This work was financially supported by the National Natural Science Foundation of China (Grants: 81522022, 51461165302). The authors would like to acknowledge all Du lab members for general assistance.

Animal experiments followed strict protocol approved by the Animal Ethics Committee on the Center of Biomedical Analysis, Tsinghua University. Under approval of Ethics Committee, human adipose tissue was obtained from Department of Plastic Surgery of Peking Union Hospital with informed consent from the patients.
1. Fabrication of 3D Microcryogels
<ol>
	<li>Design and fabrication of microstencil array chips
	<ol>
		<li>Use a commercial software to design arrays of specific geometries, such as circles, ellipses, triangles or clovers14, depending on subsequent application. 
		NOTE: Refer to section 2 of the protocol for regenerative medicine and section 3 of protocol for drug screening for design details.</li>
		<li>Import the design into the laser engraving software accompanying the laser engraving machine. Using the laser settings according to the factory recommendations for the laser engraving machine, laser-engrave the imported design onto Poly (methymethacrylate) (PMMA) sheets.</li>
		<li>Wash the microstencil array chips with deionized water to clean debris from the laser engraving. Dry the microstencil array chips at 60 &#176;C. Store in a sealed bag at room temperature for months.</li>
	</ol>
	</li>
	<li>Fabrication of microcryogel array chips
	<ol>
		<li>Place 4 microstencil array chips on the sample tray and insert into the plasma cleaner. Close the door of the cleaner and turn on the vacuum pump for 2 min, then turn on the maximum RF power (18 W) to treat the microstencil array chips in the plasma cleaner for 3 min at room temperature to increase hydrophilicity.</li>
		<li>Add 0.06 g gelatin to 1 mL deionized water to form 6% (wt/vol) gelatin precursor solution (enough to make 5 chips). Warm at 60 &#176;C in a water bath to dissolve adequately. Incubate on ice for 5 min.</li>
		<li>Add 3 &#181;L of glutaraldehyde into the gelatin precursor solution to a final concentration of 0.3%. Mix thoroughly.</li>
		<li>Pipette 200 &#181;L precursor solution with glutaraldehyde onto the upper surface of the microstencil array chip. 
		NOTE: 200 &#181;L is sufficient for a 75 mm &#215; 25 mm chip regardless of design. Increase the amount proportionally when surface area of chip is increased.</li>
		<li>Manually distribute the solution evenly over the chip by scraping back and forth with a bent glass rod 2 to 3 times. 
		NOTE: Each micro-well on the microstencil chip can be filled with precursor solution due to their hydrophilic nature after plasma treatment.</li>
		<li>Immediately place the precursor-solution-filled array chip in a -20 &#176;C freezer for 16 h for cryogelation.</li>
		<li>Adjust the lyophilizer to -40 &#176;C for 30 min. Place the array chips after cryogelation in the lyophilizer and lyophilize the array chips for 2 h in vacuum. 
		NOTE: Gelatin microcryogels with interconnected macropores are formed because ice formed during cryogelation sublimes in the lyophilizer.</li>
		<li>Proceed to section 2 for treatment of critical limb ischemia (CLI) and section 3 for high-throughput drug screening.</li>
	</ol>
	</li>
</ol>
2. Harvesting Individual Microcryogels to Form Injectable 3D Microtissues for Treatment of CLI
<ol>
	<li>Harvesting individual microcryogels
	<ol>
		<li>Design arrays (75 mm &#215; 25 mm) of 600 circles, each with 400 &#181;m diameter, in commercial software for injectable 3D microtissue construction. Use PMMA of 300 &#181;m thickness for fabrication of microstencil array chip as detailed in section 1.1.</li>
		<li>Fabricate microcryogel array chips as in section 1.2.</li>
		<li>Fabricate a PDMS Ejector Pin array with the same design as in step 2.1.1 by standard soft lithography16,17.</li>
		<li>Overlay the microcryogel array chip on top of the PDMS Ejector Pin array, aligning each microcryogel with an ejector pin on the array. Press the microcryogel array chip towards the ejector pin array to push the microcryogels out of the microwells with the protruding pins on the PDMS ejector pin array to harvest individual microcryogels.</li>
		<li>Harvest the microcryogel into water and collect them with the aid of cell strainers. Use one strainer to collect microcryogels from one chip (i.e., 600 microcryogels).</li>
		<li>Wash microcryogels with 0.1 M NaBH4 on ice for 20 min to quench any uncross-linked aldehyde residue. Use 5 mL of 0.1 M NaBH4 per chip. Discard NaBH4 and wash with 5 mL deionized water for 3 to 5 times, 15 min each time.</li>
		<li>Discard water from the cell strainer and gather microcryogels into one cluster per cell strainer and place one cluster in one 35-mm Petri dish using a curved tweezer. Add 50 - 70 &#181;L deionized water to each cluster and cover the lid of the Petri dish. Gently tap the Petri dish on the tabletop to level out all microcryogels, so that no microcryogels are lying on top of another microcryogel to form a monolayer of microcryogels on the surface of the Petri dish.</li>
		<li>Freeze the harvested microcryogels at -20 &#176;C for 4 to 16 h before lyophilizing them for 2 h(see step 1.2.7).Store microcryogels in a vacuum at room temperature until further use.</li>
	</ol>
	</li>
	<li>Characterization of microcryogels
	<ol>
		<li>Evaluate the pores of the microcryogels by scanning electron microscopy (SEM) imaging.
		<ol>
			<li>Immobilize the harvested and lyophilized microcryogels onto a sample holder by double-side adhesive tape and coat with gold with a sputter coater for 90 s, before imaging by SEM11. Evaluate and analyze the distribution of diameters of pores in the microcryogel from seven different SEM images using image analysis software.</li>
		</ol>
		</li>
		<li>Weigh one cluster of 600 lyophilized microcryogels in a 35-mm Petri dish for the weight of dried gelatin microcryogels (GMs). Add 60 &#181;L deionized water to the cluster of lyophilized microcryogels and wait for 30 s for microcryogels to fully absorb water and swell. Weigh the swollen microcryogels.
		<ol>
			<li>Determine the equilibrium swelling ratio and porosity of microcryogels, as the ratio of weight of swollen microcryogels to that of dried gelatin microcryogels and the ratio of weight of held water in microcryogels to that of hydrated microcryogels, respectively13.</li>
		</ol>
		</li>
		<li>Measure injectability of microcryogels using a programmable syringe pump integrated with a digital force gauge14.
		<ol>
			<li>Stain 600 pieces of microcryogels by Trypan blue and suspend in 600 &#181;L 15% gelatin solution to achieve homogeneous distribution. Load 1 mL microcryogel suspension in 1 mL syringe. Inject the mixture through a 27-gauge needle attached to the 1 mL syringe using a programmable syringe pump at flow rate of 1 mL/min.</li>
			<li>Monitor real-time injection force by digital force gauge testing and plot the force-time curve. After injection, observe the integrity of microcryogels under a microscope. 
			NOTE: GMs suspended in 15% (wt/vol) gelatin solution can be smoothly injected and remained intact after injection.</li>
		</ol>
		</li>
		<li>Characterize the degradability of microcryogels.
		<ol>
			<li>First, measure the weight of one cluster of 600 lyophilized microcryogels in a 35-mm Petri dish as the dry weight. Then, immerse 600 lyophilized microcryogels in 2 mL 0.025% (vol/vol) Trypsin/EDTA.</li>
			<li>Collect microcryogels from the solution using a cell strainer at different time points (i.e., 10, 20, 30, 40, 50, 60, and 70 min). Wick away excess solution with a tissue. Measure the weight of microcryogels at different time points and calculate the degradation degree, as the ratio of weight of microcryogel at a certain time point to that of microcryogel at time zero.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Autoloading of cells into microcryogels to form 3D microtissues
	<ol>
		<li>Sterilize microcryogels from step 2.1.8 by ethylene oxide sterilization system with 12 h gas exposure followed by 12 h degassing under vacuum.</li>
		<li>Choose human adipose-derived mesenchymal stromal cells (hMSCs) for treatment of mouse ischemic hindlimb. Isolate cells by following procedures as previously reported18.</li>
		<li>Culture hMSCs in the growth medium containing 2% fetal bovine serum (FBS), 10 ng/mL epidermal growth factor (EGF), 10 ng/mL platelet-derived growth factor bb (PDGF-bb), 1X insulin transferrin selenium (ITS), 10-8 M dexamethasone, 10-4 M ascorbic acid 2-phosphate, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin in DMEM/F12. Passage the cells at the ratio of 1:3 when confluent. Use cells of passage 3 to 5 in the following experiments.</li>
		<li>Harvest hMSCs with trypsin and quantify the cell number using a Fuchs-Rosenthal counting chamber and resuspend to a density of 8 x 106 cells/mL in hMSCs growth medium.</li>
		<li>Pipette 60 &#181;L hMSCs suspension onto the monolayer of 600 microcryogels with diameter of 400 &#181;m in a 35-mm dish from section 2.1. Cells are automatically absorbed into the porous micro-structures of the microcryogels.</li>
		<li>Maintain in a humidified chamber and incubate at 37 &#176;C for 2 h to allow cells to attach. After 2 h of incubation, add 2 mL culture medium. Change the medium every 2 days. Culture hMSCs-loaded microcryogels for 2 days to form 3D microtissues.</li>
		<li>After 2 days culturing, pipette 100 hMSCs-loaded microcryogels per well on a 96-well plate, add 120 &#181;L of resazurin working solution prepared according to the manufacturer&#39;s instruction into each well. Incubate for 2 h at 37 &#176;C.
		<ol>
			<li>Detect fluorescence of resazurin metabolized by viable cells in a microplate reader with an excitation light wavelength of 560 nm and emission light wavelength of 590 nm. Establish a standard curve of cell number vs. fluorescence using hMSCs to interpolate cell number from fluorescence intensity for future experiment according to the kit protocol13.</li>
		</ol>
		</li>
		<li>Assess number of hMSCs in microcryogels from day 0 to day 4 using resazurin as described in step 2.3.7 from day 0 to day 4.</li>
		<li>Stain cells in microcryogels with 1:500 dilution of Calcein AM and 1:250 dilution of Propidium Iodide (known as live/dead staining) in phosphate buffered saline (PBS) and observe under fluorescence microscope or confocal fluorescence microscope.</li>
	</ol>
	</li>
	<li>Injection of 3D microtissues in vivo for treatment of CLI in mouse model
	<ol>
		<li>Establish critical hindlimb ischemia of female BALB/c nude mice as reported11,19 to determine the therapeutic effect of 3D microtissue-based therapy.</li>
		<li>Transfer the microtissues from step 2.3.6 using a 5-mL pipette into a cell strainer and filter away the culture medium.</li>
		<li>Resuspend the hMSCs-loaded 3D microtissues in 15% gelatin solution, at a density of 100 microcryogels per 100 &#181;L solution.</li>
		<li>Before surgery, sterilize surgical tools by autoclave and perform surgery in an animal operating room in an Animal Facility Center.</li>
		<li>Place the mouse into the anesthesia induction chamber containing 1-3% isoflurane in 100% oxygen at a flow rate of 1 L/min. Apply erythromycin on the eyes of the mouse to prevent drying. Through a 1-cm-long skin incision, ligate the femoral artery and its branches with 5-10 silk sutures and excise19.</li>
		<li>Inject indocyanine green (ICG) (0.1 mL of 100 &#181;g/mL) through the tail vein injection to monitor blood flow. Perform ICG fluorescence imaging using a fluorescence imaging system (here, homemade system) in the reflectance geometry11.</li>
		<li>Intramuscularly inject microtissues into three sites of the gracilis muscle around the artery incision using a 1 mL syringe with a 23-gauge needle.</li>
		<li>After surgery, keep the mouse warm with a heated pad in the recovery cage. Inject subcutaneously meloxicam to relieve the pain and monitor continuously until awake.</li>
		<li>After 28 days, monitor the therapeutic efficacy of 3D microtissue-treated limb by fluorescence imaging11. 
		&#160;Note: Inject subcutaneously meloxicam to relieve the pain when the mouse showed spontaneous limb amputation.</li>
		<li>Euthanize mice with carbon dioxide after completion of experiments. 
		NOTE: Here, euthanasia with carbon dioxide was performed according to strict protocol approved by the Animal Ethics Committee on the Center of Biomedical Analysis, Tsinghua University.</li>
	</ol>
	</li>
</ol>
3. Assembly of Microtissue Array Chip for High-throughput Drug Screening
<ol>
	<li>Assembly of microcryogel array for on-chip cell culture
	<ol>
		<li>Modify the design in section 1.1 according to conventional multi-well plate dimensions, i.e., for 384-multi-well format, design an array of 16 &#215; 24 wells (row by column), each of 2 mm diameter and 4.5 mm center-to-center spacing.</li>
		<li>Laser engrave onto a 500 &#181;m thick PMMA sheet as described in section 1.1.</li>
		<li>Fabricate microcryogels as described in section 1.2 using the 384-multi-well array chip obtained from step 3.1.2. This microcryogel-containing array is designated as the microcryogel array chip.</li>
		<li>Wash the microcryogel array chip with 50 mL 0.1 M NaBH4 to quench any residual aldehyde uncross-linked, then repeatedly rinse with 50 mL deionized water for 3 times, 2 h each time.</li>
		<li>Discard the water, freeze the microcryogel array chip at -20 &#176;C for 4 to 16 h before lyophilizing according to step 1.2.7.</li>
		<li>Modify the 384-multi-well array design in step 3.1.1 to contain 16 &#215; 24 wells, each of 3 mm diameter and 4.5 mm center-to-center spacing.</li>
		<li>Remove one side of the backing from a sheet of ultra-thin (10 &#181;m) biocompatible double-sided adhesive tape and paste it to one side of a piece of 3-mm-thick PMMA sheet. Laser engrave the design from step 3.1.4 onto this 3-mm-thick PMMA sheet according to section 1.1. This array is designated as the reservoir array.</li>
		<li>Align the microcryogel array chip with the reservoir array chip and adhere together tightly to assemble the 3D microcryogel array chip for on-chip cell culture. Sterilize by ultra-violet radiation for 1 h.</li>
		<li>Store 3D microcryogel array chips in vacuum at room temperature for further experiments.</li>
	</ol>
	</li>
	<li>Drug screening on 3D microtissue arrays
	<ol>
		<li>Fill up a wet box with 25 mL sterile de-ionized water to serve as a humidity chamber for cell culture. Pre-heat in a humidified 5% CO2 incubator to 37 &#176;C.</li>
		<li>Harvest non-small-cell lung cancer cells (NCI-H460) and hepatocellular carcinoma cells (HepG2) from the tissue culture plates according to standard protocol and re-suspend in culture media to a final density of 1.0 x 106 cells/mL. Mix thoroughly. 
		NOTE: RPM1640 with 10% FBS, 100 U/mL penicillin and 100 &#181;g/mL streptomycin is used for NCI-H460. Use DMEM with 10% FBS, 100 U/mL penicillin and 100 &#181;g/mL streptomycin for HepG2.</li>
		<li>Remove the humidity chamber from incubator. Use tweezers to carefully place the assembled 3D microcryogel array chip from step 3.1.9 in the humidity chamber. Take caution not to wet the microcryogels with water in the chamber.</li>
		<li>Mix the cell suspension thoroughly, then aliquot 3 &#181;L of cell suspension directly onto the microcryogel into each well. 
		NOTE: The pipette tip should lightly touch the surface of the microcryogel before expelling cell suspension. Cells are auto-loaded into the microcryogel by absorption. Do not seed cells in the peripheral wells.</li>
		<li>Add 10 &#956;L of media to each well after cells are seeded into microcryogel using multi-channel pipette, such as a 96-channel liquid handler. Medium is also added to peripheral wells. 
		NOTE: Use RPM1640 media with 10% FBS, 100 U/mL penicillin and 100 &#181;g/mL streptomycin for H460 cells. Use DMEM media with 10% FBS, 100 U/mL penicillin and 100 &#181;g/mL streptomycin for HepaG2 cells.</li>
		<li>Culture the cell-loaded microcryogel array chip in the humidity chamber for 24 h in a humidified 5% CO2 incubator at 37 &#176;C to form 3D microtissue array.</li>
		<li>Dissolve doxorubicin and IMMLG-84399 in dimethyl sulfoxide (DMSO) to form a stock solution of 10 mM. Dilute drugs with culture medium to form a 10-fold dilution concentration gradient from 2 nM to 200 &#181;M.</li>
		<li>Add 10 &#181;L of drug solutions into each well. Use 0.1% DMSO (diluted in medium) as the control. Incubate the drug loaded 3D microtissue array in a humidified 5% CO2 incubator at 37 &#176;C for 24 h.</li>
		<li>Add 4 &#181;L of resazurin stock solution to each well. Incubate at 37 &#176;C for 2 h. Place the 3D microtissue array in the microplate reader and detect fluorescence of resazurin metabolized by viable cells at an excitation light wavelength of 560 nm and emission light wavelength of 590 nm.</li>
		<li>Subtract the resazurin baseline signal from all wells before further data processing. Calculate the cell viability fraction of each well by dividing its fluorescence signal by the average fluorescence signal of the control wells.</li>
		<li>Plot the dose-response curve in plotting software with the cell viability fraction as the Y-axis and the base 10 logarithm of drug concentration as the X-axis. Interpolate the 50% inhibition concentration (IC50) at the cell viability fraction of 0.5.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Regenerative medicine and in vitro models for drug screening are two important applications for tissue engineering5,6,7,8,9. While these two applications have vastly different needs, a common ground between them lies in the need for a more biomimetic culturing condition to enhance cell functions19. Only with improved cell funct...

Traditional cell culture on flattened two-dimensional (2D) surfaces, such as a culture dish or multi-well plates, can hardly elicit cell behaviors close to their native states. Accurate recapitulation of native cellular microenvironments, which comprise of various cell types, extracellular matrices and bioactive soluble factors in three-dimensional (3D) architectures1,2,3,4, is essential to construct biomimicking tissues in vitro for applications in tissue engineering, regenerative medicine, fundamental biology research and drug discovery5,6,7,8,9.
In lieu of 2D cell culture, 3D cell culture is widely used to advance biomimetic micro-architectural and functional features of cells cultured in vitro. A popular 3D cell culture method is to aggregate cells into spheroids7,8,9,10. Cellular spheroids could be injected to injured tissues with enhanced cellular retention and survival in comparison to injection of dispersed cells. However, non-uniform spheroid sizes and inevitable mechanical injury imposed on cells by fluid shear force during injection lead to poor cell therapeutic effects11,12,13. Similarly, the inherent non-uniformity during aggregation of spheroids has made their translation to 3D cell-based high-throughput drug screening challenging10.
Another method for 3D cell culture is achieved with the assistance of biomaterials, which typically encapsulates cells in aqueous hydrogels or porous scaffolds. It allows for greater flexibilities in constructing 3D architectures. For therapy, cells encapsulated in bulk scaffolds are usually delivered to animal body via surgical implantation, which is invasive and traumatic, hence restricting its wide translation to bedside. On the other hand, aqueous hydrogels enable minimally invasive therapy by injecting cells suspended in hydrogel precursor solution into animal bodies, allowing in situ gelation via thermo-, chemical or enzymatic crosslinking11. However, as cells are delivered whilst the hydrogel precursors are still in an aqueous state, they are also exposed to mechanical shear during injection. Not only so, chemical or enzymatic crosslinking during in situ gelation of hydrogel could also impose damage to cells within. For drug screening, biomaterial-assisted cell cultures face problems with uniformity, controllability and throughput. Using hydrogels, cells are typically involved during gelation, by which the process may affect cell viability and function. Gelation during cell seeding also hampers usage by most high-throughput equipment, since the hydrogel may need to be kept on ice to prevent gelation before cell seeding, and the hydrogel might jam dispensing tips, which are usually very thin to ensure accuracy for high-throughput screening. Pre-formed scaffolds could potentially separate biomaterial fabrication procedures from cell culture, however most scaffold-based products are available as bulk materials with relatively lower throughput14.
To overcome some of the shortcomings of current 3D culture methods, we have developed a microfabrication-cryogelation integrated technology to fabricate an off-the-shelf and user-friendly microcryogel array chip15. In this protocol, gelatin is selected to exemplify the microcryogel fabrication technique as it is biocompatible, degradable, cost-effective, and no further modification is required for cell attachment. Other polymers of natural or synthetic sources could also be used for fabrication, depending on the application. Via this technology, we can fabricate miniaturized and highly elastic microcryogels with controllable size, shape and layout. When loaded with a variety of cell types, 3D microtissues could be formed for various applications. These unique features enable desired injectability, cell protection and site-directed retention after injection in vivo for enhanced therapeutic effects. Not only so, the microcryogels could be further processed to form 3D microtissue arrays that are compatible with common laboratory equipment and instruments to realize high-throughput cell culture for versatile drug screening and other cellular assays. Herein, we shall detail the fabrication process of microcryogels and its post-treatment as individual 3D microtissues or 3D microtissue arrays for two important applications, cell therapy and drug screening, respectively10,15.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:199px;">Gelatin</td>
			<td style="width:211px;">sigma</td>
			<td style="width:111px;">G7041</td>
			<td style="width:283px;">All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:199px;">Glutaraldehyde&#160;</td>
			<td style="width:211px;">J&#38;K</td>
			<td style="width:111px;">902042</td>
			<td style="width:283px;">Used as crosslinker in preparation of material.</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:199px;">Glass cover slip (24X50mm)</td>
			<td style="width:211px;">CITOGLASS, China</td>
			<td style="width:111px;">10212450C</td>
			<td style="width:283px;">To scrape prcursor solution onto microstencils array chips.</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:199px;">Sodium&#160;borohydride, NaBH4</td>
			<td style="width:211px;">Beijing Chemical Works</td>
			<td style="width:111px;">116-8</td>
			<td style="width:283px;">To wash remaining glutaraldehyde away after gelation.</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:199px;">Vacuum jar</td>
			<td style="width:211px;">asperts, China</td>
			<td style="width:111px;">VC8130</td>
			<td style="width:283px;">To preserve microgels under vacuum.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Polymethylmethacrylate (PMMA) sheets&#160;</td>
			<td style="width:211px;">Sunjin Electronics Co., Ltd, China</td>
			<td style="width:111px;"></td>
			<td style="width:283px;">Ordinary PMMA sheets.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Rayjet laser system</td>
			<td style="width:211px;">Rayjet, Australia</td>
			<td style="width:111px;">Rayjet 50 C30</td>
			<td style="width:283px;">To engrave PMMA sheets to form wells.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Plasma Cleaner</td>
			<td style="width:211px;">Mycro Technologies, USA</td>
			<td style="width:111px;">PDC-32G</td>
			<td style="width:283px;">To make PMMA hyphophilic.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Lyophilizer</td>
			<td style="width:211px;">Boyikang, China</td>
			<td style="width:111px;">SC21CL</td>
			<td style="width:283px;">To lyophilize materials.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Trypan Blue solution (0.4%)</td>
			<td style="width:211px;">Zhongkekeao, China</td>
			<td style="width:111px;">DA0065</td>
			<td style="width:283px;">To dye microgels.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Doxorubicin hydrochloride</td>
			<td style="width:211px;">ENERGY CHEMICAL, China</td>
			<td style="width:111px;">A01E0801360010</td>
			<td style="width:283px;">To test drug resistance of cells in 2D or 3D microgel.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Live/dead assay</td>
			<td style="width:211px;">Dojindo Molecular Technologies (Kumamoto, Japan)</td>
			<td style="width:111px;">CS01-10</td>
			<td style="width:283px;">To distinguish alive and dead cells.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Cell Titer-Blue</td>
			<td style="width:211px;">Promega (Wisconsin, USA).</td>
			<td style="width:111px;">G8080</td>
			<td style="width:283px;">To test cell viability.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Cell strainer</td>
			<td style="width:211px;">BD Biosciences, USA</td>
			<td style="width:111px;">352360</td>
			<td style="width:283px;">To collect microgels.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">D-Luciferin</td>
			<td style="width:211px;">SYNCHEM (Germany)</td>
			<td style="width:111px;">s039</td>
			<td style="width:283px;">To tack cells.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Scanning electron microscope</td>
			<td style="width:211px;">FEI, USA</td>
			<td style="width:111px;">Quanta 200</td>
			<td style="width:283px;">To characterize microgel morphology.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">&#160;Mechanical testing machine</td>
			<td style="width:211px;">Bose, USA</td>
			<td style="width:111px;">3230</td>
			<td style="width:283px;">To measure mechanical features.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Programmable syringe pump&#160;</td>
			<td style="width:211px;">World Precision Instruments, USA</td>
			<td style="width:111px;">ALADINI 1000</td>
			<td style="width:283px;">To test injactabiliy.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Digital force gauge</td>
			<td style="width:211px;">HBO, Yueqing Haibao Instrument Co., Ltd., China</td>
			<td style="width:111px;">H-50&#160;</td>
			<td style="width:283px;">To test injactabiliy.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:199px;">Ethylene oxide sterilization system</td>
			<td style="width:211px;">Anprolene, Anderson Sterilization, Inc., Haw River, NC</td>
			<td style="width:111px;">AN74i</td>
			<td style="width:283px;">To sterilize microgels with ethylene oxide gas.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Microplate reader</td>
			<td style="width:211px;">Molecular Devices,USA</td>
			<td style="width:111px;">M5</td>
			<td style="width:283px;">To measure fluorescence intensity in micro-array.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Confocal microscope</td>
			<td style="width:211px;">Nikon, Japan</td>
			<td style="width:111px;">A1Rsi</td>
			<td style="width:283px;">To observe cell distribution in 3D.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Xenogen&#160; Lumina II imaging system</td>
			<td style="width:211px;">Caliper Life Sciences, USA</td>
			<td style="width:111px;">IVIS</td>
			<td style="width:283px;">To track cell in animals.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:199px;">Liquid work stataion</td>
			<td style="width:211px;">Apricot design,USA</td>
			<td style="width:111px;">S-pipette</td>
			<td style="width:283px;">To load medium or cell suspension high-throuputly.</td>
		</tr>
	</tbody>
</table>

3d microtissues for injectable regenerative therapy and high-throughput drug screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

Fabrication and characterization of microcryogels for 3D microtissue formation.
According to this protocol, microcryogels were fabricated to form the 3D microtissues and individual microcryogels or microcryogel arrays, and were applied to regenerative therapy and drug screening, respectively (Figure 1). Microstencil array chips fabricated from PMMA were applied as micromolds for mic...

Watch this Scientific Journal Video about 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening at JoVE.com

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

3d-microtissues-for-injectable-regenerative-therapy-high-throughput

Research

JoVE Journal

Bioengineering

10.2K Views.  Tsinghua University. This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Video: 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...