Systems and methods for assessing therapies on organs

Abstract

In some aspects, the present disclosure provides methods and systems for assessing the effects of therapies on ex vivo human organs maintained under machine perfusion.

Description

SYSTEMS AND METHODS FOR ASSESSING THERAPIES ON ORGANSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 663,743, filed June 25, 2024, the entire content of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Therapeutic agents may have unpredictable effects on human organs in clinical trials. Despite certain therapies being approved for clinical use, their specific mechanisms of action in human organs and systems may remain a challenge to understand.SUMMARY

[0003] In an aspect, the present disclosure provides a method for assessing effects of a therapy on an organ, comprising: a) providing an organ obtained from a donor subject, wherein the organ is ex-vivo with respect to the donor subject; b) subjecting the organ to machine perfusion conditions, thereby maintaining viability of the organ; c) administering the oncology therapy to the organ concurrently with the machine perfusion conditions of (b); and d) assessing the effects of the therapy on the organ, responsive to the administering in (c). In some embodiments, the therapy comprises a cancer therapy. In some embodiments, the cancer therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, radiation, a surgical resection, a laser ablation, or any combination thereof. In some embodiments, the cancer therapy comprises an antibody or an antigen binding fragment thereof, oxaliplatin, doxorubicin, taxol, bevacizumab, RNAi, or any combination thereof. In some embodiments, (b) comprises subjecting a liver to more than 30 hours of machine perfusion, subjecting a lung to more than 6 hours of machine perfusion, subjecting an intestine to more than 12 hours of machine perfusion, or subjecting a kidney to more than 12 hours of machine perfusion, or any combination thereof. In some embodiments, (b) comprises subjecting the organ to machine perfusion conditions for a duration of at least five half-lives of a drug, optionally at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty half-lives of a drug. In some embodiments, the therapy comprises a pharmacological therapy. In some embodiments, the therapy comprises a non-pharmacological therapy. In some embodiments, the therapy comprises a combination therapy. In some embodiments, the therapy comprises a small molecule drug, a large molecule drug, a biologic, a prodrug, a natural product, a cell therapy, a gene therapy, an immunotherapy, a surgery, radiation, a preventative therapy, a curative therapy, a palliative therapy, a dye, a diagnostic therapy, or any combination thereof. Insome embodiments, the effects of the therapy on the organ comprises a therapeutic effect, a pharmacodynamic response, a pharmacokinetic response, an adverse drug effect, a toxic effect, drug adsorption, drug absorption, drug distribution, drug metabolism, drug excretion, or any combination thereof. In some embodiments, the assessing in (d) further comprises assessing a molecular drug response of the organ to the therapy. In some embodiments, the assessing in (d) further comprises assessing a mechanism of toxicity of the therapy on the organ. In some embodiments, the assessing in (d) further comprises determining time-series effects of the therapy on the organ over a period of time. In some embodiments, the assessing in (d) further comprises determining a spatial assessment of the therapy on the organ. In some embodiments, the organ is unsuitable for a transplant procedure. In some embodiments, the organ comprises at least a portion of a liver, a heart, a kidney, a lung, a pancreas, a colon, an intestine, a brain, a bone, or any combination hereof. In some embodiments, the organ is at least a portion of a liver. In some embodiments, the organ has cancerous tissue. In some embodiments, the organ has a tumor. In some embodiments, the tumor is a carcinoma. In some embodiments, the carcinoma is metastatic. In some embodiments, the organ does not comprise cancerous tissue. In some embodiments, the method or system provided herein further comprises administering one or more cancer cells to the organ to simulate cancer. In some embodiments, the organ is a diseased organ obtained from a diseased subject having hepatitis, fatty liver disease, liver fibrosis, liver cirrhosis, liver failure, heart failure, cardiomyopathy, myocardial infarction, coronary artery disease, hypertension, congenital heart defects, endocarditis, Chronic Kidney Disease (CKD), diabetic nephropathy, polycystic kidney disease, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, alzheimer’s disease, parkinson’s disease, dementia, a genetic disorder, an infection disease, an autoimmune disease, or any combination thereof. In some embodiments, the assessing in (d) further comprises analyzing a sample of the organ. In some embodiments, the sample comprises a biopsy sample. In some embodiments, the biopsy sample is obtained at least in part by core needle biopsy. In some embodiments, the assessing in (d) further comprises performing an organ function test. In some embodiments, the organ is a liver and the assessing in (d) further comprises performing a liver function test. In some embodiments, the assessing in (d) further comprises imaging the organ. In some embodiments, the organ comprises a tumor and assessing in (d) further comprises determining a tumor size within the organ. In some embodiments, the assessing in (d) comprises analyzing a perfusate of the organ. In some embodiments, the organ is a liver and analyzing the perfusate comprises measuring lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate. In some embodiments, the assessing in (d) further comprises measuring pressure changes to the organ. In some embodiments, the assessing in (d) comprises measuring changes to tissue architecture. In some embodiments, the assessing in (d)comprises measuring an amount of live or dead cells. In some embodiments, the assessing in (d) comprises measuring cell type composition of the cells of the organ. In some embodiments, the assessing in (d) comprises measuring changes in gene expression of cells of the organ using spatial omics. In some embodiments, the assessing in (d) comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic, or metabolomic sequencing and / or digital pathology. In some embodiments, the organ is a brain and assessing in (d) comprises measuring neurotoxicity or blood-brain-barrier penetration. In some embodiments, the organ is a kidney and assessing in (d) comprises measuring nephrotoxicity. In some embodiments, the method or system provided herein further comprise administering at least two pharmacological therapies to the organ concurrently with the machine perfusion conditions of (b). In some embodiments, the at least two pharmacological therapies are administered sequentially to the organ. In some embodiments, the at least two pharmacological therapies are administered concurrently to the organ. In some embodiments, the method or system provided herein further comprising administering a pharmacology therapy separately from the machine perfusion conditions. In some embodiments, the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.4 weeks, or a month. In some embodiments, the perfusion conditions comprise providing a supplemented medium to the organ. In some embodiments, the perfusion conditions comprise providing oxynation to the organ. In some embodiments, the perfusion conditions comprise providing artificial blood to the organ. In some embodiments, the artificial blood comprises perfluorocarbon-based oxygen carriers (PFCs) or stabilized hemoglobin solutions (HBOCs). In some embodiments, the method or system provided herein further comprises using an automated computer-implemented system to perform(a), (b), (c), (d), or any combination thereof . In some embodiments, the method or system provided herein further comprises using an automated computer-implemented system to perform(b) and (c). In some embodiments, the method or system provided herein further comprises using an automated computer-implemented system to perform (b), (c), and (d).

[0004] In an aspect, the present disclosure provides a system for assessing effects of a therapy on an organ, comprising: a) a machine perfusion device configured to (i) receive an organ from a donor subject, wherein the organ is ex-vivo with respect to the donor subject, and (ii) subject the organ to machine perfusion conditions, thereby maintaining viability of the organ; b) a therapy dispenser configured to administer the therapy to the organ concurrently with the machine perfusion conditions of (a); and c) a computer processor programmed to assess the effects of the therapy on the organ, responsive to the administering in (b). In some embodiments, the therapy comprises a cancer therapy. In some embodiments, the cancer therapy comprises a chemotherapy,a targeted therapy, an immunotherapy, radiation, a surgical resection, a laser ablation, or any combination thereof. In some embodiments, the cancer therapy comprises an antibody or an antigen-binding fragment thereof, oxaliplatin, doxorubicin, taxol, bevacizumab, RNAi, or any combination thereof. In some embodiments, (b) comprises subjecting a liver to more than 30 hours of machine perfusion, subjecting a lung to more than 6 hours of machine perfusion, subjecting an intestine to more than 12 hours of machine perfusion, or subjecting a kidney to more than 12 hours of machine perfusion, or any combination thereof. In some embodiments, (b) comprises subjecting the organ to machine perfusion conditions for a duration of at least five halflives of a drug, optionally at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty half-lives of a drug. In some embodiments, the therapy comprises a pharmacological therapy. In some embodiments, the therapy comprises a non-pharmacological therapy. In some embodiments, the therapy comprises a combination therapy. In some embodiments, the therapy comprises a small molecule drug, a large molecule drug, a biologic, a prodrug, a natural product, a cell therapy, a gene therapy, an immunotherapy, a surgery, radiation, a preventative therapy, a curative therapy, a palliative therapy, a dye, a diagnostic therapy, or any combination thereof. In some embodiments, the effects of the therapy on the organ comprises a therapeutic effect, a pharmacodynamic response, a pharmacokinetic response, an adverse drug effect, a toxic effect, drug adsorption, drug absorption, drug distribution, drug metabolism, drug excretion, or any combination thereof. In some embodiments, the assessing in (d) further comprises assessing a molecular drug response of the organ to the therapy. In some embodiments, the assessing in (d) further comprises assessing a mechanism of toxicity of the therapy on the organ. In some embodiments, the assessing in (d) further comprises determining time-series effects of the therapy on the organ over a period of time. In some embodiments, the assessing in (d) further comprises determining a spatial assessment of the therapy on the organ. In some embodiments, the organ is unsuitable for a transplant procedure. In some embodiments, the organ comprises at least a portion of a liver, a heart, a kidney, a lung, a pancreas, a colon, an intestine, a brain, a bone, or any combination hereof. In some embodiments, the organ is at least a portion of a liver. In some embodiments, the organ has cancerous tissue. In some embodiments, the organ has a tumor. In some embodiments, the tumor is a carcinoma. In some embodiments, the carcinoma is metastatic. In some embodiments, the organ does not comprise cancerous tissue. In some embodiments, the system further comprises administering one or more cancer cells to the organ to simulate cancer. In some embodiments, the organ is a diseased organ obtained from a diseased subject having hepatitis, fatty liver disease, liver fibrosis, liver cirrhosis, liver failure, heart failure, cardiomyopathy, myocardial infarction, coronary artery disease, hypertension, congenital heart defects, endocarditis, Chronic Kidney Disease (CKD), diabetic nephropathy, polycystic kidneydisease, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, alzheimer’s disease, parkinson’s disease, dementia, a genetic disorder, an infection disease, an autoimmune disease, or any combination thereof. In some embodiments, the assessing in (d) further comprises analyzing a sample of the organ. In some embodiments, the sample comprises a biopsy sample. In some embodiments, the biopsy sample is obtained at least in part by core needle biopsy. In some embodiments, the assessing in (d) further comprises performing an organ function test. In some embodiments, the organ is a liver and the assessing in (d) further comprises performing a liver function test. In some embodiments, the assessing in (d) further comprises imaging the organ. In some embodiments, the organ comprises a tumor and assessing in (d) further comprises determining a tumor size within the organ. In some embodiments, the assessing in (d) comprises analyzing a perfusate of the organ. In some embodiments, the organ is a liver and analyzing the perfusate comprises measuring lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate. In some embodiments, the assessing in (d) further comprises measuring pressure changes to the organ. In some embodiments, the assessing in (d) comprises measuring changes to tissue architecture. In some embodiments, the assessing in (d) comprises measuring an amount of live or dead cells. In some embodiments, the assessing in (d) comprises measuring cell type composition of the cells of the organ. In some embodiments, the assessing in (d) comprises measuring changes in gene expression of cells of the organ using spatial omics. In some embodiments, the assessing in (d) comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic, or metabolomic sequencing, and / or digital pathology. In some embodiments, the organ is a brain and assessing in (d) comprises measuring neurotoxicity or blood-brain-barrier penetration. In some embodiments, the organ is a kidney and assessing in (d) comprises measuring nephrotoxicity. In some embodiments, the system further comprises administering at least two pharmacological therapies to the organ concurrently with the machine perfusion conditions of (b). In some embodiments, the at least two pharmacological therapies are administered sequentially to the organ. In some embodiments, the at least two pharmacological therapies are administered concurrently to the organ. In some embodiments, the system further comprises administering a pharmacology therapy separately from the machine perfusion conditions. In some embodiments, the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.4 weeks, or a month. In some embodiments, the perfusion conditions comprise providing a supplemented medium to the organ. In some embodiments, the perfusion conditions comprise providing oxynation to the organ. In some embodiments, the perfusion conditions comprise providing artificial blood to the organ. In some embodiments, the artificial blood comprises perfluorocarbon-based oxygen carriers (PFCs) or stabilized hemoglobin solutions (HBOCs). In some embodiments, the system further comprises using an automated computer-implemented system to perform (a), (b), (c), (d), or any combination thereof . In some embodiments, the system further comprises using an automated computer-implemented system to perform (b) and (c). In some embodiments, the system further comprises using an automated computer-implemented system to perform (b), (c), and (d).

[0005] In an aspect, the present disclosure provides a method for assessing effects of an oncology therapy on an organ, comprising: (a) obtaining an organ from a donor subject, wherein the organ is ex-vivo with respect to the donor subject; (b) subjecting the organ to machine perfusion conditions, thereby maintaining viability of the organ; (c) administering the oncology therapy to the organ concurrently with the machine perfusion conditions of (b); and (d) assessing the effects of the oncology therapy on the organ, responsive to the administering in (c).

[0006] In some embodiments, the donor subject is a candidate transplant donor and the organ is determined to be unsuitable for a transplant procedure.

[0007] In some embodiments, the organ comprises at least a portion of a liver, heart, kidney, lung, pancreas, or a brain. In some embodiments, the organ is at least a portion of the liver. In some embodiments, the organ has a tumor. In some embodiments, the tumor is a carcinoma. In some embodiments, the carcinoma is metastatic.

[0008] In some embodiments, the oncology therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, a surgical resection, a laser ablation, or any combination thereof. In some embodiments, the oncology therapy comprises administering doxorubicin, taxol, bevacizumab, RNAi, or any combination thereof.

[0009] In some embodiments, the assessing in (d) further comprises analyzing a sample of the organ. In some embodiments, the sample comprises a biopsy sample. In some embodiments, the biopsy sample is obtained at least in part by core needle biopsy. In some embodiments, the assessing in (d) further comprises an organ function test. In some embodiments, the assessing in (d) further comprises imaging the organ. In some embodiments, the assessing in (d) further comprises determining a tumor size within the organ. In some embodiments, the assessing in (d) further comprises assessing a molecular drug response of the organ to the oncology therapy. In some embodiments, the assessing in (d) further comprises assessing a mechanism of toxicity of the therapy on the organ. In some embodiments, the assessing comprises analyzing a perfusate of the organ.

[0010] In some embodiments, analyzing the perfusate comprises measuring pressure changes to the organ, lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate. In some embodiments, the assessing comprises measuring changes to tissue architectureor an amount of live or dead cells using flow cytometry. In some embodiments, the assessing comprises measuring cell type composition of cells of the organ. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using spatial omics. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic, or metabolomic sequencing, and / or digital pathology (e.g., artificial intelligence or machine learning-based predictive tools for organ toxicity and / or therapy efficacy). In some embodiments, the assessing in (d) further comprises determining time-series effects of the oncology therapy on the organ over a period of time. In some embodiments, the assessing in (d) further comprises determining a spatial assessment of the oncology therapy on the organ.

[0011] In some embodiments, the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or a month. In some embodiments, the perfusion conditions comprise providing a supplemented medium to the organ. In some embodiments, the perfusion conditions comprise providing oxygenated blood to the organ. In some embodiments, the method further comprises using an automated computer-implemented system to perform at least one of (b), (c), and (d). In some embodiments, the method further comprises using the automated computer-implemented system to perform both (b) and (c). In some embodiments, the method further comprises using the automated computer-implemented system to perform (a), (b), and (c).

[0012] In another aspect, the present disclosure provides a system for assessing effects of an oncology therapy on an organ, comprising: a machine perfusion device configured to (i) receive an organ from a donor subject, wherein the organ is ex-vivo with respect to the donor subject, and (ii) subject the organ to machine perfusion conditions, thereby maintaining viability of the organ; a therapy dispenser configured to administer the oncology therapy to the organ concurrently with the machine perfusion conditions of (a); and a computer processor programmed to assess the effects of the oncology therapy on the organ, responsive to the administering in (b).

[0013] In some embodiments, the donor subject is a candidate transplant donor and the organ is determined to be unsuitable for a transplant procedure. In some embodiments, the organ comprises at least a portion of a liver, heart, kidney, lung, pancreas, or a brain. In some embodiments, the organ is at least a portion of the liver. In some embodiments, the organ has a tumor. In some embodiments, the tumor is a carcinoma. In some embodiments, the carcinoma is metastatic. In some embodiments, the oncology therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, a surgical resection, a laser ablation, or any combination thereof. In some embodiments, the immunotherapy comprises a chimeric receptor based therapy, an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-bindingfragment thereof comprises a bi-specific antibody, a tri-specific antibody, an antibody-drug conjugate, or a bi-specific T cell engager (BiTE). In some embodiments, the oncology therapy comprises administering doxorubicin, taxol, bevacizumab, or any combination thereof.

[0014] In some embodiments, the assessing in (c) further comprises analyzing a sample of the organ. In some embodiments, the sample comprises a biopsy sample. In some embodiments, the biopsy sample is obtained at least in part by core needle biopsy. In some embodiments, the assessing in (c) further comprises an organ function test. In some embodiments, the assessing in (c) further comprises imaging the organ. In some embodiments, the assessing in (c) further comprises determining a tumor size of the organ. In some embodiments, the assessing in (c) further comprises assessing a molecular drug response of the organ to the oncology therapy. In some embodiments, the assessing comprises analyzing a perfusate of the organ. In some embodiments, analyzing the perfusate comprises measuring pressure changes to the organ, lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH. In some embodiments, the assessing comprises measuring changes to tissue architecture or an amount of live or dead cells using flow cytometry. In some embodiments, the assessing comprises measuring cell type composition of cells of the organ. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using spatial omics. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic or metabolomic sequencing, and / or digital pathology (e.g., artificial intelligence or machine learning-based predictive tools for organ toxicity and / or therapy efficacy). In some embodiments, the assessing in (c) further comprises determining time-series effects of the oncology therapy on the organ over a period of time. In some embodiments, the assessing in (c) further comprises determining a spatial assessment of the oncology therapy on the organ.

[0015] In some embodiments, the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or a month. In some embodiments, the perfusion conditions comprise providing a supplemented medium to the organ. In some embodiments, the perfusion conditions comprise providing oxygenated blood to the organ.

[0016] In some embodiments, at least one of (b), (c), and (d) are automatically performed by computer implementation. In some embodiments, both (b) and (c) are automatically performed by computer implementation. In some embodiments, (a), (b), and (c) are automatically performed by computer implementation.

[0017] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0018] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0019] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0022] FIG. 1 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.

[0023] FIG. 2 illustrates an example of a normothermic machine perfusion device (e.g., OrganOx metra®), which comprises an oxygenator, a reservoir, and a pump.

[0024] FIG. 3 is an exemplary schematic of the processes and analysis in the methods and systems provided herein.

[0025] FIGs. 4A-4D illustrate results from an experiment with liver. FIGs. 4A-4D show results of perfusion parameters, including arterial pressure (FIG. 4A), system temperature (FIG. 4B), pH (FIG. 4C), and oxygen partial pressure (FIG. 4D) measured during a seven-hour liver perfusion.

[0026] FIGs. 5A-5B show results of liver function tests in a seven-hour liver perfusion with a 30- minute acetaminophen (APAP) dose, including lactate clearance (FIG. 4E) and pH regulation (FIG. 4F)

[0027] FIGs. 6A-6D illustrate the post-APAP assessment of liver metabolism and toxicity, as measured by GSTa levels (FIG. 6A) and APAP metabolites (FIGs. 6B-6D). Reduction of APAP concentration in the liver perfusate is shown in plasma equivalent concentration (FIG. 6B) and perfusate concentration (FIG. 6C), alongside the increase of respective perfusate concentration of primary APAP metabolites (FIGs. 6C-6D). APAP: acetaminophen; APAP-S: APAP sulfate; APAP-Glu: APAP glucuronide; NAC-APAP: acetaminophen mercapturate.

[0028] FIGs. 7A-7D illustrate the post-alcohol assessment of liver metabolism and toxicity. As shown in FIG. 7A and FIG. 7B, perfusate lactate levels increased and pH dropped following ethanol challenges, indicating liver damage, though recovery occurred at later time points. FIG. 7C illustrates the detection of ethanol metabolite Ethyl Glucuronide (EtG) in the perfusate 15 minutes post ethanol exposure. FIG. 7D illustrates the overall liver metabolism of ethanol.DETAILED DESCRIPTION

[0029] Despite certain therapies being approved for clinical use, their specific mechanisms of action in human organs and systems can remain a challenge to understand. One bottleneck in understanding the mechanisms of action can involve challenges of evaluating absorption, transport, metabolism, elimination, efficacy, and toxicity of drugs in organs and systems. Some approaches may involve in vivo studies for probing isolated organs, tissues, subcellular particles, or molecular interactions. However, these methods may involve considerable amount of time, effort, and resources, especially in drug development. Even with attempts to improve the throughput of studies through the use of tissue preparations and in vitro studies, many drug candidates still fail in clinical trials due to unanticipated adverse risks of translating drug effects to real subjects.

[0030] In some aspects, the present disclosure provides improved systems and methods for evaluating a therapy to bridge the gap between non-human animal testing and human administration. In some embodiments, the methods and systems provided herein are for assessing effects of a pharmacological therapy. In some embodiments, the pharmacological therapy comprises at least one, at least two, at least three, at least four, or at least five active pharmacological agents. In some embodiments, the pharmacological therapy comprises a cancer therapy. In some embodiments, the pharmacological therapy is not a cancer therapy.

[0031] It is recognized herein that both the drug development industry and organ transplantation field face similar challenges: the necessity for better testing and preservation methods. In thecontext of drug development, the present disclosure provides improvements for moving beyond animal testing and improving human safety. In the context of organ transplantation, the present disclosure provides ability to preserve organs for longer in a state as close as possible to their functioning state in the body. More broadly, the systems and methods can be used to help understand drug response kinetics and measure the effects across normal and diseased tissue, which can help create models that more accurately reflect human (patho)physiology.

[0032] In some embodiments, the methods and systems provided herein are useful for assessing effects of a therapy on an organ for an extended period of time. In some embodiments, the methods and systems provided herein comprise subjecting an organ to long-term machine perfusion conditions. In some embodiments, the methods and systems comprise: (a) providing an organ obtained from a donor subject, wherein the organ is ex-vivo with respect to the donor subject; (b) subjecting the organ to machine perfusion conditions, thereby maintaining viability of the organ; (c) administering the therapy to the organ concurrently with the machine perfusion conditions of(b); and (d) assessing the effects of the therapy on the organ responsive to the administering in(c). In some embodiments, the methods and systems comprise subjecting the organ to long-term machine perfusion conditions.

[0033] In some embodiments, the methods and systems comprise maintaining the viability of the organ during the long-term machine perfusion conditions. In some embodiments, the duration of machine perfusion conditions depends on the type of organs used for assessing the therapy. In some embodiments, the duration of machine perfusion conditions depends on the type of therapies tested on the organ.

[0034] In some embodiments, the long-term machine perfusion conditions for a liver last for at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 10 days, at least 14 days, at least 18 days, at least 21 days, at least 25 days, at least 28 days, at least 32 days, at least 35 days, at least 39 days, at least 42 days, at least 46 days, at least 49 days, at least 53 days, at least 56 days, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, or at least 20 weeks. In some embodiments, the long-term machine perfusion conditions for a liver last for at least a month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months.

[0035] In some embodiments, the long-term machine perfusion conditions for a lung last for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, or at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 3.5 days,at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, or at least 7 days.

[0036] In some embodiments, the long-term machine perfusion conditions for an intestine last for at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least5.5 days, at least 6 days, at least 6.5 days, or at least 7 days.

[0037] In some embodiments, the long-term machine perfusion conditions for a kidney last for at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least5.5 days, at least 6 days, at least 6.5 days, or at least 7 days.

[0038] In some embodiments, the methods and systems comprise subjecting an organ to longterm machine perfusion conditions for the duration of at least one half-life, 2 half-lives, 3 halflives, 4 half-lives, 5 half-lives, 6 half-lives, 7 half-lives, 8 half-lives, 9 half-lives, 10 half-lives, 15 half-lives, 20 half-lives, 25 half-lives, or at least 30 half-lives of the drug. In some embodiments, the methods and systems comprise subjecting an organ to long-term machine perfusion conditions for the duration of a non-pharmacological procedure.

[0039] In some aspects, the present disclosure provides methods and systems of ex vivo drug testing in human organs. In some embodiments, the methods and systems can comprise testing drugs on a human liver. In some embodiments, the methods and systems described herein can use a perfusion device or system configured to extend liver preservation to improve transplantation outcomes. In some embodiments, the device or system can be fully automated, such that continuous perfusion can be performed with blood, artificial blood, nutrients, and medicines, for one or a plurality of organs. In some aspects, the present disclosure provides systems and methods for studying drug response kinetics.

[0040] In some embodiments, the methods and systems provided herein comprise assessing an effect of a therapy. In some embodiments, the effects of the therapy comprises a therapeutic effect, a pharmacodynamic response, a pharmacokinetic response, an adverse drug effect, a toxic effect, drug adsorption, drug absorption, drug distribution, drug metabolism, drug excretion, or any combination thereof. In some embodiments, assessing the effect of the therapy comprises assessing a molecular drug response of the organ to the therapy. In some embodiments, assessing the effect of the therapy comprises assessing a mechanism of toxicity of the therapy on the organ. In some embodiments, assessing the effect of the therapy comprises determining time-series effects of the therapy on the organ over a period of time. In some embodiments, assessing the effect of the therapy comprises determining a spatial assessment of the therapy on the organ.

[0041] In some embodiments, the methods and systems described herein can be used for various applications, such as mechanistic toxicology, molecular biomarker development, Al and computational modeling, cell biology, systems biology, or any combination thereof. In some embodiments, mechanistic toxicology comprises detection of hepatotoxicity mechanisms such as oxidative stress, or detection of mitochondrial dysfunction and cholestasis using functional and molecular markers. In some embodiments, the methods and systems can be used for human drug metabolism studies, such as LC-MS / MS profiling of perfusate, detailed tracking of biotransformation products, or PBPK modeling. In some embodiments, the methods and systems can be used for drug-drug interaction risk modeling, such as co-dosing with cytochrome inducers / inhibitors to evaluate of drug-drug interaction risks under controlled dynamic conditions. In some embodiments, the methods and systems can be used for donor variability and precision toxicology, which includes testing across livers from diverse donors and exploring inter-individual susceptibility to drug induced liver injury. In some embodiments, the methods and systems can be used for biomarker discovery and qualification. In some embodiments, the organs are whole organs. In some embodiments, the systems and methods capture time-resolved changes to organ functionality and / or architecture. In some embodiments, the systems and methods are used to study intact human organs for testing. In some embodiments, the systems and methods are used to longitudinally probe changes to the organ in response to perturbations. In some embodiments, the systems and methods are used to evaluate severity of hepatotoxicity through measurable changes in liver function, tissue architecture, cell death pathways, or any combination thereof. In some embodiments, the systems and methods are used to identify changes in cellular composition and / or cell-type specific gene expression. In some embodiments, the systems and methods are used to develop a spatio-temporal map of drug response. In some embodiments, the systems and methods are used to study a plurality of organs which are in fluidic or mechanical communication with one another. The plurality of organs that are in communication can be used to study systemic PK / PD. In some embodiments, the systems and methods may use biosensors for monitoring changes to liver pressure / function in real-time, etc.

[0042] In some aspects, the present disclosure provides methods and systems for assessing effects of a therapy on an organ. In some embodiments, the methods and systems comprise providing an organ that has been obtained from a donor subject. In some embodiments, the organ is ex-vivo with respect to the donor subject. In some embodiments, the method comprises subjecting the organ to machine perfusion conditions. In some embodiments, the machine perfusion conditions maintain viability of the organ. In some embodiments, the methods and systems comprise administering the therapy to the organ. In some embodiments, the administering can be concurrently with the machine perfusion conditions. In some embodiments, the administering canbe performed separately from the machine perfusion conditions. In some embodiments, the methods and systems comprise assessing the effects of the therapy on the organ. In some embodiments, the assessing can be responsive to the administering. In some embodiments, the therapy comprises a cancer therapy.

[0043] In some embodiments, the organ used in the methods and sytems provided herein is a healthy organ. In some embodiments, the organ has been obtained from a healthy donor. In some embodiments, the methods and systems further comprise administering one or more diseased cells to the organ. For example, the methods and systems can further comprise administering one or more diseased cells to the organ to simulate cancer.

[0044] In some embodiments, the organ is not a healthy organ. In some embodiments, the organ has been obtained from a donor with a disease or disorder. In some embodiments, the organ has been obtained from a donor with cancer. In some embodiments, the organ is not suitable for transplant. In some embodiments, the donor subject is a candidate transplant donor and the organ is determined to be unsuitable for a transplant procedure.

[0045] In some embodiments, the organ comprises cancerous cells or tissue. In some embodiments, the organ has a tumor. In some embodiments, the tumor is a carcinoma. For example, the organ can be a liver that has hepatocellular carcinoma. In some embodiments, the carcinoma is metastatic. For example, the organ can be a liver that has cancer cells orginicated from metastatic colon cancer or metastatic breast cancer. In some embodiments, the organ has leukemia. In some embodiments, the organ is a diseased organ obtained from a diseased subject having hepatitis, fatty liver disease, liver fibrosis, liver cirrhosis, liver failure, heart failure, cardiomyopathy, myocardial infarction, coronary artery disease, hypertension, congenital heart defects, endocarditis, Chronic Kidney Disease (CKD), diabetic nephropathy, polycystic kidney disease, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, alzheimer’s disease, parkinson’s disease, dementia, a genetic disorder, an infection disease, an autoimmune disease, or any combination thereof. In some embodiments, the organ is a fatty liver, an infected liver, a fibrotic liber, a cirrhosis liver, or any combination thereof.

[0046] In some embodiments, the organ is a whole organ. In some embodiments, the organ is a heart, a kidney, or a brain. In some embodiments, the organ comprises a portion, a segment, or a lobe of an organ. For example, the organ can be a lobe of a lung or a segment of an intestine (e.g., colon). In some embodiments, the organ comprises at least a portion of a liver, a heart, a kidney, a lung, a pancreas, a colon, an intestine, a brain, a bone, or any combination hereof. In some embodiments, the organ comprises at least a portion of a liver, a heart, a kidney, a lung, a pancreas, or a brain. In some embodiments, the organ is at least a portion of the liver. In some embodiments, the portion of the organ used by the methods and systems function the same as a whole organ.

[0047] In some embodiments, the methods and systems are for assessing a therapy. In some embodiments, the therapy comprises a cancer therapy. In some embodiments, the cancer therapy comprises a pharmacological therapy. In some embodiments, the cancer therapy comprises a non- pharmacological therapy, such as a surgery or radiation therapy. In some embodiments, the cancer therapy comprises a combination therapy. In some embodiments, the cancer therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, radiation, a surgical resection, a laser ablation, or any combination thereof. In some embodiments, the immunotherapy comprises a chimeric receptor based therapy, an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises a bi-specific antibody, a tri-specific antibody, an antibody-drug conjugate, or a bi-specific T cell engager (BiTE). In some embodiments, cancer therapy comprises oxaliplatin, doxorubicin, taxol, bevacizumab, RNAi, or any combination thereof.

[0048] In some embodiments, the methods and systems are for assessing a therapy not indicated for treating cancer. In some embodiments, the therapy comprises a small molecule drug, a large molecule drug, a biologic, a prodrug, a natural product, a cell therapy, a gene therapy, an immunotherapy, a surgery, radiation, a preventative therapy, a curative therapy, a palliative therapy, a dye, a diagnostic therapy, or any combination thereof. In some embodiments, the methods and systems are for assessing any active pharmacological agent. In some embodiments, the methods and systems are for assessing any non-pharmacological therapy. In some embodiments, the methods and systems are for assessing a combination therapy. In some embodiments, the combination therapy can comprise one or more drugs, one or more non- pharmacological therapies, or any combination thereof.

[0049] In some embodiments, the methods and systems provided herein comprise administering at least two pharmacological agents or therapies to the organ. In some embodiments, the at least two agents or therapies are administered concurrently with the machine perfusion conditions. In some embodiments, the at least two agents or therapies are administered sequentially to the organ.

[0050] In some embodiments, the at least two agents or therapies are administered concurrently to the organ. In some embodiments, the methods and systems further comprise administering a pharmacology therapy separately from the machine perfusion conditions.

[0051] In some embodiments, the methods and systems provided herein comprise assessing the effects of a therapy on an organ. In some embodiments, assessing the effect comprises analyzing a sample of the organ. In some embodiments, the sample comprises a biopsy sample. In some embodiments, the biopsy sample is obtained at least in part by a core needle or wedge biopsy.

[0052] In some embodiments, the methods and systems comprise performing a test specific to the organ to assess the effects of the therapy on the organ. For example, the methods and systems cancomprise performing a liver-specific test to assess the effects of the therapy on a liver. In some embodiments, the liver-specific test comprise a liver enzyme test. In some embodiments, the liver enzyme test comprises Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), Glutathione S-transferaes (GST), Gamma-Glutamyl Transferase (GGT), Lactate Dehydrogenase (LDH), or any combination thereof. In some embodiments, the liver-specific test further comprise albumin, bilirubin, Prothrombin Time (PT) / International Normalized Ratio (INR), an antibody test, viral serology, or any combination thereof. In some embodiments, assessing the effect comprises performing a liver function test.

[0053] In some embodiments, the methods and systems comprise performing a brain-specific test to assess the effects of the therapy on a brain. In some embodiments, the brain-specific test comprises an imaging test, an electrical activity test, a cerebrospinal fluid (CSF) analysis, genetic testing, a brain biopsy, or any combination thereof. In some embodiments, the methods and systems comprise assessing whether the drug can penetrate the brain blood barrier. In some embodiments, the methods and systems comprise assessing the permeability of the brain blood barrier.

[0054] In some embodiments, the methods and systems comprise performing a heart-specific test to assess the effects of the therapy on a heart. In some embodiments, the heart-specific test comprise electrical activity tests (e.g., electrocardiogram (ECG / EKG) or electrophysiology study), imaging tests (e.g., echocardiograms cardiac stress tests, contractility test), biomarker tests (e.g., troponins, CK / CK-MB, BNP / NT-proBNP), or any combinations thereof.

[0055] In some embodiments, assessing the effect comprises imaging the organ. In some embodiments, assessing the effect comprises determining a size of the organ. In some embodiments, assessing the effect comprises determining a size of a cancerous site on or within the organ. In some embodiments, assessing the effect comprises determining a size of a tumor. In some embodiments, imaging the organ can be performed by ultrasound, Computed Tomography (CT) Scan, Magnetic Resonance Imaging (MRI), or any combination thereof.

[0056] In some embodiments, assessing the effect comprises assessing a molecular drug response of the organ to the therapy. In some embodiments, assessing the effect comprises assessing a mechanism of liver toxicity of the therapy on the organ.

[0057] In some embodiments, the assessing comprises analyzing a perfusate of the organ. In some embodiments, analyzing the perfusate comprises measuring pressure changes to the organ. In some embodiments, analyzing the perfusate comprises measuring lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate. In some embodiments, the assessing comprises measuring changes to tissue architecture or an amount of live or dead cells using flow cytometry. In some embodiments, the assessing comprises measuring cell typecomposition of cells of the organ. In some embodiments, assessing the effect comprises performing a histological study.

[0058] In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ. In some embodiments, measuring changes in gene expression comprises performing spatial omic studies. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic or metabolomic sequencing, and / or digital pathology (e.g., artificial intelligence or machine learning-based predictive tools for organ toxicity and / or therapy efficacy). In some embodiments, the assessing further comprises determining time-series effects of the therapy on the organ over a period of time. In some embodiments, the assessing further comprises determining a spatial assessment of the therapy on the organ.

[0059] In some embodiments, the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, or a month.

[0060] In some embodiments, the perfusion conditions comprise providing a supplemented medium to the organ. In some embodiments, a supplemented medium to the organ comprises electrolytes, nutrients, oxygen carriers, buffers, oncotic agents, antioxidants, anticoagulants, antibiotics, anti-inflammatory agents, or any combination thereof.

[0061] In some embodiments, the perfusion conditions comprise providing oxygen to the organ. In some embodiments, the oxygen is provided via one or more oxygen carriers. In some embodiments, the oxygen carrier comprises a native blood component, such as a red blood cell, or an artificial blood component. In some embodiments, the oxygen carrier comprises perfluorocarbon-based oxygen carriers (PFCs) or stabilized hemoglobin solutions (HBOCs). In some embodiments, the perfusion conditions comprise providing oxygenated blood to the organ. In some embodiments, the perfusion conditions comprise providing artificial blood to the organ.

[0062] In some embodiments, the methods and systems provided herein comprises using an automated computer-implemented system. In some embodiments, the methods and systems comprises using an automated computer-implemented system to perform at least one of (b), (c), (d), or any combination thereof. In some embodiments, the method further comprises using the automated computer-implemented system to perform both (b) and (c). In some embodiments, the method further comprises using the automated computer-implemented system to perform (b), (c), and (d).

[0063] In some aspects, the present disclosure provides a system for assessing effects of an therapy on an organ. In some embodiments, the therapy comprises a cancer therapy. In some embodiments, the system comprises a machine perfusion device. In some embodiments, themachine perfusion device is configured to receive an organ from a donor subject. In some embodiments, the organ is ex-vivo with respect to the donor subject. In some embodiments, the machine perfusion device is configured to subject the organ to machine perfusion conditions. In some embodiments, the machine perfusion conditions are configured to maintain viability of the organ. In some embodiments, the system comprises a therapy dispenser. In some embodiments, the therapy dispenser is configured to administer the therapy to the organ. In some embodiments, the therapy dispenser is configured to administer the therapy concurrently with the machine perfusion conditions. In some embodiments, the therapy dispenser is configured to administer the therapy separately from the machine perfusion conditions. In some embodiments, the system comprises a computer processor programmed to assess the effects of the therapy on the organ. In some embodiments, the assessment can be responsive to the administering.

[0064] In some embodiments, the donor subject is a candidate transplant donor and wherein the organ is determined to be unsuitable for a transplant procedure. In some embodiments, the organ comprises at least a portion of a liver, heart, kidney, lung, pancreas, or a brain. In some embodiments, the organ is at least a portion of the liver. In some embodiments, the organ has a tumor. In some embodiments, the tumor is a carcinoma. In some embodiments, the carcinoma is metastatic. In some embodiments, the oncology therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, a surgical resection, a laser ablation, or any combination thereof. In some embodiments, the immunotherapy comprises a chimeric receptor based therapy, an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises a bi-specific antibody, a tri-specific antibody, an antibody-drug conjugate, or a bi-specific T cell engager (BiTE). In some embodiments, the oncology therapy comprises administering doxorubicin, taxol, bevacizumab, or any combination thereof.

[0065] In some embodiments, the assessing in (c) further comprises analyzing a sample of the organ. In some embodiments, the sample comprises a biopsy sample. In some embodiments, the biopsy sample is obtained at least in part by core needle biopsy. In some embodiments, the assessing in (c) further comprises an organ function test. In some embodiments, the assessing in (c) further comprises imaging the organ. In some embodiments, the assessing in (c) further comprises determining a tumor size of the organ. In some embodiments, the assessing in (c) further comprises assessing a molecular drug response of the organ to the oncology therapy. In some embodiments, the assessing comprises analyzing a perfusate of the organ. In some embodiments, analyzing the perfusate comprises measuring pressure changes to the organ, lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate. In some embodiments, the assessing comprises measuring changes to tissue architecture or an amount of live or dead cells using flow cytometry. In some embodiments, the assessing comprises measuring cell typecomposition of cells of the organ. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using spatial omics. In some embodiments, the assessing comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic or metabolomic sequencing, and / or digital pathology (e.g., artificial intelligence or machine learning-based predictive tools for organ toxicity and / or therapy efficacy). In some embodiments, the assessing in (c) further comprises determining time-series effects of the oncology therapy on the organ over a period of time. In some embodiments, the assessing in (c) further comprises determining a spatial assessment of the oncology therapy on the organ.

[0066] In some embodiments, the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or a month. In some embodiments, the perfusion conditions comprise providing a supplemented medium to the organ. In some embodiments, the perfusion conditions comprise providing oxygenated blood to the organ.

[0067] In some embodiments, at least one of (b), (c), and (d) are automatically performed by computer implementation. In some embodiments, both (b) and (c) are automatically performed by computer implementation. In some embodiments, (a), (b), and (c) are automatically performed by computer implementation.

[0068] In another aspect, the present disclosure provides systems and methods for improving organ preservation. Currently, one of every four patients waiting for cardiac transplantation dies due to the lack of a suitable donated organ. Heart transplantation has become increasingly feasible due to improvements in patient survival rates and the development of new immunosuppressive agents. However, there are significant obstacles in preserving the donor heart for more than four hours, which limits the geographic area from which donor hearts can be transported. The current method of hypothermic arrest and storage preservation often leads to cell swelling, intracellular acidosis, and a degradation of high-energy phosphates. Additionally, current preservation protocols do not recreate an in vzvo-like physiologic state for harvested organs.

[0069] In some aspects, the present disclosure provides systems and methods for maintaining the harvested organ in a normal functioning state. The systems and methods can be used to expand the organ donor pool and increasing the histocompatibility matching time. This extended period of preservation can dramatically improve the practices of heart transplantation by facilitating worldwide retrieval of organs, and allowing a more comprehensive assessment of each organ, reducing the risk of graft failure. Certain approaches of simulating near-normal physiological conditions may involve methods such as removing multiple donor organs in combination.Therefore, creating an extracorporeal circuit for prolonged preservation of the harvested organ at normothermic temperatures is desirable.

[0070] Computer systems

[0071] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 1 shows a computer system 101 that is programmed or otherwise configured to, for example, assess effects of a therapy on an organ.

[0072] The computer system 101 can regulate various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, assessing effects of thetherapy on an organ. The computer system 101 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0073] The computer system 101 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 105, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 101 also includes memory or memory location 110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage and / or electronic display adapters. The memory 110, storage unit 115, interface 120 and peripheral devices 125 are in communication with the CPU 105 through a communication bus (solid lines), such as a motherboard. The storage unit 115 can be a data storage unit (or data repository) for storing data. The computer system 101 can be operatively coupled to a computer network (“network”) 130 with the aid of the communication interface 120. The network 130 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet.

[0074] The network 130 in some cases is a telecommunication and / or data network. The network 130 can include one or more computer servers, which can enable distributed computing, such as cloud computing. For example, one or more computer servers may enable cloud computing over the network 130 (“the cloud”) to perform various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, assessing effects of a therapy on an organ. Such cloud computing may be provided by cloud computing platforms such as, for example, Amazon Web Services (AWS), Microsoft Azure, Google Cloud Platform, and IBM cloud. The network 130, in some cases with the aid of the computer system 101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server.

[0075] The CPU 105 may comprise one or more computer processors and / or one or more graphics processing units (GPUs). The CPU 105 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 110. The instructions can be directed to the CPU 105, which can subsequently program or otherwise configure the CPU 105 to implement methods of the present disclosure. Examples of operations performed by the CPU 105 can include fetch, decode, execute, and writeback.

[0076] The CPU 105 can be part of a circuit, such as an integrated circuit. One or more other components of the system 101 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0077] The storage unit 115 can store files, such as drivers, libraries and saved programs. The storage unit 115 can store user data, e.g., user preferences and user programs. The computer system 101 in some cases can include one or more additional data storage units that are external to the computer system 101, such as located on a remote server that is in communication with the computer system 101 through an intranet or the Internet.

[0078] The computer system 101 can communicate with one or more remote computer systems through the network 130. For instance, the computer system 101 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 101 via the network 130.

[0079] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 101, such as, for example, on the memory 110 or electronic storage unit 115. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 105. In some cases, the code can be retrieved from the storage unit 115 and stored on the memory 110 for ready access by the processor 105. In some situations, the electronic storage unit 115 can be precluded, and machine-executable instructions are stored on memory 110.

[0080] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[0081] Aspects of the systems and methods provided herein, such as the computer system 101, can be embodied in programming. Various aspects of the technology may be thought of as“products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., readonly memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0082] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0083] The computer system 101 can include or be in communication with an electronic display 135 that comprises a user interface (UI) 140 for providing, for example, (i) a visual display indicative of effects of thetherapy on an organ. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0084] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 105. The algorithm can, for example, assess effects of thetherapy on an organ.

[0085] Non-Transitory Computer Readable Storage Medium

[0086] In some aspects, the present disclosure describes a non-transitory computer-readable storage media encoded with a computer program including instructions executable by one or more processors to assess effects of a therapy on an organ, subject an organ to machine perfusion conditions, administer a therapy to an organ, obtain a biopsy of an organ, control a device or system for assessing effects of a therapy on an organ using any one of the methods disclosed herein. In some embodiments, a non-transitory computer-readable storage media may comprise assessing effects of a therapy on an organ, subjecting an organ to machine perfusion conditions, administering a therapy to an organ, obtaining a biopsy of an organ, controlling a device or system for assessing effects of a therapy on an organ. In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device.

[0087] In further embodiments, a computer readable storage medium is a tangible component of a computing device. In still further embodiments, a computer readable storage medium is optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some embodiments, the program and instructions are permanently, substantially permanently, semi-permanently, or non- transitorily encoded on the media.

[0088] Computer Program

[0089] In some aspects, the present disclosure describes a computer program product comprising a computer-readable medium having computer-executable code encoded therein, the computerexecutable code adapted to be executed to implement any one of the methods disclosed herein. In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same.

[0090] A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types. A computer program may be written in various versions of various languages. In some embodiments, APIs may comprise various languages, for example, languages in various releases of TensorFlow, Theano, Keras, PyTorch, or any combination thereof which may be implemented in various releases of Python, Python3, C, C#, C++, MatLab, R, Java, or any combination thereof.

[0091] The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.

[0092] Web Application

[0093] In some embodiments, a computer program includes a web application. In some embodiments, a user may enter a query for assessing effects of a therapy on an organ, subjecting an organ to machine perfusion conditions, administering a therapy to an organ, obtaining a biopsy of an organ, controlling a device or system for assessing effects of a therapy on an organ through a web application. In some embodiments, a user may assess effects of a therapy on an organ, subject an organ to machine perfusion conditions, administer a therapy to an organ, obtain a biopsy of an organ, control a device or system for assessing effects of a therapy on an organ through a web application. A web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of nonlimiting examples, relational, non-relational, object oriented, associative, XML, and document oriented database systems. In further embodiments, suitable relational database systems include, by way of non-limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®. A web application, in various embodiments, is written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definitionlanguages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a clientside scripting language such as Asynchronous JavaScript and XML (AJAX), Flash® ActionScript, JavaScript, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tel, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®.

[0094] Mobile application

[0095] In some embodiments, a computer program includes a mobile application provided to a mobile computing device. In some embodiments, the mobile application is provided to a mobile computing device at the time it is manufactured. In other embodiments, the mobile application is provided to a mobile computing device via the computer network described herein.

[0096] A mobile application may be created by various techniques using various hardware, languages, and development environments. Mobile applications may be written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, Java™, JavaScript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML / HTML with or without CSS, or combinations thereof.

[0097] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (los) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.

[0098] Standalone application

[0099] In some embodiments, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Standalone applications may be compiled. A compiler is a computer program(s) thattransforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications.

[0100] Software Modules

[0101] In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and / or database modules, or use of the same. Software modules may be created by various techniques using various machines, software, and languages. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, a distributed computing resource, a cloud computing resource, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, a plurality of distributed computing resources, a plurality of cloud computing resources, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, a standalone application, and a distributed or cloud computing application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.

[0102] Databases

[0103] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases, or use of the same. Many databases are suitable for storage and retrieval of information about assessing effects of a therapy on an organ, subjecting an organ to machine perfusion conditions, administering a therapy to an organ, obtaining a biopsy of an organ, controlling a device or system for assessing effects of a therapy on an organ, or any combination thereof. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entityrelationship model databases, associative databases, XML databases, document orienteddatabases, and graph databases. Further non-limiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, Sybase, and MongoDB. In some embodiments, a database is Internetbased. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing-based. In a particular embodiment, a database is a distributed database. In other embodiments, a database is based on one or more local computer storage devices.EXAMPLESExample 1: Method for testing hepatoxic drugs and cancer therapies in livers

[0104] Using methods and systems of the present disclosure, known hepatotoxic drugs (e.g., acetaminophen) and cancer therapies (chemotherapy and targeted agents - oxaliplatin, doxorubicin, taxol, bevacizumab, etc) are tested in ex-vivo livers deemed unsuitable for transplantation, with a focus on livers with hepatic tumors (primary and / or metastatic).

[0105] In a seven-day experiment, liver function is continuously monitored (e.g., perfusate lactate clearance, pH, transaminase levels, glucose metabolism, bile pH, and / or pressure changes to the organ). Core needle biopsies are collected at 3-hour intervals for molecular analysis. Initially, the biopsy data are used to examine kinetics over time within each liver by measuring changes to tissue architecture or an amount of live / dead cells using flow cytometry, cell type composition and expression changes using spatial omics, apoptotic pathway changes and novel signatures using genomic sequencing and / or epigenomic sequencing, and / or digital pathology (e.g., artificial intelligence or machine learning-based predictive tools for organ toxicity and / or therapy efficacy). The data are used to develop spatio-temporal maps of functional and molecular drug response. The new model is used to increase our understanding of drug response kinetics and develop innovative treatment strategies aimed at optimizing treatment response while reducing the incidence of adverse reactions for cancer patients.Example 2: PK / PD assessments of novel therapies in organs

[0106] Using the methods and systems of the present disclosure, PK / PD assessments of novel therapies or investigational agents in a physiologically relevant ex vivo system are performed.

[0107] The analyses described in Examples 1 and 2 can be used to create a framework for testing emerging therapeutics, including specific drug modalities and assay measurements to prioritize. High-throughput instrumentation is used to test multiple drugs, which can be delivered at different locations of one or more organs and at different times (e.g., by perfusing multiple drugs to different regions of the one or more organs).Example 3: Measuring drug response kinetics using a whole human liver model

[0108] Using methods and systems of the present disclosure, drug response kinetics are measured using a whole human liver model, including modeling kinetics of drug-induced hepatotoxicity (DILI).

[0109] The liver is one of the most prevalent organ sites for cancer development and spread. Primary liver cancers (hepatocellular carcinoma) and liver metastases originating from other sites pose significant challenges due to their notorious resistance to systemic therapies. Contributing factors include high levels of drug-metabolizing enzymes, multidrug resistance proteins, and the immunosuppressive tumor microenvironment. Novel treatment strategies are urgently needed; however, there is a scarcity of models that accurately reproduce the physical and molecular barriers associated with therapeutically targeting liver lesions.

[0110] Methods and systems of the present disclosure use a normothermic liver device that was developed to extend liver preservation. Because the system is fully automated, allowing for continuous perfusion with blood, nutrients, and medicines, it poses a suitable opportunity for human organ-level drug perfusion studies for research purposes.

[0111] Hepatotoxic drugs are tested in livers deemed unsuitable for transplantation. In a seven- day experiment, liver function is continuously monitored, and core needle biopsies are collected multiple times each day for molecular analysis. The data are used to develop spatio-temporal maps of functional and molecular drug response. After optimizing the workflows, hepatic tumors (primary and / or metastatic) are studied. The models are used to increase understanding of drug response kinetics and develop innovative treatment strategies aimed at optimizing treatment response while reducing the incidence of adverse reactions.

[0112] Various normothermic machine perfusion devices are suitable for use with the methods and systems of the present disclosure. As an example, the OrganOx metra® is used for performing normothermic machine perfusion, during which a donor liver is continuously perfused with oxygenated blood, medications, and nutrients at normal body temperature and near physiological pressures and flows. Conventional cold preservation involves storage of the liver at 4°C and aims to minimize liver degradation. In contrast, the OrganOx metra® recreates a near physiological environment by continuously perfusing the liver at near physiological pressures and flows with oxygen-carrying red blood cells at 37°C. The liver remains functional during preservation, producing bile, metabolizing glucose, and maintaining pH, allowing the objective assessment of organ performance. Via onboard blood gas analysis, the OrganOx metra® automatically measures and controls blood gases in the perfusate without user intervention. Further details of the OrganOx metra® are described at, for example, www.organox.com / metra-how-it-works.

[0113] As shown in FIG. 2, as an example, the OrganOx metra® normothermic machine perfusion device can be used. It comprises an oxygenator, a reservoir, and a pump. Oxygen isconcentrated from ambient air and supplied, on-demand to the oxygenator. The blood is also warmed by the on-board heater. Warm, oxygenated blood is stored in the soft-shell reservoir, and supplied to the liver under near-physiological pressure. Blood is drawn from the liver by a centrifugal pump, which automatically varies in speed according to changes in blood pressure. Medicines and nutrition are delivered automatically throughout perfusion, in order to minimise operator hands-on time. This ensures that the liver is functional throughout preservation, enabling functional assessment. For example, various drugs (e.g., acetaminophen, cancer therapeutics (chemotherapy, immunotherapy, etc.)) may be administered to the functional liver.

[0114] Primary outcome variables include: evaluating severity of hepatotoxicity through measurable changes in liver function, tissue architecture, and cell death pathways. Secondary outcome variabels include: identifying novel changes in cellular composition and cell-type specific gene expression.

[0115] In a first phase, non-cancerous livers are tested with acetaminophen for workflow and design. In a second phase, livers with hepatocellular carcinoma (HCC) or metastatic disease are tested with various oncology therapeutics (e.g., chemotherapy, immunotherapy, targeted therapies, siRNAs, etc.). As an inclusion criterion, the livers may have about 12 hours of delivery lead time. As exclusion criteria, livers with anatomical damage (as defined by surgical team) that may preclude pumping, DCD livers with more than 45 minutes of warm ischemic time and / or oxygen saturation <70, and donors with active infections, may be excluded from these studies.

[0116] On Day 0, an experiment may be set up with a donor liver. Once notification of an available liver is received, the donor’s medical history is reviewed. The liver is characterized at baseline. Functional assessments (perfusate lactate clearance, pH, transaminase levels, glucose metabolism, bile pH, and / or pressure changes to the organ) are performed using the OrganOx device. Baseline biopsies are obtained from the liver.

[0117] On Days 0 to 7, the liver is perfused with single doses of therapeutic. Liver function is monitored using the OrganOx device. Biopsies are continuously obtained (dynamic drug response samples) at multiple timepoints during the 24-hour day period. For example, biopsies may be collected at 3-hour intervals for up to seven days. Biopsy data are used to examine kinetics over time within each liver.

[0118] Statistical analysis is performed. For example, data are gathered to determine effect size, and data collection methods are optimized for detection of changes in outcome variables. Data points may be pooled for each day to perform a t-test comparing outcomes between baseline and last available data point. This data is used to calculate power for future studies using repeated measures of ANOVA with Dunnett’ s post hoc test to compare each available timepoint to baseline.

[0119] On Day 7, endpoint drug response is measured. Liver function is monitored using the OrganOx device. Endpoint response sample biopsies are obtained.

[0120] On Days 8+, drug response is analyzed from perfusate and tissue samples collected over the course of the experiment. Changes to tissue architecture or a number of live / dead cell types are quantified (e.g., using flow cytometry and flowjo software). Apoptotic pathway transcription is analyzed with methylation sequencing (e.g., using the methylGSA package in Bioconductor software). Spatial analysis is performed using various imaging techniques. Molecular analysis is performed (e.g., using DNA methylation-calling of tissue and blood samples, such as using Oxford Nanopore technology). Tissue samples may be frozen after the 7-day experiment for future studies and analysis.Example 4: Measuring drug metabolism and toxicity using a whole human liver model

[0121] This example describes a drug development method utilizing ex vivo machine perfusion (EVMP) of human livers to effectively model drug-induced liver injury (DILI). The method described herein allows for comprehensive evaluation of pharmacokinetic parameters (e.g., ADME) and pharmacodynamic responses. EVMP devices create stable perfusion conditions that mimic in vivo physiological environments, and the perfusate can be customized for various experimental needs, including drug metabolism studies and toxicity screening. Drugs under investigation can be directly administered into the perfusate for subsequent sampling and analysis. Furthermore, the method enables the analysis of drug metabolites, elucidation of drug toxicity mechanisms, and offers valuable insights into the variability of drug response across individuals. Tissue biopsies can also be collected for spatially resolved analysis of drug distribution and hepatic injury.

[0122] Target physiological parameters typically maintained during perfusion include: Arterial pressure: 60-90 mmHg; Temperature: 36.5-37.0°C; pH: 7.35-7.45; Oxygen partial pressure: >75 mmHg. These conditions support the liver's metabolic, synthetic, and excretory functions, such as gluconeogenesis, albumin production, and bile secretion, and are compatible with dynamic pharmacokinetic and toxicological investigations. FIGs. 4A-4D show the changes in system parameters monitored during exemplary perfusion conditions.

[0123] Results from a recent perfusion study, where a human liver was exposed to 500 mg of acetaminophen (APAP), are summarized in FIGs. 5A-5B and FIGs. 6A-6D. Functional outputs, such as bile production and glucose and lactate dynamics, were sustained throughout the experiment. Biomarkers of hepatocyte injury, like glutathione S-transferase-alpha (GST-a), were measured directly in the perfusate, providing early evidence of hepatocellular stress. This complemented kinetic metabolic profiling performed by liquid chromatography-massspectrometry (LC-MS). Additionally, tissue biopsies taken from multiple regions of the liver enabled spatially resolved analysis of drug metabolism and injury.

[0124] The study revealed clear pharmacokinetic patterns, including rapid increases in phase II conjugates (APAP -glucuronide, APAP-sulfate) and the emergence of glutathione conjugates (e.g., 3-(Cystein-S-yl) acetaminophen). A measurable reduction in APAP concentration over time, with approximately 37% conversion to identified products, was also observed.

[0125] These findings confirm the method's sensitivity in detecting drug metabolism and related biomarkers, as well as its specificity in identifying known injury signatures. Tissue biopsies, taken from multiple regions of the liver during and after perfusion, further support spatial analysis of drug distribution, histological changes, and zonated injury. This multi-compartment sampling strategy provides a robust dataset for mechanistic toxicology. Results from another recent perfusion study, where a human liver was exposed to alcohol (EtOH), are summarized in FIGs. 7A-7D. As shown in FIG. 7A and FIG. 7B, perfusate lactate levels increased and the pH dropped following ethanol challenges, indicating liver damage; however, recovery occurred at later time points. We were able to successfully cause alcohol-induced liver injury and demonstrate improvements with removal of the perturbation — highlighting that studies to evaluate drug toxicity can be conducted. Ethyl Glucuronide (EtG) is a specific biomarker for alcohol consumption; it should not be present unless alcohol consumption has occurred, as it is a direct metabolite of ethyl alcohol. Ethyl Sulfate (EtS) is a complementary biomarker, proposed as more stable and resistant to bacterial breakdown than EtG. We detected and monitored the metabolites of ethanol by liquid chromatography-mass spectrometry (LC-MS). FIG. 7C illustrates EtG detection 15 minutes post ethanol exposure. The methods and systems described herein can also be used to study drug toxicity in other organs. For example, the methods and systems can be used to study the effects of drugs on ex vivo brains. In some cases, the methods and systems comprise measuring neurotoxicity potential of novel drugs, measuring blood-brain-barrier penetration, or studying drug response in CNS metastases. In some cases, the methods and systems can be used to study drug effects on the kidneys, such as measuring nephrotoxicity potential of novel drugs using ex vivo kidneys. Multiple compounds, administered sequentially or concurrently, can be studied together in a single ex vivo organ. In some cases, cancer cells can be introduced to an organ to simulate primary or metastatic diseases. The system can also be perturbed with oncology drugs to measure drug metabolism and response.

[0126] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to theaforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for assessing effects of a therapy on an organ, comprising:(a) providing an organ obtained from a donor subject, wherein the organ is ex-vivo with respect to the donor subject;(b) subjecting the organ to machine perfusion conditions, thereby maintaining viability of the organ;(c) administering the oncology therapy to the organ concurrently with the machine perfusion conditions of (b); and(d) assessing the effects of the therapy on the organ, responsive to the administering in (c).

2. The method of claim 1, wherein the therapy comprises a cancer therapy.

3. The method of claim 2, wherein the cancer therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, radiation, a surgical resection, a laser ablation, or any combination thereof.

4. The method of claim 2 or 3, wherein the cancer therapy comprises an antibody or an antigen binding fragment thereof, oxaliplatin, doxorubicin, taxol, bevacizumab, RNAi, or any combination thereof.

5. The method of any one of the preceding claims, wherein (b) comprises subjecting a liver to more than 30 hours of machine perfusion, subjecting a lung to more than 6 hours of machine perfusion, subjecting an intestine to more than 12 hours of machine perfusion, or subjecting a kidney to more than 12 hours of machine perfusion, or any combination thereof.

6. The method of any one of the preceding claims, wherein (b) comprises subj ecting the organ to machine perfusion conditions for a duration of at least five half-lives of a drug, optionally at least ten, at least fifteen, at least twenty, at least twenty -five, or at least thirty half-lives of a drug.

7. The method of any one of the preceding claims, wherein the therapy comprises a pharmacological therapy.

8. The method of any one of the preceding claims, wherein the therapy comprises a non- pharmacological therapy.

9. The method of any one of the preceding claims, wherein the therapy comprises a combination therapy.

10. The method of any one of the preceding claims, wherein the therapy comprises a small molecule drug, a large molecule drug, a biologic, a prodrug, a natural product, a cell therapy, a gene therapy, an immunotherapy, a surgery, radiation, a preventative therapy, a curative therapy, a palliative therapy, a dye, a diagnostic therapy, or any combination thereof.

11. The method of any one of the preceding claims, wherein the effects of the therapy on the organ comprises a therapeutic effect, a pharmacodynamic response, a pharmacokinetic response, an adverse drug effect, a toxic effect, drug adsorption, drug absorption, drug distribution, drug metabolism, drug excretion, or any combination thereof.

12. The method of any one of the preceding claims, wherein the assessing in (d) further comprises assessing a molecular drug response of the organ to the therapy.

13. The method of any one of the preceding claims, wherein the assessing in (d) further comprises assessing a mechanism of toxicity of the therapy on the organ.

14. The method of any one of the preceding claims, wherein the assessing in (d) further comprises determining time-series effects of the therapy on the organ over a period of time.

15. The method of any one of the preceding claims, wherein the assessing in (d) further comprises determining a spatial assessment of the therapy on the organ.

16. The method of any one of the preceding claims, wherein the organ is unsuitable for a transplant procedure.

17. The method of any one of the preceding claims, wherein the organ comprises at least a portion of a liver, a heart, a kidney, a lung, a pancreas, a colon, an intestine, a brain, a bone, or any combination hereof.

18. The method of any one of the preceding claims, wherein the organ is at least a portion of a liver.

19. The method of any one of the preceding claims, wherein the organ has cancerous tissue.

20. The method of any one of the preceding claims, wherein the organ has a tumor.

21. The method of claim 20, wherein the tumor is a carcinoma.

22. The method of claim 21, wherein the carcinoma is metastatic.

23. The method of any one of claims 1-22, wherein the organ does not comprise cancerous tissue.

24. The method of claim 23, further comprising administering one or more cancer cells to the organ to simulate cancer.

25. The method of any one of the preceding claims, wherein the organ is a diseased organ obtained from a diseased subject having hepatitis, fatty liver disease, liver fibrosis, livercirrhosis, liver failure, heart failure, cardiomyopathy, myocardial infarction, coronary artery disease, hypertension, congenital heart defects, endocarditis, Chronic Kidney Disease (CKD), diabetic nephropathy, polycystic kidney disease, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, alzheimer’s disease, parkinson’s disease, dementia, a genetic disorder, an infection disease, an autoimmune disease, or any combination thereof.

26. The method of any one of the preceding claims, wherein the assessing in (d) further comprises analyzing a sample of the organ.

27. The method of claim 26, wherein the sample comprises a biopsy sample.

28. The method of claim 27, wherein the biopsy sample is obtained at least in part by core needle biopsy.

29. The method of any one of the preceding claims, wherein the assessing in (d) further comprises performing an organ function test.

30. The method of any one of the preceding claims, wherein the organ is a liver and the assessing in (d) further comprises performing a liver function test.

31. The method of any one of the preceding claims, wherein the assessing in (d) further comprises imaging the organ.

32. The method of any one of the preceding claims, wherein the organ comprises a tumor and assessing in (d) further comprises determining a tumor size within the organ.

33. The method of any one of the preceding claims, wherein the assessing in (d) comprises analyzing a perfusate of the organ.

34. The method of claim 33, wherein the organ is a liver and analyzing the perfusate comprises measuring lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate.

35. The method of any one of the preceding claims, wherein the assessing in (d) further comprises measuring pressure changes to the organ.

36. The method of any one of the preceding claims, wherein the assessing in (d) comprises measuring changes to tissue architecture.

37. The method of any one of the preceding claims, wherein the assessing in (d) comprises measuring an amount of live or dead cells.

38. The method of any one of the preceding claims, wherein the assessing in (d) comprises measuring cell type composition of the cells of the organ.

39. The method of any one of the preceding claims, wherein the assessing in (d) comprises measuring changes in gene expression of cells of the organ using spatial omics.

40. The method of any one of the preceding claims, wherein the assessing in (d) comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic, or metabolomic sequencing and / or digital pathology.

41. The method of any one of the preceding claims, wherein the organ is a brain and assessing in (d) comprises measuring neurotoxicity or blood-brain-barrier penetration.

42. The method of any one of the preceding claims, wherein the organ is a kidney and assessing in (d) comprises measuring nephrotoxicity.

43. The method of any one of the preceding claims, further comprising administering at least two pharmacological therapies to the organ concurrently with the machine perfusion conditions of (b).

44. The method of claim 43, wherein the at least two pharmacological therapies are administered sequentially to the organ.

45. The method of claim 43, wherein the at least two pharmacological therapies are administered concurrently to the organ.

46. The method of any one of the preceding claims, further comprising administering a pharmacology therapy separately from the machine perfusion conditions.

47. The method of any one of the preceding claims, wherein the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.4 weeks, or a month.

48. The method of any one of the preceding claims, wherein the perfusion conditions comprise providing a supplemented medium to the organ.

49. The method of any one of the preceding claims, wherein the perfusion conditions comprise providing oxynation to the organ.

50. The method of any one of the preceding claims, wherein the perfusion conditions comprise providing artificial blood to the organ.

51. The method of claim 50, wherein the artificial blood comprises perfluorocarbon-based oxygen carriers (PFCs) or stabilized hemoglobin solutions (HBOCs).

52. The method of any one of the preceding claims, further comprising using an automated computer-implemented system to perform (a), (b), (c), (d), or any combination thereof .

53. The method of any one of the preceding claims, further comprising using an automated computer-implemented system to perform (b) and (c).

54. The method of any one of the preceding claims, further comprising using an automated computer-implemented system to perform (b), (c), and (d).

55. A system for assessing effects of a therapy on an organ, comprising:(a) a machine perfusion device configured to (i) receive an organ from a donor subject, wherein the organ is ex-vivo with respect to the donor subject, and (ii) subject the organ to machine perfusion conditions, thereby maintaining viability of the organ;(b) a therapy dispenser configured to administer the therapy to the organ concurrently with the machine perfusion conditions of (a); and(c) a computer processor programmed to assess the effects of the therapy on the organ, responsive to the administering in (b).

56. The system of claim 55, wherein the therapy comprises a cancer therapy.

57. The system of claim 56, wherein the cancer therapy comprises a chemotherapy, a targeted therapy, an immunotherapy, radiation, a surgical resection, a laser ablation, or any combination thereof.

58. The system of claim 56 or 57, wherein the cancer therapy comprises an antibody or an antigen-binding fragment thereof, oxaliplatin, doxorubicin, taxol, bevacizumab, RNAi, or any combination thereof.

59. The system of any one of the preceding claims, wherein (b) comprises subjecting a liver to more than 30 hours of machine perfusion, subjecting a lung to more than 6 hours of machine perfusion, subjecting an intestine to more than 12 hours of machine perfusion, or subjecting a kidney to more than 12 hours of machine perfusion, or any combination thereof.

60. The system of any one of the preceding claims, wherein (b) comprises subjecting the organ to machine perfusion conditions for a duration of at least five half-lives of a drug, optionally at least ten, at least fifteen, at least twenty, at least twenty -five, or at least thirty half-lives of a drug.

61. The system of any one of the preceding claims, wherein the therapy comprises a pharmacological therapy.

62. The system of any one of the preceding claims, wherein the therapy comprises a non- pharmacological therapy.

63. The system of any one of the preceding claims, wherein the therapy comprises a combination therapy.

64. The system of any one of the preceding claims, wherein the therapy comprises a small molecule drug, a large molecule drug, a biologic, a prodrug, a natural product, a cell therapy, a gene therapy, an immunotherapy, a surgery, radiation, a preventative therapy, a curative therapy, a palliative therapy, a dye, a diagnostic therapy, or any combination thereof.

65. The system of any one of the preceding claims, wherein the effects of the therapy on the organ comprises a therapeutic effect, a pharmacodynamic response, a pharmacokinetic response, an adverse drug effect, a toxic effect, drug adsorption, drug absorption, drug distribution, drug metabolism, drug excretion, or any combination thereof.

66. The system of any one of the preceding claims, wherein the assessing in (d) further comprises assessing a molecular drug response of the organ to the therapy.

67. The system of any one of the preceding claims, wherein the assessing in (d) further comprises assessing a mechanism of toxicity of the therapy on the organ.

68. The system of any one of the preceding claims, wherein the assessing in (d) further comprises determining time-series effects of the therapy on the organ over a period of time.

69. The system of any one of the preceding claims, wherein the assessing in (d) further comprises determining a spatial assessment of the therapy on the organ.

70. The system of any one of the preceding claims, wherein the organ is unsuitable for a transplant procedure.

71. The system of any one of the preceding claims, wherein the organ comprises at least a portion of a liver, a heart, a kidney, a lung, a pancreas, a colon, an intestine, a brain, a bone, or any combination hereof.

72. The system of any one of the preceding claims, wherein the organ is at least a portion of a liver.

73. The system of any one of the preceding claims, wherein the organ has cancerous tissue.

74. The system of any one of the preceding claims, wherein the organ has a tumor.

75. The system of claim 74, wherein the tumor is a carcinoma.

76. The system of claim 74, wherein the carcinoma is metastatic.

77. The system of any one of claims 1-76, wherein the organ does not comprise cancerous tissue.

78. The system of claim 77, further comprising administering one or more cancer cells to the organ to simulate cancer.

79. The system of any one of the preceding claims, wherein the organ is a diseased organ obtained from a diseased subject having hepatitis, fatty liver disease, liver fibrosis, liver cirrhosis, liver failure, heart failure, cardiomyopathy, myocardial infarction, coronary artery disease, hypertension, congenital heart defects, endocarditis, Chronic Kidney Disease (CKD), diabetic nephropathy, polycystic kidney disease, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, alzheimer’s disease, parkinson’s disease,dementia, a genetic disorder, an infection disease, an autoimmune disease, or any combination thereof.

80. The system of any one of the preceding claims, wherein the assessing in (d) further comprises analyzing a sample of the organ.

81. The system of claim 80, wherein the sample comprises a biopsy sample.

82. The system of claim 81, wherein the biopsy sample is obtained at least in part by core needle biopsy.

83. The system of any one of the preceding claims, wherein the assessing in (d) further comprises performing an organ function test.

84. The system of any one of the preceding claims, wherein the organ is a liver and the assessing in (d) further comprises performing a liver function test.

85. The system of any one of the preceding claims, wherein the assessing in (d) further comprises imaging the organ.

86. The system of any one of the preceding claims, wherein the organ comprises a tumor and assessing in (d) further comprises determining a tumor size within the organ.

87. The system of any one of the preceding claims, wherein the assessing in (d) comprises analyzing a perfusate of the organ.

88. The system of claim 87, wherein the organ is a liver and analyzing the perfusate comprises measuring lactate clearance, pH, transaminase levels, glucose metabolism, or bile pH from the perfusate.

89. The system of any one of the preceding claims, wherein the assessing in (d) further comprises measuring pressure changes to the organ.

90. The system of any one of the preceding claims, wherein the assessing in (d) comprises measuring changes to tissue architecture.

91. The system of any one of the preceding claims, wherein the assessing in (d) comprises measuring an amount of live or dead cells.

92. The system of any one of the preceding claims, wherein the assessing in (d) comprises measuring cell type composition of the cells of the organ.

93. The system of any one of the preceding claims, wherein the assessing in (d) comprises measuring changes in gene expression of cells of the organ using spatial omics.

94. The system of any one of the preceding claims, wherein the assessing in (d) comprises measuring changes in gene expression of cells of the organ using genomic, epigenomic, transcriptomic, proteomic, or metabolomic sequencing, and / or digital pathology.

95. The system of any one of the preceding claims, wherein the organ is a brain and assessing in (d) comprises measuring neurotoxicity or blood-brain-barrier penetration.

96. The system of any one of the preceding claims, wherein the organ is a kidney and assessing in (d) comprises measuring nephrotoxicity.

97. The system of any one of the preceding claims, further comprising administering at least two pharmacological therapies to the organ concurrently with the machine perfusion conditions of (b).

98. The system of claim 97, wherein the at least two pharmacological therapies are administered sequentially to the organ.

99. The system of claim 97, wherein the at least two pharmacological therapies are administered concurrently to the organ.

100. The system of any one of the preceding claims, further comprising administering a pharmacology therapy separately from the machine perfusion conditions.

101. The system of any one of the preceding claims, wherein the perfusion conditions are sufficient to maintain the viability of the organ for at least 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 day, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, or a month.

102. The system of any one of the preceding claims, wherein the perfusion conditions comprise providing a supplemented medium to the organ.

103. The system of any one of the preceding claims, wherein the perfusion conditions comprise providing oxynation to the organ.

104. The system of any one of the preceding claims, wherein the perfusion conditions comprise providing artificial blood to the organ.

105. The system of claim 104, wherein the artificial blood comprises perfluorocarbon-based oxygen carriers (PFCs) or stabilized hemoglobin solutions (HBOCs).

106. The system of any one of the preceding claims, further comprising using an automated computer-implemented system to perform (a), (b), (c), (d), or any combination thereof.

107. The system of any one of the preceding claims, further comprising using an automated computer-implemented system to perform (b) and (c).

108. The system of any one of the preceding claims, further comprising using an automated computer-implemented system to perform (b), (c), and (d).

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