Name: 3d microtissues for injectable regenerative therapy and high-throughput drug screening
Uploaded: 2017-10-04T11:45:00
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 ge...

     

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 ...

The overall goal of fabricating elastic 3D macroporous microcryogels using cryogelation is to facilitate injectable regenerative therapy and in vitro high-throughput drug screening. This video presents the protocols to fabricate versatile 3D microtissues and apply them to regenerative therapy and drug screening. The regenerative therapy technique improves on stem cell retention, survival, and therapeutic functions after injection into a wound site.Drug screening is also improved because of ease of use and potential for high-throughput screening. Generally, individuals new to 3D culture struggle because of the complicated protocols. This technique simplifies 3D culture and offers researchers greater control of the resulting 3D microtissues.Assisting in the demonstration of these procedures will be Zhou Lyu, Liu Wei, Li Yaquian, and You Zhifeng, four graduate students from our laboratory. Wash the PMMA microstencil array chips with deionized water to remove the debris remaining from the laser engraving. Then, dry the microstencil array chips at 60 degrees Celsius.Next, treat the dried chips in a plasma cleaner in groups of four. First, run the vacuum pump for two minutes. Then, use 18 watts of RF power for three minutes to increase the hydrophilicity of the chips.Now, make one milliliter of gelatin precursor solution for every five chips. Dissolve the gelatin in a 60-degree Celsius water bath and incubate the solution on ice for five minutes. Once cooled slightly but still liquid, thoroughly mix glutaraldehyde into the gelatin precursor solution to a final concentration of 0.3%glutaraldehyde.Now, pipette 200 microliters of prepared solution onto the upper surface of each chip. Distribute the solution evenly over the chips using a bent glass rod. Next, immediately transfer the solution-loaded array chips into a minus 20 degree Celsius freezer for 16 hours for cryogelation.The next day, cool a lyophilizer to minus 40 degrees Celsius over about 30 minutes. Then, load the array chips, and lyophilize them for two hours under a vacuum. The ice will sublime under these conditions, and the chips will then be ready for use.Begin by harvesting individual microcryogels from the chips. Start with overlaying the fabricated microcryogel array chip on top of the fabricated PDMS ejector pin array. Align each microcryogel with an ejector pin.Then press the microcryogel array chip into the array to displace the microcryogels from their wells. Now, harvest the ejected microcryogels into a water bath, and collect them with the aid of a cell strainer. Use one strainer to collect all the microcryogels from one chip.Next, wash the microcryogels with sodium borohydride on ice. Allow the non-crosslinked aldehyde residues to be quenched for about 20 minutes. Then, discard the sodium borohydride, and wash the microcryogels with five milliliters of deionized water for 15 minutes.Perform a total of three to five water washes before proceeding. After removing the microcryogels from the last water wash, use tweezers to transfer them from the cell strainer to a 35-millimeter Petri dish. Each collection of microcryogels constitutes one cluster.Now, add 50 to 70 microliters of deionized water to each cluster of microcryogels, and cover the dish. Then, gently tap the dish to level any stacked microcryogels. Then, freeze the microcryogels at minus 20 degrees Celsius for four to 16 hours.Later, lyophilize them for two hours as before. The next step is to make the 3D microtissues. First, sterilize the microcryogels.Then, harvest the cells with trypsin, quantify their density, and resuspend them at eight million cells per milliliter in growth medium. Next, pipette 60 microliters of cell suspension onto each cluster of microcryogels. Maintain the dishes in a humidified chamber, and incubate them at 37 degrees Celsius for two hours so that the cells will absorb into the gels.After the incubation, add two milliliters of culture medium to each dish, and continue the culture, changing the medium every other day. In two days, 3D microtissues will have formed. To inject the 3D microtissues into a mouse, first transfer them into a five-milliliter pipette, and then load them into a cell strainer to filter the culture medium.Then, resuspend the 3D microtissues in a 15%gelatin solution. After preparing the animal for the implantation, intramuscularly inject the microtissues into three locations of the gracilis muscle. Each injection should contain 150 microcryogels in 150 microliters of solution.Use a one-milliliter syringe with a 23 gauge needle. After assembling the microcryogel array for on-chip cell culture, carefully place them into a preheated humidity chamber without getting them wet. Then carefully aliquot three microliters of thoroughly-mixed cell suspension directly onto each microcryogel well.Allow the pipette tip to lightly touch the surface of the microcryogel before expelling the solution. Do not seed the peripheral wells. After loading the wells, add 10 microliters of selective medium to each well, and add medium to the peripheral wells to slow evaporation using a 96-channel liquid dispenser.Then, culture the chips in the humidity chamber for 24 hours to form 3D microtissues. The next day, using the 96-channel liquid dispenser, add 10 microliters of drugs to each well in a concentration gradient four times to complete addition of all 384 wells, including DMSO controls. Then, continue the culture for 24 hours.After 24 hours of drug treatment, add four microliters of resazurin stock solution to each well, and incubate the plate for two hours, allowing the cells to metabolize the resazurin. To assay the cells'metabolism of resazurin, use a plate reader. Harvested gelatin microcryogels had predefined shapes and sizes.Analysis by SEM showed that the microcryogels contained interconnected macroporous structures with pore sizes in the range of 30 to 80 microns. The injectability of the gelatin microcryogels was quantitatively assessed by culturing them post-injection. 1, 000 per milliliter were injected at six newtons less than the clinically acceptable force of 10 newtons.The stem cells in microcryogels retained high viability and great proliferative capacity after injection during five days of culture. Using such cells, the mouse limb ischemia model was therapeutically treated, and the physiological status of the ischemic limbs was examined 28 days after surgery. Microtissue treatment with 100, 000 stem cells improved limb salvage.Only 25%of mice showed spontaneous toe amputation after 28 days. In a different application, the 3D microtissue array was used for high-throughput drug testing. Hepatocellular carcinoma cells were treated with doxorubicin, and non-small cell lung cancer cells were treated with IMMLG-8439.In both cases, drug resistance was increased by 3D culturing. After watching this video, you should have a good understanding of how to fabricate elastic 3D microporous microcryogels for off-chip 3D culture using injectable regenerative therapy or on-chip 3D cell culture arrays for in vitro high-throughput drug screening. This technique paved the way for researchers in the field of cell-based regenerative therapy and drug screening, to make use of 3D cell culturing, thus advancing both research fields.Don't forget that working with glutaraldehyde and sodium borohydride can be extremely hazardous, and precautions such as wearing proper protective garments and working under a laboratory hood should always be taken while performing this procedure.

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