Name: fabrication of a crystalline nanocellulose embedded agarose biomaterial ink for bone marrow-derived mast cell culture
Uploaded: 2021-05-11T14:00:00
Description: 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

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.

NOTE: This protocol is composed of five sections: (1) isolation of mouse bone marrow and differentiation of mouse bone marrow-derived mast cells (BMMCs), (2) fabrication of CNC/agarose/D-mannitol hydrogel substrates in a 24-well system and culture of BMMCs on the substrates, (3) removal of BMMCs from the CNC/agarose/D-mannitol hydrogel substrates and analysis of viability and biomarker expression using flow cytometry, (4) 3D bioprinting of hydrogel scaffolds from a commercially available fibrillar nanocellulose (FNC)/sod...

<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. 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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. 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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 ...

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 ...

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 ...

Research

JoVE Journal

Bioengineering

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 ...

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 ...

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 ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

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 ...

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 ...

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 ...

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

Fabrication of Inverted Colloidal Crystal Polyethylene 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 ...

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 ...

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 ...