Implementation of a High-Throughput Pilot Screen in Peptide Hydrogel-Based Three-Dimensional Cell Cultures

Cell-based high-throughput drug screening (HTS) is a common starting point for the drug discovery and development process. Currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. However, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration challenging. Here, we used the peptide-based shear-thinning hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in three dimensions and showed a preliminary implementation of the scaffold within an automated HTS setup using a pilot drug screen targeting medulloblastoma, a pediatric brain cancer. A total of 2202 compounds were screened in the 384-well format against cells encapsulated in the hydrogel as well as cells growing on traditional two-dimensional (2D) plastic. Eighty-two compounds passed the first round of screening at a single point of concentration. Sixteen-point dose–response was done on those 82 compounds, of which 17 compounds were validated. Three-dimensional (3D) cell-based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment, getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide-based hydrogel into the high-throughput format.

[1]  W. Janzen,et al.  Screening technologies for small molecule discovery: the state of the art. , 2014, Chemistry & biology.

[2]  D. Pochan,et al.  Beta Hairpin Peptide Hydrogels as an Injectable Solid Vehicle for Neurotrophic Growth Factor Delivery. , 2015, Biomacromolecules.

[3]  D. Pochan,et al.  Sustained release of active chemotherapeutics from injectable-solid β-hairpin peptide hydrogel. , 2016, Biomaterials science.

[4]  Louis Scampavia,et al.  A Cytotoxic Three-Dimensional-Spheroid, High-Throughput Assay Using Patient-Derived Glioma Stem Cells , 2018, SLAS discovery : advancing life sciences R & D.

[5]  Joel I. Pritchard,et al.  The Smo/Smo model: hedgehog-induced medulloblastoma with 90% incidence and leptomeningeal spread. , 2008, Cancer research.

[6]  Liju Yang,et al.  Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. , 2014, Assay and drug development technologies.

[7]  D. Pochan,et al.  Injectable solid hydrogel: mechanism of shear-thinning and immediate recovery of injectable β-hairpin peptide hydrogels. , 2010, Soft matter.

[8]  B. Coyle,et al.  In vitro models of medulloblastoma: Choosing the right tool for the job. , 2016, Journal of biotechnology.

[9]  A. Napper,et al.  Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in three-dimensional cell culture. , 2017, Analytical biochemistry.

[10]  J. Burdick,et al.  A practical guide to hydrogels for cell culture , 2016, Nature Methods.

[11]  I-Chi Lee,et al.  Cancer-on-a-chip for Drug Screening. , 2019, Current pharmaceutical design.

[12]  K. O'Byrne,et al.  Drug Discovery Approaches Utilizing Three-Dimensional Cell Culture. , 2016, Assay and drug development technologies.

[13]  N. Wagner,et al.  Macromolecular diffusion and release from self-assembled beta-hairpin peptide hydrogels. , 2009, Biomaterials.

[14]  Kim E. Garbison,et al.  The Minimum Significant Ratio: A Statistical Parameter to Characterize the Reproducibility of Potency Estimates from Concentration-Response Assays and Estimation by Replicate-Experiment Studies , 2006, Journal of biomolecular screening.

[15]  E. Danen,et al.  3D Cell-Based Assays for Drug Screens: Challenges in Imaging, Image Analysis, and High-Content Analysis , 2019, SLAS discovery : advancing life sciences R & D.

[16]  D. Pochan,et al.  Rheology of peptide- and protein-based physical hydrogels: are everyday measurements just scratching the surface? , 2015, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[17]  R. Tycko,et al.  Molecular structure of monomorphic peptide fibrils within a kinetically trapped hydrogel network , 2015, Proceedings of the National Academy of Sciences.

[18]  V. Virador,et al.  In vitro three‐dimensional (3D) models in cancer research: An update , 2013, Molecular carcinogenesis.

[19]  Darrin J. Pochan,et al.  Peptide Hydrogels – Versatile Matrices for 3D Cell Culture in Cancer Medicine , 2015, Front. Oncol..

[20]  Luigi Grasso,et al.  Application of 3D tumoroid systems to define immune and cytotoxic therapeutic responses based on tumoroid and tissue slice culture molecular signatures , 2017, Oncotarget.

[21]  Maddaly Ravi,et al.  3D Cell Culture Systems: Advantages and Applications , 2015, Journal of cellular physiology.

[22]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[23]  Xianting Ding,et al.  Study on a 3D Hydrogel-Based Culture Model for Characterizing Growth of Fibroblasts under Viral Infection and Drug Treatment , 2017, SLAS discovery : advancing life sciences R & D.

[24]  Matthew Pilarz,et al.  Controlling hydrogelation kinetics by peptide design for three-dimensional encapsulation and injectable delivery of cells , 2007, Proceedings of the National Academy of Sciences.

[25]  J. Vörös,et al.  Soft Hydrogels Featuring In-Depth Surface Density Gradients for the Simple Establishment of 3D Tissue Models for Screening Applications , 2017, SLAS discovery : advancing life sciences R & D.

[26]  David J. Beebe,et al.  Microscale screening systems for 3D cellular microenvironments: platforms, advances, and challenges , 2014, Cellular and Molecular Life Sciences.

[27]  Mark W. Tibbitt,et al.  Hydrogels as extracellular matrix mimics for 3D cell culture. , 2009, Biotechnology and bioengineering.

[28]  Eunice E. Cho,et al.  Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells , 2015, Apoptosis.

[29]  Ursula Graf-Hausner,et al.  Synthetic 3D multicellular systems for drug development. , 2012, Current opinion in biotechnology.

[30]  Erkki Ruoslahti,et al.  Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule , 1984, Nature.

[31]  Anna Tesei,et al.  Anticancer drug discovery using multicellular tumor spheroid models , 2019, Expert opinion on drug discovery.

[32]  Kirk Czymmek,et al.  Injectable solid peptide hydrogel as a cell carrier: effects of shear flow on hydrogels and cell payload. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[33]  D. Pochan,et al.  Encapsulation of curcumin in self-assembling peptide hydrogels as injectable drug delivery vehicles. , 2011, Biomaterials.

[34]  Sigrid A. Langhans Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning , 2018, Front. Pharmacol..

[35]  Ye Fang,et al.  Three-Dimensional Cell Cultures in Drug Discovery and Development. , 2017, SLAS discovery : advancing life sciences R & D.

[36]  A. Napper,et al.  Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology. , 2013, Assay and drug development technologies.