Novel 3D-Printed Cell Culture Inserts for Air-Liquid Interface Cell Culture

Abstract

In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of '3Rs', new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air-liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.

Keywords: 3D printing; PLA; full-thickness skin model; insert; tissue engineering.

Figure A1

Inserts in cell culture well plate with 3D tissue on the second day after seeding.

Figure A2

Drawing derivation of computer-aided design (CAD) model of the 3D-printed cell culture insert showing dimension and oblique crack.

Figure A3

Drawing derivation of computer-aided design (CAD) model of the 3D-printed cell culture insert. Detailed view.

Figure A4

Drawing derivation of computer-aided design (CAD) model of the 3D-printed cell culture insert. Detailed information on dimensions.

Figure A5

Drawing derivation of computer-aided design (CAD) model of the 3D-printed cell culture insert. Detailed information on dimensions.

Figure 1

Visualization of the 3D-printed cell culture insert. (a) Rendered insert construction. (b) Schematic of the device with upper and lower parts of the inserts. The customized membrane is indicated in between.

Figure 2

Drawing derivation of computer-aided design (CAD) model of the 3D-printed cell culture insert. Left: Front view. Right: Bird’s eye view. A more detailed plan can be found in Appendix B Figure A2, Figure A3, Figure A4 and Figure A5. Scale in mm.

Figure 3

Cell viability according to ISO 10993-5 of human dermal fibroblast (HDF) and normal human epidermal skin keratinocytes (NHEK). Viability was measured via MTT assay. The values represent means ± standard deviation of two independent experiments with six technical replicates each (n = 12).

Figure 4

Hematoxylin & eosin staining images of skin-like co-culture modelled on 3D-printed insert. Light pink-coloured collagen-based dermis harbouring distributed fibroblasts is in the bottom part of the figure. Superior to the light pink dermis, darker epidermis with recognizable layered structures can be found. Pink stratified stratum corneum desquamate detached and elongated cells can also be seen. The tissue was generated by culturing the epidermal and dermal components submerged for three days, then allowing the epidermal compartment differentiation for 19 days at ALI. Scale bar: (A) 100 µm and (B) 20 µm.

Figure 5

Immunofluorescence staining of two different skin-like co-cultures modelled in 3D-printed insert. 3D tissue was generated by culturing the epidermal and dermal components submerged for three days, then allowing the epidermal compartment differentiation for 19 days at ALI. The upper row (A–D) demonstrates the presence of fibroblasts in the lower compartment ((D), vimetin (VIM)) as well as keratinocyte differentiation ((C), keratin 10 (K10)). Cell nuclei were stained with DAPI (B) and a merged image can be seen in (A). In the lower row (E–H), a second 3D tissue was additionally stained with another marker for keratinocyte differentiation: keratin 14 ((G), K14). Furthermore, cells were positive for K10 (H) and DAPI (F). Merging all three images showed that K10 and K14 positive cells formed two layers, indicating differentiation of keratinocytes (E). Scale bar: 100 µm.