Multiple myeloma micro organ spheres

Abstract

Provided herein are micro organ spheres (MOSs) produced using cells from multiple myeloma bone marrow biopsy, as well as methods and materials for making and using such MOSs.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority benefit of U.S. Provisional Application Serial No. 63 / 404,472, filed Sep. 7, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated into the disclosure of this application by reference. Field of the Invention

[0003] This document relates to MicroOrganoSphere (MOS) generated from bone marrow biopsies containing multiple myeloma (MM) cells, and methods and materials for making and using MOS containing MM. Background of the Invention

[0004] MM is a plasma cell malignancy, with an estimated five to seven new cases per 100,000 people, making it the second most common hematologic malignancy in the United States. In 2019 alone, there were approximately 32,000 new MM cases in the United States, and 12,960 people died from MM. Despite improvements in the treatment and overall outcomes of patients with MM over the past several decades, MM remains an incurable disease, and nearly all patients will continuously experience cycles of treatment, response, and relapse. For example, nearly all patients with MM require lifelong treatment, including two to four drug combination therapies and single-agent maintenance therapies. FDA-approved drugs for treating MM include immunomodulatory agents (e.g., IMiD, thalidomide, lenalidomide, and pomalidomide), proteasome inhibitors (PI) (e.g., bortezomib, carfilzomib, and ixazomib), monoclonal antibodies (e.g., elotuzumab, daratumumab, isatuximab, and belantamab), nuclear export protein inhibitors (e.g., selinexor), doxorubicin, panobinostat, and melflufen. Steroids (e.g., dexamethasone) and alkylating agents (e.g., cyclophosphamide, melphalan, and bendamustine) are also commonly used to treat MM. Additionally, chimeric antigen receptor (CAR) T cell therapies (e.g., Abecma, Ide-cel) have been approved by the U.S. Food and Drug Administration, and bispecific antibodies may soon be used as standard treatment for MM. Summary of the Invention

[0005] This document is based at least in part on the development of methods for generating, testing, and validating patient-derived micro-organospheres (PDMOs) (also referred to herein as MM MOSs) from MM biopsies. This document is also based at least in part on the use of MM MOSs with a platform called micro-organosphere drug screening-guided care (MODEL), which can be used as a diagnostic assay to identify appropriate treatments for, e.g., relapsed / refractory (RR) MM patients and newly diagnosed MM patients such that treatment regimens can be matched to individual patients. The methods and materials provided herein can facilitate rapid diagnosis (e.g., within two weeks or less), be cost-effective and easy to use, and can provide high-throughput screening of treatments for individual MM patients. The availability of multiple therapeutic agents (e.g., the agents listed above) for treating MM makes the selection, combination, and sequential use of these agents challenging. The ability to select one or more appropriate agents and one or more appropriate drug combinations for a particular patient can facilitate the successful treatment of patients with MM.

[0006] This document provides methods and materials for generating MOSs from bone marrow biopsies obtained from mammals (e.g., humans) with MM. For example, this document provides methods that include: obtaining a bone marrow biopsy from an MM patient, preparing a population of MOSs using the obtained bone marrow tissue, and screening the MOSs for their response to various MM treatments, thereby identifying which of the tested therapies may be most effective for the patient.

[0007] As demonstrated herein, MOSs were successfully generated from fresh bone marrow biopsies from MM patients. The MOSs were contacted with several therapeutic agents to determine the effects of these drugs on the MOSs, revealing different effects on the MOSs depending on the drugs used. Additionally, further studies demonstrated that MM MOSs can withstand freezing and thawing.

[0008] In a first aspect, this document features a micro-organosphere containing bone marrow cells from a mammal with multiple myeloma (MM). The micro-organosphere can contain from about 50 to about 150 cells, from about 75 to about 125 cells, or about 100 cells. The cells can include cancer cells, stromal cells, stem cells, immune cells, or any combination thereof. The micro-organosphere can contain a ratio of cancer cells to stromal cells of less than about 1:4. The immune cells can include at least one macrophage. The micro-organosphere can contain a dissolved basement membrane matrix.

[0009] On the other hand, the present document features a composition containing micro-organ spheroids in a culture medium, wherein the micro-organ spheroids contain bone marrow cells from a mammal suffering from MM. The culture medium may include a dissolved basement membrane matrix. The composition may contain about 1% of the dissolved basement membrane matrix. The composition may further contain an immiscible fluid (such as oil).

[0010] On the other hand, the present document features a method for manufacturing micro-organ spheroids (MOS). The method may include (a) receiving a bone marrow sample from a mammal suffering from MM; (b) refining the bone marrow sample to form a refined sample; and (c) forming a population of MOS from the refined sample by: (i) driving an unpolymerized fluid mixture through one or more channels of a microfluidic device, wherein the unpolymerized fluid mixture includes the refined sample and an unpolymerized fluid matrix material, and wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the refined sample and the unpolymerized fluid matrix material travel through the one or more channels in a laminar flow manner, (ii) forming a plurality of droplets containing the unpolymerized fluid mixture within the microfluidic device, and (iii) polymerizing the fluid matrix material to form MOS, wherein each MOS has a diameter between 50 μm and 500 μm and contains between 30 and 150 cells distributed therein.

[0011] The method may further include driving an immiscible fluid through another channel of the microfluidic device such that the immiscible fluid combines with the unpolymerized fluid mixture before forming a plurality of droplets, wherein the droplets comprise the unpolymerized fluid mixture and the immiscible fluid. The immiscible fluid may be oil. Forming the MOS population may further include sorting the MOS based on cell number and / or droplet size. Sorting may include optical sorting based on cell number and / or droplet size. Forming the MOS population may include forming about 100 to about 600 MOS, forming about 600 to about 1,000 MOS, or forming more than about 1,000 MOS. The microfluidic device may maintain the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets. The microfluidic device may be configured to prevent clogging of the unpolymerized fluid mixture within one or more channels. The microfluidic device may be configured to prevent clogging by having a channel diameter of 100 μm or greater. The microfluidic device may be configured to maintain an approximately constant pressure within one or more channels. The microfluidic device may maintain a constant flow rate within one or more channels. The total length of the path taken by the unpolymerized fluid mixture before forming the plurality of droplets within the microfluidic device is less than 10 cm. The MOS within the MOS population vary in size by less than 25%. Polymerization may include crosslinking a fluid matrix material. The fluid matrix material may be chemically crosslinkable or photocrosslinkable. The bone marrow sample may include freshly biopsied cells. The bone marrow sample may be obtained from a mammal within 24 hours after forming the MOS. The bone marrow sample may include MM plasma cells, immune cells, stem cells, stromal cells, or any combination thereof. The immune cells may include one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof. The method may include flowing the unpolymerized fluid mixture through one or more channels at a flow rate of about 0.01 milliliters (mL) per minute (min) to about 100 mL / min.

[0012] In another aspect, the present document features a method for precision drug screening for personalized cancer therapy for MM. The method may include (a) receiving a bone marrow sample from a mammal suffering from MM; (b) refining the sample to form a refined sample; (c) forming a MOS population from the refined sample by: (i) driving an unpolymerized fluid mixture through one or more channels of a microfluidic device, where the unpolymerized fluid mixture includes the refined sample and an unpolymerized fluid matrix material, and where the microfluidic device controls the pressure, flow rate, or both pressure and flow rate within the one or more channels such that the refined sample and the unpolymerized fluid matrix material travel through the one or more channels in a laminar flow manner, (ii) forming a plurality of droplets containing the unpolymerized fluid mixture within the microfluidic device, and (iii) polymerizing the fluid matrix material to form MOS, where each MOS has a diameter between 50 μm and 500 μm and between 1 and 500 cells are distributed therein; (d) culturing the MOS population for between 1 and 14 days; and (e) using the MOS population to assay one or more drug therapies.

[0013] The method may further include driving an immiscible fluid through another channel of the microfluidic device such that the immiscible fluid combines with the unpolymerized fluid mixture before forming a plurality of droplets, wherein the droplets comprise the unpolymerized fluid mixture and the immiscible fluid. The immiscible fluid can be oil. The assay may include parallel determination of multiple drug therapies by exposing one or more of the MOSs to each drug therapy. The method may include characterizing the response of the MOSs to each of the multiple drug therapies based on the response of the MOSs to exposure to the multiple drug therapies. The time between receiving the bone marrow sample and characterizing the response may be less than 21 days. Forming the MOS population may further include sorting the MOSs based on cell number and / or droplet size. Sorting may include optically sorting the MOSs or based on cell number and / or droplet size. The assay may include assaying more than 10 different drug therapies. One or more drug therapies include different concentrations of one or more drugs, different combinations of two or more drugs, different ratios of two or more drugs, different carriers for one or more drugs, and / or different dosing times for one or more drugs. Forming the MOS population may include forming about 100 to about 600 MOSs, forming about 600 to about 1,000 MOSs, or forming more than 1,000 MOSs. The microfluidic device may maintain the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets. The microfluidic device may be configured to prevent clogging of the unpolymerized fluid mixture within one or more channels. The microfluidic device may be configured to prevent clogging by having a channel diameter of 100 μm or greater. The microfluidic device may be configured to maintain an approximately constant pressure within one or more channels. The microfluidic device may maintain a constant flow rate within one or more channels. The total length of the path taken by the unpolymerized fluid mixture before forming the plurality of droplets within the microfluidic device is less than 10 cm. The method may further include measuring the effect of one or more drug therapies on the cells within the MOSs. The method may further include determining that a mammal is still responsive to one of the one or more drug therapies after one or more administrations of the drug therapy by receiving a second bone marrow sample after treating the mammal with the drug therapy, forming a second MOS population from the second bone marrow sample, exposing at least some of the second MOS population to the drug therapy, and measuring the effect of the drug therapy on the cells within at least some of the second MOS population. The method may further include treating a mammal with one of the one or more drug therapies. The MOSs within the MOS population vary in size by less than 25%. Polymerization may include crosslinking the fluid matrix material. The fluid matrix material can be chemically crosslinkable or photocrosslinkable. The bone marrow sample may include freshly biopsied cells. The bone marrow sample may be obtained from a mammal within 24 hours after forming the MOSs. The bone marrow sample may include cancer cells, immune cells, stem cells, stromal cells, or any combination thereof.Immune cells may include one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof. The method may include flowing a refined bone marrow sample and an unpolymerized fluid matrix through one or more channels at a flow rate of about 0.01 mL / min to about 100 mL / min.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods, devices, and materials similar or equivalent to those described herein may be used to practice the present invention, suitable methods, devices, and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0015] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A to 1C are representative images showing that each MOS of the formed PDMO contains a single primary tissue cell, cultured for one day after formation ( Figure 1A ), cultured for three days after formation ( Figure 1B ), and cultured for seven days after formation ( Figure 1C ). These cells are derived from colorectal cancer (CRC) tissue.

[0017] Figures 2A to 2C are representative images showing that each MOS of the formed PDMO contains five primary tissue cells, cultured for one day after formation ( Figure 2A ), cultured for three days after formation ( Figure 2B ), and cultured for seven days after formation ( Figure 2C ). These cells are derived from CRC tissue.

[0018] Figures 3A to 3C are representative images showing that each MOS of the formed PDMO contains twenty primary tissue cells, cultured for one day after formation ( Figure 3A ), cultured for three days after formation ( Figure 3B ), and cultured for seven days after formation ( Figure 3C ). These cells are derived from CRC tissue.

[0019] Figures 4A to 4E include images showing a representative example in which each MOS of the formed PDMO contains ten primary tissue cells.Figure 4A Show MOS (low magnification) shortly after formation. Figure 4B Show Figure 4A Higher magnification views of some MOS, taken two days after culturing. Figure 4C Show MOS three days after culturing. Figure 4D Show MOS four days after culturing. Figure 4E Show MOS five days after culturing.

[0020] Figures 5A to 5B Are images of representative examples of MOS formed from normal mouse liver hepatocytes, cultured for one day after formation ( Figure 5A ) or cultured for ten days after formation ( Figure 5B ), and the mouse hepatocytes were obtained from the liver of a normal (non-diseased) mouse.

[0021] Figure 6 Is a schematic diagram showing the steps of a method for forming PDMO from a primary tissue (e.g., biopsy) sample.

[0022] Figure 7A Is a schematic diagram showing an example of a device for forming PDMO, including a microfluidic chip as part of the components. Figure 7B Is a Figure 7A Perspective view of an example of the microfluidic chip portion of the device shown in Figure 7C Is a schematic diagram showing a part of the microfluidic component of a device for forming PDMO (such as the device shown in Figure 7A ).

[0023] Figure 8 Is an image showing multiple PDMOs formed using a device such as Figure 7A shown in

[0024] Figure 9 Is an image of a part of a prototype microfluidic component of a device for forming PDMO, similar to that shown in Figure 7C showing the formation of PDMO.

[0025] Figure 10 Is an image showing multiple PDMOs shortly after polymerization; the PDMOs are suspended in an immiscible fluid.

[0026] Figure 11A And Figure 11B Are images showing multiple PDMOs shortly after formation and suspension in an immiscible fluid (e.g., oil) at low magnification ( Figure 11A ) and high magnification ( Figure 11B ).

[0027] Figure 12A and Figure 12B are images showing multiple PDMOs separated from immiscible fluids within hours after PDMO formation at low magnification ( Figure 12A ) and higher magnification ( Figure 12B ).

[0028] Figure 13 is an image showing another example of multiple PDMOs.

[0029] Figure 14 is a graph plotting the size distribution of the diameters of multiple PDMOs formed from an exemplary biopsy sample.

[0030] Figure 15A and Figure 15B are images showing low and higher magnification views, respectively, of an example of multiple PDMOs formed after polymerization from dissociated tissue biopsy samples and fluid matrix materials. Figure 15A is an unstained image, while in Figure 15B the MOS has been stained with trypan blue to show that the cells in the MOS are alive.

[0031] Figure 16A and Figure 16B are images showing another example of low and higher magnification views of an example of multiple PDMOs, respectively. Figure 16A is an unstained image, while in Figure 16B the MOS has been stained with trypan blue (arrows) to show that the cells in the MOS are still alive (e.g., viable) within the MOS.

[0032] Figures 17A to 17E Shows an example of a method for determining the drug response profile to multiple pharmaceutical formulations by assaying multiple PDMOs formed from a patient tumor biopsy. The procedure shown takes less than two weeks from biopsy to result.

[0033] Figure 18 Schematically shows an example of a method for treating a patient, including forming and using multiple PDMOs as part of a treatment procedure.

[0034] Figure 19 Schematically shows an example of a method for treating a patient, including multiple iterations of rapidly forming and assaying multiple PDMOs as part of a treatment procedure.

[0035] Figure 20A Includes representative images showing the formation of MM MOS from human bone marrow over a 11-day period. The MM MOS is indicated by white arrows. Figure 20BInclude representative images showing that CD138+ cells (isolated from bone marrow cells using bead selection) do not form MOS in the absence of stromal cells.

[0036] Figures 21A to 21E Show that the established MM MOS preserves the major cell populations from the bone marrow. Figure 21A Are flow cytometry plots of single cells from bone marrow biopsy samples and MOS cultured in vitro for 9 days. The myeloma cell population was shown to be CD11b- and CD38+. Figure 21B And Figure 21C Are a pair of flow cytometry plots showing that compared to bone marrow biopsy ( Figure 21B ), the major immune cell populations are retained in the MOS at day 8 ( Figure 21C ). Flow cytometry was performed on MOS at day 3 and day 8, and the percentage of total cells over time during MOS culture was plotted in the graph shown in Figure 21D . Figure 21E Include additional flow cytometry plots showing that the established MM MOS preserves the major cell populations from the bone marrow. Flow cytometry was performed on MM MOS at day 11. Plasma cells and MM cells (CD138 + and CD38 + ), T cells (CD3 + , CD4 + and CD8 + ), and dendritic cells (CD11b + ) were identified as shown.

[0037] Figure 22A Include representative images showing MOS containing 50, 70, or 100 cells at day 0 and day 7. Figure 22B Include representative images of day 7 MOS with live cell staining.

[0038] Figure 23A Is a plot showing caspase 3 / 7 signal in MM MOS after treatment with lenalidomide, bortezomib, or negative control for 92 hours. An increase in caspase 3 / 7 signal indicates death of MM MOS. Figure 23B Include representative images showing the response of MM MOS to lenalidomide as early as 24 hours after drug treatment.

[0039] Figure 24A And Figure 24B Show that MM MOS responds to carfilzomib and panobinostat treatment. Figure 24ARepresentative live / dead dye images of MOS after 4 days of treatment with 10 μM carfilzomib or 10 μM panobinostat. Calcein acetoxymethyl ester (calcein AM) (green) was used to stain live cells, and ethidium homodimer (red) was used to stain dead cells. Figure 24B Graph depicting the ratio of live / dead cells. n = 3 for each treatment group. *p < 0.05 by t-test.

[0040] Figure 25A Includes a pair of images showing MM MOS after freeze-thaw cycles, only the MOS remained viable. Figure 25B Includes a graph showing the viability of MM MOS after freeze-thaw, as measured by SYTOX TM Blue as demonstrated. Detailed Description

[0041] Cell and tissue system models, including three-dimensional (3D) aggregates such as spheroids and organoids, can be used for biological and medical research. For example, 3D cell culture models are particularly useful in developmental biology, disease pathology, regenerative medicine, drug toxicity and efficacy testing, and personalized medicine. Patient-derived cancer models (PDMCs), such as cell lines, organoids, and patient-derived xenografts (PDXs), can provide preclinical models to facilitate the identification and development of new therapies and to predict drug responses and identify novel drug combinations. For example, large-scale drug screening of cell lines and organoids derived from cancer patients can be used to identify sensitivity to a large number of potential therapies.

[0042] Multicellular tumor spheroids can be obtained by culturing cancer cell lines under non-adherent conditions. Spheroids are typically formed by cancer cell lines as free-floating cell aggregates in ultra-low attachment plates. Compared to two-dimensional (2D) cell culture, spheroids have been shown to maintain more stem cell-related properties. Organoids are in vitro-derived cell aggregates that include a population of stem cells that can differentiate into the major cell lineages. Organoids typically have a diameter greater than one millimeter and can be passaged. The growth and expansion rate of organoids is generally slower than 2D cell culture. Generating organoids from clinical samples requires a sufficient initial number of live cells (usually hundreds to thousands of cells), and thus obtaining organoids from small volume samples such as biopsies can be challenging.

[0043] Although precision medicine strategies have evolved by exploring these different PDMC models, their effective use remains hampered. Patient-derived organoids (PDOs) are considered the most accurate in depicting patient tumors, as studies have shown that the phenotypic and genotypic profiles of organoids generally show a high degree of similarity to the original patient tumors. However, there are still limitations in using PDOs to guide therapy. For example, developing and testing the drug sensitivity of organoids typically takes several months, which reduces clinical applicability, as ideally, assays should be performed within 7 - 10 days from a single-core biopsy. Additionally, the number of organoids obtained from a clinically relevant 18-core biopsy is insufficient for high-throughput drug screening.

[0044] Furthermore, it is not easy to use MM tissue to replicate tumor cells. MM cells are generally difficult to work with, and there have been few attempts to culture MM cells in 3D, especially since there is no universal recipe for 3D culture of multiple myeloma. Another challenge is that MM cells cannot survive without bone marrow stromal cells. However, as described herein, PDMOs were successfully generated from MM biopsies, which is surprising considering the above challenges. However, the techniques described herein can provide a tumor microenvironment for MM cells, thus allowing MM cells to grow. Cells within the MM immune microenvironment, such as macrophages, are also captured in the MOSs prepared by the methods provided herein, expanding the range of drugs for which the MOSs can be screened.

[0045] Thus, generally speaking, this document provides MOSs generated from bone marrow samples of mammals suffering from MM. Additionally, this document provides methods, materials, and devices for forming MM PDMOs (also referred to herein as "MM MOSs"), as well as methods, materials, and devices for using MM PDMOs (e.g., to determine the response of MM to one or more therapeutic agents). The MM MOSs described herein and the methods of making and using them can address clinical limitations such as those mentioned above.

[0046] The MM PDMOs described herein are generally spheres formed from primary cells distributed within a basal marrow material. After refining the primary cells, non-mononuclear cells (e.g., red blood cells, granulocytes, and / or platelets) are removed while retaining mononuclear cells, thereby forming a refined tissue sample containing myeloma cancer cells and stromal cells, stem cells, immune cells, or any combination thereof. These PDMOs (also referred to as "MM MOS") can have a diameter of about 50 μm and about 500 μm (e.g., about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 50 μm to about 250 μm, about 100 μm to about 500 μm, about 100 μm to about 250 μm, or about 50 μm to about 200 μm). The MM MOS can initially contain about 1 to about 1000 primary cells (e.g., about 1 to about 750, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 150, about 1 to about 100, about 25 to about 200, or about 50 to about 150 primary cells) distributed within the basal material.

[0047] Surprisingly, despite the small size (generally about 50 μm to about 250 μm) and low cell density (e.g., typically about 50 to about 200 cells per MM MOS) of the MM MOS provided herein, they can be used immediately or cultured for a relatively short period of time (e.g., 14 days or less, 10 days or less, 7 days or less, or 5 days or less), and can allow the cells within the MMMOS to survive while maintaining most or even all of the characteristics of the tissue from which they were extracted. The cell viability within the MM MOS is generally high, and the MM MOS can be cultured for days or even weeks. Also surprisingly, in some variants, the cells from the refined marrow samples within the MM MOS can even form morphological structures within the smallest MOS; although in some applications, the presence of such structures is not necessary for the use of these MOS (such as when they are used before substantial structural reorganization), but in some variants, such structures may be particularly useful.

[0048] In some cases, the methods and materials described herein for forming and using MM MOS can be used to generate many (e.g., more than 10,000) PDMOs from a single biopsy. The MOS can be used, for example, to screen various therapeutic agents to predict which / which therapeutic agents may be effective and safe for use in MM patients who have undergone a bone marrow biopsy. For example, the MM MOS can be used in toxicity screening of drugs or other chemical compositions and to determine whether one or more drug compositions may effectively treat MM patients before the patients receive drug therapy. This can allow for very rapid screening of MM patients who would otherwise receive months of chemotherapy that may be ineffective.

[0049] Accordingly, the present disclosure provides high-throughput drug screening methods and devices for using a single patient-specific MM biopsy for these methods. Droplet-forming PDMOs described herein can be formed from bone marrow samples derived from MM patients that have been refined and suspended in a basal matrix (e.g., a dissolved basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, available from Sigma). The MM MOSs can be patterned onto a microfluidic microwell array for incubation and administered drug compounds. This miniaturized assay can maximize the use of tumor samples and enable screening of more drug compounds from bone marrow aspirates at a lower cost per sample.

[0050] Although it is believed that the terms used herein are well understood by one of ordinary skill in the art, the definitions set forth herein are provided to facilitate the explanation of the presently disclosed subject matter.

[0051] The term "unpolymerized mixture" is used herein to refer to a composition containing biologically relevant materials, including refined tissue samples and a first fluid matrix material. The fluid matrix material is generally a material that can be polymerized to form a support or support network for the refined tissue samples and / or cells dispersed within the refined samples. When polymerized, the polymerized material can form a hydrogel and can be formed and / or can include proteins that form a biocompatible medium in addition to cells. A biocompatible medium suitable for the methods disclosed herein can be formed from any biocompatible material that is a gel, semi-solid, or liquid (such as a low-viscosity liquid) at room temperature (e.g., 25 °C) and can serve as a three-dimensional matrix for cells, tissues, proteins, and other biological materials of interest. Exemplary materials that can be used to form a biocompatible medium according to the presently disclosed subject matter include, but are not limited to, polymers and hydrogels containing collagen, fibrin, chitosan, (BD Biosciences, San Jose, CA), polyethylene glycol (PEG), dextran (e.g., dextran that can be chemically crosslinked or photocrosslinked), etc., as well as electrospun biological, synthetic, or biosynthetic mixtures. In some cases, the biocompatible medium can be a hydrogel.

[0052] The term "hydrogel" is used herein to refer to a two-component or multi-component gel having a three-dimensional network of polymer chains, where water serves as the dispersion medium and fills the spaces between the polymer chains. Hydrogels used in accordance with the presently disclosed subject matter can generally be selected for a particular application based on the intended use of the structure, taking into account the parameters to be used to form the MM MOS and the effect the selected hydrogel will have on the behavior and activity of the biomaterials (e.g., cells) to be incorporated into the biological suspension to be placed in the structure. Exemplary hydrogels for use with the presently disclosed subject matter include polymeric materials such as, but not limited to, alginate, collagen (including type I and type VI collagen), elastin, keratin, fibronectin, proteoglycan, glycoprotein, polylactide, polyethylene glycol, polycaprolactone, polycoconutolactide, polydioxanone, polyacrylate, polyurethane, polysulfone, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylate, polyacetate, polyester, polyamide, polycarbonate, polyanhydride, polyamino acid carbohydrate, polysaccharide and modified polysaccharide, and their derivatives and copolymers, and inorganic materials such as glass (such as bioactive glass), ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and any combination thereof.

[0053] Further with respect to the hydrogels for use in generating the MM MOS described herein, in some cases, the hydrogel can contain a member selected from the group consisting of: agarose, alginate, type I collagen, polyoxyethylene-polyoxypropylene block copolymer (e.g., F127; BASF Corporation, Mount Olive, NJ), silicone, polysaccharide, polyethylene glycol, and polyurethane. In some cases, the hydrogel can be made of alginate.

[0054] The MM MOS provided herein may also contain biologically relevant materials. The phrase "biologically relevant material" refers to a material that can be incorporated into a biocompatible medium as defined herein and subsequently interact with and / or affect a biological system. For example, in some cases, the biologically relevant material can be magnetic beads (such as beads that are magnetic per se or contain a material responsive to a magnetic field (such as iron particles)), which can be combined as part of the unpolymerized material to assist in the production of MM MOS (e.g., for the separation and purification of MOS). As another example, in some cases, the biologically relevant material can include cells in addition to refined tissue samples (e.g., biopsy materials). In the unpolymerized mixture, the refined tissue sample and the additional biologically relevant material can be present as a homogeneous mixture or as a distributed mixture (e.g., on one half or another part of the MOS, such as only in the core or only in the outer region of the formed MOS). In some cases, the additional biologically relevant material within the unpolymerized material can be suspended together with the refined tissue sample in the suspension (e.g., before the droplet polymerization of the MOS formation).

[0055] In some cases, the biologically relevant material that may optionally be included in a refined tissue sample (e.g., biopsy) can contain a variety of cell types, including preadipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells, and / or adipose tissue macrophages, as well as small blood vessel or microvessel fragments found within the stromal vascular fraction, and possibly skeletal components.

[0056] Generally, the refined tissue sample (e.g., biopsy) material included in the MM MOS described herein is from a bone marrow sample of a mammal (e.g., a human or other mammal with MM, such as a mouse, rat, or rabbit), and is typically collected by biopsy. These tissues and the resulting refined cells can be primary cells obtained from a patient biopsy (e.g., by needle aspiration biopsy). The refined cells can be incorporated into the MM MOS described herein and can include cells commonly found in the bone marrow. In this regard, exemplary cells that can be incorporated into the MM MOS include plasma cells (normal and cancerous plasma cells), stromal cells, hematopoietic stem cells, monocytes, macrophages, dendritic cells, T cells, B cells, and NK cells. Any suitable technique can be used to refine the tissue, including (but not limited to) those described herein.

[0057] In addition, it has now been found that, in some cases, the preparation and use of MOS generated from MM bone marrow biopsies can be facilitated by: (1) using about 50 to about 200 cells per MOS (e.g., about 50 to 100 cells, about 75 to about 125 cells, about 100 to about 150 cells, about 100 to about 200 cells, or about 100 cells), (2) adding the The concentration is increased to about 80%-100% (e.g., about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 80% to about 90% or about 90% to about 100%) and / or (3) use an ultra-low binding plate. Without being limited to a specific mechanism, using a relatively high number of cells can increase the likelihood that all cells from the bone marrow microenvironment (e.g., stromal cells and immune cells) are included in the MM MOS, which may be important from the perspective of drug screening. Additionally, without being limited to a specific mechanism, using a higher concentration of can help retain MM cells within the MOS, and using a low or ultra-low binding plate can reduce the likelihood of the MOS adhering to the bottom of the culture well. Additionally, in some cases, the methods for preparing MM MOS disclosed herein may include adding a small amount of (e.g., 1%) to the culture medium rather than to the MOS mixture, and / or adding one or more cytokines (e.g., IL-6, GM-CSF, IL-2, B cell activating factor (BAFF), IL-4 or a proliferation-inducing ligand (APRIL) – also known as tumor necrosis factor ligand superfamily member 13) to , regardless of whether is added to the MOS mixture or the culture medium. These strategies may also help retain MM cells within the MOS and / or prevent the MOS from adhering to the bottom of the culture well.

[0058] In view of the above, the MM MOS provided herein may have a cell number of about 50 to about 200 cells per MOS, wherein MM plasma cells, stromal cells and immune cells are included in the MOS cell population. In some cases, the ratio of MM cells to stromal cells in the MOS can be about 1:4 or less (e.g., about 1:4, about 1:5 or about 1:6). In some cases, the ratio of immune cells (e.g., macrophages) to MM cells in the MOS can be about 1:50 to about 1:200 (e.g., about 1:100). Flow cytometry targeting, for example, CD19 neg , CD38 pos , CD56 pos (and in some cases also CD138 pos ) myeloma cells can be used, and live and / or dead dyes indicating the viability of the targeted myeloma cells can be used to evaluate the number of MM cells in the MOS cell population. The number of other types of cells in the MOS cell population can be determined in a similar manner. For example, flow cytometry targeting, for example, CD3 neg , CD19 neg , CD20 neg , CD56 neg , CD45neg , CD31 neg , ALP pos Osteoblasts and / or CD3 neg , CD19 neg , CD20 neg , CD56 neg , CD11b pos , CD14 pos , RANK pos Flow cytometry of osteoblasts and evaluation of the number of stromal cells in the MOS cell population using a live and / or dead dye that indicates the viability of the targeted stromal cells. Flow cytometry targeting, for example, CD3 pos , CD4 pos or CD8 pos T cells, CD3 neg , CD56 pos NK cells, CD3 neg , CD19 pos B cells and / or CD11b pos Flow cytometry of monocytes and evaluation of the number of immune cells in the MOS cell population using a live and / or dead dye that indicates the viability of the targeted immune cells.

[0059] After formation, MM MOS can be cryopreserved and / or cultured. Generally, cultured MOS can be maintained in suspension, either static (e.g., in wells, vials, or other suitable containers) or in motion (e.g., rolling or agitation). Any appropriate technique can be used to culture MOS. Exemplary techniques can be found in, but are not limited to, the following: Freshney, Culture of Animal Cells, A Manual of Basic Techniques , 4th Edition, Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach , edited by Davis, Oxford University Press, 2002; Animal Cell Culture:A Practical Approach , edited by Masters, 2000; and U.S. Patent Nos. 5,516,681 and 5,559,022, all of which are incorporated herein by reference in their entirety.

[0060] In some cases, MM MOS can be generated by forming droplets of a refined tissue sample and an unpolymerized mixture of a fluid matrix material (in some cases, a frozen mixture) in an immiscible material such as a fluid hydrophobic material (e.g., oil). For example, MOS can be formed by combining a stream of unpolymerized material with one or more streams of immiscible material to form droplets. The density of cells present in the droplets can be determined by dilution of the refined material (e.g., cells) in the unpolymerized material. The size of the MOS may be related to the size of the droplets formed. Generally, MOS are spherical structures with a stable geometry.

[0061] Unless otherwise indicated, the methods described herein for preparing MM MOS can employ techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. Examples of such techniques are described elsewhere. See, for example, Molecular Cloning A Laboratory Manual (1989), 2nd ed., edited by Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent No. 4,683,195; DNA Cloning , Volumes I and II, edited by Glover, 1985; Oligonucleotide Synthesis , edited by Gait, 1984; Nucleic Acid Hybridization , edited by Hames and Higgins, 1984; Transcription and Translation, edited by Hames and Higgins, 1984; Culture of Animal Cells , Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes , IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning;See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells , edited by Miller and Calos, Cold Spring Harbor Laboratory, 1987; Methods in Enzymology , Volumes 154 and 155, edited by Wu et al., Academic Press Inc., N.Y.; Immunochemical Methods in Cell and Molecular Biology , edited by Mayer and Walker, Academic Press, London, 1987; and Handbook of Experimental Immunology , Volumes I - IV, edited by Weir and Blackwell, 1986.

[0062] As described above, tissue (e.g., biopsy) samples for forming MM MOS (e.g., refined samples) can be derived from bone marrow obtained from a mammal with MM. Generally, MOS contains multiple cell types resident in the originating tissue. Thus, the tissue used in the MM MOS provided herein can include cells of the immune system such as T lymphocytes, B lymphocytes, macrophages, NK cells, monocytes, and dendritic cells. The cells can include stem cells, progenitor cells, or somatic cells. The cells can be obtained directly from a subject without an intermediate step of passaging, or they can first undergo an intermediate culturing step to produce a primary culture. Any suitable method for harvesting cells from biological tissue and / or fluids containing cells can be used. For example, suitable techniques for obtaining cells from biological tissue include those described in Mahesparan, Acta Neuropathol (1999) 97:231-239.

[0063] Generally, the cell types collected (e.g., within a biopsy or aspirate) can be refined prior to forming MOS. For example, the cells in a bone marrow aspirate can be refined to isolate monocytes. Any suitable method can be used to achieve the separation of cell types from one another. For example, the cells in a bone marrow aspirate can be filtered (e.g., through a 70 μm - 100 μm coarse filter), and then subjected to Ficoll gradient purification to separate the monocytes in the aspirate from non-monocytes (e.g., red blood cells, granulocytes, and / or platelets).

[0064] In some cases, the refined tissue can be processed to remove dead / dying cells and / or cell debris. Removal of such dead and / or dying cells can be accomplished using any appropriate method (such as bead and / or antibody methods). For example, since phosphatidylserine can redistribute from the inner leaflet of the endoplasmic membrane to the outer leaflet in apoptotic or dead cells, the use of annexin V-biotin binding, followed by binding of biotin to streptavidin magnetic beads, enables the separation of apoptotic cells from live cells. Removal of cell debris can be achieved by techniques such as, for example, filtration.

[0065] Before mixing with the fluid matrix material, the refined cells can be suspended in a carrier material. Alternatively, the fluid matrix material can be referred to as the carrier material. In some cases, the carrier material can be a material having a viscosity level that delays the sedimentation of cells in the cell suspension before polymerization and formation of the MOS. In such cases, the carrier material can have sufficient viscosity to allow the cells of the refined tissue sample to remain suspended in the suspension until polymerization. The viscosity required to achieve this can be optimized by monitoring the sedimentation rate at different viscosities and selecting a viscosity that gives an appropriate sedimentation rate for the expected time delay between loading the cell suspension into the device that forms the MOS by polymerizing the unpolymerized material including the cells. In some cases, the unpolymerized material can be flowed or agitated (e.g., when using a lower viscosity material) by the device to keep the cells in suspension and / or distribute them as needed.

[0066] As described above, in some cases, the unpolymerized mixture including the refined tissue sample and the fluid matrix material can contain one or more other components, such as biologically relevant materials. Biologically relevant materials that can be included can include, but are not limited to, patient-derived serum or plasma, extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides or antibodies (e.g., to modulate any of cell survival, proliferation or differentiation), inhibitors of specific cell functions, and combinations thereof. Such biologically relevant materials can be used, for example, to increase cell viability by reducing cell death and / or activation of cell growth / replication or otherwise mimic the in vivo environment. Biologically relevant materials can include or mimic one or more of the following components: serum, interleukin, chemokine, growth factor, glucose, physiological salt, amino acid, and hormone. In some cases, biologically relevant materials can supplement one or more agents in the fluid matrix material. In some cases, the fluid matrix material can be a synthetic gel (hydrogel) and can be supplemented with one or more biologically relevant materials. In some cases, the fluid matrix can be a natural gel. Thus, the gel can include one or more extracellular matrix components, such as collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose, and / or chitosan. For example, Contains bioactive polymers important for cell survival, proliferation, development, and migration. In some cases, the matrix material can be a gel containing type I collagen (such as type I collagen obtained from rat tails). The gel can be a pure type I collagen gel or can be a gel that contains type I collagen in addition to other components (such as other extracellular matrix proteins). In some cases, the fluid matrix can be a synthetic gel that does not exist in nature. Examples of synthetic gels include gels derived from polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol (PVA), or poly(ethylene oxide) (PEO).

[0067] Images showing PDMO are presented in Figures 1A to 1C , Figures 2A to 2C , Figures 3A to 3C and Figures 4A to 4E . For example, Figures 1A to 1C includes representative images of MOS formed with each MOS being a single cell. As shown, all MOS are approximately the same size, with a diameter of about 300 μm. After three days of culture (middle figure), the cell size increases, and in some cases doubles and / or grows. After seven days of culture (right figure), the cells have doubled multiple times, showing cell clusters or cell aggregates. Figures 2A to 2C The MOS shown in Figures 3A to 3C is formed by five cells per MOS, while the MOS shown in Figures 4A to 4E is formed by 20 cells per MOS. In Figure 4A , MOS are shown immediately after formation and MOS cultured for five days, where the nearly identical MOS (e.g., having the same diameter) each include 10 cells per MOS. In Figure 4B , Figure 4C , Figure 4D and Figure 4E show MOS after two days, three days, four days, and five days, respectively. These images show that the refined tissue (cells) from biopsies within the MOS are alive and grow at a fairly consistent rate within nearly all MOS. As will be described in more detail herein, these MOS can be formed in large numbers from even a single average-sized biopsy and can produce hundreds or even thousands (e.g., about 500, about 750, about 1000, about 2000, about 5000, about 10,000, about 100 to about 600, about 600 to about 1000, more than about 1000, or more than 10,000) MOS, which contain a large number of live cells, allowing multiple rapid assays to be performed in parallel.

[0068] Figure 5A and Figure 5B show examples of MOS formed from biopsies of mouse liver as described herein. For example, showing mouse hepatocytes distributed within a polymeric fluid matrix material (in this example, ). Each MOS includes a polymeric matrix material 503 formed into a sphere with a diameter of about 300 μm, in which a set number of hepatocytes 507 are dispersed. In Figure 5A , MOS are shown one day after biopsy, tissue dissociation or refinement, and MOS formation. These MOS are then cultured for 10 days, during which the hepatocytes remain alive and grow, and in many cases double multiple times to form structure 505, asFigure 5B as shown

[0069] The MOS may generally include dissociated or purified biopsy tissues (e.g., cells) having a fixed or known cell number or concentration within the MOS (e.g., number of cells / ml or number of cells / mm 3 ). As described above, the matrix material may include one or more of natural polymers such as alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, and elastin, or may include one or more of synthetic polymers such as PEG and polyacrylamide. Both organic and inorganic synthetic polymers may be used.

[0070] In some cases, the number of cells initially included in the MOS can be selected between 1 cell and several hundred cells. Specifically, in some assays (e.g., drug toxicity assays), including about 1 to about 75 cells or about 1 to about 50 cells (i.e., a relatively small number of cells) may be beneficial. The number of cells per MOS can be set or selected by the technician producing the MOS. In some cases, as described below, the device for producing the MOS may include one or more controllers to set the number of cells from the primary tissue to be included in each MOS. The number of cells can be selected or set based on how the MOS is expected to be used. For example, MOSs with a very low number of cells (e.g., 1 cell per MOS or 1 to 5 cells per MOS) may be particularly suitable for studying clonal diversity (e.g., for tumor heterogeneity). Since each MOS is grown from a single cell, it can be observed which clones are drug-resistant and then these specific MOSs can be examined (e.g., by genomic sequencing) to determine the genomic (mutation) diversity associated with the specific clones. MOSs with a low to medium number of cells per MOS (e.g., about 3 to about 30 cells, about 5 to about 30 cells, about 5 to about 25 cells, about 5 to about 20 cells, or about 10 to about 25 cells) may be particularly suitable for rapid drug testing, including toxicity testing, because these MOSs generally grow quickly. MOSs with a larger number of cells per MOS (e.g., about 20 to about 100 cells, about 30 to about 100 cells, about 40 to about 100 cells, or more than 50 cells) may be particularly suitable for mimicking the tissue composition within each MOS because the MOSs may contain different lineages - which may include epithelial cells (e.g., cancer) and mesenchymal cells (e.g., stromal cells, immune cells, or vascular cells). It should be noted that for MM MOSs, including a relatively large number of cells per MOS (e.g., about 50 to about 150 cells per MOS) may provide different cell types for each MOS, which can serve to maintain the MM cells.

[0071] The MOS can be formed in any suitable size that can match the number of cells to be included. For example, the size of the MOA can be relatively small, having a diameter of about 20 μm to about 500 μm (e.g., an average of about 50 μm or about 100 μm or about 100 μm to about 200 μm). In some cases, the size of the MOS can be about 300 μm, in which case, about 10 to about 50 cells (e.g., about 10 to about 30 units) can be contained in each MOS. The number and size of the cells can be altered and / or controlled. In some cases, the number of cells and / or the size of the MOS can be set by one or more controls of the device used to form the MOS. For example, the size of the MOS and / or the density of the cells within the MOS can be adjusted by adjusting the flow rate and / or concentration of the refined tissue sample (e.g., cells from a biopsy).

[0072] As shown in FIGS. 1, 2, 3, Figures 4A to 4E and Figures 5A to 5B as shown, even after culturing the MOS described herein, live and healthy cells are observed throughout the volume of the MOS. The size of the MOS and / or the number of cells to be contained in the MOS can be selected based on how the MOS is expected or intended to be used. For example, in a variant where the MOS is used to examine the relationships between cells in biopsy material, the MOS can be formed to contain multiple cells (e.g., multiple types of cells or multiple cells of a single type), and can be cultured for an extended period of time (e.g., up to a week or longer).

[0073] In some cases, the PDMO can be produced by mixing a dissociated or refined tissue sample (e.g., a biopsy sample) with a fluid matrix that can polymerize in a controlled manner to form the MOS. Figure 6 A representative method for forming the PDMO is shown. Optionally, the method can include obtaining a sample from a mammal (e.g., a human with MM), such as obtaining a biopsy from patient tissue 601. A biopsy can be obtained using, for example, a biopsy needle or punch. For example, in some cases, a 14-gauge, 16-gauge, 18-gauge, 20-gauge, or 22-gauge needle inserted into a mammal to remove a biopsy can be used for a bone marrow biopsy. After removing the tissue from the mammal, the tissue can be processed to dissociate the cells and other materials (e.g., in the case of a solid tumor sample) or to refine the sample by removing certain types of cells (e.g., in the case of a bone marrow aspirate), using mechanical and / or chemical techniques. The dissociated or refined cells can be immediately used to form the PDMO as described herein, or in some cases, all or some of the cells can be modified, such as by genetically modifying the cells 603 (e.g., using transfection or electroporation to introduce one or more proteins or nucleic acids that will result in gene modification).

[0074] Further reference Figure 6, a dissociated or refined tissue sample from a biopsy material can be mixed with a fluid (e.g., liquid) matrix material to form an unpolymerized mixture 605. The unpolymerized mixture can be maintained in an unpolymerized state such that cells from the dissociated or refined tissue remain suspended within the mixture. In some cases, by keeping the cells frozen (e.g., at room temperature or lower temperature, such as a temperature of 1°C to 25°C), the cells can remain viable and unpolymerized.

[0075] The unpolymerized mixture can then be dispensed as droplets into an immiscible material (such as oil) such that the formation of the droplet size is controlled and thus the size of the formed PDMO 607 is controlled. For example, uniformly sized droplets can be formed by combining a stream of the unpolymerized material with one or more (e.g., two converging) streams of the immiscible material (e.g., oil) such that the flow rate and / or pressure of the two streams can determine how droplets of the unpolymerized material are formed when the unpolymerized material intersects the immiscible material. The droplets can be polymerized 609 to form PDMOs in the immiscible material. In some cases, the immiscible material can be heated or warmed to a temperature that causes the unpolymerized mixture (e.g., the fluid matrix material in the unpolymerized material) to polymerize. After formation, the PDMOs can be separated from the immiscible fluid. For example, the PDMOs can be washed to remove the immiscible fluid 611 and placed in a culture medium to allow the cells within the PDMOs to grow. The PDMOs can be cultured for any desired time, or can be cryopreserved, or can be assayed immediately. In some cases, the PDMOs can be cultured for a period of time (e.g., 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, 1 to 9 days, 1 to 10 days, 1 to 11 days, or 1 to 14 days). Culturing the PDMOs can allow the cells derived from the biopsy tissue to grow and / or divide (e.g., double) up to five or six passages. After culturing, the cells can be cryopreserved 615 and / or assayed 617. Examples of assays that can be used are also described herein.

[0076] In any of the methods and devices described herein, MOS can be recovered from the immiscible fluid (e.g., oil) after polymerization. For example, in some cases, MOS can be recovered by demulsification and / or reverse emulsification, such as by forming emulsified droplets and recovering the MOS after droplet formation to remove any oil (and other contaminants). This can allow the cells to grow within the polymerized droplets (MOS) without being inhibited by the immiscible fluid.

[0077] Although the methods and devices described herein illustrate a method of forming multiple droplets and thus multiple MOSs by flowing an unaggregated mixture stream into one or more streams of immiscible fluid (such as oil or other hydrophobic material), in some cases, droplets may be formed by other methods that allow control of the droplet size as described herein. For example, in some cases, droplets may be formed by printing (e.g., by printing droplets onto a surface). This can reduce or eliminate the need for additional emulsification / demulsification recovery steps. For example, droplets may be printed onto a surface (such as a flat or shaped surface) and polymerized. In any of these variants, pressure, sound, charge, or any other suitable means may be used to dispense the droplets. In some cases, an automated dispenser (e.g., a pipetting device) may be used to form droplets, which is adapted to release a small amount of unaggregated mixture onto a surface, into the air, and / or into a liquid medium (such as an immiscible fluid).

[0078] The method for forming PDMO can be automated and / or carried out using one or more devices. Specifically, the method for forming PDMO can be carried out by a device that allows selection and / or control of the size of MOSs and thus selection and / or control of the density of the number of cells. For example, Figure 7A An example of a device 700 for forming PDMO as described herein is shown.

[0079] As Figure 7A depicted, the device may include an input for receiving an unaggregated mixture of dissociated or refined tissue samples and a fluid matrix material (already mixed), or inputs for separately receiving dissociated or refined tissue samples (e.g., in a holding solution) and a fluid matrix material. In some cases, the device may include a holding chamber 706 for holding the unaggregated mixture and / or a holding chamber (not shown) for holding dissociated or refined tissue (e.g., biopsy) samples and holding the fluid matrix material. Any or all of these holding chambers may be pressurized to control and / or accelerate the flow of fluid out of these chambers and into the device. The device may receive the unaggregated mixture, or the device may receive the components and mix them. In some cases, the device may control the concentration of cells in the unaggregated mixture and may dilute the mixture (e.g., by adding additional fluid matrix material to achieve the desired density). For example, the device may include a sensor (e.g., an optical reader) for reading the density of cells (e.g., optical density) in the unaggregated mixture. The sensor may also be coupled to a controller 724, which may automatically or semi-automatically (e.g., by instructing the user) control the dilution of cells in the unaggregated mixture. The device may also include a port for receiving the unaggregated mixture. The port may include a valve or be coupled to a valve, and the valve may be controlled by the controller 724 or by a separate controller.

[0080] Device 700 may include a chamber 708 and / or ports for holding and / or receiving immiscible fluids. In some cases, immiscible fluids may be held in a pressurized chamber such that the flow rate can be controlled. Any of the pressurized chambers may be controlled by a controller 724, which may use one or more pumps 726 to control the pressure and thus the flow through the device. One or more pressure and / or flow sensors may be included in the system to monitor the flow through the device.

[0081] In Figure 7A it, the entire device 700 may be encapsulated in a housing 702, or a portion of the device 704 may be encapsulated in a housing. In some cases, the housing may include one or more openings or access portions on the device, such as for adding immiscible fluids and / or unpolymerized mixtures.

[0082] As described above, device 700 may also include one or more sensors 728 for monitoring all or critical parts of the manufacturing process. In some cases, one or more sensors may include one or more optical sensors, mechanical sensors, voltage and / or resistance (or capacitance or inductance) sensors, or force sensors. The sensors may be used to monitor the continuous operation of the components, including the formation of PDMO. Device 700 may also include one or more heat / temperature regulators 718 for controlling the temperature of the immiscible fluids and / or unpolymerized mixtures and / or fluid matrix materials.

[0083] Device 700 may also include one or more droplet formation components 720, which may be monitored (e.g., using one or more sensors), as Figure 7C and Figure 9 shown and discussed below. The droplet MOS formation component may include a dispenser (e.g., a PMOS dispenser) 722 or be coupled thereto. The dispenser may dispense droplets into, for example, a porous plate 716.

[0084] Generally, the droplet MOS formation component 720 may include one or more microfluidic chips 730 or structures for forming and controlling the flow of unpolymerized mixtures and forming actual droplets. Figure 7B An example of a microfluidic chip for forming PDMO 730 is shown. In Figure 7B it, the chip 730 includes a pair of parallel structures for forming MOS. Figure 7C The droplet formation region of a microfluidic chip for forming PMOS is shown, including an unpolymerized channel outlet 741, which opens (at a right angle in this example) into a "+" node or intersection region 737 leading to the channel outlet 741 and immiscible fluid outlets 743, 743'. In some cases, the input from one or more immiscible fluid channels may be at an angle relative to the angle (and intersection point) with the unpolymerized material. In Figure 7CIn this case, like all the figures showing dimensions in this specification, the dimensions shown are merely exemplary and are not intended to be limiting, unless otherwise specified.

[0085] In Figure 7A the microfluidic chip 730 includes an inlet (input port) 733 for immiscible fluids to enter the chip (e.g., from the inlet port or reservoir shown in Figure 7A ). A second inlet port 735 into the chip can be configured to receive the unpolymerized material and convey it along a semi-tortuous path to the node region. Similarly, the inlet port for immiscible fluids can be securely coupled to the outlet or inlet from the immiscible fluid chamber, as described above.

[0086] The inlet port 735 for the unpolymerized material to enter the chip can be coupled (as shown in Figure 7C ) through a delivery pathway 741 that connects the inlet to the node region. Similarly, the inlet 733 for immiscible fluids can be connected to two or more connection paths 743, 743' to reach the node region 737. The channels leaving the node region 737 can enable the formed MOS (in the immiscible fluid) to be transported downward along the channel to the outlet 731, which can be connected to a dispenser for dispensing the MOS into one or more chambers (e.g., for culturing and / or assay).

[0087] In Figure 7B and Figure 7C the example shown, the formed droplets that can become MOS once polymerized can be transported along a long, temperature-controlled microfluidic environment before being dispensed from the device. For example, Figure 8 shows an example of a channel region 839 shown as transparent (e.g., element 739 in Figure 7B ), which contains a plurality of MOS 803, each MOS containing a predetermined number of cells 805. It should be noted that in any of the microfluidic chips or devices described herein, the channels can be coated. For example, the channels of the microfluidic device can be coated with a hydrophobic material.

[0088] Figure 9 depicts a node region 937 shaped as described above such that the channel carrying the unpolymerized mixture 911 intersects one or more (e.g., two) channels 909 carrying a fluid (such as oil) immiscible with the unpolymerized mixture. When the unpolymerized mixture is pressured to flow out of the first channel 911 at a first rate, the immiscible fluid flowing in the cross channels 909, 909' allows a predetermined amount of the unpolymerized mixture to pass through and then pinches it off to form droplets 903 that are conveyed into the outlet channel 939. Thus, in some variations, a minced (e.g., dissociated) clinical (e.g., biopsy or excised) tissue sample having a size, for example, less than 1 mm in diameter can be combined with a temperature-sensitive gel (e.g., at 4 °C ) Mix to form an unpolymerized mixture. The unpolymerized mixture can be placed in a microfluidic device that produces droplets (e.g., water-in-oil droplets) with uniform volume and material composition. At the same time, dissociated tumor cells can be dispensed into these droplets. The gel in the unpolymerized material can solidify upon heating (e.g., 37 °C) and can form the resulting PDMO. In some cases, this method can be used to produce at least 10,000 (e.g., at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000) uniform droplets (PDMO) from tissue (e.g., biopsy material). These PDMOs are compatible with traditional 3D cell culture techniques. Figure 10 Figure 1005 shows a plurality of PDMOs formed as described above, suspended in an immiscible material 1008 (e.g., oil).

[0089] In the exemplary microfluidic chip shown above, the junctions are shown as T- or X-junctions, where the flow focusing of the microfluidics forms MOSs of a controllable size. In some cases, in addition to the microfluidic chip, droplets can be formed by robotic micro-pipetting, e.g., into an immiscible fluid and / or onto a solid or gel substrate. Alternatively, in some cases, droplets of the unpolymerized material can be generated through a micro-capillary to form with the desired size and reproducibility. Other examples of techniques that can alternatively be used to form MOSs of a specified size range and reproducibility from the unpolymerized material include colloidal manipulation, e.g., via external forces such as acoustic, magnetic, inertial, electrowetting, or gravity.

[0090] Figure 11A and Figure 11B Figure 12 shows an example of PDMOs in oil formed as described above. The cells within these MOSs derived from a single biopsy sample are alive, as visible by live dye staining ( Figures 15A to 15B and Figures 16A to 16B ). For example, Figure 12A and Figure 12B show tumor cells containing MOSs (similar to those shown in Figure 11A and Figure 11B ), which can be washed to remove the immiscible material (e.g., oil). This immiscible material can be removed relatively quickly after the formation of MOSs to prevent damage to the cells within the MOSs.

[0091] In these examples, the gel droplets can be recovered from the oil phase and resuspended in, e.g., PBS via PFO (perfluorooctanol). Centrifugation can be used to separate the immiscible fluid from the MOSs. Then the MOSs can be allowed to grow, as shown in Figures 1, 2, 3, Figures 4A to 4E and Figure 13As shown. This is important because drug screening must be performed on primary tumor cells that are viable and growing, and these cells retain the characteristics of the patient's tumor to predict the patient's outcome. The large number and uniformity of these MOSs make the screening both feasible and reliable, as described below.

[0092] Generally, the MOSs described herein are highly uniform in diameter and can have extremely low size (e.g., diameter) variation. For example, this is shown in Figure 14 a representative distribution of droplet diameter sizes.

[0093] Figure 15A and Figure 15B show another population of MOSs formed as described herein. In Figure 16A and Figure 16B these MOSs are stained with trypan blue (arrows) to show that they are alive. In some cases, MOSs formed as droplets as described herein can contain growth factors and matrix components to mimic the biological environment in which the cells in the MOSs are located. Mammalian samples (e.g., patient biopsy samples) can generally form MOSs within a few hours (e.g., about 6 to 12 hours, about 12 to 18 hours, about 18 to 24 hours, about 12 hours, about 18 hours, about 24 hours, or about 48 hours) after tissue acquisition. Each MOS can contain only a few cells (e.g., as few as 1 cell or about 4 to 6 cells, such as cancer cells when the tumor is sampled), or each MOS can contain more cells (e.g., 50 to 150 cells in the case of MM MOSs). Methods similar to those described herein have been shown to generate MOSs from a variety of types of cancer and non-cancer tissues, including colon, esophagus, melanoma, uterus, sarcoma, kidney, liver, ovary, lung, diaphragm, omentum, mediastinal lung, and breast cancer tissues (see, for example, the working examples of U.S. Publication No. 2021 / 0285054, which is incorporated herein by reference in its entirety). MOSs can be cultured for any desired period of time and typically proliferate and grow in as little as 3 to 4 days. MOSs can be maintained and passaged for months. As will be described in more detail below, MOSs can also be used to screen drug compositions as little as 4 to 14 days (e.g., 4 to 6 days, 5 to 7 days, 6 to 8 days, 7 to 10 days, 8 to 12 days, or 10 to 14 days) after tissue acquisition (e.g., biopsy).

[0094] The MOSs described herein can be stored at any time after they are formed, such as by cryopreservation. Tumor MOSs can be collected from many different patients and can be used individually or together to screen a variety of pharmaceutical formulations to determine the toxicity and / or efficacy of a particular therapeutic agent. In some cases, non-tumor cells (healthy tissue) can be biopsied, striped, and / or screened in parallel. Thus, the methods and devices provided herein can allow for high-throughput screening. In some variants, MOSs can be formed, allowed to passage twice (e.g., double in number twice), and cryopreserved. Similarly, normal, healthy tissue can also be used to form corresponding MOSs to generate hundreds, thousands, or tens of thousands of MOSs that can be used to determine drug effects, drug responses, biomarkers, proteomic signals, genomic signals, etc.

[0095] Of particular importance is that the MOSs survive in a biologically meaningful way, allowing them to provide clinically and physiologically relevant data, particularly regarding drug response. In particular, the MOSs described herein allow cells from tissue extracts / biopsies to grow extremely well and provide more representative data, especially as compared to organoids or spheroids. Without being limited to a particular mechanism, this may be because the cells in the MOSs may have a more restricted cell density, thus allowing the cells to communicate without mutual inhibition while sharing signals. The MOSs also have a very large surface-to-volume ratio, making it easier to allow the transport of growth factors and other signals to penetrate into the MOSs (e.g., the diffusion limitation of the MOSs is less).

[0096] The PDMOs described herein (e.g., MM MOSs) can be used in a variety of different assays and, in particular, can be used to determine the effect of a pharmaceutical formulation on MM tissue, including toxicity. As used herein, a pharmaceutical composition can include any drug, drug diluent, pharmaceutical formulation, composition containing multiple drugs (e.g., multiple active components), pharmaceutical formulation, drug form, drug concentration, combination therapy, etc. In some cases, a pharmaceutical formulation refers to a preparation containing a mixture of a drug and one or more inactive ingredients.

[0097] In some cases, drug screening may include applying MOS to all or some of the wells of a porous (e.g., 96-well) plate. Alternatively, a custom plate may be used (e.g., a 10,000 micro-well array can be formed by 100 x 100 wells). MOS (e.g., gel droplets) may be applied to multiple micro-well arrays or, in some cases, onto multiple micro-well arrays and incubated with a culture medium. MOS may be cultured for a process of about 3 to about 14 days. In some cases, on a selected day (e.g., day 5), a drug compound may be administered to the wells (e.g., microreactors), such as based on a set of FDA-approved anti-cancer drugs, such as drugs for treating MM, to examine the effects of the drug set. For example, the drugs being tested may be based on the screening of the National Cancer Institute (Division of Cancer Treatment and Diagnosis), which contains 147 agents and aims to achieve cancer research, drug discovery, and combination drug research. In some cases, the drugs being tested may include one or more agents selected from the following: immunomodulators (e.g., IMiD, thalidomide, lenalidomide, and pomalidomide), PIs (e.g., bortezomib, carfilzomib, and ixazomib), monoclonal antibodies (e.g., elotuzumab, daratumumab, isatuximab, and belantamab), nuclear export protein inhibitors (e.g., selinexor), doxorubicin, panobinostat, and melphalan, steroids (e.g., dexamethasone), alkylating agents (e.g., cyclophosphamide, melphalan, and bendamustine), CAR T cell therapies (e.g., Abecma and Ide-cel), and bispecific antibodies. On a subsequently selected day (e.g., day 7), the MOS may be imaged (e.g., via a standard fluorescence microscope) and ranked for drug response. In some cases, flow-based viable myeloma cell counting and any appropriate number of drug titrations (e.g., three, four, five, six, seven, eight, nine, ten, three to five, five to seven, or seven to nine titrations) and the number of repeats for each titration (e.g., two to four, two to three, three to five, or three to four repeats per titration) may be used to generate a drug IC 50 curve.

[0098] After treating MOS with one or more potential therapeutic agents, the cells therein can be evaluated to determine the level of myeloma cell killing, thereby indicating the efficacy of the one or more therapeutic agents being tested. Any suitable technique can be used to remove cells from MOS. For example, MOS can be heated to melt the substrate material (e.g., hydrogel). The cells can then be stained with antibodies specific for the biomarker to be detected. For example, antibodies against CD19, CD38, and CD58 (and optionally CD138) can be used to stain myeloma cells within the cell population. The cells can also be stained with live cell markers, dead cell markers, or both live cell markers and dead cell markers. Non-limiting examples of markers that can be used to distinguish live cells from dead cells include GHOST DYE TM ( Biosciences; Fremont, CA), LIVE / DEAD Fixable Dead Cell Stain (ThermoFisher Scientific; Waltham, MA), propidium iodide, Zombie dye (BioLegend; San Diego, CA), Phantom dye (Proteintech; Rosemont, IL), and HORIZON TM Dye (Becton Dickinson; Franklin Lakes, NJ). After labeling, the cells can be subjected to flow cytometry to detect and quantify live and / or dead cells of the target type. It should be noted that the above assays can be performed at the well level to evaluate the effect of one or more therapeutic agents on the cells within all MOS in a well or on individual MOS.

[0099] In Figures 17A to 17E an example of an assay / screening technique is shown. In this example, the screening assay can be automated, which can enable a repeatable and automated workflow, thereby increasing the number of drugs that can be tested in the screen. In Figure 17A a tumor biopsy is performed and multiple (e.g., >10,000) MOS are formed as described above (in Figure 17A the nodal region where MOS formation is shown). Thereafter, the MOS can be recovered and washed to remove immiscible materials (e.g., oil) used to form them. The MOS can then be plated into one or more microtiter plates. As Figure 17C shown, the MOS can be cultured for one or more generations (e.g., one or more passages). This is shown to occur from day 0 to day 3, day 4, or day 5. Thereafter, the MOS can be screened as Figure 17D shown, such as by applying drugs to a subset of replicate wells. Subsequently, as Figure 17E shown, the cells in the MOS can be imaged and / or automatically or manually scored to identify drug effects (e.g., drug screening and growth profiles).

[0100] Figures 17A to 17E The workflows shown can enable the integrated device to be used for growing, dosing, and / or examining MOS. In an exemplary device, a fresh biopsy or excised patient tumor sample can be dissociated and seeded into a gel with reagents to form MO as described herein. In some cases, a portion of the formed MOS can be cryopreserved. The remainder can be retrieved and incubated until seeded into a microplate for drug testing or screening as described. Growth and viability assays can be performed on the MOS, which can be imaged and tracked. The response of the MOS to drug treatment, such as IC 50 , cytotoxicity, and growth curves, can be measured to identify effective therapeutic agents against the patient's tumor (e.g., MM).

[0101] The methods and devices described herein have many advantages, including reproducibility. The sample preparation process can be automated through microfluidic sample dispensing, which can reduce the need for professionals to perform diagnostic tests and manual pipetting. This can be particularly helpful in a clinical setting. In addition, this can achieve uniformity between signal droplets, thereby improving assay sensitivity. Additionally, these assays can maximize the time required to generate MOS. In some cases, these methods can be used to generate more than 100,000 -tumor droplets (MOS) libraries in less than about 15 minutes. These methods are also highly scalable and can be multiplexed to run multiple patient biopsies in parallel.

[0102] In addition, the methods described herein are flexible and compatible with other technologies. As a research tool, for example, droplet-based microfluidics is generally compatible with a wide range of hydrogel materials, such as agarose, alginate, PEG, and hyaluronic acid. Thus, the starting gel composition can be easily modified to accompany and promote MOS growth. In addition, the droplet size can be adjusted by changing the size of the microfluidic device. In summary, these options allow for the selection of a large number of gel material compositions and microreactor sizes.

[0103] The miniaturized assays (using MOS) described herein can maximize the utility of patient tumor biopsies, enabling screening of a greater number of drug compounds. For example, a 600 μL bone marrow sample from a mammal with MM can be divided into approximately 143,000 individual microreactors, each with a volume of approximately 4 nL. By maximizing tissue samples, multiple experimental replicates can be examined, thereby increasing statistical power. These techniques can be used to examine intra-tumor heterogeneity, drug perturbations, and can identify rare cell events, such as drug resistance. MOS is generally compatible with downstream assays, such as single cell RNA transcriptome analysis and epigenetic analysis. Additionally, by maximizing the efficiency of tissue (e.g., biopsy) samples provided by MOS, a portion of the MOS can be stored (e.g., cryopreserved) for future novel drug assays and / or confirmatory analyses, including gene screening.

[0104] Figure 18 and Figure 19 Illustrations of methods of treatment using the methods and devices (including MOS) described herein are provided. For precision and personalized medicine, these methods and devices can be used as clinical metrics for selecting appropriate drugs to improve clinical outcomes and drug response. In some embodiments, a patient diagnosed with cancer (e.g., MM) can be biopsied for histopathology and multiple MOS formed from the biopsy can be screened using the methods described herein. Within approximately 7 to 10 days, screening can be performed on the biopsy to identify the most effective standard of care therapy so that the patient can begin treatment in approximately 14 days.

[0105] Figure 18 An example of this is shown. In this example, a tumor 1801 can be identified on day 0 (e.g., by CT scan), a biopsy 1803 can be performed on day 5, and hundreds, thousands, or tens of thousands of MOS can be generated on the same day. The MOS can be cultured for approximately 1 to 5 days and then screened 1805 to identify one or more drug compositions that can be used. The same steps (forming MOS and screening) can be used to guide precision medicine at multiple clinical decision points throughout the disease progression. In this example, therapy using the identified one or more drug compositions can begin on day 14 1809, and the patient can later be monitored (e.g., at a follow-up CT scan at approximately day 90) during the course of treatment to confirm that the tumor is responsive to the treatment 1811. If so, treatment can continue 1813 and ongoing progression can be monitored 1815.

[0106] Throughout the course of a patient's treatment, MOS can be reused for assays at multiple points. This is in Figure 19is shown. For example, when a patient is first diagnosed with a resectable primary tumor 1907, the technology (e.g., generation and screening of MOS 1905) can be used to determine the most effective neoadjuvant therapy 1921. Thus, a biopsy can be obtained, and hundreds, thousands, or tens of thousands of MOS can be formed and screened using a panel of potential drug compositions. Once the primary tumor is resected 1923, this technology 1905' can indicate whether adjuvant therapy should be selected and which adjuvant therapy should be selected 1925. If recurrence or metastasis occurs after surgical resection of the primary tumor 1927, the same technology (e.g., generation and screening of MOS from fresh biopsies 1905", 1905"', 1905"") can be used to guide standard of care therapies, including first-line 1929, second-line 1931, and third-line 1933 therapies. If the patient ultimately becomes refractory or resistant to all standard of care therapies, this technology 1905""' can be performed to identify off-label drugs to treat drug-resistant tumors 1935. The technology can also be used as a companion diagnostic to identify patients for specific therapies. Finally, the technology can be used to derive and preserve patient-derived MOS to establish an organoid-based living cancer bank for screening, genomic analysis, new drug discovery, drug testing, and / or clinical trial design.

[0107] Because the generation of a large number of MOS can be performed relatively minimally invasively (e.g., by resection or biopsy) and can be used to provide fairly rapid screening results, the methods provided herein can be readily adapted to the standard of care. For example, the volume of cellular material from a tissue (e.g., biopsy) input is generally rather small and can be placed in a volume, e.g., of about 10 μL to about 5 mL.

[0108] Generally speaking, screening using the MOS described herein can be performed automatically or manually. In fact, any screening technique can be used, including imaging by one or more of the following: confocal microscopy, fluorescence microscopy, liquid lenses, holography, sonar, brightfield and darkfield imaging, lasers, planar laser sheets, and image-based high-throughput assay analysis methods (e.g., using computer vision and / or supervised or unsupervised models such as CNN). Downstream screening can include sampling the culture medium and / or performing genetic or protein screening on cells from the MOS (e.g., scRNA-seq, ATAC-seq, proteomics, etc.).

[0109] Exemplary embodiments

[0110] Example 1 is a micro-organoid comprising bone marrow cells from a mammal suffering from multiple myeloma (MM).

[0111] Example 2 is the micro-organoid according to Example 1, wherein the micro-organoid comprises from about 50 to about 150 cells.

[0112] Example 3 is the micro-organ organoid according to Example 1, wherein the micro-organ organoid comprises from about 75 to about 125 cells.

[0113] Example 4 is the micro-organ organoid according to Example 1, wherein the micro-organ organoid comprises about 100 cells.

[0114] Example 5 is the micro-organ organoid according to any one of Examples 1 to 4, wherein the cells include cancer cells, stromal cells, stem cells, immune cells, or any combination thereof.

[0115] Example 6 is the micro-organ organoid according to Example 5, wherein the micro-organ organoid comprises a ratio of cancer cells to stromal cells of less than about 1:4.

[0116] Example 7 is the micro-organ organoid according to Example 5, wherein the immune cells include at least one macrophage.

[0117] Example 8 is the micro-organ organoid according to any one of Examples 1 to 6, wherein the micro-organ organoid comprises a dissolved basement membrane matrix.

[0118] Example 9 is a composition comprising the micro-organ organoid according to any one of Examples 1 to 8 in a culture medium.

[0119] Example 10 is the composition according to Example 9, wherein the culture medium comprises a dissolved basement membrane matrix.

[0120] Example 11 is the composition according to Example 10, which comprises about 1% of the dissolved basement membrane matrix.

[0121] Example 12 is the composition according to any one of Examples 9 to 11, which further comprises an immiscible fluid.

[0122] Example 13 is the micro-organ organoid according to Example 12, wherein the immiscible fluid is oil.

[0123] Example 14 is a method for manufacturing a micro-organ organoid (MOS), wherein the method comprises:

[0124] Receiving a bone marrow sample from a mammal suffering from MM;

[0125] Refining the bone marrow sample to form a refined sample; and

[0126] Forming a population of MOS from the refined sample by:

[0127] Driving an unaggregated fluid mixture through one or more channels of a microfluidic device, wherein the unaggregated fluid mixture comprises the refined sample and an unaggregated fluid matrix material, and wherein the microfluidic device controls the pressure, flow rate, or both the pressure and flow rate within the one or more channels such that the refined sample and the unaggregated fluid matrix material travel through the one or more channels in a laminar flow manner,

[0128] Forming a plurality of droplets containing the unaggregated fluid mixture within the microfluidic device, and polymerizing the fluid matrix material to form the MOSs, wherein the MOSs each have a diameter between 50 μm and 500 μm and wherein between 30 and 150 cells are distributed therein.

[0129] Example 15 is the method according to Example 14, further comprising driving an immiscible fluid through another channel of the microfluidic device such that the immiscible fluid combines with the unaggregated fluid mixture before the formation of the plurality of droplets, wherein the droplets contain the unaggregated fluid mixture and the immiscible fluid.

[0130] Example 16 is the method according to Example 15, wherein the immiscible fluid is oil.

[0131] Example 17 is the method according to any one of Examples 14 to 16, wherein forming the population of MOSs further comprises sorting the MOSs based on cell number and / or droplet size.

[0132] Example 18 is the method according to Example 17, wherein the sorting comprises optical sorting based on cell number and / or droplet size.

[0133] Example 19 is the method according to any one of Examples 14 to 18, wherein forming the population of MOSs comprises forming from about 100 to about 600 MOSs.

[0134] Example 20 is the method according to any one of Examples 14 to 18, wherein forming the population of MOSs comprises forming from about 600 to about 1,000 MOSs.

[0135] Example 21 is the method according to any one of Examples 14 to 18, wherein forming the population of MOSs comprises forming more than about 1,000 MOSs.

[0136] Example 22 is the method according to any one of Examples 14 to 21, wherein the microfluidic device maintains the viscosity of the unaggregated fluid mixture before the formation of the plurality of droplets.

[0137] Example 23 is the method according to any one of Examples 14 to 22, wherein the microfluidic device is configured to prevent clogging of the unpolymerized fluid mixture within the one or more channels.

[0138] Example 24 is the method according to Example 23, wherein the microfluidic device is configured to prevent clogging by having a channel diameter of 100 μm or greater.

[0139] Example 25 is the method according to any one of Examples 14 to 24, wherein the microfluidic device is configured to maintain an approximately constant pressure within the one or more channels.

[0140] Example 26 is the method according to any one of Examples 14 to 25, wherein the microfluidic device maintains a constant flow rate within the one or more channels.

[0141] Example 27 is the method according to any one of Examples 14 to 26, wherein the total length of the path taken by the unpolymerized fluid mixture before forming the plurality of droplets within the microfluidic device is less than 10 cm.

[0142] Example 28 is the method according to any one of Examples 14 to 27, wherein the MOS in the MOS population vary in size by less than 25%.

[0143] Example 29 is the method according to any one of Examples 14 to 28, wherein the polymerization includes crosslinking the fluid matrix material.

[0144] Example 30 is the method according to any one of Examples 14 to 29, wherein the fluid matrix material is chemically crosslinkable or photocrosslinkable.

[0145] Example 31 is the method according to any one of Examples 14 to 30, wherein the bone marrow sample includes freshly biopsied cells.

[0146] Example 32 is the method according to Example 31, wherein the bone marrow sample is obtained from the mammal within 24 hours after forming the MOS.

[0147] Example 33 is the method according to any one of Examples 14 to 32, wherein the bone marrow sample includes MM plasma cells, immune cells, stem cells, stromal cells, or any combination thereof.

[0148] Example 34 is the method according to Example 33, wherein the immune cells include one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof.

[0149] Example 35 is the method according to any one of Examples 14 to 34, which includes flowing the unpolymerized fluid mixture through the one or more channels at a flow rate of about 0.01 milliliters (mL) per minute (min) to about 100 mL / min.

[0150] Example 36 is a method for precision drug screening for personalized cancer therapy for MM, wherein the method includes:

[0151] receiving a bone marrow sample from a mammal suffering from MM;

[0152] refining the sample to form a refined sample;

[0153] forming a MOS population from the refined sample by:

[0154] driving an unpolymerized fluid mixture through one or more channels of a microfluidic device, wherein the unpolymerized fluid mixture contains the refined sample and an unpolymerized fluid matrix material, and wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the refined sample and the unpolymerized fluid matrix material travel through the one or more channels in a laminar flow manner,

[0155] forming a plurality of droplets containing the unpolymerized fluid mixture within the microfluidic device, and polymerizing the fluid matrix material to form the MOS, wherein each of the MOS has a diameter between 50 μm and 500 μm and contains between 1 and 500 cells distributed therein;

[0156] culturing the MOS population for between 1 - 14 days; and

[0157] determining one or more drug therapies using the MOS population.

[0158] Example 37 is the method according to Example 36, which further includes driving an immiscible fluid through another channel of the microfluidic device such that the immiscible fluid combines with the unpolymerized fluid mixture before the formation of the plurality of droplets, wherein the droplets contain the unpolymerized fluid mixture and the immiscible fluid.

[0159] Example 38 is the method according to Example 37, wherein the immiscible fluid is oil.

[0160] Example 39 is the method according to any one of Examples 36 to 38, wherein the determination includes parallel determination of multiple drug therapies by exposing one or more of the MOS to each drug therapy.

[0161] Example 40 is the method according to Example 39, which includes characterizing the response of the MOS to each of the multiple drug therapies based on the response of the MOS to exposure to the multiple drug therapies.

[0162] Example 41 is the method according to any one of Examples 36 to 40, wherein the time between receiving the bone marrow sample and characterizing the response is less than 21 days.

[0163] Example 42 is the method according to any one of Examples 36 to 41, wherein forming the MOS population further includes sorting the MOS based on cell number and / or droplet size.

[0164] Example 43 is the method according to any one of Examples 36 to 42, wherein the sorting includes optically sorting the MOS or based on cell number and / or droplet size.

[0165] Example 44 is the method according to any one of Examples 36 to 43, wherein the assay includes assaying more than 10 different drug therapies.

[0166] Example 45 is the method according to any one of Examples 36 to 44, wherein the one or more drug therapies include different concentrations of one or more drugs, different combinations of two or more drugs, different ratios of two or more drugs, different carriers for one or more drugs, and / or different dosing times for one or more drugs.

[0167] Example 46 is the method according to any one of Examples 36 to 45, wherein forming the MOS population includes forming from about 100 to about 600 MOS.

[0168] Example 47 is the method according to any one of Examples 36 to 45, wherein forming the MOS population includes forming from about 600 to about 1,000 MOS.

[0169] Example 48 is the method according to any one of Examples 36 to 45, wherein forming the MOS population includes forming more than 1,000 MOS.

[0170] Example 49 is the method according to any one of Examples 36 to 48, wherein the microfluidic device maintains the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets.

[0171] Example 50 is the method according to any one of Examples 36 to 49, wherein the microfluidic device is configured to prevent clogging of the unpolymerized fluid mixture within the one or more channels.

[0172] Example 51 is the method according to Example 50, wherein the microfluidic device is configured to prevent clogging by having a channel diameter of 100 μm or greater.

[0173] Example 52 is the method according to any one of Examples 36 to 51, wherein the microfluidic device is configured to maintain an approximately constant pressure within the one or more channels.

[0174] Example 53 is the method according to any one of Examples 36 to 52, wherein the microfluidic device maintains a constant flow rate within the one or more channels.

[0175] Example 54 is the method according to any one of Examples 36 to 53, wherein the total length of the path taken by the unpolymerized fluid mixture before the formation of the plurality of droplets within the microfluidic device is less than 10 cm.

[0176] Example 55 is the method according to any one of Examples 36 to 54, further comprising measuring the effect of the one or more drug therapies on the cells within the MOS.

[0177] Example 56 is the method according to any one of Examples 36 to 55, further comprising determining that the mammal remains responsive to one of the one or more drug therapies after one or more administrations of the drug therapy by receiving a second bone marrow sample after treating the mammal with the drug therapy and forming a second MOS population from the second bone marrow sample, exposing at least some of the second MOS population to the drug therapy, and measuring the effect of the drug therapy on the cells within at least some of the second MOS population.

[0178] Example 57 is the method according to any one of Examples 36 to 56, further comprising treating the mammal with one of the one or more drug therapies.

[0179] Example 58 is the method according to any one of Examples 36 to 57, wherein the MOS within the MOS population vary in size by less than 25%.

[0180] Example 59 is the method according to any one of Examples 36 to 58, wherein the polymerization comprises crosslinking the fluid matrix material.

[0181] Example 60 is the method according to any one of Examples 36 to 59, wherein the fluid matrix material is chemically crosslinkable or photocrosslinkable.

[0182] Example 61 is the method according to any one of Examples 36 to 60, wherein the bone marrow sample comprises cells from a fresh biopsy.

[0183] Example 62 is the method according to Example 61, wherein the bone marrow sample is obtained from the mammal within 24 hours after forming the MOS.

[0184] Example 63 is the method according to any one of Examples 36 to 62, wherein the bone marrow sample comprises cancer cells, immune cells, stem cells, stromal cells, or any combination thereof.

[0185] Example 64 is the method according to Example 63, wherein the immune cells comprise one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof.

[0186] Example 65 is the method according to any one of Examples 36 to 64, which comprises flowing the refined bone marrow sample and the unpolymerized fluid matrix through the one or more channels at a flow rate of about 0.01 mL / min to about 100 mL / min.

[0187] The present invention will be further described in the following examples, which do not limit the scope of the present invention described in the claims.

[0188] Examples

[0189] Example 1 - Preparation of MM MOS

[0190] Cells were isolated from fresh bone marrow samples (1 - 3 mL each) biopsied from MM patients. Briefly, red blood cells in the bone marrow samples were lysed, and 70% was added to the bone marrow cell pellet. MM MOS were generated at a density of 30 cells per MOS. The MOS medium [RPMI, 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 200 ng / mL granulocyte macrophage colony-stimulating factor (GM-CSF), and 100 ng / mL interleukin 6 (IL-6)] was changed twice a week. Figure 20A Representative images showing MM MOS formation on days 1, 6, 8, and 11 are shown, where white arrows indicate MM MOS. MM MOS on day 11 were subjected to flow cytometry to determine which cell populations were present. However, CD138+ cells isolated from bone marrow cells using bead selection did not form MOS ( Figure 20B ), indicating that the presence of stromal cells is required for MOS formation.

[0191] As Figure 21AAs shown, after 9 days in vitro, the major bone marrow cell population was maintained in the established MM MOS. The myeloma cell population was shown to be CD11b- and CD38+. Flow cytometry also showed that compared with bone marrow biopsies ( Figure 21B ), the major immune cell populations were retained in the MOS at day 8 ( Figure 21C ). The change in the overall cell percentage over time during MOS culture was plotted in the graph shown in Figure 21D . Additional studies demonstrated that populations of plasma cells and MM cells (CD138 + and CD38 + ), T cells (CD3 + , CD4 + and CD8 + ), and dendritic cells (CD11b + ) were all present in the MOS ( Figure 21E ).

[0192] Figure 22A Representative images of MOS containing 50, 70, or 100 cells shown at day 0 and day 7 are included. Figure 22B Representative images of day 7 MOS with live cell staining are included.

[0193] Each MOS containing 50, 70, or 100 cells was also prepared from bone marrow biopsy samples containing approximately 8 x 10 6 cells. Representative images showing these MOS are presented in Figure 22A , and live-dead staining of representative day 7 MOS is shown in Figure 22B , demonstrating approximately 95% viability.

[0194] Example 2 - Effect of drug treatment on MM MOS

[0195] Studies were conducted to determine the effects of various MM drugs on MM MOS. For example, MM MOS at day 11 was treated with 5 μM lenalidomide or 2 nM bortezomib for 92 hours without medium change during drug treatment. The medium contained caspase 3 / 7 green dye to monitor apoptosis and fluorescence was measured. These studies demonstrated that MM MOS death increased with lenalidomide treatment but not with negative control or bortezomib treatment ( Figure 23A ). Additionally, images taken every 2 hours during treatment in revealed that MM MOS responded to lenalidomide as early as 24 hours after treatment began ( Figure 23B ).

[0196] Starting from day 9 of MOS generation from MM patient bone marrow biopsy samples, further studies were conducted using treatment with 10 μM carfilzomib or 10 μM panobinostat. Four days after treatment, a live / dead dye (calcein AM - green and ethidium homodimer - red) was added to the wells and incubated for 30 minutes. Images were taken using an EVOS TM M7000 imaging system (ThermoFisher Scientific; Waltham, MA) ( Figure 24A ). The fluorescence signal of each individual organoid on both channels was calculated using the Xilis AI algorithm. The live / dead ratio was calculated, and the dot plot data presented in Figure 24B is the average ratio for each well. A significantly lower live / dead ratio was observed in MM MOS treated with carfilzomib or panobinostat compared to the negative control.

[0197] Example 3 - MM MOS can withstand freezing and thawing

[0198] MM MOS were frozen without disrupting the structure. After two days in liquid nitrogen, the frozen MM MOS were thawed in media in a 37 °C water bath. The droplet structure was maintained after thawing ( Figure 25A ), and viability was confirmed by SYTOX TM Blue measurement via flow cytometry ( Figure 25B ).

[0199] Other embodiments

[0200] It should be understood that although the invention has been described in connection with specific descriptions of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A micro - organ spheroid comprising bone marrow cells from a mammal suffering from multiple myeloma (MM).

2. The micro - organ spheroid according to claim 1, wherein the micro - organ spheroid comprises from about 50 to about 150 cells.

3. The micro - organ spheroid according to claim 1, wherein the micro - organ spheroid comprises from about 75 to about 125 cells.

4. The micro - organ spheroid according to claim 1, wherein the micro - organ spheroid comprises about 100 cells.

5. The micro - organ spheroid according to claim 1, wherein the cells include cancer cells, stromal cells, stem cells, immune cells or any combination thereof.

6. The micro - organ spheroid according to claim 5, wherein the micro - organ spheroid comprises a ratio of cancer cells to stromal cells of less than about 1:

4.

7. The micro - organ spheroid according to claim 5, wherein the immune cells include at least one macrophage.

8. The micro - organ spheroid according to claim 1, wherein the micro - organ spheroid comprises a dissolved basement membrane matrix.

9. A composition comprising the micro - organ spheroid according to claim 1 in a culture medium.

10. The composition according to claim 9, wherein the culture medium comprises a dissolved basement membrane matrix.

11. The composition according to claim 10, which comprises about 1% of the dissolved basement membrane matrix.

12. The composition according to claim 9, which further comprises an immiscible fluid.

13. The micro - organ spheroid according to claim 12, wherein the immiscible fluid is oil.

14. A method for manufacturing a micro - organ spheroid (MOS), the method comprising: Receiving a bone marrow sample from a mammal suffering from MM; Refining the bone marrow sample to form a refined sample; And Forming a population of MOS from the refined sample by: Driving an unpolymerized fluid mixture through one or more channels of a microfluidic device, wherein the unpolymerized fluid mixture comprises the refined sample and an unpolymerized fluid matrix material, and wherein the microfluidic device controls the pressure, flow rate or pressure and flow rate within the one or more channels such that the refined sample and the unpolymerized fluid matrix material travel through the one or more channels in a laminar flow manner, Forming a plurality of droplets containing the unpolymerized fluid mixture within the microfluidic device, and Polymerizing the fluid matrix material to form the MOS, wherein each of the MOS has a diameter between 50 μm and 500 μm and between 30 and 150 cells are distributed therein.

15. The method according to claim 14, which further comprises driving an immiscible fluid through another channel of the microfluidic device such that the immiscible fluid combines with the unpolymerized fluid mixture before the formation of the plurality of droplets, wherein the droplets contain the unpolymerized fluid mixture and the immiscible fluid.

16. The method according to claim 15, wherein the immiscible fluid is oil.

17. The method according to claim 14, wherein forming the MOS population further comprises sorting the MOS based on cell number and / or droplet size.

18. The method according to claim 17, wherein the sorting comprises optical sorting based on cell number and / or droplet size.

19. The method according to claim 14, wherein forming the MOS population comprises forming from about 100 to about 600 MOS.

20. The method according to claim 14, wherein forming the MSO population comprises forming from about 600 to about 1,000 MOS.

21. The method according to claim 14, wherein forming the MOS population comprises forming more than about 1,000 MOS.

22. The method according to claim 14, wherein the microfluidic device maintains the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets.

23. The method according to claim 14, wherein the microfluidic device is configured to prevent clogging of the unpolymerized fluid mixture within the one or more channels.

24. The method according to claim 23, wherein the microfluidic device is configured to prevent clogging by having a channel diameter of 100 μm or greater.

25. The method according to claim 14, wherein the microfluidic device is configured to maintain an approximately constant pressure within the one or more channels.

26. The method according to claim 14, wherein the microfluidic device maintains a constant flow rate within the one or more channels.

27. The method according to claim 14, wherein the total length of the path taken by the unpolymerized fluid mixture before forming the plurality of droplets within the microfluidic device is less than 10 cm.

28. The method according to claim 14, wherein the MOS within the MOS population vary in size by less than 25%.

29. The method according to claim 14, wherein the polymerization comprises crosslinking the fluid matrix material.

30. The method according to claim 14, wherein the fluid matrix material is chemically crosslinkable or photocrosslinkable.

31. The method according to claim 14, wherein the bone marrow sample comprises freshly biopsied cells.

32. The method according to claim 31, wherein the bone marrow sample is obtained from the mammal within 24 hours after forming the MOS.

33. The method according to claim 14, wherein the bone marrow sample comprises MM plasma cells, immune cells, stem cells, stromal cells, or any combination thereof.

34. The method according to claim 33, wherein the immune cells comprise one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof.

35. The method according to claim 14, which comprises flowing the unpolymerized fluid mixture through the one or more channels at a flow rate of from about 0.01 milliliters (mL) per minute (min) to about 100 mL / min.

36. A method for precision drug screening for personalized cancer therapy against MM, the method comprising: Receiving a bone marrow sample from a mammal suffering from MM; Refining the sample to form a refined sample; Forming a MOS population from the refined sample by: Driving an unpolymerized fluid mixture through one or more channels of a microfluidic device, wherein the unpolymerized fluid mixture comprises the refined sample and an unpolymerized fluid matrix material, and wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the refined sample and the unpolymerized fluid matrix material travel through the one or more channels in a laminar flow manner, Forming a plurality of droplets containing the unpolymerized fluid mixture within the microfluidic device, and Polymerizing the fluid matrix material to form the MOS, wherein each MOS has a diameter between 50 μm and 500 μm and contains between 1 and 500 cells distributed therein; Culturing the MOS population for between 1 - 14 days; and Determining one or more drug therapies using the MOS population.

37. The method according to claim 36, further comprising driving an immiscible fluid through another channel of the microfluidic device such that the immiscible fluid combines with the unpolymerized fluid mixture before the plurality of droplets are formed, wherein the droplets contain the unpolymerized fluid mixture and the immiscible fluid.

38. The method according to claim 37, wherein the immiscible fluid is oil.

39. The method according to claim 36, wherein the determination comprises parallelly determining a plurality of drug therapies by exposing one or more of the MOS to each drug therapy.

40. The method according to claim 39, comprising characterizing the response of the MOS to each of the plurality of drug therapies based on the response of the MOS to exposure to the plurality of drug therapies.

41. The method according to claim 36, wherein the time between receiving the bone marrow sample and characterizing the response is less than 21 days.

42. The method according to claim 36, wherein forming the MOS population further comprises sorting the MOS based on cell number and / or droplet size.

43. The method according to claim 36, wherein the sorting comprises optically sorting the MOS or based on cell number and / or droplet size.

44. The method according to claim 36, wherein the determination comprises determining more than 10 different drug therapies.

45. The method according to claim 36, wherein the one or more drug therapies comprise different concentrations of one or more drugs, different combinations of two or more drugs, different ratios of two or more drugs, different carriers for one or more drugs, and / or different dosing times for one or more drugs.

46. The method according to claim 36, wherein forming the MOS population comprises forming from about 100 to about 600 MOS.

47. The method according to claim 36, wherein forming the MOS population includes forming from about 600 to about 1,000 MOSs.

48. The method according to claim 36, wherein forming the MOS population includes forming more than 1,000 MOSs.

49. The method according to claim 36, wherein the microfluidic device maintains the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets.

50. The method according to claim 36, wherein the microfluidic device is configured to prevent clogging of the unpolymerized fluid mixture within the one or more channels.

51. The method according to claim 50, wherein the microfluidic device is configured to prevent clogging by having a channel diameter of 100 μm or greater.

52. The method according to claim 36, wherein the microfluidic device is configured to maintain an approximately constant pressure within the one or more channels.

53. The method according to claim 36, wherein the microfluidic device maintains a constant flow rate within the one or more channels.

54. The method according to claim 36, wherein the total length of the path taken by the unpolymerized fluid mixture before forming the plurality of droplets within the microfluidic device is less than 10 cm.

55. The method according to claim 36, further comprising measuring the effect of the one or more drug therapies on the cells within the MOS.

56. The method according to claim 36, further comprising determining that the mammal remains responsive to one of the one or more drug therapies after one or more administrations of the drug therapy by receiving a second bone marrow sample after treating the mammal with the drug therapy and forming a second MOS population from the second bone marrow sample, exposing at least some of the second MOS population to the drug therapy, and measuring the effect of the drug therapy on the cells within at least some of the second MOS population.

57. The method according to claim 36, further comprising treating the mammal with one of the one or more drug therapies.

58. The method according to claim 36, wherein the MOSs within the MOS population vary in size by less than 25%.

59. The method according to claim 36, wherein the polymerization includes crosslinking the fluid matrix material.

60. The method according to claim 36, wherein the fluid matrix material is chemically crosslinkable or photocrosslinkable.

61. The method according to claim 36, wherein the bone marrow sample includes freshly biopsied cells.

62. The method according to claim 61, wherein the bone marrow sample is obtained from the mammal within 24 hours after forming the MOS.

63. The method according to claim 36, wherein the bone marrow sample includes cancer cells, immune cells, stem cells, stromal cells, or any combination thereof.

64. The method according to claim 63, wherein the immune cells include one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof.

65. The method according to claim 36, which comprises flowing the refined bone marrow sample and the unpolymerized fluid matrix through the one or more channels at a flow rate of from about 0.01 mL / min to about 100 mL / min.

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