Integration of Mature Adipocytes to Build-Up a Functional Three-Layered Full-Skin Equivalent

Abstract

Large, deep full-thickness skin wounds from high-graded burns or trauma are not able to reepithelialize sufficiently, resulting in scar formation, mobility limitations, and cosmetic deformities. In this study, in vitro-constructed tissue replacements are needed. Furthermore, such full-skin equivalents would be helpful as in vivo-like test systems for toxicity, cosmetic, and pharmaceutical testing. Up to date, no skin equivalent is available containing the underlying subcutaneous fatty tissue. In this study, we composed a full-skin equivalent and evaluated three different media for the coculture of mature adipocytes, fibroblasts, and keratinocytes. Therefore, adipocyte medium was supplemented with ascorbyl-2-phosphate and calcium chloride, which are important for successful epidermal stratification (Air medium). This medium was further supplemented with two commercially available factor combinations often used for the in vitro culture of keratinocytes (Air-HKGS and Air-KGM medium). We showed that in all media, keratinocytes differentiated successfully to build a stratified epidermal layer and expressed cytokeratin 10 and 14. Perilipin A-positive adipocytes could be found in all tissue models for up to 14 days, whereas adipocytes in the Air-HKGS and Air-KGM medium seemed to be smaller. Adipocytes in all tissue models were able to release adipocyte-specific factors, whereas the supplementation of keratinocyte-specific factors had a slightly negative effect on adipocyte functionality. The permeability of the epidermis of all models was comparable since they were able to withstand a deep penetration of cytotoxic Triton X in the same manner. Taken together, we were able to compose functional three-layered full-skin equivalents by using the Air medium.

WAT-on-a-chip integrating human mature white adipocytes for mechanistic research and pharmaceutical applications

“…Primary mature adipocytes were isolated from subcutaneous adipose tissue samples. The isolation protocol was performed as previously described 42,43 , with slight modifications. In brief, the subcutaneous adipose tissue was rinsed twice with Dulbecco's phosphate buffered saline with MgCl 2 and CaCl 2 (PBS + ; Sigma-Aldrich Chemie GmbH), and visible blood vessels, as well as connective-tissue structures, were thoroughly removed.…”

“…The observable, mature phenotype of adipocytes suggests that the tissue-generation and -culture methods were capable of creating and maintaining physiological microenvironments and conditions. Inappropriate culture conditions have been frequently reported to induce cell dedifferentiation 43,44 which would lead to highly proliferative dedifferentiated fat cells (DFAT) with multilineage character 45 . One of the first hallmarks of dedifferentiation is the re-organization of lipids into multiple lipid droplets (i.e., a multilocular phenotype) and an increased rate of lipolysis 44 .…”

3D organ models—Revolution in pharmacological research?

“…They are typically generated by sequential cell seeding into cell culture inserts or porous 3D scaffolds. Of these, bone and cartilage [19], and epithelia such as skin [20,21], or lung [22] are currently the most advanced, while models of more complex tissues such as the brain are at earlier stages of development. Notably, organ models may vary in their complexity and, hence, predictivity.…”

WAT’s up!? – Organ-on-a-chip integrating human mature white adipose tissues for mechanistic research and pharmaceutical applications

“…The observable, mature phenotype of adipocytes suggests that the tissue-generation and -culture methods were capable of creating and maintaining physiological microenvironments and conditions. Inappropriate culture conditions have been frequently reported to induce cell dedifferentiation (38,39) which would lead to highly proliferative dedifferentiated fat cells (DFAT) with multilineage character. (40) One of the first hallmarks of dedifferentiation is the reorganization of lipids into multiple lipid droplets (i.e., a multilocular phenotype) and an increased rate of lipolysis.…”

“…Primary mature adipocytes were isolated from subcutaneous adipose tissue samples. The isolation protocol was performed as previously described (44,45), with slight modifications. In…”