Name: A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation (Video) | JoVE Journal
Uploaded: 2017-02-13T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Learn a rapid filter insert-based 3D culture system for primary prostate cell differentiation. This method enables quick biomolecule isolation and supports biologically relevant prostate cancer research.

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5 minutes

This research was supported by T32(CA 9686-18) and TL1 (TL1TR001431) postdoctoral training grant awards (LT), DOD PC140268 (CA), W81XWH-13-1-0327 (CA) TR000102-04 (CA) as well as U01 PAR-12-095 (Kumar) and P30 CA051008-21 (Weiner). Sample fixation, sectioning and staining was performed in the Lombardi Comprehensive Cancer Center Histology and Tissue Shared Resource. We thank Richard Schlegel for helpful discussions. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

1. Establishment of the 3D Cell Culture Insert System
<ol>
	<li>Place 2 polycarbonate cell culture inserts in an inverted orientation (filter side up) down in a 6 well plate (Figure 1A).</li>
	<li>Apply a thin layer of 0.1% gelatin in water to the bottom side of the insert and allow to dry (10-20 min) in a biological safety cabinet to maintain sterility.</li>
	<li>Repeat step 1.2 two more times for a total of 3 applications of 0.1% gelatin on the inserts. 
	NOTE: The gelatin coating will help limit diffusion between the chambers.</li>
	<li>To collect the primary CRCs from standard culture conditions, add 5 mL of PBS to wash the culture flask.
	<ol>
		<li>Trypsinize the cells using (1 mL for a T-25 and 2 mL for a T-75) 0.25% trypsin-EDTA for 5 min at 37 &#176;C. Gently tap the side of the flask to aid in dislodging the cells. Then proceed to stop the trypsin reaction and collect the cells by adding 5 mL of complete DMEM.</li>
		<li>Collect the cells in a 15 mL tube. Centrifuge the tube at 4 &#176;C and 188 x g and count using an automated cell counter or a hemocytometer.</li>
		<li>Re-suspend 600,000 CRCs in 250 &#956;L conditioned media (CM) and add to the properly oriented (filter side down) culture insert (Figure 1B). This is referred to as the inner chamber. 
		Note: CM is F-media conditioned by irradiated J2 cells and contains 10 &#956;m Y-27632 as previously described7,8,9,11.</li>
	</ol>
	</li>
	<li>Apply 2 mL of CM to the outer chamber of the 6 well plate and incubate at 37 &#176;C overnight (for at least 18 h).</li>
	<li>Carefully remove the CM on the inner chamber with a 1 mL or 200 &#956;L pipette and slowly add differentiation media (DM) to the inner chamber with a 1 mL pipette until full. Be careful to not disturb the cell layer.</li>
</ol>
2. Maintenance of 3D Cultures
<ol>
	<li>Check the cultures every day. Healthy cells appear as shown in Figure 2.</li>
	<li>Change the CM in the outer chamber every day or every other day depending on the metabolism of the cells.</li>
	<li>When the media of the inner chamber changes color (yellow), carefully remove the media on the inner filter chamber with a 1 mL or 200 &#956;L pipette.</li>
	<li>Aspirate the media in the outer 6 well plate chamber with an aspirating tip.</li>
	<li>Using a 1 mL pipette, slowly apply fresh DM to the inner chamber until full. Be careful not to scrape or otherwise dislodge the cell layer.</li>
	<li>Replace the media in the outer chamber with 2 mL of fresh CM.</li>
	<li>Maintain the cultures for 2 weeks, and then proceed to processing for the desired bimolecular isolation. 
	NOTE: Each row in the 6 well plate, a total of six inserts (Figure 1B), should be processed per experiment to acquire enough cells for DNA, RNA or protein for analysis.</li>
	<li>Aspirate media in the outer chamber and rinse with 2 mL phosphate buffered saline (PBS).</li>
	<li>Carefully remove media from the inner chamber with a 1 mL or 200 &#956;L pipette and add 500 &#956;L PBS. Avoid scraping or otherwise disturbing the cells on the membrane.</li>
	<li>Aspirate PBS from the outer chamber and carefully remove PBS from the inner chamber with a 1 mL or 200 &#956;L pipette. Once the PBS has been removed, proceed directly to the appropriate application detailed below. Do not allow the membrane to dry out. The culture can be kept briefly in the PBS until the application is ready to begin.</li>
</ol>
3. Processing the Filters for Pellet Banking
<ol>
	<li>Add 100 &#956;L&#160; 0.25% trypsin-EDTA to each inner chamber and incubate for 5 min at 37 &#176;C.</li>
	<li>Briefly and carefully agitate/scrape the cells on the membrane surface with a 200 &#956;L pipette while pipetting up and down (avoid puncturing the membrane). Incubate for 1 min at 37 &#176;C.</li>
	<li>Add 250 &#956;L of CM to the first inner chamber and pipette up and down gently to rinse the filter.</li>
	<li>Transfer resuspended cells to the adjacent filter chamber that contains the same media conditions and repeat pipetting up and down.</li>
	<li>Repeat this procedure for each of the inserts of a single condition. Collect and transfer cells to a 1.5 mL centrifuge tube.</li>
	<li>Rinse each inner chamber with an additional 100-200 &#956;L of CM to collect any remaining cells. Add to the 1.5 mL centrifuge tube in step 3.5.</li>
	<li>Spin the cells at 423 x g in a microcentrifuge at 4 &#176;C for 5 min.</li>
	<li>Aspirate the supernatant and wash the cell pellet once with 1000 &#956;L PBS.</li>
	<li>Spin again at 423 x g in a microcentrifuge at 4 &#176;C for 5 min.</li>
	<li>Aspirate PBS and freeze at -80 &#176;C for later use.</li>
</ol>
4. Processing the Filters for RNA or DNA Isolation
<ol>
	<li>Add 200 &#956;L of guanidinium thiocyanate-phenol-chloroform or similar extraction reagent to the inner chamber and briefly agitate the cells on the membrane surface by pipetting up and down.</li>
	<li>Incubate in the extraction reagent for 5 min at room temperature with the outer chamber dry.
	<ol>
		<li>As the filter may dissociate from the insert, wash the disassociated filter membrane by pipetting the extraction solution up and down. Collect as much of the extraction reagent as possible by tilting the 6 well plate and aspirating any residual liquid.</li>
	</ol>
	</li>
	<li>Follow the manufacturer&#39;s protocol for purification and recovery of DNA, RNA or protein.</li>
</ol>
5. Processing the Filters for Protein Isolation
<ol>
	<li>Place the filter insert in the 6 well plate on ice. To the inner chamber, add 10 &#956;L of protein lysis buffer supplemented with 1 mM sodium fluoride (NaF), 1 mM sodium vanadate (NaV), 100 mM dithiothreitol (DTT) and 100 &#956;L of anti-protease cocktail.</li>
	<li>Agitate/scrape the cells on the membrane surface with a 20 &#956;L pipette while pipetting up and down. Avoid generating bubbles in the lysis buffer.</li>
	<li>Incubate the 6 well plate with filter inserts on ice for 10 min.</li>
	<li>Repeat scraping and then rinse the membrane surface with the lysis buffer.</li>
	<li>Collect the lysates from the 6 inserts and transfer to a 1.5 mL centrifuge tube on ice.</li>
	<li>Rinse the first membrane with an additional 10 &#956;L of lysis buffer and transfer to the next filter.</li>
	<li>Repeat step 5.6 for all inserts, collecting any residual lysis buffer solution and add to the 1.5 mL tube (step 5.5).</li>
	<li>Incubate the sample on ice for an additional 5 min.</li>
	<li>Spin at maximum speed (16,873 x g in a table top centrifuge) for 15 min at 4 &#176;C.</li>
	<li>Pipette lysate supernatant into a fresh 1.5 mL tube.</li>
	<li>Proceed to the analysis of protein concentration or store lysates at -80 &#176;C.</li>
</ol>
6. Processing for H&#38;E and Immuno-staining
<ol>
	<li>Add 500 &#181;L and 1 mL of 10% NBF (neutral buffered formalin) to the inner and outer chamber and let incubate overnight at 4 &#176;C.</li>
	<li>Aspirate the NBF from the outer chamber and add 1 mL hydroxyethyl agarose (HEA) processing gel to a 1.5 mL centrifuge. Slowly melt the agarose in a microwave using low power and repeated 10-20 s pulses until the agarose is melted.</li>
	<li>Keep the HEA in a 37 &#176;C warm bath to prevent solidification until ready to use.</li>
	<li>Remove the NBF from the inner chamber and apply 25 &#956;L of molten HEA to the inner chamber and allow the agarose to solidify for 2-5 min.</li>
	<li>Wet 2 foam histology pads in 10% NBF and place one pad in an embedding cassette (see Figure 3D).</li>
	<li>Use a #11 blade scalpel (which has a very sharp, fine-pointed blade) to score the filter from the bottom side of the insert chamber, partially releasing it from the plastic insert chamber.</li>
	<li>Place a small amount (100-200 &#956;L) of NBF in a Petri dish and immerse the partially dislodged filter in the NBF (Figure 3A).</li>
	<li>With a #10 blade scalpel, gently press against the middle of the filter, from the inside of the insert chamber, to fully dislodge the filter from the insert barrel (Figure 3B). If there is any part of the filter that is still attached to the barrel, sever it with the scalpel.</li>
	<li>Cut the HEA-coated filter in half with the #10 blade scalpel (Figure 3C).</li>
	<li>Place each half of the filter onto the foam pad in the histology cassette prepared in step 6.4 (Figure 3D).</li>
	<li>Add the second 10% NBF soaked sponge to the cassette, sandwiching the filter.</li>
	<li>Snap seal the cassette, place in NBF, and incubate overnight.</li>
	<li>Process filters into paraffin blocks for sectioning and staining as described previously12.</li>
</ol>

<ol><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Organoid cultures derived from patients with advanced prostate cancer.</a> Cell. 159 (1), 176-187 (2014).</li>
<li>Chapman, S., Liu, X., Meyers, C., Schlegel, R., McBride, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+keratinocytes+are+efficiently+immortalized+by+a+Rho+kinase+inhibitor%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.</a> J Clin Invest. 120 (7), 2619-2626 (2010).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.</a> Am J Pathol. 180 (2), 599-607 (2012).</li>
<li>Yuan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+reprogrammed+cells+to+identify+therapy+for+respiratory+papillomatosis%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Use of reprogrammed cells to identify therapy for respiratory papillomatosis.</a> N Engl J Med. 367 (13), 1220-1227 (2012).</li>
<li>Signoretti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((p63+is+a+prostate+basal+cell+marker+and+is+required+for+prostate+development%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">p63 is a prostate basal cell marker and is required for prostate development.</a> Am J Pathol. 157 (6), 1769-1775 (2000).</li>
<li>Lang, S. H., Frame, F. M., Collins, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prostate+cancer+stem+cells%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">Prostate cancer stem cells.</a> J Pathol. 217 (2), 299-306 (2009).</li>
<li>Ringer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+induction+of+the+p53+tumor+suppressor+protein+bridges+the+apoptotic+and+autophagic+signaling+pathways+to+regulate+cell+death+in+prostate+cancer+cells%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">The induction of the p53 tumor suppressor protein bridges the apoptotic and autophagic signaling pathways to regulate cell death in prostate cancer cells.</a> Oncotarget. 5 (21), 10678-10691 (2014).</li>
<li>Pollock, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strigolactone+analogues+induce+apoptosis+through+activation+of+p38+and+the+stress+response+pathway+in+cancer+cell+lines+and+in+conditionally+reprogrammed+primary+prostate+cancer+cells%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.</a> Oncotarget. 5 (6), 1683-1698 (2014).</li>
<li>Crogliom, M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/26910887">Analogs of the Novel Phytohormone, Strigolactone, Trigger Apoptosis and Synergy with PARP1 Inhibitors by Inducing DNA Damage and Inhibiting DNA Repair.</a> Oncotarget. , (2016).</li>
<li>Saeed, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+Drug+Testing+of+Patient-derived+Conditionally+Reprogrammed+Cells+from+Castration-resistant+Prostate+Cancer%5BTitle%5D)+AND+%22Eur+Urol%22%5BJournal%5D)">Comprehensive Drug Testing of Patient-derived Conditionally Reprogrammed Cells from Castration-resistant Prostate Cancer.</a> Eur Urol. , (2016).</li>
<li>Palechor-Ceron, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiation+induces+diffusible+feeder+cell+factor%28s%29+that+cooperate+with+ROCK+inhibitor+to+conditionally+reprogram+and+immortalize+epithelial+cells%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells.</a> Am J Pathol. 183 (6), 1862-1870 (2013).</li>
<li>Coffey, A., Johnson, M. D., Berry, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SpOT+the+Correct+Tissue+Every+Time+in+Multi-tissue+Blocks%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">SpOT the Correct Tissue Every Time in Multi-tissue Blocks.</a> J Vis Exp. (99), e52868(2015).</li>
<li>Hidalgo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+xenograft+models%3A+an+emerging+platform+for+translational+cancer+research%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discov. 4 (9), 998-1013 (2014).</li>
<li>Drost, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+culture+systems+for+prostate+epithelial+and+cancer+tissue%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Organoid culture systems for prostate epithelial and cancer tissue.</a> Nat Protoc. 11 (2), 347-358 (2016).</li>
</ol>

The authors have nothing to disclose.

Primary cell lines are an important and rapidly developing platform for cancer research. The culture insert-based 3D culture system supports the differentiation of primary prostate CRCs within a two-week timeframe. The CRC filter method represents a new, medium throughput method for prostate research. Existing mouse PDX models are time consuming and extremely expensive, and many of the PDX samples cannot be grown in culture, limiting investigator-initiated experimentation and their usefulness. In addition, while success ...

The identification and use of cancer therapies that are personalized to individuals are a primary goal in cancer research. Recently, new approaches have been developed that allow for greater ease in the establishment of primary cell cultures, potentially providing ways of both identifying and testing personalized therapies. For example, the R-spondin-based prostate organoid approach1 allows for three-dimensional (3D) culturing of normal and metastatic prostate cancer cells in a commercial extracellular matrix (e.g., Matrigel), while the Conditionally Reprogramming of Cells (CRC) method developed at Georgetown2,3 utilizes more standard 2D culture conditions. Specifically, the combination of a Rho kinase inhibitor (Y-27632) and irradiated J2 murine fibroblast feeder cells lead to the indefinite culturing of keratinocyte CRCs2. The CRC methodology is extremely robust, with primary cell lines successfully established and maintained indefinitely from the prostate and many other normal and malignant epithelial tissues3. Importantly, our CRC technology allowed for the rapid identification of the etiological basis for recurrent respiratory papillomatosis in a patient who had failed a number of previous drug treatments. In addition, using normal and tumor-derived CRCs, the successful identification of an FDA approved drug, vorinostat, was made within two weeks of initial tissue biopsy. The patient was placed on vorinostat, resulting in the successful treatment of their disease4.
The normal prostate gland is comprised of luminal, basal and the rare neuroendocrine cells5. Luminal cells form the columnar epithelial layer of the gland and express the androgen receptor (AR), as well as other luminal markers such as cytokeratins 8 and 18 and prostate-specific antigen (PSA)6. Conversely, basal cells are localized beneath the luminal layer and express cytokeratin 5 and p63, but low levels of the AR5. We7,8,9 and others10 have successfully used prostate CRCs in preclinical mechanistic drug sensitivity investigations. However, when grown under standard 2D tissue culture conditions, these cells fail to fully engage AR signaling10. Importantly, when placed under the renal capsule of immunodeficient mice, the CRCs regained normal prostate glandular architecture and function indicating that prostate CRCs retain their lineage commitment when placed in a permissive environment. The development of the filter insert-based cell culture system described here allows for the rapid (2 weeks) in vitro differentiation of prostate CRCs as evidenced by the increased expression of the AR and AR target genes as well as decreased levels of p63.
The filter inserts used contain polycarbonate membranes (pore size, 0.4 &#181;m) that can support the culturing of mammalian cells. The system, as developed, makes use of normal and malignant prostate CRCs, 6 well culture dishes and the filter inserts. Cell culture media conditioned by J2 cells11 is placed in the bottom chamber and prostate differentiating media in the top chamber. The techniques described herein support prostate luminal cell differentiation within a 2 week timeframe, consistent with the goals of personalized medicine. It was also imperative to develop the methodologies that allow for the comprehensive molecular, genetic and cellular profiling of the cultures. Approaches for isolating DNA, RNA and protein from cells released from the filter surface have been developed and streamlined for accurate repeat sample processing. Finally, the methodology required for removing the filter to enable embedding, sectioning and for H&#38;E, immunohistochemical and immunofluorescent staining, is fully described.

<table><tbody><tr><td>Corning Costar Snapwell Culture Inserts</td><td>Corning</td><td>3801</td><td></td></tr><tr><td>Millicell Cell Culture Inserts</td><td>Millipore</td><td>PIHP01250</td><td></td></tr><tr><td>Multiwell 6 Well</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Gelatin 0.1% in water</td><td>Stemcell Technologies</td><td>7903</td><td></td></tr><tr><td>Sterile Saftey Scapel 10 Blade</td><td>Integra Miltex</td><td>4-510</td><td></td></tr><tr><td>Sterile Standard Scalpel 11 Blade</td><td>Integra Miltex</td><td>4411</td><td></td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>Thermofisher Scientific</td><td>89900</td><td></td></tr><tr><td>Sodium Fluoride</td><td>Fishcer Scientific</td><td>S299-100</td><td></td></tr><tr><td>Sodium Vanadate</td><td>Fishcer Scientific</td><td>13721-39-6</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3483123</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and Pepstatin A)</td><td>Sigma-Aldrich</td><td>P8340</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Thermofisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher Scientific</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>Glutamine</td><td>Thermofisher Scientific</td><td>25030081&amp;nbsp;</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermofisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>F-12 Nutrient Mix Media</td><td>Thermofisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Y-27632 dihydrochloride Rock inhibitor</td><td>Enzo</td><td>ALX-270-333-M025</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888</td><td></td></tr><tr><td>EGF</td><td>Thermofisher Scientific</td><td>PHG0315</td><td></td></tr><tr><td>Insulin</td><td>Thermofisher Scientific</td><td>12585-014</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C3012</td><td></td></tr><tr><td>Gentamicin</td><td>Thermofisher Scientific</td><td>15710-064</td><td></td></tr><tr><td>Fungizone</td><td>Fisher Scientific</td><td>BP264550</td><td></td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>Histogel Specimen Processing Gel (hydroxyethyl agarose (HEA))</td><td>Thermo Scientific</td><td>HG-4000-012</td><td></td></tr><tr><td>Non-Adherent Dressing</td><td>Telfa</td><td>KDL2132Z</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Sigma</td><td>SIAL0167</td><td></td></tr><tr><td>HISTOSETTE II Tissue Processing / Embedding Cassettes</td><td>Crystalgen</td><td>CG-M492</td><td></td></tr></tbody></table>

a rapid filter insert-based 3d culture system for primary prostate cell differentiation

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living biobanks&#34; of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research.

The cell culture insert based system is a relatively simple and rapid procedure for producing 3D cultures of prostate CRCs that supports luminal cell differentiation. A schematic of the system is shown (Figure 1A) highlighting the application of the gelatin coating to the bottom surface of the filter insert. The inserts are only overturned for the application of the gelatin. In Figure 1B the filters are in the appropriate orientation for culturing. Using ...

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

The overall goal of this filter insert-based 3D culture system for patient derived primary cells is to establish a more biologically relevant in vitro model system for prostate cancer research. By using primary human cells, this method can help answer key questions in prostate developmental biology as well as in prostate cancer. The main advantage of this technique is that it is a relatively rapid in vitro methodology for the establishment of differentiated primary prostate cells which also allows for the rapid isolation of biomolecules such as RNA, DNA and protein.Visual demonstration of these methods are critical as the processing of cells on the filter insert for histology or for RNA and protein collection are delicate and time sensitive. To help limit diffusion between the chambers in a biological safety cabinet, apply a thin layer of 0.1%gelatin to the bottom side of the inserts and allow them to dry. Repeat this procedure two more times.Then apply the cells to the inner chamber of the gelatin coated inserts and culture them for up to two weeks. After two weeks apply the cultured filter inserts directly to one of the appropriate downstream applications detailed in the followings. To begin this procedure, aspirate PBS from the outer chamber and carefully remove PBS from the inner chamber.The cultured inserts can be kept briefly in PBS until the application is ready to begin but do not allow the membrane to dry out. Next add 100 microliters of 0.25%Trypsin EDTA to each inner chamber. Then incubate them for five minutes at 37 degrees Celsius.Afterward briefly and carefully scrape the cells on the membrane surface with a 200 microliter pipette while pipetting up and down. Then incubate them for one minute at 37 degrees Celsius. After one minute add 250 microliters of conditioned media to the first inner chamber and pipette up and down gently to rinse the filter.Following this transfer the resuspended cells to the adjacent filter chamber that contains the same media conditions and repeat pipetting up and down. Repeat this procedure for each of the inserts of a single condition. Then collect the cells and transfer them to a 1.5 milliliter centrifuge tube.Rinse each inner chamber with an additional 100 to 200 microliters of conditioned media to collect any remaining cells and add them to the 1.5 milliliter centrifuge tube. Next spin the cells in a microcentrifuge at four degrees Celsius for five minutes. Afterward aspirate the supernatant and wash the cell pellet once with 1, 000 microliters of PBS.Spin the cells again in a microcentrifuge at four degrees Celsius for five minutes. After five minutes aspirate the PBS and freeze the sample at minus 80 degrees Celsius for later use. In this procedure add 200 microliters of guanidinium thiocyanate phenol chloroform or similar extraction reagent to the inner chamber and briefly agitate the cells on the membrane surface by pipetting up and down.Then incubate the cells in the extraction reagent for five minutes at room temperature and leave the outer chamber dry. Afterward wash the filter membrane by pipetting the extraction solution up and down. Collect as much of the extraction reagent as possible by tilting the six well plate and aspirating any residual liquid.Please note that the filter may dissociate from the insert. Wash the dissociated filter membrane if attached. Then collect as much of the extraction reagent as possible and follow the manufacturer's protocol for purification and recovery of DNA, RNA or protein.Care should be exercised when washing the cells and filter with guanidinium thiocyanate as excessive agitation can yield poor quality RNA. In order to isolate protein from the filter insert, place the filter insert in the six well plate on ice. Then add 10 microliters of protein lysis buffer to the inner chamber.Agitate and scrape the cells on the membrane surface with a 20 microliter pipette while pipetting up and down and avoid generating bubbles in the lysis buffer. Afterward incubate the six well plate with filter inserts on ice for ten minutes. Repeat scraping and then rinse the membrane surface with the lysis buffer.Collect the lysates from the six inserts and transfer them to a 1.5 milliliter centrifuge tube on ice. Next rinse the first membrane with an additional 10 microliters of lysis buffer and transfer it to the next filter. Repeat this procedure for all the inserts of a given experimental condition, collecting any residual lysis buffer solution and add to the 1.5 milliliter tube.Incubate the sample on ice for an additional five minutes. Then spin it at maximum speed for 15 minutes at four degrees Celsius. When it is done pipette the lysate into a fresh 1.5 millimeter tube.Then proceed to the analysis of protein concentration or store the lysates at minus 80 degrees Celsius. In this procedure add 500 microliters and one milliliter of 10%NBF to the inner and outer chamber respectively. Incubate them overnight at four degrees Celsius.The next day aspirate NBF from the outer chamber and add one milliliter of HEA processing gel to a 1.5 milliliter centrifuge tube. Slowly melt the agarose in a microwave using low power and repeated 10 seconds pulses until the agarose is melted. Keep the HEA in a 37 degrees Celsius warm bath to prevent solidification until ready to use.Next remove NBF from the inner chamber. Apply 25 microliters of molten HEA to the inner chamber and allow the agarose to solidify for two to five minutes. Then wet two foam histology pads in 10%NBF and place one pad in an embedding cassette.Safeguarding the integrity of the cell layers on the filter insert using HistoGel is a critical step for preservation of the intact cell layer for histological sectioning. After that use a number 11 blade scalpel to score the filter from the bottom side of the plastic insert chamber, partially releasing it. Place 100-200 microliters of NBF in a petri dish and immerse the partially dislodged filter in the NBF.With a number 10 blade scalpel, gently press against the middle of the filter from the inside of the insert chamber to fully dislodge the filter from the insert barrel. If there is any part of the filter that is still attached to the barrel, sever it with the scalpel. Subsequently cut the HEA coated filter in half.Place each half of the filter onto the foam pad in the prepared histology cassette. Then add the second 10%NBF soaked sponge to the cassette, sandwiching the filter. Next snap seal the cassette.Place it in NBF and incubate it overnight. This bright field image shows multilayered cell strata on top of the porous membrane. The individual fluorescent images for nuclei, P63 and the androgen receptor are shown, as is the merge of all three fluorescence markers.Finally a composite bright field and immunofluorescence overlay is shown, establishing the localization of the fluorescent signal relative to the cells and the filter. Here a cross sectional image of our filter insert is shown, compared to the ductal epithelial layers of an intact prostate. Once mastered this technique can be done in two weeks with a final isolation of the cells and filter insert or the desired biomolecule taking less than 30 minutes if performed properly.While performing this procedure, it's important to always remember to exercise caution when working with the filter inserts, so that no damage to the cell layer or underlying filter occurs. Following this procedure, other methods such as Western blots, RNA microarray and proteome analyses can be performed in order to answer additional questions pertaining to pathway analysis and cancer cells. This technique will help pave the way for researchers in the field of prostate cancer to better explore prostate biology as well as underpinnings of prostate cancer using primary prostate cells.After watching this video you should have an excellent understanding of how to properly set up and recover cultured primary prostate cells from this 3D culture system.

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Research

JoVE Journal

Cancer Research

In this video, we demonstrate the culturing of isolated mouse prostate epithelial cells using a matrix gel-ring based culture technique. This approach can provide complementary information about the differentiation capacity of prostate epithelial cells to form 3D glandular structures in response to genetic or pharmacological manipulation.

Prostate Organoid Assay: A Matrix Gel Ring-based Ex Vivo Culture Technique to Study the Differentiation Capacity of Prostate Epithelial Cells

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

Handling and Assessment of Human Primary Prostate Organoid Culture

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

This protocol provides a comprehensive procedure to isolate, expand, and immortalize fibroblasts from radical prostatectomies. Moreover, it describes assays developed to assess the functional effects of fibroblast-tumor cell crosstalk, taking advantage of both treatment with conditioned medium and co-culturing.

Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts

Learn a rapid filter insert-based 3D culture system for primary prostate cell differentiation. This method enables quick biomolecule isolation and supports biologically relevant prostate cancer research.

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

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