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International Journal of Bioprinting 3D-printed micro-perfused culture device

micro-perfusion chamber, was designed to provide cells Coralville, IA, USA. The Albumin Human ELISA kit
with a highly relevant microenvironment for growth. 35,41,42 used to quantify albumin secretion in hepatocyte culture
The combination of micro-fibrous scaffold and micro- medium was purchased from Abcam, Singapore.
perfusion on a microfluidic platform aims to mimic the
ECM structural support needed for cell growth and blood 2.2. Methods
41
circulation, respectively. Liver cells that reside in a highly 2.2.1. Scaffold fabrication
43
perfused environment will be used as model cells in this All electrospun scaffolds were prepared on Nanofiber
study to evaluate the effect of perfused micro-physiological Electrospinning System, Nanon-01A. Poly(lactic-co-
environment as an alternative to traditional in vitro glycolic acid) (PLGA) 80:20 (Resomer® LG 824S) was
2,44
culture. dissolved in 7:3 ratio of chloroform and dimethylformamide
(DMF) and magnetic-stirred for 24 h to obtain a 8 wt%
To achieve this, current micro/nanotechnologies, polymer solution. The electrospun polymer solution was
such as electrospinning and 3D printing, were adopted. fed at a rate of 0.5–0.8 mL/h to a flat tip stainless steel
The miniaturized 3D fibrous scaffold used in this study spinneret connected to a high-voltage power supply.
was fabricated by a previously reported electrospinning Electrospinning was carried out at ambient temperature
process. 45,46 This miniaturized 3D fibrous substrate (20°C–24°C) and a range of accelerating voltage from 16 to
was inserted onto a partially SLA-printed perfusion 18 kV was used with the collector remained earthed. The
chamber. The printing was resumed after embedding the PLGA polymer solution was pumped to a 21-gauge needle
miniaturized 3D fibrous scaffold. This fabrication approach with a collector distance optimized between 4 and 10 mm.
avoids secondary bonding process to assemble and seal The miniaturized 3D PLGA fibrous scaffold was used for
the chamber and channels for perfusion, presenting a cell viability studies on the 3D-printed perfused platform.
possibility of using additive manufacturing technology The bath solution previously optimized for the scaffold
for microfluidic device fabrication or prototyping. Huh7.5 fabrication was found to be 70:30 isopropyl alcohol and
hepatocellular carcinoma cell line was used as model deionized water for the collection of porous structure. All
46
cells to determine the effect of cell viability, proliferation, scaffolds were collected after 3 min of spinning with 1 min of
and key liver functions on perfused micro-physiological cleaning frequency and placed in freezer for approximately
environment. 24 h prior to freeze-drying. The miniaturized scaffold was
fabricated with controlled electrospinning condition to
2. Materials and methods ensure scaffold collection with consistent size. The size of
2.1. Materials the fabricated scaffold can be fitted well into the culture
Poly(lactic-co-glycolic acid) (PLGA) 80:20 (Resomer® LG chamber of MPC device without folding.
824S) used for scaffold fabrication was purchased from 2.2.2. PDMS sheet fabrication
Evonik, Germany. The chloroform and dimethylformamide The PDMS prepolymer, Sylgard 184 (Dow Corning, MI,
(DMF) solvent used to dissolve PLGA were purchased USA), was mixed with curing agent at ratio of 10:1 by
from Fisher Scientific. The PDMS prepolymer, Sylgard 184 weight. The PDMS solution was degassed and casted on a
was purchased from Dow Corning, MI, USA. NextDent Teflon mold with 0.2-mm step formed by machining. The
Ortho Clear, a biocompatible Class IIa methacrylate 3D PDMS solution was cured at 80°C for 3 h; it was removed
printing resin, was purchased from Singapore. The WST- from the Teflon mold and punched to a circular diameter
8 cell proliferation assay was purchased from Dojindo of 10 mm. The PDMS sheets were inserted at layers 1 and 2
Molecular Laboratories Inc., Kumanoto, Japan. Huh7.5 on the MPC device in order to seal up the culture chamber
human hepatocarcinoma cell line was purchased from and create a clear chamber for cell culture monitoring.
Riken, VA, Japan. Cells maintained in Dulbecco’s modified
eagle medium (DMEM) were purchased from HyClone, GE 2.2.3. Computational fluid dynamic simulation
Healthcare and Life Sciences. DMEM supplemented with The computational fluid dynamic (CFD) simulation
10% fetal bovine serum and 1% penicillin/streptomycin Flow3D was used to predict the flow behavior of medium
was purchased from Gibco, Life Technologies. The LIVE/ in the MPC device. In the simulation, the viscosity of
DEAD cell viability assay and Trizol were purchased from the medium was assumed to be the same as that of water
Life Technologies. The reaction mix used for qPCR test (0.001 Pa.s). Effect of channel dimension and flowrate on
comprises of 10 µL of SYBR Green Master Mix, 1 µL of fluid shear stress has been analyzed. This study is necessary
forward and reverse primers (5 µM), respectively, 1 µL of to determine the optimal channel dimension and flowrate
diluted cDNA and PCR grade water were purchased from to be used for the MPC device. In this study, a flowrate of
Bio-Rad Laboratories, Inc., USA. The primers of interest 0.12 mL/h was used such that sufficient medium will be
were purchased from Integrated DNA Technologies, provided to the core of scaffold for nutrients and metabolites

Volume 10 Issue 1 (2024) 145 https://doi.org/10.36922/ijb.0226