Biomimetic collagen I and IV double layer Langmuir–Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells

Abstract

Skottman, H. (2015). Biomimetic collagen I and IV double layer Langmuir-Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells. BIOMATERIALS, 51, 257-269. DOI: 10.1016/j.biomaterials.2015 AbstractThe environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaeffer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruch's membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on airsubphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of small molecular weight substance. Maturation and functionality of hESC-RPE cells on double layer collagen LS films was further assessed by RPE-specific gene and protein expression, growth factor secretion, and phagocytic activity. Here, we demonstrated that the prepared collagen LS films have layered structure with oriented fibers corresponding to architecture of the uppermost layers of Bruch's membrane and result in increased barrier properties and functionality of hESC-RPE cells as compared to the commonly used dip-coated controls.

Langmuir-Schaefer film deposition onto honeycomb porous films for retinal tissue engineering

“…In addition, collagen fibres in the inner collagenous layer are naturally disposed in a nanometre-thick multilayered structure parallel to the plane of the BM [35], and the LS deposition method is ideal to mimic such structures [14,25].…”

“…Alternatively, highly oriented collagen substrates prepared by horizontal immersion of solid substrates, or LangmuirSchafer (LS) deposition (LS) [27], have been prepared with positive effects over the adhesion and proliferation of 3T3 fibroblasts [25]. Recently, our team has also showed the potential of collagen films prepared by LS deposition onto commercial polymer substrates for RPE cell culture [14]. Even so, the use of LS films as biomaterials for tissue engineering applications is still in its infancy, and, to our knowledge, no records can be found on LS film deposition onto biodegradable polymer surfaces, as inorganic substrates such as mica, silicon and glass are the most commonly elected materials [25,28,29].…”

“…The Langmuir-Schaefer (LS) films were prepared using the KSV minitrough system as described by Sorkio et al [14]. A simplified version of the procedure is shown in Figure 1 collagen LS films, a type IV collagen film was deposited on top of a dried type I collagen film as described above (i.e.…”

“…(46, XY) were carried out using serum-free conditions as previously described [31]. Culture of the hESCs was performed on top of mitomycin (10 μg/mL; SigmaAldrich Corp., St. Louis, MO, USA) treated human foreskin fibroblast feeder cells (CRL-2429TM; ATCC, Manassas, VA, USA) as described before [14]. The hESCs were spontaneously differentiated into RPE cells in floating cell aggregates followed by enrichment on collagen IV-coated (human placenta, 5 μg cm -2…”

“…Even so, few studies so far have attempted to mimic the properties of the BM for RPE cell culture and transplantation [11,[13][14][15]. The BM is a 2-4.7 µm thick semi-permeable membrane consisting of five protein-rich layers: the basement membrane of the RPE (collagen type IV, laminin, fibronectin), the inner collagenous layer (collagens type I, III and V), the elastin layer (elastin, collagen type VI, fibronectin), the outer collagenous layer (similar to the inner collagenous layer) and the basement membrane of the choriocapillaris (laminin and collagens type IV, V and VI) [13,16].…”