Seevix s SVXgro: A Spidersilk Scaffold for Tissue Engineering

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1 Seevix s gro: A Spidersilk Scaffold for Tissue Engineering Spider dragline silk exhibits extraordinary mechanical properties that combine strength with elasticity, resulting in a toughness exceeding that of all other natural and synthetic fibers. Additionally, spidersilk is a porous biomaterial, proven to be biocompatible. Physical attributes of dragline spidersilk Strength Six times stronger than high tensile steel of the same diameter Weight Light weight: approximately 1/5 the weight of high tensile steel of the same diameter Temperature Durable from -200 o C to +230 o C Elasticity Up to 35% without tearing Specific surface area* 3.8 m 2 /gram (BET) *As measured for Seevix fibers UNIQUE PROPERTIES OF SPIDERSILK FIBERS Seevix spidersilk (gro) fibers have multiple advantages for cell culture applications. The fibers are produced by means of a unique process, resulting in microfibers composed of synthetic spider dragline silk with a proprietary composition. gro exhibits mechanical properties that are identical to those of the natural fiber. Having both micrometric and nanometric dimensions, gro fibers are comprised of nano-fibrils that greatly enhance its surface area as well as its porosity and elasticity. TEM imaging reveals an amazing structure of nano-fibrils (~10 nm diameter) that self-assemble to create the final monofilament (~230 nm diameter) (Figure 1). Coiled fiber Monofilament Nano-fibrils Microfilament 5 µm 50 nm Figure 1 Brightfield and TEM images of a gro fiber. The microfiber is comprised of 10 nm nanofibers (see inset, right) and reaches a total width of about 230 nm (middle). The length of a single microfiber is about 100 µm. Note the coiled structure on the left. gro FIBERS FOR IMPROVED CELL CULTURE gro improves the viability, function and overall health of cell spheroid cultures. gro is easy to handle and can be incorporated into spheroids by any spheroid generation technique (Figure 2A). The efficacy of gro was demonstrated in adipose-derived human mesenchymal stem cells (hmscs), rat primary hepatocytes (RPHs)**, and various, proliferating, ubiquitously used cell lines (HEK293, CHO, L929, NIH3T3), which were cultured as spheroids with and without gro. The exact mechanism of action of gro as a scaffold is not yet fully known, however, it probably involves the binding of cells to the fibers, which act as an extracellular matrix (ECM) for each cell, while adopting the shape of the cells (Figure 2B), forming aggregates that organize into stable spheroids after seeding. ** Primary hepatocytes study was performed in collaboration with Kurabo Industries LTD. Page 1

2 A B Trypsinization Cells + Cell Ultra low attachment plate (ULA) Scale bar 200 µm Figure 2 Spheroid assembly. (A) Fibers and cells are mixed and seeded on an ultra-low attachment plate. During the following days, cells form aggregates containing trapped fibers and eventually establish cell-cell interactions to form stable spheroids. (B) Primary rat hepatocytes spheroid. gro fibers integrated into the spheroids are observed on the margins of the spheroid. Separation of cells from their carrier is of great importance for clinical applications in tissue engineering and transplantation. Cells are easily separated from gro fibers by trypsinization, making them available for further analysis, treatment and eventually, therapeutic applications. gro has multiple advantages for cell culture applications Biocompatibility Cell viability Cell functionality Intricate 3D structure Spheroid integrity No toxicity in vitro and no immunogenic response in mice Prolonged cell culture health and delayed necrotic core formation even in large spheroids Improved and prolonged cell function markers Large surface area offers potential for loading of biologicals or drugs Spheroids are stable for prolonged culturing Spheroids containing gro fibers demonstrate larger size and increased viability and proliferation Primary rat hepatocytes, human mesenchymal stem cells and L929 cells, seeded as spheroids with gro fibers, show a % increase in spheroid size compared with spheroids seeded without fibers. Seeded with gro, the cells form larger, less dense spheroids, enabling better accessibility and transport of nutrients as well as improved clearance of waste products (Figure 3). Following formation, spheroids from non-proliferating cells (e.g., primary rat hepatocytes) without gro become denser and smaller, while spheroids containing fibers compact to a lesser extent. Spheroids generated from proliferating cells (e.g., hmscs) continue to grow when seeded with gro fibers, but stop proliferating when seeded without fibers. gro-containing spheroids demonstrate higher viability than control spheroids (Figure 4). Human mesenchymal stem cells spheroids cultured with gro are more viable, and the cells retain a healthy elongated phenotype, whereas without the fibers, cells are small and round and show increased cell death. This is reflected in both fluorescent live-dead imaging, as well as in an ATP quantification assay (Figure 4A). Page 2

3 A B Figure 3 Average diameter of hepatocyte and MSC spheroids. Primary rat hepatocytes (A) and human adiposederived mesenchymal stem cells (B) were seeded with or without gro fibers and consecutively cultured. Spheroids were imaged on indicated days with a light microscope (left panel), and their size was analyzed with ImageJ software (NIH) (right panel). In L929 cells, a proliferating fibroblast-like cell line, seeding with gro fibers delays formation of a necrotic core and improves the ratio between live and dead cell areas by 60% (Figure 4B). Additionally, proliferation is enhanced by approximately 300% for cells seeded with gro four days post seeding. A Control 2 ng gro/cell B Control Scale bar 100 µm 0.4 ng gro/cell Scale bar 400 µm Figure 4 Morphology of cells, proliferation and live to dead cell ratio. (A) Adipose-derived stem cell spheroids seeded with or without gro were stained with propidium iodide (PI) and Calcein AM to stain dead and live cells, respectively. The spheroids were then imaged by confocal microscopy (left). Cell-Titer Glo (Promega) assay was performed to measure cell proliferation in spheroids seeded with and without gro (right). (B) L929 spheroids seeded with and without gro were stained with propidium iodide (PI) and Calcein AM (left). Stained areas were calculated with ImageJ software (NIH). The graph shows PI staining relative to the Calcein AM staining, indicating the ratio between live and dead cells (middle). Cell proliferation (metabolic state) was quantified by alamarblue assay (BIO RAD) (right). gro fibers protect spheroid integrity Primary rat hepatocyte spheroids seeded with gro are stable up to at least 41 days, whereas control spheroids disintegrated completely by this time (Figure 5). The gro fiber scaffold supports functionality by maintaining a tissue-like 3D configuration that prevents loss of hepatocyte function. Page 3

4 Control Day 1 Day 6 Day 20 Day 41 Figure 5 Spheroid integrity. Primary rat hepatocytes were seeded with and without gro and consecutively cultured for 41 days. Spheroids were imaged with a light microscope on indicated days, and their size was analyzed with ImageJ software (NIH). Scale bar 400 µm gro fibers improve and prolong the function of cells Primary rat hepatocyte spheroids, cultured with gro, show stable prolonged albumin secretion, indicative of sustained hepatocyte function (Figure 6). In spheroids grown with gro fibers, albumin secretion is increased by about 50% during the first culturing days, an enhancement which further increases up to hundreds of percent after several weeks of culturing. In addition, albumin secretion is sustained for a much longer period: treated cultures show stabilized and high albumin secretion from 20 days post seeding up to at least 36 days post seeding, whereas control cultures show a strong reduction from two weeks down to secretion levels that are too low for detection after 27 days. Figure 6 Albumin secretion in spheroids. Primary rat hepatocytes were seeded with and without gro and consecutively cultured for 36 days. Medium was collected from spheroid cultures every two days. Albumin concentration was measured using Rat Albumin ELISA Quantitation Set (Bethy Laboratories). Human mesenchymal stem cells (hmscs) cultured with gro retain their differentiation potential. hmscs cultured with gro readily differentiate into adipocytes upon introduction of differentiating signals. Staining with Oil Red O highlights the fat droplets, indicating a differentiated adipocyte state (Figure 7). Control Figure 7 Differentiation of hmscs into adipocytes. hmscs seeded with and without gro were cultured in adipogenic differentiation medium. Spheroids were imaged with a light microscope without staining (upper panel) or following Oil Red O staining (lower panel). Arrows indicate lipid droplets, demonstrating adipogenic differentiation. Page 4

5 COMPETITIVE PRODUCTS There are several companies that have developed products for 3D culture in recent years, some of which are scaffold-based, and others which are based on scaffold-free approaches. gro has several benefits over the other approaches, including handling, chemical stability and adjustability of the scaffold for different cell types and applications. Ease of use of gro Thermal stability Scaffold adjustability and versatility Repeatability Imaging Shelf life Easy handling Withstands sterilization process Dimensions of scaffold can be adapted to cell number and type Can be directly used on desired microwell plates and used in automated systems for high repeatability Fibers are visible, yet do not interfere with light and fluorescent imaging Fibers are highly stable, also in solution Fibers can be easily added and mixed with preferred growth media and directly used TARGET MARKETS gro was thoroughly tested on different cell types, including human mesenchymal stem cells, primary rat hepatocytes and the L929 cell line. The versatility of the fibers makes them applicable for use ranging from small-scale experiments to fully automated drug screening and toxicology assays, stem cell research and tissue engineering. Academia as well as biotech and pharmaceutical companies can greatly benefit from gro, saving precious time and reducing the number of cells required for any type of experiment. SEEVIX MATERIAL SCIENCES Seevix is a privately held biotech company focused on developing innovative products based on the company's unique and proprietary man-made spidersilk technology. The company targets applications in which the combination of the fibers mechanical properties and biocompatibility can radically improve existing products and provide solutions for unmet needs. Seevix generates spidersilk fibers which are the basis for high-performance, functional materials. Upscaling technologies have been developed to meet industry demands. The company is creating a pipeline of new fibers that are being characterized and tested against its existing fibers to demonstrate specific targeted properties. In addition, Seevix is developing prototypes of composite materials, consisting of a combination of Seevix s fibers and other materials, to create functional materials that are custom-made for specific industries and products. APPLICATION OF gro FIBERS gro can be used for a variety of 3D cell culture applications for both primary cells and proliferating cell lines. Each cell type or cell line requires an initial calibration step to optimize results. gro is supplied as a concentrated stock solution to supplement cell culture media. It is recommended to add gro from the concentrated stock with each exchange of media. Page 5

6 In a multiwell plate format, the recommended quantity of gro to be added to each well is μl of 1 mg/ml stock solution per 1000 cells. For preparation of solution for a whole plate (i.e. 96 wells with 200 µl per well or 384 wells with 50 µl per well, which is a total volume of 19.2 ml per plate), to be seeded at 1000 cells/well, add µl from the stock solution to the final 19.2 ml whole plate solution. Accordingly, if another ratio of cells/well is to be used, for example, seeding 5000 cells/well, it is recommended to complete the whole plate volume of 19.2 ml with µl from stock solution ( µl/plate/1000 cells*5). Website: Contact: Dr. Shlomzion Shen shlomzi@seevix.com Page 6