Page 350 - IJB-10-3

International Journal of Bioprinting hNVU chip for brain modeling and drug screening

different chambers failed to fully replicate the intricate Co., Ltd., Shanghai, China) containing 5% fetal bovine
cellular interactions in the brain. serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco),
and EC growth supplement (ScienCell). Cell culture flasks
Compared to the linked organ chip, the multilayer
parallel hNVU chip can mimic the multilayer for hCMEC/D3 culture were coated with 150 μg/mL of a
microstructure of hNVU but also stimulate the interaction collagen solution (Meilunbio, Dalian, China).
between the BBB and brain regions. Additionally, the Human brain microvascular pericytes (HBVPs;
parallel hNVU chip facilitates the observation of drug ZQY006, Zhong Qiao Xin Zhou Biotechnology Co., Ltd.,
effects on the integrity of the BBB and the morphology and Shanghai, China) were cultured in pericyte medium
behavior of brain region cells through imaging. Bioprinting (ZQ-1321; Zhong Qiao Xin Zhou Biotechnology Co.,
technology, which allows for the three-dimensional Ltd., Shanghai, China) containing 2% FBS (Gibco),
(3D) spatial positioning of multiple cell types blended 1% penicillin–streptomycin (Gibco), and PC growth
with biometric hydrogels, offers prominent advantages supplement (ScienCell). Cell culture flasks for HBVP
in constructing multilayered parallel NVU models. 26-28 culture were coated with 20 μg/mL PLL solution (Beyotime,
Moreover, the incorporation of biomimetic hydrogels that Shanghai, China) prior to cell seeding.
resemble the native brain extracellular matrix (ECM) not
only provides biochemical and mechanical support for Immortalized human astrocytes (AC; CTCC-001-
brain cells but also simulates the barrier function of the 0398, Meisen Co., Ltd., Zhejiang, China) were cultured
basement membrane. 29,30 in astrocyte medium (Meisen Co., Ltd., Zhejiang, China)
containing 5% FBS (Gibco), 1% penicillin–streptomycin
In this study, we employed organ-on-a chip and (Gibco), and AC growth supplement (Meisen Co.,
extrusion bioprinting techniques to develop a multilayered Ltd., Zhejiang, China). The immortalized human
parallel NVU model that features the structural and cellular astrocytes were established through hTERT-dependent
composition of BBB in pediatric brain. The model architecture immortalization of a human fetal (18 weeks gestation)
would resemble a sandwich structure, with the upper and brain-derived primary astrocytes culture. Cell culture
lower layers simulating the BBB and the intermediate flasks for astrocyte culture were coated with 0.15 mg/
layer imitating the brain tissue structure (Figure 1). The mL rat tail type-I collagen solution (Meisen Co., Ltd.,
architecture is incorporated with the necessary cell types Zhejiang, China) prior to cell seeding.
of pediatric brain, such as brain microvascular endothelial
cells, brain microvascular pericytes (HBVP), embryo- HMC3 cells (CRL-3304, ATCC) were cultured
derived astrocytes and microglia (HMC3), and induced in HMC3 medium (ZQ-320, Zhong Qiao Xin Zhou
pluripotent stem cell (iPSC)-derived NPCs. We induced Biotechnology Co., Ltd., Shanghai, China) containing 10%
differentiation of NPC cells into early neurons within the FBS (Gibco) and 1% penicillin–streptomycin (Gibco). The
hydrogel matrix. This not only mimics the presence of HMC3 cell line was established through SV40-dependent
neural stem cells in brain but also incorporates neuronal immortalization of a human fetal (embryo) brain-derived
components. Furthermore, nutrient perfusion through the primary microglia culture.
upper and lower BBB structures of the chip ensured sufficient Neural precursor cells (NPCs; Nuwacell, Hefei, China)
nutrient supply. Notably, the two perfusion channels in the were cultured in NPC medium (Nuwacell, Hefei, China)
brain region could serve the dual purpose of nourishing the containing 2.5 μM blebbistatin (Nuwacell, Hefei, China).
brain tissue region and evaluating drug permeability across Cell culture flasks for astrocyte culture were coated with
the BBB as well as facilitating analysis of the metabolism of 0.5 μg/cm ncLaminin511 solution (Nuwacell, Hefei,
2
drugs in the brain region. Astroglioma cell SF188 (derived China) prior to cell seeding.
from the left temporal lobe tumor of an 8-year-old male
patient) was introduced in the model to construct an hNVU Malignant astrocytoma cells (SF188; WM-23XM167,
chip with tumor cells to mimic the pediatric brain tumors Shen Zhen Wei Sa Biotechnology Co., Ltd., Shenzhen,
for the study of chemotherapeutic drugs. China) were cultured in SF188 medium containing 10%
FBS (Gibco) and 1% penicillin–streptomycin (Gibco).
2. Materials and methods The SF188 glioblastoma cell line was derived from the left
temporal lobe tumor of an 8-year-old male patient.
2.1. Cell culture
Human cerebral microvascular endothelial cells (hCMEC/ hCMEC/D3 cells between passages 6 and 15, HBVP
D3; ZQ0961, Zhong Qiao Xin Zhou Biotechnology Co., cells between passages 8 and 15, immortalized human
Ltd., Shanghai, China) were cultured in endothelial growth astrocytes between passages 8 and 15, HMC3 cells between
medium (ZQ-1304, Zhong Qiao Xin Zhou Biotechnology passages 6 and 15, and SF188 cells between passages 6 and

Volume 10 Issue 3 (2024) 342 doi: 10.36922/ijb.1684