Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in three-dimensional cell culture

Abstract

Automated cell-based high-throughput screening (HTS) is a powerful tool in drug discovery, and it is increasingly being recognized that three-dimensional (3D) models, which more closely mimic in vivo-like conditions, are desirable screening platforms. One limitation hampering the development of 3D HTS is the lack of suitable 3D culture scaffolds that can readily be incorporated into existing HTS infrastructure. We now show that β-hairpin peptide hydrogels can serve as a 3D cell culture platform that is compatible with HTS. MAX8 β-hairpin peptides can physically assemble into a hydrogel with defined porosity, permeability and mechanical stability with encapsulated cells. Most importantly, the hydrogels can then be injected under shear-flow and immediately reheal into a hydrogel with the same properties exhibited prior to injection. The post-injection hydrogels are cell culture compatible at physiological conditions. Using standard HTS equipment and medulloblastoma pediatric brain tumor cells as a model system, we show that automatic distribution of cell-peptide mixtures into 384-well assay plates results in evenly dispensed, viable MAX8-cell constructs suitable for commercially available cell viability assays. Since MAX8 peptides can be functionalized to mimic the microenvironment of cells from a variety of origins, MAX8 peptide gels should have broad applicability for 3D HTS drug discovery.

Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning

“…Collagen and Matrigel are available in liquid form and require handling at cold temperatures to avoid premature gelation. The need for handling these hydrogels at low temperatures makes them unsuitable for common liquid handling equipment used for high-throughput screens in drug discovery (Ryan et al, 2016 ; Worthington et al, 2017 ). Fibrin is obtained through polymerization of fibrinogen, a plasma protein, and is a natural polymer formed during wound coagulation.…”

“…Current peptide hydrogels that are most successfully used for 3D culture (reviewed in Worthington et al, 2015 ) are: the yeast-derived peptides EAK16 and RADA16 (Zhang et al, 1992 , 2005 ); the peptides Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (Fluorenylmethoxycarbonyl arginine–glycine–aspartic acid) (Jayawarna et al, 2006 ; Mahler et al, 2006 ; Smith et al, 2008 ; Orbach et al, 2009 ; Zhou et al, 2009 ); the peptide hydrogel h9e that is based on the fusion of functional domains from a silk protein and a human calcium channel (Huang and Sun, 2010 ; Huang et al, 2011 , 2013 ); FEFK and FEFKEFK, which form hydrogels in the presence of a metalloprotease (Toledano et al, 2006 ; Guilbaud et al, 2010 ); and the MAX1 peptide that gelates under physiological conditions, and like h9e, has shear-thinning properties (Schneider et al, 2002 ; Haines-Butterick et al, 2007 ). A single acid substitution derivative of the MAX1 peptide, MAX8 (Haines-Butterick et al, 2007 ), with reduced gelation times has recently been reported to be compatible with liquid handling equipment making it suitable for high-throughput drug discovery (Worthington et al, 2017 ).…”

“…Self-assembling peptide hydrogels do not require covalent crosslinking reactions and can assemble into a defined hydrogel at physiological conditions. We have recently described an injectable hydrogel that flows under shear and is compatible with standard liquid automated handling equipment to form reproducible 3D cultures in 384-well plates (Worthington et al, 2017 ). The next step will be to build a 3D culture system that is versatile enough to enter mainstream drug discovery but can easily be fine-tuned to meet the tissue-specific characteristics of an in vivo -like microenvironment.…”

The Current Landscape of 3D In Vitro Tumor Models: What Cancer Hallmarks Are Accessible for Drug Discovery?

“…The most commonly used natural polymers for culture are collagen and Matrigel. Other common natural polymers are silk, chitosan,, alginate, and more recently peptide hydrogels . Common semisynthetic polymers used include hyaluronic‐acid‐based hydrogels and gelatin methylmethacrylate‐based hydrogels .…”

Development of 3D Cell Culture on Ultra-High Molecular Weight Polyethylene (UHMWPE) as the Basis of Cellular Matrix

“…Их изобретениегидрогель на основе самоорганизующегося пептида MAX8, который в физиологических условиях образует в воде сеть с порами необходимой величины для выращивания клеточной культуры. Эксперименты были поставлены с использованием культуры клеток человеческой медуллобластомы -опухоли, возникающей в мозжечке в детском возрасте [20]. Однако эти работы выявили основной недостаток использования гидрогелей, который заключается в появлении некротических участков, формирующихся в результате затруднённого оттока отходов жизнедеятельности клеток, а также ограниченного доступа питательных сред.…”