Name: Biological Standardization of Human ASC-Based Biofabrication for Reproducible Macroscale Tissue Constructs (Video) | JoVE Journal
Uploaded: 2026-06-05T13:10:00.000+00:00
Duration: 9 min 21 s
Description: Learn a standardized biofabrication workflow using hADSC spheroids and 3D-printed GelMA scaffolds to create reproducible macroscale tissue constructs for regenerative medicine applications.

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BioRender.com (https://BioRender.com/x1lxn0y) for the illustrations in the figures.

The reagents and the equipment used are listed in the Table of Materials.
1. Preparation of agarose micromolds and fabrication of GelMA 3D-printed scaffolds
<ol>
	<li>Production of the 2% agarose micromold in 0.9% Sodium Chloride (NaCl) solution (see Figure 2A) 
	NOTE: All chemical waste must be handled and disposed of in accordance with institutional chemical safety regulations and hazardous waste guidelines.
	<ol>
		<li>Prepare a 0.9% NaCl solution in ultrapure water and autoclave for 30 min at 121 &#176;C. Maintain under sterile conditions until use.</li>
		<li>Dilute 1 g of sterile ultrapure agarose in 50 mL of 0.9% NaCl solution. Homogenize and depolymerize the agarose in a microwave at 10-s intervals for 3&#8211;5 cycles, until fully dissolved.</li>
		<li>In a biological safety cabinet, position a commercially available silicone mold containing 81 circular recesses on sterile gauze.</li>
		<li>Carefully deposit 600 &#181;L of agarose in the center of the silicone mold and allow it to rest for 40 min to ensure complete polymerization.</li>
		<li>Carefully demold the fully polymerized agarose micromold and transfer it to a well of a 12-well microtiter plate.</li>
		<li>Adjust the position of the agarose micromold and fix it to the bottom of the well by filling with 600 &#181;L of agarose.</li>
		<li>Pre-incubate the micromold by incubating with Dulbecco&#8217;s Modified Eagle Medium Low Glucose (DMEM, 2 mL/well) for 15 min. Repeat this procedure twice and, in the third incubation, replace with the Three-Dimensional (3D) culture medium, composed of DMEM supplemented with ascorbic acid (50 &#181;g/mL), human serum albumin (1.25 &#181;g/mL), penicillin-streptomycin (PS, 1&#215;, from a 100&#215; stock solution), and ITS supplement (1&#215;, from a 100&#215; stock solution). 
		NOTE: The 3D culture medium differs from the monolayer expansion medium by the absence of Fetal Bovine Serum (FBS) and the inclusion of defined supplements that promote cell&#8211;cell interaction and extracellular matrix support, favoring proper spheroid self-assembly and stabilization. If mold production and cell plating are performed on different days, the procedure may be paused after the second DMEM incubation, and the molds should be stored under sterile conditions in a humidified incubator at 37 &#176;C for up to 5 days. The third incubation with 3D culture medium must be performed only on the day of plating. Before seeding the cells, completely remove the medium from both the well and the micromold.</li>
	</ol>
	</li>
	<li>Preparation of Gelatin Methacryloyl (GelMA) hydrogel 
	NOTE: The entire procedure should be performed in the dark, as the reagents are photosensitive. In this study, GelMA (gel strength 300 g Bloom, degree of substitution 60%) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were used. To prepare 10 mL of a 10% w/v GelMA solution containing 0.5% w/v LAP:
	<ol>
		<li>Dissolve 50 mg of LAP powder in 1 mL of DMEM, gently mixing with a pipette until a clear, homogeneous solution is obtained.</li>
		<li>Transfer the LAP solution to a centrifuge tube containing 9 mL of DMEM and mix thoroughly.</li>
		<li>In a biological safety cabinet, sterilize the resulting solution by filtration through a 0.22 &#181;m filter.</li>
		<li>Add 1 g of GelMA to the solution and homogenize using magnetic stirring at 50 &#176;C for approximately 3 h, or until a clear and homogeneous solution is obtained. Hold the flask up to the light to ensure that the solution is free of visible particles and turbidity.</li>
		<li>Load the resulting hydrogel into a 10 mL syringe for subsequent printing or, if not used immediately, store it at 2&#8211;8 &#176;C, protected from light, until use. 
		NOTE: The GelMA and LAP solutions were used approximately 24 h after preparation. Prolonged storage of this solution is not recommended. Both GelMA and LAP powders should be stored at 2&#8211;8 &#176;C, according to the manufacturer&#8217;s instructions. The LAP photoinitiator is classified as non-hazardous according to standards such as ABNT NBR 14725 and Regulation (EC) n&#186; 1272/2008. However, the manufacturer recommends using personal protective equipment to prevent respiratory, skin, and eye irritation. The use of safety glasses tested and approved in accordance with relevant governmental standards, such as NIOSH (National Institute for Occupational Safety and Health - US) or EN 166 (EU), as well as nitrile rubber gloves with a minimum thickness of 0.11 mm, is recommended. Respiratory protection is required only when dust is generated. The manufacturer&#8217;s recommendations regarding respiratory protection with a filter are based on standards such as DIN EN 143, DIN EN 14387, and other complementary standards related to the specific respiratory protection system in use. The manufacturer recommends using a P1-type filter.</li>
	</ol>
	</li>
	<li>3D model design, slicing, and generation of the G-code file 
	NOTE: The 3D scaffold model was designed using the online 3D computer-aided design (CAD) software Tinkercad. For this protocol, a cylindrical scaffold was generated with a diameter of 15 mm, a bottom thickness of 1 mm, and two circular pores, each with a diameter of 5.50 mm and a depth of 2 mm.
	<ol>
		<li>Once the 3D model design is completed, export it as an *.stl file (Supplementary File 1).</li>
		<li>Open the Repetier-Host V2.3.2 software, which will be used to slice the designed model.</li>
		<li>In the &#34;Slicer&#34; tab, select the desired slicer option. In this study, the CuraEngine was used.</li>
		<li>Configure the appropriate parameter profile. In this work, Speed 10 mm/s; Layer Height 0.2 mm; Infill Pattern: Grid; Nozzle Diameter 0.41 mm were used.</li>
		<li>Load the *.stl file of the 3D model to be printed.</li>
		<li>In the &#34;Slicer&#34; tab, select the previously created configuration.</li>
		<li>Click on &#34;Slice with CuraEngine&#34;. The sliced 3D model will then be visualized in the software interface.</li>
		<li>Click on &#8220;Save to File&#8221; to save the G-code file in the desired location.</li>
	</ol>
	</li>
	<li>3D Printing of Gelatin Methacryloyl (GelMA) hydrogel scaffolds for spheroid bioassembly 
	NOTE: 3D printing must be performed under sterile conditions. Therefore, before starting the printing process, check that the biological safety cabinet housing the printer is turned on and that the airflow is functioning properly. Confirm that the height of the cabinet protection glass is at the same height as the equipment sensor marking.
	<ol>
		<li>Remove the safety locks from the bioprinter.</li>
		<li>Remove the glass plate from the printing bed by releasing the metal clips.</li>
		<li>Attach an appropriately shaped acrylic guide to support fixation of the culture plate onto the printing platform, using the metal clips.</li>
		<li>Connect the printer to the power source. Note that this equipment supports dual voltage (110/220 V).</li>
		<li>Connect the printer USB cable to the computer.</li>
		<li>Press the on/off button to turn the printer on.</li>
		<li>Open Repetier-Host V2.3.2 software, which will be used to view the 3D model and print.</li>
		<li>Click the &#8220;Connect&#8221; option in the software interface.</li>
		<li>Using the software manual control, select the extruder (1 or 2) and send the printhead to the home position (X = 0, Y = 0, Z = 0).</li>
		<li>Adjust the selected extruder for proper syringe fitting using the appropriate tool of the manual control in Repetier.</li>
		<li>Carefully insert the syringe into the extruder, already loaded with hydrogel and attached to an extrusion nozzle (in this study, we used a conical polypropylene nozzle with an internal diameter of 0.41 mm).</li>
		<li>Fit the culture plate into the acrylic guide attached to the printing bed.</li>
		<li>Adjust the Z home position (Z = 0) using the Z endstop screw located on the left side of the bioprinter. That is, set the desired distance between the extrusion nozzle and the printing surface. Ideally, the nozzle should lightly touch the substrate, which in this study was the culture plate.</li>
		<li>Return the printhead to the home position.</li>
		<li>Load the previously generated G-code file.</li>
		<li>Using the appropriate manual control tool in the software, extrude some of the hydrogel from the syringe to ensure material flow through the nozzle.</li>
		<li>Start the printing process.</li>
		<li>After printing, perform photocrosslinking of the resulting scaffold using the 3D bioprinter&#39;s UV light system, equipped with two LEDs at 395-400 nm. Expose the scaffold to UV light for 5 min (light intensity of 20 mW/cm2).</li>
		<li>Use the same depolymerized 2% agarose solution used for fabricating the micromolds to attach the 3D-printed scaffolds to the wells of a 12-well culture plate. Securing the scaffolds to the bottom of the wells is necessary to provide greater stability and to prevent displacement during spheroid seeding and throughout the culture period. 
		NOTE: When performing step 4.16, check if the hydrogel forms a continuous filament during extrusion. Typically, the gelatin solution is loaded into the syringe while still in the liquid state (approximately 37 &#176;C). Within a few minutes at room temperature (22&#8211;25 &#176;C), this biomaterial begins to transition to a gel-like consistency. The material should not drip during extrusion, as this will negatively affect print quality. Continuous filament formation during extrusion indicates that the hydrogel has reached an optimal consistency for printing. The GelMA and LAP solution diluted in DMEM exhibits a slightly orange coloration. Following UV-induced photocrosslinking, a color change is commonly observed, with the material becoming whitish or translucent, as shown in Figure 3D,E. When operating the UV system, safety glasses are mandatory to ensure adequate eye protection against ultraviolet radiation.</li>
	</ol>
	</li>
	<li>Printing fidelity analysis 
	NOTE: The 3D printing fidelity analysis of GelMA scaffolds considers the following parameters: scaffold diameter, pore diameter, and scaffold thickness. ImageJ Version 1.54 software was used to perform the measurements.
	<ol>
		<li>First, capture images of the scaffolds viewed by a stereomicroscope. Use a ruler or caliper as a reference.</li>
		<li>Before analyzing the images, calibrate and set the scale in ImageJ to carry out the measurements.</li>
		<li>Use the straight-line tool to perform the measurements.</li>
		<li>Since both the scaffold and pore shapes are circular, use the arithmetic mean of the scaffold and pore diameters for statistical analysis.</li>
		<li>For printing fidelity analysis, compare the measurements obtained by ImageJ from the printed scaffolds with the values defined in the 3D model generated by Tinkercad. 
		NOTE: The measured printed scaffold thickness is compared with the sum of the pore depth and bottom thickness values projected in Tinkercad.</li>
		<li>Perform statistical analysis of the obtained data. In this case, to compare the measured scaffold values with the expected values, the Mann-Whitney U test (nonparametric) can be used. The Shapiro-Wilk test is used to assess normality. Differences are considered statistically significant when p &#60; 0.05.</li>
	</ol>
	</li>
</ol>
2. Preparation of hADSCs and spheroid formation
NOTE: Commercially available human adipose-derived mesenchymal stem cells (hADSCs) were cultured in cell-specific growth medium and passaged at 80%&#8211;90% confluence according to the manufacturer&#8217;s recommendations. Cells were used until passage 7 for this protocol. All biological waste must be properly decontaminated or sterilized before disposal and handled in accordance with institutional biosafety guidelines. For subsequent confocal visualization, the monolayer should be labeled with a fluorescent dye before spheroid formation and incubated as recommended by the manufacturer. Spheroid formation may be initiated 30 min after labeling.
<ol>
	<li>Trypsinization of the ASC monolayer
	<ol>
		<li>In a biological safety cabinet, open the culture flask and transfer its contents to a waste container.</li>
		<li>Wash the monolayer twice with 1&#215; Phosphate-Buffered Saline (PBS) and discard the solution.</li>
		<li>Add 0.125% trypsin/EDTA solution sufficient to cover the monolayer. Close the flask and incubate in a humidified incubator at 37 &#176;C for up to 5 min.</li>
		<li>Inactivate the trypsin/EDTA by adding an equal volume of DMEM supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Collect the cell suspension using a serological pipette or transfer to centrifuge tubes. Centrifuge at 400 x g for 5 min at room temperature (20&#8211;25 &#176;C).</li>
		<li>Carefully discard the supernatant.</li>
		<li>Resuspend the cell pellet in DMEM and homogenize. Take an aliquot for cell counting and viability assessment.</li>
	</ol>
	</li>
	<li>Spheroid culture (see Figure 2B)
	<ol>
		<li>Separate the appropriate number of cells to be plated for each mold into a 15 mL centrifuge tube. For hADSCs, each agarose mold containing 81 recesses should receive 1 &#215; 106 cells&#8212;one spheroid forms in each microwell, yielding 81 spheroids per micromold.</li>
		<li>Add 1&#215; PBS and centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant and add 1&#215; PBS. Repeat this step twice.</li>
		<li>After the third centrifugation, carefully discard the supernatant and dry the tube opening with sterile gauze before returning it to a vertical position.</li>
		<li>Resuspend the cell pellet in 120 &#181;L of 3D culture medium for spheroid culture and homogenize. Adjust the suspension volume to 200 &#181;L with additional 3D culture medium if necessary. 
		NOTE: Each mold with 81 resections has a capacity of 200 &#181;L.</li>
		<li>Aspirate 200 &#181;L and plate it in the center of the mold carefully, avoiding bubble formation. 
		&#8203;NOTE: This step can be automated using a robotic pipetting system, as previously described22.</li>
		<li>Leave the plate in the biological safety cabinet for 5 min to allow cells to settle into the resections.</li>
		<li>Place the plate in a humidified incubator for 40 min.</li>
		<li>After this period, carefully add 2 mL of 3D Medium to each well. Return the plate to the humidified incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Spheroids measurement and statistical analysis
	<ol>
		<li>Capture images of the spheroids positioned along the two diagonals of the agarose micromold, forming an &#8220;X&#8221; pattern, using an inverted optical microscope equipped with a digital camera and a 10&#215; objective. Use the 100 &#181;m scale bar to calibrate the image scale before measuring diameters.</li>
		<li>To determine the major and minor diameters of the spheroids, use the software&#8217;s &#8220;line&#8221; tool and perform two perpendicular measurements for each photographed spheroid, with an approximate 90&#176; angle between them.</li>
		<li>Calculate the mean diameter of each spheroid as the arithmetic mean of the two previously measured perpendicular diameters. Calculate the sphericity as the ratio of the largest to the smallest diameter of each spheroid, with values closer to 1 indicating greater uniformity and a more spherical shape.</li>
		<li>Perform statistical analyses using the obtained diameter and sphericity values.</li>
	</ol>
	</li>
</ol>
3. Seeding of spheroids on GelMA scaffolds for spheroid fusion and bioassembly
<ol>
	<li>After 24 h of spheroid formation, collect them from the agarose micromolds using a pipette with wide-bore tips and transfer them to 2 mL microtubes.</li>
	<li>When the spheroids settle at the bottom of the microtubes, discard the supernatant.</li>
	<li>Resuspend the spheroids in 50 &#181;L of 3D Medium.</li>
	<li>Using a pipette with wide-bore tips, collect the spheroids and gently embed them in the GelMA scaffold pores. 
	NOTE: In a GelMA support pore about 5.50 mm in diameter and 2 mm in depth, seed approximately 480 to 640 spheroids (corresponding to 6&#8211;8 agarose micromolds, each generating 81 spheroids with diameters ranging from 200 to 400 &#181;m) in 50 &#181;L. This estimate was based on pore volume and mean spheroid diameter, with quantification performed by the number of complete micromolds transferred rather than by individual spheroid counting, ensuring a practical and reproducible estimate of spheroid density within the pore.</li>
	<li>After depositing the spheroids into the GelMA support pore, wait 20&#8211;40 min to allow the spheroids to settle. Add 1 mL of 3D medium to each well.</li>
	<li>After 24 h, add 1 mL of 3D medium to each well.</li>
	<li>Monitor the fusion of spheroids within the pores of GelMA scaffolds by phase-contrast microscopy and stereomicroscopy. Acquire the images every 24 h, from day 0 (immediately after seeding) to day 3 (72 h).</li>
	<li>Fix the biological construction in 4% paraformaldehyde after 72 h for visualization by confocal microscopy. Handle paraformaldehyde in a certified chemical fume hood using appropriate personal protective equipment (lab coat, gloves, and eye protection) due to its toxic and irritant properties, and dispose of waste according to institutional chemical safety guidelines. For confocal observation, cells should be pre-labeled in the monolayer with a fluorescent dye before spheroid formation. 
	NOTE: If removal of the construct from the GelMA pore is required for analysis or in vivo application, carefully retrieve it using precision tweezers or an appropriately sized biopsy punch.</li>
</ol>

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J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+printing+parameters+and+cell+density+on+bioink+outcomes.">Influence of printing parameters and cell density on bioink outcomes.</a> Tissue Eng Part A. 26 (23&#8211;24), 1349-1358 (2020).</li><li>Maggiotto, F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusable+vascularized+cancer+niche+via+3D+bioprinting.">Perfusable vascularized cancer niche via 3D bioprinting.</a> Front Bioeng Biotechnol. 13, 1484738 (2025).</li><li>Shie, M. Y., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+GelMA+concentration+on+properties+and+fibroblast+behavior.">Effects of GelMA concentration on properties and fibroblast behavior.</a> Polymers (Basel). 12 (9), 1930 (2020).</li><li>Chimene, D., Lennox, K. K., Kaunas, R. R., Gaharwar, A. K. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+bioinks+for+3D+printing.">Advanced bioinks for 3D printing.</a> Adv Mater. 32 (12), 1902026 (2020).</li><li>Pepelanova, I., Kruppa, K., Scheper, T., Lavrentieva, A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GelMA+hydrogels+for+tissue+engineering.">GelMA hydrogels for tissue engineering.</a> Polymers (Basel). 10 (11), 1134 (2018).</li><li>Heinrich, M. A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+from+bench+to+translation.">3D bioprinting from bench to translation.</a> Adv Mater. 31 (23), 1805510 (2019).</li><li>Kronemberger, G. S., Chattahy, K., Spagnuolo, F. D., Karam, A. S., Kelly, D. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioassembly+of+fibrocartilage+microtissues+for+meniscal+grafts.">Bioassembly of fibrocartilage microtissues for meniscal grafts.</a> Adv Healthcare Mater. 14 (32), e02208 (2025).</li><li>Matai, I., Kaur, G., Seyedsalehi, A., McClinton, A., Laurencin, C. T. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+3D+bioprinting+for+regenerative+engineering.">Progress in 3D bioprinting for regenerative engineering.</a> Biomaterials. 226, 119536 (2020).</li><li>Decarli, M. C., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+stem+cell+spheroids+for+cartilage+engineering.">Bioprinting stem cell spheroids for cartilage engineering.</a> Adv Healthcare Mater. 12 (7), 2203021 (2023).</li><li>Zhang, Z., Liu, Y., Tao, X., Du, P., Enkhbat, M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cell+microenvironments+with+spheroids+and+hydrogels.">Engineering cell microenvironments with spheroids and hydrogels.</a> Polymers (Basel). 15 (9), 1925 (2023).</li><li>Zuliani, C. C., da Cunha, J. B., Correa, R. F., Pinto, J. u. n. i. o. r. . E. A., d&#8217;&#193;vila, M. A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+hydrogel+for+delivery+of+MSC+spheroids+in+cartilage+regeneration.">Injectable hydrogel for delivery of MSC spheroids in cartilage regeneration.</a> Artif Cells Nanomed Biotechnol. 53 (1), 453-467 (2025).</li><li>Unagolla, J. M., Jayasuriya, A. C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-based+3D+bioprinting+review.">Hydrogel-based 3D bioprinting review.</a> Appl Mater Today. 17, 100479 (2019).</li><li>Kim, M. H., Singh, Y. P., Celik, N., Yeo, M., Rizk, E., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioprinting+of+spheroids.">High-throughput bioprinting of spheroids.</a> Nat Commun. 15 (1), 10083 (2024).</li><li>Daly, A. C., Davidson, M. D., Burdick, J. A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+fusion+within+self-healing+hydrogels.">Spheroid fusion within self-healing hydrogels.</a> Nat Commun. 12 (1), 753 (2021).</li><li>Prabhakaran, V., Melchels, F. P. W., Murray, L. M., Paxton, J. Z. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+bone+tissues+via+spheroid+fusion.">Engineering bone tissues via spheroid fusion.</a> Front Endocrinol (Lausanne). 14, 1308604 (2023).</li><li>Gabriella, , et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+multicellular+tissue+fusion+of+cocultured+spheroids.">Probing multicellular tissue fusion of cocultured spheroids.</a> Adv Sci (Weinh). 8 (21), e2103320 (2021).</li><li>Kim, S., Kim, E. M., Yamamoto, M., Park, H., Shin, H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+multicellular+spheroids.">Engineering multicellular spheroids.</a> Adv Healthcare Mater. 9 (12), 2000608 (2020).</li><li>Zhang, J., Yun, S., Du, Y., Zannettino, A., Zhang, H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-based+preparation+of+cell+aggregates.">Hydrogel-based preparation of cell aggregates.</a> Appl Mater Today. 20, 100747 (2020).</li><li>Khajehmohammadi, M., Azizi Tafti, R., Nikukar, H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+porosity+on+bioprinted+scaffolds.">Effect of porosity on bioprinted scaffolds.</a> J Biomed Mater Res A. 111 (2), 245-260 (2023).</li><li>Kim, M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+nutrient+transport+in+engineered+cartilage+constructs.">Enhanced nutrient transport in engineered cartilage constructs.</a> Acta Biomater. 58, 1-11 (2017).</li><li>Chariyev-Prinz, F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC+response+to+hydrostatic+pressure+in+chondrogenesis+supportive+environments.">MSC response to hydrostatic pressure in chondrogenesis supportive environments.</a> J Biomech. 154, 111590 (2023).</li></ol>

The authors report no conflicts of interest.

The results shown in this study highlight the successful integration of quality control metrics into a standardized biofabrication workflow to ensure reproducibility. The use of 3D-printed scaffolds based on pre-established designs provides structural support for spheroid bioassembly19,23. As a proof-of-concept, a solid scaffold was designed with two central porous regions, dimensioned to support high-density spheroid loading and guided bioassembly.
<p class=...

Bioassembly and bioprinting are complementary biofabrication approaches in tissue engineering that focus on constructing complex tissues and organs at the macroscale using automated technologies1,2,3,4.
Spheroids were incorporated into biofabrication as building blocks mainly for bioassembly approaches5. Spheroids are considered microtissues that reproduce interactions between cells and the surrounding tissue microenvironment in vivo. Among the cell sources used for spheroid formation, adipose-derived stem cells (ADSCs) stand out for their ease of isolation, long-term culture maintenance, and differentiation potential6,7,8.
Due to their intrinsic capacity to form larger aggregates, spheroids can generate more complex tissue constructs when subjected to bioassembly approaches. This fusion-driven organization enhances the regenerative and differentiation potential of stem cell spheroids9,10.
Spheroid bioassembly can be supported or even guided by biomaterials11, making this strategy suitable for scalable and automated biofabrication12,13.
Existing biofabrication strategies can be broadly categorized into scaffold-free spheroid assembly approaches and hydrogel-embedded bioprinting systems2,5,14. Scaffold-free methods rely exclusively on spontaneous cellular self-assembly and inter-spheroid fusion, leveraging intrinsic cell&#8211;cell interactions. However, because they occur without external architectural guidance or geometric confinement, these approaches exhibit reduced spatial control and lower structural predictability5,15. In contrast, hydrogel-based encapsulation approaches embed cells within bulk matrices to provide structural support and predefined architecture16,17, which may partially restrict direct cell-cell contact during early fusion stages and influence cell migration dynamics18. In this framework, printable biomaterials such as Gelatin Methacryloyl (GelMA) present cell adhesion motifs and may function as scaffolds to facilitate spheroid bioassembly19. A 3D-printed GelMA scaffold is hypothesized to provide geometric confinement and spatial guidance for spheroids, thereby promoting controlled tissue formation through fusion into larger, structurally defined constructs. This approach aims to improve spatial control and structural reproducibility during spheroid bioassembly by combining scaffold-guided organization with spheroid fusion within a defined hydrogel architecture. However, the present workflow focuses on process standardization and structural biofabrication parameters and does not address long-term tissue maturation, functional differentiation, or large-scale automated production.
Biological standardization is essential not only to ensure reproducibility and scalability but also to preserve the functional integrity of biofabricated tissues19,20. Consequently, precise control of engineering, geometric, and structural parameters is critical, as these factors directly influence biological performance, including tissue function and maturation5,21. A standardized and reproducible biofabrication workflow is presented, encompassing the following key steps: (1) ASC monolayer culture; (2) ASC spheroid formation; (3) 3D printing of the GelMA scaffold; and (4) spheroid bioassembly (Figure 1). Within this workflow, objective quality control metrics were established, including spheroid uniformity and sphericity, printing fidelity, and scaffold shape integrity.

<table><tbody><tr><td>1x Phosphate-Buffered Saline (PBS)</td><td>Prepared in the laboratory</td><td></td><td>-</td></tr><tr><td>593iCAN 3D Bioprinter, Model 593 iCan2X Printhead</td><td>593iCAN</td><td>-</td><td>-</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>16500-500</td><td>-</td></tr><tr><td>Ascorbic Acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A4544-100MG</td><td>-</td></tr><tr><td>Carl Zeiss Microscopy GmbH Primovert&amp;nbsp;</td><td>Zeiss</td><td>-</td><td>415510-1101-000</td></tr><tr><td>Celldiscover&amp;nbsp;</td><td>Zeiss</td><td>-</td><td>LSM 900 with Airyscan 2</td></tr><tr><td>Celltracker</td><td>Invitrogen&amp;nbsp;</td><td>C7025</td><td>-</td></tr><tr><td>Centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>-</td><td>5702</td></tr><tr><td>Class II Biological Safety Cabinet</td><td>Streamline</td><td>-</td><td>Model SC2-4E3</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator for cell culture</td><td>Thermo Fisher Scientific</td><td>-</td><td>Series 8000 WJ&amp;nbsp;</td></tr><tr><td>Conical polypropylene nozzle 0.41 mm</td><td>593iCAN</td><td>-</td><td>-</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium Low Glucose&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D2902-10L</td><td>-</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich&amp;nbsp;</td><td>E9884</td><td>-</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco&amp;nbsp;</td><td>12657-029</td><td>-</td></tr><tr><td>Gelatin methacryloyl gel strength 300 g Bloom, degree of substitution 60%</td><td>Sigma-Aldrich</td><td>900622</td><td>Lot/Batch number: MKCS2673. Documentation for each batch, including the Safety Data Sheet (SDS) and the Certificate of Analysis (CoA), can be obtained from the manufacturer&amp;rsquo;s website.</td></tr><tr><td>GraphPad Prism 6.0 software</td><td>GraphPad Inc</td><td>-</td><td>-</td></tr><tr><td>Hot plate magnetic stirrer</td><td>IKA</td><td>Z671797</td><td>Model C-MAG HS7</td></tr><tr><td>Human serum albumin 100 mg/ml</td><td>FUJIFILM Irvine Scientific</td><td>9988</td><td>-</td></tr><tr><td>ITS (Insulin-transferrin-sodium selenite) supplement 100x&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I3146-5ML</td><td>-</td></tr><tr><td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td><td>Sigma-Aldrich</td><td>900889</td><td></td></tr><tr><td>Luxoeo 4Z Stereozoom Microscope&amp;nbsp;</td><td>Labomed</td><td>-</td><td>-</td></tr><tr><td>MicroTissues&amp;reg; 3D Petri Dish&amp;reg; micro-mold spheroids</td><td>Sigma-Aldrich</td><td>Z764019</td><td>-</td></tr><tr><td>Penicillin-streptomycin 100x</td><td>Sigma-Aldrich</td><td>15140122</td><td>-</td></tr><tr><td>Poietics&amp;trade; human adipose derived stem cells (ADSC)</td><td>Lonza&amp;nbsp;</td><td>PT-5006</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>71382-500G</td><td>-</td></tr><tr><td>Trypsin/EDTA</td><td>Gibco&amp;nbsp;</td><td>27250018</td><td>-</td></tr></tbody></table>

biological standardization of human asc-based biofabrication for reproducible macroscale tissue constructs

Biological Standardization of Human ASC-Based Biofabrication for Reproducible Macroscale Tissue Constructs

The lack of standardized protocols across laboratories poses a significant barrier to the comparability of experimental outcomes and the efficient transfer of biofabrication technologies. Rigorous biological standardization not only enhances reproducibility but also facilitates alignment with national and international regulatory frameworks, which are essential for the clinical translation of tissue engineering strategies. This study introduces a standardized biofabrication workflow comprising monolayer culture of hADSCs, spheroid formation, 3D printing of Gelatin Methacryloyl (GelMA) scaffolds, and spheroid bioassembly. Quality control metrics were integrated to ensure reproducibility. The 3D-printed GelMA scaffold was designed with two central pores, each capable of housing hundreds of spheroids. Printing fidelity was confirmed by comparing scaffold dimensions to the CAD model, with minor non-significant deviations. hADSCs formed uniform spheroids within 24 h in agarose micromolds. When seeded into scaffold pores, spheroids underwent continuous fusion over 72 h, forming macroscale constructs confirmed by confocal imaging. Scaffold design and printing accuracy ensured structural support, while spheroid uniformity enabled predictable formation of the construct. The fusion of spheroids within the scaffold pores yielded tissue constructs suitable for transplantation, with or without the scaffold, highlighting the versatility of this bioassembly approach. The findings underscore the pivotal role of biological standardization in advancing reproducible methodologies for the biofabrication of living tissue constructs, with promising implications for future clinical applications in regenerative medicine.

Human adipose-derived stem cells (hASCs) cultured in a monolayer (Figure 2C) were successfully induced to form spheroids within 24 h after seeding into agarose micromolds (Figure 2D). The resulting spheroids exhibited a compact and well-defined morphology. Morphometric analysis revealed a mean diameter of 239.8 &#181;m &#177; 87.0 &#181;m at 24 h (Figure 2E) and a sphericity value close to 1 (0.947 &#177; 0.123) (<strong class="xfig...

This study presents a reproducible biofabrication workflow using adipose-derived stem cell spheroids and 3D-printed GelMA scaffolds. It demonstrates spatial control, fusion capacity, and quality control metrics, resulting in transplantable macroscale tissue constructs with potential applications in regenerative medicine.

The lack of standardized protocols across laboratories poses a significant barrier to the comparability of experimental outcomes and the efficient ...

This work presents a standardized biofabrication workflow that uses 3D printed hydrogel templates to guide the assembly of spheroids into high density cellular micro tissues. The approach used in this study, biofabrication transplantable tissues with or without scaffolds, advances the biofabrication field for regenerative medicine.To begin, place the flask containing hADSCs monolayer in a biological safety cabinet and transfer the culture medium to a waste container. Wash the monolayer twice with 1x PBS and discard the solution. If required, label the cells with a suitable fluorescent dye. Add a 0.125% trypsin-EDTA solution sufficient to cover the monolayer and close the flask. Incubate the flask in a humidified incubator at 37 degrees Celsius for up to five minutes. After incubation, inactivate the trypsin by adding an equal volume of DMEM supplemented with 10% FBS. Collect the cell suspension using a serological pipette, transfer the cell suspension to centrifuge tubes, and spin at 400 g for five minutes at room temperature. After discarding the supernatant, resuspend the cell pellet in DMEM and take an aliquot for cell counting and viability assessment.Aliquot 1 times 10 to the

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Research

JoVE Journal

Bioengineering

This article describes a biofabrication approach:  deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Bridging the Bio-Electronic Interface with Biofabrication

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering of a Human 3D in vitro Tumor Test System

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

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.

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

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Learn a standardized biofabrication workflow using hADSC spheroids and 3D-printed GelMA scaffolds to create reproducible macroscale tissue constructs for regenerative medicine applications.

Biological Standardization of Human ASC-Based Biofabrication for Reproducible Macroscale Tissue Constructs

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