Methods and reagents for maintaining the viability of cancer cells in surgically removed tissue
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Publication Number
US-9949474-B2
Publication Date
2018-04-24
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 disclosure relates to a non-formalin cell viability reagent for a tissue sample and a method of producing the cell viability reagent. The reagent is configured to preserve the tissue sample for gene expression analysis, including maintenance of gene expression while cells remain viable. The approach uses sequentially mixed solutes including a chaotrope, a chelator, a metabolic modulator, kosmotropes, and an apoptosis substrate.
In particular, the formulation includes alpha,alpha-trehalose as a kosmotrope and sodium thiocyanate as a chaotrope. A chelator selected from EDTA, EGTA, BAPTA, imidazole, iminodiacetate, and BABIM is included at a defined concentration range, and the metabolic modulator comprises DMSO. The formulation further includes leptin as an apoptosis substrate at a defined concentration range, and the reagent is presented as enabling gene expression analysis after preservation.
Comparative results described in the disclosure indicate improved cell viability relative to formalin/EDTA/thimerosal, improved RNA integrity with RNA integrity number remaining above 6 up to longer times in preferred formulations, and maintenance of detectable mRNA copies for gene markers over time. Microarray analysis is reported to show gene expression patterns comparable to liquid nitrogen storage, and tissue slice and slice culture evaluations are described as related concepts supporting tissue use.
Claims Coverage
The independent claims include three inventive categories: one method of producing a cell viability reagent for a tissue sample and two reagent formulations for gene expression analysis. Each independent claim centers on a specific multi-component reagent set comprising alpha,alpha-trehalose, a defined chelator selection, sodium thiocyanate, leptin, and DMSO to preserve tissue for gene expression analysis.
Method producing a cell viability reagent for gene expression analysis
providing a kosmotrope as alpha,alpha-trehalose; providing a chelator selected from EDTA, EGTA, BAPTA, imidazole, iminodiacetate, and BABIM with the chelator concentration between 0.005 M and 2.0 M; providing a chaotrope as sodium thiocyanate; providing an apoptosis substrate as leptin with leptin concentration between 0.001 M and 0.5 M; providing a metabolic modulator comprising DMSO; and mixing the kosmotrope, the chelator, the chaotrope, the apoptosis substrate, and the metabolic modulator to produce the cell viability reagent capable of preserving the tissue sample for gene expression analysis.
Reagent formulation enabling gene expression analysis with specified component set
a reagent comprising a kosmotrope, a chelator, a chaotrope, an apoptosis substrate, and a metabolic modulator comprising DMSO, wherein the kosmotrope is alpha,alpha-trehalose; the chelator is selected from EDTA, EGTA, BAPTA, imidazole, iminodiacetate, and BABIM with the chelator concentration between 0.005 M and 2.0 M; the chaotrope is sodium thiocyanate; and the apoptosis substrate is leptin with leptin concentration between 0.001 M and 0.5 M, configured to allow gene expression analysis of the tissue sample.
Reagent formulation where kosmotropes comprise alpha,alpha-trehalose and glycerol
a reagent comprising a kosmotrope, a chelator, a chaotrope, an apoptosis substrate, and a metabolic modulator comprising DMSO, wherein the kosmotrope comprises alpha,alpha-trehalose and glycerol; the chelator is selected from EDTA, EGTA, BAPTA, imidazole, iminodiacetate, and BABIM with the chelator concentration between 0.005 M and 2.0 M; the chaotrope is sodium thiocyanate; and the apoptosis substrate is leptin, configured to allow gene expression analysis of the tissue sample.
Across the independent claims, the inventive core is a preservation reagent for tissue sample gene expression analysis built from alpha,alpha-trehalose, a chelator selected from a defined group at a specified concentration range, sodium thiocyanate as chaotrope, leptin as apoptosis substrate at a specified concentration range, and DMSO as metabolic modulator, with mixing producing the preservative reagent.
Stated Advantages
Improved cell viability compared to formalin/EDTA/thimerosal as reported by Trypan Blue/MTT.
Improved RNA integrity, with RNA integrity number remaining above 6 up to longer times in preferred TAG-1 formulations.
Maintained detectable mRNA copies over time as reported for gene markers by RT-PCR.
Gene expression patterns in the formulation reported as comparable to liquid nitrogen storage based on microarray analysis.
Documented Applications
Preserving surgically excised cancer tissue for gene expression analysis, including genomic analyses.
Tissue slice and slice culture evaluations as described related concepts for tissue use.
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