Microfluidics and High-Content Imaging for In-Vitro to In-Vivo Safety and Efficacy Assessments

By: David Sloan, Tim Jensen, Steve Klose, Michael McCartney, and Randall McClelland,
SciKon Innovation, Inc.

Abstract

We evaluated the utility of a newly released microfluidic culture system (SciFlow™ 1000) to support drug toxicity testing via high-content imaging and biochemical assays. Cells grown in static 2D culture wells are subjected to increasing amounts of waste products and decreasing amounts of dissolved oxygen leading to potential stress responses and toxicity not related to drug/toxicant exposures. To overcome these limitations, researchers are turning to 3D scaffolds, spheroids, hydrogels, and fluidic systems to improve the physiological relevance of the environment and enhance safety assessments. The SciFlow 1000 is designed with descending well-heights from left-to-right across the plate, each subsequent well is 0.5mm lower than the previous well. This results in a cascading flow of fluids across the system without the need for external pumps or tubes. This system sequentially links wells together to form a cascade of cell chambers through which drugs or toxicants can be applied. Toxicants interact with cells in the upstream compartments creating metabolites that will mix and interact in downstream wells forming a parent-metabolite gradient in a time-resolved fashion. Time-resolved dynamic exposure scenarios afforded by such a system are more in vivo-like and could enable more accurate assessment of adaptive vs. toxic mechanisms.

To demonstrate the value of this system we evaluated the cytotoxic effects of Aflatoxin B and acetaminophen (APAP) in the SciFlow 1000 on a metabolically competent human hepatocyte cell line (HepaRG) in comparison to static conditions using the reagents CellTox Green and CellTiter-Glo (Promega). Using a fluorescein tracer molecule we demonstrated that exposures in the fluidic system were non-linear and shaped similarly to an expected in vivo plasma curve. Furthermore, observation of shifts in the dose-response curves as a function of distance from source well provides an indicator for the role for metabolic activity in safety and efficacy assessments of these chemicals.

Technology

well height difference / source & sink wells / purple liquid / 96-well plate

Panel A shows a schematic of 5 wells in 1 row of SciFlow 1000 and the blue lines highlight the 0.5mm decrease in height for each well bottom. Panel B shows one complete row of SciFlow 1000 with the source and sink wells identified. Panel C is a picture of SciFlow 1000, with purple liquid filling one row to highlight the decreasing Z-axis across the plate. Panel D shows the whole SciFlow1000, an SBS compliant 96-well plate injection molded with tissue culture treated polystyrene.

Flow Dynamics

repeated dosing generates gradient of decreasing concentration

Repeated dosing generates a gradient of decreasing compound concentration across the connected wells of a row. Cellular metabolism further decreases the compound concentration within a given well. Fluid flow causes the metabolites and cellular responses to also flow downhill. Downstream wells are exposed to metabolites yet may never see significant concentrations of parent compound.

 

Compound Exposure

A
SciFlow 1000 dosing creates gradient concentrations vs fixed concentration

In an acellular scenario (A), SciFlow 1000 (top) dosing creates gradients of compound concentrations over time. The concentration of compound increases in each well until the equilibrium concentration is reached, and then that concentration is maintained for the duration of the experiment. Wells at different distances from the source well have very different concentration exposure profiles. Static plates (bottom) are at a fixed concentration, which only varies due to cellular metabolism.

 

B
Modeling metabolism - SciFlow dosing vs static dosing

Modelling metabolism in the SciFlow 1000 and static dosing graphs (B) produces the top two graphs. The SciFlow 1000 dosing has an increasing concentration over time, followed by an equilibrium, which is very similar to the classic in vivo plasma drug concentrations graph shown to the left. This increasing concentration over time better mimics in vivo exposure than static conditions.

 

Modeling SciFlow 1000 exposure as a cummulative exposure model

Modeling SciFlow 1000 exposure as a cumulative radiation exposure model (C), where the total exposure over time is calculated, allows us to quantify and compare the exposure to traditional methods. While the final equilibrium drug concentration is very similar across the entire plate, the total (cumulative) dosage is very different in each well.

 

Aflatoxin B Treatment

dual color images captured directly on the SciFlow 1000

HepaRG cells (Biopredic International) were cultured in SciFlow 1000 then exposed to Aflatoxin B for 4 days. CellTox Green (Promega) and Hoechst dye were present in the media allowing for the real-time assessment of cell viability, without interrupting the study. Cells were visualized on Molecular Devices ImageExpress System, multiple times/day and the percentage of viable cells was calculated. The cells exposed to the highest cumulative dose (dose x time) of Aflatoxin are those closest to the source well and they show the lowest percentage of viable cells. Those cells farther away from the source well, exposed to a lower cumulative dose have higher percentages of viable cells.

APAP Treatment: Toxic Metabolite Effect

Comparison of static and SciFlow 1000 cultured cells

Comparison of static and SciFlow 1000 cultured cells treated with acetaminophen (APAP) over 7 and 9 days. CellTiter-Glo (Promega) was used to measure viable cells. Data is normalized to vehicle (DMSO) control. Day 7: Toxicity of APAP in static plates (red) is marginally detectable only at highest concentration. SciFlow 1000 (blue) shows a downstream cell death effect which is characteristic of a toxic/reactive metabolite which is produced in the upstream cells and flows downstream. Day 9: Obvious cell death in static plate at the highest APAP concentration (5mM), and marginal effect at 2.5mM. More pronounced downstream metabolite effect in SciFlow 1000. SciFlow 1000 provides insights into mechanisms of toxic drug response. GSH was also quantified in SciFlow 1000 and depletion of GSH mirrors the downstream decrease in cell viability (data not show).

 

Results highlight concordance between visual and biochemical dataComparison of DMSO and APAP treated cells

 

Comparison of DMSO (vehicle) and APAP treated cells in the SciFlow 1000. Photos and CellTiter-Glo results highlight the concordance between visual and biochemical data. Control cells remain healthy as determined by cell number and morphology while APAP treated cells show marked decline after 10 and 14 days of drug exposure.

Conclusions

The SciFlow™ 1000 Fluidic Culture System exhibits more biologically relevant (in vivo-like) compound exposure data. SciFlow 1000 integrates the generation of parent compound and metabolite gradients across a plate with standard biochemical and high content imaging techniques. These parent compound gradients are demonstrated with the toxic effects of Aflatoxin B on HepaRG cells in a time resolved manner using high content imaging. Additionally, an APAP study demonstrates the ability to distinguish between parent compound and toxic metabolite effects. APAP treatment shows a more potent downstream effect upon cells which is a hallmark of a metabolite mediated effect, not a direct effect of the parent compound (drug). The SciFlow 1000 is a versatile fluidic culture system which is compatible with plate readers, high content imagers, and commercially available biochemical assay kits.

Acknowledgements

We would like to thank Molecular Devices for assistance with high content imaging, BMG and Tecan for allowing us to test out the Clariostar and Spark 10M multi-function plate readers. This was work was partially support by SBIR grants from the National Institutes of Health (1R43ES025970-01 and 1R43GM117954-01).

Materials

     
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