This research was supported by the Basic Science Research Program (2016R1D1A1B03934418) and the Bio &#38; Medical Technology Development Program (2018M3A9H1023141) of the NRF, and funded by the Korean government, MSIT.

1. Centrifugal microfluidic-based spheroid (CMS) culture chip fabrication
<ol>
	<li>Make PC molds for the top and bottom layers of the CMS culture chip by CNC machining. Detailed dimensions of the chip are given in Figure 1.</li>
	<li>Mix PDMS base and PDMS curing agent at a ratio of 10:1 (w/w) for 5 min and place in a desiccator for 1 h to remove air bubbles.</li>
	<li>After pouring the PDMS mixture into the molds of the CMS culture chip, remove air bubbles for 1 h more and cure in a heat chamber at 80 &#176;C for 2 h.</li>
	<li>Place them in the vacuumed plasma cleaner with the surfaces to be bonded facing up and expose them to air-assisted plasma at a power of 18 W for 30 s.</li>
	<li>Bond the two layers of the CMS culture chip and place it in the heat chamber at 80 &#176;C for 30 min to increase adhesion strength.</li>
	<li>Sterilize the CMS culture chip in an autoclave sterilizer at 121 &#176;C and 15 psi.</li>
</ol>
2. Cell preparation
<ol>
	<li>Thaw the 1 mL of the vial containing 5 &#215; 105&#8211;1 &#215; 106 hASCs or MRC-5s cells in a water bath at 36.5 &#176;C for 2 min.</li>
	<li>Add 1 mL of Dulbecco&#8217;s Modified Eagle Medium (DMEM) to a vial and gently mix with a 1,000 &#956;L pipette.</li>
	<li>Put 15 mL of the DMEM prewarmed to 36.5 &#176;C into a 150 mm diameter Petri dish using a pipette and add the cells from the vial.</li>
	<li>After 1 day, aspirate the DMEM and replace with 15 mL of fresh DMEM. Subsequently, change the media every 2 or 3 days.</li>
	<li>To detach the cells from the Petri dish, add 4 mL of trypsin to the Petri dishes and place them in an incubator at 36.5 &#176;C and 5% CO2 for 4 min.</li>
</ol>
3. Monoculture spheroid formation
<ol>
	<li>Put 2.5 mL of 4% (w/v) pluronic F-127 solution into the inlet hole of the CMS culture chip (Figure 2A) while rotating the chip at 500&#8211;1,000 rpm and then rotate the chip at 4,000 rpm for 3 min using the CMS system (Figure 2B). 
	NOTE: &#160;The pluronic coating prevents cell attachment to the inlet port while the chip rotates. Make sure air is not trapped in the microwells.</li>
	<li>Incubate the CMS culture chip filled with pluronic solution overnight at 36.5 &#176;C in 5% CO2.</li>
	<li>Remove the pluronic solution, wash out the remaining pluronic solution with DMEM, and dry the chip for 12 h on a clean bench.</li>
	<li>Add 2.5 mL of DMEM to the CMS culture chip and rotate the chip at ~4,000 rpm for 3 min for prewetting the inside of the chip.</li>
	<li>Stop the rotation and pull out 100 &#956;L of DMEM to make room for injecting the cell suspension.</li>
	<li>Add 100 &#956;L of cell suspensions that contain either 5&#160;&#215; 105 hASCs or 8&#215; 105 MRC-5s by pipetting Uniformly distribute the cells by pipetting 3&#8211;5x for resuspension.</li>
	<li>Rotate the chip at 3,000 rpm for 3 min to trap cells in each microwell by centrifugal force. 
	NOTE: &#160;Excessive rotational speed can cause cell escape through solution ejection holes.</li>
	<li>Culture the cells for 3 days in the incubator at 36.5 &#176;C, &#62;95% humidity, and 5% CO2, rotating at 1,000&#8211;2,000 rpm. Change culture medium every day.</li>
</ol>
4. Coculture spheroid formation
<ol>
	<li>Concentric spheroids formation
	<ol>
		<li>Add the first cells, 2.5 &#215; 105 hASCs, and rotate the chip at 3,000 rpm. After 3 min, add the second cells, 4 &#215; 105 MRC-5s, and rotate the chip at 3,000 rpm for 3 min. Inject a total of 100 &#956;L of cell suspensions by pipetting. When the cells are injected, shift the rotational speed to 500-1,000 rpm.</li>
		<li>Culture the cells in the incubator at 36.5 &#176;C, &#62;95% humidity%, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm. The concentric spheroids are created within 24 h. For long-term culture, change the culture medium every day.</li>
	</ol>
	</li>
	<li>Janus spheroid formation
	<ol>
		<li>Add 100 &#956;L of cell suspensions containing the first cells, 2.5 &#215; 105 hASCs, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Incubate the chip at 36.5 &#176;C, &#62;95% humidity, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm for 3 h.</li>
		<li>Add 100 &#956;L of the cell suspensions containing the second set of cells, 4 &#215; 105 MRC-5s, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Culture the cells in the incubator at 36.5 &#176;C, &#62;95% humidity, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm. The Janus spheroids are created within 24 h. For long-term culture, change the culture medium every day.</li>
	</ol>
	</li>
	<li>Sandwich spheroid formation
	<ol>
		<li>Add 100 &#956;L of cell suspensions containing the first cells, 1.5 &#215; 105 hASCs, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Incubate the chip at 36.5 &#176;C, &#62;95% humidity, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm for 3 h.</li>
		<li>Add 100 &#956;L of cell suspensions containing the second cells, 3 &#215; 105 MRC-5s, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Incubate the chip at 36.5 &#176;C, &#62;95% humidity%, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm for 3 h.</li>
		<li>Add 100 &#956;L of cell suspensions containing the third cells, 1.5 &#215; 105 hASCs, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Culture the cells in the incubator at 36.5 &#176;C, &#62;95% humidity%, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm. The sandwich spheroids are created within 12 h. For long-term culture, change the culture medium every day.</li>
	</ol>
	</li>
</ol>
5. Cell staining
<ol>
	<li>Warm the cell fluorescence dye to room temperature (20 &#176;C).</li>
	<li>Add 20 &#956;L of anhydrous dimethylsulfoxide (DMSO) per vial to make a 1 mM solution.</li>
	<li>Dilute the fluorescence to a final working concentration of 1 &#956;M using DMEM.</li>
	<li>Add the fluorescence to the cell suspension and gently resuspend using a pipette.</li>
	<li>Incubate 20 min at 36.5 &#176;C, humidity of &#62;95%, and 5% CO2.</li>
</ol>

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The authors have nothing to disclose.

The CMS is a closed system in which all injected cells enter the microwell without waste, making it more efficient and economical than conventional microwell-based spheroid generation methods. In the CMS system, the media is replaced every 12&#8211;24 h through a suction hole designed to remove the media in the chip (Figure 3A). During the media suction process, barely any media escapes from inside the microwell due to the surface tension between the media and the wall of the microwell. A us...

It is easier to simulate biological in vivo microenvironments with three-dimensional (3D) spheroid cell culture than with two-dimensional (2D) cell culture (e.g., conventional Petri dish cell culture) to produce more physiologically realistic experimental results1. Currently available spheroid formation methods include the hanging drop technique2, liquid-overlay technique3, carboxymethyl cellulose technique4, magnetic force-based microfluidic technique5, and the use of bioreactors6. Although each method has its own benefits, further improvement in reproducibility, productivity, and generating coculture spheroids is necessary. For example, while the magnetic force-based microfluidic technique5 is relatively inexpensive, the effects of strong magnetic fields on living cells must be carefully considered. The benefits of spheroid culture, particularly in the study of mesenchymal stem cell differentiation and proliferation, have been reported in several studies7,8,9.
The centrifugal microfluidic system, also known as lab-on-a-CD (compact disc), is useful for easily controlling the fluid inside and exploiting the rotation of the substrate and has thus been utilized in biomedical applications such as immunoassays10, colorimetric assays for detecting biochemical markers11, nucleic acid amplification (PCR) assays, automated blood analysis systems12, and all-in-one centrifugal microfluidic devices13. The driving force controlling the fluid is the centripetal force created by rotation. Additionally, multiple functions of mixing, valving, and sample splitting can be done simply in this single CD platform. However, compared to the above-mentioned biochemical analysis methods, there have been fewer trials applying CD platforms to culture cells, especially spheroids14.
In this study, we show the performance of the centrifugal microfluidic-based spheroid (CMS) system by monoculture or coculture of human adipose-derived stem cells (hASC) and human lung fibroblasts (MRC-5). This paper describes in detail our group&#39;s research methodology15. Thus, the spheroid culture lab-on-a-CD platform can be easily reproduced. A CMS generating system comprising a CMS culture chip, a chip holder, a DC motor, a motor mount, and a rotating platform, is presented. The motor mount is 3D printed with acrylonitrile butadiene styrene (ABS). The chip holder and rotating platform are CNC (computer numerical control) machined with the PC (polycarbonate). The rotational speed of the motor is controlled from 200 to 4,500 rpm by encoding a PID (proportional-integral-derivative) algorithm based on pulse-width modulation. Its dimensions are 100 mm x 100 mm x 150 mm and it weighs 860 g, making it easy to handle. Using the CMS system, spheroids can be generated under various gravity conditions from 1 x g to 521 x g, so the study of cell differentiation promotion under high gravity can be extended from 2D cells16,17 to 3D spheroid. Coculture of various types of cells is also a key technology for effectively mimicking the in vivo environment18. The CMS system can easily generate monoculture spheroids, as well as coculture spheroids of various structure types (e.g., concentric, Janus, and sandwich). The CMS system can be utilized not only in simple spheroid studies but also in 3D organoid studies, to consider human organ structures.

<table border="0" cellpadding="0" cellspacing="0" style="width:1056px;" width="1056">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">3D printer</td>
			<td style="width:171px;">Cubicon</td>
			<td style="width:124px;">3DP-210F</td>
			<td style="width:416px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Adipose-derived mesenchymal stem cells (hASC)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">PCS-500-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Antibiotic-Antimycotic</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:124px;">15240-062</td>
			<td>Contained 1% of completed medium and buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CellTracker Green CMFDA</td>
			<td style="width:171px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">C2925</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CellTracker Red CMTPX</td>
			<td style="width:171px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">C34552</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Computer numerical control (CNC) rotary engraver</td>
			<td style="width:171px;">Roland DGA</td>
			<td style="width:124px;">EGX-350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">DC motor</td>
			<td style="width:171px;">Nurielectricity Inc.</td>
			<td style="width:124px;">MB-4385E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:124px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Dulbecco&#39;s modified eaggle&#39;s medium (DMEM)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Dulbecco&#39;s phosphate buffered saline (D-PBS)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">30-2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Fetal bovine serum</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">30-2020</td>
			<td>Contained 10% of completed medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">human lung fibroblasts (MRC-5)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">CCL-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Inventor 2019</td>
			<td style="width:171px;">Autodesk</td>
			<td style="width:124px;"></td>
			<td>3D computer-aided design program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Petri dish &#934; 150 mm</td>
			<td style="width:171px;">JetBiofill</td>
			<td style="width:124px;">CAD010150</td>
			<td>Surface Treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Plasma cleaner</td>
			<td style="width:171px;">Harrick Plasma</td>
			<td style="width:124px;">PDC-32G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Pluronic F-127</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:124px;">11/6/9003</td>
			<td>Dilute with phosphate buffered saline to 4% (w/v) solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Polycarbonate (PC)</td>
			<td style="width:171px;">Acrylmall</td>
			<td style="width:124px;">AC15PC</td>
			<td>200 x 200 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:171px;">Dowcorning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Trypsin</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:124px;">12604021</td>
			<td></td>
		</tr>
	</tbody>
</table>

lab-on-a-cd platform for generating multicellular three-dimensional spheroids

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

The 6 cm diameter CMS culture chip (Figure 2) was successfully made following the above protocol. First, the chip was made separately from a top layer and a bottom layer and then bonded together by plasma bonding. Resulting spheroids can be easily gathered by detaching the chip. The channel of the CMS culture chip comprises an inlet port and central, slide, and microwell regions (Figure 3). The cell, medium, and pluronic solutions are injected through an inlet h...

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

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

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

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Research

JoVE Journal

Bioengineering

6.2K Views.  Chung-Ang University. We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.