This work was supported by the US National Institutes of Health grant NIH/NICHD #HD051998 (to C.A.M.).

1. Collagen Bead Preparation
<ol>
	<li>Prior to loading EVTs for 3-D cell culture, one needs to prepare the Cytodex-3 microcarrier beads:
	<ol>
		<li>Weigh out the appropriate amount of Cytodex-3 beads required for the experiment. This protocol is adapted for the 10ml RCCS vessel, in which 0.05g of beads are needed. For a 50ml RCCS vessel, scale accordingly. In a 50mL autoclavable conical tube, mix 250 mg Cytodex-3 beads with 12mL Dulbecco phosphate buffered solution (DPBS). This amount is sufficient for 5 RCCS vessels...

<ol>
	<li>Knofler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+growth+factors+and+signalling+pathways+controlling+human+trophoblast+invasion.">Critical growth factors and signalling pathways controlling human trophoblast invasion.</a> Int. J. Dev. Biol. 54, 269-269 (2010).</li><li>Cartwright, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodelling+at+the+maternal-fetal+interface:+relevance+to+human+pregnancy+disorders.">Remodelling at the maternal-fetal interface: relevance to human pregnancy disorders.</a> Reproduction. 140, 803-803 (2010).</li><li>Harris, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFPA+Gabor+Than+Award+lecture:+Transformation+of+the+spiral+arteries+in+human+pregnancy:+key+events+in+the+remodelling+timeline.">IFPA Gabor Than Award lecture: Transformation of the spiral arteries in human pregnancy: key events in the remodelling timeline.</a> Placenta. 32, S154-S154 (2011).</li><li>Whitley, G. S., Cartwright, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast-mediated+spiral+artery+remodelling:+a+role+for+apoptosis.">Trophoblast-mediated spiral artery remodelling: a role for apoptosis.</a> J. Anat. 215, 21-21 (2009).</li><li>Apps, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+profile+of+first+trimester+villous+and+extravillous+human+trophoblast+cells.">Genome-wide expression profile of first trimester villous and extravillous human trophoblast cells.</a> Placenta. 32, 33-33 (2011).</li><li>Bilban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast+invasion:+assessment+of+cellular+models+using+gene+expression+signatures.">Trophoblast invasion: assessment of cellular models using gene expression signatures.</a> Placenta. 31, 989-989 (2010).</li><li>LaMarca, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+growth+of+extravillous+cytotrophoblasts+promotes+differentiation+and+invasion.">Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion.</a> Placenta. 26, 709-709 (2005).</li><li>Jovanovic, M., Stefanoska, I., Radojcic, L., Vicovac, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-8+(CXCL8)+stimulates+trophoblast+cell+migration+and+invasion+by+increasing+levels+of+matrix+metalloproteinase+(MMP)2+and+MMP9+and+integrins+alpha5+and+beta1.">Interleukin-8 (CXCL8) stimulates trophoblast cell migration and invasion by increasing levels of matrix metalloproteinase (MMP)2 and MMP9 and integrins alpha5 and beta1.</a> Reproduction. 139, 789-789 (2010).</li><li>Husslein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+regulation+and+functional+characterization+of+matrix+metalloproteinase-3+of+human+trophoblast.">Expression, regulation and functional characterization of matrix metalloproteinase-3 of human trophoblast.</a> Placenta. 30, 284-284 (2009).</li><li>Barrila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+3D+cell+culture+models:+using+the+rotating+wall+vessel+to+study+host-pathogen+interactions.">Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions.</a> Nat. Rev. Microbiol. 8, 791-791 (2010).</li><li>Hammond, T. G., Hammond, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+suspension+culture:+the+rotating-wall+vessel.">Optimized suspension culture: the rotating-wall vessel.</a> Am. J. Physiol. Renal. Physiol. 281, 12-12 (2001).</li><li>Unsworth, B. R., Lelkes, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+tissues+in+microgravity.">Growing tissues in microgravity.</a> Nat. Med. 4, 901-901 (1998).</li><li>Schmeichel, K. L., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue-specific+signaling+and+organ+function+in+three+dimensions.">Modeling tissue-specific signaling and organ function in three dimensions.</a> J. Cell. Sci. 116, 2377-2377 (2003).</li><li>Bentrup, H. ?. ?. n. e. r. z. u., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+models+of+human+colonic+epithelium+to+study+the+early+stages+of+enteric+salmonellosis.">Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis.</a> Microbes. Infect. 8, 1813-1813 (2006).</li><li>Carterson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A549+lung+epithelial+cells+grown+as+three-dimensional+aggregates:+alternative+tissue+culture+model+for+Pseudomonas+aeruginosa+pathogenesis.">A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.</a> Infect. Immun. 73, 1129-1129 (2005).</li><li>Myers, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+phenotypic+gap+between+transformed+neuronal+cell+lines+in+culture+and+untransformed+neurons.">Closing the phenotypic gap between transformed neuronal cell lines in culture and untransformed neurons.</a> J. Neurosci. Methods. 174, 31-31 (2008).</li><li>Hjelm, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+three-dimensional+organotypic+human+vaginal+epithelial+cell+model.">Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model.</a> Biol. Reprod. 82, 617-617 (2010).</li><li>Straub, T. M. <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+cell+culture+infectivity+assay+for+human+noroviruses.">In vitro cell culture infectivity assay for human noroviruses.</a> Emerg. Infect. Dis. 13, 396-396 (2007).</li><li>Nickerson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+assemblies:+novel+models+for+the+study+of+Salmonella+enterica+serovar+Typhimurium+pathogenesis.">Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis.</a> Infect. Immun. 69, 7106-7106 (2001).</li><li>Carvalho, H. M., Teel, L. D., Goping, G., O'Brien, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+tissue+culture+model+for+the+study+of+attach+and+efface+lesion+formation+by+enteropathogenic+and+enterohaemorrhagic+Escherichia+coli.">A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli.</a> Cell. Microbiol. 7, 1771-1771 (2005).</li><li>Sainz, B., TenCate, V., Uprichard, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Huh7+cell+culture+system+for+the+study+of+Hepatitis+C+virus+infection.">Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection.</a> Virol. J. 6, 103-103 (2009).</li><li>Lelkes, P. I., Ramos, E., Nikolaychik, V. V., Wankowski, D. M., Unsworth, B. R., Goodwin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTSF-2:+a+new,+versatile+cell+culture+medium+for+diverse+normal+and+transformed+mammalian+cells.">GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.</a> In Vitro Cell. Dev. Biol. Anim. 33, 344-344 (1997).</li><li>Lelkes, P. I., Ramos, E., Nikolaychik, V. V., Wankowski, D. M., Unsworth, B. R., Goodwin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTSF-2:+a+new,+versatile+cell+culture+medium+for+diverse+normal+and+transformed+mammalian+cells.">GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.</a> In Vitro Cell. Dev. Biol. Anim. 33, 344-344 (1997).</li><li>GE Healthcare. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+-+Principles+and+Methods.">Microcarrier Cell Culture - Principles and Methods.</a> Handbooks. , (2005).</li></ol>

We have nothing to disclose.

The culture technique presented here provides investigators with highly invasive EVT-like cells. It has now been recognized that a loss of differentiation occurs in monolayers due to inhibition of cellular responses to chemical and molecular cues in three dimensions (apical, basal, and lateral cell surfaces)10,13. This technique reflects characteristics noted in utero on invading EVT cells. As the procedure mimics conventional monolayer tissue culture time kinetics, but provides cells with differentia...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Cytodex microcarrier beads</td>
 <td>Sigma-Aldrich</td>
 <td>C3275</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rotating Cell Culture System (RCCS)</td>
 <td>Synthecon</td>
 <td>RCCS-D</td>
 <td>Includes rotor base, power supply, 4 disposable RCCS units</td>
 </tr>
 <tr>
 <td>RCCS Disposable Units</td>
 <td>Synthecon</td>
 <td>Contact Synthecon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3ml Luer-Lock tip syringe</td>
 <td>BD</td>
 <td>309585</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10ml wide-tip serological pipette</td>
 <td>BD</td>
 <td>357504</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MEM Alpha</td>
 <td>Invitrogen</td>
 <td>12561-072</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 medium, powder</td>
 <td>Invitrogen</td>
 <td>41300-039</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>H2O, Endotoxin free</td>
 <td>Fisher</td>
 <td>MT-25-055-CM </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate</td>
 <td>Sigma-Aldrich</td>
 <td>S-7795</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peptone</td>
 <td>Fisher Scientific</td>
 <td>BP1420-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fructose</td>
 <td>Sigma-Aldrich</td>
 <td>F3510-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Galactose</td>
 <td>Sigma-Aldrich</td>
 <td>G5388-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G7528-250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Invitrogen</td>
 <td>15630-080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Invitrogen</td>
 <td>25030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Insulin-Transferrin-Sodium Selenite (ITS)</td>
 <td>Sigma-Aldrich</td>
 <td>I1884</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Invitrogen</td>
 <td>10437</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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16.4K ビュー.  Tulane University Medical School. 伝統的な、二次元細胞培養技術は、しばしば、分化マーカー、サイトカインおよび増殖因子に関する変更特性になる。 extravillous栄養膜細胞株を以下に示すように回転する細胞培養系における三次元細胞培養（RCCS）は、これらの要因の多くの表現を再確立します。