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. 2021 Jul 12;7(7):3332-3339.
doi: 10.1021/acsbiomaterials.1c00612. Epub 2021 Jun 25.

Fibrillar Nanomembranes of Recombinant Spider Silk Protein Support Cell Co-culture in an In Vitro Blood Vessel Wall Model

Christos Panagiotis Tasiopoulos  1 , Linnea Gustafsson  2 , Wouter van der Wijngaart  2 , My Hedhammar  1
Affiliations

Fibrillar Nanomembranes of Recombinant Spider Silk Protein Support Cell Co-culture in an In Vitro Blood Vessel Wall Model

Christos Panagiotis Tasiopoulos et al. ACS Biomater Sci Eng. .

Abstract

Basement membrane is a thin but dense network of self-assembled extracellular matrix (ECM) protein fibrils that anchors and physically separates epithelial/endothelial cells from the underlying connective tissue. Current replicas of the basement membrane utilize either synthetic or biological polymers but have not yet recapitulated its geometric and functional complexity highly enough to yield representative in vitro co-culture tissue models. In an attempt to model the vessel wall, we seeded endothelial and smooth muscle cells on either side of 470 ± 110 nm thin, mechanically robust, and nanofibrillar membranes of recombinant spider silk protein. On the apical side, a confluent endothelium formed within 4 days, with the ability to regulate the permeation of representative molecules (3 and 10 kDa dextran and IgG). On the basolateral side, smooth muscle cells produced a thicker ECM with enhanced barrier properties compared to conventional tissue culture inserts. The membranes withstood 520 ± 80 Pa pressure difference, which is of the same magnitude as capillary blood pressure in vivo. This use of protein nanomembranes with relevant properties for co-culture opens up for developing advanced in vitro tissue models for drug screening and potent substrates in organ-on-a-chip systems.

Keywords: basement membrane; cell co-culture; nanomembrane; recombinant spider silk; tissue engineering; vessel wall.

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Conflict of interest statement

The authors declare the following competing financial interest(s): M.H. has shares in Spiber Technologies AB, a company that aims to commercialize recombinant silk.

Figures

Figure 1
Figure 1
Overview of the procedure for the preparation of and cell seeding on silk membranes. Day −1: a solution of FN-4RepCT silk protein diluted in phosphate-buffered saline (PBS) is placed in an open well where the protein self-assembles into a membrane at the air–liquid interface overnight. Day 0: a holder is lowered onto the membrane, which adheres to the holder over 2 h. The holder with the membrane is lifted from the interface and placed in endothelial cell growth medium, and human dermal microvascular endothelial cells (HDMEC) are seeded on the apical side of the membrane. Day 4: the holder is reversed, smooth muscle cells (SMC) are seeded on the basolateral side of the membrane, and allowed to adhere for 30 min, after which the holder is placed in SMC growth medium and filled with endothelial cell growth medium. Day 7: HDMEC have established a confluent monolayer on the apical side, and SMC have produced a thick ECM on the basolateral side of the silk membrane. Drawing is not in scale. FN-4RepCT silk: 4RepCT silk protein functionalized with the Arg-Gly-Asp (RGD)-containing cell-binding motif from fibronectin.
Figure 2
Figure 2
Appearance of the spider silk membrane. (a) Photograph of a spider web illustration as seen through the silk membrane, showing its optical transparency. Scale bar = 1 mm. (b) Tilted SEM image of the smooth air-side (apical) and cross section. (c) SEM image of the textured liquid-side (basolateral). Scale bars = 1 μm.
Figure 3
Figure 3
ECM produced by SMC. (a) Sketch (not drawn to scale) of the silk membrane (in blue) seeded with endothelial cells (HDMEC) on the apical side (in orange) and SMC on the basolateral side and the ECM produced by the latter (in green), with the representative SEM image which has been false-colored to match the sketch. (b) Sketch (not drawn to scale) of a TC-insert (in pink) seeded with both HDMEC (in orange) and SMC and the ECM produced by the latter (in green), with the representative SEM image which has been false-colored to match the sketch. Scale bars = 2 μm. (c) Measured thickness (mean ± SD) of ECM for silk membranes and TC-inserts seeded with both HDMEC and SMC, as well as silk membranes seeded with only SMC. **P < 0.01, ns—not significant (P > 0.05). HDMEC: human dermal microvascular endothelial cells.
Figure 4
Figure 4
Mechanical properties of spider silk membranes with and without cells. Photographs of a bulging membrane under pressure differences (ΔP) of (a) 0, (b) 320, and (c) 540 Pa. The air pressure inside the holder is regulated hydrostatically using a water column, visible only in (c) (false-colored blue). Ruler is mm-scaled. Plots showing the (d) average center deflection (Δh) (mean ± SD) of the membrane and the corresponding (e) pressure difference (ΔP) (mean ± SD) at burst for silk membranes without cells, with endothelial cells (HDMEC), with SMC, and both cell types (HDMEC + SMC) after 4 (light gray) and 7 (dark gray) days in culture. **P < 0.01, *P < 0.05. HDMEC: human dermal microvascular endothelial cells.
Figure 5
Figure 5
Barrier properties of silk membranes and TC-inserts with and without cells. Normalized TEER values (mean ± SD) for (a) silk membranes and (b) TC-inserts seeded with only endothelial cells (HDMEC) and with both HDMEC and SMC (HDMEC + SMC) at different days in culture. Inserted micrographs in (a) show HDMEC stained for tight junctions (zona occludens-1, in green) and cell nuclei (DAPI, in blue) on days 4 and 7. Scale bars = 25 μm. Permeation of 10 kDa dextran (mean ± SD) through (c) silk membranes and (d) TC-inserts without cells, seeded with HDMEC, and both cell types (HDMEC + SMC). Permeation of IgG (mean ± SD) through (e) silk membranes and (f) TC-inserts for the same conditions, all after 7 days in culture. ***P < 0.001, **P < 0.01, ns—not significant (P > 0.05). HDMEC: human dermal microvascular endothelial cells.
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