Air-liquid interface (ALI) impact on different respiratory cell cultures

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

Differentiated Primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by “liquid application,” involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

The aim of this study was to establish a soft shell turtle primary culture for replication of the isolated poxvirus-like virus. To date, the primary culture cells have been successfully subcultured for more than 25 passages in a T-25 cell culture flask. Based on morphology studies, these cells were identified as mostly fibroblast cells. The results of this study showed that the L15 medium was the optimal culture medium for these cells. Interestingly, the soft shell turtle primary cells grew faster at 37°C than at temperatures below 30°C. The lysis cellular pathogenic effects (CPE) presented on the monolayer cell culture which has been inoculated with 200 μL of the tested isolated poxvirus-like viral suspension (1 × 108 TCID50/ml) on the 2nd postinoculation day. The virulence of poxvirus-like virus was significantly inhibited by 5-iodo-2’-deoxyuridine, but 5-bromo-2’-deoxyuridine had no significant effect. According to the acridine orange staining results, the virus-infected primary cells were yellow-green in color, indicating that the poxvirus-like virus was a double-stranded DNA virus. The set primary culture cells were highly capable of being successfully infected with the soft shell turtle poxvirus-like virus. This study may be the first study to determine the optimal primary culture for studying viral infections in soft shell turtle embryos. Key words: Pox-like virus, soft shell turtle, reptile, primary cell culture, replication.

The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 μg ZnO/cm2 (or 0.09 and 0.33 cm2 ZnO/cm2), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue.

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines

This thesis covers three interrelated topics: 1. The spontaneous differentiation of lens epithelial cells to elongated, thin spindle-shaped fiber-like cells in primary cell culture. 2. The interaction of primary cultured lens epithelial cells with malignant cell lines in co-culture, with regard to the establishment of a distinct proliferation border. 3. Structural reorganization processes of the microfilament and microtubule cytoskeleton during differentiation of lens epithelial to lens fiber cells. In primary cell culture, using a standard culture medium with fetal calf serum as single additionally supplement, lens epithelial cells reproducibly started a spontaneous differentiation to fiber-like cells. Accompanied by significant changes of morphology the formation of fiber-like cells appeared only in spots of cells regularly arranged in a compact monolayer. From co-culture of lens epithelial cells with non-adherent growing malignant leukemia cells, no interactions by chemo-repulsive effects could be observed. In contrast, studying a co-culture of lens epithelial cells with also adherent growing micro vascular endothelial cells, it was found that these two cell types formed a distinct proliferation border which was stable for more than two weeks. The co-culture experiments indicate that the contribution of lens epithelial cells to create a barrier for invasive growing tissues is in all probability the maintenance of a tight, compact and impenetrable cell layer. If repulsive effects (chemically or physically by contact) play a critical role at all, than probably only over very short distances within direct vicinity of the lens epithelium. The development of transparency in the mammalian eye lens includes complex structural reorganization processes during differentiation of lens epithelial to lens fiber cells. Whereas apoptotic processes cause an extensive organelle degradation to eliminate light-scattering macromolecular structures, the lens fiber cell cytoskeleton is not degraded but basically reorganized. Regarding the microfilament and microtubule cytoskeleton during lens cell differentiation dramatic reorganizations could be observed in vitro and in vivo. In lens fiber cells, alterations in the microfilament (f-actin) network resulted in an actin free cytoplasm with compact stress fibers parallel aligned to the cell membrane of these highly elongated cells. By visualizing the microtubule system in lens epithelial and cortical fiber cells the restructuring of the microtubule network from a monocentric array in epithelial to a decentralized organization in fiber cells was shown. Also the microtubule system became aligned to the central axis (from pole to pole) of the differentiating fiber cells. These effects were found to initiate already in primary cell culture during differentiation of lens epithelial to fiber-like cells. By immunofluorescence techniques using anti-gamma-tubulin antibodies, it was observed that the microtubule-organizing centre (MTOC) was separated from the cell nucleus both in primary cultured lens epithelial cells and also in isolated cortical lens fiber cells. After degradation of the cell nucleus, cortical lens fiber cells still contained gamma-tubulin complexes. Microtubules were never found to be associated with these gamma-tubulin complexes and did never radiate from one main focus. Instead, they formed a decentralized network with several crossing points, where few microtubules interlinked. The immunofluorescence and immunoblot results indicate that the reorganization of the cytoskeleton of differentiating lens fiber cells includes the separation of the MTOC from the nucleus, its immobilization at the cell membrane and subsequent disintegration. As a result the organization of the microtubule system by one main MTOC becomes lost. Disintegration of the MTOC could eliminate a light scattering center and therefore contribute to the improvement of the optical properties of the eye lens. By contrast, the cytoskeleton of cortical lens fiber cells is not degraded and might therefore be necessary for the final differentiation to a cell type which remains stable until death of the organism.

The effects of mechanical stimuli such as wall shear stresses (WSS) on cellular processes have been studied in vitro in numerous cell types. In order to study WSS effects on cells cultured under air-liquid interface (ALI) conditions, we developed a custom-designed well that can be disassembled into sub-units that allow installation of the cultured cells in a flow chamber, and then, re-assembled for further incubation or biological tests. Human nasal epithelial cells were cultured in the new wells under ALI conditions, and some of their biological characteristics were compared with those cultured in commercial Millicells. The cultured cells from both types of wells secreted the same amount of mucin and had similar cytoskeletal structures. Preliminary WSS experiments demonstrated the advantage of the new wells and provided initial indications that WSS affects the performance of ALI cultured respiratory epithelial cells.

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions.

Basic and translational (or preclinical) prostate cancer research has traditionally been conducted with a limited repertoire of immortalized cell lines, which have homogeneous phenotypes and have adapted to long-term tissue culture. Primary cell culture provides a model system that allows a broader spectrum of cell types from a greater number of patients to be studied, in the absence of artificially induced genetic mutations. Nevertheless, primary prostate epithelial cell culture can be technically challenging, even for laboratories experienced in immortalized cell culture. Therefore, we provide methods to isolate and culture primary epithelial cells directly from human prostate tissue. Initially, we describe the isolation of bulk epithelial cells from benign or tumor tissues. These cells have a predominantly basal/intermediate phenotype and co-express cytokeratin 8/18 and high molecular weight cytokeratins. Since prostatic stem cells play a major role in disease progression and are considered to be a therapeutic target, we also describe a prospective approach to specifically isolate prostatic basal cells that include both stem and transit-amplifying basal populations, which can be studied independently or subsequently differentiated to supply luminal cells. This approach allows the study of stem cells for the development of new therapeutics for prostate cancer.

The Society of Biomolecular Imaging and Informatics First Annual Conference

Differential heat shock response of primary human cell cultures and established cell lines

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two micecan reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) evoked Ca(2+) influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca(2+) concentration and ATP release. Stretch stimulation evoked intracellular Ca(2+) increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca(2+) increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca(2+) increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4alpha-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca(2+). These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

2 - INTRODUCTION TO CELL CULTURE