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At least 19 recordsLinked to original sources
PubMed2026

Single-cell transcriptomics reveals that air-liquid interface culture promotes goblet cell differentiation and inhibits glycolysis in organoid cell monolayers.

Faithfully recapitulating the cellular heterogeneity of the intestinal epithelium is essential when using organoid models. Air-liquid interface (ALI) culture has been shown to promote secretory cell differentiation, but its impact on gene expression in each epithelial cell type remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize the cellular heterogeneity of rabbit cecum-derived organoid monolayers grown under immerged or ALI conditions. We then compared these organoid cell type-specific gene expression profiles to a scRNA-seq atlas of the rabbit cecal epithelium in vivo. We selected the rabbit model notably because, unlike mice, it possesses BEST4+ epithelial cells, a newly discovered subset of mature absorptive cells. Our analysis revealed a high degree of transcriptomic similarity between in vivo and organoid-derived stem and transit-amplifying cells. ALI culture markedly enhanced the differentiation of the secretory lineage, especially goblet cells, whose transcriptome closely resembled that of in vivo goblet cells. Furthermore, ALI was the only condition allowing the detection of enteroendocrine cells. BEST4+ cells, however, were absent from organoids in immerged or ALI conditions despite their presence in vivo. In addition, ALI culture led to a consistent downregulation of hypoxia and glycolysis-associated genes across all cell types, which suggests a metabolic shift likely driven by increased oxygen availability in ALI conditions. Cell-cell communication analyses further indicated that ALI more closely mirrored in vivo patterns than immerged condition. Altogether, these results demonstrate that ALI culture allows for better recapitulation of the in vivo cellular heterogeneity and molecular signatures of the intestinal epithelium.NEW & NOTEWORTHY Using single-cell RNA sequencing, this study shows that air-liquid interface (ALI) culture enhances secretory lineage differentiation of intestinal organoid cell monolayers and improves transcriptomic similarity to the native epithelium. ALI reduced hypoxia-associated gene expression and better recapitulates in vivo-like cell-cell interactions, supporting its value for modeling intestinal epithelial heterogeneity in organoids.

Animals↗
PubMed2025

Self-organization of sinusoidal vessels in pluripotent stem cell-derived human liver bud organoids.

The induction of tissue-specific vessels in in vitro living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b+ putative liver sinusoidal progenitors by dictating developmental pathways. By devising an inverted multilayered air-liquid interface culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organize to generate and sustain hepatocyte-like cells neighboured by divergent endothelial subsets composed of CD32blowCD31high, LYVE1+STAB1+CD32bhighCD31lowTHBD-vWF- and LYVE1-THBD+vWF+ cells. WNT2 mediates sinusoidal-to-hepatic intercellular crosstalk potentiating hepatocyte differentiation and branched endothelial network formation. Intravital imaging reveals the iPS-cell-derived putative liver sinusoidal endothelial progenitor develops fully perfused human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enable correction of in vitro clotting time with Factor V-, VIII-, IX- and XI-deficient plasma, and rescues the severe bleeding phenotype in haemophilia A mice on transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.

Humans↗
PubMed1990

Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface.

It was previously reported that rat keratinocytes grown at the air-liquid interface on collagen gels or on nylon membranes produce multilayered cultures of uniformly stratified cells, comparable to the epidermis in situ by morphological and biochemical criteria. A protocol has now been developed by which primary human keratinocytes grown for two weeks submerged on microporous nylon membranes and raised to the air-liquid interface for an additional three weeks, exhibit most of the comparable characteristics of the epidermal cells in vivo. Staining with fluorescein isothiocyanate-conjugated monoclonal antibodies indicated the presence of 56,5 and 65-67 kDa keratins as well as filaggrin-type proteins in the upper cellular layers. Desmosomes, lamellar granules and keratohyalin-like granules were observed. Cultures were covered with layers of cornified cells. This study differs from the majority of other investigations on human keratinocytes in that no feeder layers or other biological substrata were used. This system should be useful in toxicological studies of chemicals which are to be applied topically to the skin.

Antibodies, Monoclonal↗
PubMed1992

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function.

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a more in vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%-h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used for in vitro research and testing and is also being tested in clinical applications.

Biological Dressings↗
PubMed2025

Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission.

Mycobacterium tuberculosis (Mtb) must withstand physical and chemical stresses during airborne transmission, including during the desiccation of aerosols small enough to reach pulmonary alveoli in a new host. There, Mtb encounters an antimicrobial pulmonary alveolar lining fluid (ALF) before it is engulfed by macrophages. To study the genes involved in Mtb's ability to survive the transition from desiccated droplet to pulmonary alveolus in an in vitro model, we formulated a model alveolar lining fluid (MALF) that mimics the composition of ALF as inferred from human bronchoalveolar lavage fluid (BALF). We compared the transcriptome of log-phase Mtb in MALF to the transcriptome of Mtb in BALF as BALF from the lungs of healthy adults was reconstituted to compensate for the dilution of ALF by lavage (rcBALF). Mtb from log-phase culture in a standard laboratory medium survived quantitatively in MALF and rcBALF for at least 24 hours. In contrast, Mtb that had passed through earlier stages of transmission began to succumb after 3 hours in MALF, past the time when particles have been observed to be phagocytized by alveolar macrophages. Screening of a genome-wide CRISPRi library of Mtb identified 35 genes as uniquely required by Mtb to survive the transition from desiccated microdroplet into rehydration in MALF. Thirty-one of these genes are non-essential under conventional laboratory conditions and seven have unknown functions. Thirteen of the 35 genes were additionally required for Mtb to survive in macrophage-like cells cultured at the air-liquid interface with pulmonary epithelial cells. This study nominates additional members of the transmission survival genome of Mtb, illustrates that different genes may contribute to the survival of Mtb at different stages of transmission, and suggests that modeled transmission can shed light on the functions of Mtb genes whose contributions have been unknown.

Journal Article↗
PubMed1991

Transforming growth factor alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures.

When cultured on plastic and treated with transforming growth factor alpha (TGF alpha), human keratinocytes exhibit an increase in proliferation at the colony periphery, apparently as a consequence of enhanced cell migration (Barrandon and Green, 1987). To investigate the effects of TGF alpha on a differentiating stratified squamous epithelium and to begin to examine the molecular basis mediating this influence, we cultured human epidermal cells on a gelled lattice of collagen and fibroblasts, floating on the air-liquid interface. Under these conditions, raft cultures differentiate and exhibit morphological and biochemical features of human skin in vivo (Asselineau et al., 1986; Kopan et al., 1987). When 3-wk-old raft cultures were treated with TGF alpha, basal cells showed a marked increase in cell proliferation. At elevated concentrations of TGF alpha, the organization of cells within the artificial tissue changed and islands of basal cells entered the collagen matrix. Biochemical analysis of the response revealed that type I collagenase and gelatinase were induced by keratinocytes within 12 h after TGF alpha treatment. In contrast, invasion of basal cells into the collagen matrix was not significant until 48-72 h post-treatment, suggesting that collagenase and gelatinase production may be a prerequisite to this phenomenon. These results have important implications for the possible role of TGF alpha in squamous cell carcinoma and tumor invasion.

Cell Differentiation↗
PubMed1990

Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface.

A fully differentiated epithelium having the features of epidermis was obtained in vitro by culturing second-passage normal human keratinocytes (NHK) in the chemically defined medium MCDB 153 on inert filter substrates at the air-liquid interface for 14 d. Vertical sections stained for histology and indirect immunofluorescence studies show a correct stratification and expression of differentiation markers. The presence of desmosomes, keratohyalin granules, and lamellar granules, and the formation of a more than ten-layers stratum corneum was evidenced by electron microscopy. Moreover, lipids typical for differentiated epidermis were present in these cultures, including ceramides, which are thought to be responsible for the relative impermeability of the stratum corneum. Under our culture conditions, i.e., in defined medium and at the air-liquid interface, the use of de-epidermized dermis as a substrate did not stimulate keratinocyte differentiation more than acetate cellulose or polycarbonate filter membrane substrates. The obtaining of a well-differentiated epidermis grown in vitro on inert filters in a chemically defined medium should be useful as a standard system for studying epidermal differentiation, re-epidermization, cytotoxicity, epidermal permeation, and transepidermal drug delivery.

Adult↗
PubMed2025

Incubator-Free Organoid Culture in a Sealed Recirculatory System.

Organoids are powerful tools for studying development and disease, offering realistic organ-like human and animal tissues and facilitating experimental observation compared to live animal models. However, traditional organoid culture methods require a humidified incubator. This requirement complicates culture due to evaporative losses and restricted access to instrumentation, hindering the potential of organoids as physiologically accurate models easily subjected to detailed experimental observation. We introduce a compact, automated, sealed, incubator-free recirculatory organoid culture platform that replaces the air-liquid interface with a nonporous polymer gas exchanger and a liquid-phase gas buffer. This design prevents evaporation and stabilizes oxygen, pH, and osmolarity without feedback control. It enables single-actuator media exchange, simplifying automation. Dispensing with the incubator, we improve access for instruments such as live cell microscopes. We demonstrate compatibility with continuous multi-week live imaging of vascular organoids and show that brain organoids in this system maintain metabolic viability, structural fidelity, and electrophysiological activity comparable to traditional shaker-based cultures in an incubator.

Journal Article↗
PubMed2026

Physical, chemical, and structural properties of human gastric organoid-derived mucus.

The gastric mucus layer protects the epithelium from gastric acid and ingested pathogens. However, studies of human gastric mucus have been limited due to poor accessibility of native human mucus and the abundance of contaminants in these samples. Here, we explored the potential of human gastric organoids as models for mucus production. Immunofluorescence staining confirmed that the organoids produced mucus containing MUC5AC and MUC6. The luminal mucus had viscoelastic properties similar to those of native human gastric mucus, as determined by particle tracking microrheology. To collect organoid-produced gastric mucus, termed bioengineered gastric mucus (BGM), organoids were cultured as monolayers at the air-liquid interface (ALI), and apically secreted mucus was harvested and analyzed by MUC5AC ELISA, proteomics, cryo-field emission scanning electron microscopy (CryoFE-SE), and rheometry. BGM contained high-molecular weight molecules also found in native gastric mucus. Proteomic analysis confirmed that BGM contained MUC5AC, MUC6, MUC1, and other stomach-specific molecules such as pepsin C, trefoil factor 2, and gastrokine. CryoFE-SE showed that both BGM and native mucus had a porous structure and a characteristic honeycomb scaffold. However, the viscosity of the BGM was generally lower than that of native human gastric mucus, and BGM failed to exhibit gelation at low pH. Collectively, these findings demonstrate the potential as well as some limitations of BGM as an accessible model system for human gastric mucus.NEW & NOTEWORTHY We demonstrate the structural and functional similarities of organoid-derived gastric mucus and native mucus collected from human patients. The bioengineered gastric mucus mimics its native counterpart in its proteomic profile and physical architecture. This work highlights the translational potential of organoid-derived mucus for functional investigations of the human gastric mucus layer.

Humans↗
PubMed1990

Reconstituted skin in culture: a simple method with optimal differentiation.

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.

Antibodies, Monoclonal↗
PubMed1992

Release of reactive oxygen species by guinea pig tracheal epithelial cells in vitro.

Regulatory and stimulatory mechanisms of H2O2 release from guinea pig tracheal epithelial cells were investigated. Cells in primary culture maintained in a previously described air-liquid interface system released H2O2 to the extracellular space only from the apical side of the cells. The rate of release was 0.044 +/- 0.003 nmol.min-1.mg protein-1. H2O2 release could be stimulated significantly during a 30-min incubation period with phorbol myristate acetate (PMA) and platelet-activating factor (PAF). A stimulatory effect of PAF was achieved at concentrations greater than 100 nM and with PMA at concentrations greater than 10 ng (16 nM). When protein kinase C was inactivated with staurosporine, the responses to both PAF and PMA were abolished, whereas the cyclooxygenase inhibitor, indomethacin, did not affect H2O2 generation. When guinea pig tracheal epithelial cells were exposed to sublethal concentrations of extracellular H2O2 (30 microM), H2O2 was detoxified from both apical and basal sides, H2O2 removal being significantly more rapid from the apical side of the cells. These results suggest that tracheal epithelial cells can be stimulated to generate reactive oxygen species into the airway lumen and that this occurs in response to inflammatory mediators that act through protein kinase C. Luminal H2O2 release may have developed as a defense mechanism against microbes, and, similarly, luminal detoxification of H2O2 could represent an important mechanism of modulation of airway inflammation in response to oxidant stress.

Analysis of Variance↗
PubMed1992

Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes.

The expression of viral genes during the productive life cycle of human papillomaviruses (HPV) is tightly coupled to the differentiation program of epithelial cells. We have examined transcription of HPV as a function of differentiation in an in vitro organotypic raft culture system which allows for epithelial stratification at the air-liquid interface. When CIN612 cells, which contain episomal copies of HPV type 31b (HPV31b), were allowed to stratify in raft cultures, they differentiated in a manner which was histologically similar to that seen in a cervical intraepithelial neoplasia I biopsy lesion. In monolayer cultures of CIN612 cells, two major polycistronic HPV31b transcripts of 1.7 kb which encode (i) E6, E7, E1-E4, and E5 and (ii) E6*, E7, E1-E4, and E5 were identified. These RNAs initiated at a promoter, P97, in the upstream regulatory region of the virus. Following differentiation in raft cultures, the relative abundance of RNAs initiated at P97 was unchanged. In contrast, the expression of a 1.3-kb RNA encoding an E1-E4 fusion protein and E5 was found to increase substantially following differentiation. This transcript was initiated at a novel promoter within the E7 gene (P742). These studies have therefore identified a constitutive viral promoter which is active throughout stratified epithelium as well as a novel promoter which is induced upon epithelial cell differentiation.

Amino Acid Sequence↗
PubMed1991

Human epithelial cells immortalized by SV40 retain differentiation capabilities in an in vitro raft system and maintain viral DNA extrachromosomally.

Carcinomas are the predominant type of cancer found in man, yet in vitro studies on the transformation of epithelial cells have been limited. In an attempt to extend our knowledge of the mechanisms involved in the development of epithelial cancers, we have examined the effects of oncogenes on keratinocytes in vitro using both the ability to immortalize and the ability to alter differentiation as criteria for transformation. SV40 T-antigen was observed to be an efficient immortalizing agent in human keratinocytes consistent with previous studies in other human cell types. Using an in vitro cell culture system (rafts) for epithelial stratification at the air-liquid interface, we observed that the morphology of rafts of SV40-immortalized keratinocytes was similar to that of untransfected epithelial cells, demonstrating that although immortal these cells retain differentiation capabilities. The ability to differentiate was lost only upon prolonged passage in culture, suggesting that this effect is separable from immortalization. In these immortalized epithelial cells, SV40 genomes were found to be maintained as a heterogeneous population of extrachromosomal molecules dependent upon the SV40 origin of replication. It is not clear whether these molecules arise continuously as a result of excision events from integrated copies or are stably maintained as episomes.

Antigens, Polyomavirus Transforming↗
PubMed1992

Reconstructed human epidermis: a model to study in vitro the barrier function of the skin.

In previous studies, we have shown that architecture and differentiation of human epidermis reconstructed by culturing dissociated keratinocytes on deepidermized dermis at the air-liquid interface resembles very much those of epidermis in vivo. The various types of epidermal layers are present with the appropriate differentiation markers, such as the bullous pemphigoid antigen, suprabasal keratins, involucrin, membrane-bound transglutaminase and filaggrin, positioned almost like in normal epidermis. At the ultrastructural level, heterogeneous keratohyalin granules and intra- and extracellular membrane-coating granules are observed. After 7 days of culture, a compact stratum corneum covers the reconstructed epidermis. In the present work, the barrier function of the reconstructed epidermis was evaluated by measuring fluxes of 3H-water. Our results show that under the various culture conditions tested, the presence of the reconstructed epidermis reduces dramatically the permeability of the deepidermized dermis although the skin equivalent exhibits a higher permeability than normal skin. Conditions that favor terminal differentiation of the keratinocytes such as maintaining the cultures in delipidized serum improve the barrier function. Reduction of the relative humidity has a similar effect, whereas the age of the culture is of no influence. Experiments are in progress to reconstruct human epidermis with a barrier function as efficient as in normal skin.

Culture Techniques↗
PubMed1992

Renewal and differentiation of keratinocytes cultured on dead de-epidermalized dermis.

Human keratinocytes grown at an air-liquid interface on dead de-epidermalized dermis exhibit a pattern of organization similar to that seen in vivo. Cell renewal is limited to the basal layer. The cell cycle time determined after 7 days of culture, using a percentage labelled mitoses (PLM) technique, was about 15 h. This result is comparable with published data for cultivated keratinocytes but is shorter than the parameter proposed for epidermis in vivo. Appearance of labelled cells in the granular layer was observed 4 days after pulse labelling. Despite this high cell renewal, a normal cell differentiation with expression of various keratinization markers was maintained.

Adult↗
PubMed1990

In vitro differentiation of epithelial cells from cervical neoplasias resembles in vivo lesions.

Cell lines derived from cervical neoplasias, as well as cells from normal cervix and neonatal foreskin were examined in an in vitro culture system (raft system) that allows for stratification and differentiation of epithelial cells at an air-liquid interface. Epithelial cells from human foreskin and ectocervix were observed to differentiate in a manner histologically similar to normal epithelium in vivo as indicated by a single layer of basal cells with a defined mid and upper zone. In contrast, cells expanded from cervical squamous carcinoma explants showed total absence of normal differentiation in the raft system with numerous cells in the upper portion of the stratum exhibiting mitotic figures. Cell cultures derived from low grade cervical intraepithelial neoplasia and condyloma acuminata exhibited partial differentiation in addition to perinuclear clearing, binucleate cells and individual cell keratinization. These studies show that in the raft system, cultured cells derived from tissue biopsies can duplicate many of the histological features observed in cervical neoplasias. In addition, epithelial cells derived from normal fetal ectocervix and electroporated with cloned human papillomavirus (HPV) type 16 DNA exhibited abnormal differentiation patterns similar to those of cervical intraepithelial neoplasia in vivo. This model system will thus be useful for examining the effects of HPV infection on epithelial maturation and for investigating the role of other factors in the progression of cervical malignancies.

Cell Differentiation↗
PubMed1992

In vitro infection of normal human keratinocytes by human papillomavirus type 1 followed by amplification of the viral genome in reconstructed epidermis.

Primary cultures of normal human keratinocytes were inoculated in vitro with human papillomavirus type 1 (HPV-1), the agent responsible for deep plantar warts. Upon transfer to dead de-epidermized dermis and growth at the air-liquid interface, keratinocytes reconstituted a pseudoepidermis. Under these highly differentiating conditions, HPV-1 DNA amplification was found to take place in the reconstructed epidermis, being detectable from 7 days after the transfer and persisting for at least 10 days thereafter. The extent of keratinocyte differentiation may be insufficient to allow a complete HPV infectious cycle.

Cell Differentiation↗
PubMed1991

Developmental and hormonal regulation of surfactant protein A (SP-A) gene expression in fetal lung.

Pulmonary surfactant is a developmentally-regulated lipoprotein synthesized and secreted by the type II cells of the pulmonary alveolus where surfactant glycerophospholipids and proteins act to reduce surface tension at the alveolar air-liquid interface. Surfactant protein A (SP-A), the major surfactant-associated protein, appears to serve an important role in surfactant function and reutilization by type II cells. SP-A synthesis and gene expression are initiated in fetal lung tissue in concert with the developmental induction of surfactant glycerophospholipid synthesis. In studies using midtrimester human fetal lung explants maintained in organ culture, we have observed that cyclic AMP and glucocorticoids have pronounced effects on morphologic development and on the levels of SP-A gene expression. Cyclic AMP analogues act primarily to induce SP-A gene transcription; whereas, glucocorticoids have complex effects at both the transcriptional and posttranscriptional levels. We also have found that human fetal lung in vitro secretes into the culture medium relatively large amounts of prostaglandins (PG) PGE2 and PGF2 alpha and the PGI2 and thromboxane A2 metabolites, 6-keto-PGF1 alpha and TxB2, respectively. The prostaglandin synthesis inhibitor, indomethacin, markedly inhibits SP-A gene expression and cyclic AMP formation by human fetal lung in culture; the inhibitory effect of indomethacin on SP-A gene expression can be prevented by simultaneous incubation with either Bt2cAMP or PGE2. These findings are suggestive that prostaglandins acting through cyclic AMP also may serve an important role in the regulation of SP-A gene expression in human fetal lung tissue.

Cell Differentiation↗