Tumor cell suspension cultures and related methods

Abstract

To provide suspension-based cell culture systems and media for timely and efficient proliferation of human tumor cell clusters from a patient, and related methods of evaluating potential responsiveness of the tumor cells and the patient to one or more therapeutic agents.SOLUTION: Embodiments of the present disclosure relate to a medium suspension (or multiple medium suspensions), comprising a population of human tumor cell clusters from a human patient, where the medium suspension provides at least about 10% proliferation of the tumor cell clusters within about 14 days. In some specific embodiments, the medium suspension provides at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% proliferation of the human tumor cell clusters within about 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 days.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to PCT / CN2016 / 075947, filed March 9, 2016, which is incorporated by reference in its entirety. [Background technology]

[0002] background Embodiments of the present disclosure relate to suspension cell culture systems and media for the timely and efficient growth of human tumor cell clusters from patients, and related methods for assessing the potential responsiveness of tumor cells and patients to one or more therapeutic agents.

[0003] Despite constant efforts to improve diagnostic and therapeutic drugs, cancer remains the leading cause of death worldwide. The diversity or heterogeneity of cancer hampers the development of new therapies and makes it difficult to identify likely responders. Furthermore, many cancer therapies suffer from primary and acquired resistance challenges, including additional point mutations and alternative pathways that bypass the target of the therapeutic agent.

[0004] The success of future therapies is likely to depend on a comprehensive analysis of the events underlying tumor progression and metastatic processes, along with the development of relevant model systems that can be easily manipulated to accurately evaluate the efficacy of chemotherapeutic drugs and combinations. As an example, primary culture of cancer cells using matrix-embedded three-dimensional (3D) cultures is increasingly being used to investigate cancer biology and predict chemotherapy sensitivity in individual patients. However, conditions for such matrix-embedded primary culture of cancer cells are currently not optimized. Moreover, such primary cultures can be cumbersome and include, among other potential problems, low chances of cancer cell survival, contamination with host cells, and results that are difficult to reproduce.

[0005] Thus, there is a need to develop methods and compositions by which primary cancer cells can be cultured in 3D as feasibly, efficiently, and in a suitable manner as cell lines. [Brief description of the drawings]

[0006] [Figure 1] We demonstrate that tumor cell clusters were successfully isolated from colon / rectal tumors surgically removed from patients and cultured in suspension in defined growth medium. [Diagram 2] It shows that tumor cells proliferated during the short-term suspension culture period as measured by Edu incorporation (Sample 1 is from FIG. 1A, Sample 2 is from FIG. 1B, Sample 3 is from FIG. 1C, and Sample 4 is from FIG. 1D). [Diagram 3] Figure 3 shows that short-term suspension cultures (within about 7 days) show the highest cell proliferation markers of tumor cell clusters. Figure 3A shows proliferation markers of four different patient samples, and Figure 3B shows proliferation markers of one exemplary patient sample over a one week period. [Figure 4] Under the test conditions (see Figure 3B), we show that the growth of tumor cell clusters is not completely inhibited by the drugs tested (sunitinib, sorafenib, erlotinib, crizotinib, oxaliplatin), which is consistent with patient-derived xenograft (PDX) tumor responses to these drugs. Summary of the Invention [Means for solving the problem]

[0007] An embodiment of the present disclosure relates to a media suspension (or a plurality of media suspensions) comprising a population of human tumor cell clusters from a human patient, the media suspension resulting in proliferation of at least about 10% of the tumor cell clusters within about 14 days.

[0008] In certain embodiments, the medium suspension results in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% growth of human tumor cell clusters within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days. In some embodiments, the medium suspension results in at least about 20-30% growth of human tumor cell clusters within about 7 days. In certain embodiments, the medium suspension results in at least about 60% growth of human tumor cell clusters within about 7 days.

[0009] In certain embodiments, the medium suspension comprises a synthetic medium, hi some embodiments, the medium suspension is serum-free or substantially serum-free.

[0010] In certain embodiments, the medium suspension includes or further includes one or more components selected from DMEM / F-12, Wnt3A, basic fibroblast growth factor (bFGF), insulin, transferrin, bovine serum albumin (BSA), cholesterol, B cell activating factor (BAFF), and 2-mercaptoethanol. In some embodiments, the medium suspension includes 2, 3, 4, 5, 6, 7, 8, or 9 of the components selected from DMEM / F-12, Wnt3A, bFGF, insulin, transferrin, bovine serum albumin (BSA), cholesterol, B cell activating factor (BAFF), and 2-mercaptoethanol. In certain embodiments, the medium suspension includes DMEM / F-12, Wnt3A, bFGF, insulin, transferrin, bovine serum albumin (BSA), cholesterol, B cell activating factor (BAFF), and 2-mercaptoethanol.

[0011] In certain embodiments, the medium suspension comprises or further comprises one or more components selected from nicotinamide, noggin, R-spondin-1, Y27632, fibroblast growth factor 10 (FGF10), and VO-OHpic. In some embodiments, the medium suspension comprises 2, 3, 4, 5, or 6 of the components selected from nicotinamide, noggin, R-spondin-1, Y27632, FGF10, and VO-OHpic. In certain embodiments, the medium suspension comprises nicotinamide, noggin, R-spondin-1, Y27632, FGF10, and VO-OHpic.

[0012] In certain embodiments, the medium suspension comprises or further comprises one or more components selected from epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), N2 supplement, B27, prostaglandin E2 (PGE-2), and N-acetyl-L-cysteine. In certain embodiments, the medium suspension comprises 2, 3, 4, 5, 6, 7, 8, or 9 of the components selected from EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, and N-acetyl-L-cysteine. In certain embodiments, the medium suspension comprises EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, and N-acetyl-L-cysteine.

[0013] In certain embodiments, the media suspension comprises or further comprises nicotinamide, noggin, R-spondin-1, Y27632, EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, N-acetyl-L-cysteine, FGF10, and VO-OHpic.

[0014] In some embodiments, the media suspension does not include extracellular matrix components, such as matrigel, collagen, laminin, or fibronectin.

[0015] In certain embodiments, the media suspension comprises human tumor cell clusters that are lung, colon, gastric, or breast cancer cells from a human patient. In some embodiments, the media suspension comprises human tumor cell clusters isolated from an immunodeficient animal xenografted with human tumor cells and / or tissue from a human patient. In certain embodiments, the media suspension comprises human tumor cell clusters isolated directly from a tumor specimen removed from a human patient, optionally selected from a surgical specimen, a biopsy, a pleural effusion, and an ascites fluid.

[0016] Also included are methods for expanding human tumor cells, comprising obtaining a population of human tumor cells from a sample and culturing the population of human tumor cell clusters in a medium suspension as described herein. Some methods comprise culturing the human tumor cell clusters in a medium suspension for about 14 days or less. Some methods comprise culturing the human tumor cell clusters in a medium suspension for about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days or less. Particular methods comprise culturing the human tumor cell clusters in a medium suspension for about 7 days or less.

[0017] 1. A method for testing the responsiveness of a human patient to a therapeutic agent, comprising: Obtaining or receiving a population of tumor cell clusters from a human patient; Cultivating a population of tumor cell clusters in a medium suspension according to any one of the preceding claims; administering a therapeutic agent to the medium suspension; measuring tumor cell proliferation and / or tumor cell apoptosis in the population of tumor cells; Also included are methods in which a reduction in tumor cell proliferation and / or induction of tumor cell apoptosis indicates responsiveness of the human patient to the therapeutic agent, and a lack of a reduction in tumor cell proliferation and / or induction of tumor cell apoptosis indicates resistance of the human patient to the therapeutic agent.

[0018] Some methods involve administering the therapeutic agent on the same day that the population of tumor cell clusters is cultured in suspension in media.

[0019] Certain methods include administering the therapeutic agent at least one day after culturing the population of tumor cell clusters in a medium suspension. Certain methods include administering the therapeutic agent about 1, 2, 3, 4, 5, 6, or 7 days or less after culturing the population of tumor cell clusters in a medium suspension.

[0020] Some methods involve measuring tumor cell proliferation and / or tumor cell apoptosis in a population of tumor cells within about 14 days of administering the therapeutic agent. Some methods involve measuring tumor cell proliferation and / or tumor cell apoptosis in a population of tumor cells within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days of administering the therapeutic agent. Particular methods involve measuring tumor cell proliferation and / or tumor cell apoptosis in a population of tumor cells within about 7, 6, or 5 days of administering the therapeutic agent.

[0021] In some embodiments, the step of measuring tumor cell proliferation comprises measuring a cell proliferation marker. In some embodiments, the cell proliferation marker is 3 The antibody is selected from one or more of 3H-thymidine, bromodeoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (Edu), Ki-67, and proliferating cell nuclear antigen (PCNA).

[0022] In certain embodiments, the step of measuring tumor cell apoptosis comprises measuring a cellular apoptosis marker, in some embodiments, the cellular apoptosis marker is selected from one or more of Fluorescently Labeled Inhibitor of Caspase (FLICA), Caspase Activation, Poly ADP-Ribose Polymerase (PARP) cleavage, DRAQ5, DRAQ7, and Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick End Labeling (TUNEL) assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Embodiments of the present disclosure relate to the discovery of defined media and associated suspension cell cultures that achieve reliable and timely growth of primary human tumor cell clusters, whether derived directly from a patient or from a patient-derived xenograft (PDX). Suspension cell cultures can be used, among other things, to assess the potential responsiveness of a patient tumor to one or more therapeutic agents, and offer the advantage of identifying optimal treatment options for a patient in a relatively short period of time, e.g., within one or two weeks from the time of culture.

[0024] definition 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, certain preferred methods and materials are described herein. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated by reference in their entirety as if each individual publication or reference was specifically and individually indicated to be incorporated by reference. For purposes of the present invention, the following terms are defined below.

[0025] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0026] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0027] Throughout this specification, unless the context indicates otherwise, the words "comprise", "comprises" and "comprising" are understood to imply the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to everything that follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the recited elements.

[0028] "Isolated" means material that is substantially or essentially free from components which normally accompany it in its native state.

[0029] The term "modulation" typically includes "increase" or "enhancement" as well as "decrease" or "reduction" in a statistically significant or physiologically significant amount when compared to a control. An "increase" or "enhancement" amount is typically a "statistically significant" amount, and is about or at least about 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000 times, or about or at least about 5%, 100% or more of the amount produced by the composition or control composition, sample, or test subject without the composition or control composition, sample, or test subject. This may include increases that are 0%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% (including all integers and ranges therebetween). A "reduction" or "reduction" amount is typically a "statistically significant" amount, and is about or at least about 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000 times, or about or at least about 5%, 100% or more of the amount produced without the composition or control composition, sample, or test subject. This may include reductions that are 0%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% (including all integers and ranges therebetween).

[0030] In certain embodiments, the "purity" of tumor cell clusters in a medium or suspension culture can be specifically defined. For example, a certain medium or suspension culture can contain tumor cell clusters that are about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% pure (including all integers and ranges therebetween) with respect to other cell types, such as non-cancerous host cells or animal cells (e.g., with respect to tumor cell clusters derived from xenografts).

[0031] "Statistically significant" means that the result is unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include p-value, which is the frequency or probability that the observed event would occur if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0032] "Substantially" or "essentially" means nearly completely or completely, e.g., 95% or more, of a given amount.

[0033] As noted above, certain embodiments relate to media suspensions comprising a population of tumor cell clusters from a human patient, which media suspensions result in proliferation of at least about 10% of the tumor cell clusters within about 14 days. Thus, in these and related embodiments, the tumor cell clusters grow in suspension culture, e.g., as clumps in suspension, rather than in adherent culture.

[0034] In some embodiments, the media suspension results in proliferation of about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (including all combinations, integers, and ranges therebetween) of the tumor cell clusters within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days.

[0035] For example, a particular medium suspension can optionally be cultured within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days, or within about 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14 days, or within about 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13 days, or within about 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 days, or within about 5-11, 6-11, 7-11, 8-11, 9-11, or or within about 5-10, 6-10, 7-10, 8-10, or 9-10 days, or within about 5-9, 6-9, 7-9, or 8-9 days, or within about 5-8, 6-8, or 7-8 days, or within about 5-7 or 6-7 days, or within about 5-6 days, , 10-95, or 10-100% proliferation of tumor cell clusters, or about or at least about 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 15-55, 15-60, 15-65, 15-70, 15-75, 15-80, 15-85, 15-90, 15-95, or 15-100% proliferation of tumor cell clusters, or about or at least about 20-25, 20-30, 20-35, 20-40, 20-45, 20-50, 20-55, 20-60, 20-65, 20-70, 20-75, 20-80, 20-95, or 20-100% proliferation of tumor cell clusters. 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, or 25-100% proliferation of tumor cell clusters or about or at least about 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85,30-90, 30-95, or 30-100% proliferation, or about or at least about 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, or 35-100% proliferation, or about or at least about 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, or 40-100% proliferation of tumor cell clusters or about or at least about 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, or 45-100% proliferation of tumor cell clusters, or about or at least about 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, or 50-100% proliferation of tumor cell clusters, or about or at least about 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, or 55-100% proliferation of tumor cell clusters, or about or at least about 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, or 60-100% proliferation of tumor cell clusters, or about or at least about 65-70, 65-75, 65-80, 65-85, 65-90, 65-95, or 65-100% proliferation of tumor cell clusters, or about or at least about 70-80, 70-85, 70 or about or at least about 75-80, 75-85, 75-90, 75-95, or 75-100% proliferation of tumor cell clusters, or about or at least about 80-85, 80-90, 80-95, or 80-100% proliferation of tumor cell clusters, or about or at least about 85-90, 85-95, or 85-100% proliferation of tumor cell clusters, or about or at least about 90-95 or 90-100% proliferation of tumor cell clusters,Alternatively, the method results in about or at least about 95-100% proliferation of the tumor cell clusters (including all combinations thereof). Particular embodiments result in at least about 20-30% proliferation of the tumor cell clusters within about 7 days, or at least about 60% proliferation of the tumor cell clusters within about 7 days.

[0036] In some embodiments, the medium suspension of tumor cell clusters comprises a "synthetic medium". In certain embodiments, a "synthetic medium" is a growth medium suitable for in vitro or ex vivo cell culture of human or animal cells, in which all of the chemical components are known. In some embodiments, the synthetic medium is "serum-free" or "substantially serum-free", that is, the medium lacks or is substantially lacking the addition of serum, for example, fetal bovine serum (FBS) or fetal calf serum (FCS).

[0037] In some embodiments, the synthetic medium comprises a basal medium such as DMEM, F12, RPMI 1640, or a combination thereof, e.g., DMEM / F12 supplemented with additional components, e.g., growth factors, antioxidants, and / or energy sources. Thus, in certain embodiments, the synthetic medium includes Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture (F12), RPMI 1640, or DMEM / F12. In some embodiments, the medium comprises DMEM / F12 in a ratio of, e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0038] In certain embodiments, the synthetic medium comprises one or more components, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 components, selected from Wnt3A, basic fibroblast growth factor (bFGF), insulin, transferrin, bovine serum albumin (BSA), cholesterol, B cell activating factor (BAFF), and 2-mercaptoethanol (including all combinations of the foregoing). Particular embodiments comprise DMEM / F-12 in combination with one or more components, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 components, selected from Wnt3A, bFGF, insulin, transferrin, bovine serum albumin (BSA), cholesterol, B cell activating factor (BAFF), and 2-mercaptoethanol. Particular embodiments comprise DMEM / F-12, Wnt3A, bFGF, insulin, transferrin, bovine serum albumin (BSA), cholesterol, B cell activating factor (BAFF), and 2-mercaptoethanol.

[0039] In certain embodiments, the concentration of Wnt3A is about 0.1 to 10,000 or 1 to 1000 ng / ml, or about 0.1 to 1000, 1 to 1000, 10 to 1000, 20 to 1000, 30 to 1000, 40 to 1000, 50 to 1000, 60 to 1000, 70 to 1000, 80 to 1000, 90 to 1000, 100 to 1000, 200 to 1000, 300 to 1000, 400 to 1000, 500 to 1000, 600 to 1000, 700 to 1000, 800 to 1000, 900 to 1000 ng / ml, or about .1-500, 1-500, 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500, 100-500, 200-500, 300-500, 400-500 ng / ml, or about 0.1-400, 1-400, 10-400, 20-400, 30-400, 50-400, 40-400, 60-400, 70-400, 80-400, 90-400, 100-400, 200-400, 300-400 ng / ml, or about 0.1-30 0, 1-300, 10-300, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, 200-300ng / ml, or about 0.1-200, 1-200, 10-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200ng / ml, or about 0.1-150, 1-150, 10-150, 20-150, 30-150, 40-150, 5 The range is 0-150, 60-150, 70-150, 80-150, 90-150, 100-150 ng / ml, or about 0.1-120, 1-120, 10-120, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120 ng / ml, or about 0.1-100, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100 ng / ml. In certain embodiments, the Wnt3A is a human or mouse Wnt3A protein, for example, a recombinant human or mouse Wnt3A protein.

[0040] In certain embodiments, the concentration of bFGF is about 0.1-100 or 1-10 ng / ml, or about 0.1-100, 0.2-100, 0.3-100, 0.4-100, 0.5-100, 0.6-100, 0.7-100, 0.8-100, 0.9-100, 1-100, 2-100, 3-100, 4-100, 5-100, 10-100, 50-100 ng / ml, or about 0.1-50, 0.2-50, 0.3-50, 0.4-50, 0.5-50, 0.6-50, 0.7-50, 0.8-5 The range is 0, 0.9-50, 1-50, 2-50, 3-50, 4-50, 5-50, 10-50 ng / ml, or about 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.6-10, 0.7-10, 0.8-10, 0.9-10, 1-10, 2-10, 3-10, 4-10, 5-10 ng / ml, or about 0.1-5, 0.2-5, 0.3-5, 0.4-5, 0.5-5, 0.6-5, 0.7-5, 0.8-5, 0.9-5, 1-5, 2-5, 3-5, 4-5 ng / ml. In certain embodiments, the bFGF is a human or mouse bFGF protein, e.g., a recombinant human or mouse bFGF protein.

[0041] In certain embodiments, the concentration of insulin is about 0.1-10,000 or 1-1000 or 100-500 μg / ml, or about 0.1-1000, 1-1000, 10-1000, 20-1000, 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 80-1000, 90-1000, 100-1000, 200-1 000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000 μg / ml, or about 0.1-500, 1-500, 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500, 100-500, 200-50 0, 300-500, 400-500 μg / ml, or about 0.1-400, 1-400, 10-400, 20-400, 30-400, 50-400, 40-400, 60-400, 70-400, 80-400, 90-400, 100-400, 200-400, 300-400 μg / ml, or about 0.1-300, 1-300, 10-300, 20-300, 30 The insulin concentration ranges from about 0.1 to 200, 1 to 200, 10 to 200, 20 to 200, 30 to 200, 40 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200 μg / ml. In certain embodiments, the insulin is a human or mouse insulin protein, e.g., a recombinant human or mouse insulin protein. In some embodiments, the insulin is a solution or powder from a pancreatic extract, e.g., a powder or solution from bovine pancreas.

[0042] In certain embodiments, the concentration of transferrin is about 0.1-10,000 or 1-1000 or 1-100 μg / ml, or about 0.1-1000, 1-1000, 10-1000, 20-1000, 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 80-1000, 90-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000 μg / ml, or about 0.1-500, 1-500, 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500, 100-500, 200-500, 300-500, 400-500 μg / ml, or about 0.1-400, 1-400, 10-400, 20-400, 30-400, 50-400, 40-400, 60-400, 70-400, 80-400, 90-400, 100-400, 200-400, 300-400 μg / ml, or about 0.1-300, 1-300, 10-300, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, 200-300 μg / ml, or about 0.1-200, 1-200, 10-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200 μg / ml, or about 0.1-150, 1-150, 10-150, 20-150, 30-150, 40-15 The range is 0, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150 μg / ml, or about 0.1-120, 1-120, 10-120, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120 μg / ml, or about 0.1-100, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100 μg / ml.

[0043] In certain embodiments, the concentration of BSA is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50%, or about 1-50%, or about 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50%, or about 5-45, 10-45, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45%, or about 5-40, 10-45, The range is 0, 15-40, 20-40, 25-40, 30-40, 35-40%, or about 5-35, 10-35, 15-35, 20-35, 25-35, 30-35%, or about 5-30, 10-30, 15-30, 20-30, 25-30%, or about 5-25, 10-25, 15-25, 20-25%, or about 5-20, 10-20, 15-20%, or about 5-15, 10-15%, or about 5-10%.

[0044] In certain embodiments, the concentration of cholesterol is, or is in the range of, about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, or is about, 01-10 times or 0.1-10 times, or about 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.6-10, 0.7-10, 0.8-10, 0.9-10, 1-10, 2-10, 5-10 times, or about 0.1-5 , 0.2-5, 0.3-5, 0.4-5, 0.5-5, 0.6-5, 0.7-5, 0.8-5, 0.9-5, 1-5, 2-5 times, or about 0.1-4, 0.2-4, 0.3-4, 0.4-4, 0.5-4, 0.6-4, 0.7-4, 0.8-4, 0.9-4, 1-4, 2-4 times, or about 0 0.1-3, 0.2-3, 0.3-3, 0.4-3, 0.5-3, 0.6-3, 0.7-3, 0.8-3, 0.9-3, 1-3, 2-3 times, or about 0.1-2, 0.2-2, 0.3-2, 0.4-2, 0.5-2, 0.6-2, 0.7-2, 0.8-2, 0.9-2, 1-2 times. Cholesterol (250X) is commercially available, for example, from ThermoFisher Scientific.

[0045] In certain embodiments, the concentration of BAFF is about 0.1-10,000 or 1-1000 ng / ml, or about 0.1-1000, 1-1000, 10-1000, 20-1000, 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 80-1000, 90-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000 ng / ml, or about 0. 1-500, 1-500, 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500, 100-500, 200-500, 300-500, 400-500 ng / ml, or about 0.1-400, 1-400, 10-400, 20-400, 30-400, 50-400, 40-400, 60-400, 70-400, 80-400, 90-400, 100-400, 200-400, 300-400 ng / ml, or about 0.1-300 , 1-300, 10-300, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, 200-300ng / ml, or about 0.1-200, 1-200, 10-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200ng / ml, or about 0.1-150, 1-150, 10-150, 20-150, 30-150, 40-150, 5 The range is 0-150, 60-150, 70-150, 80-150, 90-150, 100-150 ng / ml, or about 0.1-120, 1-120, 10-120, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120 ng / ml, or about 0.1-100, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100 ng / ml. In certain embodiments, the BAFF is a human or mouse BAFF protein, eg, a recombinant human or mouse BAFF protein.

[0046] In certain embodiments, the concentration of 2-mercaptoethanol is about 0.01-100 or 0.1-1 mM, or 0.01-10, 0.02-10, 0.03-10, 0.04-10, 0.05-10, 0.06-10, 0.07-10, 0.08-10, 0.09-10, 0.1-10, 0.5-10, 1-10, or 5-10 mM, or about 0.01-5, 0.02-5, 0.03-5, 0.04-5, 0.05-5, 0.06-5, 0 0.07-5, 0.08-5, 0.09-5, 0.1-5, 0.5-5, 1-5 mM, or about 0.01-2, 0.02-2, 0.03-2, 0.04-2, 0.05-2, 0.06-2, 0.07-2, 0.08-2, 0.09-2, 0.1-2, 0.5-2, 1-2 mM, or about 0.01-1, 0.02-1, 0.03-1, 0.04-1, 0.05-1, 0.06-1, 0.07-1, 0.08-1, 0.09-1, 0.1-1, 0.5-1 mM M, or about 0.01-0.5, 0.02-0.5, 0.03-0.5, 0.04-0.5, 0.05-0.5, 0.06-0.5, 0.07-0.5, 0.08-0.5, 0.09-0.5, or 0.1-0.5 mM, or about 0.01-0.4, 0.02-0.4, 0.03-0.4, 0.04-0.4, 0.05-0.4, 0.06-0.4, 0.07-0.4, 0.08-0.4, 0.09-0.4, or 0.1-0.4 mM, or is in the range of about 0.01-0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07-0.3, 0.08-0.3, 0.09-0.3, or 0.1-0.3 mM, or about 0.01-0.2, 0.02-0.2, 0.03-0.2, 0.04-0.2, 0.05-0.2, 0.06-0.2, 0.07-0.2, 0.08-0.2, 0.09-0.2, or 0.1-0.2 mM.

[0047] In certain embodiments, and in addition to the above components, the suspension medium comprises one or more components, for example 1, 2, 3, 4, 5, or 6 components selected from nicotinamide, noggin, R-spondin-1, Y27632, fibroblast growth factor 10 (FGF10), and VO-OHpic (including all combinations of the above). In some embodiments, the suspension medium comprises nicotinamide, noggin, R-spondin-1, Y27632, FGF10, and VO-OHpic.

[0048] In certain embodiments, the concentration of nicotinamide is about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 mM, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 mM, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 mM, or about 0.1-50, 0. or about 0.1-30, 0.5-30, 1-30, 5-30, 10-30 mM, or about 0.1-20, 0.5-20, 1-20, 5-20, 10-20 mM, or about 0.1-10, 0.5-10, 1-10, 5-10 mM, or about 0.1-5, 0.5-5, 1-5 mM, or about 0.1-1 or 0.5-1 mM.

[0049] In certain embodiments, the concentration of Noggin is about 0.1-10,000 or 1-1000 ng / ml, or about 0.1-1000, 1-1000, 10-1000, 20-1000, 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 80-1000, 90-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000 ng / ml, or about 0.1 ~500, 1~500, 10~500, 20~500, 30~500, 40~500, 50~500, 60~500, 70~500, 80~500, 90~500, 100~500, 200~500, 300~500, 400~500ng / ml, or about 0.1~400, 1~400, 10~400, 20~400, 30~400, 50~400, 40~400, 60~400, 70~400, 80~400, 90~400, 100~400, 200~400, 300~400ng / ml, or about 0.1~300 , 1-300, 10-300, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, 200-300ng / ml, or about 0.1-200, 1-200, 10-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200ng / ml, or about 0.1-150, 1-150, 10-150, 20-150, 30-150, 40-150, 5 The range is 0-150, 60-150, 70-150, 80-150, 90-150, 100-150 ng / ml, or about 0.1-120, 1-120, 10-120, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120 ng / ml, or about 0.1-100, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100 ng / ml. In certain embodiments, Noggin is a human or mouse Noggin protein, for example, a recombinant human or mouse Noggin protein.

[0050] In certain embodiments, the concentration of R-spondin-1 is about 0.1-10,000 or 1-1000 or 100-1000 ng / ml, or about 0.1-10,000, 1-10,000, 10-10,000, 20-10,000, 30-10,000, 40-10,000, 50-10,000, 60-10,000, 70-10,000, 80-10,000, 90-10,000, 100-10,000, 200-10,000, 300-10,000, 400-10 ,000, 500-10,000, 1000-10,000, 5000-10,000ng / ml, or about 0.1-5000, 1-5000, 10-5000, 20-5000, 30-10,000, 40-5000, 50-5000, 60-5000, 70-5000, 80-5000, 90-5000, 100-5000, 200-5000, 300-5000, 400-5000, 500-5000, 1000-5000ng / ml, or about 0.1-1000, 1-1000, 1 0-1000, 20-1000, 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 80-1000, 90-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000ng / ml or about 0.1-800, 1-800, 10-800, 20-800, 30-800, 40-800, 50-800, 60-800, 70 ~800, 80~800, 90~800, 100~800, 200~800, 300~800, 400~800, 500~800, 600~800, 700~800ng / ml, or about 0.1~700, 1~700, 10~700, 20~700, 30~700, 40~700, 50~700, 60~700, 70~700, 80~700, 90~700, 100~700, 200~700, 300~700, 400~700, 500~700, 600~700ng / ml, or about 0.The range is 1-600, 1-600, 10-600, 20-600, 30-600, 40-600, 50-600, 60-600, 70-600, 80-600, 90-600, 100-600, 200-600, 300-600, 400-600, 500-600 ng / ml, or about 0.1-500, 1-500, 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500, 100-500, 200-500, 300-500, 400-500 ng / ml. In certain embodiments, the R-spondin-1 is a human or mouse R-spondin-1 protein, such as a recombinant human or mouse R-spondin-1 protein.

[0051] In certain embodiments, the concentration of Y27632 is about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 μM, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 μM, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 μM, or about 0.1-50, 0. or about 0.1-30, 0.5-30, 1-30, 5-30, 10-30 μM, or about 0.1-20, 0.5-20, 1-20, 5-20, 10-20 μM, or about 0.1-10, 0.5-10, 1-10, 5-10 μM, or about 0.1-5, 0.5-5, 1-5 μM, or about 0.1-1 or 0.5-1 μM.

[0052] In certain embodiments, the concentration of FGF10 is about 0.1 to 10,000 or 1 to 1000 ng / ml, or about 0.1 to 1000, 1 to 1000, 10 to 1000, 20 to 1000, 30 to 1000, 40 to 1000, 50 to 1000, 60 to 1000, 70 to 1000, 80 to 1000, 90 to 1000, 100 to 1000, 200 to 1000, 300 to 1000, 400 to 1000, 500 to 1000, 600 to 1000, 700 to 1000, 800 to 1000, 900 to 1000 ng / ml, or about .1-500, 1-500, 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500, 100-500, 200-500, 300-500, 400-500 ng / ml, or about 0.1-400, 1-400, 10-400, 20-400, 30-400, 50-400, 40-400, 60-400, 70-400, 80-400, 90-400, 100-400, 200-400, 300-400 ng / ml, or about 0.1-30 0, 1-300, 10-300, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, 200-300ng / ml, or about 0.1-200, 1-200, 10-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200ng / ml, or about 0.1-150, 1-150, 10-150, 20-150, 30-150, 40-150, 5 The range is 0-150, 60-150, 70-150, 80-150, 90-150, 100-150 ng / ml, or about 0.1-120, 1-120, 10-120, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120 ng / ml, or about 0.1-100, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100 ng / ml. In certain embodiments, the FGF10 is a human or mouse FGF10 protein, eg, a recombinant human or mouse FGF10 protein.

[0053] In certain embodiments, the concentration of VO-OHpic is about 0.01-100 or 0.1-1 μM, or 0.01-10, 0.02-10, 0.03-10, 0.04-10, 0.05-10, 0.06-10, 0.07-10, 0.08-10, 0.09-10, 0.1-10, 0.5-10, 1-10, or 5-10 μM, or about 0.01-5, 0.02-5, 0.03-5, 0.04-5, 0.05-5, 0.06-5, 0.07 ~5, 0.08-5, 0.09-5, 0.1-5, 0.5-5, 1-5 μM, or about 0.01-2, 0.02-2, 0.03-2, 0.04-2, 0.05-2, 0.06-2, 0.07-2, 0.08-2, 0.09-2, 0.1-2, 0.5-2, 1-2 μM, or about 0.01-1, 0.02-1, 0.03-1, 0.04-1, 0.05-1, 0.06-1, 0.07-1, 0.08-1, 0.09-1, 0.1-1, 0.5-1 μM, or about 0.01-0.5, 0.02-0.5, 0.03-0.5, 0.04-0.5, 0.05-0.5, 0.06-0.5, 0.07-0.5, 0.08-0.5, 0.09-0.5, or 0.1-0.5 μM, or about 0.01-0.4, 0.02-0.4, 0.03-0.4, 0.04-0.4, 0.05-0.4, 0.06-0.4, 0.07-0.4, 0.08-0.4, 0.09-0.4, or 0.1-0.4 μM, or 0.01-0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07-0.3, 0.08-0.3, 0.09-0.3, or 0.1-0.3 μM, or about 0.01-0.2, 0.02-0.2, 0.03-0.2, 0.04-0.2, 0.05-0.2, 0.06-0.2, 0.07-0.2, 0.08-0.2, 0.09-0.2, or 0.1-0.2 μM. Specific examples of commercially available VO-OHpic include VO-OHpic trihydrate.

[0054] In certain embodiments, and in addition to the above components, the suspension medium comprises one or more components, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 components, selected from epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), N2 supplement, B27, prostaglandin E2 (PGE-2), and N-acetyl-L-cysteine ​​(including all combinations of the foregoing). In certain embodiments, and in addition to the above components, the suspension medium comprises EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, and N-acetyl-L-cysteine.

[0055] In certain embodiments, the concentration of EGF is about 0.1-1000 or 1-100 ng / ml, or about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 ng / ml, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 ng / ml, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 ng / ml, or about 0.1 The range is from about 0.1 to 40, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50 ng / ml, or about 0.1 to 40, 0.5 to 40, 1 to 40, 5 to 40, 10 to 40 ng / ml, or about 0.1 to 30, 0.5 to 30, 1 to 30, 5 to 30, 10 to 30 ng / ml, or about 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20 ng / ml, or about 0.1 to 10, 0.5 to 10, 1 to 10, 5 to 10 ng / ml, or about 0.1 to 5, 0.5 to 5, 1 to 5 ng / ml, or about 0.1 to 1 or 0.5 to 1 ng / ml. In certain embodiments, the EGF is a human or mouse EGF protein, for example a recombinant human or mouse EGF protein.

[0056] In certain embodiments, the concentration of HGF is about 0.1-1000 or 1-100 ng / ml, or about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 ng / ml, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 ng / ml, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 ng / ml, or about 0.1 The range is about 0.1-40, 0.5-40, 1-50, 5-50, 10-50 ng / ml, or about 0.1-40, 0.5-40, 1-40, 5-40, 10-40 ng / ml, or about 0.1-30, 0.5-30, 1-30, 5-30, 10-30 ng / ml, or about 0.1-20, 0.5-20, 1-20, 5-20, 10-20 ng / ml, or about 0.1-10, 0.5-10, 1-10, 5-10 ng / ml, or about 0.1-5, 0.5-5, 1-5 ng / ml, or about 0.1-1 or 0.5-1 ng / ml. In certain embodiments, the HGF is a human or mouse HGF protein, for example, a recombinant human or mouse HGF protein.

[0057] In certain embodiments, the concentration of IGF is about 0.1-1000 or 1-100 ng / ml, or about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 ng / ml, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 ng / ml, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 ng / ml, or about 0.1 In certain embodiments, the IGF is in the range of about 0.1-40, 0.5-40, 1-50, 5-50, 10-50 ng / ml, or about 0.1-40, 0.5-40, 1-40, 5-40, 10-40 ng / ml, or about 0.1-30, 0.5-30, 1-30, 5-30, 10-30 ng / ml, or about 0.1-20, 0.5-20, 1-20, 5-20, 10-20 ng / ml, or about 0.1-10, 0.5-10, 1-10, 5-10 ng / ml, or about 0.1-5, 0.5-5, 1-5 ng / ml, or about 0.1-1 or 0.5-1 ng / ml. In certain embodiments, the IGF is a human or mouse IGF protein, such as a recombinant human or mouse IGF protein.

[0058] In certain embodiments, the concentration of VEGF is about 0.1-1000 or 1-100 ng / ml, or about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 ng / ml, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 ng / ml, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 ng / ml, or about 0.1 The range is from about 0.1 to 40, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50 ng / ml, or about 0.1 to 40, 0.5 to 40, 1 to 40, 5 to 40, 10 to 40 ng / ml, or about 0.1 to 30, 0.5 to 30, 1 to 30, 5 to 30, 10 to 30 ng / ml, or about 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20 ng / ml, or about 0.1 to 10, 0.5 to 10, 1 to 10, 5 to 10 ng / ml, or about 0.1 to 5, 0.5 to 5, 1 to 5 ng / ml, or about 0.1 to 1 or 0.5 to 1 ng / ml. In certain embodiments, the VEGF is a human or mouse VEGF protein, such as a recombinant human or mouse VEGF protein.

[0059] In certain embodiments, the concentration of PDGF is about 0.1-1000 or 1-100 ng / ml, or about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 ng / ml, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 ng / ml, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 ng / ml, or about 0.1 0.1-50, 0.5-50, 1-50, 5-50, 10-50 ng / ml, or about 0.1-40, 0.5-40, 1-40, 5-40, 10-40 ng / ml, or about 0.1-30, 0.5-30, 1-30, 5-30, 10-30 ng / ml, or about 0.1-20, 0.5-20, 1-20, 5-20, 10-20 ng / ml, or about 0.1-10, 0.5-10, 1-10, 5-10 ng / ml, or about 0.1-5, 0.5-5, 1-5 ng / ml, or about 0.1-1 or 0.5-1 ng / ml. In certain embodiments, the PDGF is a human or mouse PDGF protein, e.g., a recombinant human or mouse PDGF protein.

[0060] In certain embodiments, the concentration of N2 is optionally about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10x, or 01-10x or 0.1-10x, or about 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.6-10, 0.7-10, 0.8-10, 0.9-10, 1-10, 2-10, 5-10x, or about 0.1-5, 0.2-5, 0.3-5, 0 0.4-5, 0.5-5, 0.6-5, 0.7-5, 0.8-5, 0.9-5, 1-5, 2-5 times, or about 0.1-4, 0.2-4, 0.3-4, 0.4-4, 0.5-4, 0.6-4, 0.7-4, 0.8-4, 0.9-4, 1-4, 2-4 times, or about 0.1-3, 0.2-3, 0.3-3, 0.4-3, 0.5-3, 0.6-3, 0.7-3, 0.8-3, 0.9-3, 1-3, 2-3 times, or about 0.1-2, 0.2-2, 0.3-2, 0.4-2, 0.5-2, 0.6-2, 0.7-2, 0.8-2, 0.9-2, 1-2 times. N2 supplement (100X) is commercially available from, for example, ThermoFisher Scientific and Stemcell Technologies.

[0061] In certain embodiments, the concentration of B27 is optionally about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, or 01-10 times or 0.1-10 times, or about 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.6-10, 0.7-10, 0.8-10, 0.9-10, 1-10, 2-10, 5-10 times, or about 0.1-5, 0.2-5, 0.3-5, 0 0.4-5, 0.5-5, 0.6-5, 0.7-5, 0.8-5, 0.9-5, 1-5, 2-5 times, or about 0.1-4, 0.2-4, 0.3-4, 0.4-4, 0.5-4, 0.6-4, 0.7-4, 0.8-4, 0.9-4, 1-4, 2-4 times, or about 0.1-3, 0.2-3, 0.3-3, 0.4-3, 0.5-3, 0.6-3, 0.7-3, 0.8-3, 0.9-3, 1-3, 2-3 times, or about 0.1-2, 0.2-2, 0.3-2, 0.4-2, 0.5-2, 0.6-2, 0.7-2, 0.8-2, 0.9-2, 1-2 times. B27® supplement (50x) is commercially available, for example, from ThermoFisher Scientific.

[0062] In certain embodiments, the concentration of PGE-2 is about 0.1-1000, 0.5-1000, 1-1000, 5-1000, 10-1000, 50-1000, 100-1000, 500-1000 nM, or about 0.1-500, 0.5-500, 1-500, 5-500, 10-500, 50-500, 100-500 nM, or about 0.1-100, 0.5-100, 1-100, 5-100, 10-100, 50-100 nM, or about 0.1-50, 0.5-50, 1 or about 0.1-40, 0.5-40, 1-40, 5-40, 10-40 nM, or about 0.1-30, 0.5-30, 1-30, 5-30, 10-30 nM, or about 0.1-20, 0.5-20, 1-20, 5-20, 10-20 nM, or about 0.1-10, 0.5-10, 1-10, 5-10 nM, or about 0.1-5, 0.5-5, 1-5 nM, or about 0.1-1 or 0.5-1 nM, or within these ranges.

[0063] In certain embodiments, the concentration of N-acetyl-L-cysteine ​​is about 0.1-100, 0.1-10, or 0.5-10 mM, or about 0.1-100, 0.2-100, 0.3-100, 0.4-100, 0.5-100, 0.6-100, 0.7-100, 0.8-100, 0.9-100, 1-100, 2-100, 3-100, 4-100, 5-100, 10-100, 50-100 mM, or about 0.1-50, 0.2-50, 0.3-50, 0.4-50, 0.5-50, 0.6-50, 0.7-50, 0.8-50, 0.9-50, 1-50, 2-50, 3 ~50, 4~50, 5~50, 10~50mM, or about 0.1~10, 0.2~10, 0.3~10, 0.4~10, 0.5~10, 0.6~10, 0.7~10, 0.8~10, 0.9~10, 1~10, 2~10, 3~10, 4~10, 5~10mM, or about 0.1~5, 0.2~5 , 0.3-5, 0.4-5, 0.5-5, 0.6-5, 0.7-5, 0.8-5, 0.9-5, 1-5, 2-5, 3-5, 4-5 mM, or in the range of about 0.1-2, 0.2-2, 0.3-2, 0.4-2, 0.5-2, 0.6-2, 0.7-2, 0.8-2, 0.9-2, 1-2 mM.

[0064] In certain embodiments, the suspension medium comprises nicotinamide, noggin, R-spondin-1, Y27632, EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, N-acetyl-L-cysteine, FGF10, and VO-OHpic.

[0065] In certain embodiments, the media suspension omits or is otherwise free of extracellular matrix (ECM) components. By way of example only, the media suspension does not include collagen, matrigel, laminin, and / or fibronectin.

[0066] In some embodiments, the tumor cell clusters are obtained from a human patient, e.g., primary tumor cells derived directly from a human patient. In certain embodiments, the tumor cell clusters are isolated from an immunodeficient animal xenografted with tumor cells, i.e., the tumor cells are patient-derived xenografts (PDXs). Specific examples of tumor cell clusters isolated directly from a patient or indirectly via a PDX include lung cancer cells, colon cancer cells, gastric cancer cells, and breast cancer cells. In some embodiments, the tumor cell clusters are isolated directly from tumor samples removed from a human patient, examples of which include surgical samples, biopsies, pleural effusions, and ascites fluids. The tumor cells can be obtained directly from the patient, or, for example, from a physician or other healthcare provider who plays a role in the patient's medical care and cancer treatment.

[0067] Also included are methods of expanding human tumor cells, comprising obtaining a population of human tumor cell clusters from a sample (e.g., as described herein) and culturing the population of human tumor cell clusters in a medium suspension as described herein. In some embodiments, the human tumor cell clusters are cultured for about 14 days or less, or for about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days or less, or for about 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14 days or less, or for about 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13 days or less, or for about 5-12, 6-12, 7-12, 8-12, 9-12, 10-13, 11-13, or 12-13 days or less. or within about 5-10, 6-10, 7-10, 8-10, or 9-10 days, or within about 5-9, 6-9, 7-9, or 8-9 days, or within about 5-8, 6-8, or 7-8 days, or within about 5-7 or 6-7 days.

[0068] In some embodiments, the method provides that the medium suspension results in proliferation of about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (including all combinations, integers, and ranges therebetween) of the tumor cell clusters within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days. For example, certain methods optionally include a method for determining whether or not the antibody is effective in treating a patient having a pulmonary circulation disorder, the antibody being administered within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days, or within 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14 days, or within about 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13 days, or within about 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 days. or within about 5-11, 6-11, 7-11, 8-11, 9-11, or 10-11 days, or within about 5-10, 6-10, 7-10, 8-10, or 9-10 days, or within about 5-9, 6-9, 7-9, or 8-9 days, or within about 5-8, 6-8, or 7-8 days, or within about 5-7 or 6-7 days, or within about 5-6 days, about or at least about 10-15, 10-20, 10-25, 10-30, 10-30, 10-40, 10-45, 10-50, 10-60, 10-70, 10-80, 10-90, 10-110, 10-120, 10-130, 10-140, 10-150, 10-260, 10-270, 10-310, 10-320, 10-330, 10-340, 10-350, 10-360, 10-370, 10-380, 10-390, 10-400, 10-410, 10-420, 10-430, 10-440, 10-450, 10-460, 10-470, 10-480, 10-490, 10-500, 10-510, 10-520, 10-530, 10-540, 10-550, 10-560, 10-570, 10-5 5, 10-40, 10-45, 10-50, 10-55, 10-60, 10-65, 10-70, 10-75, 10-80, 10-85, 10-90, 10-95, or 10-100% proliferation or about or at least about 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 15-55, 15-60, 15-65, 15-70, 15-75, 15-80, 15-85, 15-90, 15-95, or 15-100% proliferation of tumor cell clusters 20-70, 20-75, 20-80, 20-85, 20-90, 20-95, or 20-100% proliferation or about or at least about 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70,25-75, 25-80, 25-85, 25-90, 25-95, or 25-100% proliferation of tumor cell clusters or about or at least about 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, or 30-100% proliferation of tumor cell clusters or about or at least about 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35 or about or at least about 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, or 40-100% proliferation of tumor cell clusters, or about or at least about 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, or 45-100% proliferation of tumor cell clusters, or about or at least about 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, or 45-100% proliferation of tumor cell clusters or about or at least about 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, or 55-100% proliferation of tumor cell clusters, or about or at least about 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, or 60-100% proliferation of tumor cell clusters, or about or at least about 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, or 60-100% proliferation of tumor cell clusters. or about or at least about 65-70, 65-75, 65-80, 65-85, 65-90, 65-95, or 65-100% proliferation of the clusters, or about or at least about 70-80, 70-85, 70-90, 70-95, or 70-100% proliferation of the tumor cell clusters, or about or at least about 75-80, 75-85, 75-90, 75-95, or 75-100% proliferation of the tumor cell clusters, or about or at least about 80-85, 80-90, 80-95, or 80-100% proliferation of the tumor cell clusters,or about or at least about 85-90, 85-95, or 85-100% proliferation of tumor cell clusters, or about or at least about 90-95 or 90-100% proliferation of tumor cell clusters, or about or at least about 95-100% proliferation of tumor cell clusters. Particular methods result in at least about 20-30% proliferation of tumor cell clusters within about 7 days, or at least about 60% proliferation of tumor cell clusters within about 7 days.

[0069] As noted above, the suspension media and methods described herein may be used, inter alia, to assess the potential responsiveness of a patient tumor to one or more therapeutic agents, and provide the advantage of identifying optimal treatment options for a patient within a relatively short period of time, e.g., within one or two weeks. Certain embodiments thus include a method of testing the responsiveness of a human patient to a therapeutic agent (e.g., a drug or candidate drug), comprising obtaining a population of tumor cell clusters from a human patient, culturing the population of tumor cell clusters in a medium suspension according to any one of the preceding claims, administering a therapeutic agent or drug to the medium suspension, and measuring tumor cell proliferation and / or tumor cell apoptosis in the population of tumor cells.

[0070] In some embodiments, a decrease (e.g., a statistically significant decrease) in tumor cell proliferation indicates responsiveness of a human patient (i.e., the human patient's tumor) to a therapeutic agent, and in some embodiments, a lack of decrease (e.g., a lack of a statistically significant decrease) in tumor cell proliferation indicates the likelihood of resistance and non-responsiveness of a human patient (i.e., the human patient's tumor) to a therapeutic agent. In some embodiments, an induction (e.g., a statistically significant increase) in tumor cell apoptosis indicates the likelihood of responsiveness of a human patient (i.e., the human patient's tumor) to a therapeutic agent. In certain embodiments, a lack of increase (e.g., a lack of a statistically significant increase) in tumor cell proliferation indicates the likelihood of resistance and non-responsiveness of a human patient (i.e., the human patient's tumor) to a therapeutic agent. In certain embodiments, a decrease in tumor cell proliferation and induction of tumor cell apoptosis indicates the likelihood of responsiveness of a human patient to a therapeutic agent, and a lack of decrease in tumor cell proliferation indicates the likelihood of resistance and non-responsiveness of a human patient to a therapeutic agent.

[0071] In some embodiments, the method of testing for responsiveness includes administering the therapeutic agent on the same day (e.g., simultaneously or substantially simultaneously) as the population of tumor cell clusters is cultured in a medium suspension. Certain embodiments include administering the therapeutic agent within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the population of tumor cell clusters is cultured in a medium suspension. Also included are methods that include administering the therapeutic agent at or within about 1, 2, 3, 4, 5, 6, or 7 days after culturing the population of tumor cell clusters in media suspension, including methods that administer the therapeutic agent at or within about 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 2-7, 2-6, 2-5, 2-4, 2-3, or 3-7, 3-6, 3-5, 3-4, or 4-7, 4-6, 4-5, or 5-7, 5-6, or 6-7 days after culturing the population of tumor cell clusters in media suspension.

[0072] Certain embodiments include measuring tumor cell proliferation and / or tumor cell apoptosis in a population of tumor cells within about 14 days of administration of the therapeutic agent, e.g., within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days of administration of the therapeutic agent. Certain embodiments include measuring tumor cell proliferation and / or tumor cell apoptosis in a population of tumor cells within about 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14 days, or within about 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13 days of administration of the therapeutic agent, or within about 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 days of administration of the therapeutic agent. measuring tumor cell proliferation and / or tumor cell apoptosis in the population of tumor cells within about 5-10, 6-10, 7-10, 8-10, or 9-10 days, or within about 5-9, 6-9, 7-9, or 8-9 days, or within about 5-8, 6-8, or 7-8 days, or within about 5-7 or 6-7 days, or within about 5-6 days.

[0073] In certain embodiments, the therapeutic agent or drug for testing is a small molecule.Exemplary small molecules include cytotoxic drugs, chemotherapeutic drugs, and antiangiogenic drugs, such as those believed to be useful in treating various cancers.Specific classes of small molecules include, but are not limited to, alkylating agents, antimetabolites, anthracyclines, antitumor antibiotics, platinum, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes.

[0074] Specific examples of small molecules include chlorambrucil, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan, procarbazine, thiotepa, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib, erlotinib idarubicin, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923, and paclitaxel, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Further examples of small molecules include imatinib, crizotinib, dasatinib, sorafenib, pazopanib, sunitinib, vatalanib, geftinib, erlotinib, AEE-788, dicloacetate, tamoxifen, fasudil, SB-681323, and semaxanib (SU5416) (see Chico et al., Nat Rev Drug Discov. 8:829-909, 2009).

[0075] Further examples of small molecules include alkylating agents such as thiotepus, cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines, ethylenimines, and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine), such as benzodopa, carboquone, meturedopa, and uredopa; chlorambucil, ... Nitrogen mustards such as nafazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembikine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, autramycin, azaserine, and bleomycin. cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin antibiotics such as tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU;Androgens such as calcineurin, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elformitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenameth; pirarubicin; podophyllic acid; 2-ethylhydrazone dode; procarbazine; PSK; razoxane; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene) and Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicin; capecitabine;and pharma- ceutically acceptable salts, acids, or derivatives of any of the above.

[0076] Also included are anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY 117018, ompristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as pharmacologic acceptable salts, acids, or derivatives of any of the above.

[0077] In some embodiments, the therapeutic agent is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody or an antigen-binding fragment thereof or other polypeptide specifically binds to a cancer-associated antigen or cancer antigen, for example, a cancer antigen expressed by the tumor cell cluster being tested. Exemplary cancer antigens include cell surface proteins, such as cell surface receptors. Ligands that bind to such cell surface proteins or receptors are also included as cancer-associated antigens. In certain embodiments, the antibody or antigen-binding fragment specifically binds to an intracellular cancer antigen. In some embodiments, the cancer associated with the cancer antigen is one or more of breast cancer, metastatic brain cancer, prostate cancer, gastrointestinal cancer, lung cancer, ovarian cancer, testicular cancer, head and neck cancer, gastric cancer, bladder cancer, pancreatic cancer, liver cancer, kidney cancer, squamous cell carcinoma, CNS or brain cancer, melanoma, non-melanoma cancer, thyroid cancer, endometrial cancer, epithelial tumors, bone cancer, or hematopoietic cancer.

[0078] In certain embodiments, the antibody or antigen-binding fragment or other polypeptide is selected from the group consisting of human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33 , CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), α-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), integrin α v β3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 Specifically binds to at least one cancer-associated antigen or cancer antigen, such as pan-cancer antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), phosphatase of regenerating liver 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed in tumors of neuroectodermal origin), glypican-3 (GPC3), and / or mesothelin.

[0079] In certain embodiments, the antibody is selected from the group consisting of 3F8, 8H9, abagomomab, adecatumumab, afutuzumab, alemtuzumab, alacizumab (pegol), amatuximab, apolizumab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab (mertansine), brentuximab vedotin, cantuzumab (mertansine), cantuzumab (ravutansine), capromomab (pendetide), catumaxomab, cetuximab, sitatuzumab (bogatox), cixutumumab, clivatuzumab (tetraxetan), conatumumab, dacetuzumab, Dalotuzumab, detumomab, drozitumab, ecromeximab, edrecolomab, elotuzumab, enabatuzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, FBTA05, figitumumab, framvotumab, galiximab, gemtuzumab, ganitumab, gemtuzumab (ozogamicin), girentuximab, glembatumumab (vedotin), ibritumomab tiuxetan, icrucumab, igovomab, indatuximab ravtansine, intetumumab, inotuzumab ozogamicin, ipilimumab (M DX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab (mertansine), lucatumumab, rumiliximab, mapatumumab, matuzumab, milatuzumab, mitumomab, mogamulizumab, moxetumomab (pseudotox), nacolomab (tafenatox), naptumomab (estafenatox), narunatumab, necitumumab, nimotuzumab, nivolumab, Neuradiab® (with or without radioactive iodine), NR-LU-10, ofatumumab, olaratumab, onartuzumab, onartuzumab, Portuzumab (Monatox), oregovomab, panitumumab, patritumab, pemtumomab, pertuzumab, pritumumab, racotumomab, radletumab, ramucirumab, rilotumumab, rituximab, lobatumumab, samalizumab, sibrotuzumab, siltuximab, tabalumab, taplitumomab (Paptox), tenatumomab, teprotumumab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650, tositumomab, TRBS07, trastuzumab, tucotumab (celmoleukin), ublituximab, urelumab,Therapeutic antibodies, such as anti-cancer therapeutic antibodies, including antibodies such as veltuzumab, volociximab, votumumab, and zalutumumab. Fragments, variants, and derivatives of these antibodies are also included.

[0080] Certain embodiments include testing responsiveness to a combination of two or more therapeutic agents. Thus, certain methods include administering two or more therapeutic agents to a medium suspension that includes a combination of any two or more of the exemplary therapeutic agents listed above.

[0081] Any of a variety of methods known in the art can be used to measure tumor cell proliferation and / or tumor cell apoptosis. For example, certain methods of measuring tumor cell proliferation include measuring one or more cell proliferation markers. Exemplary cell proliferation markers include: 3 H-thymidine, bromodeoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (Edu), Ki-67, and proliferating cell nuclear antigen (PCNA). Thus, in certain embodiments, the step of measuring tumor cell proliferation optionally includes: 3 The method includes measuring a cell proliferation marker selected from one or more of 3H-thymidine, BrdU, Edu, Ki-67, and PCNA (including combinations thereof).

[0082] Similarly, any of a variety of methods known in the art can be used to measure tumor cell apoptosis. "Apoptosis" generally refers to the process of programmed cell death that occurs in multicellular organisms, including characteristic changes in cells (e.g., morphology) and biochemical events that lead to cell death. Exemplary changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. Certain methods of measuring tumor cell apoptosis include measuring cell apoptosis markers. Exemplary cell apoptosis markers include fluorochrome-labeled inhibitor of caspases (FLICA), caspase activation, poly ADP-ribose polymerase (PARP) cleavage, DRAQ5, DRAQ7, and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assays. Thus, in certain embodiments, the step of measuring tumor cell apoptosis includes measuring cell apoptosis markers, optionally selected from one or more of FLICA, PARP, DRAQ5, DRAQ7, and TUNEL assays (including combinations thereof).

[0083] In certain aspects, these responsiveness tests or methods are performed in a diagnostic laboratory, and the results are then provided to the patient or to a physician or other healthcare provider who plays a role in the patient's medical care and cancer treatment. Thus, certain embodiments include a method for providing the results of the tumor cell cluster responsiveness test to a patient, or to a physician or other healthcare provider. These results or data may be in the form of a hard copy or paper copy, or in electronic form, such as a computer readable medium. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A media suspension comprising a population of human tumor cell clusters from a human patient, the media suspension resulting in proliferation of at least about 10% of said tumor cell clusters within about 14 days. (Item 2) 2. The medium suspension of item 1, which results in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% proliferation of said human tumor cell clusters within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days. (Item 3) 3. The medium suspension according to item 1 or 2, which results in at least about 20-30% proliferation of said human tumor cell clusters within about 7 days. (Item 4) 4. The medium suspension according to any one of items 1 to 3, which results in proliferation of at least about 60% of said human tumor cell clusters within about 7 days. (Item 5) 5. The medium suspension according to any one of items 1 to 4, comprising a synthetic medium. (Item 6) 6. The medium suspension according to any one of items 1 to 5, which is serum-free. (Item 7) 7. The medium suspension of item 5 or 6, comprising one or more components selected from DMEM / F-12, Wnt3A, basic fibroblast growth factor (bFGF), insulin, transferrin, bovine serum albumin (BSA), cholesterol, B-cell activating factor (BAFF), and 2-mercaptoethanol. (Item 8) 8. The medium suspension of item 7, comprising two, three, four, five, six, seven, eight, or nine of the components selected from DMEM / F-12, Wnt3A, bFGF, insulin, transferrin, bovine serum albumin (BSA), cholesterol, B-cell activating factor (BAFF), and 2-mercaptoethanol. (Item 9) 9. The medium suspension of item 8, comprising DMEM / F-12, Wnt3A, bFGF, insulin, transferrin, bovine serum albumin (BSA), cholesterol, B-cell activating factor (BAFF), and 2-mercaptoethanol. (Item 10) 10. The medium suspension according to any one of items 5 to 9, comprising one or more components selected from nicotinamide, noggin, R-spondin-1, Y27632, fibroblast growth factor 10 (FGF10), and VO-OHpic. (Item 11) 11. The medium suspension of item 10, comprising two, three, four, five, or six of the components selected from nicotinamide, noggin, R-spondin-1, Y27632, FGF10, and VO-OHpic. (Item 12) 12. The medium suspension according to item 11, comprising nicotinamide, noggin, R-spondin-1, Y27632, FGF10, and VO-OHpic. (Item 13) 13. The medium suspension according to any one of items 7 to 12, comprising one or more components selected from epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), N2 supplement, B27, prostaglandin E2 (PGE-2), and N-acetyl-L-cysteine. (Item 14) 14. The medium suspension of item 13, comprising 2, 3, 4, 5, 6, 7, 8, or 9 of the components selected from EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, N-acetyl-L-cysteine. (Item 15) 15. The medium suspension according to item 14, comprising EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, and N-acetyl-L-cysteine. (Item 16) 16. The medium suspension according to any one of items 10 to 15, comprising nicotinamide, noggin, R-spondin-1, Y27632, EGF, HGF, IGF, VEGF, PDGF, N2, B27, PGE2, N-acetyl-L-cysteine, FGF10, and VO-OHpic. (Item 17) 17. The medium suspension according to any one of items 1 to 16, which does not contain extracellular matrix components, optionally matrigel, collagen, laminin, or fibronectin. (Item 18) 18. The culture medium suspension according to any one of items 1 to 17, wherein said human tumor cell clusters are lung cancer cells, colon cancer cells, gastric cancer cells, or breast cancer cells from a human patient. (Item 19) 19. The culture medium suspension according to any one of items 1 to 18, wherein said human tumor cell clusters are isolated from immunodeficient animals xenografted with human tumor cells and / or tissue from a human patient. (Item 20) 19. The culture suspension of any one of items 1 to 18, wherein the human tumor cell clusters are isolated directly from a tumor sample removed from a human patient, optionally selected from a surgical sample, a biopsy, a pleural effusion, and an ascites fluid. (Item 21) 21. A method for growing human tumor cells, comprising obtaining a population of human tumor cells from a sample and culturing the population of human tumor cell clusters in a medium suspension according to any one of items 1 to 20. (Item 22) 22. The method of claim 21, comprising culturing the human tumor cell clusters in the culture suspension for no more than about 14 days. (Item 23) 23. The method of claim 22, comprising culturing the human tumor cell clusters in the culture suspension for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days. (Item 24) 24. The method of claim 23, comprising culturing the human tumor cell clusters in the culture suspension for up to about 7 days. (Item 25) 1. A method for testing the responsiveness of a human patient to a therapeutic agent, comprising: Obtaining or receiving a population of tumor cell clusters from the human patient; Culturing the population of tumor cell clusters in a medium suspension according to any one of items 1 to 24; administering said therapeutic agent to said media suspension; measuring tumor cell proliferation and / or tumor cell apoptosis in said population of tumor cells; Also included are methods in which a reduction in tumor cell proliferation and / or induction of tumor cell apoptosis indicates responsiveness of the human patient to the therapeutic agent, and a lack of a reduction in tumor cell proliferation and / or induction of tumor cell apoptosis indicates resistance of the human patient to the therapeutic agent. (Item 26) 26. The method of claim 25, comprising administering the therapeutic agent on the same day that the population of tumor cell clusters is cultured in the medium suspension. (Item 27) 26. The method of claim 25, comprising administering the therapeutic agent at least one day after culturing the population of tumor cell clusters in the medium suspension. (Item 28) 28. The method of claim 27, comprising administering the therapeutic agent within about 1, 2, 3, 4, 5, 6, or 7 days after culturing the population of tumor cell clusters in the medium suspension. (Item 29) 29. The method of any one of items 1 to 28, comprising measuring tumor cell proliferation and / or tumor cell apoptosis in said population of tumor cells within about 14 days of administration of said therapeutic agent. (Item 30) 30. The method of claim 29, comprising measuring tumor cell proliferation and / or tumor cell apoptosis in said population of tumor cells within about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days of administration of said therapeutic agent. (Item 31) The method of claim 30, comprising measuring tumor cell proliferation and / or tumor cell apoptosis in the population of tumor cells within about 7, 6, or 5 days of administering the therapeutic agent. 32. The method according to any one of items 1 to 31, wherein the step of measuring tumor cell proliferation comprises measuring a cell proliferation marker. (Item 33) The cell proliferation marker is 333. The method of claim 32, wherein the antibody is selected from one or more of the following: H-thymidine, bromodeoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (Edu), Ki-67, and proliferating cell nuclear antigen (PCNA). (Item 34) 34. The method according to any one of items 1 to 33, wherein the step of measuring tumor cell apoptosis comprises measuring a cellular apoptosis marker. (Item 35) 35. The method of claim 34, wherein the cell apoptosis marker is selected from one or more of Fluorescently Labeled Inhibitors of Caspases (FLICA), Caspase Activation, Poly ADP-ribose Polymerase (PARP) cleavage, DRAQ5, DRAQ7, and Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick End Labeling (TUNEL) assays. EXAMPLES

[0084] Example 1 Isolation and analysis of tumor cell clusters After receiving patient consent, surgical specimens from colorectal cancer patients were collected from the Beijing Patient-derived xenograft tumor specimens were obtained from Nod / SCID mice inoculated with surgical tumor specimens from patients.

[0085] Briefly, non-tumor tissue and necrotic fractions were carefully removed from tumor specimens. Tumor tissue was cut into small pieces less than 1 mm in diameter using scissors. The minced tumor fraction was transferred to a sterile 100 ml triangular glass flask equipped with a magnetic stir bar. Enzyme digestion was initiated by adding 10-15 ml of digestion medium containing 0.25 U / ml Liberase DH to the minced tumor tissue. The enzyme mixture was incubated at 37°C for 1-2 h with gentle stirring. The partially digested tumor cell clusters were filtered through a 100 μm cell retention device. The filtrate was filtered again through a 40 μm cell restrainer. The tumor cell clusters retained on the 40 μm cell restrainer were collected, washed twice with HBSS, and then resuspended in synthetic growth medium supplemented with cell growth factors and small molecule inhibitors.

[0086] For ex vivo drug sensitivity assays, tumor cell clusters were allowed to recover overnight in synthetic growth medium. The synthetic growth medium was StemPro hESC SFM supplement containing nicotinamide, Wnt3A, Noggin, R-spondin-1, and Y27632. The tumor cell clusters were then exposed to drugs (e.g., docetaxel at 2 μM (Cmax)) for 24 hours before the drug was washed away. After drug exposure, tumor cell clusters were labeled with 5-ethynyl-2'-deoxyuridine (Edu) to assess tumor cell proliferation rates. Labeling began 48 hours after drug exposure and continued for 96 hours. In the control group, tumor cell clusters received no drug exposure with a medium change, but were similarly labeled with Edu. The labeled tumor cell clusters were dissociated into single cells and fixed. The fixed cells were stained with Hoechst 33342 in PBS containing 0.2% Triton X-100 overnight at 4°C. The incorporated Edu was incubated with fixed cells in 1x Click-iT in the dark. The stained cells were detected by Click-iT reaction incubated with a reaction mixture containing Edu reaction buffer, CuSO4, and azide-conjugated Alexa Fluor dye. The stained cells were washed with PBS before being dispensed into black-walled plates for image acquisition and analysis.

[0087] For image acquisition and analysis, stained tumor cells were imaged by a high content screening (HCS) platform (Thermo Scientific Cellomics ArrayScan XTi HCS reader). Images were collected using a 10x objective. 25 fields were imaged for each well, and more than 3000 cells were captured for analysis. Two fluorescent signals were obtained from the images from the HCS reader. The blue fluorescent signal recorded the nuclear signal stained with Hoechst 33342, and the green fluorescent signal detected Edu incorporated into newly synthesized DNA. The percentage of Edu positive subpopulation cells was calculated as a percentage of the total cell count. The cutoff point for drug resistance in this ex vivo assay was defined as less than 90% inhibition of Edu incorporation when tumor cell clusters were exposed to 2 μM docetaxel for 24 hours.

[0088] With regard to direct patient samples, Figures 1A-1D show that tumor cell clusters were successfully isolated from colon / rectal tumors surgically removed from patients and cultured in suspension in synthetic growth medium. Figure 2 shows that tumor cells were expanded during the short-term suspension culture period as measured by Edu incorporation (sample 1 is from Figure 1A, sample 2 is from Figure 1B, sample 3 is from Figure 1C, and sample 4 is from Figure 1D).

[0089] Figures 3A-3B show that short-term suspension cultures (within about 7 days) show the highest cell proliferation markers of tumor cell clusters. Figure 3A shows proliferation markers of four different patient samples, and Figure 3B shows proliferation markers of one exemplary patient sample over a one week period.

[0090] Figure 4 shows that under the test conditions (see Figure 3B), the growth of tumor cell clusters was not completely inhibited by the drugs tested (sunitinib, sorafenib, erlotinib, crizotinib, oxaliplatin), which is consistent with patient-derived xenograft (PDX) tumor responses to these drugs. It was confirmed that PDX-amplified tumors had a similar morphology to those isolated from patient tumor tissues when cultured in suspension with synthetic media.

Claims

[Claim 1] The invention as depicted in the drawings.

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