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>

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	<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. 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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><tbody><tr><td>0.22 &amp;micro;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 &amp;micro;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&quot; I.D. x 0.043&quot; 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&amp;nbsp; 14 Gauge</td><td>CML supply</td><td>901-14-025</td><td></td></tr><tr><td>Needles-Stainless Steel&amp;nbsp; 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 &amp;acute; 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 &amp;micro;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&quot; I.D. x 0.06&quot; O.D.)</td><td>Cole-Parmer</td><td>06419-01</td><td></td></tr><tr><td>Tygon Tubing (0.04&quot; I.D. x 0.07&quot; 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>&amp;mu;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...

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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

2.8K Views.  Mayo Clinic. Het gebied wordt beschouwd als van belang in 3D-culturen van menselijke pluripotente stamcellen vanwege de verbeterde pluripotentie en het differentiatiepotentieel dat deze cultuur heeft gevormd. We beschreven onze inkapselingsstrategie die leidt tot reproduceerbare vorming van menselijke pluripotente stamcellen sferoïden. Eenmaal ingekapseld, zijn deze stamcelsferoïden vatbaar voor teelt, hetzij in multi-well platen of geroerde bioreactoren.Onze inkapselingsstrategie is het optimali...

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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Developmental Biology

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

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.

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

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