Reconstituted human gingival epithelium: Nonsubmerged in vitro model

Summary Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type I collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.     

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

Background aimsThe aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.MethodsHAECs at passage 1–2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry.ResultsOrganotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification.ConclusionsResults from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration.     

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.     

Selective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture

We report on a three dimensional (3D)-organotypic culture in vitro for selective growth and expansion of human corneal epithelial stem cells. Limbal corneal explants were cultured on porous collagen sponges submerged in Epilife medium containing 10% fetal bovine serum. The fragments were analyzed by immunohistochemistry for the expression and distribution of a spectrum of corneal epithelium markers: p63, CK-19, CK-3, Ki-67, pan-cytokeratins and vimentin. Early in culture the epithelium began to exfoliate losing its differentiated high-zone layers into the medium, maintaining only basal and few parabasal cells (mostly both p63 and CK-19 positive), which had remained attached to the specimen. After 14 days a new epithelium was formed displaying an increasing prominence of basal and suprabasal cells that, sliding onto the whole explant, showed the tendency to underlay stromal tissue and infiltrate into the underlaying sponge. After 21 days, sponge and fragments were incubated with trypsin-EDTA and dispersed epithelial cells were pipetted on a feeder monolayer of mitomycin-c-treated murine NIH.3T3 fibroblasts. Colonies of undifferentiated epithelial cells (p63, CK-19 and Ki-67 positive, CK-3 negative) were obtained: their cells, if seeded onto a collagen matrix containing embedded primary human corneal fibroblasts as feeder, provided the basic building blocks for reconstructing in vitro a 3D-multilayered corneal epithelium.     

Growing a stratified,cornified primary culture of rat keratinocytes with epidermis-like water permeation barrier function

Summary The culture of cutaneous keratinocytes grown on a Puropore nylon microporous membrane at the air-liquid interface has been shown to be similar to the epidermis in a number of molecular and morphologic characteristics but to exhibit a significantly greater degree of tritiated water permeation. Various culture conditions have been altered in an effort to improve the water barrier properties. A Kp value in the range of 5.5±1.6×10⁻³ has been obtained for 79% of the culturea) by plating 0.9×10⁶ viable basal cells on a piece (13-mm diameter) of membrane for 7 days of submerged growth,b) by placing two membranes on two stacked glass fiber filters (47-mm extra-thick) in a culture dish (60 mm) for 14 days of growth at the air-liquid interface,c) by replacing the growth medium, i.e., 1 ml of complete minimum essential medium (CMEM) every 24 h after lifting,d) by using 10% fetal bovine serum (FBS) in the CMEM during the submerged culture period and 15% FBS in the CMEM during the lifted culture period, ande) by adding a dialysis membrane on top and a Puropore nylon membrane below the culture when the cultures were inserted in the permeation cell for testing. The percentage of cultures with this value for Kp can be increased to 90% if only cultures with yellow, smooth, and shiny surfaces are tested. This system should be useful as a replacement for skin in testing the cutaneous permeation of some chemicals. To whom correspondence should be addressed at 1528 Public Health, The University of Michigan, Ann Arbor, Michigan 48109-2029.     

Serial culturing of human bronchial epithelial cells derived from biopsies

Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.     

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 morein 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 forin vitro research and testing and is also being tested in clinical applications.     

Production of tissue-engineered three-dimensional human bronchial models

We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.     

Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers

Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl(-) current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O(2) produced similar improvements in Cl(-) current and CFTR protein as air-liquid interface culture, while increasing PO(2) from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.     

Rat tracheal epithelial cell differentiation in vitro

Summary In vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.     

Development of a Curved,Stratified, In Vitro Model to Assess Ocular Biocompatibility

Purpose

To further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing.

Methods

Immortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression.

Results

The reconstructed curved corneal epithelium was composed of 3–5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression.

Conclusions

The stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.     

Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium

We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organotypic culture of human ovarian surface epithelial cells: A potential model for ovarian carcinogenesis

Summary The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis. The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining. Two cultures from two different patients were established, HOSE-A and HOSE-B. Cultures were infected with a retrovirus expressing human papillomavirus genes E6 and E7 to extend their life span. HOSE cells were seeded onto collagen gels containing NIH3T3-J2 fibroblasts as feeder cells and grown to confluence submerged in growth medium. The collagen bed was then raised to the air-medium interface for 7 d (organotypic culture). Microscopically, fixed cultures revealed a single layer of flat cells growing on the collagen surface, reminiscent of HOSE cells in vivo. Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified. In addition, established ovarian cancer lines grown in this fashion stratified and showed malignant phenotypes. Thus, cells grown in organotypic culture resemble their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and perhaps malignant transformation of HOSE cells.     

Culture of endodermal stem/progenitor cells of the mouse tongue

The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFP(hi) cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.     

Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium.