Silk Film Culture System for <em>in vitro</em> Analysis and Biomaterial Design

Abstract

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications [3][4][5][6][7][8] . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications [9][10][11][12][13] . The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields. Video LinkThe video component of this article can be found at https://www.jove.com/video/3646/ Protocol Fabrication of Silicone Rubber Molds1. Produce or purchase a desired topographic surface for casting. For this publication a standard 100 mm etched silicon wafer will be described (Figure 1). 2. Weigh out polydimethylsiloxane (PDMS) potting (component A) and catalyst (component B) solution in a 1:9 ratio (9 g potting and 1 g catalyst) as provided in the purchased kit. 3. Mix solutions thoroughly to initiate curing process. 4. Place silicon wafer surface within a casting dish. 5. Weigh out 4.5 g of PDMS solution onto silicon wafer. 6. Spread PDMS solution as to cover a 100 mm diameter area of the wafer surface. 7. Tilt wafer to spread PDMS solution evenly. 8. Cover wafer with 100 mm diameter petri dish lid. 9. Place casting setup into 60 °C oven over night, making certain that curing is taking place on a flat surface. 10. Place cured PDMS/silicon wafer into 70% ethanol bath before removal. 11. Begin removing PDMS from wafer by using razor blade to lift edge (entire circumference) first. 12. Gently pull PDMS off using forceps within a 70% ethanol bath being careful not to tear silicone rubber casting....

Human corneal limbal epithelial cell response to varying silk film geometric topography in vitro

“…After a week in culture silk surfaces possessed similar cell numbers as glass surfaces, and indicate that silk surfaces offer a viable substrate for cell expansion. These results may also indicate that silk film surface chemistry and not the presence of patterned surface topography may inhibit cell proliferation to some degree as previously demonstrated, however it is still unclear what is causing this effect [9]. Silk films with lined surface features appeared to demonstrate an enhanced characteristic of increasing initial cell adhesion when compared to other silk and glass surfaces.…”

“…However, the use of silicon is not favorable for in vivo translation, fabrication of such materials requires a high level of expertise, and per sample production is relatively costly. Using patterned silk surfaces offers a number of advantages based on their biomaterial properties, and as a result of their straightforward and inexpensive production methods [9,25]. Therefore, silk films with both parallel line and concentric ring topographies were fabricated to assess cellular response to varying feature geometries and material surfaces.…”

“…One of the primary uses of regenerated silk materials is in the production of scaffolding material in the fields of tissue engineering and regenerative medicine due to their inherent biocompatibility [7,8]. Specifically, silk films offer a wide platform for biomaterial innovation due to their highly controlled material properties, ease of fabrication, biocompatible nature, and potential for chemical modification [5,9–11]. One interesting material property of silk films is their ability to produce topographic features on the micro and nanometer scale, which allows for the formation of surface patterned culture surfaces for cell growth [12–14].…”

“…Topographic contact guidance of cells by surface topography can have dramatic effects upon cellular development, extracellular matrix (ECM) alignment and formation, cell proliferation, cellular adhesion, and proliferation and apoptosis [13–16]. In this way silk film substrates offer an elegant system to produce customized patterned surfaces that can be designed to promote a desired cellular response with potential to elicit a clinically desirable effect [9]. …”

Novel Collagen Membranes for the Reconstruction of the Corneal Surface

“…There are some studies with silk and keratin membranes, which showed good results in vitro but so far have not been tested clinically. 12,24,25 In general collagens show a fast degradation when placed on the corneal surface. 26,27 To improve the stability and resistance of collagens different methods were utilized in the past.…”