A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function

Abstract

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors.

Intestinal Epithelial Cell Regulation of Adaptive Immune Dysfunction in Human Type 1 Diabetes

“…Thus, to determine if the phenomenon observed at the whole tissue level could be a result of altered IEC-innate immune function, primary IEC cultures were established from the crypts of duodenal tissues using our previously published method (15) (Table S1 in Supplementary Material). Here, the soluble mediator gene expression profile of resting T1D-derived IEC cultures was similar to that observed in the whole tissue, whereby 5- and 3.6-fold higher mRNA levels of BD-2 and IL-17C, respectively, were observed in T1D-derived cultures, when compared to non-TID derived cultures (Figures 4A,B), without similar upregulation in immunoregulatory mediators such as TSLP or IL-10 (Figures 4A–C).…”

“…For each case, 8.0 g of duodenal tissue was processed as follows: (1) 0.3 g of tissue was preserved for (1a) protein extraction and (1b) RNA extraction to evaluate the soluble mediator profile of the duodenum by ELISA and real-time PCR, respectively. (2) The remaining tissue was processed for (2a) intestinal immune cell isolation and (2b) intestinal crypt isolation for characterization of resident immune cell population by flow cytometry and establishment of primary IEC cultures, respectively (15). (3) Following the establishment of confluent primary IEC cultures (3a) culture supernatants and (3b) cellular mRNA were collected to evaluate the soluble mediator profile of the primary cultures by ELISA and real-time PCR, respectively.…”

“…Crypts and resident immune cells were liberated using a protocol adapted from Booth and O’Shea (15–17). Human duodenal tissues were prepared by removing the longitudinal muscle layer and washing with ice-cold Mg 2+ - and Ca 2+ -free Hank’s Balanced Salt Solution (HBSS) (Mediatech, Manassas, VA, USA) containing 100 U penicillin, 100 μg/mL streptomycin (Mediatech), 25 μg/mL gentamycin (MP Biomedicals, Solon, OH, USA), and 0.5 mM dithiothreitol (DTT) (Thermo Fisher Scientific).…”

“…Intestinal epithelial cell cultures were established from isolated crypts as previously described (15, 17) and maintained in 24-well collagen-coated culture dishes (Greiner Bio-One, Monroe, NC, USA) in 1 mL complete IEC (cIEC) media [DMEM, 5 g/L sodium pyruvate (Mediatech), 2.5% v/v FBS, 0.25 U/mL insulin (Sigma-Aldrich), 100 U penicillin, 100 µg/mL streptomycin, 25 µg/mL gentamicin, 5 µg/mL transferrin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Sigma-Aldrich)]. Cultures were left unstimulated for 24 h after which supernatants were collected and stored at −80°C.…”

“…This is in part due to the inaccessibility of sufficient and appropriate GI tissues from individuals with and without T1D. In collaboration with the Network for Pancreatic Organ Donors (nPOD), we have established methods to not only isolate and characterize immune cell populations within human duodenal tissues but to also establish primary IEC cultures from the same tissues (15). Thus, in order expand on the existing knowledge associated with the GI-immune microenvironment observed in T1D, we evaluated the soluble mediator milieu as well as the frequency and phenotype of ILC, T helper cells (CD4+), and cytotoxic T-cells (CD8+) within duodenal tissues in a cohort of organ donors with and without T1D.…”