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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

Published: May 17th, 2021

DOI:

10.3791/62353

1Department of Bioengineering and Aerospace Engineering, Universidad Carlos III de Madrid (UC3M), 2Group of Optics, Photonics and Biophotonics (GOFB). Center for Biomedical Technology, Universidad Politécnica de Madrid, 3Division of Epithelial Biomedicine, CIEMAT, 4Instituto de Investigación Sanitaria Gregorio Marañón

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Recently, advances have been made toward the development and production of in vitro human skin models for the analysis of the toxicity of cosmetic and pharmaceutical products1. Researchers in pharmaceutical and skin care industries have been using animals, mice being the most common, to test their products2,3,4,5. However, testing products on animals is not always predictive of the response in humans, which frequently leads to drug failure or adverse effects in humans and consequently to economic losses

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1. Chip design and micromachining parameters

  1. Design the microfluidic chip layers with FreeCAD open-source design software; refer to Table 1 for the dimensions of the channels. Include four 2.54 mm diameter holes in the design to use a custom-made aligner for a correct layer superposition.
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The designed chip is composed of two fluidic chambers separated by a 5 µm pore size PC membrane that allows the growth of the cell by allowing the passage of growth-promoting molecules from the lower chamber. The upper chamber holds the tissue construct, in this case, a monolayer of hKCs on a fibrin hydrogel containing hFBs.

The height of the channels is determined by the number of adhesive sheets added to each channel. The lower chamber is composed of 4 layers (380 µm) and the upper.......

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The motivation to develop this method was the desire to model skin diseases and study the effects of new and innovative therapies in a high-throughput platform. To date, this laboratory produces these dermo-epidermal equivalents by casting-either manually or with the help of the 3D bioprinting technology-the fibrin gel with fibroblasts into a cell culture insert plate and seeding the keratinocytes on top of it. Once the keratinocytes reach confluence, the 3D culture is exposed to the air-liquid interface, which induces k.......

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We sincerely thank Dr. Javier Rodríguez, Dr. María Luisa López, Carlos Matellán, and Juan Francisco Rodríguez for very helpful suggestions, discussions, and/or preliminary data. We also kindly thank the contributions of Sergio Férnandez, Pedro Herreros, and Lara Stolzenburg to this project. Special thanks go to Dr. Marta García for GFP-labelled hFBs and hKCs. Finally, we recognize the excellent technical assistance of Guillermo Vizcaíno and Angélica Corral. This work was supported by the "Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid", Project S2018/BAA-4480, Biopieltec-....

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Length (μm) Width (μm)
Name Company Catalog Number Comments
Amchafibrin Rottafarm Tranexamic acid
Antibiotic/antimycotic Thermo Scientific HyClone
Calcium chloride Sigma Aldrich
Culture plates Fisher
DMEM Invitrogen Life Technologies
Double-sided tape vynil ATP Adhesive Systems GM 107CC, 12 µm thick
Edge plotter Brother Scanncut CM900
FBS Thermo Scientific HyClone
Fibrinogen Sigma Aldrich Extracted from human plasma
Glass slide Thermo Scientific
GFP-Human dermal fibroblasts - Primary. Gift from Dr. Marta García
H2B-GFP-HaCaT cell line ATCC Immortalized keratinocytes. Gift from Dr. Marta García
Live/dead kit Invitrogen
PBS Sigma Aldrich
Polycarbonate membrane Merk TM 5 µm pore size
Polydimethylsiloxane Dow Corning Sylgard 184
Sodium chloride Sigma Aldrich
Syringes Terumo 5 mL
Thrombin Sigma Aldrich 10 NIH/vial
Transparent adhesive vinyl Mactac JT 8500 CG-RT, 95 µm thick
Trypsin/EDTA Sigma Aldrich
Tubing IDEX Teflon, 1/16” OD, 0.020” ID

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