Multiple Myeloma MicroOrganoSphere

Abstract

Provided herein are MicroOrganoSpheres (MOS) generated using cells from a multiple myeloma bone marrow biopsy, as well as methods and materials for making and using such MOS.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 404,472, filed September 7, 2022. The disclosure of this prior application is considered part of (and incorporated by reference into) the disclosure of this application.

[0002] This document relates to MicroOrganoSpheres (MOS) generated from bone marrow biopsies containing multiple myeloma (MM) cells, as well as methods and materials for making and using MM-containing MOS. [Background technology]

[0003] MM is a plasma cell malignancy with an estimated incidence of 5–7 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 cases of MM in the United States, and 12,960 deaths from MM. Despite improvements in treatment and overall outcomes for MM patients over the past several decades, MM remains an incurable disease, and nearly all patients will experience continuous cycles of treatment, response, and relapse. For example, nearly all MM patients require lifelong treatment, including two- to four-drug combination therapy as well as single-agent maintenance therapy. FDA-approved drugs for the treatment of MM include immunomodulators (e.g., IMiDs, thalidomide, lenalidomide, and pomalidomide), proteasome inhibitors (PIs) (e.g., bortezomib, carfilzomib, and ixazomib), monoclonal antibodies (e.g., elotuzumab, daratuzumab, isatuximab, and belantamab), nuclear export 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 therapy (e.g., Abecma, Ide-cel) has 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

[0004] This document is based, at least in part, on the development of methods for generating, testing, and validating patient-derived MicroOrganoSpheres (PDMOs) generated from MM biopsies (also referred to herein as MM MOSs). This document is based, at least in part, on the use of MM MOSs with a platform called the MicroOrganoSphere Drug Screen to Lead Care (MODEL), which can serve as a diagnostic assay to identify appropriate treatments for, for example, relapsed / refractory (RR) MM patients and naive MM patients, so that treatment regimens can be tailored to individual patients. The methods and materials provided herein can facilitate rapid diagnosis (e.g., within less than two weeks), are cost-effective, easy to use, and can provide high-throughput screening of treatments for individual MM patients. The availability of numerous therapeutic agents (e.g., those listed above) for treating MM makes it difficult to select, combine, and sequentially use these agents. Having the ability to select the appropriate agent(s) and appropriate drug combination(s) for a particular patient can facilitate successful management of MM patients.

[0005] This document provides methods and materials for generating MOS 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, using the obtained bone marrow tissue to prepare a population of MOS, and screening the MOS for response to various MM treatments, thus identifying which of the tested therapies may be most effectively used in the patient.

[0006] As demonstrated herein, we successfully generated MOS from fresh bone marrow biopsies from MM patients. We contacted the MOS with several therapeutic agents to determine their effects on MOS, revealing differential effects on MOS depending on the agent used. Additionally, further studies demonstrated that MM MOS can tolerate freezing and thawing.

[0007] In a first aspect, this document features a MicroOrganoSphere containing bone marrow cells from a mammal with multiple myeloma (MM). The MicroOrganoSphere can contain about 50 to about 150 cells, 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 MicroOrganoSphere can contain cancer cells and stromal cells in a ratio of less than about 1:4. The immune cells can include at least one macrophage. The MicroOrganoSphere can include solubilized basement membrane matrix.

[0008] In another aspect, this document features a composition including MicroOrganoSpheres in a culture medium, wherein the MicroOrganoSpheres comprise bone marrow cells from a mammal with MM. The culture medium can include a solubilized basement membrane matrix. The composition can include about 1% solubilized basement membrane matrix. The composition can further include an immiscible fluid (e.g., oil).

[0009] In another aspect, this document features a method for making MicroOrganoSpheres (MOSs). The method includes: (a) receiving a bone marrow sample from a mammal with MM; (b) purifying the bone marrow sample to form a purified sample; and (c) forming a population of MOSs from the purified sample by: (i) propelling an unpolymerized fluid mixture, the unpolymerized fluid mixture including the purified sample and an unpolymerized fluid matrix material, through one or more channels of a microfluidic device, where the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the purified sample and the unpolymerized fluid matrix material move through the one or more channels in a laminar flow; (ii) forming a plurality of droplets including the unpolymerized fluid mixture within the microfluidic device; and (iii) polymerizing the fluid matrix material to form MOSs, each MOS having a diameter of 50-500 μm and having 30-150 cells distributed therein.

[0010] The method may further include propelling the immiscible fluid through another channel of the microfluidic device such that the immiscible fluid is combined with the unpolymerized fluid mixture before forming the plurality of droplets, the droplets comprising the unpolymerized fluid mixture and the immiscible fluid. The immiscible fluid may be oil. Forming a population of MOS 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 a population of MOS may include forming about 100 to about 600 MOS, about 600 to about 1,000 MOS, or 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 in 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 a substantially constant pressure in one or more channels. The microfluidic device can maintain a constant flow rate within one or more channels. The total length of the path traveled by the unpolymerized fluid mixture prior to the formation of multiple droplets within the microfluidic device can be less than 10 cm. The MOS of the population of MOS can have a size variation of less than 25%. The polymerization can include crosslinking the fluid matrix material. The fluid matrix material can be chemically crosslinkable or photocrosslinkable. The bone marrow sample can include freshly biopsied cells. The bone marrow sample can be obtained from the mammal within 24 hours of the formation of the MOS. The bone marrow sample can include MM plasma cells, immune cells, stem cells, stromal cells, or any combination thereof. The immune cells can include one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof. The method can include flowing the unpolymerized fluid mixture through one or more channels at a flow rate of about 0.01 milliliters (mL) / minute (min) to about 100 mL / min.

[0011] In another aspect, this document features a method of precision drug screening for personalized cancer therapy for MM. The method includes: (a) receiving a bone marrow sample from a mammal with MM; (b) purifying the sample to form a purified sample; (c) forming a population of MOSs from the purified sample by: (i) propelling an unpolymerized fluid mixture, the unpolymerized fluid mixture including the purified sample and an unpolymerized fluid matrix material, through one or more channels of a microfluidic device, where the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the purified sample and the unpolymerized fluid matrix material move through the one or more channels in a laminar flow; (ii) forming a plurality of droplets including the unpolymerized fluid mixture within the microfluidic device; and (iii) polymerizing the fluid matrix material to form MOSs, each MOS having a diameter of 50-500 μm and having 1-500 cells distributed therein; (d) culturing the population of MOSs for 1-14 days; and (e) assaying one or more drug therapies using the population of MOSs.

[0012] The method may further include propelling the immiscible fluid through another channel of the microfluidic device such that the immiscible fluid is combined with the unpolymerized fluid mixture before forming the plurality of droplets, and the droplets include the unpolymerized fluid mixture and the immiscible fluid. The immiscible fluid may be oil. The assay may include assaying multiple drug therapies in parallel by exposing one or more of the MOS to each drug therapy. The method may include 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. The time between receiving the bone marrow sample and characterizing the response may be less than 21 days. Forming a population of MOS may further include sorting the MOS based on cell number and / or droplet size. Sorting may include optically sorting the MOS or based on cell number and / or droplet size. The assay may include assaying more than 10 different drug therapies. The one or more drug therapies may 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 administration times of one or more drugs. Forming a population of MOSs may include forming about 100 to about 600 MOSs, about 600 to about 1,000 MOSs, or more than 1,000 MOSs. The microfluidic device may maintain the viscosity of the unpolymerized fluid mixture prior to 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 a substantially 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 traveled by the unpolymerized fluid mixture prior to forming the plurality of droplets within the microfluidic device may be less than 10 cm. The method may further include measuring the effect of the one or more drug therapies on the cells within the MOSs.The method may further include determining whether the mammal remains responsive to the one or more drug therapies after one or more administrations of the one or more drug therapies by receiving a second bone marrow sample after the mammal has been treated with the one or more drug therapies, forming a second population of MOS from the second bone marrow sample, exposing at least a portion of the second population of MOS to the one or more drug therapies, and measuring the effect of the one or more drug therapies on cells within at least a portion of the second population of MOS. The method may further include treating the mammal with the one or more drug therapies. The MOS of the population of MOS may have a size variation of less than 25%. The polymerization may include crosslinking the 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 collected from the mammal within 24 hours after formation of the MOS. The bone marrow sample may include cancer cells, immune cells, stem cells, stromal cells, or any combination thereof. The immune cells can include one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocytes, and combinations thereof. The method can include flowing the purified bone marrow sample and the unpolymerized fluid matrix through one or more channels at a flow rate of about 0.01 mL / min to about 100 mL / min.

[0013] 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 the present invention can be practiced using methods, devices, and materials similar or equivalent to those described herein, suitable methods, devices, and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated 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 are not intended to be limiting.

[0014] The 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 and drawings, and from the claims. [Brief explanation of the drawings]

[0015] [Figure 1A] Representative images show PDMOs formed to contain a single primary tissue cell per MOS and cultured for 1 day after formation. These cells were derived from colorectal cancer (CRC) tissue. [Figure 1B] Representative images show PDMOs formed to contain a single primary tissue cell per MOS and cultured for 3 days after formation. These cells were derived from colorectal cancer (CRC) tissue. [Figure 1C] Representative images show PDMOs formed to contain a single primary tissue cell per MOS and cultured for 7 days after formation. These cells were derived from colorectal cancer (CRC) tissue. [Figure 2A] Representative images show PDMOs formed to contain 5 primary tissue cells per MOS and cultured for 1 day after formation. These cells are derived from CRC tissue. [Figure 2B] Representative images show PDMOs formed to contain 5 primary tissue cells per MOS and cultured for 3 days after formation. These cells are derived from CRC tissue. [Figure 2C] Representative images show PDMOs formed to contain 5 primary tissue cells per MOS and cultured for 7 days after formation. These cells are derived from CRC tissue. [Figure 3A] Representative images show PDMOs formed to contain 20 primary tissue cells per MOS and cultured for 1 day after formation. These cells are derived from CRC tissue. [Figure 3B] Representative images show PDMOs formed to contain 20 primary tissue cells per MOS and cultured for 3 days after formation. These cells are derived from CRC tissue. [Figure 3C]Representative images show PDMOs formed to contain 20 primary tissue cells per MOS and cultured for 7 days after formation. These cells are derived from CRC tissue. [Figure 4A] 1 includes images showing a representative example of a PDMO formed to contain 10 primary tissue cells per MOS. MOSs are shown (at low magnification) immediately after formation. [Figure 4B] Higher magnification views of some of the MOSs in Figure 4A taken after 2 days of culture are shown, including images showing a representative example of a PDMO formed to contain 10 primary tissue cells per MOS. [Figure 4C] Images showing a representative example of a PDMO formed to contain 10 primary tissue cells per MOS are included. MOSs are shown after 3 days of culture. [Figure 4D] Figure 4D includes images showing a representative example of a PDMO formed to contain 10 primary tissue cells per MOS. Figure 4D shows a MOS after 4 days of culture. [Figure 4E] Images showing a representative example of a PDMO formed to contain 10 primary tissue cells per MOS are included. MOSs are shown after 5 days of culture. [Figure 5A] Representative example image of MOS formed from normal mouse liver hepatocytes, cultured for 1 day after formation. Mouse hepatocytes were harvested from a normal (non-diseased) mouse liver. [Figure 5B] Representative example image of MOS formed from normal mouse liver hepatocytes, cultured for 10 days after formation. Mouse hepatocytes were harvested from a normal (non-diseased) mouse liver. [Figure 6] FIG. 1 is a schematic diagram illustrating steps in a method for forming a PDMO from a primary tissue (e.g., biopsy) sample. [Figure 7A] FIG. 1 is a schematic illustrating an example of an apparatus for forming a PDMO that includes a microfluidic chip as part of the assembly. [Figure 7B] FIG. 7B is a perspective view of an example of a microfluidic chip portion of an apparatus such as that shown in FIG. 7A. [Figure 7C]FIG. 7B is a schematic diagram illustrating a portion of a microfluidic assembly of an apparatus for forming a PDMO such as the apparatus shown in FIG. 7A. [Figure 8] 7B is an image showing multiple PDMOs formed using an apparatus such as that shown in FIG. 7A, showing the as-polymerized PDMOs suspended in channels containing an immiscible fluid (e.g., oil) before being aliquoted from the apparatus. [Figure 9] 7D is an image of a portion of a prototype microfluidic assembly of an apparatus for forming a PDMO similar to the apparatus shown in FIG. 7C, illustrating the formation of a PDMO. [Figure 10] Image showing multiple PDMOs immediately after polymerization, suspended in an immiscible fluid. [Figure 11A] 1 is an image showing multiple PDMOs immediately after formation and suspension in an immiscible fluid (eg, oil) at low magnification. [Figure 11B] 11B is an image showing multiple PDMOs immediately after formation and suspension in an immiscible fluid (eg, oil) at high magnification (FIG. 11B). [Figure 12A] 12A is an image at low magnification showing multiple PDMOs after separation from an immiscible fluid within hours of their formation (FIG. 12A). [Figure 12B] 12B is an image at high magnification showing multiple PDMOs after separation from an immiscible fluid within hours of their formation. [Figure 13] 10 is an image showing another example of multiple PDMOs. [Figure 14] 1 is a graph plotting the size distribution of diameters of multiple PDMOs formed from exemplary biopsy samples. [Figure 15A] 1A-1C are images showing low and high magnification views of several example PDMOs formed from dissociated tissue biopsies and fluid matrix materials after polymerization, respectively, and are unstained. [Figure 15B] 1A and 1B show images of low and high magnification views, respectively, of several example PDMOs formed from dissociated tissue biopsies and fluid matrix materials after polymerization. The MOSs were stained with trypan blue to show that the cells in the MOSs are viable. [Figure 16A] 1A-1C are images showing another example of low and high magnification views, respectively, of several example PDMOs; these are unstained images. [Figure 16B] 1A and 1B show images of another example of low and high magnification views, respectively, of several example PDMOs, where the MOS has been stained with trypan blue (arrows) to show that cells in the MOS remain viable (e.g., alive) within the MOS. [Figure 17A] We describe an example method for assaying multiple PDMOs formed from patient tumor biopsies to determine drug response profiles to multiple drug formulations. The described procedure takes less than two weeks from biopsy to result. [Figure 17B] We describe an example method for assaying multiple PDMOs formed from patient tumor biopsies to determine drug response profiles to multiple drug formulations. The described procedure takes less than two weeks from biopsy to result. [Figure 17C] We describe an example method for assaying multiple PDMOs formed from patient tumor biopsies to determine drug response profiles to multiple drug formulations. The described procedure takes less than two weeks from biopsy to result. [Figure 17D] We describe an example method for assaying multiple PDMOs formed from patient tumor biopsies to determine drug response profiles to multiple drug formulations. The described procedure takes less than two weeks from biopsy to result. [Figure 17E] We describe an example method for assaying multiple PDMOs formed from patient tumor biopsies to determine drug response profiles to multiple drug formulations. The described procedure takes less than two weeks from biopsy to result. [Figure 18] FIG. 1 is a schematic diagram illustrating an example method for treating a patient, including the formation and use of multiple PDMOs as part of a treatment procedure. [Figure 19] FIG. 1 is a schematic illustrating an example method for treating a patient that involves rapid formation and assay of multiple PDMOs multiple times as part of a treatment protocol. [Figure 20A] Representative images showing the formation of MM MOS from human bone marrow over an 11-day period are included, with MM MOS indicated by white arrows. [Figure 20B] Representative images are included showing that without stromal cells, CD138+ cells (isolated from bone marrow cells using bead selection) did not form MOS. [Figure 21A] Figure 1 shows that established MM MOS preserved major cell populations derived from bone marrow. Figure 2 shows a flow cytometry plot of single cells in MOS derived from a bone marrow biopsy sample and cultured in vitro for 9 days. The myeloma cell population was shown to be CD11b- and CD38+. [Figure 21B] Established MM MOS preserved major cell populations derived from bone marrow.Flow cytometry plots showing preservation of major immune cell populations in bone marrow biopsies. [Figure 21C] Established MM MOS preserved major cell populations derived from bone marrow. Flow cytometry plots showing that major immune cell populations were preserved in MOS on day 8 compared to bone marrow biopsies (FIG. 21B). [Figure 21D] This shows that established MM MOS preserved major cell populations derived from bone marrow. Flow cytometry was performed on MOS at days 3 and 8, and the percentage of total cells over time in MOS culture was plotted. [Figure 21E] Figure 1 shows that established MM MOS preserved major bone marrow-derived cell populations. Figure 2 includes additional flow cytometry plots showing that established MM MOS preserved major bone marrow-derived cell populations. Flow cytometry was performed on MM MOS on day 11. Plasma cells and MM cells (CD138+ and CD38+, respectively), T cells (CD3+, CD4+, and CD8+, respectively), and dendritic cells (CD11b+) were identified as shown. [Figure 22A] Representative images showing MOS containing 50, 70, or 100 cells on days 0 and 7 are included. [Figure 22B]Representative images of day 7 MOS stained for viable cells are included. [Figure 23A] 1 is a graph plotting caspase 3 / 7 signal in MM MOS after 92 hours of treatment with lenalidomide, bortezomib, or a negative control. MM MOS death is indicated by an increase in caspase 3 / 7 signal. [Figure 23B] Included are representative INCUCYTE® images showing MM MOS responses to lenalidomide as early as 24 hours after drug treatment. [Figure 24A] Figure 1 shows that MM MOS responded to carfilzomib and selinexor treatment. Representative live / dead dye images of MOS after 4 days of treatment with 10 μM carfilzomib or 10 μM selinexor are included. Calcein acetoxymethyl ester (calcein AM) (green) was used to stain live cells, and ethidium homodimer (red) was used to stain dead cells. [Figure 24B] MM MOS responded to carfilzomib and selinexor treatment. Graph plotting live / dead ratio. n=3 per treatment group. *p<0.05 by t-test. [Figure 25A] Included are a pair of images showing MM MOS after freeze-thaw cycles, demonstrating that the MOS remain viable. [Figure 25B] 1 includes plots showing viability of freeze-thawed MM MOS as indicated by SYTOX™ Blue measurement by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0016] Model cell and tissue systems, including three-dimensional (3D) aggregates such as spheroids and organoids, can be useful in biological and medical research. For example, 3D cell culture models are 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 facilitate the identification and development of new therapeutics and provide preclinical models for predicting drug response and identifying 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 numerous candidate therapies.

[0017] Multicellular tumor spheroids can be obtained by culturing cancer cell lines under non-adherent conditions. Spheroids are typically formed from cancer cell lines as free-floating cell aggregates in ultra-low binding plates. Spheroids have been shown to maintain more stem cell-associated properties than two-dimensional (2D) cell cultures. Organoids are in vitro-derived aggregates containing a population of stem cells capable of differentiating into cells of major cell lineages. Organoids typically have a diameter greater than 1 millimeter and can be cultured by passage. Organoids typically grow and expand more slowly than 2D cell cultures. Generating organoids from clinical samples requires a sufficient initial number of cells (generally hundreds to thousands of cells), and therefore, obtaining organoids from small samples such as biopsies can be difficult.

[0018] While the exploration of these various PDMC models has led to the development of precision medicine strategies, there are barriers to their effective use. Patient-derived organoids (PDOs) are considered the most accurate way to represent a patient's tumor, as studies have shown that the phenotypic and genotypic profiling of organoids often shows a high degree of similarity to the original patient's tumor. However, the use of PDOs to guide therapy has limitations. For example, while assays ideally should be performed from a single core biopsy within 7–10 days, it typically takes several months to develop and test drug sensitivity in organoids, reducing their clinical applicability. Additionally, the number of organoids obtained from a clinically relevant 18-gauge core biopsy is generally insufficient to perform high-throughput drug screening.

[0019] Furthermore, replicating tumor cells using MM tissue is not easy. MM cells are typically difficult to manipulate, and attempts to cultivate MM cells in 3D culture have been rare, especially because there is no universal recipe for 3D culture of multiple myeloma. An additional challenge is presented by the fact that MM cells cannot survive without bone marrow stromal cells. However, as described herein, we have successfully generated PDMOs from MM biopsies, which was surprising given the challenges mentioned above. However, the techniques described herein can provide MM cells with a tumor microenvironment, thereby enabling them to grow. Cells within the MM immune microenvironment (e.g., macrophages) can also be captured in PDMOs generated by the methods provided herein, expanding the range of drugs that can be screened on PDMOs.

[0020] Thus, generally, this document provides MOSs generated from bone marrow samples from mammals with 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 assay for MM response to one or more therapeutic agents). The MM MOSs described herein, and methods for making and using them, can address clinical limitations, such as those described above.

[0021] The MM PDMOs described herein are typically spheres formed from primary cells distributed within a bone marrow matrix after purifying the primary cells to remove non-mononuclear cells (e.g., erythrocytes, granulocytes, and / or platelets) while retaining mononuclear cells, resulting in a purified tissue sample containing myeloma cancer cells in combination with stromal cells, stem cells, immune cells, or any combination thereof. These PDMOs (also referred to as "MM MOS") can have diameters of about 50 μm to about 500 μm (e.g., about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 50 μm to about 250 μm, 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 comprise 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 substrate.

[0022] Surprisingly, despite the small size (often about 50 to about 250 μm) and low cell density (e.g., often about 50 to about 200 cells per MM MOS) of the MM MOS provided herein, the MM MOS can be used immediately or cultured for a relatively short period (e.g., 14 days or less, 10 days or less, 7 days or less, or 5 days or less), allowing the cells within the MM MOS to survive while maintaining most or all of the characteristics of the tissue from which they were extracted. The viability of cells within MM MOS is typically high, and MM MOS can be cultured for days or weeks. Also, surprisingly, in some variations, purified bone marrow sample-derived cells within MM MOS can form morphological structures within even the smallest MOS; in some applications, the presence of such structures is not necessary for the usefulness of these MOS (e.g., when used before substantial structural reorganization has occurred), although in some variations, such structures may be particularly useful.

[0023] In some cases, the methods and materials described herein for forming and using MM MOSs can be used to generate many (e.g., greater than 10,000) PDMOs from a single biopsy. MOSs can be used, for example, to screen various therapeutic agents to predict which therapeutic agent(s) may be effectively and safely used in an MM patient from whom a bone marrow biopsy was obtained. For example, MM MOSs can be used for toxicity screening of drugs or other chemical compositions to determine whether one or more drug compositions are likely to effectively treat an MM patient before the patient receives the drug therapy. This can allow for very rapid screening of MM patients before they otherwise undergo months of potentially ineffective chemotherapy.

[0024] Thus, high-throughput drug screening methods and devices for performing these methods using a single patient-specific MM biopsy are provided herein. Described herein are droplet-forming PDMOs that can be formed from purified bone marrow samples from MM patients suspended in a basement matrix (e.g., MATRIGEL®, a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, available from Sigma). MM PDMOs can be patterned onto microfluidic microwell arrays where they are incubated and administered with drug compounds. This miniaturized assay can maximize tumor sample utilization and allow more drug compounds to be screened from bone marrow aspirates at a much lower cost per sample.

[0025] Although the terms used herein are believed to be well understood by those of ordinary skill in the art, definitions are provided herein to facilitate explanation of the subject matter of the present disclosure.

[0026] The term "unpolymerized mixture" is used herein to refer to a composition containing biologically relevant materials, including a purified tissue sample and a first fluid matrix material. The fluid matrix material is typically a material that can be polymerized to form a support or support network for the purified tissue sample and / or cells dispersed within the purified sample. Upon polymerization, the polymerized material can form a hydrogel, which can form and / or contain proteins in addition to cells to form a biocompatible medium. Biocompatible media suitable for use in the methods disclosed herein can be formed from any biocompatible material that is a gel, semi-solid, or liquid (e.g., a low-viscosity liquid) at room temperature (e.g., 25°C) and can be used as a three-dimensional matrix for cells, tissues, proteins, and other biomaterials of interest. Exemplary materials that can be used to form a biocompatible medium in accordance with the presently disclosed subject matter include, but are not limited to, polymers and hydrogels, including collagen, fibrin, chitosan, MATRIGEL® (BD Biosciences, San Jose, CA), polyethylene glycol (PEG), dextran (e.g., chemically crosslinkable or photocrosslinkable dextran), and the like, as well as electrospun bio, synthetic, or bio-synthetic blends. In some cases, the biocompatible medium can be a hydrogel.

[0027] The term "hydrogel" is used herein to refer to a two- or multi-component gel having a three-dimensional network of polymer chains in which water acts as a dispersion medium, filling 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 used to form the MM MOS and the effect of the selected hydrogel on the behavior and activity of biomaterials (e.g., cells) incorporated into the biosuspension disposed within the structure. Exemplary hydrogels for use with the presently disclosed subject matter include polymeric materials such as, but not limited to, alginate, collagen (including types I and VI), elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactides, polyethylene glycols, polycaprolactones, polycolides, polydioxanones, polyacrylates, polyurethanes, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids, carbohydrates, polysaccharides, and modified polysaccharides, and derivatives and copolymers thereof, and inorganic materials such as glasses, including bioactive glasses, ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and any combination thereof.

[0028] Further with regard to the hydrogels used to generate the MM MOS described herein, in some cases, the hydrogel can comprise a material selected from the group consisting of agarose, alginate, type I collagen, polyoxyethylene-polyoxypropylene block copolymer (e.g., PLURONIC® F127, BASF Corporation, Mount Olive, NJ), silicone, polysaccharide, polyethylene glycol, and polyurethane. In some cases, the hydrogel can be made of alginate.

[0029] The MM MOSs provided herein can also include biologically relevant materials. The phrase "biologically relevant materials" refers to materials that can be included in a biocompatible medium, as defined herein, and subsequently interact with and / or affect a biological system. For example, in some cases, the biologically relevant materials can be magnetic beads (e.g., beads that are themselves magnetic or beads that include a material that responds to a magnetic field, such as iron particles) that can be combined as part of the unpolymerized material to aid in the generation of the MM MOS (e.g., for the isolation and purification of the MOS). As another example, in some cases, the biologically relevant materials can include cells in addition to the purified tissue sample (e.g., biopsy material). In the unpolymerized mixture, the purified tissue sample and the additional biologically relevant materials can be present as a homogeneous mixture or as a dispersed mixture (e.g., in one half or another portion of the MOS, e.g., only within the core or only within the outer region of the formed MOS). In some cases, the additional biologically relevant materials in the unpolymerized material can be suspended with the purified tissue sample in the suspension (e.g., prior to polymerization of the droplets that form the MOS).

[0030] In some cases, biologically relevant material that can optionally be included with a purified tissue sample (e.g., a biopsy) can include several 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 vessels or microvascular fragments found within the stromal vascular fraction, and possibly bone components.

[0031] Generally, the purified tissue sample (e.g., biopsy) material included in the MM MOS described herein is from a bone marrow sample, typically obtained by biopsy, from a mammal (e.g., a human with MM or other mammal, e.g., a mouse, rat, or rabbit). The tissue and resulting purified cells can be primary cells obtained from a patient biopsy (e.g., by needle biopsy). The purified cells can be incorporated into the MM MOS described herein and can include cells commonly found in bone marrow. In this regard, exemplary cells that can be incorporated into MM MOS include plasma cells (normal and cancerous), stromal cells, hematopoietic stem cells, monocytes, macrophages, dendritic cells, T cells, B cells, and NK cells. Tissue can be purified using any suitable technique, including, but not limited to, those described herein.

[0032] Furthermore, it has just been discovered 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 about 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) increasing the concentration of added MATRIGEL® to about 80 to 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) using ultra-low binding plates. Without being bound by any particular mechanism, the use of a relatively large number of cells can increase the likelihood that the full complement of cells from the bone marrow microenvironment (e.g., stromal and immune cells) will be included in the MM MOS, which may be important from a drug screening perspective. Additionally, without being bound by any particular mechanism, using higher concentrations of MATRIGEL® can help retain MM cells within the MOS, and using low- or ultra-low-binding plates can reduce the likelihood of MOS binding to the bottom of the culture well. Additionally, in some cases, the methods disclosed herein for generating MM MOS can include adding a small amount of MATRIGEL® (e.g., 1%) to the culture medium rather than 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 proliferation-inducing ligand (APRIL), also known as tumor necrosis factor ligand superfamily member 13) to the MATRIGEL®, regardless of whether the MATRIGEL® is added to the MOS mixture or the culture medium. These strategies can also help retain MM cells within the MOS and / or prevent MOS from binding to the bottom of the culture well.

[0033] In view of the above, the MM MOS provided herein can have a cell count of about 50 to about 200 cells per MOS, with MM plasma cells, stromal cells, and immune cells 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). The number of MM cells in the MOS cell population can be determined, for example, by CD19 - , CD38 + , CD56 + (and in some cases CD138 + ) can be assessed using flow cytometry to target myeloma cells and using live and / or dead dyes to indicate the viability of the targeted myeloma cells. The number of cells of other cell types in the MOS cell population can be determined in a similar manner. For example, the number of stromal cells in the MOS cell population can be determined using, for example, CD3 - , CD19 - , CD20 - , CD56 - , CD45 - , CD31 - , ALP + Osteoblasts and / or CD3 - , CD19 - , CD20 - , CD56 - , CD11b + , CD14 + , RANK + The number of immune cells in the MOS cell population can be assessed using flow cytometry to target osteoclasts and using live and / or dead dyes to indicate the viability of the targeted stromal cells. + , CD4 + , or CD8 + T cells, CD3 - , CD56 + NK cells, CD3 - , CD19 + B cells and / or CD11b +Assessment can be performed using monocyte cells and using live and / or dead dyes to indicate the viability of the targeted immune cells.

[0034] Once formed, the MM MOS can be cryopreserved and / or cultured. Generally, cultured MOS can be maintained in suspension either statically (e.g., in a well, vial, or other suitable container) or dynamically (e.g., by rolling or agitation). MOS can be cultured using any suitable technique. Exemplary techniques can be found in, but are not limited to, Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 4th ed., Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach, Davis, ed., Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, Masters, ed., 2000; and U.S. Patent Nos. 5,516,681 and 5,559,022, all of which are incorporated herein by reference in their entireties.

[0035] In some cases, MM MOSs can be generated by forming droplets of an unpolymerized mixture (in some cases, a cooled mixture) of a purified tissue sample and a fluid matrix material in an immiscible material, such as a fluid hydrophobic material (e.g., oil). For example, MOSs can be formed by combining a stream of unpolymerized material with one or more streams of immiscible materials to form droplets. The density of cells present in the droplets can be determined by dilution of the purified material (e.g., cells) in the unpolymerized material. The size of the MOS can be correlated to the size of the droplets formed. Generally, MOSs are spherical structures with stable geometric shapes.

[0036] Methods for producing MM MOSs described herein may employ techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, unless otherwise indicated, examples of which are described elsewhere.See, e.g., Molecular Cloning A Laboratory Manual (1989), 2nd Ed., Sambrook, Fritsch, and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, Gait, ed., 1984; Nucleic Acid Hybridization, Hames and Higgins, eds., 1984; Transcription and Translation, Hames and Higgins, eds., 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; and Methods In Enzymology (Academic Press). Press, Inc., NY), Gene Transfer Vectors For Mammalian Cells, Miller and Calos, eds., Cold Spring Harbor Laboratory, 1987, Methods in Enzymology, Vols. 154 and 155, Wu et al., eds., Academic Press Inc., NY, Immunochemical Methods in Cell and Molecular Biology, Mayer and Walker, eds., Academic Press, London, 1987, and Handbook of Experimental Immunology, Volumes I-IV, Weir and Blackwell, eds., 1986.

[0037] As described above, the tissue (e.g., biopsy) sample (e.g., purified sample) used to form the MM MOS can be derived from bone marrow obtained from a mammal with MM. Typically, the MOS contains multiple cell types present in the tissue from which it is derived. Thus, the tissue used in the MM MOS provided herein can contain 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 subculture, or they can first undergo an intermediate culture step to generate a primary culture. Any suitable method for collecting cells from biological tissue and / or cell-containing fluids can be used. For example, suitable techniques used to obtain cells from biological tissue include those described in Mahesparan, Acta Neuropathol (1999) 97:231-239.

[0038] Generally, collected cell types (e.g., those in a biopsy or aspirate) can be purified prior to forming MOS. For example, cells in a bone marrow aspirate can be purified to isolate mononuclear cells. Separating cell types from each other can be achieved using any suitable method. For example, cells in a bone marrow aspirate can be filtered (e.g., through a 70-100 μm strainer) and then subjected to Ficoll gradient purification to separate mononuclear cells in the bone marrow aspirate from non-mononuclear cells (e.g., red blood cells, granulocytes, and / or platelets).

[0039] In some cases, purified tissue can be treated to remove dead / dying cells and / or cell debris. Removal of such dead and / or dying cells can be achieved using any suitable means, such as beads and / or antibody methods. For example, because phosphatidylserine can redistribute from the inner leaflet to the outer leaflet of the plasma membrane in apoptotic or dead cells, the use of annexin V-biotin binding, followed by binding of biotin to streptavidin magnetic beads, can allow the separation of apoptotic cells from live cells. Removal of cell debris can be achieved, for example, by techniques such as filtration.

[0040] The purified cells can be suspended in a carrier material before being combined with the fluid matrix material. Alternatively, the fluid matrix material can be referred to as the carrier material. In some cases, the carrier material can be a material with a viscosity level that retards the settling of cells in the cell suspension prior to polymerization and formation of the MOS. In such cases, the carrier material can have a viscosity sufficient to allow the cells of the purified tissue sample to remain suspended in suspension until polymerization. The viscosity required to achieve this can be optimized by monitoring the settling rate at various viscosities and selecting a viscosity that provides an appropriate settling rate for the expected time delay between loading the cell suspension into an apparatus that forms the MOS by polymerizing droplets of unpolymerized material containing the cells. In some cases, the unpolymerized material can be fluidized or agitated with the apparatus (e.g., when a lower viscosity material is used) to maintain the cells in suspension and / or distribute them as desired.

[0041] As mentioned above, in some cases, the unpolymerized mixture containing the purified tissue sample and the fluid matrix material can contain one or more other components, such as biologically relevant materials. Examples of biologically relevant materials 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., for regulating 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 to otherwise mimic an in vivo environment. The biologically relevant materials can include or mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids, and hormones. In some cases, the biologically relevant materials can supplement one or more drugs 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, MATRIGEL® includes bioactive polymers important for cell viability, proliferation, development, and migration. In some cases, the matrix material can be a gel including type I collagen, such as type I collagen obtained from rat tail. The gel can be a pure type I collagen gel or a gel including 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 occur in nature.Examples of synthetic gels include gels derived from polyethylene glycol (PEG), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), or polyethylene oxide (PEO).

[0042] Images showing PDMOs are presented in Figures 1A-1C, 2A-2C, 3A-3C, and 4A-4E. For example, Figures 1A-1C include representative images of formed MOSs with a single cell per MOS. As shown, the MOSs are all approximately the same size, with a diameter of approximately 300 μm. After 3 days of culture (center panel), the cells have expanded in size and, in some cases, doubled and / or grown. By day 7 of culture (right panel), the cells have doubled multiple times, exhibiting clusters or clumps of cells. The MOSs shown in Figures 2A-2C were formed from 5 cells per MOS, while the MOSs shown in Figures 3A-3C were formed from 20 cells per MOS. Figures 4A-4E show MOSs immediately after formation and MOSs cultured for 5 days, where nearly identical MOSs (e.g., with the same diameter) each contain 10 cells per MOS. Figure 4A shows a MOS immediately after formation, still surrounded by an immiscible fluid (oil in this case) on day 0. The MOS was removed from the immiscible fluid, washed, and cultured for 5 days. Figures 4B, 4C, 4D, and 4E show MOS after 2, 3, 4, and 5 days, respectively. These images demonstrate that the tissue (cells) purified from the biopsy within the MOS are viable and growing at similar rates within nearly all MOS. As described in more detail herein, these MOSs can be formed in large quantities from even a single average-sized biopsy, resulting in hundreds or 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) of MOSs containing substantial numbers of viable cells, allowing multiple rapid assays to be performed in parallel.

[0043] Figures 5A and 5B show examples of MOSs formed as described herein from mouse liver biopsies, showing mouse hepatocytes distributed within a polymerized fluid matrix material (in this example, MATRIGEL®). Each MOS contained polymerized matrix material 503 formed into spheres with a diameter of approximately 300 μm, with a set number of hepatocytes 507 dispersed within. In Figure 5A, MOSs are shown one day after biopsy, tissue dissociation or purification, and MOS formation. These MOSs were then cultured for 10 days, during which time the hepatocytes remained viable and grew, often doubling multiple times to form structures 505, as shown in Figure 5B.

[0044] MOS generally refers to a fixed or known number of cells or cell concentration (e.g., cells / ml or cells / mm) within the MOS. 3 The matrix material may comprise biopsy tissue (e.g., cells) dissociated or purified with a cellulose membrane. As previously mentioned, the matrix material may comprise one or more natural polymers, such as alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, and elastin, or may comprise one or more synthetic polymers, such as PEG and polyacrylamide. Both organic and inorganic synthetic polymers can be used.

[0045] In some cases, the number of cells initially included in a MOS can be selected from one cell to several hundred cells. Specifically, in some assays (e.g., drug toxicity assays), it may be beneficial to include about one to about 75 cells or about one to about 50 cells (i.e., a relatively small number of cells). The number of cells per MOS can be set or selected by the technician generating the MOS. In some cases, as described below, the device for generating MOS can include one or more controls for setting the number of cells from the primary tissue included in each MOS. The number of cells can be selected or set based on how the MOS is intended to be used. For example, MOSs with very low numbers of cells (e.g., one cell per MOS or one to five cells per MOS) may be particularly suitable for studying clonal diversity (e.g., tumor heterogeneity). Because MOSs each grow from a single cell, it is possible to observe which clones are drug-resistant, and these specific MOSs can then be examined (e.g., by genome sequencing) to determine the genomic (mutational) diversity associated with specific clones. Because these MOSs typically grow rapidly, a low to moderate 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 useful for rapid drug testing (including toxicity testing). Because MOSs may contain different lineages, potentially including epithelial (e.g., cancer) cells and mesenchymal (e.g., stromal, immune, or vascular) cells, 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 of each MOS. Note that in the case of MM MOSs, including a relatively large number of cells per MOS (e.g., about 50 to about 150 cells per MOS) can provide each MOS with different cell types that can act to maintain MM cells.

[0046] MOSs can be formed in any suitable size that can accommodate the number of cells to be contained. 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 cells) can be contained in each MOS. The number and size of cells can be varied and / or controlled. In some cases, the number and / or size of cells in the MOS can be set by one or more controls on the device used to form the MOS. For example, the size of the MOS and / or the density of cells within the MOS can be adjusted by adjusting the flow rate and / or concentration of the purified tissue sample (e.g., cells from a biopsy).

[0047] As shown in Figures 1, 2, 3, 4A-4E, and 5A-5B, viable healthy cells were observed throughout the entire volume of the MOS, even after culturing the MOS as described herein. The size of the MOS and / or the number of cells contained therein can be selected based on how the MOS is expected or intended to be used. For example, in variations in which the MOS is used to examine cellular relationships in biopsied material, the MOS can be formed to contain multiple cells (e.g., cells of multiple cell types or multiple cells of a single cell type) and can be cultured for extended periods of time (e.g., up to one week or longer).

[0048] In some cases, a PDMO can be produced by combining a dissociated or purified tissue sample (e.g., a biopsy sample) with a fluid matrix that can be polymerized in a controlled manner to form a PDMO. Figure 6 illustrates an exemplary method for forming a PDMO. Optionally, the method can include collecting a sample from a mammal (e.g., a human with MM), e.g., taking a biopsy from patient tissue 601. The biopsy can be obtained, e.g., using a biopsy needle or punch. In some cases, for example, a bone marrow biopsy can be obtained using a 14-gauge, 16-gauge, 18-gauge, 20-gauge, or 22-gauge needle that is inserted into the mammal to remove the biopsy. After removing the tissue from the mammal, the tissue can be processed to dissociate cells and other material (e.g., in the case of a solid tumor sample) or to purify the sample by removing cells of certain cell types using mechanical and / or chemical techniques (e.g., in the case of a bone marrow aspirate). The dissociated or purified cells can be used immediately to form a PDMO, as described herein, or in some cases, all or a portion of the cells can be modified, for example, by genetically modifying the cells (e.g., using transfection or electroporation to introduce one or more proteins or nucleic acids that effect the genetic modification) (603).

[0049] 6, a tissue sample dissociated or purified from a biopsy can be combined with a fluid (e.g., liquid) matrix material to form an unpolymerized mixture (605). This unpolymerized mixture can be maintained in an unpolymerized state such that cells from the dissociated or purified tissue remain suspended in the unpolymerized mixture. In some cases, the cells can remain suspected and unpolymerized by keeping them cooled (e.g., at room temperature or below, e.g., at a temperature between 1°C and 25°C).

[0050] The unpolymerized mixture can then be dispensed as droplets into an immiscible material, such as oil, in a manner that controls the size of the droplets and, therefore, the size of the PDMO formed (607). For example, uniformly sized droplets can be formed by combining a stream of unpolymerized material with one or more (e.g., two converging) streams of an immiscible material (e.g., oil), such that the flow rates and / or pressures of these two streams determine how droplets of unpolymerized material form upon meeting the immiscible material. The droplets can be polymerized (609) to form a PDMO in the immiscible material. In some cases, the immiscible material can be heated or warmed to a temperature that polymerizes the unpolymerized mixture (e.g., the fluid matrix material in the unpolymerized material). Once formed, the PDMO can be separated from the immiscible fluid. For example, the PDMO can be washed to remove the immiscible fluid (611) and placed in culture medium to grow cells within the PDMO. The PDMO can be cultured for any desired time, or can be cryopreserved, or can be assayed immediately. In some cases, the PDMO can be cultured for a period of time (e.g., 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-8 days, 1-9 days, 1-10 days, 1-11 days, or 1-14 days). Culturing the PDMO can allow cells derived from the biopsied tissue to grow and / or divide (e.g., doubling) for up to five or six passages. After culture, the cells can be cryopreserved (615) and / or assayed (617). Exemplary assays that can be used are also described herein.

[0051] In any of the methods and devices described herein, the MOS can be recovered from the immiscible fluid (e.g., oil) after polymerization. For example, in some cases, the MOS can be recovered by demulsification and / or demulsification, e.g., by forming emulsified droplets and recovering the MOS after the droplets are formed to remove any oil (and other contaminants). This may allow cells to grow within the polymerized droplets (MOS) without being inhibited by the immiscible fluid.

[0052] While the methods and apparatus described herein describe forming multiple droplets, and thus multiple MOSs, by flowing the unpolymerized mixture into one or more streams of an immiscible fluid (such as oil or other hydrophobic material), in some embodiments, the droplets can be formed by other methods, which may allow the size of the droplets to be controlled, as described herein. For example, in some cases, the droplets can be formed by printing (e.g., by printing droplets onto a surface). This can reduce or eliminate the need for an additional recovery step of emulsification / demulsification. For example, droplets can be printed onto a surface, such as a flat surface or a shaped surface, and polymerized. In any of these variations, the droplets can be dispensed using pressure, sound, electric charge, or any other suitable means. In some cases, the droplets can be formed using an automated dispenser (e.g., a pipetting device) adapted to release small amounts of the unpolymerized mixture onto a surface, into air, and / or into a liquid medium, such as an immiscible fluid.

[0053] The methods for forming PDMOs can be automated and / or performed using one or more devices. Specifically, the methods for forming PDMOs can be performed by devices that allow for selection and / or control of the size of the MOS and, therefore, the density of the cell population. For example, Figure 7A illustrates one example of an apparatus 700 for forming PDMOs described herein.

[0054] As shown in FIG. 7A, the device can include an input for inputting an unpolymerized mixture of either a dissociated or purified tissue sample (already combined) and a fluid matrix material, or an input for receiving a dissociated or purified tissue sample and a fluid matrix material separately (e.g., in a holding solution). In some cases, the device can include a holding chamber 706 for holding the unpolymerized mixture and / or a holding chamber (not shown) for holding a dissociated or purified tissue (e.g., biopsy) sample and for holding a fluid matrix material. Any or all of these holding chambers can be pressurized to control and / or accelerate the flow of fluid out of the chambers and into the device. The device can either receive the unpolymerized mixture or receive its components and mix them. In some cases, the device can control the concentration of cells in the unpolymerized mixture and dilute the mixture (e.g., by adding additional fluid matrix material to achieve a desired density). For example, the device can include a sensor (e.g., an optical reader) for reading the density (e.g., optical density) of cells in the unpolymerized mixture. The sensor may also be coupled to a controller 724 that may automatically or semi-automatically (e.g., by prompting a user) control the dilution of the cells in the unpolymerized mixture. The device may also include a port for receiving the unpolymerized mixture. The port may include or be coupled to a valve, which may be controlled by the controller 724 or by a separate controller.

[0055] The apparatus 700 can include chambers 708 and / or ports for holding and / or receiving immiscible fluids. In some cases, the immiscible fluids can be held in pressurized chambers so that the flow rate can be controlled. Any of the pressurized chambers can be controlled by a controller 724, which can control the pressure and therefore the flow through the apparatus using one or more pumps 726. One or more pressure and / or flow sensors can be included in the system to monitor the flow through the device.

[0056] 7A, the entire apparatus 700 can be enclosed within a housing 702, or a portion of the apparatus 704 can be enclosed within the housing. In some cases, the housing can include one or more openings or access points on the device, for example, for adding immiscible fluids and / or unpolymerized mixtures.

[0057] As mentioned above, the apparatus 700 can also include one or more sensors 728 for monitoring all or critical portions of the manufacturing process. In some cases, the sensor(s) can include one or more optical sensors, mechanical sensors, voltage and / or resistance (or capacitance or inductance) sensors, or force sensors. These sensors can be used to monitor the ongoing operation of the assembly, including the formation of the PDMO. The apparatus 700 can also include one or more heat / temperature regulators 718 for controlling the temperature of the immiscible fluid and / or unpolymerized mixture and / or fluid matrix material.

[0058] 7C and 9 and discussed below. The droplet MOS forming assembly 720 can include or be coupled to a dispenser (e.g., a PMOS dispenser) 722. The dispenser can dispense droplets, for example, into a multiwell plate 716.

[0059] Generally, the droplet MOS formation assembly 720 can include one or more microfluidic chips 730 or structures that form and control the flow of the unpolymerized mixture and form the actual droplets. FIG. 7B illustrates an example of a microfluidic chip 730 for forming a PDMO. In FIG. 7B, the chip 730 includes a pair of parallel structures for forming a MOS. FIG. 7C illustrates the droplet formation region of a microfluidic chip for forming a PMOS, including an unpolymerized channel outlet 741 that opens (at a right angle in this example) the "+" junction or region of intersection 737 to a channel outlet 741 and immiscible fluid outlet(s) 743, 743′. In some cases, the input from the immiscible fluid channel(s) can be at an angle relative to the angle with the unpolymerized material (and the intersection). In FIG. 7C, as in all figures in this description that show dimensions, unless otherwise specified, the dimensions shown are for illustrative purposes only and are not intended to be limiting.

[0060] In Figure 7A, microfluidic chip 730 includes an inlet (input port) 733 for an immiscible fluid into the chip (e.g., from the inlet port or storage chamber shown in Figure 7A). A second inlet port 735 into the chip can be configured to receive unpolymerized material and transport it down a semi-inverted path to the junction region. Similarly, the inlet port for the immiscible fluid can be rigidly coupled to an immiscible fluid chamber or inlet-to-outlet, as described above.

[0061] An inlet port 735 for unpolymerized material into the chip can be linked via a delivery pathway 741 that connects the inlet to the junction region (as shown in FIG. 7C). Similarly, an inlet 733 for an immiscible fluid can connect two or more connecting pathways 743, 743′ to the junction region 737. A channel leaving the junction region 737 can carry the formed MOS (in the immiscible fluid) down the channel to an outlet 731 that can be connected to a dispenser for dispensing the MOS into one or more chambers (e.g., for incubation and / or assay).

[0062] In the example shown in Figures 7B and 7C, the formed droplets, which may polymerize into MOS, can be sent to a temperature-controlled microfluidic environment for an extended period before being dispensed from the device. For example, Figure 8 shows an example of a channel region 839 (e.g., element 739 in Figure 7B) shown in a transparent state, containing multiple MOSs 803, each containing a predetermined number of cells 805. Note that in any of the microfluidic chips or devices described herein, the channels can be coated. For example, the channels of a microfluidic device can be coated with a hydrophobic material.

[0063] Figure 9 shows a junction region 937 shaped as described above, where a channel carrying an unpolymerized mixture 911 intersects one or more (e.g., two) channels 909 carrying a fluid, such as oil, that is immiscible with the unpolymerized mixture. When the unpolymerized mixture exits the first channel 911 and is pressurized to flow at a first velocity, the immiscible fluid flowing through the intersecting channels 909, 909' allows a predetermined amount of the unpolymerized mixture to pass before pinching it off to form droplets 903 that are sent to the exit channel 939. Thus, in some variations, a minced (e.g., dissociated) clinical (e.g., biopsy or resection) sample of tissue, e.g., having a size less than 1 mm in diameter, can be mixed with a temperature-sensitive gel (e.g., MATRIGEL® at 4°C) to form an unpolymerized mixture. The unpolymerized mixture can be placed in a microfluidic device, which generates droplets (e.g., water-in-oil droplets) that are uniform in volume and material composition. Dissociated tumor cells can be simultaneously distributed into these droplets. The gel in the unpolymerized material can solidify upon heating (e.g., at 37°C), resulting in the formation of a PDMO. In some cases, this method can be used to generate 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 (PDMOs) from tissue (e.g., biopsy material). These PDMOs are compatible with conventional three-dimensional cell culture techniques. Figure 10 shows a plurality of PDMOs 1005 formed as described above and suspended in an immiscible material 1008 (e.g., oil).

[0064] In the exemplary microfluidic chips described above, the junctions are shown as T-junctions or X-junctions, which allow microfluidic flow focusing to form MOSs of controllable size. In some cases, rather than a microfluidic chip, droplets can be formed, for example, by robotic micropipetting into immiscible fluids and / or onto solid or gel substrates. Alternatively, in some cases, droplets of unpolymerized material can be formed with the required dimensions and reproducibility by microcapillary generation. Other examples of techniques that can alternatively be used to form MOSs of specified size ranges and reproducibility from unpolymerized materials include, but are not limited to, colloidal manipulation by external forces such as acoustic, magnetic, inertial, electrowetting, or gravity.

[0065] Figures 11A and 11B show examples of PDMOs in oil formed as described above. As seen by vital dye staining, the cells in these MOSs derived from a single biopsy sample are viable (Figures 15A-15B and 16A-16B). For example, Figures 12A and 12B show MOSs containing tumor cells (similar to those shown in Figures 11A and 11B) that can be washed to remove immiscible materials (e.g., oil). The immiscible materials can be removed relatively quickly after MOS formation to prevent harm to the cells within the MOS.

[0066] In these examples, the gel droplets can be recovered from the oil phase and resuspended in PBS, for example, via PFO (perfluorooctanol). Centrifugation can be used to separate the immiscible fluid from the MOS. The MOS can then be grown, as shown in Figures 1, 2, 3, 4A-4E, and 13. This is important because drug screening must be performed on viable, growing primary tumor cells that retain characteristics from the patient's tumor in order to predict patient outcome. As described below, the large number and uniformity of these MOS makes screening feasible and reliable.

[0067] Generally, the MOS provided herein can be very uniform in diameter and have very low size (e.g., diameter) variation, as illustrated, for example, in Figure 14, which shows a representative distribution of droplet diameter sizes.

[0068] Figures 15A and 15B show another population of MOSs formed as described herein. In Figures 16A and 16B, these MOSs were stained with trypan blue (arrows) to demonstrate their viability. In some cases, MOSs formed as droplets as described herein can contain growth factors and matrix components to mimic the biological environment from which the cells in the MOS originate. Mammalian samples (e.g., patient biopsies) can typically form into MOSs within a few hours (e.g., about 6-12 hours, about 12-18 hours, about 18-24 hours, about 12 hours, about 18 hours, about 24 hours, or about 48 hours) after tissue acquisition. MOSs can each contain just a few cells (e.g., as few as one cell or about 4-6 cells, such as a cancer cell when sampling a tumor), or they can each contain many more cells (e.g., 50-150 cells in the case of MM MOSs). Methods similar to those described herein have been shown to generate MOS from multiple types of cancerous and non-cancerous tissues, including colon cancer, esophageal cancer, melanoma, uterine cancer, sarcoma, kidney cancer, liver cancer, ovarian cancer, lung cancer, diaphragmatic cancer, omental cancer, mediastinal lung cancer, and breast cancer tissue (see, e.g., the Examples in U.S. Publication No. 2021 / 0285054, incorporated herein by reference in its entirety). MOS can be cultured for any desired period of time and typically exhibit proliferation and growth after as little as 3-4 days. MOS can be maintained and passaged for several months. As described in more detail below, MOS can also be used to screen drug compositions within as little as 4-14 days (e.g., 4-6 days, 5-7 days, 6-8 days, 7-10 days, 8-12 days, or 10-14 days) after collection of the tissue (e.g., a biopsy).

[0069] The MOS described herein can be banked at any time after their formation, for example, by cryopreservation. Tumor MOS can be collected from many different patients and used individually or collectively to screen multiple drug formulations to determine the toxicity and / or efficacy of a particular therapeutic agent. In some cases, non-tumor cells (healthy tissue) can be biopsied, banded, and / or screened in parallel. Thus, the methods and devices provided herein can enable high-throughput screening. In some variations, MOS can be formed, passaged twice (e.g., doubling), and cryopreserved. Again, normal healthy tissue can be used to form corresponding MOS to generate hundreds, thousands, or tens of thousands of MOS that can be used to assay drug effects, drug responses, biomarkers, proteomic signals, genomic signals, and the like.

[0070] It is particularly important that MOS survive in a biologically significant manner, allowing for the provision of clinically and physiologically relevant data, especially regarding drug response. Specifically, the MOS provided herein allows cells derived from tissue extracts / biopsies to grow very well and provide more representative data, especially compared to organoids or spheroids. Without being bound by a specific mechanism, this may be because cells have a more restricted cell density in MOS, allowing cells to communicate with each other without inhibiting each other while sharing signals. MOS also have a very large surface area-to-volume ratio, allowing the transmission of growth factors and other signals to penetrate MOS more easily (i.e., MOS has fewer diffusion limitations).

[0071] The PDMOs (e.g., MM MOSs) described herein can be used in a variety of different assays, and specifically can be used to determine the effects of drug formulations, including toxicity, on MM tissue. As used herein, a drug composition can include any drug, drug dilution, drug formulation, composition comprising multiple drugs (e.g., multiple active ingredients), drug formulation, drug form, drug concentration, combination therapy, etc. In some cases, a drug formulation refers to a formulation comprising a mixture of a drug and one or more inactive ingredients.

[0072] In some cases, drug screening can involve applying MOS to all or some of the wells of a multiwell (e.g., 96-well) plate. Alternatively, custom plates can be used (e.g., a 10,000-microwell array can be formed from a 100 x 100-well grid). MOS (e.g., gel droplets) can be applied to, or in some cases, onto, multiple microwell arrays and incubated with culture medium. MOS can be cultured for about 3 to about 14 days. In some cases, on a selected day (e.g., day 5), wells (e.g., microreactors) can be dosed with drug compounds to examine the effects of a drug panel, e.g., based on a set of FDA-approved anticancer drugs, such as drugs for treating MM. For example, the drugs tested can be based on the National Cancer Institute (Division of Cancer Treatment and Diagnosis) screen of 147 drugs intended to enable cancer research, drug discovery, and drug combination testing. In some cases, the drugs tested can include one or more agents selected from immunomodulatory agents (e.g., IMiDs, thalidomide, lenalidomide, and pomalidomide), PIs (e.g., bortezomib, carfilzomib, and ixazomib), monoclonal antibodies (e.g., elotuzumab, daratuzumab, isatuximab, and belantamab), nuclear export inhibitors (e.g., selinexor), doxorubicin, panobinostat, and melflufen, steroids (e.g., dexamethasone), alkylating agents (e.g., cyclophosphamide, melphalan, and bendamustine), CAR T-cell therapy (e.g., Abecma and Ide-cel), and bispecific antibodies. At a subsequent selected day (e.g., day 7), the MOS can be imaged (e.g., by standard fluorescence microscopy) and ranked based on drug response. In some cases, the drug IC 50Curves can be generated using flow-based viable myeloma cell counts with any appropriate number of drug titrations (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 5-7, or 7-9 titrations) and number of replicates per titration (e.g., 2-4, 2-3, 3-5, or 3-4 replicates per titration).

[0073] After the MOS has been treated with one or more candidate therapeutic agents, the cells within it can be evaluated to determine the level of myeloma cell death, thereby indicating the effectiveness of the tested therapeutic agent(s). The cells can be removed from the MOS using any suitable technique. For example, the MOS can be heated to melt the substrate (e.g., a hydrogel). The cells can then be stained with antibodies specific to the cellular marker to be detected. For example, antibodies against CD19, CD38, and CD58 (optionally CD138) can be used to stain myeloma cells within the cell population. The cells can also be stained with a live cell label, a dead cell label, or a live and dead cell label. Non-limiting examples of labels that can be used to distinguish between live and dead cells include GHOST DYE™ (CYTEK® 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™ dye (Becton Dickinson, Franklin Lakes, NJ). After labeling, cells can be subjected to flow cytometry to detect and quantitate the targeted type(s) of live and / or dead cells. Note that the above-described assays can be performed at the well level to assess the effect of one or more therapeutic agents on cells within all MOSs within a well, or the level of individual MOSs.

[0074] An example of an assay / screening technique is illustrated in Figures 17A-17E. In this example, the screening assay can be automated, allowing for a repeatable, automated workflow and increasing the number of drugs that can be tested in a screen. In Figure 17A, a tumor biopsy is taken, and multiple (e.g., over 10,000) MOSs are formed as described above (Figure 17A illustrates the junctional regions that form the MOS). The MOSs can then be collected and washed to remove any immiscible materials (e.g., oil) from which they formed. The MOSs can then be plated into one or more microwell plates. As shown in Figure 17C, the MOSs can be cultured for one or more generations (e.g., one or more passages). This is shown to occur from day 0 to day 3, day 4, or day 5. The MOSs can then be screened, for example, by applying drugs to a subset of replicate wells, as shown in Figure 17D. As shown in Figure 17E, cells in the MOS can then be imaged and / or scored automatically or manually to identify drug effects (e.g., drug screening and growth profiling).

[0075] The workflow described in Figures 17A-17E can enable an integrated device to be used for the growth, dosing, and / or screening of MOS. In an exemplary device, a freshly biopsied or resected patient tumor sample can be dissociated and seeded into a gel with a reagent to form the MOS described herein. In some cases, a portion of the formed MOS can be cryopreserved. The remainder can be harvested and incubated until seeded into microwell plates for drug testing or screening, as described. Growth and viability assays can be performed on the MOS, which can be imaged and tracked. IC 50 MOS responses to drug treatment, such as cytotoxicity, growth curves, and the like, can be measured to identify effective therapeutic agents for a patient's tumor (eg, MM).

[0076] The methods and devices described herein have numerous advantages, including reproducibility. The sample preparation process can be automated using microfluidic sample dispensing, thereby reducing the need for specialized personnel to perform diagnostic testing and manual pipetting. This can be particularly useful in clinical settings. Furthermore, this can enable uniformity between signal droplets, increasing assay sensitivity. Additionally, these assays can minimize the time required to generate MOS. In some cases, these methods can be used to generate libraries of over 100,000 MATRIGEL® tumor droplets (MOSs) in less than about 15 minutes. These methods are also highly scalable, allowing for multiplexing to perform multiple patient biopsies in parallel.

[0077] Additionally, the methods described herein are flexible and compatible with other techniques. As a research tool, droplet-based microfluidics is generally compatible with a wide range of hydrogel materials, such as agarose, alginate, PEG, and hyaluronic acid. Therefore, the starting gel composition can be easily modified to accompany and promote MOS growth. Furthermore, droplet size can be adjusted by changing the size of the microfluidic device. Taken together, these options allow for a wide selection of gel material compositions and microreactor sizes.

[0078] The miniaturized assay described herein (using MOS) can maximize the utility of patient tumor biopsies, allowing more drug compounds to be screened. For example, a 600 μL bone marrow sample from a mammal with MM can be distributed into approximately 143,000 individual microreactors, each with a volume of approximately 4 nL. By optimizing tissue sample utilization, multiple experimental replicates can be examined, increasing statistical power. These techniques enable testing of intratumor heterogeneity, drug perturbations, and the identification of rare cellular events such as drug resistance. MOS may generally be compatible with downstream assays such as single-cell RNA transcriptome analysis and epigenetic profiling. Additionally, by maximizing the efficiency of tissue (e.g., biopsy) samples provided by MOS, a portion of the MOS can be banked (e.g., by cryopreservation) for future novel drug assays and / or confirmatory analyses, including genetic screening.

[0079] Figures 18 and 19 provide a description of a treatment method using the methods and devices including the MOS described herein. In the case of precision medicine and personalized medicine, these methods and devices can be used as clinical indicators for appropriate drug selection to improve clinical outcomes and drug response. In some embodiments, a patient diagnosed with cancer (e.g., MM) has a biopsy taken for histopathology and screening of multiple MOSs formed from the biopsy using the methods described herein. Within approximately 7-10 days, screening can be performed from the biopsy to identify the most effective standard of care therapy, allowing the patient to begin treatment within approximately 14 days.

[0080] An example of this is illustrated in Figure 18. In this example, a tumor can be identified (e.g., by CT scan) on day 0 (1801), a biopsy can be taken on day 5 (1803), and hundreds, thousands, or tens of thousands of MOS can be generated that same day. The MOS can be cultured for approximately 1-5 days and then screened (1805) to identify one or more drug compositions that can be used. This same step (MOS formation and screening) can be used to guide precision medicine at multiple clinical decision points throughout disease progression. In this example, therapy using the identified one or more drug compositions can be initiated on day 14 (1809), and the patient can then be monitored during the course of treatment (e.g., by a follow-up CT scan on about day 90) to confirm that the tumor is responding to treatment (1811). If so, therapy can be continued (1813), and ongoing progression can be monitored (1815).

[0081] The use of MOS in assays can be repeated at multiple time points throughout the course of patient treatment. This is illustrated in Figure 19. For example, when a patient is initially diagnosed with a resectable primary tumor (1907), this technique (e.g., generating and screening MOS 1905) can be used to determine the most effective neoadjuvant therapy (1921). In this manner, a biopsy can be taken, and hundreds, thousands, or tens of thousands of MOSs can be formed and screened with a panel of candidate drug compositions. Once the primary tumor is resected (1923), this technique 1905' can indicate whether and which adjuvant therapy should be selected (1925). If recurrence or metastasis occurs after surgical removal of the primary tumor (1927), the same technique (e.g., generating and screening MOS from fresh biopsies 1905'', 1905''', 1905''') can be used to guide standard of care therapy, including first-line therapy 1929, second-line therapy 1931, and third-line therapy 1933. If a patient ultimately becomes resistant or refractory to all standard treatment therapies, this technique 1905 can be performed to identify off-label drugs to treat the resistant tumors 1935. This technique can also be used as a companion diagnostic to identify patients for specific treatments. Finally, this technique can be used to obtain patient-derived MOS, which can be stored to establish organosphere-based live cancer banks for screening, genomic profiling, drug discovery, drug testing, and / or clinical trial design.

[0082] Because the generation of large numbers of MOSs can be performed relatively minimally invasively (e.g., by excision or biopsy) and they can be used to provide reasonably rapid results from screening, the methods provided herein can be readily adapted to standard of care. For example, the volume of cellular material from tissue (e.g., biopsy) input is typically very small, e.g., can be placed in a volume of about 10 μL to about 5 mL.

[0083] Generally, the use of MOS described herein for screening can be automated or performed manually. Virtually any screening technique can be used, including imaging by one or more of confocal microscopy, fluorescence microscopy, liquid lens, holography, sonar, bright-field and dark-field imaging, laser, planar laser sheet, and high-throughput embodiments of image-based 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 gene or protein screening (e.g., scRNA-seq, ATAC-seq, proteomics, etc.) on MOS-derived cells.

[0084] Illustrative Embodiments Embodiment 1 is a MicroOrganoSphere comprising bone marrow cells from a mammal with multiple myeloma (MM). Embodiment 2 is a MicroOrganoSphere according to embodiment 1, wherein the MicroOrganoSphere comprises about 50 to about 150 cells. Embodiment 3 is a MicroOrganoSphere according to embodiment 1, wherein the MicroOrganoSphere comprises about 75 to about 125 cells. Embodiment 4 is the MicroOrganoSphere of embodiment 1, wherein the MicroOrganoSphere comprises about 100 cells. Embodiment 5 is a MicroOrganoSphere according to any one of Embodiments 1 to 4, wherein the cells comprise cancer cells, stromal cells, stem cells, immune cells, or any combination thereof. Embodiment 6 is the MicroOrganoSphere of embodiment 5, wherein the MicroOrganoSphere comprises cancer cells and stromal cells in a ratio of less than about 1:4. Embodiment 7 is a MicroOrganoSphere of embodiment 5, wherein the immune cells comprise at least one macrophage. Embodiment 8 is a MicroOrganoSphere of any one of embodiments 1-6, wherein the MicroOrganoSphere comprises a solubilized basement membrane matrix. Embodiment 9 is a composition comprising the MicroOrganoSpheres of any one of embodiments 1-8 in a culture medium. Embodiment 10 is the composition of embodiment 9, wherein the culture medium comprises a solubilized basement membrane matrix. Embodiment 11 is the composition of embodiment 10, comprising about 1% of the solubilized basement membrane matrix. Embodiment 12 is the composition of any one of embodiments 9-11, further comprising an immiscible fluid. Embodiment 13 is a MicroOrganoSphere according to embodiment 12, wherein the immiscible fluid is an oil. Embodiment 14 is a method for making MicroOrganoSpheres (MOS), comprising: receiving a bone marrow sample from a mammal having MM; purifying the bone marrow sample to form a purified sample; A population of MOS is isolated from the purified sample. forcing an unpolymerized fluid mixture, the unpolymerized fluid mixture comprising the purified sample and the unpolymerized fluid matrix material, through one or more channels of a microfluidic device, wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the purified sample and the unpolymerized fluid matrix material move through the one or more channels in a laminar flow; forming a plurality of droplets comprising the unpolymerized fluid mixture within the microfluidic device; and and polymerizing the fluid matrix material to form the MOSs, each having a diameter of 50-500 μm and having 30-150 cells distributed therein. Embodiment 15 is the method of embodiment 14, further comprising combining an immiscible fluid with the unpolymerized fluid mixture prior to forming the plurality of droplets, and propelling the immiscible fluid through another channel of the microfluidic device such that the droplets comprise the unpolymerized fluid mixture and the immiscible fluid. Embodiment 16 is the method of embodiment 15, wherein the immiscible fluid is oil. Embodiment 17 is the method of any one of embodiments 14 to 16, wherein forming a population of the MOS further comprises sorting the MOS based on cell number and / or droplet size. Embodiment 18 is the method of embodiment 17, wherein the sorting comprises optical sorting based on cell number and / or droplet size. Embodiment 19 is the method of any one of embodiments 14 to 18, wherein forming the population of MOSs comprises forming about 100 to about 600 MOSs. Embodiment 20 is the method of any one of embodiments 14 to 18, wherein forming the population of MSOs comprises forming about 600 to about 1,000 MSOs. Embodiment 21 is the method of any one of embodiments 14 to 18, wherein forming the population of MOSs comprises forming more than about 1,000 MOSs. Embodiment 22 is the method of any one of embodiments 14 to 21, wherein the microfluidic device maintains the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets. Embodiment 23 is the method of any one of embodiments 14 to 22, wherein the microfluidic device is configured to prevent clogging of the unpolymerized fluid mixture in the one or more channels. Embodiment 24 is the method of embodiment 23, wherein the microfluidic device is configured to prevent clogging by having a channel diameter of 100 μm or more. Embodiment 25 is the method according to any one of embodiments 14 to 24, wherein the microfluidic device is configured to maintain a substantially constant pressure in the one or more channels. Embodiment 26 is the method according to any one of embodiments 14 to 25, wherein the microfluidic device maintains a constant flow rate in the one or more channels. Embodiment 27 is the method of any one of embodiments 14 to 26, wherein the total length of the path traveled by the unpolymerized fluid mixture prior to the formation of the plurality of droplets in the microfluidic device is less than 10 cm. Embodiment 28 is the method of any one of embodiments 14 to 27, wherein the MOSs in the population of MOSs have a size variation of less than 25%. Embodiment 29 is the method of any one of embodiments 14 to 28, wherein the polymerizing comprises crosslinking the fluid matrix material. Embodiment 30 is the method of any one of embodiments 14 to 29, wherein the fluid matrix material is chemically crosslinkable or photocrosslinkable. Embodiment 31 is the method of any one of embodiments 14 to 30, wherein the bone marrow sample comprises freshly biopsied cells. Embodiment 32 is the method of embodiment 31, wherein the bone marrow sample is obtained from the mammal within 24 hours after formation of the MOS. Embodiment 33 is the method of any one of embodiments 14 to 32, wherein the bone marrow sample comprises MM plasma cells, immune cells, stem cells, stromal cells, or any combination thereof. Embodiment 34 is the method of embodiment 33, wherein the immune cells comprise one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocyte cells, and combinations thereof. Embodiment 35 is the method of any one of embodiments 14 to 34, comprising flowing the unpolymerized fluid mixture through the one or more channels at a flow rate of about 0.01 milliliters (mL) / minute (min) to about 100 mL / min. Embodiment 36 is a method of precision drug screening for personalized cancer therapy of MM, comprising: receiving a bone marrow sample from a mammal having MM; purifying the sample to form a purified sample; A population of MOS is isolated from the purified sample. forcing an unpolymerized fluid mixture, the unpolymerized fluid mixture comprising the purified sample and the unpolymerized fluid matrix material, through one or more channels of a microfluidic device, wherein the microfluidic device controls the pressure, flow rate, or pressure and flow rate within the one or more channels such that the purified sample and the unpolymerized fluid matrix material move through the one or more channels in a laminar flow; forming a plurality of droplets comprising the unpolymerized fluid mixture within the microfluidic device; and polymerizing the fluid matrix material to form the MOSs, each having a diameter of 50-500 μm and having 1-500 cells distributed therein; Culturing the population of MOS for 1 to 14 days; and assaying one or more drug therapies using the population of MOS. Embodiment 37 is the method of embodiment 36, further comprising combining an immiscible fluid with the unpolymerized fluid mixture prior to forming the plurality of droplets, and propelling the immiscible fluid through another channel of the microfluidic device such that the droplets comprise the unpolymerized fluid mixture and the immiscible fluid. Embodiment 38 is the method of embodiment 37, wherein the immiscible fluid is oil. Embodiment 39 is the method of any one of embodiments 36 to 38, wherein the assay comprises assaying multiple drug therapies in parallel by exposing one or more of the MOSs to each drug therapy. Embodiment 40 is the method of embodiment 39, comprising 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. Embodiment 41 is the method of any one of embodiments 36 to 40, wherein the time between receiving the bone marrow sample and characterizing the response is less than 21 days. Embodiment 42 is the method of any one of embodiments 36 to 41, wherein forming a population of the MOS further comprises sorting the MOS based on cell number and / or droplet size. Embodiment 43 is the method of any one of embodiments 36 to 42, wherein the sorting comprises optically sorting the MOS or based on cell number and / or droplet size. Embodiment 44 is the method of any one of embodiments 36 to 43, wherein the assay comprises assaying more than 10 different drug therapies. Embodiment 45 is the method of any one of embodiments 36 to 44, 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 administration times of one or more drugs. Embodiment 46 is the method of any one of embodiments 36 to 45, wherein forming the population of MOSs comprises forming about 100 to about 600 MOSs. Embodiment 47 is the method of any one of embodiments 36 to 45, wherein the population of MOSs comprises forming about 600 to about 1,000 MOSs. Embodiment 48 is the method of any one of embodiments 36 to 45, wherein forming the population of MOSs comprises forming more than 1,000 MOSs. Embodiment 49 is the method of any one of embodiments 36 to 48, wherein the microfluidic device maintains the viscosity of the unpolymerized fluid mixture before forming the plurality of droplets. Embodiment 50 is the method of any one of embodiments 36 to 49, wherein the microfluidic device is configured to prevent clogging of the unpolymerized fluid mixture in the one or more channels. Embodiment 51 is the method of embodiment 50, wherein the microfluidic device is configured to prevent clogging by having a channel diameter of 100 μm or more. Embodiment 52 is the method according to any one of embodiments 36 to 51, wherein the microfluidic device is configured to maintain a substantially constant pressure in the one or more channels. Embodiment 53 is the method according to any one of embodiments 36 to 52, wherein the microfluidic device maintains a constant flow rate in the one or more channels. Embodiment 54 is the method of any one of embodiments 36 to 53, wherein the total length of the path traveled by the unpolymerized fluid mixture prior to the formation of the plurality of droplets in the microfluidic device is less than 10 cm. Embodiment 55 is the method of any one of embodiments 36 to 54, further comprising measuring the effect of the one or more drug therapies on cells within the MOS. Embodiment 56 is the method of any one of embodiments 36-55, further comprising determining that the mammal remains responsive to one of the one or more drug therapies after one or more administrations of the one or more drug therapies by receiving a second bone marrow sample after the mammal has been treated with the one drug therapy, forming a second population of MOS from the second bone marrow sample, exposing at least a portion of the second population of MOS to the one drug therapy, and measuring the effect of the one drug therapy on cells within the at least a portion of the second population of MOS. Embodiment 57 is the method of any one of embodiments 36 to 56, further comprising treating the mammal with one of the one or more medications. Embodiment 58 is the method of any one of embodiments 36 to 57, wherein the MOSs in the population of MOSs have a size variation of less than 25%. Embodiment 59 is the method of any one of embodiments 36 to 58, wherein the polymerizing comprises crosslinking the fluid matrix material. Embodiment 60 is the method of any one of embodiments 36 to 59, wherein the fluid matrix material is chemically crosslinkable or photocrosslinkable. Embodiment 61 is the method of any one of embodiments 36 to 60, wherein the bone marrow sample comprises freshly biopsied cells. Embodiment 62 is the method of embodiment 61, wherein the bone marrow sample is obtained from the mammal within 24 hours after formation of the MOS. Embodiment 63 is the method of any one of embodiments 36 to 62, wherein the bone marrow sample comprises cancer cells, immune cells, stem cells, stromal cells, or any combination thereof. Embodiment 64 is the method of embodiment 63, wherein the immune cells comprise one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocyte cells, and combinations thereof. Embodiment 65 is a method according to any one of embodiments 36 to 64, comprising flowing the purified 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.

[0085] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0086] Example 1 - Preparation of MM MOS Cells were isolated from fresh bone marrow samples (1-3 mL each) derived from MM patient biopsies. Briefly, red blood cells were lysed in the bone marrow samples, and 70% MATRIGEL® was added to the bone marrow cell pellet. MM MOS were generated at a density of 30 cells per MOS. MOS culture 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 weekly. Representative images showing MM MOS formation on days 1, 6, 8, and 11 are shown in Figure 20A, with MM MOS indicated by white arrows. Day 11 MM MOS 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 (FIG. 20B), indicating that MOS formation requires the presence of stromal cells.

[0087] As shown in Figure 21A, major myeloid cell populations were preserved in the established MM MOS after 9 days in vitro. The myeloma cell population was shown to be CD11b- and CD38+. Flow cytometry also showed that major immune cell populations were preserved in the day 8 MOS (Figure 21C) compared to the bone marrow biopsy (Figure 21B). The total cell percentage over time in the MOS culture is plotted in the graph shown in Figure 21D. Additional studies have shown that the plasma cell and MM cell populations (CD138, respectively) are distinct from each other. + and CD38 + ), and T cell populations (CD3 + , CD4 + , and CD8 + ), as well as dendritic cell populations (CD11b + ) were all present in MOS (Figure 21E).

[0088] Figure 22A includes representative images showing MOS containing 50, 70, or 100 cells on days 0 and 7. Figure 22B includes representative images of MOS on day 7 stained for viable cells.

[0089] MOS containing 50, 70, or 100 cells, respectively, were prepared using approximately 8 × 10 6 MOS were also prepared from bone marrow biopsy samples containing 1000 cells. A representative image showing these MOS is presented in Figure 22A, and a representative live-dead staining of MOS at day 7 is shown in Figure 22B, demonstrating approximately 95% viability.

[0090] Example 2 - Effect of Drug Treatment on MM MOS Studies were conducted to determine the effects of various MM drugs on MM MOS. For example, day 11 MM MOS were treated with 5 μM lenalidomide or 2 nM bortezomib for 92 hours without changing the medium during drug treatment. Caspase 3 / 7 green dye was included in the medium to monitor apoptosis, and fluorescence was measured. These studies showed that MM MOS death was increased with lenalidomide treatment, but not with the negative control or bortezomib treatment (Figure 23A). Additionally, INCUCYTE® images taken every 2 hours over the course of treatment revealed that MM MOS responded to lenalidomide as early as 24 hours after treatment initiation (Figure 23B).

[0091] Further studies were performed using 10 μM carfilzomib or 10 μM selinexor treatment starting on day 9 of generating MOS from bone marrow biopsy samples from MM patients. After 4 days of treatment, live / dead dyes (calcein AM—green and ethidium homodimer—red) were added to the wells and incubated for 30 minutes. Images were captured using an EVOS™ M7000 Imaging System (ThermoFisher Scientific, Waltham, MA) (Figure 24A). The Xilis AI algorithm was used to calculate the fluorescent signal of individual organoids on both channels. Live / dead ratios were calculated, and dot plot data are presented in Figure 24B as the average ratio from each well. A significant decrease in the live / dead ratio was observed in MM MOS treated with either carfilzomib or selinexor compared to the negative control.

[0092] Example 3 - MM MOS can withstand freezing and thawing. MM MOS were frozen without disrupting the MATRIGEL® structure. After 2 days in liquid nitrogen, the frozen MM MOS were thawed in culture medium in a 37°C water bath. The droplet structure was maintained after thawing (Figure 25A), and viability was confirmed by SYTOX™ Blue measurement by flow cytometry (Figure 25B).

[0093] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A MicroOrganoSphere comprising bone marrow cells derived from a mammal with multiple myeloma (MM).

2. 10. The MicroOrganoSphere of claim 1, wherein the MicroOrganoSphere comprises about 50 to about 150 cells.

3. 10. The MicroOrganoSphere of claim 1, wherein the MicroOrganoSphere comprises about 75 to about 125 cells.

4. 10. The MicroOrganoSphere of claim 1, wherein the MicroOrganoSphere comprises about 100 cells.

5. The MicroOrganoSphere of claim 1 , wherein the cells comprise cancer cells, stromal cells, stem cells, immune cells, or any combination thereof.

6. 6. The MicroOrganoSphere of claim 5, wherein the MicroOrganoSphere comprises cancer cells and stromal cells in a ratio of less than about 1:

4.

7. The MicroOrganoSphere of claim 5 , wherein the immune cells comprise at least one macrophage.

8. The MicroOrganoSphere of claim 1 , wherein the MicroOrganoSphere comprises a solubilized basement membrane matrix.

9. A composition comprising the MicroOrganoSphere of claim 1 in a culture medium.

10. The composition of claim 9 , wherein the culture medium comprises a solubilized basement membrane matrix.

11. 11. The composition of claim 10, comprising about 1% of the solubilized basement membrane matrix.

12. The composition of claim 9 further comprising an immiscible fluid.

13. The MicroOrganoSphere of claim 12 , wherein the immiscible fluid is an oil.

14. 1. A method for making a MicroOrganoSphere (MOS), comprising: receiving a bone marrow sample from a mammal with MM; purifying the bone marrow sample to form a purified sample; extracting a population of MOS from the purified sample; propelling an unpolymerized fluid mixture, the unpolymerized fluid mixture comprising the purified sample and the unpolymerized fluid matrix material, through one or more channels of a microfluidic device, wherein the microfluidic device controls the pressure, the flow rate, or the pressure and the flow rate within the one or more channels such that the purified sample and the unpolymerized fluid matrix material move through the one or more channels in a laminar flow; forming a plurality of droplets comprising the unpolymerized fluid mixture within the microfluidic device; and and polymerizing the fluid matrix material to form the MOSs, each having a diameter of 50-500 μm and having 30-150 cells distributed therein.

15. 15. The method of claim 14, further comprising propelling the immiscible fluid through another channel of the microfluidic device such that an immiscible fluid is combined with the unpolymerized fluid mixture prior to forming the plurality of droplets, the droplets comprising the unpolymerized fluid mixture and the immiscible fluid.

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

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

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

19. 15. The method of claim 14, wherein forming the population of MOSs comprises forming about 100 to about 600 MOSs.

20. 15. The method of claim 14, wherein forming the population of MOSs comprises forming about 600 to about 1,000 MOSs.

21. 15. The method of claim 14, wherein forming the population of MOSs comprises forming greater than about 1,000 MOSs.

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

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

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

25. The method of claim 14 , wherein the microfluidic device is configured to maintain a substantially constant pressure within the one or more channels.

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

27. 15. The method of claim 14, wherein the total length of the path traveled by the unpolymerized fluid mixture prior to the formation of the plurality of droplets within the microfluidic device is less than 10 cm.

28. 15. The method of claim 14, wherein the MOSs in the population of MOSs have a size variation of less than 25%.

29. The method of claim 14 , wherein the polymerizing comprises crosslinking the fluid matrix material.

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

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

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

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

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

35. 15. The method of claim 14, comprising 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. 1. A method of precision drug screening for personalized cancer therapy for MM, comprising: receiving a bone marrow sample from a mammal with MM; purifying the sample to form a purified sample; extracting a population of MOS from the purified sample; propelling an unpolymerized fluid mixture, the unpolymerized fluid mixture comprising the purified sample and the unpolymerized fluid matrix material, through one or more channels of a microfluidic device, wherein the microfluidic device controls the pressure, the flow rate, or the pressure and the flow rate within the one or more channels such that the purified sample and the unpolymerized fluid matrix material move through the one or more channels in a laminar flow; forming a plurality of droplets comprising the unpolymerized fluid mixture within the microfluidic device; and polymerizing the fluid matrix material to form the MOSs, each having a diameter of 50-500 μm and having 1-500 cells distributed therein; Culturing the population of MOS for 1 to 14 days; and assaying one or more drug therapies using said population of MOS.

37. 37. The method of claim 36, further comprising propelling an immiscible fluid through another channel of the microfluidic device such that an immiscible fluid is combined with the unpolymerized fluid mixture prior to forming the plurality of droplets, the droplets comprising the unpolymerized fluid mixture and the immiscible fluid.

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

39. 37. The method of claim 36, wherein the assay comprises assaying multiple drug therapies in parallel by exposing one or more of the MOS to each drug therapy.

40. 40. The method of claim 39, comprising characterizing a 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.

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

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

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

44. 37. The method of claim 36, wherein the assay comprises assaying more than 10 different drug therapies.

45. 37. The method of 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 administration times of one or more drugs.

46. 37. The method of claim 36, wherein forming the population of MOSs comprises forming about 100 to about 600 MOSs.

47. 37. The method of claim 36, wherein said population of MOSs comprises forming about 600 to about 1,000 MOSs.

48. 37. The method of claim 36, wherein forming the population of MOSs comprises forming more than 1,000 MOSs.

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

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

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

52. 37. The method of claim 36, wherein the microfluidic device is configured to maintain a substantially constant pressure within the one or more channels.

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

54. 37. The method of claim 36, wherein the total length of the path traveled by the unpolymerized fluid mixture prior to the formation of the plurality of droplets within the microfluidic device is less than 10 cm.

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

56. 37. The method of 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 one or more drug therapies by receiving a second bone marrow sample after the mammal has been treated with the one drug therapy, forming a second population of MOS from the second bone marrow sample, exposing at least a portion of the second population of MOS to the one drug therapy, and measuring the effect of the one drug therapy on cells within the at least a portion of the second population of MOS.

57. 37. The method of claim 36, further comprising treating the mammal with one of the one or more medications.

58. 37. The method of claim 36, wherein the MOSs in the population of MOSs have a size variation of less than 25%.

59. 37. The method of claim 36, wherein the polymerizing comprises crosslinking the fluid matrix material.

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

61. 37. The method of claim 36, wherein the bone marrow sample comprises freshly biopsied cells.

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

63. 37. The method of claim 36, wherein the bone marrow sample comprises cancer cells, immune cells, stem cells, stromal cells, or any combination thereof.

64. 64. The method of claim 63, wherein the immune cells comprise one or more of T cells, B cells, macrophages, dendritic cells, NK cells, monocyte cells, and combinations thereof.

65. 37. The method of claim 36, comprising flowing the purified 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.

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