Page 370 - IJB-9-6

International Journal of Bioprinting Aflatoxin B1-induced cancer stem cells

only enhance cell–substrate interaction and cell response to proteoglycan , can be similarly created by a mixture of
[27]
stress, but also increase the level of resistance against cancer biocompatible and natural polysaccharide and protein.
therapies [3,9] . Because a certain set of markers uniquely Moreover, ECM stiffness, which is critical for metastasis ,
[28]
appear in CSCs rather than normal cancer cells, these can be mimicked by cross-linking nature-derived protein
markers can be utilized to distinguish and isolate CSCs or polymer. Furthermore, the 3D spheroid culture system
from the entire population of various cancer cell lines [10-13] . is highly biologically relevant to native in vivo states in that
3D spheroids have the vital characteristics such as natural
Aflatoxin B1 is a mycotoxin derived from Aspergillus
flavus and Aspergillus parasiticus. It is widely known as cell shape, a heterogeneous interface with the surrounding
a carcinogen, particularly for liver cancer, by inducing medium, and similar gene and protein expressions with
[29,30]
a mutation in p53 gene. Its metabolite by cytochrome the natural in vivo cancers . Therefore, the results
P450 enzyme, exo-aflatoxin B1-8,9-epoxide, can link obtained in 3D cell culture model have been observed
with guanine and generate an adduct with DNA. The to resemble in vivo context, which is reflected by aspects
[31,32]
interference to normal DNA structure by the DNA like higher cell viability , higher yield of extracellular
[34]
[33]
adducts can inhibit the activity of tumor suppressor genes. vesicles , more active drug metabolism , or higher
As a result, cell growth is no longer properly controlled, levels of stem-like properties and epithelial–mesenchymal
and cancer is initiated . Aflatoxin B1 has been found transition (EMT) markers, such as NANOG, SOX2,
[14]
[35]
in a wide range of foods, such as corn , peanuts , and CD44, and CD133 , compared to 2D cell culture. To
[15]
[16]
rice . Therefore, it is likely that humans absorb aflatoxin date, a number of 3D cell culture techniques have been
[17]
B1 through their daily diet. Previously, the correlation developed, which can be classified into scaffold-based and
[36,37]
between aflatoxin B1 and liver cancer pathogenesis (or scaffold-free approaches . Meanwhile, 3D bioprinting
[38-40]
CSC) has been mainly studied using two-dimensional has been widely used in energy harvesting , food
[42-44]
[41]
[45-51]
(2D) cell culture model . However, several drawbacks industry , tissue engineering , and cell biology .
[18]
of 2D cell culture have been highlighted. For example, in In the present study, a scaffold-based 3D bioprinting
2D cell culture, Birgersdotter et al. indicated that there is in vitro model was developed to quantitatively determine
a loss in cell polarity, a pivotal feature of tissue, as well as drug-resistant single cancer cells formed in HepG2 tumor
an alteration in gene expression . Moreover, it failed to spheroids following the exposure to aflatoxin B1. The
[19]
preserve the natural morphology of cells and represented HepG2 cancer cell-laden hydrogel consisting of alginate
a similarity in the accessibility of cells to media and drug and gelatin was 3D-bioprinted such that single cancer cells
solutions, as observed in in vivo studies. This explains the grown into tumor spheroids were uniformly distributed in
significant difference between the results of in vitro studies the hydrogel. By using 3D bioprinting, size and shape of
using 2D cell culture and in vivo studies; it also highlights the hydrogel could be precisely controlled, and the porous
the need to develop a model relevant to physiological structure of hydrogel after crosslinking could serve as an
conditions to simulate biological processes in the body. artificial ECM, where nutrients and soluble factors could
be stored. The hydrogel entrapping cancer cells is allocated
In contrast, three-dimensional (3D) cell culture into a consistent cross structure with similar amount by
has been proven to effectively simulate physiological 3D bioprinting, which not only makes the development
conditions, particularly the tumor microenvironment. of in vitro model less labor-intensive, but also remarkably
As the 3D-cultured cancer cells grow into a spheroid enhances reproducibility. Owing to these advantages,
structure, they maintain their natural shape while forming the proposed model is expected to mimic physiological
a tumor-like cluster with inner and outer layers wherein conditions. The present model is based on the assumption
oxygen, nutrients, and drug gradients form, making that human liver cancer patients, who are not aware of the
them less accessible to cancer cells in the inner layers . tumor in their bodies, may be exposed to the carcinogen
[20]
Additionally, by allowing cancer cells to grow in a gel-like aflatoxin B1 through their diet. Particularly, this approach
structure, 3D cell culture also mimics extracellular matrix is greatly significant because it quantitatively evaluates the
(ECM) . Previous studies have stated the important extent to which drug-resistant CSCs, which are not easily
[21]
role of ECM in tumor microenvironment (TME) , in killed by anticancer agents, can be formed in single tumor
[22]
which it is the major component of the TME and supports spheroids when exposed to the carcinogen aflatoxin B1.
unique characteristics of CSCs, such as drug resistance [23,24]
and tumorigenesis . Unlike 2D cell culture, which 2. Materials and methods
[25]
obviously lacks ECM, 3D cell culture, where cells are
grown in a 3D structure, has a potential to simulate key 2.1. Cell culture
features of ECM . Firstly, ECM, which is composed Liver cancer cell line HepG2 from Korea Cell line bank
[26]
by various macromolecules like laminin, collagen, and (No. 30022, Seoul, Korea) was employed for the experiment

Volume 9 Issue 6 (2023) 362 https://doi.org/10.36922/ijb.0985