Name: 3d microtissues for injectable regenerative therapy and high-throughput drug screening
Uploaded: 2017-10-04T11:45:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening at JoVE.com

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><tbody><tr><td>Gelatin</td><td>sigma</td><td>G7041</td><td>All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.</td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>J&amp;amp;K</td><td>902042</td><td>Used as crosslinker in preparation of material.</td></tr><tr><td>Glass cover slip (24 x 50 mm)</td><td>CITOGLASS, China</td><td>10212450C</td><td>To scrape prcursor solution onto microstencils array chips.</td></tr><tr><td>Sodium&amp;nbsp;borohydride, NaBH&lt;sub&gt;4&lt;/sub&gt;</td><td>Beijing Chemical Works</td><td>116-8</td><td>To wash remaining glutaraldehyde away after gelation.</td></tr><tr><td>Vacuum jar</td><td>asperts, China</td><td>VC8130</td><td>To preserve microgels under vacuum.</td></tr><tr><td>Polymethylmethacrylate (PMMA) sheets&amp;nbsp;</td><td>Sunjin Electronics Co., Ltd, China</td><td></td><td>Ordinary PMMA sheets.</td></tr><tr><td>Rayjet laser system</td><td>Rayjet, Australia</td><td>Rayjet 50 C30</td><td>To engrave PMMA sheets to form wells.</td></tr><tr><td>Plasma Cleaner</td><td>Mycro Technologies, USA</td><td>PDC-32G</td><td>To make PMMA hyphophilic.</td></tr><tr><td>Lyophilizer</td><td>Boyikang, China</td><td>SC21CL</td><td>To lyophilize materials.</td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Zhongkekeao, China</td><td>DA0065</td><td>To dye microgels.</td></tr><tr><td>Doxorubicin hydrochloride</td><td>ENERGY CHEMICAL, China</td><td>A01E0801360010</td><td>To test drug resistance of cells in 2D or 3D microgel.</td></tr><tr><td>Live/dead assay</td><td>Dojindo Molecular Technologies (Kumamoto, Japan)</td><td>CS01-10</td><td>To distinguish alive and dead cells.</td></tr><tr><td>Cell Titer-Blue</td><td>Promega (Wisconsin, USA).</td><td>G8080</td><td>To test cell viability.</td></tr><tr><td>Cell strainer</td><td>BD Biosciences, USA</td><td>352360</td><td>To collect microgels.</td></tr><tr><td>D-Luciferin</td><td>SYNCHEM (Germany)</td><td>s039</td><td>To tack cells.</td></tr><tr><td>Scanning electron microscope</td><td>FEI, USA</td><td>Quanta 200</td><td>To characterize microgel morphology.</td></tr><tr><td>&amp;nbsp;Mechanical testing machine</td><td>Bose, USA</td><td>3230</td><td>To measure mechanical features.</td></tr><tr><td>Programmable syringe pump&amp;nbsp;</td><td>World Precision Instruments, USA</td><td>ALADINI 1000</td><td>To test injactabiliy.</td></tr><tr><td>Digital force gauge</td><td>HBO, Yueqing Haibao Instrument Co., Ltd., China</td><td>H-50&amp;nbsp;</td><td>To test injactabiliy.</td></tr><tr><td>Ethylene oxide sterilization system</td><td>Anprolene, Anderson Sterilization, Inc., Haw River, NC</td><td>AN74i</td><td>To sterilize microgels with ethylene oxide gas.</td></tr><tr><td>Microplate reader</td><td>Molecular Devices,USA</td><td>M5</td><td>To measure fluorescence intensity in micro-array.</td></tr><tr><td>Confocal microscope</td><td>Nikon, Japan</td><td>A1Rsi</td><td>To observe cell distribution in 3D.</td></tr><tr><td>Xenogen&amp;nbsp; Lumina II imaging system</td><td>Caliper Life Sciences, USA</td><td>IVIS</td><td>To track cell in animals.</td></tr><tr><td>Liquid work stataion</td><td>Apricot design,USA</td><td>S-pipette</td><td>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

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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

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Cancer Research

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

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

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

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

Summary

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Chapters in this video

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models