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).

<ol>
	<li>Jing, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hypoxia+in+cancer+therapy+by+regulating+the+tumor+microenvironment.">Role of hypoxia in cancer therapy by regulating the tumor microenvironment.</a> Molecular Cancer. 18 (1), 157 (2019).</li><li>Al Tameemi, W., Dale, T. P., Al-Jumaily, R. M. K., Forsyth, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-modified+cancer+cell+metabolism.">Hypoxia-modified cancer cell metabolism.</a> Frontiers in Cell and Developmental Biology. 7, 4 (2019).</li><li>Petrova, V., Annicchiarico-Petruzzelli, M., Melino, G., Amelio, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic+tumour+microenvironment.">The hypoxic tumour microenvironment.</a> Oncogenesis. 7 (1), 1-13 (2018).</li><li>Hockel, M., Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+hypoxia:+definitions+and+current+clinical,+biologic,+and+molecular+aspects.">Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects.</a> Journal of the National Cancer Institute. 93 (4), 266-276 (2001).</li><li>Kim, H., Lin, Q., Glazer, P. M., Yun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic+tumor+microenvironment+in+vivo+selects+the+cancer+stem+cell+fate+of+breast+cancer+cells.">The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells.</a> Breast Cancer Research. 20 (1), 16 (2018).</li><li>Jeong, G. S., Lee, J., Yoon, J., Chung, S., Lee, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+lithography+for+fabricating+self-organizing+soft+micro-honeycomb+structures+with+ultra-high+aspect+ratios.">Viscoelastic lithography for fabricating self-organizing soft micro-honeycomb structures with ultra-high aspect ratios.</a> Nature Communications. 7 (1), 1-9 (2016).</li><li>Razian, G., Yu, Y., Ungrin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+large+numbers+of+size-controlled+tumor+spheroids+using+microwell+plates.">Production of large numbers of size-controlled tumor spheroids using microwell plates.</a> Journal of Visualized Experiments:JoVE. (81), e50665 (2013).</li><li>Nunes, A. S., Barros, A. S., Costa, E. C., Moreira, A. F., Correia, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+spheroids+as+in+vitro+models+to+mimic+in+vivo+human+solid+tumors+resistance+to+therapeutic+drugs.">3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs.</a> Biotechnology and Bioengineering. 116 (1), 206-226 (2019).</li><li>Wan, L., Neumann, C., LeDuc, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-on-a-chip+for+integrating+a+3D+tumor+microenvironment:+chemical+and+mechanical+factors.">Tumor-on-a-chip for integrating a 3D tumor microenvironment: chemical and mechanical factors.</a> Lab on a Chip. 20 (5), 873-888 (2020).</li><li>Nam, H., Funamoto, K., Jeon, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+migration+and+cancer+drug+screening+in+oxygen+tension+gradient+chip.">Cancer cell migration and cancer drug screening in oxygen tension gradient chip.</a> Biomicrofluidics. 14 (4), 044107 (2020).</li><li>Palacio-Casta&#241;eda, V., Kooijman, L., Venzac, B., Verdurmen, W. P., Le Gac, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+switching+of+tumor+cells+under+hypoxic+conditions+in+a+tumor-on-a-chip+model.">Metabolic switching of tumor cells under hypoxic conditions in a tumor-on-a-chip model.</a> Micromachines. 11 (4), 382 (2020).</li><li>Ronaldson-Bouchard, K., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organs-on-a-chip:+a+fast+track+for+engineered+human+tissues+in+drug+development.">Organs-on-a-chip: a fast track for engineered human tissues in drug development.</a> Cell Stem Cell. 22 (3), 310-324 (2018).</li><li>Mi, S., Du, Z., Xu, Y., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crossing+and+integration+between+microfluidic+technology+and+3D+printing+for+organ-on-chips.">The crossing and integration between microfluidic technology and 3D printing for organ-on-chips.</a> Journal of Materials Chemistry B. 6 (39), 6191-6206 (2018).</li><li>Yi, H. -. G., Lee, H., Cho, D. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+of+organs-on-chips.">3D printing of organs-on-chips.</a> Bioengineering. 4 (1), 10 (2017).</li><li>Yi, H. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioprinted+human-glioblastoma-on-a-chip+for+the+identification+of+patient-specific+responses+to+chemoradiotherapy.">A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy.</a> Nature Biomedical Engineering. 3 (7), 509-519 (2019).</li><li>Kang, T. -. Y., Hong, J. M., Jung, J. W., Yoo, J. J., Cho, D. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+assessment+of+a+microfluidic+network+system+for+oxygen+transport+in+engineered+tissue.">Design and assessment of a microfluidic network system for oxygen transport in engineered tissue.</a> Langmuir. 29 (2), 701-709 (2013).</li><li>Woo Jung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effective+diffusivity+of+a+freeform+fabricated+scaffold+using+computational+simulation.">Evaluation of the effective diffusivity of a freeform fabricated scaffold using computational simulation.</a> Journal of Biomechanical Engineering. 135 (8), (2013).</li><li>Brown, A. C., De Beer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+stereolithography+(STL)+slicing+and+G-code+generation+algorithm+for+an+entry+level+3-D+printer.">Development of a stereolithography (STL) slicing and G-code generation algorithm for an entry level 3-D printer.</a> 2013 Africon (IEEE). , 1-5 (2013).</li><li>Shim, J. -. H., Lee, J. -. S., Kim, J. Y., Cho, D. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+of+a+mechanically+enhanced+three-dimensional+dual+cell-laden+construct+for+osteochondral+tissue+engineering+using+a+multi-head+tissue/organ+building+system.">Bioprinting of a mechanically enhanced three-dimensional dual cell-laden construct for osteochondral tissue engineering using a multi-head tissue/organ building system.</a> Journal of Micromechanics and Microengineering. 22 (8), 085014 (2012).</li><li>Gillispie, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+methodologies+for+extrusion-based+bioink+printability.">Assessment methodologies for extrusion-based bioink printability.</a> Biofabrication. 12 (2), 022003 (2020).</li><li>Kim, B. S., Das, S., Jang, J., Cho, D. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix-based+bioinks+for+engineering+tissue-and+organ-specific+microenvironments.">Decellularized extracellular matrix-based bioinks for engineering tissue-and organ-specific microenvironments.</a> Chemical Reviews. 120 (19), 10608-10661 (2020).</li></ol>

The authors have no disclosures.

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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Cells</td>
		</tr>
		<tr>
			<td>Human umbilical vein endothelial cells</td>
			<td>Promocell</td>
			<td>C-12200</td>
			<td></td>
		</tr>
		<tr>
			<td>U-87 MG cells</td>
			<td>ATCC</td>
			<td>ATCC HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Disposable</td>
		</tr>
		<tr>
			<td>0.2 &#956;m syringe filter</td>
			<td>Sartorius</td>
			<td>16534-K</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL disposable syringe</td>
			<td>Jung Rim</td>
			<td>10ml 21G32</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL glass vial</td>
			<td>Hubena</td>
			<td>A0039</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>91010</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SPL lifescience</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>18G plastic needle</td>
			<td>Musashi engineering</td>
			<td>PN-18G-B</td>
			<td></td>
		</tr>
		<tr>
			<td>20G plastic tapered dispense tip</td>
			<td>Musashi engineering</td>
			<td>TPND-20G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>22x50 glass cover</td>
			<td>MARIENFIELD</td>
			<td>0101142</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>90125</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL disposable syringes</td>
			<td>HENKE-JET</td>
			<td>4020-X00V0</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>91005</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>SPL lifescience</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>90150</td>
			<td></td>
		</tr>
		<tr>
			<td>50N precision nozzle</td>
			<td>Musashi engineering</td>
			<td>HN-0.5ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>SINKWANG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tips</td>
			<td>Gilson</td>
			<td>CP1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-scrapper</td>
			<td>SPL lifescience</td>
			<td>90030</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal dish</td>
			<td>SPL lifescience</td>
			<td>200350</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-coated histology slide</td>
			<td>MATSUNAMI</td>
			<td>MAS-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>SPL lifescience</td>
			<td>23050</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 cell culture flask</td>
			<td>SPL lifescience</td>
			<td>70075</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>3DX printer</td>
			<td>T&#38;R Biofab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>JEIOTECH</td>
			<td>AC-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuger</td>
			<td>Cyrozen</td>
			<td>1580MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser microscopy</td>
			<td>Olympus Life Science</td>
			<td>FV 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>FISHER SCEINTIFIC</td>
			<td>O221S366</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Korea Ace Scientific</td>
			<td>HC.203-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand tally counter</td>
			<td>KTRIO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>MARIENFIELD</td>
			<td>0650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Panasonic</td>
			<td>MCO-170AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow cabinet</td>
			<td>DAECHUNG SCIENCE</td>
			<td>CB-BMMS C-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal syringe</td>
			<td>IWASHITA engineering</td>
			<td>SUS BARREL 10CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>Hirose</td>
			<td>HC.13-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>JEIOTECH</td>
			<td>OF-12, H070023</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive displacement pipette</td>
			<td>GILSON</td>
			<td>NJ05652</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>SAMSUNG</td>
			<td>CRFD-1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltex Mixer</td>
			<td>DAIHAN scientific</td>
			<td>VM-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>DAIHAN SCIENTIFIC</td>
			<td>WB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Water purifier</td>
			<td>WASSER LAB</td>
			<td>DI-GR</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>0.25 % Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Intron</td>
			<td>IBS-BP007a</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Biosesang</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Daejung</td>
			<td>4018-4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HIF-1 alpha antibody</td>
			<td>Abcam</td>
			<td>ab16066</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SHMT2/SHMT antibody</td>
			<td>Abcam</td>
			<td>ab88664</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SOX2 antibody</td>
			<td>Abcam</td>
			<td>ab75485</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Thermo scientific</td>
			<td>J10857-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from porcine skin</td>
			<td>Dalim tissen</td>
			<td>PC-001-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermofisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium-2</td>
			<td>Promocell</td>
			<td>C22011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Theromofisher</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>Theromofisher</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>High-glucose Dulbecco&#8217;s Modified Eagle Medium(DMEM)</td>
			<td>Hyclone</td>
			<td>SH30243-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311413-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead assay kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control</td>
			<td>Abcam</td>
			<td>ab170190</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene-vinyl acetate)&#160;</td>
			<td>Poly science</td>
			<td>06108-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane</td>
			<td>Dowhitech</td>
			<td>sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG, polyclonal - Isotype Control</td>
			<td>Abcam</td>
			<td>ab37415</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>Samchun</td>
			<td>S0610</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Biosesang</td>
			<td>TRI020-500-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Software</td>
		</tr>
		<tr>
			<td>COMSOL Multiphysics 3.5a</td>
			<td>COMSOL AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMS beamer</td>
			<td></td>
			<td></td>
			<td>in-house software</td>
		</tr>
		<tr>
			<td>SolidWorks Package</td>
			<td colspan="2">Dassault Systems SolidWorks Corporation</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d cell-printed hypoxic cancer-on-a-chip for recapitulating pathologic progression of solid 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&#160;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 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...

Watch this Scientific Journal Video about 3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer at JoVE.com

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

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, ...

3d-cell-printed-hypoxic-cancer-on-chip-for-recapitulating-pathologic

Research

JoVE Journal

Bioengineering

4.6K Views.  Pohang University of Science and Technology (POSTECH). 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.

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

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...