Cryopreservation of human colorectal carcinomas prior to xenografting

Abstract

BackgroundMolecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity.MethodsFourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days).ResultsTake rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation.ConclusionsOur procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.

Patient-derived xenograft cryopreservation and reanimation outcomes are dependent on cryoprotectant type

“…18,37 First, when water freezes in the cell suspending solution, solutes become more concentrated in the remaining liquid phase 21,37 that leads to cellular osmotic dehydration of the biological sample and injury/death. 38,39 Second, as cells freeze, intracellular ice crystals can form and directly puncture and damage the plasma membrane 18,19,24,39–41 , In order to prevent such freezing injury various cryoprotectants such as glycerol, ethanediol, propanediol and dimethyl sulfoxide (DMSO) are used for stable freezing and long-term preservation of established cell lines and tissues. 18,20,25,40–42 These solutions are non-toxic and easily soluble and are thought to prevent such injury via several proposed mechanisms.…”

“…22–27 Cryopreservation of primary patient tumor and subsequent PDX has been investigated previously, but the efficacy of reanimation has typically been low, or not reported. 23,24,28,29 Cryopreservation of composite tissues, such as PDX tumors, is more complex than for homogenous cell lines as a three-dimensional architecture needs to be maintained during both freezing and thawing. As such, tissues are composed of a variety of cell types with varying degree of vulnerability to the cryopreservation process.…”

Colon cancers carrying BRAF V600E and β-catenin T41A activating mutations are resistant to numerous common anticancer drugs

“…There have been several publications on the establishment of PDCCX mouse models (21)(22)(23)(24). However, these studies did not systemically characterize the genetic alterations of the established PDCCX lines, thereby limiting the applicability of the lines, and also lacked the clarity required for defining the targeted patient population for the therapeutics developed using those lines.…”

Single nucleotide polymorphism array analysis of microsatellite-stable, diploid/near-diploid colorectal carcinomas without the CpG island methylator phenotype

“…Two dinucleotide markers located at 3p14.2 (D3S1234, D3S1300) were added in the present study. For the control cases, the majority of microsatellite analyses were conducted using DNA from the cell lines; only in a minority of cases was DNA from corresponding xenografts used (details of the xenografting procedures have been published previously) (9). Microsatellite instability was tested with the Bethesda markers.…”