David A. Fluri

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture–reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor–expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell– and small aggregate–based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.

The food additive vanillic acid controls transgene expression in mammalian cells and mice

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

3D spherical microtissues and microfluidic technology for multi-tissue experiments and analysis

Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue-tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.

High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1 × 10⁷  cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day⁻¹ with a perfusion rate (D) of 5.0 day⁻¹. A twofold increase in maximum cell density (to greater than 2.5 × 10⁷  cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture.

96-Well Format-Based Microfluidic Platform for Parallel Interconnection of Multiple Multicellular Spheroids

In this article, we present a microfluidic platform, compatible with conventional 96-well formats, that enables facile and parallelized culturing and testing of spherical microtissues in a standard incubator. The platform can accommodate multiple microtissues (up to 66) of different cell types, formed externally by using the hanging-drop method, and enables microtissue interconnection through microfluidic channels for continuous media perfusion or dosage of substances. The platform contains 11 separate channels, and each channel has six tissue compartments. Primary rat liver tissues were cultured over 8 days, and multiple tumor tissues (HCT116) were exposed to various concentrations of 5-fluorouracil for platform characterization.

A model-based assay design to reproduce in vivo patterns of acute drug-induced toxicity.

Lars Kuepfer1 · Olivia Clayton2 · Christoph Thiel1 · Henrik Cordes1 · Ramona Nudischer2 · Lars M. Blank1 · Vanessa Baier1 · Stephane Heymans3,4 · Florian Caiment5 · Adrian Roth2 · David A. Fluri6 · Jens M. Kelm6 · José Castell7 · Nathalie Selevsek8 · Ralph Schlapbach8 · Hector Keun9 · James Hynes10 · Ugis Sarkans11 · Hans Gmuender12 · Ralf Herwig13 · Steven Niederer14 · Johannes Schuchhardt15 · Matthew Segall16 · Jos Kleinjans5

Simultaneous impedance spectroscopy and stimulation of human iPS-derived cardiac 3D spheroids in hanging-drop networks

Here, we present electrical impedance spectroscopy (EIS) data of human iPS-derived cardiac 3D spheroids with electric stimulation integrated in a hanging drop network. Microscopy videos of the beating spheroids were correlated with synchronously obtained EIS recordings. For stimulation, the spheroid was exposed to a continuous sinusoidal electric field - in contrast to traditional pulse trains. This stimulating field was supplied via the same electrodes that were used for the EIS recordings. Our measurements revealed a beating frequency modulation upon tuning the stimulation signal amplitude.

Abstract B75: High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds

Background 3D tumor microtissues or spheroids are widely accepted as a more representative biological model in phenotypic drug discovery. Size and morphology are important determinants to evaluate the biological behavior of 3D spheroids, particularly in development of anti-cancer drugs where monitoring cell growth is a particularly critical endpoint. The increasing number of applications of 3D tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including high-throughput image analysis. In this study, we assessed the growth and morphology changes of HCT116 colorectal cancer spheroids in response to two chemotherapy drugs-gemcitabine and docetaxel- using a high throughput, bright-field spheroid imager (Cell3iMager, SCREEN Holdings Ltd., Inc.). Methods Tumor spheroids consisting of the HCT116 colorectal cancer cell line were aggregated in InSphero9s GravityPLUS™ plate (a scaffold-free hanging drop platform). The aggregated spheroids were transferred into a GravityTRAP™ plate prior to dosing. Spheroids were dosed with anti-cancer drugs-gemcitabine and docetaxel (0.8-20nM) at day 0 and re-dosed at day 3. The plates were scanned and spheroid size and morphology were analyzed daily for 7 days. Endpoint analysis of ATP content was carried out at day 7 utilizing a luminescence-based ATP assay (CellTiter-Glo® 2.0 Assay, Promega) Results HCT116 spheroids were measured daily for 7 days to determine dose-response effects of gemcitabine and docetaxel treatment (0.8-20nM) on spheroid area (um 2 ). A size increase was observed for the control and the lowest compound concentration treated spheroids whereas the spheroid growth was inhibited at the higher concentrations tested (>4nM). Among the tested compounds, HCT116 spheroids showed less sensitivity to docetaxel compared to gemcitabine. At day 7 the treatment with 20nM gemcitabine resulted in more than 50% reduction in tumor area (um2) which was positively correlated with the decrease in cell viability, determined by ATP content. Using HCT116 tumor microtissues cultured in InSphero9s GravityTRAP™ plates, we demonstrate that the measurement of tumor size with the Cell3iMager could be a suitable endpoint compared to that of ATP content for drug induced cytotoxicity in tumor spheroids, without lysing cells or otherwise interfering with long term culture of spheroids. Conclusions Label-free measurement of changes in 3D microtissue size and morphology using the Cell3iMager enables efficient assessment of phenotypic endpoints without disrupting tumor growth, and can be used to focus selection of therapeutic targets and treatment strategies before costly and tedious testing in animal models. Citation Format: Leena Mol Thuruthippallil, Jens M. Kelm, David Fluri. High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B75.