Page 119 - IJB-9-1

International Journal of Bioprinting Fabrication of 3D breast tumor model for drug screening

breast cancer to liver. The liver-derived dECM contains Ltd. (Shantou, China). EDTA, glutaraldehyde, and ethyl
collagen, fibronectin, laminin, glycosaminoglycan alcohol were provided by Damao Chemical Reagent Factory
(GAG), proteoglycan (PG), and a variety of insoluble (Tianjin, China). Hematoxylin and eosin (H&E) dye,
growth factors, which can mimic the liver environment, Masson dye, and pepsin were provided by Beijing Coolaber
provide support and anchorage, and regulate intercellular Science & Technology Co., Ltd. (Beijing, China). Hoechst
communication for liver metastatic breast cancer cells . 33324, Calcein-AM, and propidium iodide (PI) were
[31]
However, although dECM has good biocompatibility, it is provided by Shanghai Beyotime Biotechnology Co., Ltd.
difficult to effectively fabricate tissue-engineered scaffolds (Shanghai, China). α-DMEM and penicillin-streptomycin
by 3D printing using dECM alone due to its low viscosity. were provided by Sigma-Aldrich Inc. (St. Louis, MO,
Therefore, it needs to be modified to make dECM more USA). RPMI 1640 was provided by Procell Life Science
suitable for 3D printing . Gelatin is a hydrolytic product of & Technology Co., Ltd. (Wuhan, China). CCK-8 kit was
[32]
collagen, which has good biocompatibility and temperature provided by Dojindo Laboratories (Kumamoto, Japan).
sensitivity. The viscosity of dECM solution would increase
after combining with gelatin under warm bath, and gelation 2.2. Cell culture
can be formed at lower temperature, which is conducive Mouse fibroblasts L929 were cultured in Dulbecco’s
to fabricate scaffolds with high-resolution 3D printing . modified eagle medium (DMEM), supplemented with
[11]
Sodium alginate is an easily available natural polymer with 10% fetal calf serum and 1% penicillin-streptomycin at
good biocompatibility. It can be crosslinked with divalent 37°C with 5% CO . The medium was changed once every
2
cations to form hydrogels under mild conditions. When 2 or 3 d, and when cells covered 90% of culture flask, they
combine with other biomolecules, it can significantly were digested by 2 mL 0.25% trypsin, and divided into
change the properties of the gel and make it suitable for 3–5 new culture flasks. Mouse breast tumor cells 4T1
different applications. were cultured in Roswell Park Memorial Institute medium
(RPMI) supplemented with 10% fetal calf serum and 1%
In this study, dECM derived from porcine liver, penicillin-streptomycin at 37°C with 5% CO . The medium
2
gelatin, and sodium alginate were used as materials to was changed every day, and when cells covered 80% of
prepare tissue-engineered scaffolds. Using appropriate culture flask, they were digested by 2 mL 0.25% trypsin and
decellularization methods, the extracellular matrix divided into 3–5 new culture flasks.
components in the tissue and its original structure could
be preserved while completely removing the cells at the 2.3. Preparation of dECM and decellularization
same time. dECM was resolved and mixed with different efficiency
concentration of gelatin and sodium alginate to prepare Fresh porcine liver purchased from the supermarket was
bioinks, among which the printable bioinks were selected firstly washed with distilled water until most blood stains
to fabricate tissue-engineered scaffolds. The porosity, on the tissue had been removed. After that, it was cut into
swelling, mechanical properties, and biocompatibility of pieces with the size of 10 mm × 10 mm × 5 mm and stirred
scaffolds were tested, and metastatic mouse breast cancer in distilled water for 2 h. The water was replaced every
cells 4T1 were seeded on scaffolds. The current study 0.5 h. The porcine liver pieces were then subjected to the
aimed to construct a metastatic tumor model to provide first step of decellularization treatment with 2.5% trypsin-
a platform simulating the in vivo environment of tumor EDTA solution and stirred at 37°C for 6 h. The liver pieces
tissues for anti-cancer drug screening and the delineation were then placed into a 2% Triton X-100 solution with
of the mechanism underlying tumor progression. 1.25% NH ·H O and stirred for 72 h. Finally, the liver
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pieces were decellularized by stirring in 0.1% SDS solution
2. Materials and methods until they turned white, and the dECM was obtained after
2.1. Materials washing with distilled water for 24 h to remove the residual
Fresh porcine liver was purchased from the supermarket. reagents.
L929 cell line and 4T1 cell line were donated by Zhengzhou The microstructures of the dECM were observed by
Institute of Emerging Industrial Technology (Zhengzhou, scanning electron microscopy (SEM; QUANTA 450, FEI,
China) and Cancer Hospital of China Medical University USA) to evaluate the decellularization efficiency. Briefly,
(Shenyang, China), respectively. NaCl, KCl, KH PO , the dECM (native tissue) was cut into the size of 3 mm
2
4
and Na HPO ·12H O were provided by Tianjin Kemiou × 3 mm × 1 mm and fixed with 2.5% glutaraldehyde
2
4
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Chemical Reagent Co., Ltd. (Tianjin, China). Triton X-100, solution for 3 h. Gradient dehydration was carried out
sodium dodecyl sulfate (SDS), and trypsin were provided with 50%, 70%, 90%, and 100% alcohol successively for
by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, 0.5 h. Then the specimens were observed with SEM after
China). NH ·H O was provided by Xilong Scientific Co., metal spraying. The specimens were also stained with H&E
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Volume 9 Issue 1 (2023) 111 https://doi.org/10.18063/ijb.v9i1.630