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. 2018 Jun 7;5(2):43.
doi: 10.3390/bioengineering5020043.

Bioengineering of a Full-Thickness Skin Equivalent in a 96-Well Insert Format for Substance Permeation Studies and Organ-On-A-Chip Applications

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Bioengineering of a Full-Thickness Skin Equivalent in a 96-Well Insert Format for Substance Permeation Studies and Organ-On-A-Chip Applications

Katharina Schimek et al. Bioengineering (Basel). .
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Abstract

The human skin is involved in protecting the inner body from constant exposure to outer environmental stimuli. There is an evident need to screen for toxicity and the efficacy of drugs and cosmetics applied to the skin. To date, animal studies are still the standard method for substance testing, although they are currently controversially discussed Therefore, the multi-organ chip is an attractive alternative to replace animal testing. The two-organ chip is designed to hold 96-well cell culture inserts (CCIs). Small-sized skin equivalents are needed for this. In this study, full-thickness skin equivalents (ftSEs) were generated successfully inside 96-well CCIs. These skin equivalents developed with in vivo-like histological architecture, with normal differentiation marker expressions and proliferation rates. The 96-well CCI-based ftSEs were successfully integrated into the two-organ chip. The permeation of fluorescein sodium salt through the ftSEs was monitored during the culture. The results show a decreasing value for the permeation over time, which seems a promising method to track the development of the ftSEs. Additionally, the permeation was implemented in a computational fluid dynamics simulation, as a tool to predict results in long-term experiments. The advantage of these ftSEs is the reduced need for cells and substances, which makes them more suitable for high throughput assays.

Keywords: 96-well cell culture insert; full thickness skin equivalents; multi-organ chip; substance permeation.

Conflict of interest statement

Uwe Marx is the CEO and shareholder and Roland Lauster and Gerd Lindner are shareholder of TissUse GmbH, a company manufacturing and commercializing the multi-organ-chip (MOC) technology. Other authors declare no conflict of interests regarding the publication of this paper.

Figures

Figure 1
Figure 1
Schematic diagram of the ftSE cultivation. (a) Punches are cut out of the Matriderm™ matrix and (b) degassed in an exsiccator. (c) Then the matrix is seeded with human dermal fibroblasts. (d) On day nine, the ftSE is transferred into a cell culture insert system. (e) After four days of cultivation, the skin equivalent is sealed with a fibrin gel. (f) One day later, normal human keratinocytes are seeded onto the top of the equivalent. (g) On day 20, the equivalents are lifted to the air-liquid interface and cultivated further for ~10 days.
Figure 2
Figure 2
Pictures of the 48-well plate Transwell® holder. (a) The Transwell® can fitted in the polycarbonate (PC) holder plate and (b) positioned in a 48-well plate.
Figure 3
Figure 3
Exploded diagram of the Two-organ chip system. The blue box shows a zoom on the fluid channel with the position of the micro pumps, insert 1 and insert 2.
Figure 4
Figure 4
Schematic side view of the 2OC. The acceptor, where the samples are taken, is located at Insert 1. The 96-well CCI system with ftSE is positioned on Insert 2. The donator is the investigated substance on the ftSE. Black arrows indicate the direction of the fluid flow, and the red arrows the permeation.
Figure 5
Figure 5
Geometry of the numerical simulation. The green cylinder shows the volume of the acceptor, and the blue cylinder shows the volume of the donator. At the bottom of the acceptor the ftSE is located. The zoom shows the position of the in- and outflow for the fluid simulation.
Figure 6
Figure 6
The mash of the numerical simulation. To generate the grit, the auto mash function with “normal” tetrahedral element size was used.
Figure 7
Figure 7
Hematoxylin and eosin (HE) staining of full-thickness skin equivalents (ftSEs). The ftSE morphology was evaluated at different time points. Images show representative cuts of ftSEs of (a,g) dermis only, (b,h) dermis with fibrin gel added on top, and (c,i) after seven days, (d,j) 10 days, (e,k) 17 days, and (f,l) 24 days of ALI culture. The skin equivalents developed a multilayered and stratified epithelium during this 24-day period. (af) Scale bar: 500 µm. (gl) Scale bar: 50 µm.
Figure 8
Figure 8
Immunohistochemistry of ftSEs. Expression of skin-specific markers was observed over time. Representative images of (a,d) dermis only and (e,f) dermis with added fibrin gel, as well as ftSEs after (il) seven days air lift (ALI), (mp) 10 days ALI, (qt) 17 days ALI, and (ux) 24 days ALI were selected for further analysis. Column 1 (a,e,i,m,q,u) shows keratin 10 (red)/15 (green) double immunostaining. Column 2 (b,f,j,n,r,v) shows collagen IV, Column 3 (c,g,k,o,s,w) shows filaggrin, and Column 4 (d,h,l,p,t,x) shows vimentin staining. An increase in epidermal differentiation and basement membrane marker expressions at different time points indicates a physiologically relevant skin development. Counterstaining of cell nuclei with DAPI (blue). Scale bar: 50 µm.
Figure 9
Figure 9
TUNEL-Ki67 stain of ftSEs. TUNEL-positive apoptotic cells are stained in green, and proliferative Ki67 positive cells are stained in red. A considerable number of proliferating cells within the basal layer of the ftSEs and a few apoptotic cells are found after (a) 7, (b) 10, (c) 17 and (d) 24 days of ALI cultivation. Counterstaining of cell nuclei with DAPI (blue). Scale bar: 100 µm.
Figure 10
Figure 10
HE staining and immuno-labeling of ftSE cultured for seven days in the 2OC. (a,b) HE stain of the ftSE showed preserved morphology comprising a multilayered epidermis. (c) TUNEL-Ki67 (green and red) staining of the ftSE revealed a considerable number of proliferating cells within the basal layer of the epidermis, while only a few apoptotic cells could be detected. (d) Keratin 10 (red)/15 double stain, (e) collagen IV (red), (f) filaggrin (red), and (g) vimentin (green) staining indicate advanced levels of differentiation within the ftSE cultured in the 2OC. Counterstaining of cell nuclei with DAPI (blue). (a) Scale bar: 500 µm. (b,dg) Scale bar: 50 µm. (c) Scale bar: 100 µm.
Figure 11
Figure 11
Macroscopic view and HE staining of the ftSE (ad,j) with high and (eh,k) low trans-epithelial electrical resistance (TEER). About 40% of the ftSE showed severe contraction. (i) Performance of the skin barrier was additionally evaluated by TEER. (d,h) Scale bar: 500 µm. (j,k) Scale bar: 50 µm.
Figure 12
Figure 12
Permeation coefficient over the time of the ftSE. The permeation of fluorescein sodium salt through the ftSEs was observed over time. The ALI was executed at day 0. The days before pre-culture are declared with a minus. A continuous decrease of permeation over time can be seen (gray arrow).
Figure 13
Figure 13
Comparison of the simulation with the permeation experiment. The diagrams show the donator concentration over time. The permeation of fluorescein sodium salt through the ftSE on day 0 (day of the ALI) and day 24 were determinant. The diffusion was calculated with numerical simulation by parameter adaption with Fick’s law.

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