High-dimensional immune phenotyping and transcriptional analyses reveal robust recovery of viable human immune and epithelial cells from frozen gastrointestinal tissue

Abstract

Simultaneous analyses of peripheral and mucosal immune compartments can yield insight into the pathogenesis of mucosal-associated diseases. Although methods to preserve peripheral immune cells are well established, studies involving mucosal immune cells have been hampered by lack of simple storage techniques. We provide a cryopreservation protocol allowing for storage of gastrointestinal (GI) tissue with preservation of viability and functionality of both immune and epithelial cells. These methods will facilitate translational studies allowing for batch analysis of mucosal tissue to investigate disease pathogenesis, biomarker discovery and treatment responsiveness.

Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

“…Small intestinal biopsies were collected at the time of endoscopy and transported in R10 (RPMI-1640 [Lonza] + 10% FCS [Sigma-Aldrich] + 1% penicillin/streptomycin [Sigma-Aldrich]), before cryopreservation in freezing medium (90% FCS [Sigma-Aldrich], 10% DMSO [Sigma-Aldrich]). This method preserves immune cell viability, surface marker expression, and function ( Konnikova et al., 2018 ). When required, samples were rapidly thawed in a 37°C water bath and washed in 20 mL R10 before tissue dissociation.…”

An intestinal organoid–based platform that recreates susceptibility to T-cell–mediated tissue injury

“…Most biopsies were collected from Crohn's disease patients because of their higher probability of harboring the ATG16L1 T300A risk allele and the availability of small intestinal biopsies. Viable organoids can be generated from frozen tissue, allowing parallel experiments with banked immune cells 60 or, in our case, coculture. T cells were sorted from PBMCs obtained from the same individuals as above or from an independent cohort of 20 healthy donors.…”

The Pediatric Cell Atlas: Defining the Growth Phase of Human Development at Single-Cell Resolution

“…While protocols for single-cell analysis suggest processing either newly acquired or cryopreserved tissues, there have been advances in processing single-cell DNA from formalin-fixed paraffin-embedded tissues to study whole-genome copy number variation in cancer (Martelotto et al, 2017), as well as recovery of transcriptomic data from cryopreserved tissues (Konnikova et al, 2018), both of which open up opportunities for similar analyses utilizing samples from pediatric tissue repositories. The PCA could assist projects by linking them to other well-established biobanks such as those founded by the Children’s Oncology Group (https://www.childrensoncologygroup.org/), the Children’s Brain Tumor Tissue Consortium (https://cbttc.org/), and Alex’s Lemonade Stand (http://www.cccells.org/).…”