Various studies have shown that in vitro skin micro-tissues comprising of a fibroblast layer containing dermis topped by a stratified layer of keratinocytes can be formed on polycarbonate transwells using air-liquid interface cultures. The total duration to obtain the full thickness skin models can vary from 28–30 days from seeding the fibroblasts, followed by keratinocytes and differentiation.

 

The RAFTTM 3D Cell Culture System could provide an advantage of shortening the culture period to 22–24 days. Our air-liquid interface format provides a scaffold for differentiating keratinocytes, embedding fibroblasts and possibly additional skin cell types.

The skin provides a vitally important protective separation between the internal and the external environments1. There are three structural layers to the skin: the epidermis, the dermis and subcutis. The epidermis is the outer layer, serving as the physical and chemical barrier between the interior body and the exterior environment. The dermis is a deeper layer providing the structural support of the skin. Subcutis is made up of loose connective tissue and fat, which can be up to 3 cm thick on the abdomen. 

Epidermis is a stratified squamous epithelium consisting of several cell types. The most abundant cell type of epithelial layer of the skin is the keratinocytes which synthesize the protein keratin2. Protein bridges called desmosomes connect the keratinocytes, which are in a constant state of transition from the deeper layers to the superficial. The epidermis varies in thickness based on the tissue of origin. The four separate layers of the epidermis are stratum basale (basal or germinativum cell layer), stratum spinosum (spinous or prickle cell layer), stratum granulosum (granular cell layer) and stratum corneum. These layers are formed by the differing stages of keratinocyte maturation.

Normal Human Skin Tissue. Image courtesy of Wikimedia.

Normal Human Skin Tissue. Image courtesy of Wikimedia.

 

Dermal fibroblasts are cells within the dermis layer of skin which are responsible for generating connective tissue and allowing the skin to recover from injury.3 Using organelles (particularly the rough endoplasmic reticulum), dermal fibroblasts generate and maintain the connective tissue which unites separate cell layers. Furthermore, these dermal fibroblasts produce the protein molecules including laminin and fibronectin which comprise the extracellular matrix. By creating the extracellular matrix between the dermis and epidermis, fibroblasts allow the epithelial cells of the epidermis to affix the matrix, thereby allowing the epidermal cells to effectively join together to form the top layer of the skin.

 

Epidermal-dermal skin substitutes are currently the most utilized tissue-engineered skin model that closely resembles the structure of native human skin. The presence of both keratinocytes and fibroblasts within the epidermal-dermal skin substitutes leads to the production of a variety of growth factors and cytokines which expedite wound healing, highlighting the importance of epithelial-mesenchymal interactions. These epidermal-dermal skin substitutes have been utilized for wound healing assays, toxicological research, co-culture studies, tissue engineering research. 

Primary Human Keratinocytes and Fibroblasts Cultured in RAFTTM System

In our recent white paper, we describe the procedure in constructing full thickness (FT) skin models using the RAFTTM 3D Culture System with primary Human Neonatal Epidermal Keratinocytes (NHEK) and primary Human Neonatal Dermal Fibroblasts (NHDF). The RAFTTM Skin Model consists of a compressed collagen-type-I-based hydrogel that closely mimics the human dermis. This layer is topped with differentiated epidermal keratinocytes in an ALI mimicking the epidermis. We show analyses of the FT skin model to resemble the native skin by immunohistochemistry and immunofluorescence validated using key markers for epidermis and dermis.

Download the white paper to read the full study:
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Construction of a Full Thickness Skin Model Using RAFTTM 3D Cell Culture System

In this Technical Note, we describe the procedure in constructing full thickness (FT) skin models using the RAFTTM 3D Culture System with primary Human Neonatal Epidermal Keratinocytes (NHEK) and primary Human Neonatal Dermal Fibroblasts (NHDF).
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Related Products

  • Human Neonatal Epidermal Keratinocytes (NHEK) and KGM-GoldTM Keratinocyte Growth Medium
  • Human Neonatal Dermal Fibroblasts (NHDF) and FGM-2 BulletKitTM Medium
  • Buy a RAFTTM Trial Kit today in one of these formats - 24-well, 24-well cell culture insert, 96-well

 

 

References

  1. Richard LE and Ellen AR.  Molecular biology of keratinocyte differentiation. Environmental Health Perspectives (1989); 80: 109-116 2.
  2. Skin Structure and Function 
  3. Darling, David (10 September 2011). "Hypodermis". Encyclopedia of Science.

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