Immunofluorescence microscopy of 3 dimensional, bilayered Cocultures.

<p>DAPI staining of cell nuclei in blue, Involucrin as specific marker for HaCaT cells in red. bar = 100 µm.</p

MEF cells, cultivated on spidersilk alone.

<p>1^st (A) and 4^th (B) day after seeding. By comparison, MEF cells reach confluence earlier than HaCaT cells. bar = 100 µm.</p

Force testing.

<p>The diagrams show graphs describing the generated force of the particular test samples of the control group (n = 5) (A), after a 1 day culture period (n = 4) (B), after a 5 days culture period (n = 8) (C) and after a 21 days culture period (n = 5) (D).</p

SEM of a confluent MEF cell layer on a frame with spider silk.

<p>The brink of the meshwork, where spider silk fibres do not cross each other is just rarely colonized, whereas a confluent cell-layer can be observed in the central meshwork. Culturing period took 5 days. bar = 1 mm.</p

3-dimensional cell culturing.

<p>Skin equivalents, cultivated under different medium conditions: Addition of choleratoxin (A), Hydrocortisone (B) or A-2-P (B). bar = 100 µm.</p

Frame design.

<p>Stainless steel straight wires with a diameter of 0,7 mm were bended to frames with side lengths of 1–1,5 cm and wound with spider dragline silk (A). Spider silk was woven in cross pattern to reach a mesh size between 10–100 µm (B). bar = 100 µm.</p

Cultivation at the air liquid interface.

<p>Frames with spider silk, seeded with MEF and HaCaT cells, are lifted on a silicone scale which is filled up with polymer fibres. The surface of the nutrient media is below the altitude of the frame. Media and frame are connected by the polymer fibres, which allow unilateral nutrient supply and metabolite evacuation by diffusion.</p

H&E stained section of a frame, which was cultured at the air liquid interphase without any special media supplements or serum reduction.

<p>Development of two continuously separated strata could not be achieved. Arrows mark cross sectioned spider silk fibres. (A) bar = 40 µm Immunofluorescence of the same frame, DAPI staining of all cells nuclei in blue, Involurin as specific marker for HaCaT cells in red (B).</p

Maximum forces.

<p>The mean value of maximum generated force seems to decrease in all groups over culture time.</p

HaCaT cells, cultivated on spidersilk alone.

<p>1^st (A) and 4^th (B) day after seeding. From the corners of the meshwork, cells spread into the meshes and reach confluence within 1 week. bar = 100 µm.</p