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* These authors contributed equally
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.
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.
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 hyperplasia and metastasis2,3. Subsequently, hypoxia in cancer cells causes the destruction of neighboring normal tissues. Furthermore, hypoxia is strongly associated with the therapeutic resistance of solid tumors in multifactorial manners. Hypoxia may severely impede radiotherapy, as radiosensitivity is limited owing to reactive oxygen species1,4. In addition, it decreases pH levels of cancer microenvironments, which decreases drug accumulation1. Therefore, reproducing pathological features related to hypoxia in vitro is a promising strategy for scientific and pre-clinical findings.
Modeling a specific microenvironment of cancer is essential for understanding cancer development and exploring appropriate treatments. Although animal models have been widely used because of their strong physiological relevance, issues related to species differences and ethical problems exist5. Furthermore, although conventional 2D and 3D models allow for the manipulation and real-time imaging of cancer cells for an in-depth analysis, their architectural and cellular complexity cannot be fully recapitulated. For example, cancer spheroid models have been widely used, as cancer cell aggregation in a spheroid can naturally generate hypoxia in the core. Moreover, large numbers of cellular spheroids of uniform size have been produced using plastic- or silicone-based multi-well systems6,7. However, the lower flexibility with regard to capturing the exact heterogeneous structure of cancerous tissues with conventional platforms has required the establishment of an advanced biofabrication technology to build a highly biomimetic platform to improve cancer research8.
3D microphysiological systems (MPSs) are useful tools to recapitulate the complex geometry and pathological progression of cancer cells9. As cancer cells sense the biochemical gradient of growth factors and chemokines and the mechanical heterogeneity reproduced on the system, important features of cancer development can be investigated in vitro. For instance, cancer viability, metastatic malignancy, and drug resistance depending on the varying oxygen concentrations has been studied using MPSs10,11. Despite recent advancements, generating hypoxic conditions of in vitro models relies on complex fabrication procedures, including connection with physical gas pumps. Therefore, simple, and flexible methods to build cancer-specific microenvironments are needed.
3D cell printing technology has gained considerable attention because of its precise control of the spatial arrangement of biomaterials to recapitulate native biological architectures12. In particular, this technology overcomes the existing limitations of 3D hypoxia models owing to its high controllability and feasibility for building the spatial features of the cancer microenvironment. 3D printing also facilitates computer-aided manufacturing through a layer-by-layer process, thereby providing a rapid, accurate, and reproducible construction of complex geometries to mimic actual tissue architectures. In addition to the advantages of existing manufacturing strategies for 3D MPSs, the pathophysiological features of cancer progression can be reproduced by patterning the biochemical, cellular, and biophysical components13,14.
Herein, we present a 3D cell-printing strategy for a hypoxic cancer-on-a-chip for recapitulating the heterogeneity of a solid cancer (Figure 1)15. The fabrication parameters were determined via a computational simulation of central hypoxia formation in the system. Cancer-stroma concentric rings were printed using collagen bioinks containing glioblastoma cells and endothelial cells to emulate the pathophysiology of glioblastoma, a type of solid cancer. The formation of a radial oxygen gradient aggravated cancer malignancy, indicating strengthened aggressiveness. Furthermore, we indicate future perspectives for the applications of the chip to patient-specific preclinical models. The proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models of cancer.
1. Computer simulation of oxygen gradient formation
2. Cell culture of cancer cells and stromal cells
3. Preparation of collagen pre-gel solution
4. 3D printing of gas-permeable barrier
5. Preparation of cell-encapsulated collagen bio-inks
6. 3D cell-printing of cancer-stroma concentric rings
7. Evaluation of post-printing cell viability
8. Immunofluorescence to validate the formation of central hypoxia and its effect on cancer malignancy
9. Statistical analysis
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...
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 ...
The authors have no disclosures.
This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2020R1A6A1A03047902 and NRF-2018H1A2A1062091) and the Korea government (MSIT) (No. NRF-2019R1C1C1009606 and NRF-2019R1A3A3005437).
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 |
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