Comparison of Surgical and Cadaveric Intestine as a Source of Crypt Culture in Humans

Human small intestinal crypts are the source of intestinal stem cells (ISCs) that are capable of undergoing self-renewal and differentiation to an epithelial layer. The development of methods to expand the ISCs has provided opportunities to model human intestinal epithelial disorders. Human crypt samples are usually obtained from either endoscopic or discarded surgical samples, and are thereby exposed to warm ischemia, which may impair their in vitro growth as three-dimensional culture as spheroids or enteroids. In this study we compared duodenal samples obtained from discarded surgical samples to those isolated from whole-body preserved cadaveric donors to generate in vitro cultures. We also examined the effect of storage solution (phosphate-buffered saline or University of Wisconsin [UW] solution) as well as multiple storage times on crypt isolation and growth in culture. We found that intestinal crypts were successfully isolated from cadaveric tissue stored for up to 144 h post-procurement and also were able to generate enteroids and spheroids in certain media conditions. Surgical samples stored in UW after procurement were sufficiently viable up to 24 h and also allowed the generation of enteroids and spheroids. We conclude that surgical samples stored for up to 24 h post-procurement in UW solution allowed for delayed crypt isolation and viable in vitro cultures. Furthermore, in situ, hypothermic preservation in cadaveric duodenal samples permitted crypt/ISC isolation, and successful culture of spheroids and enteroids from tissues held for up to 6 days post-procurement.

Molecular Analysis of Single Tumor Glands Using the Crypt Isolation Method in Endometrial Carcinomas

ObjectiveEndometrial adenocarcinomas are characterized by the presence of many single tumor glands in which multiple genetic changes have accumulated. To elucidate the differences in molecular abnormalities among single tumor glands, individual tumor glands were analyzed and microsatellite alterations (loss of heterozygosity (LOH) and microsatellite instability [MSI]) were examined using the crypt isolation method in glands from each tumor from patients with endometrial carcinoma.MethodsTwenty-five patients with endometrial adenocarcinoma who underwent surgery were included in this study. We obtained cancerous individual isolated tumor glands from each patient using the crypt isolation method. For LOH and MSI analyses, we used 15 microsatellite markers (3p, 5q, 10q, 13q, 17p, 18q, BAT25, and BAT26) and the promoter regions of 6 genes (transforming growth factor beta receptor II, BAX, insulin-like growth factor II receptor, E2F4, MutS homolog 3, and MSH6).ResultsLoss of heterozygosity was detected in 8 (32%) of 25 patients, and MSI was detected in 9 (36%) of 25 patients. Some MSI-positive carcinomas had LOH in single tumor gland samples, and the coexistence of LOH and MSI was confirmed. In 16 (64%) of 25 cases, intratumoral genetic heterogeneity among single tumor gland samples was detected.ConclusionsBy analyzing multiple single tumor glands within the same tumor, we found that endometrial adenocarcinoma was composed of various tumor glands with different molecular abnormalities, even in a limited region within the same tumor.

A Unique Method for Mutation Analysis of Tumor Suppressor Genes in Colorectal Carcinomas Using a Crypt Isolation Technique

Background.—Contamination of nontumor tissue makes genetic analysis difficult. For this reason, it is important to obtain pure tumor tissue to ensure accurate genetic analysis. Objective.—To accurately assess the incidence of mutation of tumor suppressor genes (p53: exon 5–8; APC: mutated cluster region; NF-2 gene: all exons) in 45 colorectal carcinomas. Methods.—We developed an application of the polymerase chain reaction–single-strand conformation polymorphism and DNA sequence by coupling them with crypt isolation. Results.—Mutations of p53 and APC genes were found in 24 and 22 of 45 colorectal carcinomas, respectively. No mutation of the NF-2 gene was observed in this cancer. Single-strand conformation polymorphism using a crypt isolation technique showed a clear migrating band and no false-positive data. Conclusions.—The crypt isolation technique is a useful method for accurately analyzing genetic alterations. Furthermore, our proposed method confirmed the morphological findings obtained before the genetic analysis.