We thank the faculty, fellows, residents, and physician assistants of the Brigham and Women's Hospital, Department of Pathology for making tissue available for these studies. This work was supported by research grants from the NIH/National Cancer Institute (P50 CA105009, K08 CA108748, U01 CA152990), Ovarian Cancer Research Fund, The May Kay Foundation, Novartis Pharmaceuticals, Robert and Deborah First Fund, Randi and Joel Cutler Ovarian Cancer Research Fund, Marsha Rivkin Foundation - Scientific Scholar Award, AACR - George and Patricia Sehl Fellowship for Cancer Genetics Research, and the American Physician Fellowship for Medicine in Israel - Claire and Emmanuel G. Rosenblatt Foundation Grant.

1. Collagen Preparation and Filter Coating.
<ol>
	<li>To prepare human placental collagen, tweeze out 30 mg of human placental collagen and put on top of 50 ml of distilled water (dH2O) in a 500 ml beaker with stir bar.</li>
	<li>Add 100 &mu;ls of glacial acetic acid directly onto collagen to make it easier to dissolve. Warm to 37&deg;C and stir it to dissolve the collagen. It should dissolve in 20-30 minutes. If collagen strands remain in solution, filter sterilization will be difficult.</li>
	<li>Dilute the 50 ml stock with 450 ml of dH2O and filter-sterilize with a 0.2 &mu;m membrane. This is the final working solution. Store at 4&deg;C.</li>
	<li>Prepare Transwell filter membranes by coating them with collagen (50-100 &mu;l) overnight. Chilled collagen must be warmed to RT and then added to the upper compartment to cover the membrane overnight and at RT. Before plating the dissociated primary fallopian tube (FT) epithelium (see below) onto the Transwell filters, wash the filters three times in 1X phosphate buffered saline (PBS) at least 1 hour before use to remove excess collagen.</li>
</ol>
2. Tissue Collection and Dissociation.
<ol>
<li>Fresh fallopian tube (FT) fimbria should be collected in sterile 1X PBS in a 50 ml centrifuge tube and kept on ice prior to prompt processing in a sterile tissue culture hood.</li>
	<li>Wash the tissue sample once in 20 ml chilled 1X PBS (if sample has been collected in another type of media or is bloody wash at least 3X to get rid of as much blood and foreign media as possible as this might interfere with plating efficiency).</li>
	<li>Using sterile tweezers transfer FT to a 10 cm tissue culture dish with a little PBS and, using a sterile scalpel and sterile tweezers, cut longitudinally to maximize the exposure of the epithelial cells. Open up the fimbria so that it is no longer a tube but an almost flat sheet of tissue. Then cut into smaller but still retrievable pieces (about 3 mm in diameter).</li>
	<li>Transfer pieces to 45 ml of chilled dissociation media (MEM containing 1.4 mg/ml Pronase and 0.1 mg/ml DNAse) in a 50 ml tube.</li>
	<li>Rock gently at 4&deg;C for 24 to 72 hours. (from experience, 48 hours is optimal). To check amount of dissociation, put a drop of dissociation media on a slide and check amount of clumping and tissue viability (beating ciliated cells). You want to see both single cells and some small clumps.</li>
</ol>
3. Fallopian Tube Plating.
<ol>
<li>When the amount of epithelial cell dissociation is optimal, inactivate the media by adding 10% volume of FBS. Invert a few times to dislodge the cell suspension into smaller aggregates.</li>
	<li>Allow large tissue clumps (stromal tissue) to settle in the bottom the tube. Remove media containing epithelial cells into a second 50 ml tube.</li>
	<li>Add 50 ml of cold MEM (no FBS) to the original 50 ml tube. Invert to collect additional cells. Collect media into a third 50 ml tube (if the sample is very large and a high number of cells is expected, this step can be repeated to maximize cell collection). The large pieces of stromal/extracellular tissue can now be discarded. You now have two 50 ml tubes of cell suspension. Centrifuge at 1000 rpm for 5 minutes and aspirate dissociation media from cell pellets.</li>
	<li>Resuspend in about 5-10 ml of USG media (DMEM:F12 supplemented with 2% USG and 1% Pen Strep) warmed to 37&deg;C. Pipette GENTLY. Be careful not to pipette too vigorously as this may damage the epithelial cells and reduce the quality of cultures. There should be single cells and small clumps (Figure 1).</li>
	<li>Transfer onto Primaria culture dishes and incubate for a minimum of 1 hour or up to 3 hours. Fibroblasts and red blood cells will stick to the plastic but the FT epithelial cells will not. Take a look at the plate after an hour to see if anything is sticking. The presence of ciliated cells can be seen by noting the beating of the cilia.</li>
	<li>During this incubation, collagen coated filters can be washed and made ready for cell plating (See step 1.4)</li>
	<li>After 1-3 hours of incubation on the Primaria plates, transfer the cell suspension to a 15 ml tube and rinse the Primaria plate with 5 ml USG media, transfer this media to the tube to make a final volume of 15 ml. Centrifuge at 1000 rpm for 5 minutes.</li>
	<li>Carefully aspirate media and avoid disrupting the cell pellet. Judging by the pellet size, resuspend in the appropriate amount of USG media. (Usually between 1-3 ml) Less is better in the beginning so that you can dilute further if you need to. Resuspend gently.</li>
	<li>Add 10 &mu;l of the resuspended cells to a hemacytometer and determine the cell density. Tips: You are looking for epithelial cells that have a distinctly dark cell membrane and are non-columnar. You can also count ciliated cells that are definitely moving. Smaller cells that have a halo appearance are red blood cells and should not be counted. There will be some clumping, so you need to estimate that into you cell count. The seeding target is 1.65 x105 cells/membrane, or at least 75% filter coverage (Figure 1). If cells are plated too sparsely, cells will not be able to form a complete epithelial layer.</li>
	<li>Add 500 &mu;l of the USG media to bottom of the well of a 24 well plate and insert Transwell. The required volume of FT cell suspension (see above) can then be added onto each membrane, this is typically 100 &mu;l.</li>
	<li>Incubate and grow for 1-2 days without disturbing top of membrane. You can change the media on the basal side during those days if necessary. After 1-2 days, you can carefully rinse the top with USG media and add a small amount (50-100 &mu;l) of media on top of membrane. It is easier to determine the confluence of cell cultures if media is removed from the apical side of the filters before microscopy. The lower compartment should always contain media. Once cultures are fully confluent (normally within 5 days), the amount of media that travels up through the filters from the basal to apical sides should be negligible.</li>
	<li>Change the basal media once every two days for the first 10 days. The lower compartment must always have media to keep cells alive. Once the cells have formed a tight epithelial layer (Figures 1 and 2) they can be used in further studies, such as immunofluorescence (IF) or immunohistochemistry (IHC).</li>
</ol>
4. Membrane Processing.
<ol>
<li>Membrane processing is dependent on the type of study that will be performed, but typically involves removal of the filter from the Transwell inserts.</li>
	<li>The filters are typically washed 3 times in 200 &mu;l of 1X PBS before removal.</li>
	<li>Aspirate PBS from apical and basal side of the filters. This should be done 1 well at a time to prevent the membranes from drying out.</li>
	<li>Remove the insert from the plate, flip it upside down and use a sterile scalpel to cut around the membrane. Try to remove the membrane in one piece by making straight cuts around the membrane edge, instead of dragging the scalpel around the edge.</li>
	<li>Use tweezers to remove the filter from the insert, keeping track of which side the cells are on. The filter can then be processed for IF, IHC, etc. as appropriate.</li>
	<li>Example: for IF studies, the filter (after fixation, permeabilization, blocking and incubation with appropriate antibodies, according to standard IF procedures) should be placed on a slide, cells facing UP, Vectashield with DAPI is dropped onto the filter and covered with a glass coverslip.</li>
</ol>
5. Representative Results:
The transwell filters can be readily removed to examine the ex vivo epithelium by both immunohistochemistry (IHC) and immunofluorescence (IF). Using lineage specific markers, one can image and quantify the secretory (Pax8 positive) and ciliated (Sall2 positive) cell compartments in these cultures (Figure 3). Moreover, using these markers, one can monitor how each cell type responds to different physiologic cues 10. This system has been used to characterize the changes in the secretome and intracellular phosphoproteome of FT epithelium in response to various stimuli.
<img src="/files/ftp_upload/2728/2728fig1.jpg" alt="Figure 1" /> 
 Figure 1. Illustration depicting how ex vivo cultures are generated from primary human fallopian tube tissue. Fallopian tube fimbrial tissue is obtained from the surgical suite and minced to generate small fragments 1 that are washed and incubated with dissociation media for 24-72 hours 2. After dissociation is complete, the tissue fragments are allowed to settle to the bottom of the incubation tube and the media containing the dissociated epithelial cells is harvested 3. The efficiency of dissociation can be monitored by examination under a phase-contrast microscope 4. The dissociated epithelial cells are then cultured on Primaria plates to help remove fibroblast and hematopoietic cells that invariably admixed with the epithelial cells 5. Once the non-epithelial cells are adequately removed, the epithelial cells are seeded onto transwell filters that are coated with human placental collagen 5. The media is provided by diffusion through the transwell from below. The cultures are incubated for 24-48 hours and then the apical media is removed. The ex vivo cultures are then allowed to grow for 5-8 days to form a full, complete lawn on the transwell filters. The ex vivo cultures can be maintained viably in this state for up to 4 weeks. The cartoon 5 shows a representation of the fully grown ex vivo culture with an example of a filter removed from the insert and stained with hematoxylin and eosin (H&amp;E) to demonstrate the polarity and architecture of the ex vivo epithelium.
<img src="/files/ftp_upload/2728/2728fig2.jpg" alt="Figure 2" /> 
 Figure 2. Bright field microscopy of FT ex vivo cultures. Transwell inserts are coated with human placental collagen 0.4&mu;m pores are visible (a). FT epithelial cells are cultured on the collagen coated inserts (b), where they form an epithelial layer (c). Some debris is normally observed adhering to cells in the epithelial culture (indicated by arrows), most often to ciliated cells.
<img src="/files/ftp_upload/2728/2728fig3.jpg" alt="Figure 3" /> 
 Figure 3. FT ex vivo culture immunofluorescence (IF). Examples of IF images of ex vivo cultures fixed and stained with antibodies against (a) secretory (Pax8) and (d) ciliated cell (Sall2) markers. DAPI is used as a control for location of cell nuclei (blue) (b and e) and merged antibody and DAPI staining is also shown (c and f). The number of cells depends on the length of time the cells are in culture (Pax8 staining, 7 days; Sall2 staining, 3 days).

<ol>
	<li>Jemal, A., Siegel, R., Xu, J., Ward, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J. Clin. 60, 277-300 (2010).</li><li>Markman, M., Walker, 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=Intraperitoneal+chemotherapy+of+ovarian+cancer:+a+review,+with+a+focus+on+practical+aspects+of+treatment.">Intraperitoneal chemotherapy of ovarian cancer: a review, with a focus on practical aspects of treatment.</a> J. Clin. Oncol. 24, 988-994 (2006).</li><li>Liu, J., Matulonis, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+advances+in+ovarian+cancer.">New advances in ovarian cancer.</a> Oncology (Williston Park). 24, 721-728 (2010).</li><li>Cannistra, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+of+the+ovary.">Cancer of the ovary.</a> N. Engl. J. Med. 351, 2519-2529 (2004).</li><li>Kurman, R. J., Shih, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+pathogenesis+of+epithelial+ovarian+cancer:+a+proposed+unifying+theory.">The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.</a> Am. J. Surg. Pathol. 34, 433-443 (2010).</li><li>Fong, M. Y., Kakar, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+mouse+models:+a+summary+of+current+models+and+their+limitations.">Ovarian cancer mouse models: a summary of current models and their limitations.</a> J Ovarian Res. 2, 12-12 (2009).</li><li>Shan, W., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+ovarian+cancer:+focus+on+genetics+and+animal+models.">Epithelial ovarian cancer: focus on genetics and animal models.</a> Cell Cycle. 8, 731-735 (2009).</li><li>Levanon, K., Crum, C. P., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+pathogenesis+of+serous+ovarian+cancer+and+its+clinical+impact.">New insights into the pathogenesis of serous ovarian cancer and its clinical impact.</a> J. Clin. Oncol. 26, 5284-5293 (2008).</li><li>Karst, A. M., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+pathogenesis:+a+model+in+evolution.">Ovarian cancer pathogenesis: a model in evolution.</a> J Oncol. 2010, 932371-932371 (2010).</li><li>Levanon, K., Ng, V., Piao, H. Y., Zhang, Y., Chang, M. C., Roh, M. H., Kindelberger, D. W., Hirsch, M. S., Crum, C. P., Marto, J. A., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ex+vivo+cultures+of+human+fallopian+tube+epithelium+as+a+model+for+serous+ovarian+carcinogenesis.">Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis.</a> Oncogene. 25, 1103-1113 (2010).</li><li>Landen, C. N., Birrer, M. J., Sood, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+events+in+the+pathogenesis+of+epithelial+ovarian+cancer.">Early events in the pathogenesis of epithelial ovarian cancer.</a> J. Clin. Oncol. 26, 995-1005 (2008).</li><li>Vermeer, P. D., Einwalter, L. A., Moninger, T. O., Rokhlina, T., Kern, J. A., Zabner, J., Welsh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+receptor+and+ligand+regulates+activation+of+epithelial+growth+factor+receptor.">Segregation of receptor and ligand regulates activation of epithelial growth factor receptor.</a> Nature. 422, 322-326 (2003).</li></ol>

Dr. Drapkin is a consultant to Novartis Pharmaceuticals. No other authors declare any potential conflicts of interest.

The identification of the FT as a candidate site of origin for SOC provides the opportunity for basic and translational research aimed at deciphering the mechanisms linking known risk factors and the actual serous carcinogenic process. Critical for this is the development of tractable model systems that will enable us to begin testing the hypothesis that the FTSEC is a cell-of-origin for pelvic serous carcinomas. The ex vivo culture model described herein is a novel system that permits the isolation and co-culture of primary FT secretory and ciliated cells in a manner that preserves the morphology and biology of native FT epithelium. Using this system, we recently characterized the secretome of this epithelium and how the epithelium responds to mechanical and genotoxic injury 10. Ovulation, a major risk factor associated with ovarian tumorigenesis, is characterized by a combination of tissue injury, inflammatory mediators, growth factors, and hormones 11. This system could be used to study the effect of an ovulatory milieu on the response and viability of secretory and ciliated cells. In addition, the impact of other cell types (i.e. inflammatory cells) could be examined to allow a better understanding of the events that drive cell-type differentiation and the factors that contribute to the neoplastic transformation of these cells. Similar models have been described in other tissues where polarized epithelium is present. For instance, in polarized primary cultures of airway epithelium, the effect of heregulin on cell growth and response to cellular injury was assessed 12. These types of model systems provide a new and useful way to study the initiation and pathogenesis of tumors which arise from epithelial tissues, and this is of critical importance in tissues such as the FT where no cell lines currently exist, and where there is a great need for the identification of key pathways and development of novel treatment strategies.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Collagen from human placenta (Bornstein and Traub Type IV)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7521</td>
<td>&nbsp;</td>
<tr>
<td>DMEM:F12 media</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>15-090-CV</td>
<td>&nbsp;</td>
<tr>
<td>Ultroser G</td>
<td>&nbsp;</td>
<td>Crescent Chemical Company</td>
<td>67042</td>
<td>Add 20 ml of DMEM:F12 to stock bottle and use 10mls per 500 ml of media (2% final concentration)</td>
</tr>
<tr>
<td>Pen Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>BD Primaria culture plates</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>353802</td>
<td>&nbsp;</td>
<tr>
<td>24 well Transwell Permeable supports</td>
<td>&nbsp;</td>
<td>Corning (Costar)</td>
<td>3470</td>
<td> Clear, 6.5 mm insert, 0.4 &mu;m pore size, treated polyester </td>
</tr>
<tr>
<td>MEM (Minimal Essential Media)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-010-CV</td>
<td>&nbsp;</td>
<tr>
<td>Pronase</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11459643001</td>
<td>&nbsp;</td>
<tr>
<td>DNAse</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>DN25</td>
<td>&nbsp;</td>
<tr>
<td>Sall2 antibody</td>
<td>&nbsp;</td>
<td>Dr T. Benjamin, Harvard</td>
<td>Gift</td>
<td>1:20 dilution</td>
</tr>
<tr>
<td>Pax8 antibody</td>
<td>&nbsp;</td>
<td>Proteintech</td>
<td>10336-1-AP</td>
<td>1: 1000 dilution</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells at JoVE.com

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 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 ...

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19.6K vistas.  Dana-Farber Cancer Institute. La trompa de Falopio (FT) se está convirtiendo en un sitio alternativo de origen para el carcinoma de ovario seroso (SOC). Este protocolo describe un nuevo método para el aislamiento y Ex vivo Cultivo de células epiteliales del tubo de Falopio. Este recapitula el sistema In vivo y permite el estudio de la patogénesis del SOC.

Video: Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

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

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.

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

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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.

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.

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

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

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.

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