We thank Alberta Innovates for providing the CNC and Ken Harris and Jae-Young Cho for their technical advice when preparing the CNC/agarose/D-mannitol matrix. We also thank Ben Hoffman, Heather Winchell and Nicole Diamantides for their technical advice and support with the setup and calibration of the INKREDIBLE+ 3D bioprinter.

<ol>
	<li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnology and Bioengineering. 103 (4), 655-663 (2009).</li><li>Drury, J. L., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+tissue+engineering:+Scaffold+design+variables+and+applications.">Hydrogels for tissue engineering: Scaffold design variables and applications.</a> Biomaterials. 24 (24), 4337-4351 (2003).</li><li>Lee, K. Y., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+tissue+engineering.">Hydrogels for tissue engineering.</a> Chemical Reviews. 101 (7), 1869-1879 (2001).</li><li>Halib, N., Ahmad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanocellulose:+Insight+into+health+and+medical+applications.">Nanocellulose: Insight into health and medical applications.</a> Handbook of Ecomaterials. , 1345-1363 (2019).</li><li>Alonso-Lerma, B., 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=High+performance+crystalline+nanocellulose+using+an+ancestral+endoglucanase.">High performance crystalline nanocellulose using an ancestral endoglucanase.</a> Communications Materials. 1 (1), 57 (2020).</li><li>Tummala, G. K., Lopes, V. R., Mihranyan, A., Ferraz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+of+nanocellulose-reinforced+PVA+hydrogel+with+human+corneal+epithelial+cells+for+ophthalmic+applications.">Biocompatibility of nanocellulose-reinforced PVA hydrogel with human corneal epithelial cells for ophthalmic applications.</a> Journal of Functional Biomaterials. 10 (3), 35 (2019).</li><li>Fey, C., 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=Bacterial+nanocellulose+as+novel+carrier+for+intestinal+epithelial+cells+in+drug+delivery+studies.">Bacterial nanocellulose as novel carrier for intestinal epithelial cells in drug delivery studies.</a> Materials Science and Engineering: C. 109, 110613 (2020).</li><li>Ojansivu, M., 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=Wood-based+nanocellulose+and+bioactive+glass+modified+gelatin-alginate+bioinks+for+3D+bioprinting+of+bone+cells.">Wood-based nanocellulose and bioactive glass modified gelatin-alginate bioinks for 3D bioprinting of bone cells.</a> Biofabrication. 11 (3), 035010 (2019).</li><li>Jonsson, M., 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=Neuronal+networks+on+nanocellulose+scaffolds.">Neuronal networks on nanocellulose scaffolds.</a> Tissue Engineering Part C: Methods. 21 (11), 1162-1170 (2015).</li><li>Samulin Erdem, 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=Cellulose+nanocrystals+modulate+alveolar+macrophage+phenotype+and+phagocytic+function.">Cellulose nanocrystals modulate alveolar macrophage phenotype and phagocytic function.</a> Biomaterials. 203, 31-42 (2019).</li><li>Menas, A. L., 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=Fibrillar+vs+crystalline+nanocellulose+pulmonary+epithelial+cell+responses:+Cytotoxicity+or+inflammation.">Fibrillar vs crystalline nanocellulose pulmonary epithelial cell responses: Cytotoxicity or inflammation.</a> Chemosphere. 171, 671-680 (2017).</li><li>Halova, I., Draberova, L., Draber, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+chemotaxis+chemoattractants+and+signaling+pathways.">Mast cell chemotaxis chemoattractants and signaling pathways.</a> Frontiers in Immunology. 3, 1-19 (2012).</li><li>Groll, 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=A+definition+of+bioinks+and+their+distinction+from+biomaterial+inks.">A definition of bioinks and their distinction from biomaterial inks.</a> Biofabrication. 11 (1), 013001 (2019).</li><li>Schwab, A., 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=Printability+and+shape+fidelity+of+bioinks+in+3D+bioprinting.">Printability and shape fidelity of bioinks in 3D bioprinting.</a> Chemical Reviews. 120 (19), 11028-11055 (2020).</li><li>Jungst, T., Smolan, W., Schacht, K., Scheibel, T., Groll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+molecular+design+criteria+for+3D+printable+hydrogels.">Strategies and molecular design criteria for 3D printable hydrogels.</a> Chemical reviews. 116 (3), 1496-1539 (2016).</li><li>Sasaki, D. T., Dumas, S. E., Engleman, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+of+viable+and+non-viable+cells+using+propidium+iodide+in+two+color+immunofluorescence.">Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence.</a> Cytometry. 8 (4), 413-420 (1987).</li><li>Usov, I., 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=Understanding+nanocellulose+chirality+and+structure-properties+relationship+at+the+single+fibril+level.">Understanding nanocellulose chirality and structure-properties relationship at the single fibril level.</a> Nature Communications. 6 (1), 7564 (2015).</li></ol>

This work was supported by the National Research Council Canada and Alberta Innovates.

The fabrication of 3D biomimetic tissues requires the successful amalgamation of the bioink, which mimics components of the extracellular matrix, with the cellular component(s) to create physiological analogs of in vivo tissues. This necessitates the use of primary cells, and not transformed cells, when fabricating physiological biomimetic tissues. Primary immunological cells, such as mast cells, however, are particularly susceptible to cytotoxic effects and phenotypic changes that may be elicited by the bioink matrix itself, which is undesirable. Therefore, the ability to rapidly assess the effects of the candidate biomaterial ink on the viability and phenotype (surface receptor expression) of the mast cells is highly advantageous, especially prior to 3D bioprinting of complex tissues containing different cell types embedded within the bioink matrix, which is costly and time-consuming.
The approach of this protocol involves culturing BMMCs, an example of primary mast cells, on the surface of preformed candidate hydrogel bioink substrates (1-12.5% CNC embedded in agarose), which permits the easy retrieval of the BMMCs for subsequent analysis of cell viability and surface receptor expression by flow cytometry. The isolation of bone marrow from mouse femur and tibia requires precision and attention to detail due to the fragility and small size of these bones. After successful isolation and deposition of the bone marrow into cell culture medium, it is essential to maintain the cell culture with 30 ng/mL mouse recombinant IL-3 continuously for 4 weeks, which ensures sustained stimulation to differentiate the hematopoietic progenitor cells into mast cells that are double-positive for the Kit (CD117) and Fc&#949;RI cell surface receptors.
Flow cytometric analysis of mast cells for the surface expression of Kit (CD117) and Fc&#949;RI receptors should employ isotype antibody controls to assist with differentiating non-specific background signal from the specific signals of the antibodies used to target these receptors, as demonstrated in Figure 4A(ii),B(ii). It is also essential to gate on a well-defined mast cell population to exclude cell debris as shown in Figure 4A(i) and 4B(i), which can also non-specifically bind antibodies. Gentle micropipetting should be used when retrieving the BMMCs from the surface of the bioink hydrogel substrates to reduce the shear forces exerted on the cells, which could damage the cell membrane and result in artificially elevated staining with PI. The PI-stained BMMCs should be analyzed by flow cytometry immediately after the staining incubation period ends as the PI staining medium, which is based on PBS-0.5% w/v BSA, is not intended to sustain cells for extended periods of time outside of cell culture medium. Because PI only permeates cells with compromised cell membranes, it is able to stain necrotic cells with high selectivity and sensitivity. However, PI is unable to detect apoptotic cells, which maintain intact cell membranes.
The PI-exclusion flow cytometric assay described in this protocol may be augmented with Annexin-V-fluorescein isothiocyanate (FITC), which specifically binds to the phospholipid, phosphatidylserine, that is translocated to the outer leaflet of the cell membrane of apoptotic cells. However, compensation is necessary to account for the fluorescence emission bleed-through of FITC into the detector channel used to acquire fluorescence emission from PI and vice versa. In preparing the bioprinter for 3D bioprinting from the provided 24-well plate G-code, it is essential that the starting point calibration be performed accurately, whereby the x- and y-coordinates of the center of well D1 are recorded, and the G-code file is updated with these x- and y-coordinates on line 1. If these initial calibration steps are not performed accurately, the printheads will not move to the correct starting point at the commencement of printing. Correct calibration of the z-axis is equally important as failure to do so can result in the nozzle of the printhead colliding with the printbed or the 24-well plate at the commencement of printing.
The schematic diagram of the 5 x 5 x 1 mm 2-layer rectilinear grid construct (Figure 5C(iii)), illustrates the presence of pores between the extruded bioink filaments that form the substrate. The size of the pores and diameter of the filaments are dependent on three parameters: (i) the travel speed of the print nozzle, (ii) the extrusion pressure applied to the bioink within the cartridge, and (iii) the print nozzle diameter. Bioink substrates with large filament diameters and small pores can be printed using a slower print nozzle speed, higher extrusion pressure (&#62;12 kPa), and larger diameter print nozzle (22 G). Conversely, a faster print nozzle speed, lower extrusion pressure (&#60;12 kPa), and smaller diameter print nozzle (27 G) will result in substrates with finer filaments and larger pores. However, an insufficiently high extrusion pressure will result in inconsistent clumps of bioink being extruded instead of continuous filaments, which will adversely affect the quality and structural integrity of the 3D bioprinted substrate.
The CNC/agarose/D-mannitol candidate bioink presented in this protocol undergoes thermal gelation when agarose cools down to room temperature. In contrast, the commercial bioink used for 3D bioprinting in this protocol undergoes ionic crosslinking with CaCl2 due to the inclusion of sodium alginate in the bioink formulation. It is, therefore, necessary to immerse the bioprinted FNC/alginate/D-mannitol scaffolds in 50 mM CaCl2 solution after bioprinting to facilitate crosslinking (gelation) of the substrate. Omission of the ionic crosslinking step with CaCl2 will result in the dissolution of the FNC/alginate/D-mannitol scaffolds when they are immersed in cell culture medium. The foremost limitation of the CNC/agarose/D-mannitol bioink formulation in its application in actual 3D bioprinting is the exceptionally high melting temperature of agarose (90-95 &#176;C). Even though the printheads of the 3D bioprinter used in this study can reach a maximum temperature of 130 &#176;C, the extruded filaments of CNC/agarose/D-mannitol will likely disperse rapidly as successive layers are printed to construct the substrate. This limitation of the CNC/agarose/D-mannitol bioink formulation can be circumvented by either printing the CNC/agarose/D-mannitol bioink directly onto a cooled printbed to accelerate gelation, or substituting agarose with sodium alginate, which can be extruded at room temperature and undergoes rapid ionic crosslinking with 50 mM CaCl2 within 5 min.
This protocol offers a reliable approach to rapidly screen and exclude bioink formulations that exhibit poor biochemical compatibility with sensitive immunological cells, such as mast cells, that should not be subjected to pneumatic-extrusion 3D bioprinting. Furthermore, this protocol is cost-effective as it requires relatively low quantities of cells to perform a multiplexed screening assay. In contrast, pre-formulated bioink-cell mixtures need to be bioprinted at very high cell densities (&#62;107 cells/mL), which can prove to be costly and time-consuming to perform a biocompatibility screening assay especially when culturing primary cells that are dependent on expensive recombinant growth factors.

The recent interest in 3D culture systems and 3D bioprinting has focused attention on hydrogels and hydrogel composites. These composites serve as viscous yet porous biomimetics and can be composed of up to 99% water content by weight, which is comparable to biological tissues1,2,3. These features of hydrogel composites thereby permit the growth of cells without affecting their viability and function. One such composite is crystalline nanocellulose (CNC), which has been used as a reinforcing material in hydrogel composites, cell scaffolds in the development of biomaterial implants, and in two-dimensional (2D) and 3D in vitro cell culture4,5. For the most part, matrices composed of CNC are not overtly cytotoxic to human corneal epithelial cells6, intestinal epithelial cells7, human bone marrow-derived mesenchymal stem cells8, or neuron-like cells9. However, metabolic activity and proliferation of human bone marrow-derived mesenchymal stem cells decreases in correlation with the increased viscosity of wood-based nanocellulose composites, suggesting that the composition of the matrix must be carefully tested for its deleterious effects on cell functions8.
Similarly, CNC can induce inflammatory responses in macrophages upon internalization, which could have serious consequences in 3D immune cell culture systems10,11. In fact, there is very little data available on how CNC may influence other immune cell responses, particularly allergic inflammatory responses that are initiated by mast cells. Mast cells are granulated leukocytes that express the high-affinity IgE receptor, FceRI, responsible for activating inflammatory responses to allergens. Their proliferation and differentiation are dependent-upon stem cell factor (SCF), which binds the tyrosine receptor, Kit. Mast cells are derived from bone marrow progenitor cells that enter the circulation and subsequently migrate peripherally to disperse ubiquitously in all human tissues12. As mast cells function in a 3D tissue environment, they are an ideal immune cell candidate for studying immunological processes in in vitro 3D tissue models. However, to date, there is no viable in vitro 3D tissue model containing mast cells.
Due to the highly sensitive nature of mast cells and their propensity to elicit pro-inflammatory responses to external stimuli, careful consideration of the 3D matrix constituents and the bioprinting method of introducing mast cells into the 3D scaffold is required, as discussed further. Tissue constructs can be biofabricated from two broad categories of biomaterials, i.e., bioinks and biomaterial inks. The distinction lies in the fact that bioinks are cell-laden hydrogel composites, whereas biomaterial inks are hydrogel composites that are devoid of cells, as defined by Groll et al.13,14. Hence, 3D constructs printed with bioinks contain cells pre-embedded within the hydrogel matrix, whereas 3D constructs printed with biomaterial inks need to be seeded with cells post-printing. The biofabrication of culture scaffolds from hydrogel-based bioinks/biomaterial inks is most commonly performed using extrusion 3D bioprinters, which extrude the bioink/biomaterial ink through a microscale nozzle under pressure via either a pneumatically or mechanically driven piston14. Extrusion bioprinters fabricate 3D scaffolds by depositing the bioink in 2D cross-sectional patterns that are sequentially stacked upon each other in a &#39;bottom-up&#39; approach.
To be compatible with extrusion bioprinting, the hydrogel-based bioink/biomaterial ink must possess thixotropic (shear-thinning) properties, whereby the constituent hydrogel polymers of the bioink/biomaterial ink flow like a fluid through a microchannel nozzle when subjected to shear stress, but revert to a viscous, gel-like state upon removal of the shear stress15. Due to their high water content, the polymers of hydrogel-based bioinks/biomaterial inks must be crosslinked, either physically or covalently, to maintain the architecture and structural integrity of the 3D bioprinted structure. In the case of cell-laden bioinks, the cells are directly subjected to chemical stresses during the crosslinking process. The process of extruding cells encapsulated within the bioink hydrogel matrix also subjects the cells to shear stress, which can lead to reduced viability and/or cell death. Once the 3D tissue model has been bioprinted, it is difficult to discriminate between the levels of cytotoxicity elicited by the hydrogel matrix itself and the extrusion and crosslinking processes, respectively. This is particularly challenging in the context of 3D scaffolds where the cells are pre-embedded within the hydrogel matrix, thus making it difficult to remove the cells for subsequent analyses, which would be detrimental to the viability of mast cells.
A gentler approach to generating 3D tissue constructs containing mast cells involves seeding the cells into pre-printed, porous biomaterial ink 3D scaffolds from a cell culture suspension, which leverages the innate ability of mast cells to migrate from the circulation into peripheral tissues. The benefits of this cell seeding approach are two-fold: (i) the mast cells are not subjected to shear and chemical stresses from the extrusion and crosslinking processes, respectively, and (ii) the cells can be easily removed from the 3D scaffold after exposure by gentle washing for analysis without adversely affecting their viability. The additional benefit of seeding and analyzing the cell viability of mast cells on 3D bioprinted, porous hydrogel scaffolds as opposed to 2D hydrogel discs is that the 3D bioprinted hydrogel scaffolds recapitulate microscale topographical features of in vivo tissues, which are not present in bulk, 2D planar hydrogel discs. This approach is a suitable, rapid, and cost-effective approach to determine the potentially catastrophic cytotoxic effects of candidate bioink hydrogel matrices on mast cells, as well as other immunological cells, prior to investment in costly 3D tissue engineering experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid (glacial)</td>
			<td>Sigma Aldrich</td>
			<td>AX0074-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose (OmniPur)</td>
			<td>EMD Millipore Corporation</td>
			<td>2125-500GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Armenian Hamster IgG Isotype Control, APC (Clone: eBio299Arm)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-4888-82</td>
			<td></td>
		</tr>
		<tr>
			<td>B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Fisher Scientific</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Nicotinamide adenine dinucleotide sodium salt (NAD)</td>
			<td>Sigma Aldrich</td>
			<td>N0632-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 5 mL Syringe (Luer-Lok Tip)</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 26G x 1/2 in</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLite 24 Well Multidish</td>
			<td>Thermo Fisher Scientific</td>
			<td>930-186</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLite 96 Well Multidish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130-188</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLite 175 cm2 Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130-191</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet Class II</td>
			<td>Microzone Corp., Canada</td>
			<td>BK-2-6-B3</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, Fraction V (OmniPur)</td>
			<td>EMD Millipore Corporation</td>
			<td>2930-100GM</td>
			<td></td>
		</tr>
		<tr>
			<td>C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>CD117 (c-Kit) Monoclonal Antibody, PE (Clone: 2B8)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-1171-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK BIOINK (3 x 3 mL Cartridge)</td>
			<td>CELLINK LLC</td>
			<td>IK1020000303</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK CaCl2 Crosslinking Agent - Sterile Bottle 1 x 60 mL</td>
			<td>CELLINK LLC</td>
			<td>CL1010006001</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK Empty Cartridges 3cc with End and Tip Caps</td>
			<td>CELLINK LLC</td>
			<td>CSC0103000102</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK HeartWare for PC</td>
			<td>CELLINK LLC</td>
			<td>Version 2.4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK INKREDIBLE+ 3D BIOPRINTER</td>
			<td>CELLINK LLC</td>
			<td>S-10003-001</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK Sterile Standard Conical Bioprinting Nozzles 22G</td>
			<td>CELLINK LLC</td>
			<td>NZ4220005001</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK Sterile Standard Conical Bioprinting Nozzles 25G</td>
			<td>CELLINK LLC</td>
			<td>NZ4250005001</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLINK Sterile Standard Conical Bioprinting Nozzles 27G</td>
			<td>CELLINK LLC</td>
			<td>NZ4270005001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit II (XTT) (Roche)</td>
			<td>Sigma Aldrich</td>
			<td>11465015001</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (Benchtop)</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar&#160;96 Well Clear Flat-Bottom Non-Treated PS Microplate</td>
			<td>Sigma Aldrich</td>
			<td>CLS3370</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Binder GmbH, Germany</td>
			<td>9040-0113</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>A00-1-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>D</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-mannitol (MilliporeSigma Calbiochem)</td>
			<td>Fisher Scientific</td>
			<td>44-390-7100GM</td>
			<td></td>
		</tr>
		<tr>
			<td>F</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Polystyrene Conical Tubes, Sterile</td>
			<td>Corning</td>
			<td>352095</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Polystyrene Conical Tubes, Sterile</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>FceR1 alpha Monoclonal Antibody, APC (Clone: MAR-1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-5898-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), qualified, heat inactivated</td>
			<td>Thermo Fisher Scientific</td>
			<td>12484028</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>Becton Dickinson &#38; Co. USA</td>
			<td>Version 10.6.2</td>
			<td></td>
		</tr>
		<tr>
			<td>G</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, LLC</td>
			<td>Version 8.4.3</td>
			<td></td>
		</tr>
		<tr>
			<td>H</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer (Improved Neubauer 0.1 mmm deep levy)</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Sodium Salt</td>
			<td>Fisher Scientific</td>
			<td>BP410-500</td>
			<td></td>
		</tr>
		<tr>
			<td>I</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodonitrotetrazolium chloride (INT)</td>
			<td>Sigma Aldrich</td>
			<td>I10406-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium L-lactate</td>
			<td>Sigma Aldrich</td>
			<td>L2250-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>M</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids 100 mL 100x (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Methoxy-5-methylphenazinium methyl sulfate (MPMS)</td>
			<td>Sigma Aldrich</td>
			<td>M8640</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes (1.7 mL clear)</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes (2.0 mL clear)</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ Academic (for producing MilliQ ultrapure water)</td>
			<td>Millipore</td>
			<td>ZMQS60001</td>
			<td></td>
		</tr>
		<tr>
			<td>N</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow 90 mm Filter Unit (0.2 mm Pore size, 500 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Syringe filter (0.2 mm PES, 25 mm)</td>
			<td>Thermo Fisher Scientific</td>
			<td>725-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>P</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin 100 mL (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4, No Calcium/Magnesium, 500 mL (Gibco)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide, 1.0 mg/mL (Invitrogen)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>R</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PE (Clone: eB149/10H5)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-3</td>
			<td>PeproTech, Inc.&#160;</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium 1X + 2.05 mM L-Glutamine (HyClone)</td>
			<td>GE Healthcare</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>S</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sarstedt 96 well round base PS transparent micro test plate (82.1582.001)</td>
			<td>Fisher Scientific</td>
			<td>NC9913213</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide, 500 g</td>
			<td>Fisher Scientific</td>
			<td>BP922I-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM) 100X (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>T</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base (2-amino-2(hydroxymethyl)-1,3-propanediol)</td>
			<td>Sigma Aldrich</td>
			<td>252859</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution (0.4%, for microscopy)</td>
			<td>Sigma Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr>
			<td>V</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VARIOSKAN LUX Microplate Spectrophotometer (Type: 3020)</td>
			<td>Thermo Fisher Scientific</td>
			<td>VLBL00D0</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a crystalline nanocellulose embedded agarose biomaterial ink for bone marrow-derived mast cell culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

One of the most crucial characteristics of a successful biomaterial ink or culture substrate is that of biocompatibility. Primarily, the substrate must not induce cellular death. There are several microtiter-based and flow cytometric methods of quantifying cell viability and necrosis; however, these methods are not amenable to analyzing cells embedded within a hydrogel matrix. In this protocol, the above mentioned limitation is circumvented by seeding the BMMCs onto the hydrogel substrate or bioprinted scaffold. After a specific incubation period (6-48 h in this study), the BMMCs are easily harvested by micropipetting without the need for mechanical disruption or hydrolysis of the hydrogel substrate or bioprinted scaffold, which would otherwise cause physical damage to the cells. The viability of the BMMCs is then rapidly analyzed using the PI permeability method via flow cytometry. PI is a membrane-impermeant dye that is excluded from viable cells with intact cell membranes and fluoresces only upon intercalating between the bases pairs of DNA16.
As such, only cells with compromised cell membranes are permeable to PI and therefore, will fluoresce. PI permeability analysis demonstrated no changes in BMMC viability when they are cultured on the CNC/agarose/D-mannitol substrate. In fact, none of the CNC concentrations tested (up to 12.5% w/v) elicited any adverse effects on BMMC viability as compared to the BMMCs cultured in the absence of the CNC/agarose/D-mannitol hydrogel substrates (Figure 3B,C). It is also vital that the bioink or culture substrate does not alter the morphology of the cells. Based on the flow cytometric FSC vs. SSC plots, the hydrogel substrate with the highest CNC concentration (12.5% w/v) did not alter the native size (FSC) or granularity (SSC) of the BMMCs as compared to the BMMCs cultured in the absence of the CNC/agarose/D-mannitol hydrogel substrates (Figure 3A(i), (ii)).
Another important characteristic of a biomaterial ink or culture substrate is that it must possess the ability to maintain the differentiation and phenotype of the cells it supports. Two of the most quintessential biomarkers of mast cells are the high-affinity IgE receptor, Fc&#949;RI, which facilitates BMMC responses to antigens and the stem cell factor receptor, Kit (CD117), which is required for mast cell survival and differentiation. Mature mast cells are defined by the expression of these two surface receptors, and for a bioink substrate to maintain them in culture, it must not significantly modify the expression of these two receptors. The CNC/agarose/D-mannitol substrate appeared to increase Fc&#949;RI and Kit expression at CNC concentrations &#8805; 2.5% (Figure 4A(iii),B(iii)). Interestingly, the elevated Fc&#949;RI and Kit expression levels remained relatively consistent between 2.5% and 12.5% CNC, which is indicative of a plateau effect and suggests an effect that is not dependent upon the concentration of CNC, but on some other parameter of the hydrogel composite.
The 3D bioprinted hydrogel biomaterial ink scaffolds generated in this study consist of fibrillar nanocellulose (FNC), instead of crystalline nanocellulose, and sodium alginate as the gelator, instead of agarose. Fibrillar nanocellulose exhibits distinct nanoscale topographical features as compared to CNC17&#160;which, in addition to the microscale architecture of the 3D bioprinted FNC hydrogel bioink substrates, could potentially affect the viability of the BMMCs. Following the 3D bioprinting and ionic crosslinking of FNC/alginate/D-mannitol bioink scaffolds (Figure 6), BMMCs were cultured either alone or on the 3D bioprinted hydrogel scaffolds for 6, 18, 24, and 48 h, respectively, in order to assess the dynamic changes in the viability of the BMMCs in response to the FNC/alginate/D-mannitol scaffolds, if any, over extended periods of time. The XTT assay indicated that the metabolic activity of the BMMCs cultured on the 3D bioprinted hydrogel scaffolds remained relatively consistent at ~100% across all tested time points when compared to BMMCs cultured alone (Figure 7A). The lysis of cells results in the release of LDH into the cell culture medium, which the LDH assay detects as a function of its oxido-reductive enzymatic activity.
The BMMCs cultured on the 3D bioprinted hydrogel scaffolds exhibited a gradual time-dependent increase in LDH release when compared to BMMCs cultured alone; however, this trend had a non-significant standard deviation of less than 9% (Figure 7B). PI staining of the BMMCs cultured on the 3D bioprinted hydrogel scaffolds revealed no significant changes in viability compared to BMMCs cultured alone at each time point (Figure 7C).&#160;Notably, the MFI of PI-stained BMMCs cultured on the 3D bioprinted hydrogel scaffolds remained relatively consistent (PI-MFI of 7000) across all time points and was similar to the BMMCs cultured on the CNC/agarose/D-mannitol substrates, which exhibited a consistent PI-MFI of 7000 across all CNC concentrations (2.5-12.5%). Collectively, these data demonstrate that the FNC/alginate/D-mannitol hydrogel scaffolds do not adversely affect the viability of the BMMCs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62519/62519fig01.jpg" /> 
Figure 1:&#160;Anatomy of mouse leg, depicting the tibia and femur from which bone marrow is isolated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig01large.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/62519/62519fig02.jpg" /> 
Figure 2:&#160;Preparation of the CNC/agarose/D-mannitol hydrogel substrate. (A) Schematic of CNC/agarose/D-mannitol hydrogel substrate preparation protocol. (B) CNC/agarose/D-mannitol preparations (0, 1, 2.5, 5, 10, and 12.5% (w/v) CNC in PBS/agarose/D-mannitol) were loaded onto a 24-well plate in quadruplicate as illustrated. Abbreviations: PBS = phosphate-buffered saline; CNC = crystalline nanocellulose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig02large.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/62519/62519fig03.jpg" /> 
Figure 3:&#160;Flow cytometric analysis of BMMC viability via propidium Iodide exclusion following incubation on CNC/agarose/D-mannitol substrates. BMMCs were removed from the CNC/agarose/D-mannitol hydrogel substrates, washed twice, resuspended in PBS-0.5% w/v BSA, stained with PI for 1 h at 4 &#176;C, and analyzed by flow cytometry (n=4). A total of 20,000 cells per sample were acquired including PI fluorescence emission detection in the PE channel. Data analysis was performed using flow cytometry analysis software. (A) Forward scatter (x-axis) versus side scatter (y-axis) dot plot analysis of total cell population from (i)&#160;untreated control BMMC (0% CNC/agarose/D-mannitol) and (ii) BMMCs cultured on 12.5% CNC/agarose/D-mannitol, depicting the gated cell population used for data analysis. (B) Histogram overlay profile of gated cells (unstained or stained with PI), from untreated control BMMCs (0% CNC/agarose/D-mannitol) and BMMCs cultured on 12.5% CNC/agarose/D-mannitol. (C) BMMCs were cultured on different CNC/agarose/D-mannitol substrates (1-12.5% (w/v) CNC) for 18 h, and cell viability was determined by flow cytometric analysis of PI-stained cells. Graphical representation of PI MFIs for BMMCs incubated on different CNC/agarose/D-mannitol substrates (1-12.5% (w/v) CNC) relative to BMMCs that were untreated and stained with PI. Abbreviations: BMMCs = bone marrow-derived mast cells; CNC = crystalline nanocellulose; PBS = phosphate-buffered saline; BSA = bovine serum albumin; PI = propidium iodide; PE = phycoerythrin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig03large.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/62519/62519fig04.jpg" /> 
Figure 4:&#160;Flow cytometric analysis of Fc&#949;RI and Kit (CD117) cell surface receptor expression. BMMCs were cultured either in the absence or presence of CNC/agarose/D-mannitol substrates for 18 h, removed, and analyzed for receptor expression. Data is representative of 4 replicates. Forward scatter (x-axis) versus side scatter (y-axis) dot plot of the total cell population in the untreated BMMC sample (0% CNC/agarose/D-mannitol), stained with (A)(i) the isotype control antibody-APC or (B)(i) the isotype control antibody-PE, and the gated cell population used for data analysis.&#160;Histogram overlay profiles of gated BMMCs (stained with isotype control antibody or (A)(ii) anti-Fc&#949;RI-APC antibody or (B)(ii) anti-Kit-PE antibody), incubated either alone (0% CNC/agarose/D-mannitol) or on 12.5% CNC/agarose/D-mannitol substrates. BMMCs were cultured for 18 h on different CNC/agarose/D-mannitol substrates (1-12.5% (w/v) CNC) followed by Fc&#949;RI and Kit surface receptor expression analysis, respectively, via flow cytometry (n=4). Graphical representation of MFIs of BMMCs stained with (A)(iii)&#160;anti-Fc&#949;RI-APC or (B)(iii)&#160;anti-Kit-PE antibodies, respectively, following culture on different CNC/agarose/D-mannitol substrates (1-12.5% (w/v) CNC) relative to cells that were untreated (0% CNC) and stained similarly. Abbreviations: CNC = crystalline nanocellulose; APC = allophycocyanin; PE = phycoerythrin; BMMCs = bone marrow-derived mast cells; MFI = mean fluorescence intensities. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62519/62519fig05.jpg" /> 
Figure 5: 3D bioprinter equipment and consumables required to bioprint 5 x 5 x 1 mm 2-layer rectilinear bioink hydrogel scaffolds in a 24-well plate format. (A)(i) A pneumatic-extrusion 3D bioprinter with the printhead assembly position at its resting position upon completing the homing of its x-y-z axes. (A)(ii) Starting point calibration of printhead 1 with a bioink cartridge installed and placement of the print nozzle directly over the middle of well D1 in the x-y-z dimensions. (A)(iii) Position of PH1 following z-axis calibration and the extrusion pressure of PH1 set to 12 kPa. (B)(i)&#160;Bioink cartridge (3 mL) containing NFC/Alginate/D-mannitol biomaterial ink formulation. (B)(ii) Droplet dispenser of 50 mM CaCl2 crosslinking solution. (C)(i)&#160;Schematic representation of a print layout from a G-code file encoding the printing of 5 x 5 x 1 mm 2-layer rectilinear bioink hydrogel scaffolds in all wells of a 24-well plate. (C)(ii) Schematic representation of a print layout from a G-code file encoding the printing of 5 x 5 x 1 mm 2-layer rectilinear bioink hydrogel scaffolds only in wells A1-3, B1-3, C1-3, and D1-3 of a 24-well plate. (C)(iii)&#160;Expanded view of the 5 x 5 x 1 mm 2-layer rectilinear bioink hydrogel scaffold pattern in the slicing program, Slic3r. (D)Topview of actual 3D bioprinted 5 x 5 x 1 mm 2-layer rectilinear bioink hydrogel scaffolds in a 24-well plate format immersed in PBS. Abbreviations: 3D = three-dimensional; PH1 = printhead 1; NFC = nanofibrillar cellulose; G-code = geometric code; PBS = phosphate-buffered saline. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62519/62519fig06.jpg" /> 
Figure 6:&#160;Schematic diagram depicting workflow of 3D bioprinting and BMMC culture on FNC/alginate/D-mannitol hydrogel scaffolds. 3D bioprinting of hydrogel scaffolds with FNC/Alginate/D-mannitol bioink from a G-code file encoding a 5 x 5 x 1 mm 2-layer grid pattern, crosslinking of the hydrogel scaffolds with CaCl2, and culturing of BMMCs on the crosslinked FNC/Alginate/D-mannitol hydrogel constructs. Abbreviations: 3D = three-dimensional; 2D = two-dimensional; G-code = geometric code; FNC = fibrillar nanocellulose; BMMCs = bone-marrow-derived mast cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62519/62519fig07.jpg" /> 
Figure 7: Flow cytometric (PI) and microtiter (XTT and LDH) assays of BMMC viability following incubation on FNC/alginate/D-mannitol scaffolds. (A) Cell proliferation (XTT) metabolic assay data for BMMCs cultured on FNC/Alginate/D-mannitol bioink substrates for 6, 18, 24, and 48 h, respectively. Values are presented as a percentage of the XTT metabolic data for BMMCs cultured alone for 6, 18, 24, and 48 h, respectively. Error bars indicate standard deviation (n=3). (B) LDH enzyme release assay data for BMMCs cultured on FNC/Alginate/D-mannitol bioink scaffolds for 6, 18, 24, and 48 h, respectively. Values are presented as fold-changes relative to the LDH enzyme released by BMMCs cultured alone for 6, 18, 24, and 48 h, respectively. Error bars indicate standard deviation (n=3). (C) Flow cytometric data of PI-stained BMMCs that were cultured either alone or on FNC/Alginate/D-mannitol bioink scaffolds for 6, 18, 24, and 48 h, respectively. Error bars indicate standard deviation (n=3). Abbreviations: LDH = lactate dehydrogenase; BMMCs = bone marrow-derived mast cells; FNC = fibrillar nanocellulose; PI = propidium iodide; MFI = mean fluorescence intensity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/62519fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.1.1. 500 mL bottle of RPMI (HyClone, GE Healthcare, USA)</td>
		</tr>
		<tr>
			<td>1.1.2. 4 mM L-glutamine (Gibco, Waltham Massachusetts, USA)</td>
		</tr>
		<tr>
			<td>1.1.3. 50 mM &#946;-Mercaptoethanol (Fisher Scientific, Hampton, New Hampshire, USA)</td>
		</tr>
		<tr>
			<td>1.1.4. 1 mM Sodium Pyruvate (Gibco)</td>
		</tr>
		<tr>
			<td>1.1.5. 100 U/mL penicillin (Gibco)</td>
		</tr>
		<tr>
			<td>1.1.6. 100 &#181;g/mL streptomycin (Gibco)</td>
		</tr>
		<tr>
			<td>1.1.7. 0.1 mM MEM non-essential amino acids (Gibco)</td>
		</tr>
		<tr>
			<td>1.1.8. 25 mM HEPES (Fisher)</td>
		</tr>
		<tr>
			<td>1.1.9. 10% heat inactivated FBS (Gibco)</td>
		</tr>
		<tr>
			<td>1.1.10. 30 ng/mL mouse recombinant IL-3 (Peprotech, Rocky Hill, New Jersey, USA)&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Complete RPMI-1640 media supplements.
Supplemental File 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/Supplemental File 1_24-well plate_6x4Grid_5x5-2Layer.gcode" target="_blank">Please click here to download this File.</a>
Supplemental File 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/Supplemental File 2_24-well plate_3x4Grid_5x5-2Layer.gcode" target="_blank">Please click here to download this File.</a>
Supplemental File 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62519/Supplemental File 3_LDH Assay.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture at JoVE.com

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

fabrication-crystalline-nanocellulose-embedded-agarose-biomaterial

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.0K Views.  National Research Council Canada. This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (FcεRI).

Video: Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.