RESUMO
Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Dano ao DNA , Exodesoxirribonucleases/metabolismo , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , Senescência Celular , Colágeno/metabolismo , Progressão da Doença , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Membrana Nuclear/ultraestrutura , Proteólise , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (µMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the µMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.
Assuntos
Separação Imunomagnética/métodos , Leite/química , Salmonella/isolamento & purificação , Animais , Escherichia coli/isolamento & purificação , Imunoglobulina G/química , Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microesferas , Salmonella/citologia , Salmonella/imunologiaRESUMO
A new sample treatment approach for sensitive determination of three amyloid-ß peptides (Aß 1-42, Aß 1-40 and Aß 1-38) with capillary electrophoresis coupled with laser induced fluorescent detection is reported herein. These Aß peptides are considered an important family of biomarkers in the cerebrospinal fluid (CSF) for early diagnosis of Alzheimer's disease (AD). Due to their extremely low abundance in CSF (down to sub nM ranges), batch-wise preconcentration via magneto-immunocapture with enrichment factors up to 100 was implemented. The Aß peptides were first captured onto magnetic micro-beads. Then, on-beads fluorescent labeling of the captured Aß peptides were carried out to avoid the unwanted presence of extra fluorescent dye in the eluent as in the case of in-solution labeling. Finally thermal elution was performed and eluted labeled peptides were analyzed off line with CE-LIF. The Aß-capturing efficiencies of different commercially available antibodies grafted onto magnetic beads were tested. Aß peptides in CSF samples collected from AD's patients and healthy persons (used as controls) were measured and evaluated. As a proof of concept, the developed strategy was adapted into a miniaturized fluidized bed configuration that has the potential for coupling with a microchip separation system.
Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Técnicas Analíticas Microfluídicas/métodos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Espectrometria de Fluorescência , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/isolamento & purificação , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Eletroforese Capilar , Corantes Fluorescentes/química , Humanos , Lasers , Magnetismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Espectrometria de Fluorescência/instrumentaçãoRESUMO
In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line.
Assuntos
Separação Imunomagnética/métodos , Imãs , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Molécula de Adesão da Célula Epitelial , Humanos , Separação Imunomagnética/instrumentação , Microesferas , Ácidos Polimetacrílicos/químicaRESUMO
In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.
Assuntos
Apoptose/efeitos dos fármacos , Axônios/metabolismo , Caspases/metabolismo , NAD/farmacologia , Sirtuína 3/metabolismo , Animais , Axônios/efeitos dos fármacos , Camundongos , Microfluídica , Resveratrol , Estilbenos/farmacologiaRESUMO
In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.
Assuntos
DNA/química , Rad51 Recombinase/química , Trifosfato de Adenosina/metabolismo , Fenômenos Biomecânicos , DNA/metabolismo , DNA/ultraestrutura , Humanos , Ligação Proteica , Rad51 Recombinase/metabolismoRESUMO
The metastatic cascade includes a blood circulation step for cells detached from the primary tumor. This stage involves significant shear stress as well as large and fast deformation as the cells circulate through the microvasculature. These mechanical stimuli are well reproduced in microfluidic devices. However, the recovery dynamics after deformation is also pivotal to understand how a cell can pass through the multiple capillary constrictions encountered during a single hemodynamic cycle. The microfluidic system developed in this work allows single cell recovery to be studied under flow-free conditions following pressure-actuated cell deformation inside constricted microchannels. We used three breast cancer cell lines - namely MCF-7, SK-BR3 and MDA-MB231 - as cellular models representative of different cancer phenotypes. Changing the size of the constriction allows exploration of moderate to strong deformation regimes, the latter being associated with the formation of plasma membrane blebs. In the regime of moderate deformation, all cell types display a fast elastic recovery behavior followed by a slower viscoelastic regime, well described by a double exponential decay. Among the three cell types, cells of the mesenchymal phenotype, i.e. the MDA-MB231 cells, are softer and the most fluid-like, in agreement with previous studies. Our main finding here is that the fast elastic recovery regime revealed by our novel microfluidic system is under the control of cell contractility ensured by the integrity of the cell cortex. Our results suggest that the cell cortex plays a major role in the transit of circulating tumor cells by allowing their fast morphological recovery after deformation in blood capillaries.
Assuntos
Técnicas Analíticas Microfluídicas , Humanos , Linhagem Celular Tumoral , Técnicas Analíticas Microfluídicas/instrumentação , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Células MCF-7RESUMO
In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).
Assuntos
Ácidos Nucleicos Livres , Dispositivos Lab-On-A-Chip , Humanos , Ácidos Nucleicos Livres/isolamento & purificação , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Reação em Cadeia da Polimerase/métodos , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Magnetismo/métodos , Neoplasias/sangue , Neoplasias/genética , Neoplasias/diagnósticoRESUMO
At first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field.
Assuntos
Separação Celular/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/patologia , Antígenos de Superfície/análise , Células Sanguíneas/citologia , Adesão Celular , Movimento Celular , Separação Celular/instrumentação , Centrifugação , Eletroforese , Células Endoteliais/citologia , Filtração , Fluorescência , Humanos , Magnetismo , Técnicas Analíticas Microfluídicas , Microfluídica/instrumentaçãoRESUMO
We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.
Assuntos
Separação Celular/métodos , Imageamento Tridimensional/métodos , Magnetismo , Microfluídica/métodos , Modelos Teóricos , Algoritmos , Linhagem Celular Tumoral , Separação Celular/instrumentação , Citometria de Fluxo , Humanos , Imunofenotipagem , Células Jurkat , Microfluídica/instrumentação , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
3D in vitro biological systems are progressively replacing 2D systems to increase the physiological relevance of cellular studies. Microfluidics-based approaches can be powerful tools towards such biomimetic systems, but often require high-end complicated and expensive processes and equipment for microfabrication. Herein, a drug screening platform is proposed, minimizing technicality and manufacturing steps. It provides an alternate way of spheroid generation in droplets in tubes. Droplet microfluidics then elicit multiple droplets merging events at programmable times, to submit sequentially the spheroids to chemotherapy and to reagents for cytotoxicity screening. After a comprehensive study of tumorogenesis within the droplets, the system is validated for drug screening (IC50) with chemotherapies in cancer cell lines as well as cells from a patient-derived-xenografts (PDX). As compared to microtiter plates methods, our system reduces the initial number of cells up to 10 times and opens new avenues towards primary tumors drug screening approaches.
Assuntos
Microfluídica , Neoplasias , Humanos , Microfluídica/métodos , Avaliação Pré-Clínica de Medicamentos , Detecção Precoce de Câncer , Esferoides Celulares , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológicoRESUMO
Correction for 'Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions' by Marine Verhulsel et al., Lab Chip, 2021, 21, 365-377, https://doi.org/10.1039/d0lc00672f.
RESUMO
The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5 degrees . Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 A per base pair) and untwisted by 18.2 degrees per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65 degrees +/- 5 degrees. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.
Assuntos
DNA/metabolismo , Nucleoproteínas/metabolismo , Polímeros/metabolismo , Rad51 Recombinase/metabolismo , DNA/química , Humanos , Magnetismo , Conformação de Ácido Nucleico , Nucleoproteínas/química , Polímeros/química , Rad51 Recombinase/químicaRESUMO
Microfluidics has provided clinicians with new technologies to detect and analyze circulating tumor biomarkers in order to further improve their understanding of disease mechanism, as well as to improve patient management. Among these different biomarkers, circulating tumor cells have proven to be of high interest for different types of cancer and in particular for breast cancer. Here we focus our attention on a breast cancer subtype referred as HER2-positive breast cancer, this cancer being associated with an amplification of HER2 protein at the plasma membrane of cancer cells. Combined with therapies targeting the HER2 protein, HER2-HER3 dimerization blockade further improves a patient's outcome. In this work, we propose a new approach to CTC characterization by on-chip integrating proximity ligation assay, so that we can quantify the HER2-HER3 dimerization event at the level of single CTC. To achieve this, we developed a microfluidic approach combining both CTC capture, identification and HER2-HER3 status quantification by Proximity Ligation Assay (PLA). We first optimized and demonstrated the potential of the on-chip quantification of HER2-HER3 dimerization using cancer cell lines with various levels of HER2 overexpression and validated its clinical potential with a patient's sample treated or not with HER2-targeted therapy.
RESUMO
Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Fenômenos Mecânicos , Fenômenos Biomecânicos , Cromatina/química , Montagem e Desmontagem da Cromatina , Histonas/química , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Conformação Proteica , RotaçãoRESUMO
The detection of unknown mutations remains a serious challenge and, despite the expected benefits for the patient's health, a large number of genes are not screened on a routine basis. We present the diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis(®) , Fluigent, Paris, France), a novel method based on heteroduplex analysis by capillary electrophoresis using innovative matrices. BRCA1 and BRCA2 were screened for point mutations and large rearrangements in 1,525 unrelated patients (372 for the validation step and 1,153 in routine diagnosis) using a single analytical condition. Seven working days were needed for complete BRCA1/2 screening in 30 patients by one technician (excluding DNA extraction and sequencing). A total of 137 mutations were found, including a BRCA2 duplication of exons 19 and 20, previously missed by Comprehensive BRACAnalysis(®) . The mutation detection rate was 11.9%, which is consistent with patient inclusions. This study therefore suggests that EMMA represents a valuable short-term and midterm option for many diagnostic laboratories looking for an easy, reliable, and affordable strategy, enabling fast and sensitive analysis for a large number of genes.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Análise Mutacional de DNA/métodos , Genes BRCA1 , Genes BRCA2 , Testes Genéticos/métodos , Mutação Puntual , Proteína BRCA1/análise , Proteína BRCA2/análise , Neoplasias da Mama/genética , Aberrações Cromossômicas , Análise Custo-Benefício , Análise Mutacional de DNA/economia , DNA Recombinante , Eletroforese Capilar , Feminino , Mutação da Fase de Leitura , Ensaios de Triagem em Larga Escala/métodos , Humanos , Mutação de Sentido Incorreto , Neoplasias Ovarianas/genéticaRESUMO
Magnetic tweezers were used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin three-dimensional architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, corresponding to different crossing statuses of the entry/exit DNAs (positive, null or negative, respectively). Torsional strain displaces that equilibrium, leading to an extensive reorganization of the fiber's architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin and may provide the basis to better understand the dynamic binding of chromatin-associated proteins.Note: In the supplementary information initially published online to accompany this article, Supplementary Figure 2 was mistakenly replaced by Supplementary Equation 2. The error has been corrected online.
Assuntos
Cromatina/química , Cromatina/metabolismo , Materiais Biomiméticos/metabolismo , Cromatina/efeitos dos fármacos , DNA/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Cloreto de Sódio/farmacologiaRESUMO
We present a purely hydrodynamic method for the high-throughput encapsulation of single cells into picoliter droplets, and spontaneous self-sorting of these droplets. Encapsulation uses a cell-triggered Rayleigh-Plateau instability in a flow-focusing geometry, and self-sorting puts to work two extra hydrodynamic mechanisms: lateral drift of deformable objects in a shear flow, and sterically driven dispersion in a compressional flow. Encapsulation and sorting are achieved on-flight in continuous flow at a rate up to 160 cells per second. The whole process is robust and cost-effective, involving no optical or electrical discrimination, active sorting, flow switching, or moving parts. Successful encapsulation and sorting of 70-80% of the injected cell population into drops containing one and only one cell, with <1% contamination by empty droplets, is demonstrated. The system is also applied to the direct encapsulation and sorting of cancerous lymphocytes from a whole blood mixture, yielding individually encapsulated cancer cells with a >10,000-fold enrichment as compared with the initial mix. The method can be implemented in simple "soft lithography" chips, allowing for easy downstream coupling with microfluidic cell biology or molecular biology protocols.
Assuntos
Separação Celular/métodos , Microfluídica/métodos , Células Sanguíneas/citologia , Desenho de Equipamento , Humanos , Linfócitos/patologia , Ciência de Laboratório Médico/instrumentação , Ciência de Laboratório Médico/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Neoplasias/patologia , ÁguaRESUMO
Organoids are widely used as a model system to study gut pathophysiology; however, they fail to fully reproduce the complex, multi-component structure of the intestinal wall. We present here a new gut on chip model that allows the co-culture of primary epithelial and stromal cells. The device has the topography and dimensions of the mouse gut and is based on a 3D collagen I scaffold. The scaffold is coated with a thin layer of laminin to mimic the basement membrane. To maintain the scaffold structure while preserving its cytocompatibility, the collagen scaffold was rigidified by threose-based post-polymerization treatment. This treatment being cytocompatible enabled the incorporation of primary intestinal fibroblasts inside the scaffold, reproducing the gut stromal compartment. We observed that mouse organoids, when deposited into crypts, opened up and epithelialized the scaffold, generating a polarized epithelial monolayer. Proper segregation of dividing and differentiated cells along the crypt-villus axis was achieved under these conditions. Finally, we show that the application of fluid shear stress allows the long-term culture of this intestinal epithelium. Our device represents a new biomimetic tool that captures key features of the gut complexity and could be used to study gut pathophysiology.