Tao Jiang thanks the China Scholarship Council (201403170354) and McGill Engineering Doctoral Award (90025) for their scholarship funding. Jose G. Munguia-Lopez thanks CONACYT (250279, 290936 and 291168) and FRQNT (258421) for their scholarship funding. Salvador Flores-Torres thanks CONACYT for their scholarship funding (751540). Joseph M. Kinsella thanks the National Science and Engineering Research Council, the Canadian Foundation for Innovation, the Townshend-Lamarre Family Foundation, and McGill University for their funding. We would like to thank Allen Ehrlicher for allowing us to use his rheometer, Dan Nicolau for allowing us to use his confocal microscope, and Morag Park for granting us access to fluorescently labeled cell lines.

<ol>
	<li>Cui, X., Hartanto, Y., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+multicellular+spheroids+formation.">Advances in multicellular spheroids formation.</a> Journal of the Royal Society Interface. 14 (127), (2017).</li><li>Yip, D., Cho, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicellular+3D+heterospheroid+model+of+liver+tumor+and+stromal+cells+in+collagen+gel+for+anti-cancer+drug+testing.">A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing.</a> Biochemical and Biophysical Research Communications. 433 (3), 327-332 (2013).</li><li>Breslin, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+using+3D+cell+cultures,+in+addition+to+2D+monolayer+cultures,+when+evaluating+breast+cancer+drug+sensitivity+and+resistance.">The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.</a> Oncotarget. 7 (29), 45745-45756 (2016).</li><li>Yue, X., Lukowski, J. K., Weaver, E. M., Skube, S. B., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+and+phosphoproteomic+comparison+of+2D+and+3D+colon+cancer+cell+culture+models.">Quantitative proteomic and phosphoproteomic comparison of 2D and 3D colon cancer cell culture models.</a> Journal of Proteome Research. 15 (12), 4265-4276 (2016).</li><li>Priwitaningrum, D. 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The authors have nothing to disclose.

Cell-laden structures can be compromised if contamination (biological or chemical) occurs at any point in the process. Usually, biological contamination is seen after two or three days of culture as a color change in the culture media or the bioprinted structure. Therefore, the sterilization (physical and chemical disinfection) is a key step for all the cell-related processes. Noteworthy, autoclaving gelatin changes its gelling properties, which made it gel slower in the trials we conducted. Therefore, we sterilized the alginate and gelatin power via UV exposure. Due to the very limited penetration capability of UV light, a very thin layer (&#60; 0.5 mm) should be used. The concentration of alginate and gelatin can be arbitrarily changed to tune the mechanical and biological properties32. In the present work, we chose A3G7, because it provides desirable printability during its printing window, and the crosslinked hydrogel provides a high stability through the 30-day experiment. Heating to 60 &#176;C while dissolving powder into DPBS helps to enhance the liquefication of gelatin, which eases the stirring. A lower temperature can also be used; however, a longer stirring time will then be required.
The rheological temperature sweep gives a sol-gel point at 30.6 &#176;C, which is compatible with other publications33. Theoretically, any temperature higher than 30.6 &#176;C can liquefy the precursor. We chose 37 &#176;C to simplify the work, as cell culture medium is pre-warmed in a 37 &#176;C water bath. Based on the gelation kinetics (before the precursor gels), there is approximately 17 min for cell mixing; after this time, mixing the cells and the hydrogel precursor is difficult due to the increased viscosity and moduli, causing a non-homogeneous cell-laden bioink. Therefore, we suggest handling the cell-mixing work within the first 10 min of gelling. The yield stress is not considered to be a material attribute that affects printability until recently34; regardless, it has been extensively recognized by polymer scientists as a marker of pasty/granular fluid31,35,36,37. The existence of yield stress helps to build up a structural integrity to withstand its own weight. A high yield stress can also require an increased startup pressure in extrusion; thus, this may result in damage to the cells due to excessive shear stress32. The model fidelity can be compromised if the extrusion process occurs outside of the optimal printing window. Common indicators of this problem are shown in the final bioprinted structure: it will either be too rough or too liquid at the edges. The A3G7 precursor exhibits an optimal printing window during 50 - 90 min of gelling that satisfies both the structural stability and the cell survival and can be used as the medium to build heterogeneous tumor models14.
The total height of the propeller model is 600 &#181;m as it is generated from four layered 150 &#181;m filaments. This design allows nutrient and gas exchange as cells are at most 300 &#181;m from the surface contacting the media during the incubation. Higher models are achievable in fabrication but are bottlenecked by the limited diffusion distance of the nutrients in the hydrogel38, which can result in a low cell survival in the core region.
Different strategies have been developed to form MCTS in vitro, such as hanging drop, microfluidic chip, assembly, and bioprinting39. However, some of these methodologies can alter the cell physiology and biochemistry, generating different cell behaviors that occur in normal tumor tissue. For instance, the hanging drop method forces single cells to stay as cell aggregates through physical confinement40. Also, the chemical induction to form MCTS by a peptide addition can alter the biochemistry of the spheroids41. The hydrogel composite described here allows an MCTS formation by creating a biomimetic environment without external stressors. Starting out as single well-dispersed cells, after 7 days of cultures, cells reorganize as small MCTS (more than 6 cells per spheroid), increasing their size and quantity during 30 days of culture.
In the results of this research, we found that MDA-MB-231-GFP spheroids decrease the cell viability at day 21, correlating this phenomenon with the formation of large spheroids. A solid tumor is comprised of three different layers, whereby the external layer presents a high proliferation rate compared to the middle layer and the internal necrotic or senescent core of the spheroid18. This could explain the viability decrease in the results.
This limitations of this protocol are (1) bioink dynamics and (2) cell type compatibility. Bioink dynamics refers to the varying material properties during the gelation process, resulting in an optimal printing window beyond which the printed structure is defective. Cell type compatibility refers to the incapacity of certain cell types (cell line or primary culture) to exhibit a native in vivo behavior.
The A3G7 hydrogel achieves a high stability and cell viability. 3D bioprinting can be used to build 3D heterogeneous disease models with high-throughput, low cost, and high reproducibility as a more realistic alternative to traditional cell culture and small animal tumor models.

Although 2D cell culture is widely used in cancer research, limitations exist as the cells are grown in a monolayer format with a uniform concentration of nutrients and oxygen. These cultures lack important cell-cell and cell-matrix interactions present in the native tumor microenvironment (TME). Consequently, these models poorly recapitulate physiological conditions, resulting in aberrant cell behaviors, including unnatural morphologies, irregular receptor organization, membrane polarization, and abnormal gene expression, among other conditions1,2,3,4. On the other hand, 3D cell culture, where cells are expanded in a volumetric space as aggregates, spheroids, or organoids, offers an alternative technique to create more accurate in vitro environments to study fundamental cell biology and physiology. 3D cell culture models can also encourage cell-ECM interactions that are critical physiological characteristics of the native TME in vitro1,4,5. The emerging 3D bioprinting technology provides possibilities to build models that mimic the heterogeneous TME.
3D bioprinting is derived from rapid prototyping and enables the fabrication of 3D microstructures that are capable of mimicking some of the complexities of living tissue samples6,7. The current bioprinting methods include inkjet, extrusion, and laser-assisted printing8. Among them, the extrusion method allows the heterogeneity to be controlled within the printed matrices by precisely positioning distinct types of materials at different initial locations. Therefore, it is the best approach to fabricate heterogeneous in vitro models involving multiple types of cells or matrices. Extrusion bioprinting has been successfully used to build auricular shaped scaffolds9, vascular structures10,11,12, and skin tissues13, resulting in high printing fidelity and cell viability. The technology also features versatile material selections, the ability to deposit materials with cells embedded with a known density, and high reproducibility14,15,16,17. Natural and synthetic hydrogels are frequently used as bioinks for 3D bioprinting due to their biocompatibility, bioactivity, and their hydrophilic networks that can be engineered to structurally resemble the ECM7,18,19,20,21,22,23.Hydrogels are also advantageous since they can include adhesive sites for cells, structural elements, permeability for nutrients and gases, and the appropriate mechanical properties to encourage cell development24. For instance, collagen hydrogels offer integrin anchorage sites that cells can use to attach to the matrix. Gelatin, denatured collagen, retains similar cell adhesion sites. In contrast, alginate is bioinert but provides mechanical integrity by forming crosslinks with divalent ions25,26,27,28.
In this work, we developed a composite hydrogel as a bioink, comprised of alginate and gelatin, with similarities to the microscopic architecture of a native tumor stroma. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed via an extrusion-based bioprinter to create a 3D model that mimics the in vivo microenvironment. The engineered 3D environment allows cancer cells to form multicellular tumor spheroids (MCTS) with a high viability for long periods of cell culture (&#62; 30 days). This protocol demonstrates the methodologies of synthesizing composite hydrogels, characterizing the materials&#39; microstructure and printability, bioprinting cellular heterogeneous models, and observing the formation of MCTS. These methodologies can be applied to other bioinks in extrusion bioprinting as well as to different designs of heterogeneous tissue models with potential applications in drug screening, cell migration assays, and studies that focus on fundamental cell physiological functions.

<table><tbody><tr><td>Sodium alginate</td><td>FMC BioPolymer</td><td>CAS-No: 9005-38-3</td><td>Protanal LF 10/60 FT</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td>Type B gelatin from bovine skin</td></tr><tr><td>Dubelcco&#039;s phosphate buffered saline (DPBS 1X)</td><td>Gibco</td><td>LS14190136</td><td>1&amp;times;, w/o calcium, w/o magnesium</td></tr><tr><td>Magnetic hotplate</td><td>Corning&amp;nbsp;</td><td>N/A</td><td>Stirrer/hot plate model PC-420</td></tr><tr><td>50 mL centrifuge tubes</td><td>Corning</td><td>352098</td><td>Falcon&amp;reg; 50mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile</td></tr><tr><td>Centrifuge</td><td>GMI</td><td>N/A</td><td>Sorvall RT6000D, GMI, USA</td></tr><tr><td>Calcium chloride anhydrous</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>MilliQ water</td><td>Millipore</td><td>N/A</td><td></td></tr><tr><td>Millipore 0.22 &amp;micro;m filters</td><td>Millipore</td><td>SLGS033SB</td><td>Millex-GS Syringe Filter Unit, 0.22&amp;nbsp;&amp;micro;m, mixed cellulose esters, 33&amp;nbsp;mm, ethylene oxide sterilized</td></tr><tr><td>Oscillation rheometer MCR 302</td><td>Anton Paar</td><td>N/A</td><td></td></tr><tr><td>Rheometer measuring tool CP25</td><td>Anton Paar</td><td>79038</td><td>Conical plate geometry for rheometer</td></tr><tr><td>RheoCompass</td><td>Anton Paar</td><td>N/A</td><td>Software controlling rheometer MCR 302</td></tr><tr><td>Scanning electron microscope</td><td>Hitachi</td><td>N/A</td><td>SEM, Hitachi SU-3500 Variable Pressure</td></tr><tr><td>Paraformaldehyde, 96%, extra pure</td><td>Acros Organics</td><td>416785000</td><td></td></tr><tr><td>Dulbecco modified eagle medium (DMEM)</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>Antibiotic/Antimycotic solution (100X) stabilized</td><td>Sigma</td><td>A5955</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Wisent Bioproducts</td><td>080-150</td><td></td></tr><tr><td>Cell culture T-75 flasks</td><td>Sigma-Aldrich</td><td>CLS430641</td><td>75 cm&lt;sup&gt;2&lt;/sup&gt; TC-Treated surface treatment</td></tr><tr><td>3D bioprinter BioScaffolder 3.1</td><td>GeSiM</td><td>N/A</td><td></td></tr><tr><td>GeSim software</td><td>GeSiM</td><td>N/A</td><td>Software controlling BioScaffolder 3.1</td></tr><tr><td>10cc cartridge UV resist</td><td>EFD Nordson</td><td>7012126</td><td></td></tr><tr><td>End cap</td><td>EFD Nordson</td><td>7014472</td><td></td></tr><tr><td>Tip cap</td><td>EFD Nordson</td><td>7014469</td><td></td></tr><tr><td>Piston&amp;nbsp;</td><td>EFD Nordson</td><td>7012182</td><td></td></tr><tr><td>Stainless nozzle G25</td><td>EFD Nordson</td><td>7018345</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>N/A</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td>Bioreagent, for molecular biology</td></tr><tr><td>Costar 6-well plates&amp;nbsp;</td><td>Corning</td><td>3516</td><td>TC-Treated Multiple Well Plates, Individually Wrapped, Sterile&amp;nbsp;</td></tr><tr><td>Confocal spinning disk inverted microscope</td><td>Olympus Life Science</td><td>N/A</td><td>Olympus IX83</td></tr><tr><td>MTS assay kit</td><td>Promega</td><td>G3582</td><td>CellTiter 96&amp;reg; AQueous&amp;nbsp;One Solution Cell Proliferation Assay&amp;nbsp;</td></tr><tr><td>Live/Dead viability cytotoxicity kit</td><td>Molecular Probes,ThermoFisher Scientific</td><td>L3224</td><td></td></tr><tr><td>Trypsin 0.25/EDTA 1X</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>Corning 96-well plate</td><td>Corning</td><td>3595</td><td>Clear Flat Bottom Polystyrene TC-Treated Microplate, Individually Wrapped, with Low Evaporation Lid, Sterile</td></tr><tr><td>Autoclave Tuttnauer</td><td>Heidolph Brinkmann</td><td>N/A</td><td>Heidolph Tuttnauer 2540E Autoclave Sterilizer Electronic Model with 4 Stainless Steel Trays, 23L Capacity</td></tr><tr><td>Trypan blue</td><td>Invitrogen&amp;nbsp;</td><td>T10282</td><td>0.4% solution</td></tr><tr><td>Ethanol</td><td>Commercial Alcohols</td><td>P016EA95</td><td>Greenfield Speciality Alcohols</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Panasonic</td><td>N/A</td><td>MCO 19AIC-PA</td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>SP Scientific</td><td>N/A</td><td>Virtis Sentry 2.0</td></tr><tr><td>SolidWorks</td><td>Dassault Systems</td><td>N/A</td><td>A CAD software used to build demostrative propeller-like model</td></tr><tr><td>MATLAB</td><td>The MathWorks</td><td>N/A</td><td>A programming software used to generate G-code for BioScaffolder 3.1</td></tr></tbody></table>

bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

The temperature sweep shows a distinct difference of the A3G7 precursor at 25 &#176;C and 37 &#176;C. The precursor is liquid at 37 &#176;C and has a complex viscosity of 1938.1 &#177; 84.0 mPa x s, which is validated by a greater G&#34; over G&#39;. As the temperature decreases, the precursor undergoes physical gelation due to the spontaneous physical entanglement of the gelatin molecules into a tri-helix formation29,30. Both the G&#39; and the G&#34; increase and converge at 30.6 &#176;C, indicating a sol-gel transition. The moduli continue to increase, with decreased temperature, reaching 468.5 &#177; 34.2 Pa of G&#39; and 140.7 &#177; 9.3 Pa of G&#34; at 25 &#176;C (Figure 1a). Based on the results of the temperature sweep, we chose 25 &#176;C as the printing temperature. The gelation kinetics experiment simulates the temperature change that occurs during the sample preparation and handling (i.e., removing the sample from the 37 &#176;C water bath and placing it into the 25 &#176;C bioprinter chamber). G&#39;, G&#34;, and |&#951;*| increase with time at the lowered temperature, and the sol-gel transition happens at ~ 17 min at 25 &#176;C, when G&#39; &#8776; G&#34; &#8776; 64.3 Pa and the loss factor equals 1 (Figure 1b and 1c). The precursor continues to stiffen and reaches a printing window after 50 - 90 min of gelation. Within this printing window, the precursor can be smoothly extruded using a G25 cylindrical nozzle with a pressure of 200 kPa. The existence of yield stress implies a solid-like behavior of the material and raises the structural integrity after the extrusion to withstand its own weight31. The stress ramp recognizes the yield stress at different times of the gelation. Results show that the yield stress increases with the increasing gelation time. At 50 min of gelation, the yield stress reaches 325.9 Pa, assuring that the model is stable after the extrusion.
The printed propeller model is shown in Figure 2a. Confocal microscopy confirms the initial extrusion locations of both the MDA-MB-231-GFP and the IMR-90-mCherry cells (Figure 2b). The MDA-MB-231-GFP cells begin to develop spheroids 15 days into the culture period, followed by increased size and numbers of spheroids until day 30 (Figure 2c - 2f). Some of the IMR-90-mCherry cells also form agglomerations (Figure 2g - 2j). Noticeably, after 30 days of culture, IMR-90-mCherry cells are observed in the region initially occupied by the MDA-MB-231-GFP cells (Figure 2f), implying possible migration events in the model. Likewise, migrated MDA-MB-231-GFP cells can also be observed in the IMR-90-mCherry dominated region on day 30 (Figure 2j).
The disk model is shown in Figure 3a. Confocal microscopy confirms the homogeneous cell distribution within the disk (Figure 3b). The MDA-MB-231-GFP cells behave similarly in the disk model as they did when printed as a propeller model. Representative images are displayed in Figure 3c - 3f. SEM imaging reveals an MDA-MB-231-GFP spheroid-laden hydrogel after 21 days of culture (Figure 4a). The generation, and presence, of the spheroid is validated by comparing the SEM images with cell-free hydrogels (Figure 4b).
The cell viability of both cell types is demonstrated in Figure 4c. MDA-MB-231-GFP cells express a higher cell viability compared to IMR-90-mCherry cells. Interestingly, the MDA-MB-231-GFP cells exhibit an increased trend of viability before day 15, with a tripling of the number of viable cells compared to day 0. This is followed by a decrease on day 21, before recovering again on day 30. In contrast, the IMR-90-mCherry cells have minimal fluctuations in viability during the entirety of the culture period. The decreasing values in viability of the MDA-MB-231-GFP cells at day 21 correspond to a large MTCSs formation, which has developed necrotic cores.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57826/57826fig1.jpg" /> 
Figure 1: Rheological characterization of the hydrogel precursor. (a) A temperature sweep shows the sol-gel transition at 30.6 &#176;C. (b-c) The gelation kinetics of the A3G7 precursor shows an increase of G&#39;, G&#34;, and |&#951;*| at time of gelation, and the sol-gel transition happens at approximately 17 min at 25 &#176;C. (d) This panel shows the yield stress of the precursor versus the time of gelation. An increase of yield stress is observed with a longer gelling time. The results are shown in mean &#177; SD, n&#8805; 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57826/57826fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57826/57826fig2.jpg" /> 
Figure 2: MCTS formation within a 3D bioprinted in vitro model consisting of IMR-90-mCherry myofibroblast and MDA-MB-231-GFP breast cancer cells. (a) These photographs show the bioprinted in vitro sample (left) and the CAD model (right). (b) This representative confocal time-lapse image shows the MDA-MB-231-GFP (green) and IMR-90-mCherry (red) cells bioprinted within the model. (c - f) These zoom-ins show the MDA-MB-231-GFP cell regions (white dotted boxes). (g - j) These zoom-ins show the IMR-90-mCherry cell regions (yellow dotted boxes). The scale bars are 2 mm in panel 2a, 1 mm in panel 2b, and 500 &#181;m in panels 2c - 2j for selected areas, and the magnification is 10X. Capital &#34;D&#34; in the images means &#34;days of culture&#34;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57826/57826fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57826/57826fig3.jpg" /> 
Figure 3: MCTS formation within a 3D MDA-MB-231-GFP-laden hydrogel disk. (a) This photograph shows a hydrogel disk. (b) This representative confocal image shows MDA-MB-231-GFP cells embedded in the composite. (c-f) These zoom-ins show the MDA-MB-231-GFP cell region (white dotted box in panel 3b) at (c) 0, (d) 7, (e) 15, and (f) 21 days of culture. The scale bars are 2 mm in panel 3a, 1 mm in panel 3b, and 500 &#181;m in panels 3c - 3f for selected areas, and the magnification is 10X. Capital &#34;D&#34; in the images means &#34;days of culture&#34;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57826/57826fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57826/57826fig4.jpg" /> 
Figure 4: Morphology and cell viability of MTCSs. (a) This SEM image shows an MDA-MB-231-GFP MCTS within the gel after 21 days of culture, showing small MCTS (arrow-pointed). (b) This SEM image shows an alginate/gelatin hydrogel without cells. The magnification is 350X. (c) This panel shows the cell viability of MDA-MB-231-GFP and IMR-90-mCherry during 30 days of culture within the hydrogel. The results were analyzed as the mean &#177; SD and statistically analyzed using a two-way ANOVA and a Bonferroni&#39;s post-test. p (*) &#60; 0.05, n &#8805; 3. The data was normalized to the cell density used to create the samples on day 0. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57826/57826fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

bioprintable-alginategelatin-hydrogel-3d-vitro-model-systems-induce

Nanyang Technological University

Research

JoVE Journal

Bioengineering

18.5K Views.  McGill University Montreal. Wij ontwikkeld een heterogene borst kanker model bestaande uit vereeuwigd tumor en fibroblast cellen ingebed in een bioprintable alginaat/gelatine bioink. Het model recapituleert de tumor in vivo communicatie en vergemakkelijkt de vorming van meercellige tumor spheroïden, inzicht in de mechanismen van tumorvorming rijden oplevert.

Video: Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Biology

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

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