המחברים מודים מאוד ד&quot;ר ריצ&#39;רד Inculet למתן הרקמות הסימפונות. מחקר אתיקה האישורים מועצת התקבלו המילטון בריאות סנט ג&#39;וזף של אוניברסיטת מערב אונטריו / לונדון למדעי הבריאות מרכז (ד&quot;ר דוד מקורמק), רקמות ארכיון הוועדה, המחלקה לפתולוגיה. אנו מודים גם ארני שפיצר (מיקרוסקופית אלקטרונים, אוניברסיטת מקמאסטר) למתן TEMs של תרבויות עלי שלנו, פרקש דניאלה למתן חלק מהחומרים הדרושים immunostaining. עבודה זו מומנה על ידי מענק בלוק טווח מהחברה אונטריו בית החזה, ד&quot;ר אסמא יגי נתמכה על ידי מלגה FSORC, בריאות סנט ג&#39;וזף, המילטון, אונטריו, קנדה.

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	<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>

במחקר זה הצגנו שיטות מפורטות תרבות והרחבה של האדם תאים ראשוניים אפיתל הסימפונות. הראינו כיצד explants והשתלות של רקמת הסימפונות, תרבותי בתקשורת אשר לקדם גדילת תאים אפיתל, יכולים לספק מקור מתמיד של תאים אפיתל דרכי הנשימה האנושית ללימודי הלא מובחן מודלים תא (שקוע) הבדיל (ג?...

דרישות אחרות: חממה, בטיחות ביולוגית הממשלה (BSC), צנטריפוגות, צלחות תרבות 100mm, צינורות סטרילי (15 מ&quot;ל, 50 מ&quot;ל, ו - 2 מ&quot;ל), טיפים פיפטה סטרילית, ידית האזמל להבי, מספריים חדות קטן, חלוקי מעבדה וכפפות. אלו ניתן להשיג מהספקים המועדפת עליך.

primary human bronchial epithelial cells grown from explants

האדם תאים אפיתל הסימפונות יש צורך מודלים התא של המחלה כדי לחקור את ההשפעה של excipients וסוכני תרופתי על תפקוד ומבנה של תאים אפיתל אנושיים. כאן אנו לתאר בפירוט את השיטה של ​​גידול בתאי אפיתל הסימפונות מרקמות דרכי הנשימה הסימפונות כי הוא שנקטפו על ידי המנתח על פי ניתוחים הריאות (סרטן ריאות למשל או ניתוח הקטנת נפח הריאה). באישור אתיקה הסכמה מדעת, המנתח לוקח את מה שדרוש על פתולוגיה מספק לנו חלק הסימפונות כי הוא מרוחק מאזורי החולות. הרקמה משמשת אז כמקור explants כי ניתן להשתמש לגידול העיקרי לתאי האפיתל הסימפונות בתרבות. קטעי הסימפונות על 0.5-1cm ארוך ≤ 1cm בקוטר הם שטופים עם EBSS קר רקמות parenchymal עודף מוסר. מגזרי נחתכים פתוח טחון לתוך 2-3mm<sup&gt; 3</sup&gt; פיסות רקמה. חתיכות משמשים כמקור של תאים ראשוניים. לאחר 100mm ציפוי צלחות תרבות שעות 1-2 עם שילוב של קולגן (30 מיקרוגרם / מ&quot;ל), פיברונקטין (10 מיקרוגרם / מ&quot;ל), ו - BSA (10 מיקרוגרם / מ&quot;ל), צלחות הם שרוטים באזורים 4-5 ורקמות חתיכות ממוקמים באזורים שרוט, אז בינוני תרבות (DMEM / חם ה-F-12 עם תוספים) מתאים צמיחת תאים אפיתל מתווסף וצלחות ממוקמים באינקובטור ב 37 מעלות צלזיוס CO 5%<sub&gt; 2</sub&gt; האוויר humidified. בינוני התרבות משתנה כל 3-4 ימים. לתאי האפיתל לצמוח מן החלקים ויוצרים כ -1.5 ס&quot;מ קוטר הטבעות 3-4 שבועות. Explants ניתן שימוש חוזר עד 6 פעמים על ידי העברת אותם מראש מצופה צלחות חדשות. תאים הם הרימו באמצעות טריפסין / EDTA, ונקווה, נספר, מחדש מצופה ב T75 צלוחיות Bind Cell להגדיל את מספרם. T75 seeded צלוחיות עם 2-3 מיליון תאים לגדול מפגש 80% ב 4 שבועות. מורחבות תאים ראשוניים אפיתל האדם יכול להיות תרבותי מותר להבדיל על ממשק אוויר נוזלי. השיטות המתוארות כאן לספק מקור שופע של האדם תאים אפיתל הסימפונות מרקמות מבודדים טרי ולאפשר לחקר התאים הללו כמודלים של המחלה ועל פרמקולוגיה סינון הרעלים.

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

כאן אנו מתארים את שיטת מפורט לגידול העיקרי בתאים אנושיים אפיתל הסימפונות מ explants של רקמה אנושית דרכי הנשימה הסימפונות כולל גידול הבדיל על ממשק אוויר נוזלי. שיטה זו מספקת מקור שופע של תאים ראשוניים על חקירת תפקידו של אפיתל דרכי הנשימה בריאות מחלת ריאות האדם.

ראשי תאים אנושיים אפיתל הסימפונות גדלה מ Explants

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

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24.0K Views.  McMaster University. כאן אנו מתארים את שיטת מפורט לגידול העיקרי בתאים אנושיים אפיתל הסימפונות מ explants של רקמה אנושית דרכי הנשימה הסימפונות כולל גידול הבדיל על ממשק אוויר נוזלי. שיטה זו מספקת מקור שופע של תאים ראשוניים על חקירת תפקידו של אפיתל דרכי הנשימה בריאות מחלת ריאות האדם.

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.

בידוד תרבות למבוגרים גזע תאים אפיתל של העור האנושי

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

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 התרבות של האדם תאים ראשוניים החצוצרה אפיתל

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&#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.

בידוד תאי גזע בוגרים נאיבי מתרבויות פיברובלסטים אנושיות הראשוניות

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.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

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.

אופטימלית Lentivirus ייצור תרבית תאי תנאים הכרחיים בהצלחת transduce האדם הראשי תאי אפיתל סימפונות

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

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

שיטות לחקר תפקוד החלבון התחבורה אפיתל וביטוייה המעי Native ו קאקו-2 תאים שגודלו ב -3 D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

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.

מודל אדם רב-תאי המורכב מתאי אפיתל ותאי מערכת החיסון העיקריים להערכת סיכונים

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.

דור של מונוליירים אפיתל המעי הגס הראשי מורין מקריפטות מעיים

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 ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

ניתוח יעיל ותרבות של תאי אפיתל פיגמנט רשתית העכבר הראשי

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

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.

קביעת הרעילות של קרינת UV וכימיקלים על תאי אפיתל ראשוניים ואימורטליים של הקרנית האנושית

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