The authors would like to thank Brooke Hjelm for her technical expertise and Andrew Larsen for his protein analysis. This work was funded in part by the Alternatives Research Development Foundation (MMHK) Grant and the NIH NIAID Sexually Transmitted Infections and Topical Microbicides Cooperative Research Center IU19 AI062150-01(MMHK). We gratefully acknowledge Biology of Reproduction for reuse of figures.

All steps should be performed under BSL-2 conditions in a Laminar flow hood.
1. Preparing the STLV Bioreactor
<ol>
 <li>Assemble the STLV bioreactor according to manufacturer's protocol and perform detoxification protocol to ensure sterility of the bioreactor. Cover open ports with luer caps and fill the STLV with 95% ethanol for 24 h.</li>
 <li>Remove ethanol and fill the STLV with sterilized distilled water for 24 h.</li>
 <li>Repeat step 1.2 with sterilized distilled water only.</li>
 <li...

<ol>
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J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Hyg. 27, 493-497 (1938).</li><li>Beer, B. E., 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=In+vitro+preclinical+testing+of+nonoxynol-9+as+potential+anti-human+immunodeficiency+virus+microbicide:+a+retrospective+analysis+of+results+from+five+laboratories.">In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories.</a> Antimicrob Agents Chemother. 50, 713-723 (2006).</li><li>Hickey, D. K., Patel, M. V., Fahey, J. V., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immunity+at+mucosal+surfaces+of+the+female+reproductive+tract:+stratification+and+integration+of+immune+protection+against+the+transmission+of+sexually+transmitted+infections.">Innate and adaptive immunity at mucosal surfaces of the female reproductive tract: stratification and integration of immune protection against the transmission of sexually transmitted infections.</a> J. Reprod. Immunol. 88, 185-194 (2011).</li><li>Andersch-Bjorkman, Y., Thomsson, K. A., Holmen Larsson, J. M., Ekerhovd, E., Hansson, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+identification+of+proteins,+mucins,+and+their+O-glycosylation+in+the+endocervical+mucus+during+the+menstrual+cycle.">Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle.</a> Mol. Cell Proteomics. 6, 708-716 (2007).</li><li>Barrila, 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=3D+cell+culture+models:+Innovative+platforms+for+studying+host-pathogen+interactions.">3D cell culture models: Innovative platforms for studying host-pathogen interactions.</a> Nature Reviews Microbiology. 8, 791-801 (2010).</li><li>Vamvakidou, A. P., 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=Heterogeneous+breast+tumoroids:+An+in+vitro+assay+for+investigating+cellular+heterogeneity+and+drug+delivery.">Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery.</a> J. Biomol Screen. 12, 13-20 (2007).</li><li>Jin, F., 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=Establishment+of+three-dimensional+tissue-engineered+bone+constructs+under+microgravity-simulated+conditions.">Establishment of three-dimensional tissue-engineered bone constructs under microgravity-simulated conditions.</a> Artif Organs. 34, 118-125 (2010).</li><li>Vertrees, R. A., 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=Development+of+a+three-dimensional+model+of+lung+cancer+using+cultured+transformed+lung+cells.">Development of a three-dimensional model of lung cancer using cultured transformed lung cells.</a> Cancer Biol Ther. 8, 356-365 (2009).</li><li>Hwang, Y. 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=The+use+of+murine+embryonic+stem+cells,+alginate+encapsulation,+and+rotary+microgravity+bioreactor+in+bone+tissue+engineering.">The use of murine embryonic stem cells, alginate encapsulation, and rotary microgravity bioreactor in bone tissue engineering.</a> Biomaterials. 30, 499-507 (2009).</li><li>Pei, M., He, F., Kish, V. 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The authors have nothing to disclose.

Utilization of the RWV bioreactor technology presented here may provide researchers with the capability to advance their current cell culture system to a more physiologically relevant organotypic cell culture model. The RWV bioreactor cell culture system provides a low shear microenvironment that enables cells to form 3-D cellular aggregates with in vivo-like characteristics, including tight junctions, mucin production, extracellular processes (i.e. microvilli), and cellular polarity. The majority of the data an...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21131</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>FACSDiva</td>
			<td>BD</td>
			<td>&nbsp;</td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>&beta;-tublin antibody</td>
			<td>Calbiochem</td>
			<td>654162</td>
			<td>Used at 1:5000 dilution</td>
		</tr>
		<tr>
			<td>Bio-Plex 2000</td>
			<td>BioRad</td>
			<td>171-000205</td>
			<td>v5 software</td>
		</tr>
		<tr>
			<td>Bioreactor and components</td>
			<td>Synthecon</td>
			<td>RCCS-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>40&mu;m pore size</td>
		</tr>
		<tr>
			<td>Conical tube (50mL)</td>
			<td>Corning</td>
			<td>5-538-60</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48366067</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cytokine bead array kits</td>
			<td>BioRad</td>
			<td>Custom human kit</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cytodex beads</td>
			<td>Sigma</td>
			<td>C3275</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>ED-500G</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Epithelial specific antibody (ESA)</td>
			<td>Chemicon</td>
			<td>CBL251</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10438</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>HARV (Disposable)</td>
			<td>Synthecon</td>
			<td>D-405</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>258148</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Involucrin antibody</td>
			<td>Sigma</td>
			<td>I 9018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>16004-368</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MTT reagent</td>
			<td>MP Biomedicals, LLC</td>
			<td>194592</td>
			<td>3-(4,5-Dimethylthiazolyl 1-2)-2,5-Diphenyl Tetrazolium Bromide</td>
		</tr>
		<tr>
			<td>MUC1 antibody (microscopy)</td>
			<td>Santa Cruz</td>
			<td>Sc-7313</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>MUC1 antibody (flow cytometry)</td>
			<td>BD Pharmingen</td>
			<td>559774</td>
			<td>Also called CD227, use 20&mu;L per test</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Diluted to 4% in DPBS</td>
		</tr>
		<tr>
			<td>Petri dish (small)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube with filter</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene flow tube</td>
			<td>BD Falcon</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PR antibody</td>
			<td>DAKO</td>
			<td>M3569</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td>Mounting media with DAPI</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74903</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma</td>
			<td>71725</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterilization pouch</td>
			<td>VWR</td>
			<td>11213-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stopcocks (one-way)</td>
			<td>Medex</td>
			<td>MX5061L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe (10mL)</td>
			<td>BD</td>
			<td>309604</td>
			<td>Luer-lock tip</td>
		</tr>
		<tr>
			<td>Syringe (5mL)</td>
			<td>BD</td>
			<td>309603</td>
			<td>Luer-lock tip</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vp5 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13525</td>
			<td>HSV-2 antibody Clone 6F10; used at 1:5000 dilution</td>
		</tr>
	</tbody>
</table>

culturing and applications of rotating wall vessel bioreactor derived 3d epithelial cell models

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Watch this Scientific Journal Video about Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models at JoVE.com

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

culturing-applications-rotating-wall-vessel-bioreactor-derived-3d

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Biology

26.1K Görüntüleme.  University of Arizona College of Medicine - Phoenix. Epitel hücreleri 3-D hücre toplam oluşumu ile sonuçlanarak, fizyolojik koşullar altında büyüyebilir olanak sağlayan bir döner hücre kültürü sistemi tarif edilmektedir. Agregalar ekran üretilen In vivo-Gibi özellikleri, geleneksel kültür modellerinde gözlenen ve bilimsel araştırmalar bir çok için daha doğru bir organotipik model sistem olarak hizmet edemez.