If the acidic collagen solution has a protein concentration of 4 mg/mL (in 0.02 N-acetic acid), the components necessary for preparation of 75 mL neutralization buffer are the following:?10 PBS (Corning 46-013-CM), 10 mL; HEPES (1 N, ThermoFisher 15630080, pH?= 7.4), 2 mL; sodium bicarbonate (7.5% w/v, Corning 25-035-CI, pH?= 8), 6 mL; sodium hydroxide (1 N), 575 L; and deionized water, 56.4?mL. The buffer was sterile filtered and stored at room temperature until use. the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. Conclusions This study demonstrates that a self-renewing 2-D murine and human Umeclidinium bromide monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies. organoid ISCs Umeclidinium bromide exhibit their defining properties by self-renewing and giving rise to progenitors that differentiate into absorptive colonocytes (water and electrolyte uptake), goblet cells (mucus production), enteroendocrine cells (hormones), and Paneth cells (antimicrobial and stem cell niche functions).5 By virtue of their non-transformed condition, 3-D organoids represent a physiologically relevant model enabling novel assays and pharmaceutical and dietary compound screens that are not currently possible with colon cancer cell lines such as Caco-2.3, 11, 12 Although organoid culture technology has had a major positive impact on the in?vitro study of primary gut epithelium, the 3-D geometry of organoids prevents access to the apical aspect of the epithelium, producing a number of challenges to physiologically relevant studies. The apical surface of the organoid is analogous to the lumen of the gut where digested contents and microbial communities interact with the epithelium. The spheroidal architecture of the organoids prevents access of exogenous compounds to the luminal epithelial surface, limiting studies focused Umeclidinium bromide on apical transporters, receptors, metabolic enzymes, and microbiota.13 Matrigel embedded organoids exist in multiple planes, making collection of experimental readout by using conventional microscopy exceptionally challenging.14, 15 Unfolding the spherical organoid into a two-dimensional (2-D) RPS6KA5 planar tissue construct is a solution that addresses these major challenges and has the potential to further transform in?vitro study of the gut epithelium. We have previously demonstrated that primary intestinal epithelial cells can be cultured on polydimethylsiloxane (PDMS) and other artificial surfaces in the absence of a hydrogel.4 Although they are supplied with the requisite soluble growth factors for growth within Matrigel, culture of primary epithelium on non-hydrogel surfaces produced a short-lived, non-proliferative monolayer of cells. Dissociated 3-D small intestinal and colonic organoids have been cultured on a porous membrane (coated with 0.1% gelatin or 10 g/cm2 collagen) to form a monolayer, but these monolayers were not self-renewing, suggesting that stem cells were lost from the monolayers over time and a self-renewing ISC compartment was not supported.16, 17 The failure of existing 2-D culture methods to produce long-term monolayers suggests that a biochemical environment composed of media and soluble growth factors alone is not adequate to sustain a self-renewing monolayer containing both stem and differentiated cells. To overcome the limitations in monolayer culture duration, we sought to identify parameters that would support self-sustaining monolayers. Materials and Methods Isolation of Crypts From Mouse Colon and Human Rectal Biopsies Male mice were used at age 6C10 weeks. All experiments were performed in Umeclidinium bromide compliance with the relevant laws and institutional guidelines at the University of North Carolina (UNC). All experiments and animal usage were approved by the Institutional Animal Care and Use Committee (IACUC) at UNC. Mice were humanely killed by lethal dose of isoflurane, followed by cervical dislocation under the approved UNC IACUC-approved protocol #13-200. A cytomegalovirus enhancer plus chicken actin promoter (CAG)-DsRed mouse model in which all cells expressed the DsRed fluorescent protein was used to monitor the proliferation of colonic epithelial cells by fluorescence microscopy. CAG-DsRed heterozygous mice were bred on a CD-1 background, and wild-type mice were bred on a C57BL/6 background. Wild-type mice were used for fluorescence-based assays and compound screens. An Lgr5EGFPCreERT2xR26 confetti mouse was used for lineage tracing experiments on the 2-D monolayer. The confetti mouse was injected with 5 mg tamoxifen at 48 hours before death Umeclidinium bromide and isolation of crypts from the large intestine.18 Human rectal biopsies were obtained from UNC Hospitals Meadowmont Endoscopy Center with consent of the patient (under the approved UNC Institutional Review Board #14-2013). Isolation buffer was composed of 5.6 mmol/L Na2HPO4 (Sigma S7907; Sigma-Aldrich, St Louis, MO), 8.0 mmol/L.
