic device, e.g., a device having a plurality of converging channels into which the unpolymerized mixture and the immiscible fluid interact to form droplets having a precisely controlled volume.
• the droplets are formed (e.g., pinched off) in an excess of the immiscible material, and the droplets may be concurrently and/or subsequently polymerized to form the Patient-Derived Micro-Organosphere.
• the region in which the streams converge may be configured to polymerize the unpolymerized mixture after the droplet has been formed, e.g., by heating, and/or the regions downstream may be configured to polymerize the unpolymerized mixture after the droplets have been formed and are surrounded by the immiscible material.
• the immiscible material is heated (or alternatively cooled) to a temperature that promotes polymerization of the unpolymerized material, forming the Patient-Derived Micro- Organospheres.
• polymerizing may comprise heating the droplet to greater than 35 degrees C.
• forming the droplet may include forming the droplet in a fluid that is immiscible with the unpolymerized mixture. Further, any of these methods may include separating the immiscible fluid from the Patient-Derived Micro-Organospheres. For example, and of these methods may include removing the immiscible fluid from the Patient- Derived Micro-Organosphere.
• an immiscible fluid may include a liquid (e.g., oil, polymer, etc.), including in particular a hydrophobic material or other material that is immiscible with the unpolymerized (e.g., aqueous) material.
• the fluid matrix material may be a synthetic or non- synthetic unpolymerized basement membrane material.
• the unpolymerized basement material may comprise a polymeric hydrogel.
• the fluid matrix material may comprise a MATRIGEL.
• combining the dissociated tissue sample and the fluid matrix material may comprise combining the dissociated tissue sample with a basement membrane matrix.
• the tissue sample may be combined with the fluid matrix material within six hours of removing the tissue sample from the patient or sooner (e.g., within about 5 hours, within about 4 hours, within about 3 hours, within about 2 hours, within about 1 hour, etc.).
• a 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 having less than a 25% variation in a size of the droplets; polymerizing the droplets to form a plurality of Patient-Derived Micro-Organospheres having a diameter of between 50 and 700 pm with between 1 and 1000 dissociated cells distributed therein; and assaying or cryopreserving the plurality of Patient-Derived Micro-Organospheres.
• a 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; polymerizing the droplets to form a plurality of Patient-Derived Micro- Organospheres each having a diameter of between 50 and 500 pm with between 1 and 200 dissociated cells distributed therein; and cryopreserving or assaying the plurality of Patient- Derived Micro-Organospheres within 15 days, wherein the microoganoids are assayed to determine the effect of one or more agents on the cells within the Patient-Derived Micro- Organospheres.
• a method may include: combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets having less than a 25% variation in a size of the droplets by converging a stream of the unpolymerized mixture with one or more streams of a fluid that is immiscible with the unpolymerized mixture; polymerizing the droplets by warming to form Patient-Derived Micro-Organospheres each having a diameter of between 50 and 500 pm with between 1 and 200 dissociated cells distributed therein; and assaying or cryopreserving the Patient-Derived Micro-Organospheres before six passages, whereby heterogeneity of the cells within the Patient-Derived Micro- Organospheres is maintained, further wherein assaying comprises assaying in order to determine the effect of one or more agents on the cells within the Patient-Derived Micro-Organosphere.
• the plurality of Patient-Derived Micro-Organospheres may be cryopreserved or assayed before six passages, whereby heterogeneity of the cells within the Patient-Derived Micro-Organospheres is maintained. Any of these methods may further include modifying the cells within the dissociated tissue sample prior to forming the droplets.
• Forming the droplet may include forming a plurality of droplets of the unpolymerized mixture of uniform size with less than about 25% variation in size (e.g., less than about 20%, less than about 15%, less than about 10%, less than about 7%, less than about 5%, etc.).
• Any of these methods may include culturing the Patient-Derived Micro- Organospheres for an appropriate length of time, as mentioned above (e.g., culturing the Patient- Derived Micro-Organospheres for between 2-14 days before assaying). For example, these methods may include removing the immiscible fluid from the Patient-Derived Micro- Organospheres before culturing.
• culturing the Patient-Derived Micro- Organospheres comprises culturing the Patient-Derived Micro-Organospheres in suspension.
• assaying the Patient-Derived Micro-Organospheres may comprise genomically, transcriptomically, epigenomically and/or metabolically analyzing the cells in the Patient-Derived Micro-Organospheres before and/or after assaying or cryopreserving the Patient- Derived Micro-Organospheres. Any of these methods may include assaying the Patient-Derived Micro-Organosphere by exposing the Patient-Derived Micro-Organosphere to a drug (e.g., drug composition).
• a drug e.g., drug composition
• assaying may comprise visually assaying the effect of the one or more agents on the cells in the Patient-Derived Micro-Organosphere either manually and/or automatically. Any of these methods may include marking or labeling cells in the Patient- Derived Micro-Organospheres for visualization. For example, assaying may include
• a composition of matter may comprise a plurality of cryopreserved Patient-Derived Micro-Organospheres, wherein each Patient-Derived Micro-Organosphere has a spherical shape having a diameter of between 50 pm and 500 pm and comprises a polymerized base material, and between about 1 and 1000 dissociated primary cells distributed within the base material that have been passaged less than six times, whereby heterogeneity of the cells within the Patient-Derived Micro-Organospheres is maintained.
• compositions of matter comprising a plurality of cryopreserved Patient-Derived Micro-Organospheres, wherein each Patient-Derived Micro- Organosphere has a spherical shape having a diameter of between 50 pm and 500 pm, wherein the Patient-Derived Micro-Organospheres have less than a 25% variation in size, and wherein each Patient-Derived Micro-Organosphere comprises a polymerized base material, and between about 1 and 500 dissociated primary cells distributed within the base material that have been passaged less than six times, whereby heterogeneity of the cells within the Patient-Derived Micro-Organospheres is maintained.
• the primary cells may be primary tumor cells.
• the dissociated primary cells may have been genetically or biochemically modified.
• the plurality of cryopreserved Patient-Derived Micro-Organospheres may have a uniform size with less than 25% variation in size.
• the plurality of cryopreserved Patient-Derived Micro-Organospheres may comprise Patient-Derived Micro-Organospheres from various sources. In any of these Micro-Organospheres, the majority of cells in each Micro-Organosphere may comprise cells that are not stem cells.
• the primary cells comprise metastatic tumor cells.
• the primary cells may comprise both cancer cells and stroma cells.
• the primary cells comprise tumor cells and one or more of: mesenchymal cells, endothelial cells, and immune cells.
• the primary cells may be distributed within the polymerized base material at a density of less than, e.g., 5 x 10 7 cells/ml, 1 x 10 7 ells/ml, 9 x 10 6 cells/ml, 7 x 10 6 cells/ml, 5 x 10 6 cells/ml, 1 x 10 6 cells/ml, 9 x 10 5 cells/ml, 7 x 10 5 cells/ml, 5 x 10 5 cells/ml, 1 x 10 5 cells/ml, etc.
• the polymerized base material may comprise a basement membrane matrix (e.g., MATRIGEL).
• the polymerized base material comprises a synthetic material.
• the microoganoids may have a diameter of between 50 pm and 1000 pm, or more preferably between 50 pm and 700 pm, or more preferably between 50 pm and 500 pm, or between 50 pm and 400 pm, or between 50 pm and 300 pm, or between 50 pm and 250 pm, etc. (e.g., less than about 500 pm, less than about 400 pm, less than about 300 pm, less than about 250 pm, less than about 200 pm, etc.).
• the Patient-Derived Micro-Organospheres described herein may include any appropriate number of primary tissue cells initially in each Patient-Derived Micro- Organosphere, for example 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 cell, or less than about 5 cells, etc.).
• each Patient-Derived Micro-Organospheres includes between about 1 and 500 cells, between about 1-400 cells, between bout 1-300 cells, between about 1-200 cells, between about 1-150 cells, between about 1-100 cells between about 1-75 cells, between about 1-50 cells, between about 1-30 cells, between about 1-25 cells, between about 1-20 cells, etc.
• methods of operating a Patient-Derived Micro-Organosphere forming apparatus comprising: receiving an unpolymerized mixture comprising a chilled mixture of a dissociated tissue sample and a first fluid matrix material in a first port; receiving a second fluid that is immiscible with the unpolymerized mixture in a second port; combining a stream of the unpolymerized mixture with one or more streams of the second fluid to form droplets of the unpolymerized mixture having a uniform size that varies by less than 25%; and polymerizing the droplets of the unpolymerized mixture to form a plurality of Patient-Derived Micro-Organospheres.
• a method of operating a Patient-Derived Micro-Organosphere forming apparatus may include: receiving an unpolymerized mixture comprising a chilled mixture of a dissociated tissue sample and a first fluid matrix material in a first port; receiving a second fluid that is immiscible with the unpolymerized mixture in a second port; combining a stream of the unpolymerized mixture at a first rate with one or more streams of the second fluid at a second rate to form droplets of the unpolymerized mixture having a uniform size that varies by less than 25%, wherein the droplets are between 50 pm and 500 pm diameter; and polymerizing the droplets of the unpolymerized mixture to form a plurality of Patient-Derived Micro- Organospheres.
• any of these methods may include coupling a first reservoir containing the unpolymerized mixture in fluid communication with the first port.
• the method may include combining the dissociated tissue sample and the first fluid matrix material to form the unpolymerized mixture.
• the method includes adding the unpolymerized mixture to a first reservoir in fluid communication with the first port.
• These methods may include coupling a second reservoir containing the second fluid in fluid communication with the second port. Any of these methods may include adding the second fluid to a second reservoir in fluid communication with the second port.
• receiving the second fluid comprises receiving an oil.
• these methods may include separating the second fluid (e.g., the immiscible fluid) from the plurality of Patient-Derived Micro-Organospheres.
• This fluid may be manually or automatically separated.
• the second (immiscible) fluid may be removed by washing, filtering, or any other appropriate method.
• Combining the streams may comprise driving the stream of the unpolymerized mixture at a first flow rate across one or more streams of the second fluid which is traveling a second flow rate.
• the first flow rate is greater than the second flow rate.
• the flow rate and/or the amount of material e.g., the unpolymerized mixture may be present in smaller amount than the second fluid, so that the unpolymerized mixture is encapsulated in a precisely-controlled droplet, as described herein, that may then be
• combining the streams comprises driving the stream of the unpolymerized mixture across a junction into which the one or more streams of the second fluid also converge.
• Polymerizing the droplets may comprise heating the droplets to greater than a temperature at which the unpolymerized material polymerizes (e.g., greater than about 25 degrees C, greater than about 30 degrees C, greater than about 35 degrees C, etc.).
• Any of these methods may include aliquoting the plurality of Patient-Derived Micro- Organospheres. For example, aliquoting into a multi-well dish.
• a method may include:
• determining, within 2 weeks of taking of the biopsy, that the tumor will respond to a drug formulation by: forming, from the patient biopsy, a plurality of micro-organo spheres having a diameter of between 50 and 500 pm with between 1 and 200 dissociated tumor cells distributed through a polymerized base material, and exposing at least some of the Patient-Derived Micro-Organospheres to the drug formulation before the dissociated tumor cells have undergone more than five passages; and measuring an effect of the drug formulation on the cells within the at least some of the micro-organospheres to determine if the drug will treat the tumor based on the determined effect.
• these methods may include determining that the tumor is still responding 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-Organospheres from the second patient biopsy, exposing at least some of the second plurality of Patient-Derived Micro-Organospheres to the drug formulation, and measuring the effect of the drug formulation on cells within the at least some of the second plurality of micro-organospheres.
• Determining that the tumor will respond to a drug formulation may include exposing at least some of the Patient-Derived Micro-Organospheres to a plurality of drug formulations, and reporting the measured effects for each of the drug formulations. In some variations, determining further comprises dispensing the micro-organospheres into a multi- well plate prior to assaying the Patient-Derived Micro-Organosphere.
• any of these methods may include biopsying the patient to collect the patient biopsy (or otherwise taking a tissue sample from a patient or a sample of a patient-derived tissues or cells) and/or treating the patient with the drug formulation, or assisting a physician in treating the patient (e.g., advising the physician as to which drug formulations would be effective).
• the time between receiving the biopsy and reporting may be less than about 21 days (e.g., less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, etc.).
• FIGS. 1A to 1C illustrate Patient-Derived Micro-Organospheres formed as described herein to include a single dissociated primary tissue cell per Micro-Organosphere, cultured for one day after forming (FIG. 1A), cultured for three days after forming (FIG. IB), and cultured for seven days after forming (FIG. 1C).
• the cells originate from colorectal cancer (CRC) tissue.
• FIGS. 2A to 2C illustrate Patient-Derived Micro-Organospheres formed as described herein to include five dissociated primary tissue cells per Micro-Organosphere, cultured for one day after forming (FIG. 2A), cultured for three days after forming (FIG. 2B), and cultured for seven days after forming (FIG. 2C).
• the cells originate from colorectal cancer (CRC) tissue.
• FIGS. 3 A to 3C illustrate Patient-Derived Micro-Organospheres formed as described herein to include twenty dissociated primary tissue cells per Micro-Organosphere, cultured for one day after forming (FIG. 3A), cultured for three days after forming (FIG. 3B), and cultured for seven days after forming (FIG. 3C).
• the cells originate from colorectal cancer (CRC) tissue.
• FIGS. 4A to 4E illustrate examples of Patient-Derived Micro-Organospheres formed as described herein to include ten dissociated primary tissue cells per Micro-Organosphere.
• FIG. 4A shows the Micro-Organospheres shortly after formation (at low magnification).
• FIG. 4B shows a higher magnification view of some of the Micro-Organospheres of FIG. 4A taken after culturing for two days.
• FIG. 4C shows the Micro-Organospheres after culturing for three days.
• FIG. 4D shows the Micro-Organospheres after culturing for four days.
• FIG. 4E shows the Micro- Organospheres after culturing for five days.
• FIGS. 5 A to 5B illustrate examples of Micro-Organospheres formed as described herein from normal mouse liver hepatocytes, cultured for one day after forming (FIG. 1A), cultured for ten days after forming (FIG. IB), The mouse hepatocytes are taken from a normal (e.g., non-diseased) mouse liver.
• FIG. 6 illustrates on the method of forming Patient-Derived Micro-Organospheres from primary tissue (e.g., biopsy) samples, as described herein.
• FIG. 7A schematically illustrates one example of an apparatus for forming Patient- Derived Micro-Organospheres as described herein, including a microfluidic chip as part of the assembly.
• FIG. 7B is a perspective view of one example a microfluidics chip portion of an apparatus such as that shown in FIG. 7A.
• FIG. 7C schematically illustrates a portion of a microfluidics assembly for an apparatus for forming Patient-Derived Micro-Organospheres, such as the one shown in FIG. 7 A.
• FIG. 8 shows one example of an image showing a plurality of Patient-Derived Micro- Organospheres formed using an apparatus such as that shown in FIG. 7A, showing the Patient- Derived Micro-Organospheres shortly after polymerizing, suspended within a channel containing the immiscible fluid (e.g., oil) prior to being aliquoted from the apparatus.
• the immiscible fluid e.g., oil
• FIG. 9 is an image of a portion of a prototype microfluidics assembly for an apparatus for forming Patient-Derived Micro-Organospheres, similar to that shown in FIG. 7C, illustrating the formation of Patient-Derived Micro-Organospheres.
• FIG. 10 illustrates a plurality of Patient-Derived Micro-Organospheres as described herein, shortly after polymerization; the Patient-Derived Micro-Organospheres are suspended in the immiscible fluid.
• FIGS. 1 lA-1 IB illustrate another example of a plurality of Patient-Derived Micro- Organospheres shortly after formation and suspended in the immiscible fluid (e.g., oil) at low magnification (FIG. 11A) and higher magnification (FIG. 11B).
• immiscible fluid e.g., oil
• FIGS. 12A-12B show a plurality of Patient-Derived Micro-Organospheres following separation from the immiscible fluid within a few hours of formation of the Patient-Derived Micro-Organospheres at low magnification (FIG. 12A) and higher magnification (FIG. 12B).
• FIG. 13 is another example of an image showing a plurality of Patient-Derived Micro-Organospheres formed as described herein.
• FIG. 14 is a chart illustrating the size distribution of the diameters from a plurality of Patient-Derived Micro-Organospheres formed from an exemplary biopsy sample.
• FIGS. 15A-15B illustrate a low and higher magnification views, respectively, of one example of a plurality of Patient-Derived Micro-Organospheres formed from a dissociated tissue biopsy sample and a fluid matrix material, after polymerizing.
• FIG. 15A is an unstained image, while in FIG. 15B the Organospheres have been stained with Trypan blue to show that the dissociated cells in the Micro-Organospheres are alive.
• FIGS. 16A-16B is another example, similar to that shown in FIG. 15A-15B, showing low and higher magnification views, respectively, of one example of a plurality of Patient- Derived Micro-Organospheres.
• FIG. 16A is an unstained image, while in FIG. 16B the
• Organospheres have been stained with Trypan blue (arrows) to show that the dissociated cells in the Micro-Organospheres indicated that the cell remail viable (e.g., living) within the Micro- Organo sphere.
• FIGS. 17A-17E illustrates one example of a method of assaying a plurality of Patient- Derived Micro-Organospheres, in this example formed from a patient tumor biopsy, to determine a drug-response profile to multiple drug formulations.
• the illustrated procedure takes less than two weeks (e.g., approximately one week) from biopsy to results.
• FIG. 18 schematically illustrates an example of a method for treating a patient including the formation and use of a plurality of Patient-Derived Micro-Organospheres as part of the treatment procedure.
• FIG. 19 schematically illustrates an example of a method for treating a patient including multiple iterations of rapidly forming and assaying a plurality of Patient-Derived Micro-Organospheres as part of the treatment procedure.
• FIG. 20 schematically illustrates one variation of a portion of an apparatus for forming a plurality of Patient-Derived Micro-Organospheres as described herein.
• FIG. 21 schematically illustrates a method of operating an apparatus for forming a plurality of Patient-Derived Micro-Organospheres similar to that shown in FIG. 20.
• FIGS. 22A-22D illustrate one example of a validation of a methods of using a plurality of Patient-Derived Micro-Organospheres as described herein to identify drug resistance.
• FIG. 22A illustrates the use of traditional (“2D”) tumor cell assay methods, predicting drug resistance.
• FIG. 22B illustrates the use of one example of a Patient-Derived Micro- Organospheres method as described herein, to assay for drug resistance, predicting drug sensitivity.
• FIGS. 22C and 22D show that the Patient-Derived Micro-Organosphere based method accurately predicted the actual response of the tumor (drug responsive), unlike traditional cultured cells.
• FIGS. 23A-23D illustrates another example validating the use of Patient-Derived Micro-Organospheres as described herein to identify drug resistance, showing the predicted drug response to both Oxaliplatin and Irinotecan as consistent with actual tumor response following treatment with these drugs.
• FIG. 24 illustrates one example of a drug screen using the Patient-Derived Micro- Organospheres as described herein, in which a single tumor biopsy may generate a plurality of nearly-identical Micro-Organospheres in large quantities extremely fast (e.g., within less than two weeks) and be quickly tested against a large number of drug formulations (e.g., 27 are shown) in parallel.
• FIGS. 25A-25B illustrate examples of mouse liver Micro-Organospheres formed from a mouse liver tissue, having diameters of 300 pm, and 1 cell per Organosphere.
• FIG. 25A shows the Micro-Organo spheres at day 1
• FIG. 25B shows the Micro-Organospheres at day
• FIGS 26A-26B illustrate examples of mouse liver Micro-Organospheres formed from the partial hepatectomy mouse liver tissue, having diameters of 300 pm, and 25 cells per Organosphere similar to those shown in FIGS. 25A-25B .
• FIG. 26A shows the Micro- Organospheres at day 1
• FIG. 26B shows the Micro-Organospheres at day 10.
• FIGS. 27A-27C illustrate examples of human liver Micro-Organospheres formed from human liver tissue.
• FIG. 27 A shows the Micro-Organospheres at day 1, seeded with 40 cells/droplet.
• FIGS. 27B and FIG. 27C show the Micro-Organospheres at day 18.
• the Micro-Organospheres are hepatocyte-like structures, while FIG. 27C shows Cholangiocyte- like Micro-Organospheres.
• FIGS. 28A-28D show examples of Micro-Organospheres generated from a patient derived xenograft tumor line, having diameters of 300 pm, and 1 cell per Organosphere FIG.
• FIG. 28 A shows the Micro-Organospheres at day 1
• FIG. 28B shows the Micro-Organospheres at day 3
• FIG. 28C shows the Micro-Organospheres at day 5
• FIG. 28D shows the Micro- Organospheres at Day 7.
• FIGS. 29A-29D show examples of Micro-Organospheres generated from a patient- derived xenograft model, having diameters of 300 pm, and 5 cell per Organosphere.
• FIG. 29A shows the Micro-Organospheres at day 1
• FIG. 29B shows the Micro-Organospheres at day 3
• FIG. 29C shows the Micro-Organospheres at day 5
• FIG. 29D shows the Micro- Organospheres at Day 7.
• FIG. 30 is a graph comparing the responses of traditional organoids and Micro- Organospheres formed from a colorectal cancer patient-derived organoid to Oxalipalatin, showing a comparable response for the traditional organoids and Micro-Organospheres.
• FIG. 31 is a graph comparing the responses of traditional organoids and Micro- Organospheres formed from two colorectal cancer patient-derived xenograft models to SN38 (7- Ethyl-10-hydroxy-camptothecin), showing comparable responses.
• FIG. 32 is a graph comparing the responses of traditional organoids and Micro- Organospheres formed from a colorectal cancer patient-derived xenograft model to 5-FU (Fluorouracil), showing comparable responses.
• FIGS. 33 A and 33B show examples of toxicity assays using mouse liver Micro-
• FIG. 33A shows that the sizes of the tissue in the mouse liver Micro- Organospheres in the control group are relatively large (as indicated by the arrows). In contrast, in FIG. 33 A, showing the acetaminophen (10 mM) treatment group, the tissue in most of the Micro-Organospheres is smaller and contains many dead cells.
• FIGS. 34A and 34B show examples of toxicity assays using human liver Micro- Organospheres.
• FIG. 34A shows typical human liver Micro-Organospheres observed in the control group including tissue structures (indicated by the arrows).
• FIG. 34B shows Micro- Organospheres in an acetaminophen (10 mM) treatment group, showing atypical tissue structures (arrows) and debris.
• the Patient-Derived Micro-Organospheres described herein are typically spheres formed from dissociated primary cells distributed within the base material.
• These Patient- Derived Micro-Organospheres (“PMOSs” or“Organospheres”) may have a diameter of between about 50 pm and about 500 pm (e.g., between about 50 pm and about 400 pm, about 50 pm and about 300 pm, about 50 pm and about 250 pm, etc.), and may initially contain between about 1 and 1000 dissociated primary cells distributed within the base material (e.g., between about 1 and 750, between about 1 and 500, between about 1 and 400, between about 1 and 300, between about 1 and 200, between about 1 and 150, between about 1 and 100, between about 1 and 75, between about 1 and 50, between about 1 and 40, between about 1 and 30, between about 1 and 20, etc.).
• these Micro-Organosphere may be used immediately or cultured for a very brief period of time (e.g., 14 days or less, 10 days or less, 7 days or less, 5 days or less, etc.) and may allow the cells within the Micro-Organosphere to survive while maintaining much, if not all, of the characteristics of the tissue, including tumor tissue, from which they were extracted.
• the survival rate of the cells within the Micro- Organospheres is remarkably high, and the Micro-Organospheres may be cultured for days (or weeks) through multiple passages, in which the cells will divide, cluster and form structures similar to the parent tissue.
• the cells from the dissociated tissue within the Micro-Organosphere forms morphological structures inside even the smallest Micro-Organospheres; although in some applications, the presence of such structures is not necessary for the utility of these Micro-Organospheres (e.g., they may be used before substantial structural reorganization has occurred) in some variations they may be particularly useful.
• Micro- Organospheres may be used to create many (e.g., greater than 10,000) Patient-Derived Micro- Organospheres from a single biopsy. These Micro-Organospheres may be used screen for drug compositions that may predict what therapies may be effectively applied to the patient from whom the biopsy was taken. This may be useful, for example, in toxicity screen for drugs or other chemical compositions, from healthy normal tissue and/or from cancerous (e.g., tumor) tissue.
• the Patient-Derived Micro-Organospheres, methods and apparatuses for forming them and methods and apparatuses for testing them may be used for screening to identify one or more drug compositions that may effectively treat the patient (e.g., a cancer patient) prior to undergoing the drug therapy.
• This may allow, for example, very rapid screening of a cancer patient before they would otherwise undergo months of chemotherapy that may not be effective for them.
• Described herein are high-throughput drug screening methods (and apparatuses for performing these methods) using a single patient-specific biopsy (or other appropriate tissue/cell source).
• Described herein are droplet formed Patient-Derived Micro-Organospheres that may be formed from patient-derived tumor samples that have been dissociated and suspended in a basement matrix (e.g., MATRIGEL).
• the Micro-Organospheres can be patterned onto a microfluidic microwell array, to be incubated, and dosed with drug compounds. This miniaturized assay maximizes the use of tumor samples, and enables more drug compounds to be screened from a core biopsy at much lower cost per sample.
• PDMC patient-derived models of cancer
• PDXs patient-derived xenografts
• PDXs patient-derived xenografts
• large-scale drug screens of cell lines and organoids derived from cancer patients have been used to identify sensitivity to a large number of potential therapeutics.
• PDXs are also used to predict drug response and identify novel drug combinations.
• precision medicine strategies are in development through the exploration of these various PDMC models, there are substantial barriers to their effective use.
• PDO patient derived organoids
• studies have shown that phenotypic and genotypic profiling of organoids often show a high degree of similarity to the original patient tumors.
• PDO patient derived organoids
• it takes several months to develop and test drug sensitivity in organoids, which decreases the clinical applicability.
• the number of organoids obtained from a clinically relevant 18- gauge core biopsy is not sufficient to perform high throughput drug screen.
• an assay should be performed from a single core biopsy within 7-10 days.
• the Micro-Organospheres and methods of making and using them described herein may address these clinical limitations.
• an unpolymerized mixture is used herein to refer to a composition comprising biologically-relevant materials, including a dissociated tissue sample and a first fluid matrix material.
• the fluid matrix material is typically a material that may be polymerized to form a support or support network for the dissociated tissue (cells) dispersed within it. Once polymerized, the polymerized material may form a hydrogel and may be formed or and/or may include proteins forming the biocompatible medium, in addition to the cells.
• a suitable biocompatible medium for use in accordance with the presently-disclosed subject matter can typically be formed from any biocompatible material that is a gel, a semi- solid, or a liquid, such as a low-viscosity liquid, at room temperature (e.g., 25° C.) and can be used as a three- dimensional substrate for cells, tissues, proteins, and other biological materials of interest.
• a biocompatible material that is a gel, a semi- solid, or a liquid, such as a low-viscosity liquid, at room temperature (e.g., 25° C.) and can be used as a three- dimensional substrate for cells, tissues, proteins, and other biological materials of interest.
• Exemplary materials that can be used to form a biocompatible medium in accordance with the presently-disclosed subject matter include, but are not limited to, polymers and hydrogels comprising collagen, fibrin, chitosan, MATRIGELTM (BD Biosciences, San Jose, Calif.), polyethylene glycol, dextrans including chemically crosslinkable or photo-crosslinkable dextrans, and the like, as well as electrospun biological, synthetic, or biological- synthetic blends.
• the biocompatible medium is comprised of a hydrogel.
• hydrogel is used herein to refer to two- or multi-component gels comprising a three-dimensional network of polymer chains, where water acts as the dispersion medium and fills the space between the polymer chains.
• Hydrogels used in accordance with the presently-disclosed subject matter are generally chosen for a particular application based on the intended use of the structure, taking into account the parameters that are to be used to form the Micro-Organospheres, as well as the effect the selected hydrogel will have on the behavior and activity of the biological materials (e.g., cells) incorporated into the biological suspensions that are to be placed in the structure.
• Exemplary hydrogels of the presently-disclosed subject matter can be comprised of polymeric materials including, but not limited to: alginate, collagen
• the hydrogel is comprised of a material selected from the group consisting of agarose, alginate, collagen type I, a polyoxyethylene-polyoxypropylene block copolymer (e.g., Pluronic® F127 (BASF Corporation, Mount Olive, N.J.)), silicone, polysaccharide, polyethylene glycol, and polyurethane.
• the hydrogel is comprised of alginate.
• the Micro-Organospheres described herein may also include biologically-relevant materials.
• biologically-relevant materials may describe materials that are capable of being included in a biocompatible medium as defined herein and subsequently interacting with and/or influencing biological systems.
• the biologically-relevant materials are magnetic beads (i.e., beads that are magnetic themselves or that contain a material that responds to a magnetic field, such as iron particles) that can be combined as part of the unpolymerized material to produce Micro-Organosphere that can be used in the methods and compositions (e.g., for the separation and purification of Micro- Organospheres).
• the biologically-relevant materials may include additional cells, in addition to the dissociated tissue sample (e.g., biopsy) material.
• the dissociated tissue sample and the additional biologically relevant material in a uniform mixture or as a distributed mixture (e.g., on just one half or other portion of the Micro-Organosphere, including just in the core or just in the outer region of the formed Micro-Organosphere).
• the additional biologically- relevant material within the unpolymerized material may be suspended with the dissociated tissue sample in suspension, e.g., prior to polymerization of the droplet forming the Micro- Organosphere.
• the biologically relevant material that may be included with the dissociated tissue sample (e.g., biopsy) material may contain a number of cell types, including preadipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells, and adipose tissue macrophages, as well as small blood vessels or microvascular fragments found within the stromal vascular fraction.
• these tissues may be any appropriate tissue from a patient, typically taken by biopsy.
• these tissues and the resulting dissociated cells
• Tissues may be from a healthy tissue biopsy or from cancerous (e.g., tumor) cell biopsy.
• the dissociated cells may be incorporated into a Micro-Organosphere of the presently-disclosed subject matter, based on the intended use of that Micro-Organosphere.
• relevant tissues may typically include cells that are commonly found in that tissue or organ (or tumor, etc.).
• exemplary relevant cells that can be incorporated into Micro-Organosphere of the presently-disclosed subject matter include neurons, cardiomyocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, biliary epithelial cells, and the like. These types of tissues may be dissociated by conventional techniques known in the art.
• Suitable biopsied tissue can be derived from: bone marrow, skin, cartilage, tendon, bone, muscle (including cardiac muscle), blood vessels, corneal, neural, brain, gastrointestinal, renal, liver, pancreatic (including islet cells), lung, pituitary, thyroid, adrenal, lymphatic, salivary, ovarian, testicular, cervical, bladder, endometrial, prostate, vulval and esophageal tissue.
• Normal or diseased (e.g., cancerous) tissue may be used.
• the tissue may arise from tumor tissue, including tumors originating in any of these normal tissues.
• Micro-Organospheres may be cryopreserved and/or cultured.
• Cultured Micro-Organospheres may be maintained in suspension, either static (e.g., in a well, vial, etc.) or in motion (e.g., rolling or agitated).
• the Micro-Organosphere may be cultured using known culturing techniques. Exemplary techniques can be found in, among other places;
• the Micro-Organospheres are formed by forming a droplet of the unpolymerized mixture (e.g., in some variations a chilled mixture) of a dissociated tissue sample and a fluid matrix material in an immiscible material, such as a fluid hydrophobic material (e.g., oil).
• a Micro-Organosphere may be formed by combining a stream of
• the density of the cells present in the droplet may be determined by the dilution of the dissociated material (e.g., cells) in the unpolymerized material.
• the size of the Micro- Organosphere may correlate to the size of the droplet formed. In general, the Micro- Organosphere is a spherical structure having a stable geometry.
• a drug composition may include any drug, drug dilution, drug formulation, compositions including multiple drugs (e.g., multiple active ingredients), drug formulations, drug forms, drug concentrations, combination therapies, and the like.
• a drug formulation refers to a formulation comprising a mixture of a drug and one or more inactive ingredients.
• the term“passaged” may refer to the average number of doublings of the cells within the Micro-Organospheres.
• traditional passage number refers to the transfer or subculture of cells from one culture vessel to another, the cells within a Micro- Organospheres may be stably retained within the same Micro-Organospheres, and may continue to grow and divide.
• the passage number as used herein typically refers to the average number of doublings undergone by the dissociated cells from the biopsied tissue within the Micro-Organospheres.
• the population doubling number is the approximate number of doublings that the cell population has undergone since isolation (e.g., since forming of the Micro- Organospheres from the freshly dissociated biopsy tissue).
• the Micro-Organospheres described herein may be cultured for a short period of time relative to the growth, e.g., doublings, of some or all of cells within the Micro-Organospheres (e.g., fewer than 10 passages, fewer than 9 passages, fewer than 8 passages, fewer than 7 passages, fewer than 6 passages, fewer than 5 passages, fewer than 4 passages, fewer than 3 passages, etc.).
• some or all of cells within the Micro-Organospheres e.g., fewer than 10 passages, fewer than 9 passages, fewer than 8 passages, fewer than 7 passages, fewer than 6 passages, fewer than 5 passages, fewer than 4 passages, fewer than 3 passages, etc.
• the cells from the dissociated, biopsied tissue in the Micro- Organospheres can aggregate, cluster or assemble within the Micro-Organospheres. Aggregates of cells may be highly organized, and may form defined morphology or may be a mass of cells that have clustered or adhered together. The organization may reflecting the tissue of origin. Although in some variations the Micro-Organospheres may contain a single cell type
• Micro-Organospheres may contain more than one cell type (heterotypic).
• the tissue used to form the Patient-Derived Micro- Organospheres may be derived from a normal or healthy biological tissue, or from a biological tissue afflicted with a disease or illness, such as a tissue or fluid derived from a tumor.
• the tissue used in the Micro-Organospheres may include cells of the immune system, such as T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages and dendritic cells.
• the cells may be stem cells, progenitor cells or somatic cells.
• the tissue may be mammalian cells such as human cells or cells from animals such as mice, rats, rabbits, and the like.
• these tissue may generally be taken from a biopsy to form the Micro-Organospheres.
• the tissue may be derived from any of a biopsy, a surgical specimen, an aspiration, a drainage, or a cell-containing fluid.
• Suitable cell-containing fluids include any of blood, lymph, sebaceous fluid, urine, cerebrospinal fluid or peritoneal fluid.
• ovarian or colon cancer cells may be isolated from peritoneal fluid.
• cervical cancer cells may be taken from the cervix, for example by large excision of the transformation zone or by cone biopsy.
• Micro-Organospheres will contain multiple cell types that are resident in the tissue or fluid of origin.
• the cells may be obtained directly from the subject without intermediate steps of subculture, or they may first undergo an intermediate culturing step to produce a primary culture.
• Methods for harvesting cells from biological tissue and/or cell containing fluids are well known in the art. For example, techniques used to obtain cells from biological tissue include those described by R. Mahesparan (Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro. Acta Neuropathol (1999) 97:231-239).
• the cells are first dissociated or separated from each other before forming the Micro-Organospheres.
• Dissociation of cells may be accomplished by any conventional means known in the art.
• the cells are treated mechanically and/or chemically, such as by treatment with enzymes.
• mechanically we include the meaning of disrupting connections between associated cells, for example, using a scalpel or scissors or by using a machine such as an homogenizer.
• enzymes we include the meaning of treating the cells with one or more enzymes disrupt connections between associated cells, including for example any of collagenase, dispases, DNAse and/or hyaluronidase.
• One or more enzymes may be used under different reaction conditions, such as incubation at 37° C. in a water bath or at room temperature.
• the dissociated tissue may be treated to remove dead and/or dying cells and/or cell debris.
• the removal of such dead and/or dying cells may be accomplished by any conventional means known to those skilled in the art, for example using beads and/or antibody methods. It is known, for example, that phosphatidylserine is redistributed from the inner to outer plasma membrane leaflet in apoptotic or dead cells.
• the use of Annexin V-Biotin binding followed by binding of the biotin to streptavidin magnetic beads enables the separation of apoptotic cells from living cells.
• removal of cell debris may 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.
• the carrier material may be a material that has a viscosity level that delays sedimentation of cells in a cell suspension prior to polymerization and formation of the Micro- Organospheres.
• a carrier material may have sufficient viscosity to allow the dissociated biopsy tissue cells to remain suspended in the suspension until polymerization.
• the viscosity required to achieve this can be optimized by the skilled person by monitoring the sedimentation rate at various viscosities and selecting a viscosity that gives an appropriate sedimentation rate for the expected time delay between loading the cell suspension into the apparatus forming the Micro- Organospheres forming the Micro-Organospheres by polymerizing the droplets of the
• unpolymerized material including the cells.
• the unpolymerized material may be flowed or agitated by the apparatus even where lower viscosity materials are used, in order to keep the cells in suspension and/or distributed as desired.
• the unpolymerized mixture including the dissociated tissue sample and the fluid matrix material may include one or more components, e.g., biologically-relevant materials.
• a biologically-relevant material that may be included may include any of: an extracellular matrix protein (e.g. fibronectin), a drug (e.g. small molecules), a peptide, or an antibody (e.g., to modulate any of cell survival, proliferation or differentiation); and/or an inhibitor of a particular cellular function.
• Such biologically-relevant materials may be used, for example, to increase cell viability by reducing cell death and/or activation of cell growth/replication or to otherwise mimic the in vivo environment.
• the biologically-relevant materials may include or may mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids and hormones.
• the biologically-relevant materials may supplement one or more agents in the fluid matrix material.
• the fluid matrix material is a synthetic gel (hydrogel) and may be supplemented by one or more biologically-relevant materials.
• the fluid matrix is a natural gel.
• the gel may be comprised of one or more extracellular matrix components such as any of collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose and chitosan.
• the matrix material may be a gel that comprises collagen type 1 such as collagen type 1 obtained from rat tails.
• the gel may be a pure collagen type 1 gel or may be one that contains collagen type 1 in addition to other components, such as other extracellular matrix proteins.
• a synthetic gel may refer to a gel that does not naturally occur in nature. Examples of synthetic gels include gels derived from any of polyethylene glycol (PEG), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), poly ethylene oxide (PEO).
• FIGS. 1A-1C, 2A- 2C, 3A-3C and 4A-4E Examples of Patient-Derived Micro-Organospheres are shown in FIGS. 1A-1C, 2A- 2C, 3A-3C and 4A-4E.
• FIGS. 1A-1C illustrate Micro-Organospheres formed having a single cell per Micro-Organosphere. As shown, the Micro-Organospheres are all approximately the same size, e.g., approximately 300 pm diameter.
• FIG. IB shows Micro- Organospheres formed at the same time after 3 days in culture. The cells have expanded in size, in some cases doubling and/or growing. By seven days in culture, as shown in FIG. 1C, the cells have doubled multiple times, showing clusters or masses of cells.
• FIGS. 2A-2C and 3A-3C show Micro-Organospheres formed from five cells per Micro-Organosphere or 20 cells per Micro-Organospheres, respectively.
• FIGS. 4A-4E the Micro-Organospheres are shown immediately after formation, and cultured for five days, in which nearly-identical Micro-Organospheres (e.g., having the same diameter) each include 10 cells per Micro-Organosphere.
• the Micro-Organospheres are shown immediately after forming, still surrounded by the immiscible fluid, in this case, oil, at day 0. The Micro-Organospheres are removed from the immiscible fluid and washed, and cultured for five days.
• FIG. 4B shows the Micro-Organospheres after 2 days
• FIG. 4C shows the Micro-Organospheres after 3 days
• FIGS. 4D and 4E show the Micro-Organospheres at 4 and 5 days, respectively.
• FIGS. 4A-4E show that the dissociated tissue (cells) from the biopsy within the Micro-Organospheres are viable and growing within nearly all of the Micro- Organospheres at comparable rates.
• Micro- Organospheres may be formed in large amounts from even a single, average-sized biopsy and may result in many hundreds or thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000 or more) of Micro-Organospheres that include a significant number of viable cells, allowing multiple rapid assays to be performed in parallel.
• FIG. 5A and 5B illustrate an example of Micro-Organospheres formed as described herein from a dissociated biopsy of mouse liver, e.g., showing mouse hepatocytes distributed within a polymerized fluid matrix material (in this example, MATRIGEL).
• Each Micro- Organospheres includes the polymerized matrix material 503 formed into a sphere having a diameter, e.g., of about 300 pm, in which a set number of hepatocytes 507 are dispersed.
• the Micro-Organospheres are shown one day after biopsying, dissociation and forming of the Micro-Organospheres.
• These Micro-Organospheres 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 FIG. 5B.
• the Micro-Organospheres may generally include the dissociated, e.g. biopsy, tissue (e.g., cells) in a fixed or known number of cells and/or concentration (cells/ml or cells/mm 3 ) within the Micro-Organospheres.
• this matrix material may be natural polymers, such as one or more of: alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, elastin; or a synthetic polymer, such as one or more of: polyethylene glycol (PEG) and polyacrylamide. Both organic and inorganic synthetic polymers may be used.
• the number of cells initially included in the Micro-Organospheres may be selected from between 1 cell up to several hundred.
• assays e.g., drug toxicity assays
• the number of cells per Micro-Organosphere may be set or selected by the user.
• the apparatus will include one or more controls to set the number of cells from the primary tissue to include in each Micro- Organosphere. The number of cells may be chosen or set based on how the user intends to use the Micro-Organospheres.
• Micro-Organospheres having very low number of cells may be particularly suitable for studying clonal diversity (e.g., for tumor heterogeneity). Since each Micro- Organosphere grows from a single cell, we can observe which clones are drug resistant and these specific Micro-Organospheres may be examined (e.g., by genomic sequencing) to determine the genomic (mutation) diversity related to the particular clone.
• a low to moderate number of cells per Micro-Organosphere may be particularly useful for rapid drug testing, including toxicity testing as these Micro-Organospheres typically grow quickly.
• a larger number of cells per Micro-Organosphere e.g., between about 20-100 cells, e.g., 30-100 cells, 40-100 cells, greater than 50 cells, etc.
• the Micro-Organosphere may contain different lineages, potentially including epithelial (or cancer, etc.) and mesenchymal (or stromal, immune, blood vessel, etc.) cells.
• the Micro-Organospheres may be formed in any appropriate size, which may be matched to the number of cells to be included.
• the size may be as small as about 20 pm, up to 500 pm in diameter (e.g., 50 or 100 pm on average, e.g., between about 100-200 pm, etc.).
• the size is about 300 pm in which between about 10-50 cells (e.g., between about 10-30 cells) are included in each Micro-Organosphere.
• the number of cells and the size may be varied and/or may be controlled.
• the number of cells and/or the size of the Micro-Organospheres may be set by one or more controls on the apparatus forming the Micro-Organospheres.
• the size of the Micro-Organospheres and/or the density of cells within the Micro-Organospheres may be adjusted by adjusting the flow rates and/or the concentration of the dissociated tissue sample (e.g., the cells from a biopsy).
• the dissociated tissue sample e.g., the cells from a biopsy.
• the Micro-Organospheres described herein allow for viable and healthy cells through the entire volume of the Micro-Organosphere.
• the size of the Micro-Organospheres and/or the number of cells to be included in the Micro- Organospheres may be selected based on how the Micro-Organospheres are expected or intended to be used. For example, in variations in which the Micro-Organospheres is to be used to examine relationships between cells of the biopsied material the Micro-Organospheres may be formed having multiple cells and may be cultured for extended periods of time (e.g., up to one week or more).
• the Patient-Derived Micro-Organospheres described herein may be made by combining a dissociated tissue sample, e.g., a biopsy sample, with a fluid matrix that may be polymerized in a controlled manner to form the Micro-Organospheres.
• FIG. 6 illustrates one method of forming Patient-Derived Micro-Organospheres.
• the method may include taking the sample from a patient, such as taking a biopsy from a patient tissue 601.
• the biopsy may be taken, e.g., using a biopsy needle or punch.
• the biopsy may be taken with a 14-gauge, a 16-gauge, an 18-gauge, etc.
• the tissue may be processed to dissociate the material, either mechanically and/or chemically.
• the dissociated cells may be immediately used to form the Patient-Derived Micro-Organospheres, as described; in some variations, all or some of the cells may be modified, such as by genetically modifying the cells 603, for example, by transfection, electroporation, etc.
• the dissociated tissue sample from the biopsy material may be combined with the fluid (e.g., liquid) matrix material to form the unpolymerized mixture 605.
• This unpolymerized mixture may be held in an unpolymerized state, so that the cells from the dissociated tissue may remain suspended within the mixture.
• the cell may remain suspected and unpolymerized by keeping them chilled, e.g., at room temperature of below (e.g., between 1-25 degrees C).
• the unpolymerized mixture may then be dispensed as droplets, e.g., into an immiscible material, such as an oil, in a manner that controls the formation of the size of the droplets and therefore the size of the Patient-Derived Micro-Organospheres formed 607.
• an immiscible material such as an oil
• uniformly-sized droplets may be formed by combining a stream of the unpolymerized material into one or more (e.g., two converging) streams of the immiscible material (e.g., oil) so that the flow rates and/or pressures of the two streams may determine how droplets of the unpolymerized material are formed as they intersect the immiscible material.
• the droplets may be polymerized 609 to form the Patient-Derived Micro-Organospheres (PMOSs) in the immiscible material.
• the immiscible material may be heated or warmed to a temperature that causes the unpolymerized mixture (e.g., the fluid matrix material in the unpolymerized material) to polymerize.
• the Patient-Derived Micro-Organospheres may be separated from the immiscible fluid, e.g., the PMOSs may be washed to remove the immiscible fluid 611, and placed in a culture media to allow the cells within the Patient-Derived Micro-Organospheres to grow.
• the Patient-Derived Micro-Organospheres may be cultured for any desired time, or may be cryopreserved and/or assayed immediately.
• the Patient-Derived Micro-Organospheres may be cultured for a brief period of time (e.g., for between 1-3 days, between 1-4 days, between 1-5 days, between 1-6 days, between 1-7 days, between 1-8 days, between 1-9 days, between 1-10 days, between 1-11 days, between 1-14 days etc.).
• This may allow the cells derived from the dissociated biopsy tissue to grow and/or divide (e.g., double) for up to five or six passages.
• the cells may be either or both cryopreserved 615 and/or assayed 617. Examples of assays that may be used are also described herein.
• the Micro-Organospheres may be recovered from the immiscible fluid (e.g., oil) after polymerization.
• the Micro-Organospheres may be recovered by demulsficiation and/or de- emulsification, for example, by forming emulsified droplets and recovering the Micro- Organospheres after the droplets are formed to remove any oil (and other contaminants). This may allow the cells to grow within the polymerized droplet (the Micro-Organosphere) without being inhibited by the immiscible fluid.
• the methods and apparatuses described herein illustrate methods of forming the plurality of droplets, and thus the plurality of Micro-Organospheres, by streaming the unpolymerized mixture into one or more streams of immiscible fluid (such as an oil or other hydrophobic material), ins some variations the droplets may be formed by other methods that may allow for the size of the droplet to be controlled as described herein. For example in some variations the droplets may be formed by printing (e.g., by printing droplets onto a surface). This may reduce or eliminate the need for an additional recovery step of emulsification/de
• the droplets may be printed onto a surface, such as a flat or shaped surface, and polymerized.
• the droplets may be dispensed using pressure, sound, charge, etc.
• the droplets may be formed using an automatic dispenser (e.g., pipetting device) adapted to release the small amount of the unpolymerized mixture onto a surface, into the air, and/or into a liquid medium (including an immiscible fluid).
• the method for forming the Patient-Derived Micro-Organospheres may be automated, or performing using one or more apparatuses.
• the method of forming the Patient-Derived Micro-Organospheres may be performed by an apparatus that allows the selection and/or control of the size of the Patient-Derived Micro-Organospheres (and therefore the density of the number of cells).
• FIG. 7A illustrates one example of an apparatus 700 for forming Patient-Derived Micro-Organospheres as described.
• the apparatus typically includes an input for inputting either the unpolymerized mixture of the dissociated tissue sample and a fluid matrix material (already combined) or may separately receive the dissociated tissue sample, e.g., in a holding solution, and a fluid matrix material.
• the apparatus include a holding chamber 706 for holding the unpolymerized mixture, and/or holding chambers (not shown) for holding the dissociated tissue (e.g., biopsy) sample and holding the fluid matrix material. Any or all of these holding chambers may be pressurized to control and/or speed up fluid flow out of the chambers and into the device.
• the apparatus may either receive the unpolymerized mixture or it may receive the components and mix it. In some variations the apparatus may control the
• the apparatus may include a sensor (e.g., an optical reader) for reading the density (e.g., the optical density) of the cells in the unpolymerized mixture (not shown).
• the sensor may also be coupled to the controller 724, which may automatically or semi-automatically (e.g., by indicating to a user) control the dilution of the cells in the unpolymerized mixture.
• the apparatuses may also include a port for receiving the unpolymerized mixture.
• the port may include a valve or may be coupled to a valve and the valve may be controlled by the controller 724 (or a separate controller).
• the apparatus 700 may include a chamber 708 and/or port for holding and/or receiving the immiscible fluid.
• the immiscible fluid may be held in a pressurized chamber so that the flow rate may be controlled. Any of the pressurized chambers may be controlled by the controller 724 which may use one or more pumps 726 to control the pressure and therefore the flow through the apparatus.
• One or more pressure and/or flow sensors may be included in the system to monitor the flow through the device.
• the entire apparatus 700 may be enclosed in a housing 702 or a portion of the apparatus 704 may be enclosed in a housing.
• the housing may include one or more openings or access portions on the device, e.g., for adding the immiscible fluid and/or the unpolymerized mixture.
• any of these apparatuses 700 may also include one or more sensors 728 for monitoring all or key portions of the manufacturing process.
• the sensors may include optical sensors, mechanical sensors, voltage and/or resistance (or capacitance, or inductance) sensors, force sensors, etc. These sensors may be used to monitor the ongoing operation of the assembly, including the formation of the Patient-Derived Micro- Organospheres.
• the apparatus 700 may also include one or more thermal/temperature regulators 718 for controlling the temperatures of either or both the immiscible fluid and/or the
• any of these apparatuses may also include one or more droplet forming assemblies 720 that may be monitored (e.g., using one or more sensors) as will be illustrated below in FIGS. 7C and 9.
• the droplet Micro-Organosphere forming assembly may include (or may be coupled with, a dispenser (e.g., a PMOS dispenser) 722.
• the dispenser may dispense, for example, in to a multi- well plate 716.
• the droplet Micro-Organosphere forming assembly 720 may include one or more microfluidic chips 730 or structures that forms and controls the streams of the
• FIG. 7B illustrates one example of a microfluidic chip for forming Patient-Derived Micro-Organospheres 730.
• the chip 730 includes a pair of parallel structures for forming Micro-Organospheres.
• FIG. 7C illustrates the droplet-forming region of the microfluidic chip for forming PMOSs, including an
• unpolymerized channel outlet 741 that opens (in this example, as a right angle) a“+” junction or region of intersection 737 to the channel outlet 741 and the immiscible fluid outlet(s), 743, 743’.
• the input from the immiscible fluid channel(s) may be at an angle relative to the angle (and point of intersection) with the unpolymerized material.
• FIG. 7C as in all figures in this description showing dimensions, the dimensions shown are exemplary only, and are not intended to be limiting, unless they otherwise specify.
• the microfluidics chip 730 include an inlet (input port) 733 for the immiscible fluid into the chip (e.g., from the inlet port or storage chamber shown in FIG 7A).
• a second inlet port 735 into the chip may be configured to receive the unpolymerized material and transport it down a semi-tortious path to the junction region.
• the inlet port for the immiscible fluid may be securely coupled to the outlet from the immiscible fluid chamber or inlet, described above.
• the inlet port 735 for the unpolymerized material into the chip may be coupled through a delivery pathway 741 connecting the inlet 275 to the junction region (as shown in FIG. 7C.
• the inlet 733 for the immiscible fluid may connect to two (or more) connecting paths 743, 743’ to the junction region 737.
• a channel leaving the junction region 737 may pass the formed Micro-Organospheres (in the immiscible fluid) down the channel to an outlet 731 that may connect to a dispenser (not show) for dispensing from the Micro-Organospheres into one or more chambers, e.g., for culture and/or assaying.
• FIGS. 7B and 7C the formed droplets, which may become Micro-Organospheres once polymerized, may be transmitted down a long, temperature controlled microfluidics environment, prior to being dispensed from the apparatus (not shown).
• FIG. 8 illustrates one example of a channel region 839 (e.g., element 739 in FIG. 7B) that is shown transparent, containing a plurality of Micro-Organospheres 803 each containing a predetermined number of cell 805.
• the junction region 937 is shaped as described above, so that the channel carrying the unpolymerized mixture 911 intersects one or more (e.g., two) channels 909 carrying a fluid, such as an oil, that is immiscible with the unpolymerized mixture.
• a fluid such as an oil
• the unpolymerized mixture is pressurized to flow at first rate out of the first channel 911, the flowing immiscible fluid in the intersecting channels, 909, 909’, permit a predefined amount of the unpolymerized mixture to pass before pinching it off to form a droplet 903 that is passed into the outlet channel 939.
• a minced (e.g., dissociated) clinical (e.g., biopsy or resected) sample of tissue such as ⁇ 1 mm in diameter
• a temperature-sensitive gel i.e. MATRIGEL, at 4 degrees C
• This unpolymerized mixture may be placed into the microfluidic device that may generates droplets (e.g., water-in-oil droplets) that are uniform in volume and material composition.
• the dissociated tumor cells may be partitioned into these droplets.
• the gel in the unpolymerized material may solidify upon heating (e.g., at 37 degrees C), and the resulting Patient-Derived Micro- Organospheres may be formed.
• this method may be used to produce over 10,000 (e.g., over 20,000, over 30,000, over 40,000, over 50,000, over 60,000, over 70,000, over 80,000, over 90,000, over 100,000) uniform droplets (Patient-Derived Micro-Organospheres) from the tissue (e.g., biopsy material).
• These Patient-Derived Micro-Organospheres are compatible with traditional 3D cell culture techniques.
• FIG. 10 illustrates a plurality of Patient- Derived Micro-Organospheres 1005 formed as described above, suspended in the immiscible material 1008 (e.g., oil).
• the junction is shown as a T- or X-junction in which the flow focusing of the microfluidics forms the controllable size of the Micro-Organospheres.
• the droplets may be formed by robotic micro-pipetting, e.g., into an immiscible fluid and/or onto a solid or gel substrate.
• the droplets of unpolymerized material may be formed in the requisite dimensions and reproducibility by micro -capillary generation.
• Micro-Organospheres in the specified size range and reproducibility from the unpolymerized material may include colloid manipulation, e.g., via external forces such as acoustics, magnetics, inertial, electro wetting, or gravitational.
• FIGS. 11A and 11B shows examples of Patient-Derived Micro-Organospheres in oil formed as described above.
• the cells within these Patient-Derived Micro-Organospheres, derived from a single biopsy sample, are viable, as seen by vital dye staining, as shown in FIGS. 15A-15B and 16A-16B.
• FIG. 12A-12B illustrates Micro-Organospheres having tumor cells (similar to those shown in FIG. 1 lA-1 IB) that may be washed to remove the immiscible material (e.g., oil). This immiscible material may be removed relatively quickly after forming the Micro-Organospheres in order to prevent harm to the cells within the Micro- Organo sphere.
• immiscible material e.g., oil
• the gel droplets are recovered from the oil phase and resuspended, e.g., into PBS via PFO (perfluoro octanol) and centrifugation. This may separate the immiscible fluid from the Micro-Organospheres.
• PFO perfluoro octanol
• these Micro-Organospheres, including tumor-based Micro-Organospheres can be successfully grown, as shown in FIGS. 1A-1C, 2A-2C, 3A-3C and 4A-4E, above, and in FIG. 13. This is an important improvement, as drug screening has to be performed on viable and growing primary tumor cells that retain their properties from patient tumors to predict patient outcomes.
• the channels may be coated.
• the channel of the microfluidic device may be coated with a hydrophobic material.
• the Micro-Organospheres described herein are highly uniform in diameter, and may have a very low size, e.g., diameter, variance. This is illustrated, for example, in FIG.
• FIGS. 15A-15B shows Micro-Organospheres formed as described herein; in FIG. 16A-16B, these Micro-Organospheres have been stained with Trypan blue (arrowheads), showing that they are alive.
• the Micro-Organospheres formed as droplets in this manner may contain growth-factors and matrix to mimic the biological environment from which the tissue arose.
• Patient samples e.g., biopsy samples
• Micro- Organospheres including hundreds, thousands, or tens of thousands of Micro-Organospheres
• the Micro-Organospheres may be cultured for any desired period of time, and typically show proliferation and growth in as few as 3-4 days. They may be maintained and passaged for months. As will be described in greater detail below, they may be used to screen thousands of drug compositions within as few as 4-6 days from taking the tissue (e.g., biopsy).
• the Micro-Organospheres described herein may, at any point after they are formed, be banked, e.g., by cryopreserving them.
• Tumor Micro-Organospheres may be collect from many different patients and may be used individual or collectively to screen multiple drug formulations to determine toxicity and/or efficacy.
• Non-tumorous cells healthy tissue
• Non-tumorous cells healthy tissue
• these methods and apparatuses may allow for high throughput screening.
• the Micro-Organospheres may be formed and allowed to passage twice (e.g., two doublings), and cryopreserved.
• Micro-Organospheres may be used to form these same Micro-Organospheres to generate hundreds, thousands, or tens of thousands of Micro-Organospheres that may be used for assaying drug effects, drug response, biomarkers, proteoimic signals, genomic signals, etc.
• the Micro-Organospheres described herein permit tissue extract/biopsy originated cells to grow exceptionally well and provide more representative data, especially as compared to organoids or spheroids. Without being bound by a particular theory, this may be because the cells may have a more constrained cell density in the Micro-Organospheres, permitting cells to communicate without inhibiting each other while sharing signals.
• the Micro- Organospheres also have a very large surface to volume ratio, more readily permitting transmission of growth factors and other signals to penetrate into the Micro-Organospheres (e.g., the Micro-Organospheres are less diffusion limited).
• the Patient-Derived Micro-Organospheres described herein may be used in a variety of different assays, and in particular may be used to determine drug formulation effects, including toxicity, on normal and/or abnormal (e.g., cancerous) tissue.
• drug screening may include applying Micro-Organospheres into all or some wells of multi-well (e.g., a 96-well) plate. Alternatively custom plates may be used (e.g., a 10,000 micro-well array may be formed of a 100 x 100 wells).
• the Micro-Organospheres e.g., gel droplets
• the Micro-Organospheres may be cultured over the course of 3-5 days.
• the wells e.g., micro-reactors
• the wells may then be dosed with drug compounds, e.g., based on a set of FDA-approved anticancer drugs, to examine the effects of the drug panel.
• drug compounds e.g., based on a set of FDA-approved anticancer drugs
• the drugs texted may be based on the National Cancer Institute (Division of Cancer Treatment and Diagnosis (11)) screen, consisting of 147 agents intended to enable cancer research, drug discovery and combination drug studies.
• the Micro-Organospheres may be imaged via standard fluorescent microscopy and ranked based on drug response.
• FIGS. 17A-17E An example of this assaying technique is shown in FIGS. 17A-17E.
• the screening assay may be automated. This may enable repeatable and automated workflow, which may increase the number of drugs screened from a few to hundreds.
• FIGS. 17A-17E illustrate one example of this workflow.
• a tumor biopsy is taken and a plurality (e.g., >10,000) Micro-Organospheres are formed as described above (in FIG. 17A the junction region forming the Micro-Organospheres is illustrated). Thereafter, the Micro-Organospheres may be recovered and washed (e.g., to remove the immiscible (e.g., oil) material in which they were formed. The Micro-Organospheres may then be plated into one or more microwell plates. As shown in FIG.
• the Micro-Organospheres may be cultured for one or more generations (e.g., one or more passages. This is shown occurring from day 0 to days 3, 4 or 5. Thereafter, the Micro-Organospheres may be screened, as shown in FIG. 17D, e.g., by applying drugs to a subset of the replicant wells. Thereafter, as shown in FIG. 17E, on day 7, the cells in the Micro-Organospheres may be imaged and/or automatically or manually scored to identify drug effects (e.g., drug screening and growth profiling).
• generations e.g., one or more passages. This is shown occurring from day 0 to days 3, 4 or 5.
• the Micro-Organospheres may be screened, as shown in FIG. 17D, e.g., by applying drugs to a subset of the replicant wells.
• FIG. 17E on day 7, the cells in the Micro-Organospheres may be imaged and/or automatically or manually scored to identify drug effects (e.
• the workflow shown in FIGS. 17A-17E may enable an integrated device to be used for growing, dosing and/or reviewing the Micro-Organospheres.
• freshly biopsied or resected patient tumor samples may be disassociated and seeded into gel with regents to form the Micro-Organospheres (as described above).
• a portion of the Micro- Organospheres formed may be cryopreserved. The rest may be recovered and incubated until seeded into microwell plates for drug testing or screening as just described.
• Growth and viability assays may be performed on the Micro-Organospheres, which may be imaged and tracked. Their response to drug treatments, such as IC-50, cytotoxicity, and growth curves, may be measured to identify effective therapeutics against the patient’s tumor.
• the methods and apparatuses described herein have numerous advantages, including reproducibility.
• the sample preparation process may be automated by the microfluidic sample partitioning which may reduce the need for specialized personnel for diagnostic testing and manual pipetting. This may be particularly helpful in a clinical setting. Moreover, this may enable uniformity among signal droplets, increasing assay sensitivity. In addition, these assays may minimize the time required to generate Micro-Organospheres. Based on preliminary data, these methods may be able to generate a library of over 100,000 MATRIGEL-tumor droplets (Micro-Organospheres) in less than about 15 minutes. These methods are also highly scalable, and can be multiplexed to run multiple patient biopsies in parallel.
• droplet-based microfluidics is generally compatible with a wide range of hydrogel materials such as agarose, alginate, PEG, and hyaluronic acid).
• hydrogel materials such as agarose, alginate, PEG, and hyaluronic acid.
• the starting gel composition can easily be modified to accompany and encourage Micro-Organospheres growth.
• the droplet-size can be adjusted by modifying the size of our microfluidic device. Together, these allow a large selection of gel material composition and micro-reactor sizes.
• the miniaturized assays described here may maximizes the patient tumor biopsy, enabling more drug compounds to be screened.
• a 600 uL tumor sample can be partitioned into ⁇ 143,000 individual micro-reactors that are ⁇ 4 nL in volume.
• By maximizing the tissue sample multiple experimental replicates may be examined, increasing statistical power.
• These techniques may allow the inspection of intra-tumor heterogeneity, drug perturbation and identify rare cellular events, such as drug resistance.
• the Micro-Organospheres may generally be compatible with downstream assays including single cell RNA transcriptome analysis and epigenetic profiling.
• a portion of the Micro- Organospheres may be banked (e.g., by cryopreservation for biobanking) for future novel drug assays and/or for confirmation analysis, including genetic screening.
• FIGS. 18 and 19 illustrate example of therapeutic methods that use the methods and apparatuses, including the Micro-Organospheres, described herein.
• these methods and apparatuses can be used as a clinical indicator for appropriate drug selection to improve clinical outcome and drug response.
• a patient diagnosed with metastatic cancer will take a biopsy for histopathology and for screening of a plurality of Micro-Organospheres formed from a biopsy as described herein. Within 7-10 days, the screening may be performed from the biopsy to identify the most effective standard-of- care therapy so the patient can start treatment around 14 days.
• the tumor may be identified at day 0 (e.g., by CT scan) 1801, and a biopsy taken 1805 at day 5, and on the same day hundreds, thousands, or tens of thousands of Micro-Organospheres can be formed and cultured for 1-5 days and screened 1805 to identify one or more drug compositions that can be used.
• This same step (forming the Micro-Organospheres and screening) may be used to guide precision medicine at multiple clinical decision points throughout disease progression.
• therapy using the identified one or more drug compositions may be started on day 14 1809, and the patient may later be monitoring during the course of treatment (e.g., a follow-up CT scan on about day 90) to confirm that the tumor is responding to the treatment 1811. If so, the therapy may be continued 1813 and the ongoing progress monitored 1815.
• course of treatment e.g., a follow-up CT scan on about day 90
• Micro-Organospheres to assay may be repeated at multiple point throughout treatment during the course of the treatment. This is illustrated in FIG. 19.
• this technique e.g., generation of Micro-Organospheres and screening 1905
• a biopsy may be taken and hundreds, thousands, or tens of thousands of Micro-Organospheres may be formed and screened with a panel of potential drug compositions.
• this technique 1905’ may indicate whether and which adjuvant therapy should be chosen 1925.
• the same technique e.g., generating and screening Micro-Organospheres from a fresh biopsy 1905”, 1905”’, 195”
• this technique 1905”” can be performed to identify off-label drugs to treat resistant tumors 1935.
• This technique can also be used as companion diagnostics to identify patients for a specific treatment.
• the technique can be used to derive and preserve patient-derived Micro-Organospheres to establish Organosphere-base living cancer bank for screening, genomic profiling, new drug discovery, drug testing and clinical trial design.
• Micro- Organospheres may be done relatively low-invasively (e.g., by resection or biopsy), to provide reasonably fast results from the screening, these methods may be easily adapted for standard of care.
• the volume of cellular material from the tissue (e.g., biopsy) input is quite small, and may be dissociated into a volume of, e.g., between 10pL to 5 ml.
• Micro-Organospheres described herein for screening may be automated or manually performed.
• Virtual any screening technique may be used, including imaging by one or more of: confocal microscopy, fluorescent microscopy, liquid lens, holography, sonar, bright and dark field imaging, laser, planar laser sheet, including high- throughput embodiments of image-based analysis methods (e.g., using computer vision, and/or supervised or unsupervised model, e.g., CNN).
• Downstream screening may include sampling the culture media and/or performing genetic or protein screening (e.g., scRNA-seq, ATAC-seq, proteomics, etc.) on cells from the Micro-Organospheres.
• genetic or protein screening e.g., scRNA-seq, ATAC-seq, proteomics, etc.
• FIGS. 20 and 21 illustrate another example of an apparatus for forming a plurality of Micro-Organospheres as described herein.
• the apparatus may include a plurality of Micro-Organosphere forming junctions, in which the immiscible material (e.g., oil) 2002 may be added to a reservoir and/or port 2004 in the device.
• the unpolymerized material 2006 in this example, including the dissociated biopsy cells and the fluid matrix material
• a second or additional material e.g., a biologically active agent
• a junction similar to that described above
• a droplet in the immiscible material may be polymerized into the Micro-Organospheres.
• FIG. 20 three (or more) parallel junctions with corresponding inputs and output are shown.
• FIGS. 21 illustrates the method of forming the Micro-Organospheres using an apparatus as shown in FIG. 20.
• the resulting Micro-Organospheres includes both the target (e.g., tumor) biopsy cells but also one or more additional biologically active agents that are combined to form the Micro-Organosphere.
• a first channel 2103 may include the unpolymerized material (including the dissociated biopsy cells and the matrix material)
• a second channel 2107 includes an additional active biological material
• a pair of intersecting channels 2109, 2109’ carrying the immiscible material e.g., oil
• the additional active biological material may be, e.g., freezing medium (e.g., to aid in banking the Micro-Organospheres), and/or co-cultures with additional cells (e.g., immune cells, stromal cells, endothelial cells, etc.), additional supportive network molecules (e.g., ECM, collagen, enzymes, glycoproteins, biomimetic scaffolds, etc.), additional growth factors, and/or drug compounds.
• additional cells e.g., immune cells, stromal cells, endothelial cells, etc.
• additional supportive network molecules e.g., ECM, collagen, enzymes, glycoproteins, biomimetic scaffolds, etc.
• the Patient-Derived Micro-Organospheres and methods of using them to screen for drug compositions may be used to accurately predict the response of a patient tumor to one or more drug therapies.
• the use of Micro-Organospheres may provide accurate results where traditional cultured drug screening does not accurately predict drug response.
• FIG. 22A-22D the Micro-Organospheres, but not a cell line, was able to correlate with patient response.
• a traditional cell line dosed with drugs e.g., Oxalipalitin
• FIG. 22B For comparison a plurality of Micro-Organospheres were generated from a patient biopsy, as shown in FIG. 22B.
• the Patient-Derived Micro-Organospheres showed significant decreases in cell survival from the tumor Micro-Organospheres, predicting drug sensitivity.
• the tumor responded to treatment, as shown in FIG. 22C (before treatment) and 22D (post treatment).
• FIG. 23A shows the effect of a first drug (Oxalipalatin) on these Micro- Organospheres, showing no change in the percent survival of the Micro-Organospheres in the presence of the drug, predicting drug resistance.
• a second drug Irinotecan
• FIG. 23C shows the lack of effect on the Micro-Organospheres, predicting drug resistance, as shown in FIG. 23C.
• the patient was treatment with both Oxalipalatin and Irinotecan, and, after 6 months of treatment, showed no response.
• the Micro-Organospheres correlated strongly with patient response to the standard of care drugs.
• the patient endured six months of side effects and toxicities that may have been avoided by the predicted response from the Micro- Organospheres, indicating (within 7-10 days from the biopsy) that the tumor would not respond to these drugs.
• Example 4 Multi-Drug screening
• FIG. 24 shows an example of a panel of drug (e.g., chemotherapeutic agents) that may be generated using a Patient Derived plurality of Micro-Organospheres as described herein.
• a drug screen using the Patient-Derived Micro-Organospheres was run by dosing a plurality of replicates for each of a plurality of (27) drugs.
• a single tumor biopsy was used to generate a plurality of Micro-Organospheres in large quantities extremely fast (e.g., within less than two weeks) and these Micro-Organospheres were tested against the panel of drug formulations (e.g., 27 formulations are shown). This testing was done in parallel and could be automatically quantified (e.g., by optical detection and quantification.
• the drug showing the largest toxicity was Pazopanib for this particular tumor.
• Materials an apparatus for forming the Micro-Organospheres, as described above, including a droplet microfluidic chip (200um); Bio-rad Droplet Generation Oil for EvaGreen (catalog #186-4006), 3-5 mL per run, Perfluoro octanol (PFO), Sigma, 10% Perfluoro octanol (PFO) in Novec HFE 7500, PBS, Cell culture media (i.e. RPMI w/ 10% FBS and 1% PenStrep), 70um or lOOum filters, 50 mL conical, Petri dish.
• a droplet microfluidic chip 200um
• Bio-rad Droplet Generation Oil for EvaGreen catalog #186-4006
• PFO Perfluoro octanol
• Sigma 10% Perfluoro octanol
• PFO Perfluoro octanol
• Biopsy sample dissociation using a biopsy sample (human/animal) to generate a dissociated sample (i.e. single cell tissue) from patient. Coat the microfluidic chip, and assemble the microfluidic chip and holder. Connect microfluidic tubing and fitting to an output (e.g., multiwall plate, 15mL Eppendorf, etc.) for the Micro-Organospheres and the waste oil.
• a biopsy sample human/animal
• a dissociated sample i.e. single cell tissue
• Micro-Organospheres may be formed from biopsied renal tissue.
• instruments used may include: a tube rotator or 100 pm and 70 pm cell strainer, 15 mL conical tubes, 50 mL conical tubes, Razor blades, Tweezers and surgical scissors, Petri dish (100 x 15 mm) or tissue culture dish.
• the reagents may include: EBM-2 media, Collagenase (5 mg/mL stock), Hank’s Balanced Salt Solution (HBSS), Calcium Chloride (10 mM stock solution), Phosphate Buffer Solution (IX PBS), MATRIGEL, 0.4% Trypan Blue solution and Trypsin.
• domes of MATRIGEL-cell suspension in the center of wells in a pre-warmed 24-well flat bottom plate. Transfer the plate to a 37 ⁇ cell incubator and incubate for at least 20 minutes. Confirm that domes are polymerized. Gently add 500 pL of prewarmed EBM-2 media down the wall of the well. Incubate in 37 DC incubator. Perform a full media change every 2 days to expand Micro-Organospheres.
• Micro-Organospheres may be formed from normal (e.g., non- cancerous) and/or abnormal tissue.
• FIGS. 25A-25 and 26A-26B illustrate one example of Micro-Organospheres formed from a mouse liver tissue that has been dislocated, combined with a fluid matrix material to form an unpolymerized mixture, then a droplet of the unpolymerized mixture was polymerized to form the Micro-Organospheres.
• the Micro-Organospheres have a diameter of about 300 pm.
• the Micro- Organospheres were formed with a single cell per droplet.
• the Micro- Organospheres were formed with 25 cells per droplet.
• FIG. 25A the Micro-Organospheres are shown one day after forming;
• FIG. 25B shows the Micro-Organospheres after ten days in culture. Cells in some of the Micro-Organospheres have divided, forming clusters exhibiting structure; other Micro-Organospheres included cells that were slower to divide or that did not divide.
• FIGS. 26A-26B the Micro-Organospheres initially including about 25 cells in each Micro-Organospheres. After ten days in culture, some of the Micro-Organospheres showed a great deal of cell growth, forming structures, while other Micro-Organospheres showed only modest growth. In both cases, the cells within the Micro-Organospheres have been found to exhibit properties characteristic of the original tissue (e.g., liver cells) from which they originated.
• the original tissue e.g., liver cells
• FIGS. 27A-27C The same procedure was successfully performed on human liver tissue, as shown in FIGS. 27A-27C.
• the Micro-Organospheres were initially formed with about fifty cells, as shown in FIG. 27A.
• day 18 in culture some of the Micro-Organospheres showed cells having clusters and forming structures, while others had smaller structures or the cells did not divide.
• Micro-Organospheres may be formed from cultured cells or cells, including either 2D cultured cells or 3D cultured cells.
• the Micro-Organospheres may be formed from cell lines grown as part of a Patient Derived Xenograft (PDX).
• FIGS. 28A-28D illustrate Micro-Organospheres formed from cultured PDX240 cells.
• PDX240 cells are a Patient Derived Xenograft (PDX) tumor cell line (numbered 240 based on patient source) that were human tumors grown in immunodeficient mice (PDX) to form in vivo tumors.
• the xenograft tissue was extracted dissociated, and used to form Micro-Organospheres as described above. In this example a single cell was included in each Micro-Organospheres as it was formed.
• FIG. 28A shows the Micro-Organospheres after one day in culture
• FIG. 28B shows the Micro- Organospheres after three days in culture
• FIGS. 28C and 28D show the Micro- Organospheres after five and seven days in culture, respectively.
• at least some of the Micro-Organospheres show the cells dividing and forming structures.
• FIGS. 29A-29D show a similar experiment in which five PDX240 cells were initially included in each droplet forming each of the Micro-Organospheres. With time in culture (e.g., from day 1, day 3, day 5 and day 7, as shown in FIGS. 29A-29, respectively) the cells may divide and form structures.
• Organoids were formed from Patient Derived Xenograft cells (including the
• the organoids were formed using conventional techniques in which a large mass of MATRIGEL in a well or dish was seeded with cells and cultured until growth was confirmed. Micro-Organospheres were generated from the traditional organoids.
• FIGS. 30 and 31 were generated, and show similar response curves.
• the drug response curves of PD019187 bulk organoids and Micro-Organospheres showed similar response curves to Oxaliplatin concentration, as did PDX240 bulk organoids and Micro-Organospheres.
• the drug response curves for both PDX19187 and PDX240 also showed similar results for both bulk organoids and Micro- Organospheres for SN38.
• FIG. 32 shows response curves for another anit- cancer drug, 5-FU (Fluorouracil), again showing similar drug response curves for both PDZ- 19187 and PDX-240 traditional organoids and Micro-Organospheres.
• 5-FU Fluorouracil
• the Micro-Organospheres described herein which may be formed more quickly and reliably, and which may have a higher overall survival rate as compared to traditional organoids, may provide drug responses that are comparable to those of bulk organoids formed using the same cells.
• the Micro-Organospheres may be used more quickly and may be formed in much larger numbers.
• the Micro-Organospheres described herein may be used to peroform one or more assays, including toxicity assays. Any appropriate assay may be performed, as the results determined by analysis of the tissue (e.g., cells, tissue structures) suspended within the Micro-Organosphere.
• the Micro-Organospheres described herein may be assayed or analysed optically, chemically, electrically, genetically, or in any other manner known in the art.
• Optical (either manual or automatic) detection may be particularly useful and may include optically analyzing the effects of one or more drug formulations on the tissue (including cells, clusters of cells, structures of cells, etc.) within the Micro-Organospheres.
• the drug formulation may be assayed for cell death (e.g., number and/or size of tissues within the Micro-Organospheres tested.
• the Micro- Organosphere may be assayed for cell growth, including reduction in the size, type and/or rate of growth.
• the Micro-Organosphere may be assayed for changes in the tissue structures formed.
• FIG. 33A-33B illustrate the effect of one drug formulation, in this example, acetaminophen (10 mM) on mouse liver Micro-Organospheres.
• FIG. 33 A is a control group, in which the Micro-Organospheres were not treated, showing tissue within the Micro- Organosphers (arrows) grown when cultured.
• FIG. 33B shows a similar set of Micro- Organosphere formed from mouse liver that were instead treated with 10 mM acetaminphin.
• the tissue structures within the Micro-Organosphere are relatively large as compared with the treatment group.
• the tissue in most of the Micro-Organospheres of the acetaminiphin is smaller and contains many dead cells.
• FIGS. 34A-34B also show toxicity assays using human liver Micro- Organospheres.
• FIG. 34A shows typical human liver Micro-Organospheres observed in the control group including tissue structures (indicated by the arrows) formed therein.
• FIG. 34B shows the treatment group, in which the human liver Micro-Organospheres are treated with acetaminophen (10 mM). The tissue in the treated Micro-Organospheres showed a significant increase atypical tissue structures (arrows) and debris, as compared to the control group.
• Any of these reviews may be scored, graded, ranked, or otherwise quantified.
• the results of these two assays may be quantified to indicate the size difference, number of live/dead cells/tissue, and the like.
• scoring may be automated.
• Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to control perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.
• a processor e.g., computer, tablet, smartphone, etc.
• the device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
• the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
• first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.
• any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and/or steps may alternatively be exclusive, and may be expressed as“consisting of’ or alternatively“consisting essentially of’ the various components, steps, sub-components or sub-steps.
• a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc.
• Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then “about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.
• one or more method steps may be skipped altogether.
• Optional features of various device and system embodiments may be included in some embodiments and not in others.

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