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Cytotoxicity evaluation of sodium lauryl sulfate in

Cite this: DOI: 10.1039/d2ay00161f
a paper-based 3D cell culture system†
Young Ju Lee,a Yong Jin Ahnb and Gi-Ja Lee *ab

Received 28th January 2022
Accepted 27th March 2022
DOI: 10.1039/d2ay00161f
rsc.li/methods
Because three-dimensional (3D) cell culture is more similar to in vivo cell microenvironments than two-
dimensional (2D) cell culture, various 3D cell culture systems have been developed. Recently, paper has
been used as a promising material for 3D cell culture and tissue models due to its flexibility, ease of
manufacture, low cost, and widespread accessibility. In this study, we fabricated a paper-based 3D cell
culture platform consisting of a hydrophilic region for cell attachment and a hydrophobic region printed
with wax. Using this paper platform for 3D culture of L929 cells, we evaluated the cytotoxicity of
a model substance, sodium lauryl sulfate (SLS), using water-soluble tetrazolium salt, Live/Dead, and
luminescence assays. Then we compared those cytotoxicity results with results from a conventional 3D
cell culture kit and 2D cell culture. We found that 3D cultured cells on paper responded more sensitively
to SLS than 2D cultured cells, and the cytotoxicity of SLS to cells grown on the paper-based 3D cell
culture platform was similar to that of cells grown using a commercially available 3D cell culture kit.
Therefore, we expect that our paper-based 3D cell culture platform can be applied as a simple and facile
tool for cell viability evaluation.

testing, mainly due to a lack of clinical efficacy or unacceptable

1. Introduction
Cell-based analysis techniques are widely used in various elds,
including basic research, new drug discovery, and testing of
pharmacokinetic medicines, because they are simple, fast, and
cost-effective tools that can replace large-scale, cost-intensive
animal experiments.1 To date, most cell-based assays use two-
dimensional (2D) cell monolayers cultured on at and rigid
substrates. Although 2D cell cultures have been the primary
tools for cell-based research, they have a signicant limitation:
in vivo, cells are surrounded by other cells and extracellular
matrix (ECM) in a three-dimensional (3D) microenvironment,
and 2D cell culture cannot adequately represent the natural 3D
environment of cells. Consequently, 2D cell culture-based tests
sometimes provide misleading and unpredictable data about in
vivo reactions.1–3 In drug discovery, for example, the standard
procedure for high-throughput screening of new compounds
begins with 2D cell culture-based tests and then proceeds from
animal model tests to clinical trials. Only about 10% of those
compounds successfully pass the nal clinical trial, and many
others fail during later clinical trials.4,5 Many compounds fail
during phase III, which is the most expensive stage of clinical
a
Department of Biomedical Engineering, College of Medicine, Kyung Hee University, 26
Kyungheedae-ro, Seoul 02447, Korea. E-mail: gjlee@khu.ac.kr; Fax: +82 2 6008 5535;
Tel: +82 2 961 2305
b
Department of Medical Engineering, Kyung Hee University Graduate School, Seoul
02447, Korea
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d2ay00161f
toxicity.6,7 Some portion of those failures can be attributed to
data collected from 2D cell monolayers, in which the cellular
response to drugs was altered by the unnatural microenviron-
ment. Besides, testing the safety of any biomaterials or medical
devices which are used in direct and indirect contact with
patient is critical for the safe use in clinical practice. Thus, the
evaluation of biocompatibility using the best method is an
important concern for manufacturing companies and certi-
cation authorities.8 Recently, growing evidence has indicated
that 3D cell culture systems more accurately represent the
actual microenvironment of cells within tissues.7,9,10 The spatial
and physical aspects of 3D cell culture affect the transmission of
signals from outside cells to inside cells and the resulting gene
expression11,12 and cellular behavior.13,14 Cell responses in 3D
culture have been shown to be more similar to their in vivo
behavior than those in 2D culture.15,16
Paper has attracted attention as a substrate for various
biomedical applications, including paper-based analytical
devices17–19 and cell culture platforms.20–26 Paper can be used as
an alternative cell culture substrate due to its essential
biocompatibility, ease of chemical and physical modication,
cost efficiency, eco-friendliness, easy availability and ease of
large-scale manufacturing, as well as simplicity and ease of use.
The most important advantage of paper substrate as a cell
culture platform is its intrinsic 3D conguration that can mimic
the native pathophysiological cellular microenvironment.23–28
Thus, paper-based 3D cell culture systems can produce physi-
ologically relevant uid ows and oxygen and nutrient gradients

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that reect the native microenvironment better than conven-
tional 2D cell culture systems.27,29 In addition, the thickness,
porosity, and exibility of paper can easily be controlled to
simulate specic in vivo environments.20 A variety of commer-
cially available papers including Whatman lter paper,20,23,24
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nitrocellulose membrane,30 Kimwipes31 and weighing paper32
are currently used in cell culture and biomedical applications.
Among them, Whatman lter paper is the mostly used for 3D
cell culture because it is less expensive and easy to obtain the
suitable pore size.
In this study, we fabricated a paper-based 3D cell culture
platform with patterned hydrophobic wax barriers and hydro-
philic regions for cell attachment by using a simple wax-
printing method. First, we evaluated the toxicity of the manu-
factured paper platform itself and chemically modied the
surface of the paper platform with cell adhesion molecules.
Then, we seeded L929 cells in the paper-based 3D culture
platform and treated them with sodium lauryl sulfate (SLS),
a model toxic substance. L929 cells were used according to the
International Standards Organization (ISO) guidelines (UNE-EN
ISO 10993-5:2009 Part 5 In Vitro Cytotoxicity Test) which
provides a specic in vitro test method for cytotoxicity evalua-
tion of various types of materials. Colorimetric, uorescence,
and luminescence assays were used to compare its cytotoxicity
to L929 cells 3D cultured on our paper platform, 2D cultured,
and cultured in a commercialized 3D cell culture kit. The
fabrication process of 3D cell culture paper and experimental
scheme for the evaluation of cytotoxicity are presented in Fig. 1.
2. Experimental procedures
2.1. Materials and chemicals
Whatman grade 1 lter paper was purchased from GE Health-
care Bio-Sciences (Pittsburgh, PA, USA). Collagen I (type I
solution from rat tail), bronectin (from human plasma), poly-L-
lysine, ECM gel (from Engelbreth-Holm-Swarm murine
sarcoma), and SLS were purchased from Sigma Aldrich (St.
Fig. 1 Schematic diagrams of (A) the fabrication process of paper-based 3D cell culture platform and (B) the cytotoxicity evaluation of sodium
lauryl sulfate on L929 cells 3D cultured on our paper platform, 2D cultured, and cultured in a commercialized 3D cell culture kit.
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Paper
Louis, MO, USA). The water soluble tetrazolium salt (WST) assay
was purchased from Daeil Lab Service (EZ-CyTox colorimetric
assay kit, Seoul, Korea). A Live/Dead™ viability/cytotoxicity kit
was purchased from Molecular Probes (Eugene, OR, USA). A
CellTiter-Glo® luminescent cell viability assay kit was
purchased from Promega Corporation (Madison, WI, USA). A
3D collagen culture kit was purchased from Merck Millipore
(Darmstadt, Germany).
2.2. Fabrication of paper-based 3D cell culture platform
To prepare our paper-based 3D cell culture platforms, we used
a wax-printing technique to create hydrophobic regions on the
paper. The wax pattern was designed using AutoCAD soware
and printed onto Whatman grade 1 lter paper with a Xerox
ColorQube 8570 N solid-wax printer (Fuji Xerox, Tokyo, Japan).
The paper substrate for 3D cell culture contains four circular
zones, each of 5 mm diameter. To cultivate the cells within only
these four zones, all the background area was printed with wax.
Then, the wax-printed paper sheet was baked in a BF-150C
drying oven (DAIHAN Scientic, Seoul, Korea) oven at 130
C
for 150 s, which melted the printed wax and allowed it to
penetrate into the paper and form the hydrophobic and insu-
lating patterns. The wax-printed paper was subjected to UV
sterilization and then used for cytotoxicity testing (Fig. 1A). To
increase cell adhesion, the chemical properties of the paper
surface were changed using various cell adhesion molecules:
100 mg ml1 of collagen type I, 0.1% of bronectin, 0.1 mg ml1
of poly-L-lysine, and 8–12 mg ml1 of ECM gel. Cell adhesion
and cell growth on our paper-based 3D cell culture platform
were colorimetrically evaluated. An Epson Perfection V700
Photo atbed scanner (SeikoEpson, Nagano, Japan) and ImageJ
soware were used for image readout and analysis.
2.3. Cell culture
Mouse L929 cells were obtained from the Korea Cell Line Bank
(Seoul, South Korea). The cells were routinely maintained in
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Paper
complete growth medium consisting of RPMI 1640 (GIBCO,
Grand Island, NY, USA) supplemented with L-glutamine, 10%
heat-inactivated fetal bovine serum (FBS), and 1% penicillin/
streptomycin at 37
C in an incubator with a humidied 5%
CO2 atmosphere.
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2.4. Cytotoxicity evaluation of the wax-printed paper-based
3D cell culture platform
To determine whether our wax-printed paper-based 3D cell
culture platform induced cytotoxicity, the wax-printed paper
was UV sterilized and then immersed in RPMI-1640 medium
without FBS to collect eluates for 24, 48, and 72 h. The control
sample was cell culture medium that was not exposed to the
waxed paper. L929 broblast cells (4  104 cells per ml) were
seeded in 48-well plates and incubated for 24 h. The cells were
treated with eluates containing 10% FBS for 24, 48, and 72 h.
Then, cytotoxicity was measured using the WST (EZ-CyTox
colorimetric assay kit) assay. OD450 was measured with
a multi-microplate reader (Synergy HT multi-mode microplate
instrument, BioTek, Winooski, VT, USA). The results of each
sample were averaged and expressed as a percentage of the
control.
2.5. Cytotoxicity evaluation using WST assay
In the 2D cell culture, L929 broblasts (1  105 cells per ml)
were inoculated into 96-well plates and cultured in cell culture
medium for 24 h. In the 3D cell culture, the same number of
cells (1  105 cells per ml) was inoculated onto our paper-based
3D cell culture platform coated with collagen. It was incubated
in a 5% CO2 incubator at 37
C for 20 min to allow the cells to
stabilize on the paper and then incubated in a solid cell culture
medium containing 0.5% agarose for 24 h. The cells were then
treated with various concentrations of SLS (0, 0.02, 0.04, 0.06,
0.08, 0.1, 0.12, and 0.14 mg ml1) and further incubated in the
agarose medium for 24 h at 37
C in a 5% CO2 incubator. Aer
the 24 h incubation, cell cytotoxicity in the 2D cell culture of 96-
well plates was measured using an EZ-CyTox colorimetric cell
viability assay and the multi-microplate reader. To evaluate cell
cytotoxicity on the paper-based 3D cell culture platform, the EZ-
CyTox colorimetric cell viability assay solution was adminis-
tered directly to the paper, and the changes in color intensity of
the cell paper were analyzed quantitatively using an Epson
scanner and ImageJ soware, as described previously.33 Briey,
aer the images were split into red, green, and blue channels,
the blue channel intensity of the whole area of each cell zone
was analyzed to minimize the deviation between each paper.
The change in blue channel intensity was dened as the blue
channel intensity of the blank zone minus that of the cell zone.
2.6. Cytotoxicity evaluation using the Live/Dead™ assay
For 2D cell culture, 1  105 L929 cells were seeded in a 35 mm
glass bottom dish, and for 3D cell culture, the same number of
cells was seeded on our paper-based 3D cell culture platform
and then incubated for 24 h in agarose culture medium. Aer
exposure to various concentrations of SLS (0, 0.02, 0.04, 0.06,
0.08, 0.1, 0.12, and 0.14 mg ml1) for 24 h, the cells were
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incubated with a mixture of 8 M ethidium homodimer and 2 M
calcein acetoxymethyl in phosphate-buffered saline for 30–
45 min at room temperature. The numbers of viable (green) and
non-viable (red) cells were counted manually from images
collected using a Zeiss LSM-700 confocal microscopy system
(Thornwood, NY, USA).
2.7. Cytotoxicity evaluation using CellTiter-Glo®
luminescent cell viability assay
Mouse L929 broblasts (1  105 cells per ml) were seeded in
black 96-well plates and the paper-based 3D cell culture plat-
form and incubated for 24 h. The cells were then treated with 0,
0.02, 0.04, 0.06, 0.08, 0.1, 0.12, or 0.14 mg ml1 of SLS and
further incubated at 37
C in a 5% CO2 incubator for 24 h. To
measure the luminescence signal in the paper-based 3D cell
culture platform, each paper containing cells was cut and
transferred to a black 96-well plate. CellTiter-Glo® reagent in
the same volume as the cell culture medium present in each
well was added and reacted in an orbital shaker for 2 min to
induce cell lysis. Aer stabilizing the luminescence signal by
incubating the plate for 10 min at room temperature, the
luminescence signal was measured by the multi-microplate
reader.
2.8. Cytotoxicity evaluation in conventional 3D collagen
culture kit
An L929 cell suspension (1  106 cells per ml) was mixed with
chilled collagen solution under sterile conditions so that 1 
105 cells/100 ml of cells were seeded in 96-well plates, followed
by polymerization of the collagen in a 37
C incubator for 1 h to
form a gel. Cell culture medium was added to cover the collagen
gel and incubated for 24 h. The cells were treated with 0, 0.02,
0.04, 0.06, 0.08, 0.1, 0.12, or 0.14 mg ml1 of SLS. Following
incubation for 24 h, cytotoxicity was measured using the WST
and Live/Dead™ assays.
2.9. Statistical analysis
The data are presented as mean  the standard deviation.
Independent t-test was used to analyze differences between the
two groups and was conducted using the IBM® SPSS (ver. 25.0;
IBM Corporation, Armonk, NY, USA), and p-values less than
0.05 were regarded as statistically signicant. The half maximal
inhibitory concentration (IC50) values for SLS in each assay
were obtained using the Quest Graph™ IC50 Calculator, AAT
Bioquest, Inc (https://www.aatbio.com/tools/ic50-calculator)
online resource. Values were normalized dividing by the largest
response.
3. Results and discussion
3.1. Characterization of the paper-based 3D cell culture
platform
Paper-based analytical platforms have several advantages,
including low cost, simple fabrication, and ease-of-use, that
make them attractive for many applications, such as biochem-
ical and analytical sensors.34–36 Paper can easily be made with
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a desired pattern by wax printing with a non-toxic reagent. In
addition, paper is generally made of cellulose, which makes it
compatible with biological systems. Paper is suitable for
colorimetric detection and provides strong contrast with
colored substrates, allowing researchers to see the results
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on cell viability of L929 cells, 3  104 cells were seeded on three
types of paper (collagen-coated) and cultured for 24 h, which
was followed by Live/Dead™ analysis. As shown in Fig. 2B,
grade 1 paper showed the best cell viability. Thus, we selected
Whatman grade 1 paper as the substrate for culture of L929

directly with the naked eye.37 Therefore, we physically modied
paper using hydrophobic patterning to make the inner area of
the paper hydrophilic and provide a close space for cells to grow
and then evaluated cell viability by culturing cells and directly
processing cytotoxicity reagents on that paper.
To optimize the paper-based 3D cell culture platform, we
rstly evaluated the cytotoxicity of wax-patterned paper itself
according to the type of Whatman® lter paper used (grades 1,
2, or 114). The characteristics of each paper are listed in Table
S1 of the ESI.† Aer sterilizing the paper with UV irradiation for
2 h or more, we immersed each paper in cell growth medium for
24, 48, or 72 h and conducted WST toxicity testing with L929
cells (4  104 cells per ml). As shown in Fig. 2A, grade 1 paper
patterned with wax did not induce cytotoxicity for 72 h. And the
cell viability aer treatment of grade 2 paper was >90% for 72 h.
But the wax-patterned grade 114 paper induce cytotoxicity for
24, 48 and 72 h. Grade 114 paper have a high wet strength (Table
S1†) due to the addition of a small quantity of chemically stable
resin. The cell toxicity of grade 114 paper might be attributed to
these impurities. To conrm the effect of paper substrate type
cells. In addition, we conrmed cell survival and proliferation of
L929 cell over time (up to 7 d) in the paper-based 3D cell culture
platform using a Live/Dead™ assay (Fig. S1†).
Because cell adhesion to paper can play an important role in
cell proliferation and differentiation, we modied the chemical
surface of the wax-patterned Whatman grade 1 paper with three
types of cell adhesion molecules, poly-L-lysine, bronectin, and
collagen, and compared them with the ECM gels commonly
used for 3D cell culture. First, UV-sterilized paper was coated
with 8–12 mg ml1 of ECM gel, 0.1 mg ml1 poly-L-lysine, 0.1%
bronectin, or 100 mg ml1 collagen type I, and then dried. L929
cells (1  105 cells per zone) were dropped on the dried paper
surface and cultured in an incubator. Our results show that the
cells grew well in all cases. With the ECM gel, the cells tended to
clump and grow, whereas in the poly-L-lysine and bronectin,
the cells spread out individually. With collagen, the cells tended
to clump and grow, similar to their behavior in the ECM gel.
Collagen is one of the major components of ECM, and its main
function is related to many biological mechanisms, from the
maintenance of homeostasis38,39 and cell adhesion and

Fig. 2 Optimization of the paper-based 3D cell culture platform. (A) Cytotoxicity evaluation of the fabricated paper platform according to the
type of Whatman filter paper (grades 1, 2, or 114) and elution time (24, 48, or 72 h) (n ¼ 3). (B) Representative fluorescence images of Live/Dead
staining of L929 cells (3  104 cells per zone) cultured in the various paper platform for 24 h. The scale bar represents 100 mm. (C) L929 cell
growth (1  105 cells per zone for 24 h) fluorescence images of Live/Dead staining after modifying grade 1 paper surface with cell adhesion
molecules: (a) collagen type I, (b) fibronectin, (c) poly-L-lysine, and (d) ECM gel. The scale bar represents 100 mm. (D) Correlation between WST
color intensity (change in blue channel intensity) and number of LNCaP cells (5  103, 1  104, 5  104 and 1  105 cells per zone) seeded onto
the grade 1 paper for 3D cell culture (n ¼ 3). The error bars represent standard deviations.

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Paper
survival40 to cell differentiation.41 When we additionally
considered economic feasibility, collagen was deemed to be the
most suitable material for the chemical surface modication to
provide inexpensive 3D cell culture (Fig. 2C).
Finally, we evaluated the efficiency of cell seeding of L929 in
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the paper-based 3D cell culture platform. First, we seeded
varying numbers of L929 cells (0, 5  103, 1  104, 5  104, or 1
 105 cells per zone) on the collagen-coated paper, cultured
them on the agarose medium for 24 h, and then performed
a WST assay. Fig. 2D shows the effect of the number of paper-
based 3D-cultured L929 cells on WST color intensity (change
in blue channel intensity). The L929 cell number was linearly
proportional to the color intensity with R2 ¼ 0.985. This result
indicated that L929 cells were viable on the paper-based 3D cell
culture platform. Therefore, we conrmed that our paper-based
3D cell culture platform contains no cytotoxic substances and
provides an appropriate cell culture environment that offers
both cell adhesion and a nutritious medium supply.
3.2. Comparison of toxicity assessment in 2D and 3D cell
culture systems using colorimetric, uorescent, and
luminescent analyses
In vitro cytotoxicity testing is an indispensable method for
establishing the safety of medical devices or identifying poten-
tial cytotoxic side effects of new drug compounds. They have
been routinely used in toxicology due to their high sensitivity,
reproducibility, and cost-effectiveness, compared to in vivo
methods.42 Most cytotoxicity assays assess membrane integrity
and/or cells functions with colorimetric or uorescence dyes.
Among them, the WST assay is one of the most popular
methods to measure cell viability through mitochondrial
activity with a colorimetric dye, WST. WST is reduced by dehy-
drogenases in cells to give a yellow-colored formazan product,
which is soluble in cell culture medium.43 And the Live/Dead™
assay simultaneously detects live and dead cells with uores-
cent dyes which measure intracellular esterase activity and
plasma membrane integrity. In addition, the CellTiter-Glo®
luminescent cell viability assay uses rey luciferase, which
reacts with available cellular adenosine triphosphate (ATP), an
indicator of metabolically active cells, to produce a biolumi-
nescent signal proportional to the number of live cells present
in the assay.44 Using three types of cytotoxicity assays including
colorimetric, uorescence, and luminescence assays, we evalu-
ated the cytotoxicity of SLS to L929 cells in both 2D culture and
on our paper-based 3D culture systems. SLS is an anionic
detergent that is commonly used as a positive control for
toxicity testing.
The commonly used WST test was performed as our colori-
metric cytotoxicity evaluation. Cell viability in the 2D cell
culture of 96-well plates was measured using a multi-microplate
reader at a wavelength of 450 nm, and cell viability on our
paper-based 3D cell culture platform was evaluated through
a color analysis on the paper platform itself. As the WST reagent
is only active in metabolically intact cells, the intensity of the
reacted product directly relates with the number of viable
cells.45 As the paper provided strong contrast with colored
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prints, allowing us to see the results directly with the naked eye,
the WST method is very useful and easy to use for analysis of
paper-based cell culture platform. Aer coating the wax-
patterned Whatman grade 1 paper with collagen type I (100
mg ml1), we incubated L929 cells in agarose medium for 24 h by
dropping 1  105 cells per zone. Various concentrations of SLS
(0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14 mg ml1) were admin-
istered for 24 h. The next day, 10–20 ml of WST reagent was
added and processed for 1 h in a 37
C incubator, which
changed the color of the paper depending on the degree of
toxicity. To quantify the toxicity represented by the degree of
color change, we completely dried the paper and analyzed
quantitatively using an Epson scanner and ImageJ soware.
As cell density plays an important role in cellular responses
to drugs or chemicals, we rstly tested the cytotoxicity of SLS to
L929 cells with two different cell density – 1  104 and 1  105
cells per ml or zone in both 2D and paper-based 3D culture
systems. As shown in Fig. S2,† cellular responses to SLS were
different depending on cell density. We calculated the IC50 for
SLS from a dose–response curve using the Quest Graph™ IC50
Calculator. The estimated IC50 for SLS in 1  104 L929 cells
were 0.056 mg ml1 in 2D culture and 0.060 mg ml1 in paper-
based 3D culture, which were not within the 95% condence
interval (CI) according to the ISO 10993-5 guidelines (0.070–
0.116 mg ml1). But the IC50 for SLS in 1  105 L929 cells were
0.116 mg ml1 in 2D culture and 0.102 mg ml1 in paper-based
3D culture. Therefore, we performed cytotoxicity evaluation of
SLS in 1  105 L929 cells using 2D culture, paper-based 3D
culture systems. As shown in Fig. 3, SLS treatment inhibited
L929 cell viability (1  105 cells per ml or zone) in a dose
dependent manner in both 2D and paper-based 3D culture
systems. But when we compared the cytotoxicity according to
SLS concentration in two cell culture systems, the response of
the 3D cells (80.9  1.74% in 0.8 mg ml1 SLS) on paper
appeared to be more sensitive than the response of the 2D cells
(95.7  1.35% in 0.8 mg ml1 SLS).
Next, the Live/Dead™ assay was performed to evaluate
cytotoxicity using uorescence. For the 2D cell culture, the cells
were seeded in 35 mm glass bottom dishes, and for the 3D cell
culture, the cells were seeded on collagen type I-coated paper
that formed our 3D cell culture platform and then incubated in
agarose medium for 24 h. Aer treatment with various
concentrations of SLS, the cell morphology and cytotoxicity
were conrmed using a Live/Dead™ assay. On the paper-based
3D cell culture platform, the L929 cells grew in spatial
arrangements that were well connected with one another,
whereas the 2D cultures attached to plastic surfaces showed
a at and round morphology (Fig. 4A). Aer treatment with the
toxic SLS at 0.08 mg ml1, the L929 cells in 2D cell culture at
showed no difference in cell morphology or cell viability (97.8 
1.64%), but both cell viability (77.5  0.53%) and morphological
changes, such as the disappearance of lopodia and circular
deformation, were observed on the paper-based 3D cell culture
platform (Fig. 4B). The estimated IC50 means for SLS were
0.113 mg ml1 in 2D culture and 0.097 mg ml1 in paper-based
3D culture, respectively. These results support that 3D cultured
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Fig. 3 Toxicity evaluation in (A) monolayer 2D culture and (B) paper-based 3D cell culture systems using a colorimetric analysis. Mouse L929
fibroblast cells (1  105 cells per ml or zone) were treated with various concentrations of SLS and incubated for 24 h. Cell cytotoxicity was
measured using the WST assay. (A) Absorbance was measured at 450 nm using a multi-microplate reader and converted into the cell survival
percentage compared with control cells (n ¼ 4). (B) For cells cultured on paper, cell viability was assessed by placing the WST reagent directly on
the paper (n ¼ 4). The error bars represent standard deviations.

Fig. 4 Fluorescent images from Live/Dead™ assays of L929 cells (1  105 cells per ml or zone) grown in (A) a 2D monolayer culture and (B) the
paper-based 3D cell culture system and treated with various concentrations of SLS (n ¼ 3). Images were collected using a Zeiss LSM-700
confocal microscopy system. The numbers of viable (green) and non-viable (red) cells in the images were counted automatically. The error bars
represent standard deviations.

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Fig. 5 CellTiter-Glo® luminescent cell viability assay with L929 cells (1  105 cells per ml or zone). The graph shows cell viability data for L929
cells in (A) 2D and (B) paper-based 3D cell culture systems (n ¼ 3). The error bars represent standard deviations.

cells on paper are more sensitive to toxic substances than 2D
cultured cells.
Finally, we performed the CellTiter-Glo® luminescent assay
to evaluate cytotoxicity using luminescence. ATP is precisely
regulated in healthy cells, but it rapidly degrades at cell death.
Luciferase catalyzes the reaction between ATP and the substrate
luciferin, producing light. ATP measurement is thus a powerful
tool for measuring cell proliferation and enables accurate
measurement of the proliferation and inhibition of cells in
culture.46 Mouse L929 broblasts were seeded in black 96-well
plates and the paper-based 3D cell culture platform and incu-
bated for 24 h. To process the 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12,
and 0.14 mg ml1 SLS concentrations and measure the lumi-
nescent signal on the paper-based 3D cell culture platform, each
paper containing cells was cut and transferred to a black 96-well
plate. The luminescent signals were measured by a multi-
microplate reader. As with the results of the luminescence
analysis, the response of cells 3D cultured on paper was more
sensitive than the response of the 2D cultured cells (Fig. 5). The
estimated IC50 means for SLS were 0.113 mg ml1 in 2D culture
and 0.099 mg ml1 in paper-based 3D culture, respectively.
The results of three different cytotoxicity assays show that
the response of cells cultured on the paper-based 3D cell culture
system to SLS was more sensitive than that of cells in the 2D
culture. Unlike monolayer cells growing on a xed and rigid 2D
matrix in a culture dish, the environmental factors of 3D culture
reect spatial differences in the extracellular composition
surrounding the cells.27 Their greater sensitivity to the toxic SLS
is thought to result from the cells' access to soluble factors,
including O2, nutrients, and toxic substances, which affects the
distribution of biomolecules inside the cells.

Fig. 6 Comparison of SLS toxicity in L929 cells (1  105 cells per ml or zone) grown on the paper-based 3D cell culture system (A)–(C) and
a commercialized 3D collagen cell culture kit (D)–(F). Cytotoxicity was measured using three cell viability assays, (A) and (D) WST assay or, (B) and
(E) Live/Dead™ assay (C) and (F) CellTiter-Glo® luminescent cell viability assay (n is at least 3). The error bars represent standard deviations.

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Table 1 Comparison of IC50 values for sodium lauryl sulfate (SLS) to L929 cells (1  105 cells per ml or zone) in 2D and paper-based 3D culture,
and commercial 3D collagen kit, evaluated by WST assay. The IC50 values for individual repeats (3 sets) are given as mean and 95% confidence
interval (CI) [95% CI, +95% CI]

Culture methods IC50 mean [95% CI] for SLS (mg ml1)

2D culture using 96 well plates 0.116 [0.108, 0.124]

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Paper-based 3D culture 0.097 [0.084, 0.109]a

3D collagen culture using commercial kits 0.098 [0.080, 0.115]b
a
p < 0.01 vs. 2D culture. b p < 0.05 vs. 2D culture.

3.3. Evaluation of cell toxicity between the paper-based 3D
cell culture platform and a commercialized 3D cell culture kit
We next compared the cytotoxicity of SLS between our new
paper-based 3D cell culture platform and a commercialized 3D
cell culture kit. In the commercially available 3D cell culture kit
containing collagen-rich ECM, the cells grew into a circular
form,47,48 and on the paper-based 3D cell culture platform, the
cells attached to and diffused across the surface in response to
its topographical features (Fig. 4B). We used the same three cell
viability assays (WST, Live/Dead™, and CellTiter-Glo® lumi-
nescence) to assess the cytotoxicity of various concentrations of
SLS, and all samples showed similar cytotoxicity results (Fig. 6).
We compared the IC50 mean values for SLS in 2D and paper-
based 3D culture systems of L929 cells, as well as commercial-
ized 3D cell culture kit. As shown in Table 1, the IC50 mean for
SLS in 2D cultured L929 cells was 0.116 mg ml1 (95% CI ¼
0.108 to 0.124 109 mg ml1), which was signicantly different
with those in both 3D culture kit and paper-based 3D cell
culture system. But the IC50 mean (0.097 mg ml1; 95% CI ¼
0.084 to 0.109 mg ml1) for SLS in paper-based 3D cultured
L929 cells was not signicantly different with that in commer-
cialized 3D cell culture kit (0.098 mg ml1; 95% CI ¼ 0.080 to
0.115). Though a commercial 3D cell culture kit showed the
similar cytotoxicity of cell grown in our paper-based 3D cell
culture system, they were expensive, and should be kept under
refrigeration due to the relatively short shelf life. Besides, it was
necessary to additional time more than 1 h for the polymeri-
zation between cells and collagen matrix. Therefore, our paper-
based 3D cell culture platform, which can be simply produced at
low cost, can cultivate cells while mimicking the in vivo micro-
environment, and we expect it to be used to efficiently evaluate
cell viability, which can be detected directly using the color of
the paper.
4. Conclusion
It is becoming increasingly clear that 3D cell culture models are
better than conventional 2D monolayer cultures because of
improved cell–cell interactions and cell–ECM interactions and
cell populations and structures that are similar to in vivo
structures. Paper has emerged as a promising cell culture
substrate because of its low cost, porosity, exibility, and
biocompatibility. In this study, we designed a paper-based 3D
cell culture system that allows for easy and convenient mass
production of cell culture environments that mimic in vivo
conditions, and we compared toxicity assessments using cells
grown on 2D and 3D cell culture systems. Cells grown on our 3D
cell culture platform were more sensitive to the toxic test
substance than those grown in the 2D cell culture. In addition,
the paper-based 3D cell culture platform and a conventional
commercialized 3D cell culture kit showed similar cytotoxicity
results. In summary, a simple and facile colorimetric assay can
monitor the viability of cells grown on a wax-patterned, paper-
based, 3D cell culture platform. We expect that it can be
applied as a new strategy for drug screening and toxicity anal-
yses, together with compatible readout techniques.
Author contributions
Conceptualization, G. L.; methodology, Y. L. and Y. A.; valida-
tion, Y. L.; formal analysis, Y. L. and Y. A.; investigation, Y. L.
and G. L.; writing—original dra preparation, Y. L.; writing—
review and editing, G. L.; visualization, Y. L. and G. L.; super-
vision, G. L.; project administration, G. L.; funding acquisition,
G. L. All authors have read and agree to the published version of
the manuscript.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This research was supported by the Korea Medical Device
Development Fund grant funded by the Korean government
(Ministry of Science and ICT, the Ministry of Trade, Industry,
and Energy, the Ministry of Health & Welfare, the Ministry of
Food and Drug Safety) (Project Number:
KMDF_PR_20200901_0023, 1711137928) and National
Research Foundation grants funded by the Korean government
(MSIP) (No. NRF-2018R1A2B6007635).
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