Multilayer spheroids to quantify drug uptake and diffusion in 3D.

There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil's inhibition of Pgp and GJIC were similar (IC50: 8 µM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.

Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

Advances in the formation, use and understanding of multi-cellular spheroids.

INTRODUCTION: Developing in vitro models for studying cell biology and cell physiology is of great importance to the fields of biotechnology, cancer research, drug discovery, toxicity testing, as well as the emerging fields of tissue engineering and regenerative medicine. Traditional two-dimensional (2D) methods of mammalian cell culture have several limitations and it is increasingly recognized that cells grown in a three-dimensional (3D) environment more closely represent normal cellular function due to the increased cell-to-cell interactions, and by mimicking the in vivo architecture of natural organs and tissues. AREAS COVERED: In this review, we discuss the methods to form 3D multi-cellular spheroids, the advantages and limitations of these methods, and assays used to characterize the function of spheroids. The use of spheroids has led to many advances in basic cell sciences, including understanding cancer cell interactions, creating models for drug discovery and cancer metastasis, and they are being investigated as basic units for engineering tissue constructs. As so, this review will focus on contributions made to each of these fields using spheroid models. EXPERT OPINION: Multi-cellular spheroids are rich in biological content and mimic better the in vivo environment than 2D cell culture. New technologies to form and analyze spheroids are rapidly increasing their adoption and expanding their applications.

Quantification of the kinetics and extent of self-sorting in three dimensional spheroids.

The self-sorting of cells into distinct compartments in three-dimensional (3D) microtissues is a process critical to developmental biology, cancer metastasis, and tissue engineering. Although self-sorting has been studied since the 1950s, little quantitative data exist that describe this dynamic process. Here, we describe a recently developed assay designed to quantify the extent and kinetics of self-sorting in 3D. Mixtures of fluorescently labeled normal human fibroblasts (NHF) and hepatocyte (H35) cells were fluorescently labeled, red and green respectively, and seeded onto micro-molded non-adhesive hydrogels. The cells self-assembled into a spheroid and self-sorted with NHFs forming the central core and H35s forming the outer shell. A time course of fluorescent images was used to analyze the ratio of red (NHFs) and green (H35s) fluorescence in concentric hollow cylinders throughout a spheroid and was statistically compared with the fluorescent ratio of the perfectly sorted spheroid. We found that NHFs and H35s, at a 1:1 ratio, sorted to a final extent of 88±3% at an initial rate of 0.36±0.06% per minute and reached 50% self-sorted at 2.7±0.3 h. Studies with varying ratios of NHFs and H35s show that self-sorting and self-assembly are coincident in time when the proportion of NHFs are varied over a 6-fold range (14% to 85%). This method can, thus, be used to characterize the sorting behavior of additional pairs of cells, the effect of drugs, and growth factors that may change the kinetics of the process, and bring an understanding to the cellular mechanisms which control self-sorting.