3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer

Summary

Hypoxia is a hallmark of tumor microenvironment and plays a crucial role in cancer progression. This article describes the fabrication process of a hypoxic cancer-on-a-chip based on 3D cell-printing technology to recapitulate a hypoxia-related pathology of cancer.

Abstract

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, and chemoresistance. Although several in vitro models have been developed to study hypoxia-related cancer pathology, the complex interplay of the cancer microenvironment observed in vivo has not been reproduced yet owing to the lack of precise spatial control. Instead, 3D biofabrication approaches have been proposed to create microphysiological systems for better emulation of cancer ecology and accurate anticancer treatment evaluation. Herein, we propose a 3D cell-printing approach to fabricate a hypoxic cancer-on-a-chip. The hypoxia-inducing components in the chip were determined based on a computer simulation of the oxygen distribution. Cancer-stroma concentric rings were printed using bioinks containing glioblastoma cells and endothelial cells to recapitulate a type of solid cancer. The resulting chip realized central hypoxia and aggravated malignancy in cancer with the formation of representative pathophysiological markers. Overall, the proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models for cancer research.

Introduction

The cancer microenvironment is a critical factor driving cancer progression. Multiple components, including biochemical, biophysical, and cellular cues, determine the pathological features of cancer. Among these, hypoxia is strongly associated with cancer survival, proliferation, and invasion¹. Due to the unlimited growth and division of cancer cells, nutrients and oxygen are continuously depleted, and a hypoxic gradient is generated. Under low-oxygen conditions, cells activate hypoxia-inducible transcription factor (HIF)-associated molecular cascade. This process induces a necrotic core, triggers metabolic changes, and initiates blood vessel h....

Protocol

1. Computer simulation of oxygen gradient formation

1. Generation of a 3D geometry model for hypoxic cancer-on-a-chip printing
   1. Run a 3D CAD software.
   2. Sketch the geometry model of hypoxic cancer-on-a-chip. Click on Sketch and select the desired plane to draw the geometry. Refer to the drawing (Figure 2A) for the detail scale of each part.
   3. Set the thickness of the geometry by clicking on Feature-Protrusion Boss/Base. Enter the desired thickness (refer to Figure 2A) in the empty box and select the green check icon to fo....

Results

The hypoxic cancer-on-a-chip was developed using computer-aided 3D cell-printing technology to recapitulate hypoxia and cancer-related pathology (Figure 1). Oxygen transportation and consumption were simulated using the 3D geometry model. The chip was designed in the form of concentric rings to mimic the radial oxygen diffusion and depletion, in cancer tissues (Figure 2A). After defining the control volume of a space where oxygen.......

Discussion

In this study, we describe the fabrication process of a hypoxic cancer-on-a-chip based on 3D cell-printing technology. The formation of the hypoxic gradient in the designed chip was predicted through computer simulations. The environment that can induce a heterogeneous hypoxic gradient was reproduced via a simple strategy combining the 3D-printed gas-permeable barrier and the glass cover. The hypoxia-related pathological features of glioblastoma, including pseudopalisade and a small population of cancer stem cells, were .......

Materials

Name	Company	Catalog Number	Comments
Cells
Human umbilical vein endothelial cells	Promocell	C-12200
U-87 MG cells	ATCC	ATCC HTB-14
Disposable
0.2 μm syringe filter	Sartorius	16534-K
10 mL disposable syringe	Jung Rim	10ml 21G32
10 mL glass vial	Hubena	A0039
10 mL Serological pipette tip	SPL lifescience	91010
15 mL conical tube	SPL lifescience	50015
18G plastic needle	Musashi engineering	PN-18G-B
20G plastic tapered dispense tip	Musashi engineering	TPND-20G-U
22x50 glass cover	MARIENFIELD	0101142
25 mL Serological pipette tip	SPL lifescience	90125
3 mL disposable syringes	HENKE-JET	4020-X00V0
40 µm cell strainer	Falcon	352360
5 mL Serological pipette tip	SPL lifescience	91005
50 mL conical tube	SPL lifescience	50050
50 mL Serological pipette tip	SPL lifescience	90150
50N precision nozzle	Musashi engineering	HN-0.5ND
Aluminum foil	SINKWANG
Capillary tips	Gilson	CP1000
Cell-scrapper	SPL lifescience	90030
Confocal dish	SPL lifescience	200350
Parafilm	Bemis	PM996
Pre-coated histology slide	MATSUNAMI	MAS-11
Reservoir	SPL lifescience	23050
T-75 cell culture flask	SPL lifescience	70075
Equipment
3DX printer	T&R Biofab
Autoclave	JEIOTECH	AC-12
Centrifuger	Cyrozen	1580MGR
Confocal laser microscopy	Olympus Life Science	FV 1000
Fluorescence microscope	FISHER SCEINTIFIC	O221S366
Forcep	Korea Ace Scientific	HC.203-30
Hand tally counter	KTRIO
Hemocytometer	MARIENFIELD	0650030
Incubator	Panasonic	MCO-170AIC
Laminar flow cabinet	DAECHUNG SCIENCE	CB-BMMS C-001
Metal syringe	IWASHITA engineering	SUS BARREL 10CC
Operating Scissors	Hirose	HC.13-122
Oven	JEIOTECH	OF-12, H070023
Positive displacement pipette	GILSON	NJ05652
Refrigerator	SAMSUNG	CRFD-1141
Voltex Mixer	DAIHAN scientific	VM-10
Water bath	DAIHAN SCIENTIFIC	WB-11
Water purifier	WASSER LAB	DI-GR
Materials
0.25 % Trypsin-EDTA	Gibco	25200-072
10x PBS	Intron	IBS-BP007a
4% Paraformaldehyde	Biosesang
70% Ethanol	Daejung	4018-4410
Anti-CD31 antibody	Abcam	ab28364
Anti-HIF-1 alpha antibody	Abcam	ab16066
Anti-SHMT2/SHMT antibody	Abcam	ab88664
Anti-SOX2 antibody	Abcam	ab75485
Bovine Serum Albumin	Thermo scientific	J10857-22
Collagen from porcine skin	Dalim tissen	PC-001-1g
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermofisher	D1306
Endothelial Cell Growth Medium-2	Promocell	C22011
Fetal bovine serum	Gibco	12483-020
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Theromofisher	A-11001
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594	Theromofisher	A-11012
High-glucose Dulbecco’s Modified Eagle Medium(DMEM)	Hyclone	SH30243-0
Hydrochloric acid	Sigma-Aldrich	311413-100ML
Live/dead assay kit	Invitrogen	L3224
Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control	Abcam	ab170190
Penicillin/streptomycin	Gibco	15140-122
Phenol red solution	Sigma-Aldrich	P0290-100ML
Poly(ethylene-vinyl acetate)	Poly science	06108-500
Polydimethylsiloxane	Dowhitech	sylgard 184
Rabbit IgG, polyclonal - Isotype Control	Abcam	ab37415
Sodium hydroxide solution	Samchun	S0610
Triton X-100	Biosesang	TRI020-500-50
Trypan Blue	Sigma-Aldrich	T8154
Software
COMSOL Multiphysics 3.5a	COMSOL AB
IMS beamer			in-house software
SolidWorks Package	Dassault Systems SolidWorks Corporation