RepliGut® Crypt
A self-renewing intestinal epithelium for evaluating long-term drug exposure and epithelium repair capability.
RepliGut® Crypt
The primary subunit of the gut epithelium is the crypt, which at its base contains stem cells that differentiate into multiple cell types as they migrate to the epithelium surface. It’s essential to utilize models that accurately replicate the intestinal crypt environment, as they better simulate the complex interactions between cell types.
RepliGut® Crypt is the first primary stem cell-derived platform that recapitulates cell turnover in the gut (proliferation, differentiation, and migration). Using engineered cell culture inserts, primary intestinal cells establish a 2D self-organized tissue spatially segregated into a stem cell niche alongside proliferative, differentiating, and mature cells of absorptive and secretory lineages. This innovative solution represents a valuable advancement, offering unparalleled relevance to human biology and advancing our understanding of intestinal health.
Key Features of RepliGut® Crypt systems
The standardized plate format with access to both apical and basal surfaces enables wide range of assays to suit your needs.
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Mimicking the in vivo crypt
Unlike a standard Transwell membrane that is highly permeable, RepliGut® Crypt is cultured on an impermeable substrate laser drilled with 91 microholes arranged in a precise hexagonal array. Primary human epithelial stem cells derived from post-mortem colonic crypts are grown on hydrogel-coated inserts until cells reach confluence. Growth factors in the basal media produce a growth factor gradient within the hydrogel scaffold, promoting self-organized tissue development. Cells that reside over the microholes maintain a proliferative status (EdU+) while cells adjacent to the holes differentiate into secretory (MUC2+) and absorptive (ALP+) lineages.
RepliGut® Crypt. (A) Localization of key cell types within the intestinal crypt. (B) Schematic of the engineered cell culture insert and crypt array comprised of 91 crypt units. Each crypt unit is centered around a microhole and contains an EdU+ proliferative zone surrounded by a MUC2+ and ALP+ positive differentiated zone. (C) Schematic of proliferative and differentiated zones.
Applications
Our innovative platform is versatile, making it ideal for a range of applications, including:
Cell proliferation studies
Drug safety assessment
Drug efficacy and lead optimization
Epithelial damage or repair capabilities
Inflammatory disease modeling
Cell proliferation studies
The self-renewing capability of RepliGut® Crypt allows for assessment of pro- or anti-proliferative effects of compounds.
IL-22 induces a reversible shift to cell populations. (A) EdU+ and (B) ALP percent positive area. A 7-day treatment with the cytokine IL-22 significantly reduced ALP+ enterocytes and increased EdU+ cells. These effects reversed after a 7-day washout. Each data point represents one crypt unit. Black line indicates mean. Signficance was determined using a Kruskal-Wallis test. N =3 crypt arrays.
Why choose RepliGut® Crypt?
Relevance to human biology
RepliGut® Crypt closely mimics the architecture of the human intestinal epithelium, offering better human relevance than standard cell lines.
Dynamic cell behavior
The juxtaposition of proliferative and differentiated cells enables the study of dynamic cellular behaviors, such as differentiation, migration, and response to stimuli.
Enhanced cell functionality
Combining stem and differentiated cells better represents the intestinal environment, facilitating the exploration of cellular interactions.
Capabilities Summary
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Apparent permeability (Papp) and efflux ratios
Human jejunum epithelial stem cells are cultured and differentiated on semi-permeable membrane inserts in a 96-well plate to facilitate access to both the apical and basal sides of the cell monolayer. Test compounds are introduced into either compartment to assess their transport in both directions. Accurate mass spectrometry analysis of compound concentrations in each compartment allows for the calculation of permeability (Papp) and efflux ratios. Controls such as atenolol and propranolol, representing low and high permeability respectively, can be included. Additionally, transporter inhibitors can be applied to assess compound interactions with specific transporters. These assays are adaptable to specific research needs.
Typical study design | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Cell Culture Timeline | 10-11 days | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Number of Replicates | Typically 3 wells per treatment | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Incubation Time | Up to 120 min | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Incubation Buffer | HBSS + 10mM HEPES + 10mM Glucose, pH 7.4 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Barrier Integrity Assessment | Automated TEER using EVOM™ Auto (WPI) measured before and after compound incubation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Control Drugs | Atenolol and propranolol | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Analysis Method | Accurate mass spectrometry measurement using LC/MS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Data Readout | Papp (apparent permeability coefficient), Efflux ratio, TEER | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Permeability markers tested in RepliGut® Planar-Jejunum
Absorption Group | Drug | RepliGut® A→B Papp (10-6 cm/s) | Human Fraction Absorbed (Fa) |
|---|---|---|---|
High absorption (Fa ≥ 85%) | Antipyrine | 14.7 ±2.3 | 97% |
Metoprolol | 10.5 ±5.3 | 95% | |
Propranolol | 22.1 ±1.0 | 90% | |
Carbamazepine | 12.8 ±3.2 | 85% | |
Moderate absorption (Fa = 50 – 84%) | Ranitidine | 0.98 ±0.2 | 50% |
Atenolol | 0.97 ±0.9 | 50% | |
Low absorption (Fa < 50%) | Nadolol | 0.47 ±0.29 | 34% |
Lisinopril | 0.17 ±0.14 | 25% |
Absorption Group | Drug | Transporter | Efflux Ratio |
|---|---|---|---|
Efflux Substrates | Antipyrine | P-gp | 12.5 ±5.8 |
Estrone-3-sulphate | BCRP | 30.7 ±14.4 |
