Name: a rapid filter insert-based 3d culture system for primary prostate cell differentiation
Uploaded: 2017-02-13T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+keratinocytes+are+efficiently+immortalized+by+a+Rho+kinase+inhibitor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+reprogrammed+cells+to+identify+therapy+for+respiratory+papillomatosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p63+is+a+prostate+basal+cell+marker+and+is+required+for+prostate+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+cancer+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+the+p53+tumor+suppressor+protein+bridges+the+apoptotic+and+autophagic+signaling+pathways+to+regulate+cell+death+in+prostate+cancer+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analogs+of+the+Novel+Phytohormone,+Strigolactone,+Trigger+Apoptosis+and+Synergy+with+PARP1+Inhibitors+by+Inducing+DNA+Damage+and+Inhibiting+DNA+Repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Drug+Testing+of+Patient-derived+Conditionally+Reprogrammed+Cells+from+Castration-resistant+Prostate+Cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+induces+diffusible+feeder+cell+factor(s)+that+cooperate+with+ROCK+inhibitor+to+conditionally+reprogram+and+immortalize+epithelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpOT+the+Correct+Tissue+Every+Time+in+Multi-tissue+Blocks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+culture+systems+for+prostate+epithelial+and+cancer+tissue.">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

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 ...

Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

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

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 ...

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

Research

JoVE Journal

Cancer Research

8.5K Views.  Lombardi Comprehensive Cancer Center. 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.

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

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

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

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

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.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

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

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

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.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

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

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

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.

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

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

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

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.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

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.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Bioengineering

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

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

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.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

Biology

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

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

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.

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...