Methods and reagents for maintaining the viability of cancer cells in surgically removed tissue
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Publication Number
US-9565852-B2
Publication Date
2017-02-14
Expiration Date
Abstract
A composition and method for generating reagents and the composition of these reagents for the stabilization and preservation of viability of cancer tissue which has been surgically excised and the suspension and/or termination of apoptosis (cell death) by significant modulation of cell metabolism by low molar concentrations of synergistic chemistries and hormonal growth enhancers while maintaining normal gene expression patterns of the surgically excised tissue.
Core Innovation
The invention relates to preserving a biological sample by contacting a solid tumor sample with a cell preservation reagent. The reagent comprises a chelator and leptin, and preservation is provided for at least 72 hours after contacting.
The disclosed reagent further includes a kosmotrope, where the kosmotrope comprises α,α-trehalose, together with a chelator and leptin for preserving surgically excised cancer tissue samples. Preservation is described as maintaining gene expression-related characteristics after continuous contacting for at least 72 hours or at least 96 hours.
The disclosure also characterizes the chelator as selected from EDTA, EGTA, and BAPTA, and associates the preservation reagent with additional formulation components, including buffer options within a specified pH range and a metabolic penetrant and apoptosis substrate that include leptin. The documented comparisons describe improved cancer cell viability and preserved RNA integrity and gene expression over time after preservation.
Claims Coverage
The independent claims cover methods of preserving biological samples, particularly solid tumor and surgically excised cancer tissue samples, by contacting the sample with a cell preservation reagent. Across the independent claims, the inventive features include chelator plus leptin, defined constraints for leptin concentration, defined chelator selection, and, in a separate independent claim, inclusion of a kosmotrope comprising α,α-trehalose.
Chelator plus leptin for solid tumor preservation
A method of preserving a biological sample by contacting the biological sample with a cell preservation reagent comprising a chelator and leptin, wherein the biological sample comprises a solid tumor, and wherein the biological sample is preserved for at least 72 hours after contacting.
Chelator, α,α-trehalose kosmotrope, and leptin for surgically excised cancer tissue
A method of preserving a biological sample by contacting the biological sample with a cell preservation reagent comprising a chelator, a kosmotrope comprising α,α-trehalose, and leptin, wherein the biological sample comprises a surgically excised cancer tissue sample.
Leptin concentration constrained chelator plus leptin for solid tumor preservation
A method of preserving a biological sample by contacting the biological sample with a cell preservation reagent comprising a chelator and leptin, wherein the biological sample comprises a solid tumor, and wherein a concentration of the leptin is between about 0.001 M to about 2 M.
Chelator selection and defined leptin concentration for solid tumor preservation
A method of preserving a biological sample by contacting the biological sample with a cell preservation reagent comprising a chelator and leptin, wherein the biological sample comprises a solid tumor, wherein the biological sample is preserved for at least 72 hours after contacting, wherein a concentration of the leptin is between about 0.001 M to about 2 M, and wherein the chelator is selected from the group consisting of EDTA, EGTA, and BAPTA.
Overall, the independent claim set is directed to preserving solid tumor or surgically excised cancer tissue by contacting with a cell preservation reagent centered on chelator plus leptin, with additional coverage for inclusion of an α,α-trehalose kosmotrope and for constrained leptin concentration and chelator selection (EDTA/EGTA/BAPTA).
Stated Advantages
Improved cancer cell viability after preservation compared with controls.
Preserved RNA integrity and gene expression over time after preservation.
Retention of RNA Integrity Number at long aging times.
Improved preservation of gene expression measured by microarray correlation across timepoints.
Documented Applications
Ex vivo handling/preservation of surgically excised cancer tissue samples (solid tumors) using a cell preservation reagent.
Gene expression analysis of preserved biological samples, including RNA/protein measurement as described in the document.
Comparative preservation performance assessment over time using assays and analyses including RNA Integrity Number retention, mRNA detection (RT-PCR), and microarray correlation across timepoints, with comparisons to formalin and flash-frozen (liquid nitrogen) controls.
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