3D bioprinting of functional human skin: production and in vivo analysis

Abstract

Abstract. Significant progress has been made over the past 25 years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin, or for the establishment of in vitro human skin models. In this sense, laboratory-grown skin substitutes containing dermal and epidermal components offer a promising approach to skin engineering. In particular, a human plasma-based bilayered skin generated by our group, has been applied successfully to treat burns as well as traumatic and surgical wounds in a large number of patients in Spain. There are some aspects requiring improvements in the production process of this skin; for example, the relatively long time (three weeks) needed to produce the surface required to cover an extensive burn or a large wound, and the necessity to automatize and standardize a process currently performed manually. 3D bioprinting has emerged as a flexible tool in regenerative medicine and it provides a platform to address these challenges. In the present study, we have used this technique to print a human bilayered skin using bioinks containing human plasma as well as primary human fibroblasts and keratinocytes that were obtained from skin biopsies. We were able to generate 100 cm 2 , a standard P100 tissue culture plate, of printed skin in less than 35 minutes (including the 30 minutes required for fibrin gelation). We have analyzed the structure and function of the printed skin using histological and immunohistochemical methods, both in 3D in vitro cultures and after long-term transplantation to immunodeficient mice. In both cases, the generated skin was very similar to human skin and, furthermore, it was indistinguishable from bilayered dermo-epidermal equivalents, handmade in our laboratories. These results demonstrate that 3D bioprinting is a suitable technology to generate bioengineered skin for therapeutical and industrial applications in an automatized manner.

3D bioprinting for cell culture and tissue fabrication

“…It was demonstrated that cell viability in the skin-like construct was above 90% in 1 week [74]. Moreover, Cubo et al created a human plasma-based bilayer skin-like tissue containing plasma-derived fibrin, fibroblasts and keratinocytes, which had successfully used for the treatment of burnt, traumatic and surgical wounds in many patients [75]. The results revealed that the printed skin-like tissue, both in vitro culture and in transplantation to immunedeficient mice, showed similar characteristics with human skin [75].…”

Beyond 2D: 3D bioprinting for skin regeneration

“…Nieves Cubo et al used primary human FBs and human KCs as well as human plasma to print a human bi‐layered skin. According to the histological and immunohistochemical in vivo and in vitro analyses, they demonstrated that the printed skin was very similar to normal human skin and successfully used the in clinical . Byoung Soo Kim et al used FBs and KCs to fabricate a human skin model with a functional transwell system by using extrusion and inkjet modules synchronously .…”

“…It is more suitable for the bioprinting of 3D scaffolds in most solid organs, blood vessels, and hard tissues. The potential applied advantages of a microextrusion bioprinter make it a promising printer in the construction of bioprinting skin substitutes . Its main drawback is the low resolution (below 50 μm) and the shear stress that may affect cell viability .…”

“…In addition, novel materials for ideal bioink must also be considered. Currently, gelatin hydrogels are widely used as bioink for their high biocompatibility . However, the printed construct's disadvantages of severe contraction, fast degradation, and limited lifespan cannot be ignored .…”

3D Miniaturization of Human Organs for Drug Discovery

“…These results demonstrate that 3D bioprinting is a suitable technology to generate bioengineered skin for therapeutical and industrial applications in an automatized manner. [106] Koch et al selected different skin cells (fibroblasts, keratinocytes) and hMSC based on their high potential in regeneration of human skin and possible applications in stem cell therapy for their laser printing experiments. [107] This group generated a viable skin tissue free of any DNA damage.…”