This study was supported in part by the grants from the Mayo Clinic Center for Regenerative Medicine, J. W. Kieckhefer Foundation, Al Nahyan Foundation, Regenerative Medicine Minnesota (RMM 101617 TR 004), and NIH (DK107255).

1. Device fabrication
<ol>
	<li>Make the designs for the microencapsulation device and dissociation device using CAD software10,11.</li>
	<li>Spin-coat the three layers of SU-8 photoresist sequentially on a silicon wafer (Figure 2A) to achieve structures with the desired heights: 60, 100, and 150 &#181;m. 
	NOTE: The process for the top and bottom molds is identical.
	<ol>
		<li>Spin coat a clean 10 cm silicon wafer with SU-8 2025 negative photoresist at 1,100 rpm to create the first 60 &#181;m layer. After a soft bake at 65 &#176;C for 3 min and 95 &#176;C for 10 min on a hot plate, expose the mold using a maskless aligner by uploading the design file of the core channel pattern to the &#181;PG 101 PC. Then, proceed with the post-exposure bake at 65 &#176;C for 3 min and at 95 &#176;C for 10 min.</li>
		<li>Spin coat the second SU-8 2025 layer (40 &#181;m) at 1,500 rpm and expose by uploading the appropriate CAD file for the shell channel pattern. 
		NOTE: Soft and post-exposure bakes were similar to the previous step.</li>
		<li>Spin coat the third SU-8 2025 layer at 1,400 rpm to achieve 50 &#181;m height and repeat the above process but expose the structures for the oil phase.</li>
		<li>Develop the mold with all three layers in a single step by submersion in SU-8 developer until all the unexposed photoresist is removed.</li>
		<li>Hard bake the mold on a hot plate at 160 &#176;C for 10 min to improve adhesion and seal minor cracks that could appear after the development.</li>
		<li>Expose the mold to chlorotrimethylsilane to avoid elastomer bonding in the next step and place it in 15 cm Petri dishes until use.</li>
	</ol>
	</li>
	<li>Prepare the dissociation device mold in the same manner, as described in Figure 1D and Figure 2B. Spin coat one layer of SU-8 photoresist on a silicon wafer, as described earlier in step 1.2.3, to achieve structures with the desired height: 50 &#181;m. Conduct the soft and post-exposure bake, development, and hard bake similarly to the previous step. 
	NOTE: An array of triangular-shaped posts covered the whole chamber, with spacing between posts decreasing as chamber width decreased toward the outlet. Triangle posts were 200 &#181;m per side with a pitch ranging from 400 &#181;m (at the inlet) to 30 &#181;m (at the outlet).</li>
	<li>Prepare the molds by soft lithography using polydimethylsiloxane (PDMS).
	<ol>
		<li>Mix PDMS elastomer base and elastomer curing agent in a 10:1 w/w ratio in a planetary centrifuge. Pour the mixture onto both microencapsulation master molds and dissociation master mold to create a 3-4 mm layer. 
		NOTE: A mixture of 50 g of elastomer base with 5 g of curing agent is sufficient to cover a master mold with 3 mm thickness.</li>
		<li>Place the Petri dishes containing the molds in a vacuum desiccator for 15 min to de-gas the uncured PDMS.</li>
		<li>Move the Petri dishes to an oven and bake at 80 &#176;C for a minimum of 60 min.</li>
		<li>Remove the master molds from the oven and allow them to cool down. Using a precision knife, carefully cut the PDMS following the shape of the wafer and peel out the PDMS pieces from the master mold.</li>
		<li>Cut out the PDMS slabs by trimming the edges of each device with a scalpel, then punch out inlet/outlet fluidic ports in the dissociation device and only in the top microfluidic device using stainless steel needles. Cover the devices with magic tape to avoid contamination. 
		NOTE: The needle gauge is 14 G and 15 G for inlet and outlet ports, respectively.</li>
		<li>For bonding, treat the patterned sides of the top and bottom PDMS pieces with O2 plasma on a plasma etcher for 2 min.</li>
		<li>Align both PDMS parts under a stereoscope after adding a thin separating layer of distilled water directly in the microchannels (2 &#181;L).</li>
		<li>Place the aligned PDMS device in the oven at 80 &#176;C overnight to complete the bonding and restore the PDMS to its original hydrophobic state. Store the devices until further use. 
		NOTE: This results in a coaxial flow-focusing device with three different heights of 120 &#181;m for core, 200 &#181;m for shell, and 300 &#181;m for oil channels.</li>
		<li>In order to use the device right after the plasma bonding, carefully but firmly inject 30 &#181;L of hydrophobic coating solution into the fluidic channels to achieve hydrophobic coating. Then, immediately purge air into the channels until no residues of the hydrophobic coating solution are observed in the device.</li>
		<li>Bond the dissociation device by first treating the patterned side of the device and a cleaned slide glass with O2 plasma for 2 min, and then assemble them for further curing in the oven at 80 &#176;C overnight. 
		&#8203;NOTE: Devices can be stored until further use.</li>
	</ol>
	</li>
</ol>
2. Preparation of solutions
<ol>
	<li>Stock solutions. First, prepare stock solutions (concentrated solutions) that will be diluted to lower concentrations for actual use. 
	NOTE: Stock solutions are used to save preparation time, conserve materials, reduce storage space, and improve accuracy when preparing a working solution in lower concentrations.
	<ol>
		<li>Prepare 30 mL of 2x core solution (16% PEG and 34% density gradient media): mix 4.8 g of 35 kDa PEG, 10.2 mL of density gradient media, and 19.8 mL of DMEM/F12 media. Filter this core solution using a 0.22 &#956;m syringe filter.</li>
		<li>Prepare 50 mL of mineral oil with 3% surfactant: mix 48.5 mL of mineral oil and 1.5 mL of surfactant. Keep in a sterile condition after filtration through a 0.22 &#181;m syringe filter.</li>
		<li>Prepare 50 mL of mineral oil with 0.5% surfactant: mix 49.75 mL of mineral oil and 0.25 mL of surfactant. Keep in a sterile condition.</li>
		<li>Prepare 1 M Dithiotheritol (DTT): dissolve 1.5425 g of DTT in 10 mL distilled water. Keep in a sterile condition.</li>
		<li>Prepare 1 M Triethanolamine (TEA): add 66.4 &#181;L of TEA in 433.6 &#181;L of distilled water. Keep in a sterile condition.</li>
		<li>Prepare 1% Pluronic F127 (PF127): dissolve 100 mg of PF127 in 10 mL of distilled water. Keep in a sterile condition.</li>
	</ol>
	</li>
	<li>Working solutions
	<ol>
		<li>Prepare a crosslinker emulsion by mixing 4.5 mL of mineral oil with 3% surfactant and 300 &#181;L of 1 M DTT (1:15 ratio). Vortex the solution for 2 min and sonicate for 45-60 min in an ultrasonic bath at 20 &#176;C. Load this solution to a 5 mL syringe with a 27 G needle. 
		NOTE: Emulsion is generated by sonicating oil/water mixture.</li>
		<li>Prepare shielding oil solution by loading the mineral oil with 0.5% surfactant to a 5 mL syringe with a 27 G needle.</li>
		<li>Prepare shell (8% w/v PEG-4-Mal and 15 mM TEA) solution by adding 32 mg of PEG-4-Mal in 400 &#181;L of 1x DPBS to form 8% w/v solution, and vortex the solution for 2 min. Then, add 6 &#181;L of 1 M TEA (final concentration 15 mM TEA). Finally, centrifuge at 13,000 x g for 5 min through a spin-filter and keep it in the dark on the rocker for 30 min. Then, load this solution to a 1 mL syringe with a 27 G needle. 
		NOTE: Leave the PEG-4-Mal container to sit at room temperature for 10 min before opening the lid, and blow N2 gas to replace the O2 with N2 before closing the lid. Vortex TEA before adding to the solution.</li>
	</ol>
	</li>
	<li>Prepare the cell suspension in core solution
	<ol>
		<li>Treat the hPSCs with trypsin for 5 to 10 min at 37 &#176;C. Then, collect them from plates. Quench the trypsin with the medium after cell detachment, and then transfer the cells to a 15 mL conical tube.</li>
		<li>Centrifuge the bulk cell suspension (400 x g, 5 min). Carefully aspirate the supernatant and then resuspend the cell pellet using a minimal volume of growth medium. Count the cells using an automated cell counter. 
		NOTE: Typical cell aliquots contain 12-20 x 106 cells and are enough to make 400 &#181;L of the core solution.</li>
		<li>Take 12-20 x 106 cells from the bulk cell suspension and centrifuge the cell/media suspension (400 x g, 5 min).</li>
		<li>Carefully aspirate media from the cell pellet. Then, add 200 &#181;L media and 200 &#181;L of 2x PEG solution (final concentration 30-50 x 106 cell/mL) and mix gently. 
		NOTE: Low cell concentrations (less than 20 x 106 cells/mL) resulted in many empty capsules.</li>
		<li>Add 30 &#181;L of 1% PF127 to the solution.</li>
		<li>Insert a micro stirrer bar (2 mm x 7 mm) into a 1 mL syringe, and then load the cell containing core solution to the syringe with a 27 G needle. Keep the solution cold with ice during the whole encapsulation process.</li>
	</ol>
	</li>
</ol>
3. Experimental setup
<ol>
	<li>Place bonded PDMS droplet device, four pieces of 20 cm long and one piece of 5 cm long inlet micro tubes (0.38 mm I.D. x 1.1 mm O.D.), one piece of 10 cm outlet tube (0.5 mm I.D. x 1.5 mm O.D.), and six pieces of 0.5 cm fitting tubes (1 mm I.D. x 1.8 mm O.D.) beneath a germicidal light source (typically built into biological safety cabinets) and sterilize for 30 min.</li>
	<li>Use forceps to fit the tubing into the fluidic inlet ports and the dissociation ports.</li>
	<li>Use the three pieces of 20 cm long inlet microtubes for the shell, oil, crosslinker emulsion inlets, and one piece for the inlet of the dissociation device. Then, insert the opposite end of the tubes to the needle of the syringe with the corresponding solution. Meanwhile, connect the core inlet of the microfluidic device and the outlet of the dissociation device with one 5 cm microtube (Figure 1C). 
	NOTE: The inlet microtubes should be tightly secured within the fitting tubes, which were inserted in the last step.</li>
	<li>Insert one outlet tube into the fluidic outlet port and place the opposite end into a collection reservoir.</li>
	<li>Place the device on a microscope stage and attach the tubing to avoid moving. Align and focus the microscope on the device nozzle for flow inspection.</li>
	<li>Place each syringe on different syringe pumps and secure properly (Figure 1B). Program each pump according to manufacturer&#39;s protocols to the following flow rates: core (3-5 &#181;L/min), shell (3-5 &#181;L/min), shielding oil (20-80 &#181;L/min), and crosslinking oil (40-60 &#181;L/min). Start the flow in all pumps.</li>
	<li>Wait for each fluid to enter the device and fill up the channels while pushing out the remaining air. 
	NOTE: If there is a large amount of air in the inlet tubing, temporarily increase each flow rate until the air is completely removed. Be mindful that excessive flow rate could lead to PDMS-to-PDMS bond failure.</li>
	<li>When the capsule formation is stabilized (Figure 3), place the opposite end of the collection tube to a new conical tube with 5 mL of mTeSR media. 
	NOTE: The media contains 10 &#956;M ROCK inhibitor, 100 U/mL penicillin, and 100 &#956;g/mL streptomycin.</li>
	<li>After a sufficient number of capsules are collected, incubate at 37 &#176;C with 5% CO2 for 10 min. Capsules residing in the oil phase will be at the top of the tube (see Figure 4A). Gently tapping on the tube causes capsules to sediment to the bottom. After this, most of the oil and media may be aspirated.</li>
	<li>Collect the capsules from the bottom of the conical tube, place them on a cell strainer (100 &#956;m pore size), and wash with copious amounts of media. Then, collect microcapsules using 2 mL media in a 6-well culture plate by running the media through the filter as it is inverted on top of the well-plate (see Figure 4A).</li>
</ol>
4. hPSC culture and analysis in microcapsules
<ol>
	<li>Basic culture
	<ol>
		<li>Incubate the stem cell capsules in a 6-well plate culture dish at 37 &#176;C with 5% CO2.</li>
		<li>Change the media every other day by collecting the capsules on a cell strainer filter, washing them with excess media, and resuspending them to a new well in 6-well plates with 2 mL media as was done in step 3.10.</li>
		<li>Culture the capsules for at least 10 days.</li>
	</ol>
	</li>
	<li>Perform the live/dead assay to determine viability of encapsulated stem cells.
	<ol>
		<li>Thaw live/dead assay solutions (ethidium homodimer-1 and calcein AM).</li>
		<li>Add 4 &#181;L of the 2 &#956;M ethidium homodimer-1 and 1 &#181;L of the 4 &#956;M calcein AM solutions to 2 mL of sterile DPBS. Vortex to ensure complete mixing.</li>
		<li>Collect approximately 100 microcapsules on a cell strainer and rinse them with sterile DPBS to allow diffusion of medium out from the capsules.</li>
		<li>Collect them again to a 12 well culture plate by using 1 mL of the solution prepared in step 4.2.2. Incubate at 37 &#176;C for 20 min.</li>
		<li>Visualize the cells under a fluorescence microscope. 
		NOTE: Cell viability is quantified by calculating the percentage of live cells, which are visualized as green, among the total number of cells.</li>
	</ol>
	</li>
	<li>Spheroid size characterization.
	<ol>
		<li>When desired, place the well plate with microcapsules under the microscope and measure the diameter of spheroids shown on the related software with proper scale bars.</li>
		<li>Measure and analyze the spheroid size.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zhu, Z., Huangfu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cells:+an+emerging+model+in+developmental+biology.">Human pluripotent stem cells: an emerging model in developmental biology.</a> Development. 140 (4), 705-717 (2013).</li><li>Liu, G., David, B. T., Trawczynski, M., Fessler, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+pluripotent+stem+cells:+history,+mechanisms,+technologies,+and+applications.">Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.</a> Stem Cell Reviews and Reports. 16 (1), 3-32 (2020).</li><li>Chan, S. W., Rizwan, M., Yim, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+methods+for+enhancing+pluripotent+stem+cell+expansion.">Emerging methods for enhancing pluripotent stem cell expansion.</a> Frontiers in Cell and Developmental Biology. 8, 70 (2020).</li><li>Lei, Y., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fully+defined+and+scalable+3D+culture+system+for+human+pluripotent+stem+cell+expansion+and+differentiation.">A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (52), 5039-5048 (2013).</li><li>Olmer, R., 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=Suspension+culture+of+human+pluripotent+stem+cells+in+controlled,+stirred+bioreactors.">Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors.</a> Tissue Engineering Part C: Methods. 18 (10), 772-784 (2012).</li><li>Kraehenbuehl, T. P., Langer, R., Ferreira, L. S. <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+biomaterials+for+the+study+of+human+pluripotent+stem+cells.">Three-dimensional biomaterials for the study of human pluripotent stem cells.</a> Nature Methods. 8 (9), 731-736 (2011).</li><li>Fattahi, 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=Core-shell+hydrogel+microcapsules+enable+formation+of+human+pluripotent+stem+cell+spheroids+and+their+cultivation+in+a+stirred+bioreactor.">Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor.</a> Scientific Reports. 11 (1), 1-13 (2021).</li><li>Siltanen, 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=Microfluidic+fabrication+of+bioactive+microgels+for+rapid+formation+and+enhanced+differentiation+of+stem+cell+spheroids.">Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids.</a> Acta Biomaterialia. 34, 125-132 (2016).</li><li>Agarwal, 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=One-step+microfluidic+generation+of+pre-hatching+embryo-like+core-shell+microcapsules+for+miniaturized+3D+culture+of+pluripotent+stem+cells.">One-step microfluidic generation of pre-hatching embryo-like core-shell microcapsules for miniaturized 3D culture of pluripotent stem cells.</a> Lab on a Chip. 13 (23), 4525-4533 (2013).</li><li>Siltanen, 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=One+step+fabrication+of+hydrogel+microcapsules+with+hollow+core+for+assembly+and+cultivation+of+hepatocyte+spheroids.">One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids.</a> Acta Biomaterialia. 50, 428-436 (2017).</li><li>Rahimian, A., Siltanen, C., Feyzizarnagh, H., Escalante, P., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulated+immunoassays+for+detection+of+cytokines+in+human+blood.">Microencapsulated immunoassays for detection of cytokines in human blood.</a> ACS Sensors. 4 (3), 578-585 (2019).</li><li>Kim, M., Lee, J., Jones, C. N., Revzin, A., Tae, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-based+hydrogel+as+a+matrix+for+encapsulation+and+cultivation+of+primary+hepatocytes.">Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes.</a> Biomaterials. 31, 3596-3603 (2010).</li><li>Shin, D. 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=Photodegradable+hydrogels+for+capture,+detection,+and+release+of+live+cells.">Photodegradable hydrogels for capture, detection, and release of live cells.</a> Angewandte Chemie International Edition. , (2014).</li><li>You, 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=Bioactive+photodegradable+hydrogel+for+cultivation+and+retrieval+of+embryonic+stem+cells.">Bioactive photodegradable hydrogel for cultivation and retrieval of embryonic stem cells.</a> Advanced Functional Materials. , (2015).</li></ol>

The authors have nothing to disclose.

The encapsulation process described here results in reproducible formation of hPSC spheroids. The microcapsule format makes it easy to dispense spheroids into wells of a microtiter plate for experiments aimed at improving/optimizing differentiation protocols or testing therapies. Encapsulated stem cell spheroids may also be used in suspensions cultures where hydrogel shell protects cells against shear-induced damage7.
There are several critical steps within the protocol...

There is considerable interest in 3D cultures of human pluripotent stem cells (hPSCs)&#160;due to the improved pluripotency and differentiation potential afforded by this culture format1,2,3. hPSCs are typically formed into spheroids or other 3D culture formats by means of bioreactors, microwells, hydrogels, and polymeric scaffolds4,5,6. Encapsulation offers another means for organizing single hPSCs into spheroids. Once encapsulated hPSC spheroids may be handled with ease and transferred into microtiter plates for differentiation, disease modeling, or drug testing experiments. Encasing hPSCs in a hydrogel layer also protects cells against shear damage and allows to culture spheroids in a bioreactor at high rates of stirring7.
Our methodology for stem cell encapsulation evolved over time. First, we focused on solid hydrogel microparticles and demonstrated successful encapsulation and cultivation of mouse embryonic stem cells (mESCs)8. However, it was noted that human embryonic stem cells (hESCs) had low viability when encapsulated in such hydrogel microparticles, presumably due to the greater need for these cells to re-establish cell-cell contacts after the encapsulation. We reasoned that heterogeneous microcapsule, possessing an aqueous core, may be better suited for encapsulation of cells that rely on rapid re-establishment of cell-cell contacts. The concept of co-axial flow focusing microfluidic device for making aqueous core/hydrogel shell microcapsules was adapted from He et al.9, but instead of alginate employed in the original approach, a PEG-based hydrogel was incorporated into the shell. We first demonstrated successful encapsulation and spheroid formation of primary hepatocyte in core-shell microcapsules10 and most recently described encapsulation of hES and iPS cells7. As outlined in Figure 1A, capsules are fabricated in a flow focusing device where the shell and core flow streams transition from side-to-side to co-axial flow before ejection into the oil phase. The core flow contains cells and additives that increase the viscosity of the solution (non-reactive PEG MW 35kD and iodixanol - commercial name OptiPrep) while the shell stream contains reactive molecules (PEG-4-Mal). Continuous co-axial flow stream is discretized into droplets that retain core-shell architecture. The core-shell structure is made permanent by exposure to di-thiol crosslinker (DTT), which reacts with PEG-4-Mal via click chemistry and results in formation of a thin (~10 &#181;m) hydrogel skin or shell. After the emulsion is broken and capsules are transferred into an aqueous phase, molecules of PEG diffuse from the core and are replaced by water molecules. This results in aqueous core and hydrogel shell microcapsules.
Provided below are step-by-step instructions on how to make microfluidic devices, how to prepare cells, and how to carry out encapsulation of hPSCs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Syringe Filters</td>
			<td>Genesee Scientific</td>
			<td>25-244</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe luer-lock tip</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1x DPBS</td>
			<td>Corning</td>
			<td>23220003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-arm PEG maleimide, 10kDa</td>
			<td>Laysan Inc.</td>
			<td>164-68</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe luer-lock tip</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>6-WELL NON-TREATED PLATE</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquapel Applicator Pack</td>
			<td>Aquapel Glass Treatment</td>
			<td>47100</td>
			<td></td>
		</tr>
		<tr>
			<td>CAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD v2020</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL STRAINER 100 &#181;m pore size</td>
			<td>cardinal</td>
			<td>335583</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane</td>
			<td>Aldrich</td>
			<td>386529-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Life technology</td>
			<td>A27974</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital hot plate</td>
			<td>Dataplate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>215370</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Gibco</td>
			<td>15230-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiotheritol (DTT)</td>
			<td>Sigma</td>
			<td>D0632-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Fisher scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand accuSpin Micro 17 Microcentrifuge</td>
			<td>live</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACELL VIOS 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Motorized Microscope</td>
			<td>Olympus</td>
			<td>Olympus IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Laurell Spin Coaters</td>
			<td>Laurell Technologies</td>
			<td>WS-650MZ-23NPPB</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead mammalian staining kit</td>
			<td>Fisher</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Magic tape</td>
			<td>Staples</td>
			<td>483535</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Medical Tubing (0.015&#34; I.D. x 0.043&#34; O.D.)</td>
			<td>Scientific Commodities, Inc</td>
			<td>BB31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro stir bar</td>
			<td>Daigger Scientific</td>
			<td>EF3288E</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Filter Forceps</td>
			<td>Fisher scientific</td>
			<td>XX6200006P</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M8410-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR 1 Basal Medium</td>
			<td>STEMCELL TECHNOLOGY</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles-Stainless Steel&#160; 14 Gauge</td>
			<td>CML supply</td>
			<td>901-14-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles-Stainless Steel&#160; 15 Gauge</td>
			<td>CML supply</td>
			<td>901-15-050</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>STEMCELL TECHNOLOGY</td>
			<td>7820</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Thermo Scientific</td>
			<td>HERA THERM Oven</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin:Streptomycin (10,000 U/mL Penicillin G, 10mg/mL Streptomycin)</td>
			<td>Gemini</td>
			<td>400-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 150X20 Sterile Vent</td>
			<td>Sarstedt, Inc.</td>
			<td>82.1184.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaning System</td>
			<td>Yield Engineering System, Inc.</td>
			<td>YES-G500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) 35kDa</td>
			<td>Sigma</td>
			<td>94646-250G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 27G</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock inhibitor Y-27632 dihydrocloride</td>
			<td>SELLECK CHEM</td>
			<td>S1049-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer 100mm</td>
			<td>University Wafer</td>
			<td>452</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide glass (75mm &#180; 25mm)</td>
			<td>CardinalHealth</td>
			<td>M6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Span 80</td>
			<td>Sigma</td>
			<td>S6760-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedMixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-X Centrifuge Tube Filter (0.22 &#181;m)</td>
			<td>Costar</td>
			<td>8160</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2025</td>
			<td>Kayaku Advanced Materials</td>
			<td>Y111069 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>Y020100 4000L1PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design Royaltek Stainless Steel Surgical Scalpel Blades</td>
			<td>fisher scientific</td>
			<td>22-079-684</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD TM 184 Silicone Elastomer Kit (PDMS)</td>
			<td>Dow Corning</td>
			<td>2065622</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump System, Inc</td>
			<td>NE-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine</td>
			<td>Sigma-aldrich</td>
			<td>T58300-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Tubing (0.02&#34; I.D. x 0.06&#34; O.D.)</td>
			<td>Cole-Parmer</td>
			<td>06419-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Tubing (0.04&#34; I.D. x 0.07&#34; O.D.)</td>
			<td>Cole-Parmer</td>
			<td>06419-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner FS20D</td>
			<td>Fisher Scientific</td>
			<td>CPN-962-152R</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Bel-Art</td>
			<td>F42025-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Stemi DV4 Stereo Microscope 8x-32x</td>
			<td>ZEISS</td>
			<td>435421-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;PG 101 laser writer</td>
			<td>Heidelberg Instruments</td>
			<td>HI 1128</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic fabrication of core-shell microcapsules carrying human pluripotent stem cell spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

By following the above-mentioned protocol, the reader will be able to fabricate microfluidic devices and produce cell-carrying microcapsules. Figure 3A shows examples of optimal and suboptimal microcapsules fabricated using microfluidic droplet generation. Different formulations of PEG-4-Mal resulted in capsules of varying morphologies - wrinkled capsules were associated with poor gelation, low mechanical integrity, and did not withstand cultivation in a stirred bioreactor. Smooth capsules o...

Watch this Scientific Journal Video about Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids at JoVE.com

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

microfluidic-fabrication-core-shell-microcapsules-carrying-human

Research

JoVE Journal

Bioengineering

2.7K Views.  Mayo Clinic. This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

Video: Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

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 ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...