The authors would like to acknowledge the National Health and Medical Research Council (Grant no. GNT1111694 and GNT1141602) and the Australian Research Council (Grant no. FT180100417, FL150100060, and CE14100036). The authors would like to acknowledge the Biomedical Imaging Facility at the University of New South Wales. Figures were created with Biorender.com, Adobe Photoshop, and Adobe Illustrator and have been exported under a paid subscription.

1. Bone-ink fabrication
<ol>
	<li>Synthesis of &#945;-tricalcium phosphate
	<ol>
		<li>Weigh out calcium hydrogen phosphate (CaHPO4) and calcium carbonate (CaCO3) powders in a 3:2 Ca:P molar ratio. Using a spatula, thoroughly homogenize the two powders.</li>
		<li>Add the calcium hydrogen phosphate-calcium carbonate powder mixture to a zirconia crucible such that it is no more than 75% full. 
		NOTE: To avoid contamination, use a new crucible or a crucible previously used to make the same material. To clean, rinse with 100% ethanol and air-dry in a fume hood until completely dry before adding the powders.</li>
		<li>Transfer the crucible to a furnace. Heat to 1,400 &#176;C at a rate of 5 &#176;C/min and hold for 3 h.</li>
		<li>Quench the reaction by removing the crucible from the furnace and leave it atop a refractory block. Allow it to cool completely before handling. 
		NOTE: Use crucible tongs of an appropriate length and ensure adequate heat protection.</li>
		<li>Use a mortar and pestle to break and grind the &#945;-TCP cake such that the resulting granules have a maximum size of 200 &#181;m. 
		NOTE: Use a standard stainless-steel sieve to ensure correct particle size.</li>
		<li>Further grind the granules using a planetary mill in two stages. First, add 3 mm yttria-stabilized zirconia balls in a weight ratio of 8:1 balls:powder, then 100% ethanol in a weight ratio of 3:1 ethanol:powder. Secure the lid and grind for 2 h at 180 rpm.</li>
		<li>Collect the suspension and separate the balls, using 100% ethanol for washing.</li>
		<li>Dry the suspension in an oven at 120 &#176;C for 24 h.</li>
		<li>Add the dried powder to milling jars with 1 mm zirconia balls and 100% ethanol in the same weight ratios as in the first stage. Grind for 2 h at 180 rpm, separate, and dry. 
		NOTE: The entire synthesis procedure is represented in Figure 1A.</li>
	</ol>
	</li>
	<li>Formulation of bone-ink
	<ol>
		<li>To make the bone-ink, add 2 g of &#945;-TCP powder to a ball mill jar that contains 630 &#181;L of glycerol and 130 &#181;L of polysorbate 80 whilst continuously stirring with a spatula.</li>
		<li>Add 100 mg of ammonium phosphate dibasic ((NH4)2HPO4, APD) and stir to combine. 
		NOTE: Excessive residue of liquid phases left on the spatula will result in a ratio imbalance of the ink components and, hence, the kinetics of setting.</li>
		<li>Add a 25 mm zirconia ball, secure the lid, and place it inside a planetary mill for 60 min at 180 rpm, pausing halfway to scrape down the sides of the jar with a spatula.</li>
		<li>Using a spatula, load the ink into a 1 mL syringe. Wrap adequately to avoid contact with moisture. Store at &#8722;20 &#176;C if not used immediately.</li>
	</ol>
	</li>
	<li>Bone-ink microstructure characterization
	<ol>
		<li>Print the bone-ink in deionized water and allow to set for 5 min.</li>
		<li>Wash the sample 3x with 100% ethanol and allow it to dry completely.</li>
		<li>Coat with a thin layer (15 nm thickness) of gold.</li>
		<li>Capture micrographs using a field emission scanning electron microscope at an acceleration voltage of 5 kV.</li>
	</ol>
	</li>
</ol>
2. Fabrication of microgel suspensions for printing
<ol>
	<li>Synthesis of GelMA 
	NOTE: This procedure has been tested for batch sizes consisting of 10 g and 20 g of gelatin. This method details measurements for a batch using 10 g.
	<ol>
		<li>Make a 10% w/w solution of gelatin type A (porcine, Bloom strength 300) in 1x phosphate-buffered saline (PBS) by weighing out 10 g of gelatin and adding it to a conical flask with 90 mL of PBS. Heat to 50 &#176;C while stirring until the gelatin is completely dissolved.</li>
		<li>Add 5.796 mL of methacrylic anhydride. Place a rubber cap on the conical flask and continue stirring in the dark at 50 &#176;C for 90 min. 
		CAUTION: Methacrylic anhydride is toxic if inhaled or swallowed and is a skin and eye irritant. Handle only inside a fume hood and use appropriate PPE.</li>
		<li>Quench the reaction by diluting the conical flask&#39;s contents twofold with PBS.</li>
		<li>Decant into 50 mL tubes and centrifuge at 3,000 &#215; g at room temperature for 3 min to remove unreacted methacrylic anhydride.</li>
		<li>Dialyze the supernatant inside 14 kDa cutoff cellulose dialysis tubes against deionized water at 40 &#176;C for 5 days while gently stirring. Replace the deionized water every day.</li>
		<li>Prepare for storage by decanting into 50 mL tubes, securing the cap, and placing it in the refrigerator for 12 h. Store in a refrigerator for up to 7 days.</li>
		<li>Freeze using liquid nitrogen and immediately lyophilize for 5 days at &#8722;54 &#176;C and 0.4 mbar. 
		NOTE: Ensure that the tubes contain no more than 40 mL of liquid when freezing. Once frozen, replace the cap with a covering that allows gas exchange such as a delicate task wipe secured with an elastic band.</li>
		<li>Store the resulting foam in a freezer at &#8722;20 &#176;C until required for microgel suspension synthesis.</li>
	</ol>
	</li>
	<li>Synthesis of GelMA microgels 
	NOTE: The microgels are synthesized using a water-in-oil emulsion method13 (Figure 2). This method has been tested for GelMA solution volumes of 1-10 mL. The same protocol can be used to synthesize gelatin microgels used to print standalone bone-prints.
	<ol>
		<li>Make a 10% w/w GelMA solution in PBS by weighing the lyophilized GelMA, adding it to a tube with PBS, and heating in a water bath at 50 &#176;C until fully hydrated.</li>
		<li>Add 37 mL of oil per 1 mL of GelMA solution to a beaker, ensuring it is no more than 65% full.</li>
		<li>Set up a double-beaker system on a hot plate with magnetic stirring by placing the beaker containing oil inside a larger beaker. 
		NOTE: The size of the two beakers should be such that ice can be easily dropped into the space between their walls. The setup is shown in Figure 2.</li>
		<li>Heat to 40 &#176;C while stirring. 
		NOTE: Ensure the vortex is not turbulent and has a depth of approximately 1/3 of the height of the oil in the beaker.</li>
		<li>Load the GelMA solution into a syringe and add it dropwise into the stirring oil through a 0.45 &#181;m filter. Allow the emulsion to equilibrate for 10 min.</li>
		<li>Reduce the temperature of the emulsion to 15 &#176;C to thermally stabilize the spheres by adding crushed ice into the space between the two beakers.</li>
		<li>Add acetone to the spinning emulsion in a volume ratio of 1:11 GelMA solution to acetone. 
		NOTE: Add the acetone gently through a funnel to avoid disrupting the emulsion. Stir for 60 min.</li>
		<li>Decant the contents of the beaker into 50 mL tubes, making sure to wash the walls of the beaker with acetone. Leave for 20 min to allow the dehydrated microgels to settle to the bottom.</li>
		<li>Discard the supernatant and wash at least 2x with acetone. 
		NOTE: The supernatant should be clear.</li>
		<li>Consolidate into one tube, top up with acetone, and sonicate for 10 s. Wash 2x with acetone.</li>
		<li>Store in acetone at room temperature until required for printing.</li>
	</ol>
	</li>
	<li>Preparing the GelMA microgel suspension for printing
	<ol>
		<li>Prepare a 1% w/w solution of GelMA in Dulbecco&#39;s Modified Eagle Medium (DMEM) by weighing lyophilized GelMA into a tube, adding DMEM, and heating in a water bath at 50 &#176;C until fully hydrated.</li>
		<li>Evaporate the acetone from the dehydrated microgels and weigh the resulting powder into a tube. Add acetone and transfer to a sterile environment.</li>
		<li>To form the microgel suspension, evaporate the acetone and add DMEM, 1% w/w GelMA solution in DMEM, and 2.5% w/w lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) initiator solution to achieve a final packing fraction of 30%. Allow to fully hydrate for at least 12 h at room temperature. Store in a refrigerator for up to 7 days. Allow to come up to room temperature before use. 
		NOTE: The volumes of these reagents are based on the dry weight of the microgels and can be calculated using the equations in Table 1.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Equation</td>
		</tr>
		<tr>
			<td></td>
			<td>x = weight of dry microgels (mg)</td>
		</tr>
		<tr>
			<td>Volume of 1% w/w GelMA in DMEM, a (&#181;L)</td>
			<td>a = 21.93x</td>
		</tr>
		<tr>
			<td>Volume of DMEM, b (&#181;L)</td>
			<td>b = 8.773x</td>
		</tr>
		<tr>
			<td>Volume of 2.5% w/w LAP solution, c (&#181;L)</td>
			<td>c = 0.6267x</td>
		</tr>
		<tr>
			<td>Total volume of microgel suspension produced (&#181;L)</td>
			<td>a + b + c</td>
		</tr>
	</tbody>
</table>
Table 1: Equations for calculating the volumes of reagents required to hydrate the GelMA microgel suspensions.&#160;Abbreviations: GelMA = gelatin methacrylate; LAP = lithium phenyl-2,4,6-trimethylbenzoylphosphinate.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63943/63943fig02.jpg" /> 
Figure 2: Schematic of the oil-emulsion method used for microgel synthesis. The double-beaker setup shows a beaker containing the stirring (indicated by arrow) emulsion placed inside a larger beaker to allow cooling. Abbreviation: GelMA = gelatin methacrylate&#160;<a href="https://www.jove.com/files/ftp_upload/63943/63943fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Printing bone-ink into cell suspensions
NOTE: Gelatin-based microgels support the adhesion of many different cell types, which makes this approach amenable to single and multiple cells within the microgel matrix. This protocol describes the procedure for using adipose-derived mesenchymal stem cells (ADSCs), as this is a popular and robust cell type for musculoskeletal tissue engineering.
<ol>
	<li>Culture the ADSCs in low-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 &#176;C and 5% CO2 until confluent.</li>
	<li>Detach the ADSCs from the tissue culture flask by removing the medium, washing with sterile PBS, and incubating at 37 &#176;C and 5% CO2 with 0.25% trypsin for 3 min.</li>
	<li>Pellet the cells by centrifuging at 150 &#215; g at room temperature for 5 min.</li>
	<li>Count the cells and calculate 5 &#215; 105 cells for each 1 mL of GelMA microgels. Allocate the required volume of cell suspension to a separate tube and pellet as above.</li>
	<li>Carefully remove as much supernatant as possible using a pipette, leaving only the cell pellet. Add the required volume of microgel suspension to the pellet and aspirate gently to ensure even cell distribution. 
	NOTE: If there are excess air bubbles in the suspension, gently centrifuge to remove and pipette up and down to redistribute the cells.</li>
	<li>Load the cell-laden microgel suspension into a reactor using a pipette. 
	NOTE: In the present study, 10 mm x 10 mm x 3 mm reactors with a volume capacity of 100 &#181;L were 3D printed.</li>
	<li>Deposit bone-ink using a 1 mL syringe fitted with a 23 G needle. 
	NOTE: This can be done with a 3D printer either by retrofitting an extrusion system that allows printing directly from the 1 mL syringe or by loading the bone-ink directly into the printer&#39;s extrusion cartridge (Figure 3).</li>
	<li>Crosslink the cell- and bone-ink-laden GelMA microgel construct with a UV-crosslinker lamp (405 nm) for 90 s. Immediately transfer to an appropriately sized well plate and cover with complete DMEM. 
	&#8203;NOTE: The aforementioned 3D-printed reactors fit inside 24-well cell culture plates.</li>
	<li>Incubate at 37 &#176;C and 5% CO2. Replace the culture medium after 24 h, then every 48-72 h as required.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63943/63943fig03.jpg" /> 
Figure 3: Schematic representation of the COBICS procedure showing the hydration of microgels, incorporation of cells, and subsequent printing of bone-ink in the cell-laden microgel suspension.&#160;Abbreviation: COBICS = ceramic omnidirectional bioprinting in cell-suspensions; GelMA = gelatin methacrylate.&#160;<a href="https://www.jove.com/files/ftp_upload/63943/63943fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Cell viability and proliferation assessment
<ol>
	<li>To assess bone-ink cytotoxicity, keep cell-laden COBICS constructs in complete culture medium. Perform a Live Dead assay at 24 h, 72 h, and 120 h (or relevant time points).</li>
	<li>At each time point, wash the constructs with PBS, then add a solution of phenol-free DMEM containing 4 mM calcein and 2 mM ethidium bromide. Incubate for 1 h at 37 &#176;C and 5% CO2.</li>
	<li>Wash with PBS and transfer to a glass-bottomed dish for imaging with a confocal microscope at Ex/Em spectra of 494/517 nm and 528/617 nm.</li>
</ol>

<ol>
	<li>Bates, P., Ramachandran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+injury,+healing+and+grafting.">Bone injury, healing and grafting.</a> Basic Orthopaedic Sciences. The Stanmore Guide. , 123-134 (2007).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+extracellular+matrix+in+bone+formation+and+regeneration.">The bone extracellular matrix in bone formation and regeneration.</a> Frontiers in Pharmacology. 11, 757 (2020).</li><li>Reznikov, N., 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+materials+science+vision+of+extracellular+matrix+mineralization.">A materials science vision of extracellular matrix mineralization.</a> Nature Reviews Materials. 1, 16041 (2016).</li><li>Kang, H. W., 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+3D+bioprinting+system+to+produce+human-scale+tissue+constructs+with+structural+integrity.">A 3D bioprinting system to produce human-scale tissue constructs with structural integrity.</a> Nature Biotechnology. 34 (3), 312-319 (2016).</li><li>Lin, K., 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=3D+printing+of+bioceramic+scaffolds-Barriers+to+the+clinical+translation:+From+promise+to+reality,+and+future+perspectives.">3D printing of bioceramic scaffolds-Barriers to the clinical translation: From promise to reality, and future perspectives.</a> Materials. 12 (17), 2660 (2019).</li><li>Qu, 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=Multi-dimensional+printing+for+bone+tissue+engineering.">Multi-dimensional printing for bone tissue engineering.</a> Advanced Healthcare Materials. 10 (11), 2001986 (2021).</li><li>Lode, 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=Fabrication+of+porous+scaffolds+by+three-dimensional+plotting+of+a+pasty+calcium+phosphate+bone+cement+under+mild+conditions.">Fabrication of porous scaffolds by three-dimensional plotting of a pasty calcium phosphate bone cement under mild conditions.</a> Journal of Tissue Engineering and Regenerative Medicine. 8 (9), 682-693 (2014).</li><li>Bernal, P. N., 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=Volumetric+bioprinting+of+complex+living-tissue+constructs+within+seconds.">Volumetric bioprinting of complex living-tissue constructs within seconds.</a> Advanced Materials. 31 (42), 1904209 (2019).</li><li>Diloksumpan, P., 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=Combining+multi-scale+3D+printing+technologies+to+engineer+reinforced+hydrogel-ceramic+interfaces.">Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces.</a> Biofabrication. 12 (2), 025014 (2020).</li><li>Thrivikraman, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+fabrication+of+vascularized+and+innervated+cell-laden+bone+models+with+biomimetic+intrafibrillar+collagen+mineralization.">Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization.</a> Nature Communications. 10 (1), 3520 (2019).</li><li>Romanazzo, S., 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=Synthetic+bone-like+structures+through+omnidirectional+ceramic+bioprinting+in+cell+suspensions.">Synthetic bone-like structures through omnidirectional ceramic bioprinting in cell suspensions.</a> Advanced Functional Materials. 31 (13), 2008216 (2021).</li><li>Hinton, T. 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=Three-dimensional+printing+of+complex+biological+structures+by+freeform+reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.</a> Science Advances. 1 (9), 1500758 (2015).</li><li>Phromsopha, T., Baimark, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+starch/gelatin+blend+microparticles+by+a+water-in-oil+emulsion+method+for+controlled+release+drug+delivery.">Preparation of starch/gelatin blend microparticles by a water-in-oil emulsion method for controlled release drug delivery.</a> International Journal of Biomaterials. 2014, 829490 (2014).</li><li>Moreno, D., 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=Solid-state+synthesis+of+alpha+tricalcium+phosphate+for+cements+used+in+biomedical+applications.">Solid-state synthesis of alpha tricalcium phosphate for cements used in biomedical applications.</a> Bolet&#237;n de la Sociedad Espa&#241;ola de Cer&#225;mica y Vidrio. 59 (5), 193-200 (2020).</li></ol>

The authors declare that they do not have any conflicts of interest to disclose.

The 3D printing technique COBICS was developed to enable the fabrication of mineralized bone-like structures via extrusion into a crosslinkable microgel suspension containing live cells. The technique has been applied to a degradable microgel suspension, and cells show good viability, spreading, and osteogenic differentiation ability within the system11. A key determinant of the success of constructs created using this technique is the proper synthesis of the &#945;-TCP-based bone-ink. First, the ...

Bone has remarkable regeneration abilities as one of the few structures in the body that can heal by recreating its normal cellular composition, orientation, and mechanical strength up until a critical defect size, when endogenous healing capacity is compromised1. Bone, together with cartilage and ligament, supports and facilitates body movement, while also storing minerals and fats and producing blood cells. As a hard, dense connective tissue, bone is mainly composed of an inorganic phase, water, and organic material composed primarily of collagen fibers2. Cells are embedded within this highly mineralized matrix of collagen I fibers and hydroxyapatite (HA) crystals, forming a hierarchical structure3.
The complex organization of this tissue makes the fabrication of synthetic alternatives to replicate the heterogeneous bone micro- and nano-environments exceptionally challenging3. For this purpose, a variety of materials, including bioceramics, cell-laden hydrogels, and synthetic materials have been proposed as solutions to create bone matrices. Among the scaffold fabrication techniques, 3D printing-based techniques have recently emerged and received much attention from the tissue engineering community owing to their remarkable ability to allow the fabrication of highly sophisticated and precise structures with great promise of patient-specific treatment4,5,6. Hydrogels have been the most popular choice of matrix mimics and bio-inks since they can be printed together with cells and bioactive molecules, generating functional constructs6. However, hydrogels lack the functional properties of bone, such as mechanical strength and a highly calcified, inorganic phase containing metabolically active cells.
3D printed ceramic scaffolds typically require postprocessing steps, including sintering, high-temperature treatments, or using harsh chemicals that must be thoroughly washed before in vitro or in vivo applications5. To address these limitations, Lode et al.7 recently developed an &#945;-tricalcium phosphate-based paste formed by hydroxyapatite, which can be printed and set under physiological conditions. However, this material still cannot be printed together with live cells as it requires post-treatment in a humid environment and subsequent aqueous solution immersion for a long period.
Alternatively, cell-laden hydrogels with inorganic particles incorporated have been proposed as a replacement for 3D bone matrix8,9. Despite their great ability to support cell viability, they are not able to recapitulate the densely mineralized bone tissue environment. Thrivikarman et al.10 adopted a biomimetic approach in which a supersaturated calcium and phosphate medium was used with a non-collagenous protein analog to better mimic the nanoscale apatite deposition. However, their constructs still cannot generate rigid 3D constructs with micro- and macro-scale architecture resembling bone.
The present study addresses these shortcomings through the development of a printing strategy to fabricate bone-mimicking constructs, in inorganic and organic phases, that are able to integrate both cells and growth factors11. COBICS uniquely recapitulates the mineral and cellular structure of bone using a microgel-based bioprinting technique. The protocol herein describes the process of synthesizing the ceramic bone-ink and gelatin-based microgels and then combining cells that enable COBICS. The process begins with the synthesis of the main precursor material of the bone-ink. The cross-linkable hydrogel is then synthesized and formed into microgels. Lastly, the bone-ink is deposited omnidirectionally in a support bath of the microgels laden with cells (Figure 1).
The bone-ink may be printed into any suspension of microgels that have the appropriate yield-stress characteristics, that is, the ability to fluidize at a specific shear rate and subsequently support the deposited structure. Two flexible approaches have been demonstrated: a suspension consisting of gelatin microgels and a suspension consisting of gelatin methacrylate (GelMA) microgels. The former suspension dissolves when the temperature is raised to 37 &#176;C, the freeform reversible embedding of suspended hydrogels (FRESH) technique12, while the latter can be photocrosslinked after printing, effectively &#34;stitching&#34; the microgels together and locking the printed bone-ink in place. The present study focuses on using GelMA as the matrix as it provides the unique advantage of being able to support cell growth with in situ printing of complex bone mimetic structures. Ultimately, this approach enables the generation of complex tissue models with high levels of biomimicry and broad implications for disease modeling, drug discovery, and regenerative engineering.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63943/63943fig01.jpg" /> 
Figure 1: Schematic of the workflow. (A) The bone-ink is synthesized starting from &#945;-tricalcium phosphate synthesis and its subsequent combination with glycerol, polysorbate 80, and ammonium phosphate dibasic. (B) GelMA microgels are fabricated by the water-in-oil emulsion method. The obtained microgels are then (C) hydrated and (D) combined with cells. Cell-microgel composites are then used as a granular bath in which the bone-ink is deposited. (E) The whole construct is then UV-crosslinked and transferred to the incubator for culture. Abbreviations:&#160;&#945;-TCP =&#160;&#945;-tricalcium phosphate; GelMA =gelatin methacrylate.&#160;<a href="https://www.jove.com/files/ftp_upload/63943/63943fig01large.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>3D Printer Extruder</td>
			<td>Hyrel3D</td>
			<td>EMO-25</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Falcon</td>
			<td>BDAA352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol 100% Denatured</td>
			<td>Chem-Supply</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Chem-Supply</td>
			<td>154871</td>
			<td></td>
		</tr>
		<tr>
			<td>Alumina crucible</td>
			<td>Coors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium phosphate dibasic (NaHPO4)</td>
			<td>Sigma</td>
			<td>A5764</td>
			<td></td>
		</tr>
		<tr>
			<td>Autodesk Fusion 360</td>
			<td>Autodesk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet level 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium carbonate</td>
			<td>Sigma</td>
			<td>239216</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydrogen phosphate (CaHPO4)</td>
			<td>Sigma</td>
			<td>C7263</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>various volumes used</td>
		</tr>
		<tr>
			<td>Cellulose Dialysis Tubes, 14 kDa cut-off</td>
			<td>Sigma</td>
			<td>D9777</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td>3-16KL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensing Tip, 23 G</td>
			<td>Nordson</td>
			<td>7018302</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>Thermo FIsher</td>
			<td>11885084</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo FIsher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Elevator furnace</td>
			<td>Labec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Engine HR Multihead Printer</td>
			<td>Hyrel3D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Bovogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin type A, from porcine skin</td>
			<td>Sigma</td>
			<td>G2500</td>
			<td></td>
		</tr>
		<tr>
			<td>General Purpose Stainless Steel Tips</td>
			<td>Nordson EF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Human adipose derived stem cells</td>
			<td>ATCC</td>
			<td>PCS-500-011</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 800 Confocal Microscope</td>
			<td>ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer (Alpha 1-4 LDplus)</td>
			<td>Christ</td>
			<td>101541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic hot plate and stirrer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylic anhydride</td>
			<td>Sigma</td>
			<td>276685</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini 2 Desktop 3D Printer</td>
			<td>LulzBot</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm sealing film</td>
			<td>Parafilm</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo FIsher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary ball mill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary ball mill jar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylenesorbitan monooleate Tween-80</td>
			<td>Sigma</td>
			<td>P6224</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>FEI Nova NanoSEM 450 FE-SEM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Science Kimwipes Delicate Task Wipers</td>
			<td>Kimtech</td>
			<td>18813156</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel standard test sieve</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower Oil</td>
			<td>Community Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% phenol red</td>
			<td>Thermo FIsher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Microscope Software</td>
			<td>ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead viability/ cytotoxicity kit for mammalian cells </td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, low glucose, no phenol red </td>
			
			<td>Thermo Fisher</td>
			<td>11054020</td>
<td></td>
		</tr>
	</tbody>
</table>

ceramic omnidirectional bioprinting in cell-laden suspensions for the generation of bone analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering) which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. To demonstrate COBICS formation of complex bone structures and the co-printing of cells in microgel suspensions, representative images of bone-like composites made of the bone-ink were taken, and a semi-quantitative analysis of ADSC viability...

Watch this Scientific Journal Video about Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs at JoVE.com

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

ceramic-omnidirectional-bioprinting-cell-laden-suspensions-for

Research

JoVE Journal

Bioengineering

1.8K Views.  University of New South Wales. This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Video: Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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

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

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

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

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

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

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

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

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

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...