Organotypic epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents

Commonly used acronym: OERCs

Scope of the method

The Method relates to
  • Animal health
  • Human health
The Method is situated in
  • Basic Research
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Humans - lambs
Type of cells/tissues/organs
Keratinocytes derived from neonatal foreskins and epithelial cells from tonsils


Method keywords
  • Organotypic epithelial raft cultures
  • Keratinocyte differentiation
  • Skin mimic
  • Viral replication
  • Tumor cell growth
Scientific area keywords
  • Skin equivalents
  • 3D keratinocytes-tumor cells co-cultures
  • virus growth
  • Antiviral/antitumor activitity
Method description

Organotypic epithelial raft cultures accurately reproduce the process of epithelial differentiation in vitro and can be prepared from normal keratinocytes, explanted epithelial tissue, or established cell lines. Normal primary human keratinocytes (PHKs) stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while transformed cell lines exhibit dysplastic morphologies similar to the (pre)neoplastic lesions seen in vivo. This three-dimensional (3D) culture system provides an essential tool for either alone or co-cultured with PHKs, tumor biology and selectivity of antitumor agents can be analyzed. For the preparation of dermal equivalents, a collagen matrix solution is made with rat-tail collagen mixed on ice with 10-fold concentrated Ham's F-12 medium, 10-fold concentrated reconstitution buffer, and 3T3 J2 murine fibroblasts. One milliliter of the collagen matrix solution is poured into 24-well plates. After equilibration of the gel with 1 ml of growth medium overnight at 37°C, 2.5 × 105 PHKs isolated from neonatal foreskins are seeded on the tops of the gels and maintained submerged for 24 to 48h. The collagen rafts are raised and placed onto stainless steel grids at the interface between the air and the liquid culture medium. The raft cultures can be infected with epitheliotropic viruses at different stages of epithelial cell differentiation. The epithelial cells are then allowed to stratify for 10 to 12 days. The cultures can be harvested at 10-12 days post-lifting and analyzed to check epithelial differentiation by histological examination and viral replication. Cultures can also be prepared using primary epithelial cells derived from tonsillectomies. Tumor cells can be used to prepare the raft cultures, for instance, cell lines transformed with human papillomavirus (HPV) transformed cells (such as HeLa and SiHa) or Merkel cell carcinomas (MCCs), melanoma cell lines, etc. The tumor cells are seeded alone on top of the gels or in combination with PHKs, resulting, respectively, in the production of 3D cultures with dysplastic morphology (tumor cells monoculture) or 3D cultures with patches of dysplastic morphology in the differentiated epithelial cells.

Lab equipment
  • Cell culture equipped laboratory:
  • - Laminar airflow ;
  • - CO2 incubator ;
  • - Microscope ;
  • - Water-bath ;
  • - Centrifuge.
Method status
  • History of use
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential

  • - Faithfully reproduces epithelial cell growth.
  • - Allows multiple applications in virology and tumor biology.
  • - Permits the study of virus-host cell interactions in stratified epithelia and the investigations of tumor biology in an ex-vivo system that closely resembles the in vivo situation.
  • - Usefulness demonstrated by several reports analyzing viruses that target epithelial cells at least during a part of their life cycles epithelial cells as well as in tumor biology.
  • - Tricky method, includes various crucial steps.
  • - Experienced and trained personnel required.
  • The method can be further modified by the addition of other cell types such as endothelial cells and mononuclear cells.
  • The method can be adapted for epithelial cells derived from different animal species.
Future & Other applications

The methodology can be adapted for the study of any virus targeting epithelial cells as well as for the study of co-infections. It also allows the growth of patient-derived biopsies and can be adapted for the (co)culture of different tumor cells.

References, associated documents and other information


Andrei, G., Duraffour, S., Van den, O.J., Snoeck, R., 2010. Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents. Antiviral Res 85, 431-449.

Andrei, G., Lisco, A., Vanpouille, C., Introini, A., Balestra, E., Van den, O.J., Cihlar, T., Perno, C.F., Snoeck, R., Margolis, L., Balzarini, J., 2011. Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication. Cell Host Microbe 10, 379-389.

Andrei, G., van den Oord, J., Fiten, P., Opdenakker, G., De Wolf-Peeters, C., De Clercq, E., Snoeck, R., 2005. Organotypic epithelial raft cultures as a model for evaluating compounds against alphaherpesviruses. Antimicrob Agents Chemother 49, 4671-4680.

Balzarini, J., Andrei, G., Balestra, E., Huskens, D., Vanpouille, C., Introini, A., Zicari, S., Liekens, S., Snoeck, R., Holy, A., Perno, C.F., Margolis, L., Schols, D., 2013. A multi-targeted drug candidate with dual anti-HIV and anti-HSV activity. PLoS Pathog 9, e1003456.

Dal Pozzo, F., Andrei, G., Lebeau, I., Beadle, J.R., Hostetler, K.Y., De Clercq, E., Snoeck, R., 2007. In vitro evaluation of the anti-orf virus activity of alkoxyalkyl esters of CDV, cCDV and (S)-HPMPA. Antiviral Res 75, 52-57.

Duraffour, S., Snoeck, R., Krecmerova, M., Van den, O.J., De Vos, R., Holy, A., Crance, J.M., Garin, D., De Clercq, E., Andrei, G., 2007. Activities of several classes of acyclic nucleoside phosphonates against camelpox virus replication in different cell culture models. Antimicrob. Agents Chemother 51, 4410-4419.

Lebeau, I., Andrei, G., Dal Pozzo, F., Beadle, J.R., Hostetler, K.Y., De Clercq, E., van den, O.J., Snoeck, R., 2006. Activities of alkoxyalkyl esters of cidofovir (CDV), cyclic CDV, and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine against orthopoxviruses in cell monolayers and in organotypic cultures. Antimicrob. Agents Chemother 50, 2525-2529.

Snoeck, R., Holy, A., Dewolf-Peeters, C., Van Den Oord, J., De Clercq, E., Andrei, G., 2002. Antivaccinia activities of acyclic nucleoside phosphonate derivatives in epithelial cells and organotypic cultures. Antimicrob Agents Chemother 46, 3356-3361.

Contact person

Graciela Andrei


Katholieke Universiteit Leuven (KUL)
Microbiology, Immunology & Transplantation
Flemish Region