Peer Reviewed Articles from SLAS Technology February 2019

Special Collection on Advances in 3D and Organoid Cell Culture
Article and Original Research Abstracts

Article Abstracts

Human iPS Cell-derived Patient Tissues and 3D Cell Culture Part 1: Target Identification and Lead Optimization
Abstract: Human induced pluripotent stem cells (HiPSCs), and new technologies to culture them into functional cell types and tissues, are now aiding drug discovery. Patient derived HiPSCs can provide disease models that are more clinically relevant and, so more predictive than the currently available animal-derived or tumor cell-derived cells. These cells, consequently, exhibit disease phenotypes close to the human pathology, particularly when cultured under conditions that allow them to recapitulate the tissue architecture in three dimensions (3D) systems.

A key feature of HiPSCs is that they can be cultured under conditions that favor formation of multicellular spheroids or organoids. By culturing and differentiating in systems mimicking the human tissue in vivo, the HiPSC microenvironment further reflects patient in vivo physiology, pathophysiology, and ultimately pharmacological responsiveness.

We assess the rationale for using HiPSCs in several phases of preclinical drug discovery, specifically in disease modeling, target identification and lead optimization. We also discuss the growing use of HiPSCs in compound lead optimization, particularly in the profiling compounds for their potential metabolic liability and off-target toxicities. Collectively, we contend that both approaches i.e., HiPSCs and 3D cell culture, when used in concert, have the exciting potential for the development of novel medicines.
Human iPS Cell-derived Patient Tissues and 3D Cell Culture Part 2: Spheroids, Organoids and Disease Modeling
Abstract: Human induced pluripotent stem cells (HiPSCs) provide several advantages for drug discovery, but principally as they provide a source of clinically relevant tissue. Furthermore, the use of HiPSCs cultured in 3 dimensional (3D) systems, as opposed to traditional 2D culture approaches, better represents the complex tissue architecture in vivo. The use of HiPSCs in 3D spheroid and organoid culture is now growing but particularly when using myocardial, intestinal enteric nervous system, and retinal cell lines. However, organoid cell culture is perhaps making the most notable impact in research and drug discovery, in which 3D neuronal cell cultures, allow direct modeling of cortical cell layering and neuronal circuit activity. Given the specific degeneration seen in discrete neuronal circuitry in Alzheimer’s (AD) and Parkinson’s disease (PD), HiPSC culture systems are proving to be a major advance. In the second part of this two-part review, we discuss novel methods in which 3D cell culture systems (principally organoids) are now being used to provide insights into disease mechanisms. The use of HiPSCs is target identification, in general, was reviewed in detail in Part 1 of this review.
Original Research Abstracts
Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy
Abstract: Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1.

Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high throughput drug screen for cell viability using a 1,912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp-90 and proteasome inhibitors. JHH-136 was uniquely sensitive to Topoisomerase 1 inhibitors while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 Kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. This data shows these GBM spheroid lines are amenable to high throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.
Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation
Abstract: Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and by endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform 3- and 4-factor, 2-level full-factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.
A Single-step Self-assembly Approach for the Fabrication of Aligned and Multilayered Three-Dimensional Tissue Constructs Using Multidomain Peptide Hydrogel
Abstract: Hydrogels are homogenous materials that are limited in their ability to form oriented multilayered architecture in three-dimensional (3D) tissue constructs. Current techniques have led to advancements in this area. Such techniques often require extra devices and/or involve complex processes that are inaccessible to many laboratories. Here is described a one-step methodology that permits reliable alignment of cells into multiple layers using a self-assembling multidomain peptide (MDP) hydrogels. We characterized the structural features, viability, and molecular properties of dental pulp cells fabricated with MDP and demonstrated that manipulation of the layering of cells in the scaffolds was achieved by decreasing the weight by volume percentage (w/v%) of MDP contained within the scaffold. This approach allows cells to remodel their environment and enhanced various gene expression profiles, such as cell proliferation, angiogenesis, and extracellular matrix (ECM) remodeling-related genes. We further validated our approach for constructing various architectural configurations of tissues by fabricating cells into stratified multilayered and tubular structures. Our methodology provides a simple, rapid way to generate 3D tissue constructs with multilayered architectures. This method shows great potential to mimic in vivo microenvironments for cells and may be of benefit in modeling more complex tissues in the field of regenerative medicine.