Patent US5726009A successfully refreshed from Google Patents.
US5726009A
Expired / Lapsed
US
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Native-state method and system for determining viability and proliferative capacity of tissues in vitro
Assignee: AMERICAN COMMERCIAL INDUSTRIES Inc A CORP OF DELAWARE
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Filing: 1994-06-29
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Granted: 1998-03-10
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Expires: 2014-06-29
Abstract
Abstract The present invention relates to a method of using an in vitro culture system to measure the cellular proliferative capacity cellular viability of human tissues, particularlytumor tissues. The invention also describes the use of the method to evaluate the effectiveness of an antineoplastic drug in inhibiting tumor cell proliferation of viability.
Inventors
- 👤 Kenneth M. Connors
- 👤 Anna Monosov
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 16:57
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; G01N33/5008; G01N33/5011; G01N2800/00; G01N2800/52; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
193 words
An in vitro method for predicting the effectiveness of treating a preselected tumor tissue of a subject in vivo with an anti-tumor agent, which method comprises: a) histoculturing, in separate containers, first, second, third and fourth portions of a tumor tissue sample; b) exposing said first and second portions of said tumor tissue sample to said agent; c) determining the percentage of viable cells within said first and third portions of said histocultured tumor tissue sample to produce a viable cell index comprising a ratio of viable to non-viable cells in said first and third portions; d) determining the percentage of proliferating cells within said second and fourth portions of said histocultured tumor tissue sample to produce a growth fraction index comprising a ratio of proliferating to non-proliferating cells in said second and fourth portions; and e) wherein a reduced viable call index in said first portion as compared to said third portion combined with a reduced growth fraction index in said second portion as compared to said fourth portion substantially increases the reliability of the in vitro as say as predicting in vivo effectiveness of said antitumor agent against said tumor tissue.
Claim 2
Dependent Claim
193 words
An in vitro method for predicting the effectiveness of treating a preselected tumor tissue of a subject in vivo with an anti-tumor agent, which method comprises: a) histoculturing, in separate containers, first, second, third and fourth portions of a tumor tissue sample; b) exposing said first and second portions of said tumor tissue sample to said agent; c) determining the percentage of viable cells within said first and third portions of said histocultured tumor tissue sample to produce a viable cell index comprising a ratio of viable to non-viable cells in said first and third portions; d) determining the percentage of proliferating cells within said second and fourth portions of said histocultured tumor tissue sample to produce a growth fraction index comprising a ratio of proliferating to non-proliferating cells in said second and fourth portions; and e) wherein a reduced viable call index in said first portion as compared to said third portion combined with a reduced growth fraction index in said second portion as compared to said fourth portion substantially increases the reliability of the in vitro as say as predicting in vivo effectiveness of said antitumor agent against said tumor tissue.
Claim 3
Dependent Claim
34 words
The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a protein synthesis marker.
Claim 4
Dependent Claim
13 words
The method of claim 2 wherein said protein synthesis marker is 35 S-methionine.
Claim 5
Dependent Claim
35 words
The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a viable cell specific dye.
Claim 6
Dependent Claim
51 words
The method of claim 4 wherein said viable cell specific dye is selected from the group consisting of 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM); glycine-N,N'-((3',6'-dihydroxy-3-oxospiro(isobenzofuran-l(3H),9'-(9H)xanthene)-2,7-diyl)bis(methylene))bis(N-carboxymethyl)-acetoxymethyl ester (calcein-AM); 5-carboxyfluorescein diacetate (CFDA); 3,6-acridinediamine, N,N,N',N'-tetramethyl-monohydrochloride (acridine orange); 7-hydroxy-4-methylcoumarin-8-methyleneiminodiacetic acid (calcein blue); 5-oxazolecarboxylic acid 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benxofuranyl)-(acetyloxy)methyl ester (fura-2AM), fluorescein diacetate and 7-hydroxy-4-carboxymethylcoumarin-8-methyleneiminodiacetic acid acetoxymethyl ester (carboxy calcein blue-AM).
Claim 7
Dependent Claim
26 words
The method of claim 2 wherein said process further comprises measuring the number of cells in said histocultured tissue sample that incorporate a dead-cell specific dye.
Claim 8
Dependent Claim
24 words
The method of claim 6 wherein said dead-cell specific dye is selected from the group consisting of propidium iodide, ethidium bromide and ethidium homodimer.
Claim 9
Dependent Claim
34 words
The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that metabolically convert a detectable marker.
Claim 10
Dependent Claim
21 words
The method of claim 8 wherein said detectable marker that is metabolically converted by said viable cells is a tetrazolium salt.
Claim 11
Dependent Claim
14 words
The method of claim 9 wherein said tetrazolium salt is 3- 4,5-dimethylthiazol-2-yl!-2,5-diphenyltetrazolium bromide (MTT).
Claim 12
Dependent Claim
33 words
The method of claim 1 wherein the percentage of proliferating cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a DNA-synthesis marker.
Claim 13
Dependent Claim
27 words
The method of claim 11 wherein said DNA-synthesis marker is selected from the group consisting of 3 H-thymidine, 3 H-deoxyadenosine, 3 H-deoxyguanosine, 3 H-deoxycytidine and 3 H-deoxyuridine.
Claim 14
Dependent Claim
66 words
The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a fluorescent dye indicative of viability and the percentage of proliferating cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate the DNA-synthesis marker 3 H-thymidine.
Claim 15
Dependent Claim
60 words
A kit for in vitro prediction of the effectiveness of in vivo treatment of a tumor with an anti-minor agent, which kit comprises a viable cell-specific marker for measuring cell viability in vitro and a cell proliferation marker for measuring cell proliferation in vitro, said markers each present in an mount sufficient to perform at least one in vitro determination.
Claim 16
Dependent Claim
60 words
A kit for in vitro prediction of the effectiveness of in vivo treatment of a tumor with an anti-minor agent, which kit comprises a viable cell-specific marker for measuring cell viability in vitro and a cell proliferation marker for measuring cell proliferation in vitro, said markers each present in an mount sufficient to perform at least one in vitro determination.
Claim 17
Dependent Claim
26 words
The kit of claim 14 wherein said viable cell-specific marker is the fluorescent dye 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and said cell proliferation marker is 3 H-thymidine.
Claim 18
Dependent Claim
129 words
An in vitro method for predicting the effectiveness of treating a preselected tumor tissue of a subject in vivo with an antitumor agent which method comprises determining the viability of tumor cells in a histocultured sample of said tumor in the presence and absence of said agent; determining the proliferation of tumor cells in a histocultured sample of said tumor tissue in the presence and absence of said agent; wherein a reduction of viability of said cells in the presence as compared to the absence of said agent combined with a reduction in proliferation of said cells in the presence as compared to the absence of said agent substantially increases the reliability of the in vitro as predicting in vivo effectiveness of said antitumor agent against said tumor tissue.
Claim 19
Dependent Claim
129 words
An in vitro method for predicting the effectiveness of treating a preselected tumor tissue of a subject in vivo with an antitumor agent which method comprises determining the viability of tumor cells in a histocultured sample of said tumor in the presence and absence of said agent; determining the proliferation of tumor cells in a histocultured sample of said tumor tissue in the presence and absence of said agent; wherein a reduction of viability of said cells in the presence as compared to the absence of said agent combined with a reduction in proliferation of said cells in the presence as compared to the absence of said agent substantially increases the reliability of the in vitro as predicting in vivo effectiveness of said antitumor agent against said tumor tissue.
References Cited (Backward Citations)
US4060081A
Cited
WO1990011371A1
Cited
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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