This work was supported by the Beijing Natural Science Foundation (3212007), Tsinghua University Initiative Scientific Research Program (20197050024), Tsinghua University Spring Breeze Fund (20201080760), the National Natural Science Foundation of China (51805294), National Key Research and Development Program of China (2018YFA0703004), and the 111 Project (B17026).

1. Cell culture
<ol>
	<li>Supplement high-glucose Dulbecco&#39;s modified Minimum Essential Medium (H-DMEM) with 10% fetal bovine serum (FBS), 1% nonessential amino acid solution (NEAA), 1% penicillin G and streptomycin, and 1% Glutamine supplement as culture media for A549 cells.</li>
	<li>Culture A549 cells in a CO2 incubator at 37 &#176;C and with 5% CO2</li>
	<li>Dissociate cells for subculture using trypsin at approximately 80% confluence.
	<ol>
		<li>Use 3 mL of trypsin to treat the cells in...

<ol>
	<li>Chen, A. K., Reuveny, S., Oh, S. K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+human+mesenchymal+and+pluripotent+stem+cell+microcarrier+cultures+in+cellular+therapy:+Achievements+and+future+direction.">Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: Achievements and future direction.</a> Biotechnol Advances. 31, 1032-1046 (2013).</li><li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past,+present,+and+future+of+microcarrier-based+tissue+engineering.">Past, present, and future of microcarrier-based tissue engineering.</a> Journal of Orthopaedic Translation. 3, 51-57 (2015).</li><li>Badenes, S. M., Fernandes, T. G., Rodrigues, C. A. V., Diogo, M. M., Cabral, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier-based+platforms+for+in+vitro+expansion+and+differentiation+of+human+pluripotent+stem+cells+in+bioreactor+culture+systems.">Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.</a> Journal of Biotechnol. 234, 71-82 (2016).</li><li>de Soure, A. M., Fernandes-Platzgummer, A., Da Silva, C. L., Cabral, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+microcarrier-based+manufacturing+of+mesenchymal+stem/stromal+cells.">Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.</a> Journal of Biotechnol. 236, 88-109 (2016).</li><li>Naqvi, S. 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=Living+cell+factories+-+electrosprayed+microcapsules+and+microcarriers+for+minimally+invasive+delivery.">Living cell factories - electrosprayed microcapsules and microcarriers for minimally invasive delivery.</a> Advanced Materials. 28, 5662-5671 (2016).</li><li>Sarkar, 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=Chitosan:+A+promising+therapeutic+agent+and+effective+drug+delivery+system+in+managing+diabetes+mellitus.">Chitosan: A promising therapeutic agent and effective drug delivery system in managing diabetes mellitus.</a> Carbohydrate Polymers. 247, (2020).</li><li>Sulaiman, S. B., Idrus, R. B. H., Hwei, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin+microsphere+for+cartilage+tissue+engineering:+current+and+future+strategies.">Gelatin microsphere for cartilage tissue engineering: current and future strategies.</a> Polymers. 12, (2020).</li><li>Huang, L., Abdalla, A. M. E., Xiao, L., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopolymer-based+microcarriers+for+three-dimensional+cell+culture+and+engineered+tissue+formation.">Biopolymer-based microcarriers for three-dimensional cell culture and engineered tissue formation.</a> International Journal of Molecular Sciences. 21, (2020).</li><li>Isiklan, N., Tokmak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+thermo/pH-responsive+chitosan+coated+pectin-graft-poly(N,+N-diethyl+acrylamide)+microcarriers.">Development of thermo/pH-responsive chitosan coated pectin-graft-poly(N, N-diethyl acrylamide) microcarriers.</a> Carbohydrate Polymers. 218, 112-125 (2019).</li><li>Lau, T. T., Wang, C., Wang, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+delivery+with+genipin+crosslinked+gelatin+microspheres+in+hydrogel/microcarrier+composite.">Cell delivery with genipin crosslinked gelatin microspheres in hydrogel/microcarrier composite.</a> Composites Science &amp; Technology. 70, 1909-1914 (2010).</li><li>Lau, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-microcarrier+composite+systems+for+cell+delivery+in+tissue+engineering.">Hydrogel-microcarrier composite systems for cell delivery in tissue engineering.</a> Acta Biomaterialia. 10, 1646-1662 (2014).</li><li>Kwon, Y. J., Peng, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-alginate+gel+bead+cross-linked+with+gelatin+as+microcarrier+for+anchorage-dependent+cell+culture.">Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture.</a> Biotechniques. 33, 218 (2002).</li><li>Leach, J. B., Bivens, K. A., Patrick, C. W., Schmidt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocrosslinked+hyaluronic+acid+hydrogels:+natural,+biodegradable+tissue+engineering+scaffolds.">Photocrosslinked hyaluronic acid hydrogels: natural, biodegradable tissue engineering scaffolds.</a> Biotechnology &amp; Bioengineering. 82, 578-589 (2003).</li><li>Kurisawa, M., Chung, J. E., Yang, Y. Y., Gao, S. J., Uyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+biodegradable+hydrogels+composed+of+hyaluronic+acid-tyramine+conjugates+for+drug+delivery+and+tissue+engineering.">Injectable biodegradable hydrogels composed of hyaluronic acid-tyramine conjugates for drug delivery and tissue engineering.</a> Chemical Communications. 34, 4312-4314 (2005).</li><li>Yao, R., Alkhawtani, A. Y. F., Chen, R., Luan, J., Xu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+in+vivo+angiogenesis+directed+by+electro-assisted+bioprinting+of+alginate/collagen+microspheres+with+human+umbilical+vein+endothelial+cell+coating+layer.">Rapid and efficient in vivo angiogenesis directed by electro-assisted bioprinting of alginate/collagen microspheres with human umbilical vein endothelial cell coating layer.</a> International Journal of Bioprinting. 5, 194 (2019).</li><li>Mahou, R., Vlahos, A. E., Shulman, A., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpenetrating+alginate-collagen+polymer+network+microspheres+for+modular+tissue+engineering.">Interpenetrating alginate-collagen polymer network microspheres for modular tissue engineering.</a> Acs Biomaterials Science &amp; Engineering. 4 (11), 3704-3712 (2017).</li><li>Aftab, 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=Microfluidic+platform+for+encapsulation+of+plant+extract+in+chitosan+microcarriers+embedding+silver+nanoparticles+for+breast+cancer+cells.">Microfluidic platform for encapsulation of plant extract in chitosan microcarriers embedding silver nanoparticles for breast cancer cells.</a> Applied Nanoscience. 10, 2281-2293 (2020).</li><li>Park, 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=Microfluidic-printed+microcarrier+for+in+vitro+expansion+of+adherent+stem+cells+in+3D+culture+platform.">Microfluidic-printed microcarrier for in vitro expansion of adherent stem cells in 3D culture platform.</a> Macromolecular Bioscience. 19, (2019).</li><li>Chui, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrosprayed+genipin+cross-linked+alginate-chitosan+microcarriers+for+ex+vivo+expansion+of+mesenchymal+stem+cells.">Electrosprayed genipin cross-linked alginate-chitosan microcarriers for ex vivo expansion of mesenchymal stem cells.</a> Journal of Biomedical Materials Research Part A. 107, 122-133 (2019).</li><li>Min, N. G., Ku, M., Yang, J., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+production+of+uniform+microcarriers+with+multicompartments+through+phase+separation+in+emulsion+drops.">Microfluidic production of uniform microcarriers with multicompartments through phase separation in emulsion drops.</a> Chemistry of Materials. 28 (5), 1430-1438 (2016).</li><li>Park, W., Jang, S., Kim, T. W., Bae, J., Lee, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic-printed+microcarrier+for+in+vitro+expansion+of+adherent+stem+cells+in+3D+culture+platform.">Microfluidic-printed microcarrier for in vitro expansion of adherent stem cells in 3D culture platform.</a> Macromolecular Bioscience. 19, (2019).</li><li>Xu, T., Kincaid, H., Atala, A., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Production+of+Single-Cell+Microparticles+Using+an+Inkjet+Printing+Technology.">High-Throughput Production of Single-Cell Microparticles Using an Inkjet Printing Technology.</a> Journal of Manufacturing Science &amp; Engineering. 130, 137-139 (2008).</li><li>Rao, 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=Enhanced+enrichment+of+prostate+cancer+stem-like+cells+with+miniaturized+3D+culture+in+liquid+core-hydrogel+shell+microcapsules.">Enhanced enrichment of prostate cancer stem-like cells with miniaturized 3D culture in liquid core-hydrogel shell microcapsules.</a> Biomaterials. 27 (27), 7762-7773 (2014).</li><li>Choi, C. H., Weitz, D. A., Lee, C. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+engineering+microparticles+and+their+nascent+utilization+in+biomedical+delivery+and+diagnostic+applications.">Recent advances in engineering microparticles and their nascent utilization in biomedical delivery and diagnostic applications.</a> Lab On A Chip. 17 (4), 591-613 (2017).</li><li>Liu, T., Pang, Y., Zhou, Z., Yao, R., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+cell+printing+system+for+the+construction+of+heterogeneous+tissue+models.">An integrated cell printing system for the construction of heterogeneous tissue models.</a> Acta Biomaterialia. 95, 245-257 (2019).</li><li>Hassan, 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=Functional+inks+and+extrusion-based+3D+printing+of+2D+materials:+a+review+of+current+research+and+applications.">Functional inks and extrusion-based 3D printing of 2D materials: a review of current research and applications.</a> NANOSCALE. 12, 19007-19042 (2020).</li><li>Vithani, 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=An+overview+of+3D+printing+technologies+for+soft+materials+and+potential+opportunities+for+lipid-based+drug+delivery+systems.">An overview of 3D printing technologies for soft materials and potential opportunities for lipid-based drug delivery systems.</a> Pharmaceutical Research. 36, (2019).</li></ol>

The protocol described here provides instructions for the preparation of multi-types of hydrogel microcarriers and subsequent cell seeding. Compared to microfluidic chip and inkjet printing methods, AVIFJ approach to constructing microcarriers offers greater flexibility and biocompatibility. An independent nozzle enables a wide range of lightweight nozzles, including glass micropipettes, to be used in these printing systems. The highly controllable processing enables parameters including the volume of the reservoir, the ...

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area and are commonly used for large-scale culture of cells1,2. Their outer surface provides abundant growth sites for cells, and the interior provides a support structure for spatial proliferation. The spherical structure also provides convenience in monitoring and controlling parameters, including pH, O2, and concentration of nutrients and metabolites. When used in combination with stirred tank bioreactors, microcarriers can achieve higher cell densities in a relatively small volume compared to conventional cultures, thereby providing a cost-effective way to achieve large-scale cultures3. Microcarrier culture technology has become one of the main techniques in cytological research, and much progress has been made in the field of large-scale expansion of stem cells, hepatocytes, chondrocytes, fibroblasts, and other structures4. They have also been found to be ideal drug delivery vehicles and bottom-up units, therefore taking on an increasingly important role in clinical drug screening and in vitro tissue engineering repair5.
To meet mechanical property requirements in different scenarios, multiple types of hydrogel materials have been developed for use in the construction of microcarriers6,7,8,9,10,11. Alginate and hyaluronic acid (HA) hydrogels are two of the most used microcarrier materials owing to their good biocompatibility and crosslinkability12,13. Alginate can be easily cross-linked by calcium chloride, and its mechanical properties can be modulated by changing the cross-linking time. Tyramine-conjugated HA is cross-linked by the oxidative coupling of tyramine moieties catalyzed by hydrogen peroxide and horseradish peroxidase14. Collagen, due to its unique spiral structure and cross-linked fiber network, is often used as an adjuvant to mix into the microcarriers to further promote cell attachment15,16.
Current methods for preparing microcarriers include microfluidic chips, inkjet printing, and electrospray17,18,19,20,21,22,23. Microfluidic chips have been proven to be fast and efficient in producing uniform-sized microcarriers24. However, this technology relies on a complex flow channel design and fabrication process25. High temperature or excessive extrusion forces during inkjet printing, as well as intense electric fields in the electrospray approach, may adversely affect the properties of the material, especially its biological activity19. Besides, when applied to various biomaterials and diameters, the customized nozzles used in these methods result in limited processing complexity, high cost, and low flexibility.
To provide a convenient method for microcarrier preparation, a 3D printing technique called alternating viscous-inertial forces jetting (AVIFJ) has been applied to construct hydrogel microcarriers. The technique utilizes downward driving forces and static pressure generated during vertical vibration to overcome the surface tension of the nozzle tip and thus form droplets. Instead of severe forces and thermal conditions, small rapid displacements act directly on the nozzle during printing, causing a minor effect on the physicochemical properties of the bioink and presenting great attraction for bioactive materials. Utilizing the AVIFJ method, microcarriers of multiple biomaterials with diameters of 100-300 &#181;m were successfully formed. Besides, the microcarriers were further proven to bind cells well and provide a suitable growth environment for adhered cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>Human non-small cell lung cancer cell line</td>
		</tr>
		<tr>
			<td>Bright field microscope</td>
			<td>Olympus</td>
			<td>DP70</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>TI-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, FBS</td>
			<td>BI</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>SIGMA</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass micropipettes</td>
			<td>sutter instrument</td>
			<td>b150-110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>H-DMEM</td>
			<td>GIBCO</td>
			<td>11960-044</td>
			<td>Dulbecco&#39;s modified eagle medium</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase powder</td>
			<td>SIGMA</td>
			<td>P6782</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic agent</td>
			<td>3M</td>
			<td>PN7026</td>
			<td>Follow the manufacturer&#39;s instructions and use after dilution</td>
		</tr>
		<tr>
			<td>Micro-forge device</td>
			<td>narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids, NEAA</td>
			<td>GIBCO</td>
			<td>11140-050</td>
			<td>non-essential amino acids</td>
		</tr>
		<tr>
			<td>Penicillin G and streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SIGMA</td>
			<td>P5731-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>sutter instrument</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>SIGMA</td>
			<td>A0682</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>GIBCO</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Type I collagen solution from rat tail</td>
			<td>SIGMA</td>
			<td>C3867</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d printing of in vitro hydrogel microcarriers by alternating viscous-inertial force jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

Printheads of varied convergence rates and diameters were fabricated to achieve the printing of multiple types of materials. The nozzles obtained with increasing pull strength are shown in Figure 1B. The nozzles were divided into three areas: reservoir (III), contraction (II), and printhead (I). The reservoir was the unprocessed part of the nozzle, in which the liquid provided static pressure and bioink input for printing. The contraction area was the main part for generating downward drivin...

Watch this Scientific Journal Video about 3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting at JoVE.com

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

We offer a mild bioprinted process for preparing microcarriers with a good controllability and biocompatibility. After encapsulation by cells, the carriers can also be used in in vitro cell expansion and functional microtissue construction. Limited displacement, rather than severe forces under similar conditions act on the nozzle during the printing process, maintaining the original physical/chemical properties of the bio-ink to the maximum extent.In addition to conventional microcarriers, units with internal cell distribution or tritosin-encapsulated capsule structures can be constructed and applied to the assembly of different tissue structures. For the nozzle preparation, load glass micropipettes onto the puller according to the manufacturer's instructions and set the pulling parameters for the puller. Use a microforge to cut off the nozzle at the designated diameter to obtain the specific tip diameter for the experiment, and sterilize the nozzle in alcohol for five minutes.Then, rinse the nozzle three times with sterile water to remove any residual alcohol. To prepare the hydrogel bio-ink, dilute sterilized 4%sodium alginate stock solution in 0.9%sodium chloride at 0.5, 1, 1.5, and 2%weight-by-volume concentrations. For microdroplet formation, load five milliliters of bio-ink into a disposable sterile syringe and install the syringe onto a syringe pump.Using a one-millimeter inner-diameter hose, connect the syringe and printing nozzle. Tighten the clamping screw to secure the nozzle into place and rapidly depress the syringe pump to load the bio-ink into the nozzle. Set the signal generator parameters and preset the motion path and trigger mode of the vibration to drop on demand.Then print the droplets following the pre-designed patterns. For microcarriers formation, add five milliliters of crosslinking solution to a Petri dish, and place the dish under the printing nozzle as the substrate. After printing, crosslink the microcarriers for three minutes before transferring the microcarrier suspension to a centrifuge tube.Then, enrich the microcarriers by centrifugation and resuspend them in an appropriate culture medium at approximately 600 microcarriers per milliliter. Before their inoculation, replace the supernatant of an A549 cell culture with five milliliters of serum-free medium supplemented with 10-micromolar cell tracker green CMFDA dye for a 30-minute incubation in the cell culture incubator. At the end of the incubation, replace the dye solution with fresh medium and resuspend the cells at a density of 1.6 times 10 to the six cells per milliliter of medium.Add one milliliter of microcarrier suspension to the cells and one milliliter of A549 cells to each well of a low-adherent six-well culture plate. Place the plate onto a shaker in the cell culture incubator at 30 revolutions per minute. At the end of the incubation, remove the plate from the shaker and allow the microcarriers to settle for 30 minutes before observing and measuring the printing nozzle, Petri dishes, and microcarrier-inoculated cells by bright-field and confocal fluorescence microscopy.Using a 30-micrometer-tip nozzle, multiple types of bio-ink, including PBS, 1.5%alginate, and 1.5%gelatin can be stably printed onto Petri dishes. As the inks increase in viscosity, the printing process becomes increasingly more difficult and greater driving forces are needed to obtain larger droplets. The printing parameters can be set to print the droplets in specific 2D arrangements, as observed in this printing experiment.The diameter of the nozzle tip has a direct impact of the size of microcarrier that can be printed. As observed in these bright-field and confocal images, A549 cells adhere to the alginate-collagen microcarriers after two days of culture. After six days, the A549 cells almost fully cover the microcarrier surfaces.Following this procedure, we have coated the microcarrier surface with chitosan, and used gluconate to dissolve the alginate gel to form a cystic structure that can be applied to construct hollow structures such as alveoli. The printing process for preparing microcarriers is quite gentle, thus, this method can be widely used for printing cell-containing microcarriers.

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JoVE Journal

Bioengineering

2.7K visualizações.  Tsinghua University. Oferecemos um leve processo bioimpresso para preparar microcarriers com boa controlabilidade e biocompatibilidade. Após o encapsulamento por células, os portadores também podem ser usados na expansão celular in vitro e na construção de microtissue funcionais. O deslocamento limitado, em vez de forças severas em condições semelhantes, atuam no bocal durante o processo de impressão, mantendo as propriedades físicas/químicas originais da bioindálua na medida máxima.Além das...