SEACSAFETY & ENVIRONMENTAL ASSURANCE CENTRE

High-throughput and non-depletive quantification in 3D liver microtissue in vitro assay

SAFETY SCIENCE IN THE 21ST CENTURYFor more information visit www.tt21c.org

Non-specific binding to equipment

Protein/serum binding

Free/available

Adsorption to labware

KPlastic

PrecipitationKBSA(serum)

KCell

KAW

LogS

Metabolism / degradation

Non-animal approaches for assessing toxicological and environmental risk of ingredients require more advanced and realistic in vitro models as well as a better understanding of a chemical’s distribution and extrapolation from in vitro to in vivo. Here we applied a novel ultra-thin SPME fibersampling method to analyse the fate of Diclofenac in a standard in vitro model as well as measure the metabolic depletion of Diclofenac in a 3D liver cell model .

1) Novel ultra-thin SPME fibers for non-depletive extraction of small volumes1

Background

Realistic models Exposure characterization

QIVIVE

Methods and Materials

❑ HLB Coating – hydrophilic lipophilic balance❑ 2.0 mm length ❑ Non-depletive extraction❑ Clean-up step❑ Cfree

❑ Automated concept sampling system

❑ 96 well plate format

2) Testing with standard cell model – HT1080

% free in cell media 5% FBS

Methods Diclofenac

SPME 9 ± 0.4%

RED 9 ± 1%

2) Metabolism of Diclofenac over 48h with liver microtissues (InSphero 3D InSightTM)

Results 1) Fiber optimization

Diclofenac

3) Experimental design

1 - Boyacı, E., et al. (2018). "High-throughput analysis using non-depletive SPME: challenges and applications to the determination of free and total concentrations in small sample volumes." Scientific Reports 8(1): 1167.

3) Metabolism in InSphero liver microtissues

Traditional 2D in vitro models are an indispensable tool that allow for robust and high-throughput testing. Their simplicity is also their main drawback as they often fail to replicate relevant physiological behaviours such as cell-cell signalling, inflammation response and in general tend to have reduced function compared to their in vivo homologues.

Historically, results from in vitro assays have quoted nominal concentrations of test chemicals which can lead to significant error in defining dose-response relationships and hinder the quantitative in vitro to in vivo extrapolation. For some chemicals, partitioning into other phases in the system may cause nominal concentrations to be significantly higher than the free (Cfree) and effective concentration.

Alexandre Teixeiraa, Ezel Boyacib, Mi-Young Leea, Beate Nicola

a) Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, Bedford MK44 1LQ, United Kingdom

b) Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada

Extrapolation to in vivo

The ultimate goal in a risk assessment is to predict the in vivointernal dose and relevant biologically perturbed pathways in order to determine a safe exposure level. To reduce uncertainty associated with the extrapolation from in vitro to the complex in vivo system, representative cell models are required as well as understanding of the biokinetic behaviour of the molecule of interest .

Pawliszyn Research Group

Diclofenac Acyl Glucuronide

Absent in human hepatocytes

Major in vivo in human

4’ hydroxydiclofenac

High in human hepatocytes

Major in vivo in human

Traditional method

❑ Time and work intensive

❑ Low throughput

❑ Higher sensitivity

Automated SPME❑ High throughput❑ Cost saving❑ Lower sensitivity❑ Additional method

development

Conditioning

Aqueous solution (e.g. PBS)

Extraction

37 °C, 70 µL sample, 100 rpm agitation, 5 min extraction time

Rinsing

Pure water, with agitation, 10 s

Desorption 1

100 µL ACN/H2O with 0.1% FA (80/20, v/v),with agitation, 2 min

LC-MS/MS

Optimized SPME sample extraction protocol Plate format (nominal dose levels in µM)

2%

4%

6%

8%

10%

12%

14%

0 1 2 3 4

% f

ree

Hours

Diclofenac in HT1080 cell culture

Graph 2 - SPME extractions were carried from the 96 well plate containing HT1080 cells over a period of 4h. Cells were supplemented in Eagles essential media with 5% FBS. Extraction time 5min at 100rpm.

Proof of concept experiment to demonstrate applicability of system to “live” cell cultures. The technique measures Cfree which in this case amounted to 9% of nominal due to binding to protein in media. Each extraction depletes the system by ~4%

Graph 3 –Metabolic depletion of diclofenac was monitored by SPME. Liver microtissues were cultured in serum-free media with three different concentrations (1, 5, 10 uM) of diclofenac over a period of 48h (A). The formation of two main metabolites was also measured, but only DAG was detected (LoD – 0.2uM) and levels of hydroxy metabolite were to low for detection with SPME method (10nM vs. LoD – 1uM)

Ksf

Nitinol wire

Vs.

Graph 4 – Concentration of Diclofenacmeasured after 48h by “traditionalmethod” of direct analyses ofsupernatant by LC-MS/MS vs. SPME.

Graph 1 - Fiber-water optimization for Diclofenac performed in PBS solution. For (A) a 300ng/mL solution was used, equilibrium was not reached after 30min. For (B) extraction time was 5min for each concentration, linear between 0.03 – 3uM (r2 = 0.988). Figures of merit in Boyaci et al. 2018.

0.0

1.0

2.0

3.0

4.0

5.0

0 10 20 30 40Am

ou

nt

ex

tra

cte

d (

ng

)

Extraction time (min)

A - Time to equilibrium

0

0.5

1

1.5

2

2.5

3

0.00 10.00 20.00 30.00 40.00

Are

a R

ati

o

uM

B - SPME calibration

0.4

0.6

0.8

1

1.2

1.4

0 2 24 48

me

as

ure

d / n

om

ina

l

Hours

A- Metabolic depletion of Diclofenac

1uM 5uM 10uM

0%

20%

40%

60%

80%

100%

120%

0 2 24 48

% v

ari

ati

on

Hours

B – Stability of Diclofenac Acyl Glucuronide

10uM 5uM

Conclusion and future work ❑ Novel in vitro systems can provide a more in vivo-relevant approach for use in next generation risk

assessment. However the complexity of these models can lead to increases in cost and labour.❑ The novel SPME fibers and automated system presented can significantly reduce number of cells

required and analysis time.❑ Method also allows direct measurement of Cfree, which is the relevant effect dose.❑ Next step is to test fibers with a wide range of chemicals in order to define a chemical space applicability❑ Develop fibers with different coatings (e.g. C18)

M – media only, DM - DMSO

Kuepfer et al, 2016, CPT: Pharmac. & Sys. 5(10):516-531

From mpbio.com, MP Biomedicals, LLC

InSphero 3D InSight™ Human Liver Microtissue Functionality © InSphero AG. Reprinted with permission.

Solid phase micro-extraction (SPME) fibers

LOD = 0.03uM

0

1

2

3

4

5

6

7

8

1 µM 5 µM 10 µM

measu

red

µM

SPME Supernatant

Improved endogenous and xenobiotic metabolic stability of primary human hepatocytes in 3D co-cultured liver spheroids, allows evaluation of the metabolic profiles and intrinsic clearance rates of low-clearance compounds something not possible with more standard approaches such as microsomes and primary human hepatocyte suspensions.

Vorrink et al., 2017, FASEB J 31 (6): 2696-2708.

Tang, Wei., 2013, CDM 4 (6):319-329

Cell