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Materials

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Bioengineering

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

Published: May 11th, 2021

DOI:

10.3791/62519

1Nanotechnology Research Center, National Research Council Canada, 2Department of Medical Microbiology and Immunology, University of Alberta

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).

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 (FcεRI) 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.

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 imp....

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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.......

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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 .......

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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 .......

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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.

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

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