# US 8,771,930 B2 · Ex vivo methods for testing toxicity of substances using donated human organs or tissues > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,930 B2 - Document ID: US8771930B2 - Status: Granted - Kind: B2 - Application number: 11/802,059 - Filed: 2007-05-18 - Published: 2008-11-20 - Granted: 2014-07-08 - Earliest claimed: 2007-05-18 - Assignees: Lifeline Scientific, Inc. - Applicants: Gerald Curtis; John Brassil; David Kravitz - Inventors: Gerald Curtis; John Brassil; David Kravitz - Web page: https://patentgrep.com/patent/US8771930B2 ## CPC - G01N 33/5082: {Supracellular entities, e.g. tissue, organisms} - G01N 33/5014: {for testing toxicity} - G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} - G01N 33/575: for cancer - G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} - G06Q 20/102: {Bill distribution or payments} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G06Q: INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR ## Abstract Properties such as toxicity of substances may be determined by assaying properties, fates and effects of substances in an ex vivo metabolically active human organ or tissue under normothermic perfusion with a fluid containing a test substance. The data can be used as, for example, part of a submission to a government regulatory organization. Preferred methods use perfused endocrine gland organs or tissues to evaluate hormone or other bodily chemical disruption caused by substances and pre-donation diseased or injured organs or tissues. ## Claims ### Claim 1 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions and physiological oxygenation, osmolality, electrolyte balance and pH conditions through at least one metabolically active ex vivo donated human organ or tissue that was injured or diseased before donation, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate under normothermic conditions and physiological oxygenation, osmolality, electrolyte balance and pH conditions that does not contain the substance through the at least one donated human organ or tissue before and/or after passing the at least one donated human organ or tissue with the perfusate comprising the substance, wherein the at least one donated human organ or tissue acts as its own control, the perfusate that does not contain the substance comprises the positive and negative controls, and the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); collecting data derived from the perfused organ or tissue, wherein collecting data derived from the perfused organ or tissue comprises: assaying an effluent from the perfused organ or tissue, and monitoring one or more parameters selected from the group consisting of cell death, apoptosis, genetic mutation, changes in gene expression, biochemical inhibition, reduction in metabolism, induction and oxidative damage, hormone disruption, and bodily chemical disruption; and analyzing the data to assess the toxicity of the substance, wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics; wherein said at least one metabolically active ex vivo donated human organ or tissue is a glandular organ or tissue. ### Claim 2 (depends on 1) The method of claim 1, wherein the data are collected by electrophysiological testing, medical diagnostic imaging, spectroscopic testing, microdialysis, solid state tissue probe testing or a combination thereof. ### Claim 3 (depends on 1) The method of claim 1, wherein the substance is selected from the group consisting of industrial products, building materials and cleaning products. ### Claim 4 (depends on 3) The method of claim 3, wherein the cleaning products are selected from the group consisting of detergents, bleaches, soaps, shampoos and conditioners. ### Claim 5 (depends on 1) The method of claim 1, wherein the substance is selected from the group consisting of tobacco products and cosmetics. ### Claim 6 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions through at least one metabolically active ex vivo donated human organ or tissue that was injured or diseased before donation, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate that does not contain the substance through the donated human organ or tissue before and/or after perfusing the donated human organ or tissue with the perfusate comprising the substance, wherein the donated human organ or tissue acts as its own control, the perfusate that does not contain the substance comprises the positive and negative controls, the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); and but for the presence of the substance, the perfusate comprising the substance is compositionally the same as the perfusate that does not contain the substance; collecting data derived from the perfused organ or tissue, wherein collecting data derived from the perfused organ or tissue comprises: assaying an effluent from the perfused organ or tissue, and monitoring one or more parameters selected from the group consisting of cell death, apoptosis, genetic mutation, changes in gene expression, biochemical inhibition, reduction in metabolism, induction and oxidative damage, hormone disruption, and bodily chemical disruption; and analyzing the data to assess the toxicity of the substance, wherein the at least one metabolically active ex vivo donated human organ or tissue is an intact organ or tissue, and data are used to analyze at least one of the following: hormone level, target cell-receptor mediation, glandular secretion function, hormone function, organ function and a combination thereof; wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics; wherein said at least one metabolically active ex vivo donated human organ or tissue is a glandular organ or tissue. ### Claim 7 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions through at least one metabolically active ex vivo donated human organ or tissue that was injured or diseased before donation, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate that does not contain the substance through the donated human organ or tissue before and/or after perfusing the donated human organ or tissue with the perfusate comprising the substance, wherein the donated human organ or tissue acts as its own control, and the perfusate that does not contain the substance comprises the positive and negative controls, wherein the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); collecting data derived from the perfused organ or tissue, wherein collecting data derived from the perfused organ or tissue comprises assaying an effluent from the perfused organ or tissue, and analyzing the data to assess the toxicity of the substance, wherein the organ or tissue is selected from the group consisting of endocrine glands and exocrine glands, and analyzing the data comprises determining a concentration of the substance at which the secretion ability of said glands decreases, increases or is otherwise altered; wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics. ### Claim 8 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions through at least one metabolically active ex vivo donated human organ or tissue that was injured or diseased before donation, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate that does not contain the substance through the donated human organ or tissue before and/or after perfusing the donated human organ or tissue with the perfusate comprising the substance, wherein the donated human organ or tissue acts as its own control, and the perfusate that does not contain the substance comprises the positive and negative controls, wherein the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); collecting data derived from the perfused organ or tissue, wherein collecting data derived from the perfused organ or tissue comprises assaying an effluent from the perfused organ or tissue; and analyzing the data to assess the toxicity of the substance, wherein the organ or tissue is an organ selected from the group consisting of pancreas, thymus, male reproductive organs, female reproductive organs, thyroid gland and adrenal gland, and analyzing the data comprises determining the concentration of substance at which the rate of production of secretory products or concentration of secretory products of the organ decreases, increases or is otherwise altered; wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics. ### Claim 9 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions through at least one metabolically active ex vivo donated human organ or tissue that was injured or diseased before donation, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate that does not contain the substance through the donated human organ or tissue before and/or after perfusing the donated human organ or tissue with the perfusate comprising the substance, wherein the donated human organ or tissue acts as its own control, and the perfusate that does not contain the substance comprises the positive and negative controls, wherein the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); collecting data derived from the perfused organ, or tissue, wherein collecting data derived from the perfused organ or tissue comprises assaying an effluent from the perfused organ or tissue; analyzing the data to assess the toxicity of the substance; and quantifying a functional state of the donated human organ or tissue that was injured or diseased before donation, wherein the passing comprises perfusing the organ or tissue with a first perfusate that does not contain the substance, followed by perfusing the organ or tissue with a second perfusate that contains the substance; wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics; wherein said at least one metabolically active ex vivo donated human organ or tissue is a glandular organ or tissue. ### Claim 10 (depends on 9) The method of claim 9, wherein the organ or tissue is an entire organ or tissue and has been injured by mechanical injury. ### Claim 11 (depends on 9) The method of claim 9, wherein the organ or tissue is an entire organ or tissue and diseased with a disease that the substance comprised in the perfusate is intended to treat. ### Claim 12 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions through at least one metabolically active ex vivo donated human organ or tissue that was injured or diseased before donation, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate that does not contain the substance through the donated human organ or tissue before and/or after perfusing the donated human organ or tissue with the perfusate comprising the substance, wherein the donated human organ or tissue acts as its own control, and the perfusate that does not contain the substance comprises the positive and negative controls, wherein the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); collecting data derived from the perfused organ or tissue, wherein collecting data derived from the perfused organ or tissue comprises assaying an effluent from the perfused organ or tissue; analyzing the data to assess the toxicity of the substance; quantifying a functional state of the donated human organ or tissue that was injured or diseased before donation with respect to a hormone level, wherein the data are analyzed to assess whether the hormone level is unaltered or is increased, decreased or otherwise altered in response to passing the perfusate comprising the substance under normothermic conditions through said at least one metabolically active ex vivo donated human organ or tissue; wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics; wherein said at least one metabolically active ex vivo donated human organ or tissue is a glandular organ or tissue. ### Claim 13 (depends on 12) The method of claim 12, wherein the substance is selected from the group consisting of industrial products, building materials, tobacco products and cleaning products. ### Claim 14 A method of testing the toxicity of a substance, comprising: passing a perfusate comprising the substance under normothermic conditions through at least one metabolically active ex vivo donated human endocrine gland or endocrine gland tissue under normothermic conditions, wherein perfusate comprising the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); passing a perfusate that does not contain the substance through the human endocrine gland or endocrine gland tissue before and/or after perfusing the human endocrine gland or endocrine gland tissue with the perfusate comprising the substance, wherein the human endocrine gland or endocrine gland tissue acts as its own control, and the perfusate that does not contain the substance comprises the positive and negative controls, wherein the perfusate that does not contain the substance further comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human, serum albumin, N-acetylcysteine, and adenosine monophosphate (AMP); collecting data derived from the perfused gland or gland tissue, wherein collecting data derived from the perfused gland or gland tissue comprises assaying an effluent from the perfused gland or gland tissue; quantifying a functional state of the at least one metabolically active ex vivo donated human endocrine gland or endocrine gland tissue with respect to hormone uptake or production; and analyzing the data to assess effects of the substance on the hormone uptake or production by said gland or gland tissue; wherein the substance is selected from the group consisting of agricultural products, industrial products, building materials, tobacco products, cleaning products and cosmetics. ### Claim 15 (depends on 14) The method of claim 14, wherein the data are collected by electrophysiological testing, medical diagnostic imaging, spectroscopic testing, microdialysis, solid state tissue probe testing or a combination thereof. ### Claim 16 (depends on 14) The method of claim 14, wherein the gland is selected from the group consisting of pancreas, thymus, male reproductive organs, female reproductive organs, thyroid gland and adrenal gland. ### Claim 17 (depends on 14) The method of claim 14, wherein the gland or gland tissue has been injured by prolonged warm ischemia. ### Claim 18 (depends on 14) The method of claim 14, wherein the gland or gland tissue has been injured by prolonged hypothermic storage. ### Claim 19 (depends on 14) The method of claim 14, wherein the gland or gland tissue has been injured by mechanical injury. ### Claim 20 (depends on 14) The method of claim 14, wherein the gland or gland tissue is diseased. ### Claim 21 (depends on 14) The method of claim 14, wherein the data are analyzed to assess whether a hormone level is unaltered or is increased, decreased or otherwise altered. ### Claim 22 (depends on 14) The method of claim 14, wherein the substance is selected from the group consisting of industrial products, building materials and cleaning products. ### Claim 23 (depends on 14) The method of claim 14, wherein the substance is selected from the group consisting of tobacco products and cosmetics.