The utilization of 3D cell culture for spheroid formation holds significant implications in cancer research, contributing to a fundamental understanding of the disease and aiding drug development. Conventional methods such as the hanging drop technique and other alternatives encounter limitations due to smaller drop volumes, leading to nutrient starvation and restricted culture duration. In this study, we present a straightforward approach to creating superhydrophobic paper cones capable of accommodating large volumes of culture media drops. These paper cones have sterility, autoclavability, and bacterial repellent properties. Leveraging these attributes, we successfully generate large spheroids of ovarian cancer cells and, as a proof of concept, conduct drug screening to assess the impact of carboplatin. Thus, our method enables the preparation of flexible superhydrophobic surfaces for laboratory applications in an expeditious manner, exemplified here through spheroid formation and drug screening demonstrations.

1.
K. L.
Schmeichel
and
M. J.
Bissell
, “
Modeling tissue-specific signaling and organ function in three dimensions
,”
J. Cell Sci.
116
,
2377
2388
(
2003
).
2.
C.
Jensen
and
Y.
Teng
, “
Is it time to start transitioning from 2D to 3D cell culture?
,”
Front. Mol. Biosci.
7
,
33
(
2020
).
3.
J. W.
Haycock
,
3D Cell Culture: A Review of Current Approaches and Techniques
(
Springer
,
2011
).
4.
R. M.
Sutherland
,
J. A.
McCredie
, and
W. R.
Inch
, “
Growth of multicell spheroids in tissue culture as a model of nodular carcinomas
,”
J. Natl. Cancer Inst.
46
,
113
120
(
1971
).
5.
D.
Antoni
,
H.
Burckel
,
E.
Josset
, and
G.
Noel
, “
Three-dimensional cell culture: A breakthrough in vivo
,”
Int. J. Mol. Sci.
16
,
5517
5527
(
2015
).
6.
E.
Fennema
,
N.
Rivron
,
J.
Rouwkema
,
C.
van Blitterswijk
, and
J.
De Boer
, “
Spheroid culture as a tool for creating 3D complex tissues
,”
Trends Biotechnol.
31
,
108
115
(
2013
).
7.
K. M.
Yamada
and
M.
Sixt
, “
Mechanisms of 3D cell migration
,”
Nat. Rev. Mol. Cell Biol.
20
,
738
752
(
2019
).
8.
E.
Cukierman
,
R.
Pankov
,
D. R.
Stevens
, and
K. M.
Yamada
, “
Taking cell-matrix adhesions to the third dimension
,”
Science
294
,
1708
1712
(
2001
).
9.
A.
Ivascu
and
M.
Kubbies
, “
Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis
,”
SLAS Discovery
11
,
922
932
(
2006
).
10.
S. S.
Chen
,
W.
Fitzgerald
,
J.
Zimmerberg
,
H. K.
Kleinman
, and
L.
Margolis
, “
Cell-cell and cell-extracellular matrix interactions regulate embryonic stem cell differentiation
,”
Stem Cells
25
,
553
561
(
2007
).
11.
S.
Gerecht-Nir
,
S.
Cohen
, and
J.
Itskovitz-Eldor
, “
Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation
,”
Biotechnol. Bioeng.
86
,
493
502
(
2004
).
12.
F.
Pampaloni
,
E. G.
Reynaud
, and
E. H.
Stelzer
, “
The third dimension bridges the gap between cell culture and live tissue
,”
Nat. Rev. Mol. Cell Biol.
8
,
839
845
(
2007
).
13.
G. M.
Keller
, “
In vitro differentiation of embryonic stem cells
,”
Curr. Opin. Cell Biol.
7
,
862
869
(
1995
).
14.
T.-M.
Achilli
,
J.
Meyer
, and
J. R.
Morgan
, “
Advances in the formation, use and understanding of multi-cellular spheroids
,”
Expert Opin. Biol. Ther.
12
,
1347
1360
(
2012
).
15.
R.
Edmondson
,
J. J.
Broglie
,
A. F.
Adcock
, and
L.
Yang
, “
Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors
,”
Assay Drug Dev. Technol.
12
,
207
218
(
2014
).
16.
Y.
Fang
and
R. M.
Eglen
, “
Three-dimensional cell cultures in drug discovery and development
,”
SLAS Discovery
22
,
456
472
(
2017
).
17.
H.
Sasaguri
,
P.
Nilsson
,
S.
Hashimoto
,
K.
Nagata
,
T.
Saito
,
B.
De Strooper
,
J.
Hardy
,
R.
Vassar
,
B.
Winblad
, and
T. C.
Saido
, “
APP mouse models for Alzheimer’s disease preclinical studies
,”
EMBO J.
36
,
2473
2487
(
2017
).
18.
M. P.
Lutolf
and
J.
Hubbell
, “
Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering
,”
Nat. Biotechnol.
23
,
47
55
(
2005
).
19.
P. G.
Layer
,
A.
Robitzki
,
A.
Rothermel
, and
E.
Willbold
, “
Of layers and spheres: The reaggregate approach in tissue engineering
,”
Trends Neurosci.
25
,
131
134
(
2002
).
20.
G.
Rijal
and
W.
Li
, “
A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening
,”
Sci. Adv.
3
,
e1700764
(
2017
).
21.
G.
Mehta
,
A. Y.
Hsiao
,
M.
Ingram
,
G. D.
Luker
, and
S.
Takayama
, “
Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy
,”
J. Controlled Release
164
,
192
204
(
2012
).
22.
J.
Ahn
,
D.-H.
Kim
,
D.-J.
Koo
,
J
Lim
,
T.-E.
Park
,
J.
Lee
,
J.
Ko
,
S.
Kim
,
M.
Kim
,
K.-S.
Kang
,
D.-H.
Min
,
S.-Y.
Kim
,
Y.
Kim
, and
N. L.
Jeon
, “
3D microengineered vascularized tumor spheroids for drug delivery and efficacy testing
,”
Acta Biomater.
165
,
153
167
(
2023
).
23.
R.
Foty
, “
A simple hanging drop cell culture protocol for generation of 3D spheroids
,”
J. Vis. Exp.
51
,
e2720
(
2011
).
24.
Y.-C.
Tung
,
A. Y.
Hsiao
,
S. G.
Allen
,
Y.-S.
Torisawa
,
M.
Ho
, and
S.
Takayama
, “
High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array
,”
Analyst
136
,
473
478
(
2011
).
25.
J. M.
Kelm
,
N. E.
Timmins
,
C. J.
Brown
,
M.
Fussenegger
, and
L. K.
Nielsen
, “
Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types
,”
Biotechnol. Bioeng.
83
,
173
180
(
2003
).
26.
A. M.
Rabie
,
A. S.
Ali
,
M. A.
Al-Zeer
,
A.
Barhoum
,
S.
El-Hallouty
,
W. G.
Shousha
,
J.
Berg
,
J.
Kurreck
, and
A. S.
Khalil
, “
Spontaneous formation of 3D breast cancer tissues on electrospun chitosan/poly (ethylene oxide) nanofibrous scaffolds
,”
ACS Omega
7
,
2114
2126
(
2022
).
27.
L.
Rabiet
,
L.
Arakelian
,
N.
Jeger-Madiot
,
D. R.
García
,
J.
Larghero
, and
J.-L.
Aider
, “
Acoustic levitation as a tool for cell-driven self-organization of human cell spheroids during long-term 3D culture
,”
Biotechnol. Bioeng.
121
,
1422
(2024).
28.
J. H.
Park
,
J.-R.
Lee
,
S.
Park
,
Y.-J.
Kim
,
J.-K.
Yoon
,
H. S.
Park
,
J.
Hyun
,
Y. K.
Joung
,
T. I.
Lee
, and
S. H.
Bhang
, “
Subaqueous 3D stem cell spheroid levitation culture using anti-gravity bioreactor based on sound wave superposition
,”
Biomater. Res.
27
,
51
(
2023
).
29.
K.
Kwapiszewska
,
A.
Michalczuk
,
M.
Rybka
,
R.
Kwapiszewski
, and
Z.
Brzózka
, “
A microfluidic-based platform for tumour spheroid culture, monitoring and drug screening
,”
Lab Chip
14
,
2096
2104
(
2014
).
30.
S.
Lopa
,
F.
Piraino
,
G.
Talò
,
V. L.
Mainardi
,
S.
Bersini
,
M.
Pierro
,
L.
Zagra
,
M.
Rasponi
, and
M.
Moretti
, “
Microfluidic biofabrication of 3D multicellular spheroids by modulation of non-geometrical parameters
,”
Front. Bioeng. Biotechnol.
8
,
366
(
2020
).
31.
B.
Patra
,
M.
Sharma
,
W.
Hale
, and
M.
Utz
, “
Time-resolved non-invasive metabolomic monitoring of a single cancer spheroid by microfluidic NMR
,”
Sci. Rep.
11
,
53
(
2021
).
32.
A.
Tevlek
,
S.
Kecili
,
O. S.
Ozcelik
,
H.
Kulah
, and
H. C.
Tekin
, “
Spheroid engineering in microfluidic devices
,”
ACS Omega
8
,
3630
3649
(
2023
).
33.
M. B.
Oliveira
,
A. I.
Neto
,
C. R.
Correia
,
M. I.
Rial-Hermida
,
C.
Alvarez-Lorenzo
, and
J. F.
Mano
, “
Superhydrophobic chips for cell spheroids high-throughput generation and drug screening
,”
ACS Appl. Mater. Interfaces
6
,
9488
9495
(
2014
).
34.
A.
Wang
,
L. A.
Madden
, and
V. N.
Paunov
, “
Advanced biomedical applications based on emerging 3D cell culturing platforms
,”
J. Mater. Chem. B
8
,
10487
10501
(
2020
).
35.
V.
Velasco
,
S. A.
Shariati
, and
R.
Esfandyarpour
, “
Microtechnology-based methods for organoid models
,”
Microsyst. Nanoeng.
6
,
76
(
2020
).
36.
K.
Białkowska
,
P.
Komorowski
,
M.
Bryszewska
, and
K.
Miłowska
, “
Spheroids as a type of three-dimensional cell cultures—examples of methods of preparation and the most important application
,”
Int. J. Mol. Sci.
21
,
6225
(
2020
).
37.
D.
Banerjee
,
Y. P.
Singh
,
P.
Datta
,
V.
Ozbolat
,
A.
O’Donnell
,
M.
Yeo
, and
I. T.
Ozbolat
, “
Strategies for 3D bioprinting of spheroids: A comprehensive review
,”
Biomaterials
291
,
121881
(
2022
).
38.
K.
Bloch
,
H.
Smith
, and
V. E. A.
van Hamel Parsons
, “
Metabolic alterations during the growth of tumour spheroids
,”
Cell Biochem. Biophys.
68
,
615
(
2014
).
39.
T.
Tidwell
,
G.
Røsland
, and
K. E. A.
Tronstad
, “
Metabolic flux analysis of 3D spheroids reveals significant differences in glucose metabolism from matched 2D cultures of colorectal cancer and pancreatic ductal adenocarcinoma cell lines
,”
Cancer Metab.
10
,
9
(
2022
).
40.
X.
Deng
,
L.
Mammen
,
H.-J.
Butt
, and
D.
Vollmer
, “
Candle soot as a template for a transparent robust superamphiphobic coating
,”
Science
335
,
67
70
(
2012
).
41.
R.
Iqbal
,
B.
Majhy
, and
A. K.
Sen
, “
Facile fabrication and characterization of a pdms-derived candle soot coated stable biocompatible superhydrophobic and superhemophobic surface
,”
ACS Appl. Mater. Interfaces
9
,
31170
31180
(
2017
).
42.
B. N.
Sahoo
,
S.
Nanda
,
J. A.
Kozinski
, and
S. K.
Mitra
, “
PDMS/camphor soot composite coating: Towards a self-healing and a self-cleaning superhydrophobic surface
,”
RSC Adv.
7
,
15027
15040
(
2017
).
43.
C.
Thamaraiselvan
,
E.
Manderfeld
,
M. N.
Kleinberg
,
A.
Rosenhahn
, and
C. J.
Arnusch
, “
Superhydrophobic candle soot as a low fouling stable coating on water treatment membrane feed spacers
,”
ACS Appl. Bio. Mater.
4
,
4191
4200
(
2021
).
44.
E.
Fadeeva
,
V. K.
Truong
,
M.
Stiesch
,
B. N.
Chichkov
,
R. J.
Crawford
,
J.
Wang
, and
E. P.
Ivanova
, “
Bacterial retention on superhydrophobic titanium surfaces fabricated by femtosecond laser ablation
,”
Langmuir
27
,
3012
3019
(
2011
).
45.
P.
Tang
,
W.
Zhang
,
Y.
Wang
,
B.
Zhang
,
H.
Wang
,
C.
Lin
, and
L.
Zhang
, “
Effect of superhydrophobic surface of titanium on staphylococcus aureus adhesion
,”
J. Nanomater.
2011
,
1
8
(
2011
).
46.
C. R.
Crick
,
S.
Ismail
,
J.
Pratten
, and
I. P.
Parkin
, “
An investigation into bacterial attachment to an elastomeric superhydrophobic surface prepared via aerosol assisted deposition
,”
Thin Solid Films
519
,
3722
3727
(
2011
).
47.
X.
Zhang
,
L.
Wang
, and
E.
Levänen
, “
Superhydrophobic surfaces for the reduction of bacterial adhesion
,”
RSC Adv.
3
,
12003
12020
(
2013
).
48.
J.
Ma
,
Y.
Sun
,
K.
Gleichauf
,
J.
Lou
, and
Q.
Li
, “
Nanostructure on taro leaves resists fouling by colloids and bacteria under submerged conditions
,”
Langmuir
27
,
10035
10040
(
2011
).
49.
J. E.
George
,
S.
Chidangil
, and
S. D.
George
, “
A study on air bubble wetting: Role of surface wettability, surface tension, and ionic surfactants
,”
Appl. Surf. Sci.
410
,
117
125
(
2017
).
50.
L.
Torres
and
M.
Weislogel
, “
The ejection of large non-oscillating droplets from a hydrophobic wedge in microgravity
,”
npj Microgravity
7
,
52
(
2021
).
51.
X.
Zhang
,
S.
Ben
,
Z.
Zhao
,
Y.
Ning
,
Q.
Li
,
Z.
Long
,
C.
Yu
,
K.
Liu
, and
L.
Jiang
, “
Lossless and directional transport of droplets on multi-bioinspired superwetting v-shape rails
,”
Adv. Funct. Mater.
33
,
2212217
(
2023
).
52.
W.
Xu
,
Z.
Lan
,
B.
Peng
,
R.
Wen
,
Y.
Chen
, and
X.
Ma
, “
Directional movement of droplets in grooves: Suspended or immersed?
,”
Sci. Rep.
6
,
18836
(
2016
).
53.
T.
Han
,
H.
Noh
,
H. S.
Park
, and
M. H.
Kim
, “
Effects of wettability on droplet movement in a v-shaped groove
,”
Sci. Rep.
8
,
16013
(
2018
).
54.
A.
Behera
,
R.
Ashraf
,
A. K.
Srivastava
, and
S.
Kumar
, “
Bioinformatics analysis and verification of molecular targets in ovarian cancer stem-like cells
,”
Heliyon
6
,
e04820
(
2020
).
55.
E.
Gheytanchi
,
M.
Naseri
, and
F. E. A.
Karimi-Busheri
, “
Morphological and molecular characteristics of spheroid formation in HT-29 and CaCo-2 colorectal cancer cell lines
,”
Cancer Cell Int.
21
,
204
(
2021
).

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