Los autores están muy agradecidos con el Dr. Richard Inculet para proporcionar los tejidos bronquiales. Junta de Ética de Investigación aprobaciones se obtuvieron de Salud de San José Hamilton y la Universidad de Western Ontario / Londres Centro de Ciencias de la Salud (Dr. David McCormack), Tejidos y Comité de Archivos del Departamento de Patología. También agradecemos a Ernie Spitzer (Microscopía Electrónica de la Universidad McMaster) para proporcionar TEM de nuestras culturas ALI, y Farkas Daniela para proporcionar algunos de los materiales necesarios para la inmunotinción. Este trabajo fue financiado por una subvención plazo de bloques de la Sociedad Torácica de Ontario, el Dr. Asma Yaghi fue apoyado por una beca FSORC, Salud de San José, Hamilton, Ontario, Canadá.

<ol>
	<li>Phillips, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+Cells+on+Transwell+Inserts+-+Tips+and+Techniques+[Internet].">Growing Cells on Transwell Inserts - Tips and Techniques [Internet].</a> Corning Life Sciences, Technical Resources, Online Training. , (2008).</li><li>Turi, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+activates+anion+exchange+protein+2+and+AP-1+in+airway+epithelial+cells.">Oxidative stress activates anion exchange protein 2 and AP-1 in airway epithelial cells.</a> Am J Physiol Lung Cell Mol. Physiol. 283, L791-L798 (2002).</li><li>Lechner, J. F., Haugen, A., McClendon, I. A., Pettis, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+growth+of+normal+adult+human+bronchial+epithelial+cells+in+a+serum-free+medium.">Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium.</a> In Vitro. 18, 633-642 (1982).</li><li>Yoshisue, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+ciliated+bronchial+epithelium+1,+a+ciliated+cell-associated+gene+induced+during+mucociliary+differentiation.">Characterization of ciliated bronchial epithelium 1, a ciliated cell-associated gene induced during mucociliary differentiation.</a> Am. J. Respir. Cell Mol. Biol. 31, 491-500 (2004).</li><li>Wetering, S. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+secretory+leukocyte+proteinase+inhibitor+(SLPI)+production+by+human+bronchial+epithelial+cells:+increase+of+cell-associated+SLPI+by+neutrophil+elastase.">Regulation of secretory leukocyte proteinase inhibitor (SLPI) production by human bronchial epithelial cells: increase of cell-associated SLPI by neutrophil elastase.</a> J. Investig. Med. 48, 359-366 (2000).</li><li>Tristram, D. A., Hicks, W., Hard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+syncytial+virus+and+human+bronchial+epithelium.">Respiratory syncytial virus and human bronchial epithelium.</a> Arch. Otolaryngol. Head Neck Surg. 124, 777-783 (1998).</li><li>Mattinger, C., Nyugen, T., Schafer, D., Hormann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+serum-free+culture+conditions+for+primary+human+nasal+epithelial+cells.">Evaluation of serum-free culture conditions for primary human nasal epithelial cells.</a> Int. J. Hyg. Environ. Health. 205, 235-238 (2002).</li><li>Freshney, R. I., Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of epithelial cells. , 1-23 (1992).</li><li>Freshney, R. I., Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+bronchial+epithelial+cell+cultures+in+Culture+of+specialized+cells+series.">Normal human bronchial epithelial cell cultures in Culture of specialized cells series.</a> Culture of epithelial cells. , 181-196 (1992).</li></ol>

En este estudio se presentan métodos detallados para la cultura y la expansión de la enseñanza primaria humanos células epiteliales bronquiales. Hemos demostrado cómo los explantes y el trasplante de tejido bronquial, cultivadas en los medios de comunicación que promueven el crecimiento de las células epiteliales, pueden proporcionar una fuente continua de las células epiteliales de las vías respiratorias humanas para los estudios de la no-diferenciados modelos celulares (sumergido) y diferenciado (cultivados e...

Otros requisitos: incubadora, del gabinete de seguridad biológica (BSC), centrífuga, las placas de 100 mm de la cultura, tubos estériles (15 ml, 50 ml y 2 ml), puntas de pipeta, bisturí y hojas, pequeñas tijeras afiladas, batas y guantes. Estas se pueden obtener de sus proveedores preferidos.

primary human bronchial epithelial cells grown from explants

Células epiteliales bronquiales humanas son necesarios para los modelos de celulares de la enfermedad y para investigar el efecto de los excipientes y agentes farmacológicos sobre la función y estructura de las células epiteliales humanas. A continuación se describe en detalle el método de cultivo de células del epitelio bronquial de las vías aéreas bronquiales tejido que es recogido por el cirujano en el momento de la cirugía pulmonar (por ejemplo, el cáncer de pulmón o una cirugía de reducción de volumen pulmonar). Con la aprobación de la ética y el consentimiento informado, el cirujano obtiene lo que se necesita para la patología y nos proporciona una parte bronquial que está alejado de las áreas afectadas. El tejido se utiliza como fuente de explantes que se pueden utilizar para el cultivo de células epiteliales bronquiales primarias en la cultura. Segmentos bronquial 0,5-1cm de longitud y 1 cm de diámetro ≤ se enjuagan con EBSS frío y tejidos del parénquima se elimina el exceso. Los segmentos son a cielo abierto y picado en 2-3mm<sup&gt; 3</sup&gt; Piezas de tejido. Las piezas se utilizan como fuente de células primarias. Después de placas de revestimiento de la cultura de 100mm de 1-2 horas con una combinación de colágeno (30 mg / ml), la fibronectina (10 mg / ml) y BSA (10 mg / ml), las placas están rayados en las zonas 4-5 y tejidos piezas se colocan en las áreas de rayado, entonces el medio de cultivo (DMEM / HAM F-12 con aditivos) adecuadas para el crecimiento de las células epiteliales se agrega y las placas se colocan en una incubadora a 37 ° C en 5% CO<sub&gt; 2</sub&gt; Aire humidificado. El medio de cultivo se cambió cada 3-4 días. Las células epiteliales crecer a partir de las piezas que forman los anillos alrededor de 1,5 cm de diámetro en 3-4 semanas. Los explantes se puede volver a utilizarse hasta 6 veces por su traslado a los nuevos pre-revestidos placas. Las células se levantó con tripsina / EDTA, agrupados, se cuentan y re-chapada en frascos T75 celular de enlace para aumentar su número. T75 frascos sembrados con 2-3 millones de células de crecer hasta la confluencia del 80% en 4 semanas. Ampliado primaria células epiteliales humanas pueden ser cultivadas y permite diferenciar en la interfaz aire-líquido. Los métodos descritos aquí proporcionan una fuente abundante de células epiteliales bronquiales de los tejidos recién aisladas y permitir el estudio de estas células como modelos de enfermedad y de la farmacología y la investigación de la toxicología.

Watch this Scientific Journal Video about Primary Human Bronchial Epithelial Cells Grown from Explants at JoVE.com

Primary Human Bronchial Epithelial Cells Grown from Explants

A continuación se describe un método detallado para el cultivo de primaria células epiteliales bronquiales humanas a partir de explantes de tejido humano vías respiratorias bronquiales incluyendo el crecimiento diferenciado en una interfase aire-líquido. Este método proporciona una fuente abundante de células primarias para investigar el papel del epitelio de las vías respiratorias en la salud pulmonar y la enfermedad humanas.

Primaria células epiteliales bronquiales Cultivada a partir de explantes

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

primary-human-bronchial-epithelial-cells-grown-from-explants

Research

JoVE Journal

Biology

24.0K vistas.  McMaster University. A continuación se describe un método detallado para el cultivo de primaria células epiteliales bronquiales humanas a partir de explantes de tejido humano vías respiratorias bronquiales incluyendo el crecimiento diferenciado en una interfase aire-líquido. Este método proporciona una fuente abundante de células primarias para investigar el papel del epitelio de las vías respiratorias en la salud pulmonar y la enfermedad humanas.

Video: Primary Human Bronchial Epithelial Cells Grown from Explants

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Investigación

Biología

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Lentivirus óptimo de producción y el cultivo celular condiciones necesarias para la transducción de éxito células epiteliales bronquiales humanos primarios

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Modelo alveolar humano multicelular compuesto por células epiteliales y células inmunes primarias para la evaluación de riesgos

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

Generación De Monocapas Epiteliales Murinas Primarias De Colon De Criptas Intestinales

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

Determinación de la toxicidad de la radiación UV y los productos químicos en las células epiteliales corneales humanas primarias e inmortalizadas

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Aislamiento de células epiteliales del folículo dental humano

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

Células epiteliales nasales humanas primarias: biobancos en el contexto de la medicina de precisión

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...

Methods Article

Primaria células epiteliales bronquiales Cultivada a partir de explantes