1Department of Microbiology & Immunology, University of North Carolina-Chapel Hill, 2Lineberger Cancer Center, University of North Carolina-Chapel Hill
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Anacker, D., Moody, C. Generation of Organotypic Raft Cultures from Primary Human Keratinocytes. J. Vis. Exp. (60), e3668, doi:10.3791/3668 (2012).
The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.
1. Preparation for Organotypic Raft Cultures
2. Preparation of Collagen Gels
3. Preparing Keratinocytes for Differentiation
4. Making the Raft Cultures
5. Representative Results
When the protocol is performed correctly with primary keratinocytes, this procedure will result in a well-differentiated epithelium where the different layers of the skin are evident. This be achieved through histological examination by staining sections of raft cultures for hematoxylin and eosin (H&E), as shown in Figure 1B. H&E staining is also useful to examine the morphology of rafts and the effect of viruses or antiviral compounds on the ability of cells to stratify and differentiate. For example, rafts made from HPV-immortalized primary human keratinocytes (HFK-31) are notably thicker compared to rafts made from normal human foreskin keratinocytes (HFK), reflecting an increased rate of proliferation and the ability of HPV proteins to maintain differentiating cells active in the cell cycle (Figure 1B)10. This results in retention of nuclei throughout the epithelium, whereas normal keratinocytes exit the cell cycle upon differentiation, resulting in a breakdown of the nuclear envelope. To more closely examine differentiation, immunohistochemistry can be performed to examine specific differentiation markers, including cytokeratins, as well as involucrin, and filaggrin. Different cytokeratins are expressed at specific stages of differentiation7. As shown in Figure 2, cytokeratin 10 (K10) is not expressed in the basal layer, and is only found in the suprabasal layers that consist of cells progressively differentiating. In HPV positive rafts, the expression of K10 is delayed compared to that of normal keratinocytes, again reflecting the influence of HPV proteins on keratinocyte differentiation. Immunohistochemistry can also be used to examine the expression profile of viral genes. Shown in Figure 3 is a representative staining examining the expression of the HPV protein E1^E4, which is a late protein whose expression is restricted to the uppermost layers of the epithelium. As expected, no staining is observed in raft cultures from normal human foreskin keratinocytes.
Figure 1. A) Outline of method for preparing organotypic raft cultures from primary keratinocytes or epithelial cell lines. B) Hematoxylin and eosin staining of organotypic raft cultures generated from human foreskin keratinocytes stably maintaining HPV-31 (HFK-31) episomes, or from normal human foreskin keratinocytes (HFK). The individual epithelial layers are identified, as well as the collagen plug.
Figure 2. Cytokeratin 10 (K10) expression is restricted to the suprabasal layers of the epithelium. Immunohistochemistry was performed on cross sections of organotypic raft cultures generated from HFK-31 cells, as well as normal HFKs using an antibody to K10. Cellular DNA was counterstained with DAPI. Images were captured using confocal fluorescence microscopy. Arrows indicate the basal layer of the epithelium.
Figure 3. The HPV E1^E4 protein is produced late in the productive phase of the viral life cycle. Immunohistochemistry was performed on cross sections of organotypic raft cultures generated from HFK-31 cells, as well as normal HFKs using antibodies to E1ˆE4. Cellular DNA was counterstained with DAPI. Images were captured using confocal fluorescence microscopy. Arrows indicate the basal layer of the epithelium.
We describe here a method that can be used to study epithelial differentiation in general, but can also easily be adapted to study viral pathogenesis, as well as the efficacy of potential therapeutics. Many viruses target epithelial cells either as the primary site of infection, as in the case of HPV, or at some point in the viral life cycle, as with herpesviruses. Although growing organotypic raft cultures is time consuming, the ability to faithfully recapitulate in vivo epithelial differentiation provides an extremely useful method to examine virus:host cell interactions. To successfully grow a fully differentiated epithelium there are some critical steps that must be acknowledged. In order to retain the ability to differentiate in raft cultures, one must have a sufficient number of fibroblast feeders in the collagen gel to maintain the keratinocyte monolayer. Poor differentiation in raft cultures can also be due to too low a density of keratinocytes on the collagen:fibroblast gel, improper raft construction, or failure to change the media everyday. One additional parameter that must be considered to ensure the quality of epithelial differentiation is the type of fibroblast feeder used. For the procedure described here, mouse 3T3 J2 fibroblasts were used. While other fibroblasts can be used as feeders, it is recommended that fibroblasts that divide rapidly, or could potentially migrate up to the dermal surface not be used7. Although this protocol calls for harvesting of the rafts after 14 days, rafts can be harvested before this time point, as well as after. However, after 14 days the rafts will progressively become thinner. In addition to sectioning raft cultures for immunohistochemical analysis, rafts can also be harvested for RNA and DNA, as well as virus production.
We have nothing to disclose.
The authors would like to thank Sally Roberts (University of Birmingham, Birmingham UK) for the kind gift of the E1ˆE4 antibody. This work was supported by a grant from the National Cancer Institute (4R00CA137160-03).
|Rat Tail Collagen type 1 (4-5mg/ml)||BD Biosciences||354236|
|DMEM without NaHC03||Invitrogen||12100-061|
|Stainless Steel metal grids||Williams and Mettle Co.||CR-03063-040-100-S|
|Epidermal Growth Factor||BD Biosciences||354010|