Fabrication of Human Keratinocyte Cell Clusters for Skin Graft Applications by Templating Water-in-Water Pickering Emulsions

Abstract

Most current methods for the preparation of tissue spheroids require complex materials, involve tedious physical steps and are generally not scalable. We report a novel alternative, which is both inexpensive and up-scalable, to produce large quantities of viable human keratinocyte cell clusters (clusteroids). The method is based on a two-phase aqueous system of incompatible polymers forming a stable water-in-water (w/w) emulsion, which enabled us to rapidly fabricate cell clusteroids from HaCaT cells. We used w/w Pickering emulsion from aqueous solutions of the polymers dextran (DEX) and polyethylene oxide (PEO) and a particle stabilizer based on whey protein (WP). The HaCaT cells clearly preferred to distribute into the DEX-rich phase and this property was utilized to encapsulate them in the water-in-water (DEX-in-PEO) emulsion drops then osmotically shrank to compress them into clusters. Prepared formulations of HaCaT keratinocyte clusteroids in alginate hydrogel were grown where the cells percolated to mimic 3D tissue. The HaCaT cell clusteroids grew faster in the alginate film compared to the individual cells formulated in the same matrix. This methodology could potentially be utilised in biomedical applications.

Advanced biomedical applications based on emerging 3D cell culturing platforms

“…This technique initially confines the cells within emulsion droplets and then increases the concentration of the constant phase to impose osmotic pressure to fabricate spheroids within a very short time. 62,63 This results in the encapsulation of the cells in the DEX emulsion drops due to the higher affinity of the cells to the DEX phase, as mentioned above. Cell-cell interactions are strongly promoted with the emulsion droplets, which assists the formation of 3D cell clusters, termed clusteroids.…”

“…53 Another promising application of APTS to generate spheroid was reported by Celik et al, where generally, they embedded significant amounts of spheroids in alginate gel, which allowed the formation of skin-like tissue. 63 These examples set the scene for the following research on the utilization of ATPS in the biomaterial field.…”

“…Spheroid transplants have been utilized in almost all human systems including the cardiovascular system, 90 digestive system, 91 musculoskeletal system, 92 and skin. 63,93 However, spheroid implantation remains a relatively undiscovered field with limited in vivo published result. Thus, there is unexplored potential for the in vivo applications of spheroids.…”

Bovine Decellularized Amniotic Membrane: Extracellular Matrix as Scaffold for Mammalian Skin

“…Although several studies recommend the use of cell lines in this kind of experiments [66][67][68][69][70][71], it is of paramount importance to indicate that in vitro studies have evidenced that in a standard cell culture, fibroblast positively influence keratinocyte growth, most likely due to the fact that these cells secrete soluble growth factors. In natural skin, the interaction is relevant as well.…”

“…The amniotic membrane has structures, which are histologically similar to the skin, i.e., composed of a multilayer epithelium and the basic membrane, and the structure might be considered as a good support for wound healing, reepithelialization and inhibition of scar formation and bacterial growth [50,52,75,76]. Although several studies recommend the use of cell lines in this kind of experiments [66][67][68][69][70][71], it is of paramount importance to indicate that in vitro studies have evidenced that in a standard cell culture, fibroblast positively influence keratinocyte growth, most likely due to the fact that these cells secrete soluble growth factors. In natural skin, the interaction is relevant as well.…”