Cell viability assessment using the Alamar blue assay: A

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Cell viability assessment using the Alamar blue assay: A Cell viability assessment using the Alamar blue assay: A

comparison of 2D and 3D cell culture models comparison of 2D and 3D cell culture models

Franck Bonnier

Technological University Dublin

, Fr[email protected]

Mark Keating

Technological University Dublin

, [email protected]

Tomasz P. Wrobel

Jagiellonian University Cracow

See next page for additional authors

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Recommended Citation Recommended Citation

Bonnier, F. et al. (2015) Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D

cell culture models,

Toxicology in Vitro,

29, pp. 124-131. doi:10.1016/j.tiv.2014.09.014

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Authors Authors

Franck Bonnier, Mark Keating, Tomasz P. Wrobel, Katarsyna Majzner, Malgorzata Baranska, Amaya Garcia,

Alfonso Blanco, and Hugh Byrne

This article is available at ARROW@TU Dublin: https://arrow.tudublin.ie/nanolart/57

Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D cell

culture models

F. Bonnier

, M. Keating

, T. Wróbel

2,3

K. Majzner

, M. Baranska

, A. Garcia

, A. Blanco

H.J. Byrne

1. Focas Research Institute, Dublin Institute of Technology (DIT), Camden Row,

Dublin 8, Ireland

2. Jagiellonian Univiversity, Fac Chem, PL-30060 Krakow, Poland

3. Jagiellonian Univiversity, JCET, PL-30348 Krakow, Poland

4. UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

*Corresponding Author: Franck Bonnier,

Focas Research Institute, Dublin Institute of Technology,

Kevin Street, Dublin 8, Ireland.

E mail address: [email protected]

Ph: +353 1 4027917

Fax: +353 1 4027904

Abstract

Comparisons of 2D and 3D cell culture models in literature have indicated differences in

cellular morphology and metabolism, commonly attributed the better representation of in

vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D

collagen gels for in vitro analysis has been growing. Although comparative studies to date

have indicated an enhanced resistance of cells on collagen matrices against different

toxicants, in the present study it is demonstrated that non-adapted protocols can lead to

misinterpretation of results obtained from classical colorometric dye-based cytotoxic

assays. Using the well established Alamar Blue assay, the study demonstrates how the

transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin

itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that

the cell viability is unaffected when cells are grown on collagen matrices, thus the

difference seen in the fluorescence is a result of a dilution of the resazurin dye in the

collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the

surrounding environment, facilitating more effective diffusion through the cellular

membrane. The results are supported by a rate equation based simulation, verifying that

differing uptake kinetics can result in apparently different cell viability. Finally, this work

highlights the feasibility to apply classical dye-based assays on collagen based 3D cell

culture models. However, the diffusion and bioavailbility of test substances in 3D

matrices used in in vitro toxicological assays must be considered and adaption of the

protocols is necessary for direct comparison with the traditional 2D models. Moreover,

the observations made based on the resazurin dye can be applied to drugs or nanoparticles

which freely diffuse through the collagen matrices, thus affecting the effective

concentration exposed to the cells.

Keywords: Alamar blue assay, 3-D Cell culture, collagen gels, extracellular matrix, cell

viability, flow cytometry,

1. Introduction

Significant efforts have been devoted towards the development of more realistic in vitro

cell culture models, better mirroring in vivo conditions. For example, it is well accepted

that, in an environment mirroring the in vivo conditions encountered by the cells, the

observations made are more representative of the cancerous cell phenotype compared to

those found on conventionally used 2D cultures (Breslin and O'Driscoll, 2012; Elliott and

Yuan, 2011; Kimlin et al., 2011). Moreover, providing a microenvironment with

adequate adhesion and proliferation has been reported to allow a more accurate

investigation of cellular homeostasis, differentiation and migration (Kim, 2005; Kim et

al., 2004). The use of collagen gels and more complex multi-component systems such as

Matrigel has become increasingly popular, as they provide the cells with a matrix which

more accurately represents the extra cellular matrix (ECM) (Petersen et al., 1992;

Prestwich, 2008; Weaver et al., 1995). The impact of such matrices on the cell phenotype

and metabolism has been already documented in the literature. For example, it has been

shown that antibodies against B1-integrins exhibit different behaviour when tested in 2D

compared to 3D models (Wang et al., 1998); induced doxorubicin-resistance by the extra

cellular matrix in human osteosarcoma and HT1080 cells

has been demonstrated (Fourre

et al., 2008; Harisi et al., 2007); reduced radiation induced toxicity when cells are grown

in a 3D environment has also been reported (Sowa et al., 2010). The use of collagen

matrices as 3-D cell culture matrices has greatly increased in the last few years and

numerous other publications can be found in the literature describing modification of the

cell phenotype, metabolism or composition when grown in a 3D matrix, compared to 2-D

environments (Lupanova et al., 2010; Wu et al., 2009).

Although 3D matrices are increasingly routinely employed for cellular analysis, the

impact of the ECM like microenvironment on the cells is still under investigation. The

degree of modification of the cellular behaviour and metabolism remains unclear and the

comparison between conventional 2D substrates and 3D models is still ongoing.

However, the use of a 3D cell culture environment could lead to difficulties in adapting

established cytological protocols as well as in the interpretation of the results. Although

already intensively used in cancer research, collagen gels have not been adapted to other

fields such as cyto- or nano- toxicology. Moreover, although efforts have been devoted to

explaining the difference of cell behavior when grown on 3D substrates such as collagen

gels, no consideration has been given to the differing cell geometry and more specifically

the cell surface exposed to the microenvironment.

In the present study, the Alamar blue (AB) in vitro cytotoxicity assay has been used as a

model to demonstrate the link between the cell substrate used and the cell geometry,

ultimately also influencing the outcome of the colorimetric assay employed, independent

of the cell viability or proliferation. The results are supported by a rate equation based

simulation, verifying that differing uptake kinetics can result in apparently different cell

viability (Supplementary Information).

2. Materials and Methods

2.1. HeLa Cell line

HeLa cells, immortalized human cervical cells, were obtained from the ATTC (Manassas,

VA, USA). Cells were cultured in DMEM supplemented with 1% L-glutamine (200 mM)

and 10% foetal bovine serum and maintained in a humidified atmosphere containing 5%

at 37ºC. HeLa cells were cultured until they reached approximately 80% confluency

before preparing the plates for the cytotoxicity assay. Cells were seeded at a density of 2

x 10

cells per well in 6-well-plates (Nunc Lab-Tek

). Half the plates were prepared with

collagen gels at a concentration of 2.5 mg/mL, and the other half were directly grown on

the plastic base of the plate as controls. The experiments were performed in triplicate and

readings of the cell viability using the AB assay were performed after 24h. Moreover, it

is important to point out that the whole experiment (in triplicate) has been repeated twice

within three weeks interval in order to confirm the reproducibility of the observations

made.

2.2. Collagen gels

Solutions of collagen I from rat tail tendons (Gibco) were used for preparation of the

collagen gels. The 5mg/mL solution was mixed with sterile 10X phosphate buffered

saline (PBS), sterile distilled water (dH

O) and 1M NaOH. The appropriate relative

quantities of these components is determined by the final concentration (2.5 mg/mL) and

volume needed. All the steps were carried out on ice to slow the gelation process. After

mixing, 500 μL of the solution were either placed in 6-wells-plates for the AB

cytotoxicity assay or 1 mL of the solution was placed in a 25 flask for the flow

cytometric analysis, before incubation at 37 °C degrees in a 95% humidity incubator until

a solid gel was formed (about 30 minutes).

2.3. Alamar blue assay

The Alamar Blue® assay is designed to quantitatively measure the proliferation of human

and animal cell lines, bacteria and fungi (Kuda et al., 2003; Mosmann, 1983; O'Brien et

al., 2000; Pettit et al., 2005; S.Al-Nasiry et al., 2007). Over the past 50 years, the AB

assay has been widely used in studies of cell viability and cytotoxicity for biological and

environmental applications (Rampersad, 2012; Vega-Avila and Pugsley, 2011; White et

al., 1996). The bioassay can also be used to establish the relative cytotoxicity of agents

within various chemical classes (Bopp and Lettieri, 2008; Borra et al., 2009; Mikus and

Steverding, 2000; Miret et al., 2006). Using the REDOX indicator resazurin (oxidised

form), it is possible to spectrophotometrically measure the cellular proliferation.

Resazurin is blue and non-fluorescent, whereas resorufin (reduced form) is red and highly

fluorescent. Thus, measuring the changes in the fluorescence of the dye in the

intracellular environment, modifications in the number of metabolic active cells can be

detected. Tetrazolium salts can deliver similar information regarding cell growth, but

present incompatibility problems, the most limiting being the high toxicity of the DMSO

or HCl/isopropanol required for reading the results (Mosmann, 1983). Thus, the AB

assay is generally preferred for kinetic studies. The oxidation-reduction potential of

resazurin is +380 mV at pH 7.0, 25 °C, which means it can be reduced by NADPH (Eo =

320 mV), FADH (Eo = 220 mV), FMNH (Eo = 210 mV), NADH (Eo = 320 mV), as well

as cytochromes (Eo = 290 mV to +80 mV), all part of the cellular respiration metabolic

reactions. However, other enzymes such as the diaphorases (dihydrolipoamine

dehydrogenase (Matsumoto et al., 1990)), NAD(P)H:quinoneoxidoreductase (Belinsky

and Jaiswal, 1993) and flavin reductase (Chikuba et al., 1994) located in the cytoplasm

and the mitochondria can also reduce Resazurin. Therefore, AB reduction is the result of

multiple metabolic reactions and does not necessarily specifically indicate a

mitochondrial dysfunction, but remains a suitable indicator of the cellular health and

viability (Ahmed et al., 1994).

The AB assay was carried out according to manufacturer’s instructions. Briefly, control

medium was removed; the cells were rinsed with PBS and 2 mL of an AB solution (5%

[v/v] solution of AB dye) prepared in fresh medium (without FBS or supplements) were

added to each well. Following 3 hours incubation, AB fluorescence was quantified at the

respective excitation and emission wavelength of 540 and 595nm using a Tecan Genios

microplate reader. The results were averaged over 3 different independent experiments

(n=3, each conducted with one week interval) with 3 replicates per experiment (3 x 6 well

plates), each replicate being prepared from different T75 flasks in order to take into

account the biological variability. Finally, for each plate the reading was also done in

triplicate (values obtained from 3 different wells averaged) in order include the technical

variability due to the efficiency of AB assay, sensitivity of the plate reader or simply

related to the sample preparation. For each experiment, wells containing only the AB

solution without cells were also prepared and incubated for 3h. The fluorescence

measured in those was used as a background and subtracted. However, although this

protocol is routinely used in the literature, some variations in the AB solution

concentration and exposure time have been made purposely throughout this study. These

modifications will be clearly highlighted and discussed at the appropriate points in the

manuscript, for clarity. Again, each experiment was conducted in triplicates (3 x 6 well

plates) which have been prepared from different flasks in order to have independent

replicate based on different cell populations.

2.4 Flow cytometry assays

2.4.1. Apoptosis assay

This study was performed using a Partec CyFlow® Space Flow Cytometer (Partec UK

Limited, Germany). The default Partec FloMax® flow cytometry software has been used

for the analysis of the samples, but the Beckman Coulter Summit software and the

FCSExpress Research Edition have been used for the reanalysis of the samples. The QC

control of the instrument was performed using Spherotech 6 and 8 peak beads. HeLa cells

(1x10

cells//flask) were seeded in T-25 flasks and incubated in a 5% CO

incubator at

37°C for 24 hours. These conditions were selected to avoid cells becoming over

confluent, which might lead to misinterpretation of the apoptosis assay due to cellular

death caused by the stress of the culture conditions and medium depletion causing cell

starvation. The experiments have been conducted in triplicate. However, in order to keep

replicates as independent from each other as possible, 3 T75 flasks were initially seeded

and from each one of them two T25 were prepared for flow cytometry, with only one

containing a collagen gel as described above. Following incubation, the cells were

washed twice with pre-warmed PBS and were collected by trypsinization, after which the

trypsin was removed by centrifugation. The cells were then washed twice with pre-

warmed PBS and stained with the YOPRO1/Propidium iodide (PI) dyes (Life

Technologies), whereby 1 μL of YOPRO1 dye (100 µm) and 1 µL of PI (1mg/mL) were

used to stain 1x10

cell/ml. After staining of a cell population, apoptotic cells show a

green fluorescence, whereas necrotic cells show green and red fluorescence. After

incubation on ice for 30 min, the cells were analysed by flow cytometry within 30

minutes, using 488 nm excitation and reading the fluorescence of YoPro with a IBP

527/30G filter and PI with a IBP 682 B filter. Unstained and single control samples have

been used in order to set up the instrument and gates in order to visualize 3 groups: live

cells, apoptotic cells and necrotic cells. Samples with over-confluent cell populations

leading to medium depletion which is ultimately associated with a high cellular death

have been used as biological controls. 15,000 single cells per sample have been analysed.

3. Results

3.1. Cell viability monitoring using the AB assay

Conventional in vitro cytotoxicity assays such as AB, Neutral Red and MTT (3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) have been developed for rapid

screening of cellular proliferation, viability, metabolic activity, lysosomal and

mitochondrial activity (Davoren et al., 2007; Herzog et al., 2007; Mukherjee et al., 2010;

Naha et al., 2010). AB is widely used for cytotoxicity assay and has been selected for the

low induced cytotoxicity from the redox indicator giving more freedom in the

concentration of reazurin used.

Figure 1: Comparison of Alamar blue assay results obtained after compensation of the AB solution

concentration for cells grown on collagen gels (blue) and plastic (red) for 24hrs using both 5% and 6.7%

AB solutions. The experiments have been repeated twice in triplicates and the error bars indicate the

overall variability observed

The viabilities of the HeLa cells grown on a 2D plastic surface and a 3D collagen matrix

have been evaluated using the AB assay according to the standard protocol described in

Section 2.3. Figure 1 displays the AB fluorescence measured after 24h for cells cultured

on both plastic substrates and collagen gels. The Y axis is the fluorescence intensity after

subtraction of background signal of the AB with no cells but incubated for 3 hr.

After 24h incubation, the cells grown on collagen gels exhibit significantly higher

(~21%) fluorescence intensity than those grown on the 2-D substrate (p<0.001). The

statistical variations represented by the error bars show small variance in the fluorescence

read at 24h. The difference in the fluorescence measured indicates a higher degree of

reduction of the AB dye by the cells grown on collagen gels. In absence of exogenous

agents with toxic properties for the cells, the difference in the readings indicates an

increased viability compared to the cells seeded directly on plastic. Therefore, in order to

validate this observation, further approaches have been used.

3.2 Flow cytometry

As collagen gels are purported to better represent in vivo conditions, a possible cause for

the strong increase of the cell viability obtained using the AB assay is a better

biocompatibility of the cells with the 3D matrix. In order to validate the observations

made using the AB assay, additional assays were needed to give more information

regarding the cellular behaviour when grown on plastic compared to the collagen gels.

Flow cytometry is a powerful tool for the analysis of large cell population based on

fluorescent dyes. Moreover, the main advantage for this study was the possibility to label

the cells while in suspension rather than still attached and thus the labeling process

should be independent of the growth substrate.

For the purpose of this work, the apoptosis assay has been selected to visualize the

amount of live / dead cells in the suspension to get an indication of the possible higher

toxicity/better biocompatibility of the substrates.

Figure 2: Flow cytometry apoptosis assay using the Yo-pro/PI dyes. A: Debris have been excluded by

gating them out based on the scatted characteristics (FSC vs SSC), gate 1, B: Aggregated events have been

excluded with gate 2 in FSC-Area vs FSC-Height; C and D: Cells characterized as alive respectively from

plastic (2C) and collagen gels substrates (2D). The experiments have been repeated in triplicate from 3

different T25 flasks.

The results of the apoptosis assay performed after 24h incubation are presented in figure

2. The plot presents the fluorescence measured for both the YOPRO1 (figure 2C and D -

X axis) and the PI (figure 2C and D - Y axis) dyes. The distribution of the data indicates

the proportion of live versus dead cells present in the suspensions harvested from the

collagen gels (figure 2C) and plastic (figure 2D). Apoptotic and necrotic cells incorporate

different levels of the two fluorescent dyes, as well as scatter changes as can be observed

in the scatter bivariate histogram. In the present study, and for comparison with the AB

assay, only the viable cells are of interest. As shown in figure 2, in the case of the cells

grown both on collagen and plastic, the cluster is mainly located in the bottom left area

(with no uptake of any of the dyes), indicating a high proportion of live cells. After

performing the experiments in triplicate from 3 different sets of T25 flasks, the

percentage of live cells has been evaluated to be 84.6% (+/- 5.2%) for those harvested

from the collagen gels whereas for the plastic substrate it was found to be 87% (+/-

2.9%). Therefore, neither of the substrates used has a statistically significant toxicity that

could possibly affect the outcome of the AB assay after 24h incubation, and notably,

according to the standard deviation calculate from the triplicates, there is no significant

difference between the cell populations grown on 2D substrates and 3D matrices.

3.3 Effect of the collagen gels on the AB solution concentration

The observations made using flow cytometry clearly demonstrate that the apparent

difference in the cell viability indicated by the AB assay is not linked to differences in

cell viability. Thus, the question remains as to how to understand and interpret the origin

of the differences in the fluorescence readings of the AB assay.

The collagen matrices are prepared from a solution of collagen initially at 5 mg/mL

which is diluted for the needs of the experiments to 2.5 mg/mL. All the reagents used are

water based and, after gelation and formation of the collagen fibers, the matrices are

similar to wet sponges. Thus, the first concern is about any reduction of the effective

concentration of the AB solution, as presented to the cells. Wells free of cells and only

containing the collagen gels covered with 2 mL of AB solution (5%) were prepared and

incubated for 1h, 2h and 3h. After each time point, the AB was pipetted out of the wells

and the absorbance at 570 nm was measured. The results are expressed as a percentage of

the absorbance obtained from the AB solution placed in the well without any collagen.

After 1h, the absorbance is equal to 76% of the control, after 2h 74% and after 3h 76%.

This demonstrates how quickly the dye from the AB solution can diffuse through the

collagen gels and therefore how the concentration of the AB solution used can be affected

by dilution of the dye solution by the water contained in the matrix. As the collagen gel is

mostly water, it is important to take into account the volume of collagen used to form the

matrix into the calculation of the solution used for the assay.

The effect of the AB solution concentration on the assay can be easily visualised. Based

on the absorbance measurements, it can be estimated that the effective concentration of

the nominally 5% AB solution is reduced by ~25% in the collagen gel, resulting in the

cells being exposed to only 3.75% AB solution. To compensate the dilution of the dyes

into the collagen gels, the assays have been run in parallel using 6.7% AB solutions.

Figure 1 presents the results obtained after 24h for both AB solutions tested on cells

grown directly on plastic and collagen gels. According to the observations made on the

dilution of the dyes in the collagen gels, the results obtained from the cells grown on

plastic with a 5% AB solution should be compared to those obtained from the cells grown

on collagen gels exposed to a solution at 6.7%. Under these conditions, the difference in

the cell viability found is even greater, with a fluorescence intensity 49% more intense

after 24h incubation.

3.4 Understanding the Alamar blue assay

Figure 3: Alamar blue performed on cells grown on collagen gels with high concentration of AB solution

ranging from 25% to 50%. Cells have been exposed to the AB solution for 3h (top) and 5h (bottom). The

error bars indicate the variability observed over 3 different replicates.

The cell viability as measured using flow cytometry highlighted that there is no increased

cell death on the plastic substrates compared to the collagen gels, which rules out

potential toxicity due to the environment of the cells. Thus, two hypotheses could explain

the difference in the fluorescence measured using the AB assay. Firstly, an increased

metabolic activity of the cells when grown on the collagen gels leading to an increased

conversion rate of the resazurin to its fluorescent form. Secondly and more simply,

different rates of uptake of the AB dye by the cells in the two culture environments. The

organization of the collagen matrix can be compared to a network formed by the

polymerized fibers. In this work, the cells have been seeded onto the collagen gels, but,

as discussed above, the dyes from the AB solution freely diffuse through the gels. Uptake

of the dye by the cells occurs by diffusion through the cytoplasmic membrane. Whereas

the cells seeded on plastic have only one side exposed to the surrounding medium, when

seeded on the collagen matrix, the underside of the cells also have direct contact with the

AB solution and thus present a significantly larger surface (membrane) area through

which the dye can diffuse. A higher uptake rate of the AB solution by the cells would

result in a higher fluorescence reading, indicative of a higher metabolic activity or larger

cell population.

Ideally, the measurement of the actual amount of AB dye present in the cells would be

the best approach to investigate differing uptake rates by the cells. However, the dye

diffuses passively through the membrane of the cells and is continuously reduced in the

cytosol. A measurement of the absorbance would register the unreacted, resazurin

(oxidized) form of the dye, whereas fluorescence registers the reacted, resorufin

(reduced) form of the dye.

In order to estimate the effects of different uptake rates of the dye by the two cell culture

models, the protocol for the AB assay has been modified to operate in the regime of

saturated uptake, in which different uptake rates do not affect the quantity of dye uptaken.

One of the advantages of the AB assay is the low cytotoxicity of the dye at low

concentrations which allows the kinetics of the cellular viability over a prolonged time to

be monitored. However, for the purpose of this study, the concentration of the AB

solution was increased up to 50% in order to visualize a point of saturated dye

uptake/conversion, as indicated by the fluorescence intensity reaching a maximum.

Figure 3A presents the results obtained for cells grown on collagen exposed to 25%,

30%, 35%, 40%, 45% and 50% AB solution for 3h. Although, for concentrations up to

35%, the fluorescence slightly increases, it appears that higher concentrations do not

induce a statistically significant increase of the fluorescence. This indicates either

saturation of the dye uptake or of the reduction rate of the resazurin into resorufin.

For varying AB concentrations, measurement of the fluorescence was made every 45

minutes for 7 different time points. After 5h exposure to the AB solution (figure 3B), the

fluorescence still exhibits a maximum intensity at 35% AB solution, but for higher

concentrations, a slight decrease can be seen, likely related to the toxicity of the AB

solution at high concentrations. Similar observations can be made for the cells grown on

plastic substrates (data not shown). A 35% AB concentration was therefore selected in

order to avoid interferences from the cytotoxicity of the solution on the cells which could

make the interpretation of the results more difficult. Figure 4 shows the fluorescence

measured for incubation periods from 3h to 7h15 min (7 time points). Although a small

difference can be seen, with a slightly higher fluorescence for the cells grown on

collagen, it is not comparable to the 21 % difference observed for the standard assay

protocol (3hr, 5% AB). For more precise visualisation, figure 5 displays the difference in

the fluorescence intensities, but also this difference expressed as a percentage of the total

fluorescence measured. The fluorescence is between 1% and 5% higher for the cells

cultured on collagen gels, which is considerably less than the 21% difference found when

working with 5% AB solution. This highlights that, using a higher concentration, the

system has reached saturation. The fact that the fluorescence exhibits similar intensities

also indicates that the cells have reached their maximum reduction rate, which,

importantly, is the same for cells grown on both collagen and plastic. Therefore, the

metabolic activity of the cells as determined by the AB assay is the same for both cell

cultures. This is confirmed in figure 4 as the gap between the two curves remains

constant over time, indicating identical reduction rates of the resazurin into resorufin in

the two cell culture models. If the metabolic activity was higher for cells grown on

collagen, the difference would increase over time. However, the fluorescence difference

between the two substrates remains similar with a maximum difference of about 5% after

4h 15 min. These observations demonstrate that the metabolic activity of the cells in the

two cultures is comparable and therefore the difference in the fluorescence observed

under the standard assay conditions is not related to higher cell viability or proliferation

but to a higher uptake of the dye by the cells. The larger surface area exposed to the

medium from the cells grown on 3D collagen gels increases the passive diffusion into the

cytosol inducing the slightly higher reading obtained which reflects the delay between the

2 models until reaching the saturation point.

The impact of the differences in the effective cell surface areas in 2D vs 3D cell culture

models can be further illustrated using a rate equation model as described in the

Supplementary Information. The model clearly demonstrates how the apparent

differences in cell viability as indicated by the AB assay can be affected by differing

uptake rates of the dye, as a result of different effective cell surface areas (see

supplementary materials). Notably, such a numerical approach could potentially be

employed as a guide to adapting 2D protocols to 3D cultures.

Figure 4: Kinetic of the fluorescence obtained using an AB solution at 35% for cells grown on plastic

(blue) and collagen gels (red) from 3h exposure up to 7h15mins. Reading s have been performed every 45

mins. The values presented are mean fluorescence calculated from experiments conducted in triplicate.

Figure 5: Representation of the difference of the fluorescence obtained for cell grown on plastic and

collagen gels when performing the AB assay using a 35% solution for each time point. The histogram

represents the difference in the fluorescence intensity while the curve is the difference expressed in

percentage. The results presented have been obtained from experiments conducted in triplicate and

averaged.

4. Discussion

According to the observations made throughout this study, there is no evidence that the

transfer from the 2D plastic to the 3D collagen gels has any influence on the cell health

and viability. Clearly, however, the effects of diffusion of the active dyes through the

matrices and their subsequent bioavailability to the cells can lead to misinterpretation of

the results obtained. The concern addressed in the present study relates to the relevancy

of the change of cell behavior, proliferation or resistance against active agents

documented in the literature. The AB assay has been used as a model to highlight the

importance of the cell culture model geometry on the outcome of cytotoxic investigation.

While the cell surface exposed to the surrounding environment will have an impact of the

uptake rates, it is essential to take into consideration the added volume added to the cell

environment associated with the use of 3D protein based substrates. Importantly, the

observations made regarding the resazurin can be applied to other models. Thus, both the

dilution of the solution by the gel and the different uptake routes will also apply to all

solutions/dispersions in vitro such as toxicants, chemotherapeutic agents and

nanoparticles. The 3D matrix acts as a sponge which results in a dilution factor of the

medium and thus of any active agent, drug or nanoparticle in suspension. However, this

effect is often neglected in in vitro studies investigating the cytotoxicity of drugs or

toxicants on cells (Godugu et al., 2013; Lee et al., 2008; Millerot-Serrurot et al., 2010).

Although the collagen gels may appear to have a significant impact on the cell behavior

and metabolism, when comparing IC

‘s or EC

’s calculated from 2D and 3D models,

neglect of the dilution effect in 3D matrices makes the observations questionable.

Apparent systematic variations in viability can be a result of a reduced effective

concentration of the agent or assay used in 3D matrices compared to 2D cultures,

resulting in a dilution factor which could account for the difference in the IC

calculated(Godugu et al., 2013; Lee et al., 2008; Millerot-Serrurot et al., 2010) and/or

increased cell resistance to toxic/chemotherapeutic agents

Conclusion

The use of 3D collagen gels as growth substrates has been reported to affect the cell

phenotype and behavior, exhibited as different degrees of resistance to toxicants or

modified levels of metabolic activity. In the literature, many studies aim to understand

and explain how the use of 3D matrices can induce considerable changes in the results

obtained when testing toxicants, chemotherapeutic agents or nanoparticles compared to

the well known 2D surfaces. Although the interaction of the cells with their surrounding

environment can trigger different signaling pathways in the cells, modifying their

behavior, the present study demonstrates that adaption or comparison of the results

obtained from 2D models with 3D matrix cell culture systems requires consideration of

the geometry and morphology of the cell/substrate interface. Therefore, the diffusion and

bioavailability of test substances in 3D matrices used in in vitro toxicological assays must

be considered and adaption of the protocols is necessary for direct comparison with the

traditional 2D models. The example of the well established AB cytotoxicity assay

highlights that, what can be first interpreted as an increase of the cell viability, is in fact a

result of a reduction of the effective concentration of the assay by dilution in the collagen

matrix, and a difference in the cell area exposed to the surrounding environment of the

cells, resulting in a higher rate of dye uptake by the cells. Thus, in order to clearly

understand how the 3D cell culture model affects the cell resistance and survival, the

protocols used for 2D models need to be carefully improved and adapted to allow direct

comparison of the results obtained.

Acknowledgement

This research was supported by the National Biophotonics and Imaging Platform (NBIP)

Ireland, Higher Education Authority PRTLI (Programme for Research in Third Level

Institutions) Cycle 4, co-funded by the Irish Government and the European Union

Structural Fund and by Science Foundation Ireland Principle Investigator Award

11/PI/1108.A. Blanco would like to acknowledge the ISAC Scholars Program.

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Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D cell

culture models

F. Bonnier

, M. Keating

, T. Wróbel

2,3

K. Majzner

, M. Baranska

, A. Garcia

, A Blanco

H.J. Byrne

1. Focas Research Institute, Dublin Institute of Technology (DIT), Camden Row,

Dublin 8, Ireland

2. Jagiellonian Univ, Fac Chem, PL-30060 Krakow, Poland

3. Jagiellonian Univ, JCET, PL-30348 Krakow, Poland

4. UCD Conway Institute, Unversity College Dublin, Belfield, Dublin 4, Ireland

Supplementary Information

S.1 Mathematical model describing the relation between uptake rate and cell surface

area

The system can be simply modeled using a rate equation approach, by which the

differences between low dose and high dose responses, as well as the impact of AB

solution dilution by the collagen matrix and the greater uptake rate of cells in 3D gels

compared to 2D substrates may be more easily visualized. The diffusive uptake and

release of the AB dye by the cell is described by

int

= k

.A.B.D – k

.A.N

int

(t) Equation 1

where N

int

is the amount of dye internalized by the cells, k

is the rate of internalization,

D is the % dose of the dye, and k

is the rate of reverse diffusion from the cells. A is a

factor which normalizes for the effective exposure of the cells, depending on the

substrate, and B normalizes for the reduction of the effective concentration of the dye

solution in the 3D gels.

The saturated uptake of the dye is simulated by introducing a concentration of receptors

(Black and Leff, 1983), N

recp

, which are occupied according to

recp

= k

recp

(t).N

int

(t) Equation 2

where k

is a receptor/dye binding rate. The fluorescence reading, I

, from the AB can

then be represented by

= k

recp

(t).N

int

(t)- k

tox

int

(t) Equation 3

The second term allows for a reduction of AB fluorescence due to dye toxicity.

Using such a model, the complete time evolution and concentration dependence of the

AB fluorescence can be simulated. To simulate the differences between the 3D and 2D

cell culture environments, the effective area parameter A, is reduced from 1 for 3D to 0.5

for 2D, mimicking a 50% reduction of the effective area of the cells exposed to the AB

solution. In the 3D environment, the effective concentration of the AB solution is reduced

by 25%, and so a value of B =0.75 is used. For the purpose of demonstrating the

appropriateness of the model for this study, Figure S.1 shows, for example, the dose

dependent %fluorescence predicted for 3hrs and 5 hrs. For collagen gels, values of A=1

and B=0.75 were employed. The figure qualitatively reproduces the behavior observed in

figure 5, whereby an increasing %fluorescence is observed in the range 25-50%

concentration of AB solution after 3hrs, whereas a maximum in %fluorescence is

observed at a concentration of ~30% after 5hrs.

Figure S.1: Comparison of the simulated AB fluorescence after 3 and 5 hrs as a function of

%Concentration of AB solution for the 3D gels (A = 1, B =0.75)

Using the same parameters, the simulated AB fluorescence from the 3D matrices (A = 1,

B = 0.75) after 3hrs can be compared with that of the 2D substrates (A = 0.5, B = 1), as

shown in Figure S.2. It can be seen that, although there is a significant difference of

~25% between the viability values for the two cell culture substrates at an AB

concentration of 5%, this has reduced to ~5% at an AB concentration of 35%. Although it

cannot be considered a precise description of the system, the numerical simulation does

indicate that the observed differences in AB fluorescence can be accounted for simply by

the differences in the cell surface area which is accessible by the dye for diffusion.

Figure S.2: Comparison of the simulated AB fluorescence after 5 hrs as a function of %Concentration of

AB solution for the 3D gels (A = 1, B =0.75) and the 2D substrates (A = 0.5 B =1)