The authors wish to acknowledge support from a National Research Foundation Fellowship (NRF -NRFF2011-01) and Competitive Research Programme (NRF-CRP10-2012-07).

1. ICC Scaffold Fabrication (Figure 1)
<ol>
	<li>Prepare the polystyrene (PS) lattices (diameter = 6 mm; 8-13 layers of beads).
	<ol>
		<li>To prepare the mold, cut the tips off from 0.2 ml boil-proof microcentrifuge tubes at the 40 &#181;l level. Adhere the top of the cut-tubes to 24 x 60 mm2 microscope cover glass slips with water-proof glue.</li>
		<li>Put the PS spheres (diameter = 140 &#181;m) contained within a water suspension into a 20 ml vial, carefully pipette out the water suspension, and add 18 ml of 70% ethanol solution into the vial. Put the sphere solution into an ultrasonic bath to loosen aggregated spheres. Repeat this washing step several times in order to remove water and water-soluble components completely.</li>
		<li>Pipette 100 &#181;l of ethanol into the molds.</li>
		<li>Cut the top of a 200 &#181;l micropipette tip by 4 mm. Pipette 25 &#181;l of the spheres into the mold twice using the 200 &#181;l micropipette to achieve a total volume of 50 &#181;l in each mold.</li>
		<li>Place the molds on a rocking shaker at 120 rpm overnight.</li>
		<li>Check the arrangement of the spheres in each mold under an optical microscope. If the spheres are not ordered hexagonally, add 50 &#181;l of 70% ethanol and shake manually in the longitudinal and lateral axis direction to correct the arrangement.</li>
		<li>Let the ethanol evaporate at room temperature (RT) for two nights. Place the mold and bead complex in a 130 &#176;C furnace for 6 hr to anneal the PS beads.</li>
	</ol>
	</li>
	<li>Preparing bare and ECM-coated PEGDA scaffolds.
	<ol>
		<li>Synthesize PEGDA macromers using established protocols45,46 for acrylating linear PEG macromers (Mw = 4.6 kDa).</li>
		<li>Prepare 50% (w/v) PEGDA solution in de-ionized (DI) water and allow the macromer to properly dissolve by centrifuging at 4,713 x g until it is completely dissolved.
		<ol>
			<li>For ECM conjugated ICC scaffolds, dissolve an additional 10% (w/v) Acryloyl-PEG-NHS (Mw=3.4 kDa) in the 50% PEGDA solution.</li>
		</ol>
		</li>
		<li>Prepare a 20% (w/v) stock solution of 2-hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenone (PI) in 70% ethanol.</li>
		<li>Add 50 &#181;l of 20% (w/v) PI stock solution per 1 ml of 50 % (w/v) of PEGDA. Adjust the needed amount of PI stock solution based on the molecular weight of PEGDA.</li>
		<li>Vortex the mixture in centrifuge tube for 1 min to reach a homogenous solution.</li>
		<li>Peel the molds from the glass slide (from step 1.1.7), remove the glue from the molds, push the lattices out carefully using a spatula and place each of them into a 1.5 ml tube. Pipette 300 &#181;l of the PEGDA solution and centrifuge at 845 x g for 5 min to allow proper PEGDA solution infiltration into the lattice.</li>
		<li>Remove the lattice from the tube using tweezers and carefully blot dry excess PEGDA solution on gloves. Place the lattice on a paraffin film-covered glass with the flat circular surface facing up.</li>
		<li>Expose the PEGDA solution infiltrated scaffold to 365 nm ultraviolet (UV) light (10.84 mW/cm2) for 5 min using a UV spot lamp.</li>
		<li>Place PEGDA-polymerized crystal lattices in new vials (around 10 lattices per vial) and add 20 ml of tetrahydrofuran (THF). Shake the vials on an orbital shaker at 300 rpm. Change THF at least 3 times with an interval of 1-2 hr. 
		Note: Do not remove THF completely when changing the THF in order to prevent bubbles from entering the scaffolds, which in turn can cause incomplete removal of PS. Leave enough solution to cover the lattices and add new THF. 
		Caution: THF is toxic. Wear gloves, a lab coat and goggles. Avoid inhalation by operating under the fume hood.</li>
		<li>Check if PS spheres are dissolved by putting water into the used THF solution and observing the solution color. Repeat step 1.2.9 if the PS spheres are not properly dissolved. 
		Note: The solution color will change to white if there are any remaining PS spheres.</li>
	</ol>
	</li>
	<li>Clean the scaffolds in the biosafety cabinet (BSC).
	<ol>
		<li>To sterilize the scaffolds, prepare a 50 ml centrifuge tube with 2 ml of 70% ethanol per scaffold and place the scaffolds in the tube using a spatula. Allow the scaffolds to soak in ethanol for 1 hr. From this step forward, conduct all the procedures in the BSC.</li>
		<li>Carefully pour the ethanol out and replace with phosphate buffer saline (PBS) (2 ml per scaffold) and centrifuge at 524 x g for 3 min to remove bubbles. Keep it in the refrigerator and change the PBS a few times with an interval of 1-2 hr.
		<ol>
			<li>For Collagen type I-coated scaffolds, prepare another 50 ml centrifuge tube containing Collagen type 1 stock solution (1 ml per scaffold), transfer the sterilized scaffolds to this tube using a spatula, and centrifuge at 524 x g for 3 min. Shake the scaffolds at 400 rpm on an orbital shaker for 30 min and keep the tube in the refrigerator overnight.</li>
			<li>Wash the scaffolds with PBS twice before use by submerging the scaffolds in fresh PBS and then aspirating the PBS. 
			Note: Other ECM proteins can also be used instead of Collagen type I because the NHS chemistry requires an amine group to form the bond (Figure 2).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54331/54331fig1.jpg" /> 
Figure 1. Overview of ICC fabrication. PEG-based ICC scaffolds are fabricated using microfabrication techniques with and without ECM-functionalization. ECM-coated ICC scaffolds require PEG-NHS as well as PEGDA (as detailed in Figure 2). The PS lattice has a diameter of 6 mm and a height of 8-13 bead layers. PS, polystyrene; PEGDA, poly (ethylene glycol) diacrylate; UV, ultraviolet; THF, tetrahydrofuran; ECM, extracellular matrix. This figure has been modified and used with permission from Wiley47. <a href="https://www.jove.com/files/ftp_upload/54331/54331fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

2. ICC Structure Characterization
<ol>
	<li>To analyze ICC structure with or without conjugated proteins, use scanning electron microscopy (SEM)47.
	<ol>
		<li>Fix the scaffolds with 4% paraformaldehyde (PFA), serially dehydrate them in 25, 50, 75, 95 and 100% ethanol solutions, and store them at -80 &#176;C until the ethanol evaporates completely.</li>
		<li>Dry samples in a freeze drier for 48 hr.</li>
		<li>Affix the sample onto sample holder using carbon tape and place in a sputter coater.</li>
		<li>After automatic vacuuming, coat it with a Pt film of 10 nm thickness by sputtering for 60 sec at 20 mA.</li>
		<li>Image ICC scaffolds using SEM at a voltage of 5 kV (Figure 3A, Figure 4A).</li>
	</ol>
	</li>
	<li>To measure the pore and interconnection diameter of cavities, analyze SEM micrographs using image analysis software48 (e.g., ImageJ; Figure 3B,C).</li>
	<li>To visualize the conjugated collagen to the scaffold without cells, fluorescently tag the collagen using antibodies (1:100) against the collagen type I and image with confocal laser scanning microscopy47 (CLSM; Figure 4B).</li>
</ol>
3. Huh-7.5 Cell Culture and Seeding
<ol>
	<li>Culture Huh-7.5 cells at a seeding density of 2-2.5 x 106 cells/ml in 100 mm cell culture dishes with 10 ml Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin (growth media) at 37 &#176;C and 5% CO2. Change the media every three days in the BSC until they have reached 75-80% confluency.</li>
	<li>Prepare the scaffolds for cell seeding in the BSC.
	<ol>
		<li>Carefully place the scaffolds in a 24-well plate with the flat surface facing up.</li>
		<li>To wash the scaffold, pipette 2 ml PBS to each well containing a scaffold. Aspirate the PBS and pipette 2 ml fresh PBS into each well.</li>
		<li>Aspirate the PBS and pipette 2 ml of growth media (see step 3.1) and leave for 30 min. Aspirate the media and allow the scaffold to dry for 1 hr.</li>
	</ol>
	</li>
	<li>Detach confluent Huh-7.5 cells (from step 3.1) from the culture plate in the BSC using the trypsin digestion method.
	<ol>
		<li>Aspirate media from plate, add 4 ml PBS to wash adherent cells and then aspirate PBS.</li>
		<li>Pipette 0.75-1 ml 0.25% trypsin and place in an incubator at 37 &#176;C, 5% CO2 for 3 min.</li>
		<li>Remove plate from incubator and pipette 5 ml media to stop the trypsin reaction. Pipette media, detached cells, and trypsin mixture into a 15 ml tube.</li>
		<li>Centrifuge at 524 x g for 3 min, remove the supernatant and resuspend the pellet in 5 ml media.</li>
	</ol>
	</li>
	<li>Count cells using a hemocytometer and calculate the volume of the cell suspension that contains cells at target number, N0, per 25 &#181;l (for standard experiment, N0 is 1 x 106 cells). 
	Volume of cell suspension=(target number of cells)/(concentration of cell suspension)</li>
	<li>Slowly pipette 25 &#181;l of cell suspension (containing N0 cells) directly on top of each scaffold (from step 3.1.4). Place the 24-well plate in the incubator.</li>
	<li>After 12 hr, transfer the scaffolds carefully with the use of a spatula into a new 24-well plate and pipette 2 ml of media into each well. Place the 24-well plate in the incubator.</li>
	<li>Change the media every 3 days or depending on when media is collected for protein secretion analysis (see Step 5.1).</li>
</ol>
4. Cell Viability
<ol>
	<li>To qualitatively analyze cell viability, use fluorescent live/dead staining kits to stain the cells and image using CLSM.
	<ol>
		<li>Following kit instruction, prepare a solution with 4 &#181;M calcein AM and 8 &#181;M ethidium homo-dimer-1 in media (see step 3.1.3). 
		Note: Optimize depending on the cell number. Use double the amount of reagent if cells proliferate and double in number (around 2 million).</li>
		<li>In the BSC, aspirate media in each well with a scaffold (step 3.7) and pipette 500 &#181;l of the prepared solution. Incubate samples in a 37 &#176;C incubator for 1 hr.</li>
		<li>Cover the plates in foil to protect the samples from light when removing the plate out of the incubator. Image samples using CLSM49.</li>
	</ol>
	</li>
	<li>To quantitatively assess cell viability, measure enzymatic activity (in live cells) using colorimetric assays50 (i.e., 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (monosodium-salt reagent)).
	<ol>
		<li>Create a standard curve for the ICC platform (i.e., a graph of absorbance (OD) versus given cell number). 
		Note: Cell seeding is not 100% efficient in comparison to other platforms, since cells can pass through the cavities of scaffolds.
		<ol>
			<li>Determine the cell seeding numbers, N0 that will be used to make the standard curve. 
			Note: Choose a range that includes cell numbers that are estimated in the experiment. For example, if the initial cell number is 5 x 105 cells and there is an estimated ~3 fold increase by the last day of the experiment, choose 2.5 x 105, 5 x 105, 1 x 106, and 2 x 106 cells as N0.</li>
			<li>Perform cell seeding in the ICC scaffolds (one N0 per scaffold with seeding volume of 25 &#181;l) as described in steps 3.1-3.6.</li>
			<li>After 6 hr, transfer the scaffolds to another 24-well plate well. Select a time that allows cell adherence but not cell proliferation.</li>
			<li>Perform cell counting on the transferred ICC scaffold.
			<ol>
				<li>Dilute 10x monosodium-salt reagent (MSR) solution to 1x with media in the BSC and pipette 500 &#181;l 1x MSR solution into each well with a scaffold. Incubate the 24-well plate at 37 &#176;C for 1 hr.</li>
				<li>From each well, transfer 100 &#181;l into a 96-well plate well. As a blank, pipette 100 &#181;l of fresh 1x monosodium-salt reagent solution into different wells on the 96-well plate. Manually remove any bubbles present using a dry pipette tip and cover the 96-well plate in foil to protect it from light.</li>
				<li>Measure OD at &#955; = 450 nm reading using a spectrophotometer. Subtract the blank OD from other values to find the accurate OD51.</li>
			</ol>
			</li>
			<li>Count the number of cells (NL) remaining in the well after transferring the scaffold (step 4.2.1.3) using a hemocytometer. 
			Note: Use 300 &#181;l of trypsin to trypsinize the cells.</li>
			<li>Calculate the actual cell number, NA. 
			actual = initial - left in well 
			NA=N0-NL</li>
			<li>Make standard curve by plotting OD obtained in Step 4.2.1.4.3 vs. actual cell number (NA) and use this to estimate the cell number in the experiments. 
			Note: Make a new standard curve if any ICC parameters (i.e., porogen size, dimensions of the scaffold, ECM protein, etc.) are changed.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Cell Function
<ol>
	<li>Analyze protein secretion by the Huh-7.5 cells (i.e., albumin, urea) from the collected media (from step 3.7) by enzyme linked immunosorbent assay (ELISA)52. 
	Note: Dilute the media, depending on the number of cells seeded and the amount of media collected. For 5 x 105 cells seeded in ICC, use a ~1:25 ratio, before introducing it to the antibody-precoated wells.</li>
	<li>To qualitatively analyze cell function, immunostain specific intracellular proteins (i.e., albumin), enzymes (i.e., CYP450), stain structural components (i.e., cellular actin) as well as the nuclei and image using CLSM49.
	<ol>
		<li>Aspirate media (from step 3.7) and pipette 2 ml PBS to wash the cell-laden ICC scaffolds.</li>
		<li>Pipette 1 ml of 4% PFA and incubate for 5 min at room temperature for fixation.</li>
		<li>Wash 3x with 2 ml PBS.</li>
		<li>Permeabilize membranes by incubating the scaffolds in 1 ml of 0.1% 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (surfactant) for 30 min.</li>
		<li>Wash 3x with 2 ml PBS to remove any leaking proteins.</li>
		<li>Pipette 500 &#181;l 1% bovine serum albumin (BSA) and incubate at RT for 1 hr to block non-specific binding.</li>
		<li>Prepare diluted primary antibody (i.e., albumin, CYP450) solution.
		<ol>
			<li>Pipette 500 &#181;l of 1% BSA solution into a 15 ml tube and add 4.5 ml of 0.1% surfactant solution to prepare a total 5 ml of 0.1% BSA solution.</li>
			<li>Pipette 98 &#181;l of the 0.1% BSA solution into a 200 &#181;l microcentrifuge tube and 2 &#181;l of the primary antibody to produce a 1:50 (primary antibody: 0.1% BSA) primary antibody solution.</li>
		</ol>
		</li>
		<li>Pipette 40 &#181;l of the primary antibody solution on the scaffold and cover the substrate with paraffin film. Wrap the 24 well plate with aluminum foil and store the dish at 4 &#176;C overnight.</li>
		<li>Wash 3x with 2 ml PBS and shake the plate gently in between washing.</li>
		<li>Prepare diluted biotinylated secondary antibody (i.e., Anti-mouse antibody) stock solution.
		<ol>
			<li>Pipette 198 &#181;l 0.1% BSA solution and 2 &#181;l second antibody to produce a 1:100 (secondary antibody: 0.1% BSA) secondary antibody solution.</li>
			<li>Prepare a 0.1% stock solution of rhodamine or fluorescein labeled phalloidin (to stain the cellular actin filaments) in the 0.1% BSA solution.</li>
			<li>Pipette 25 &#181;l of each solution in a tube and mix well.</li>
		</ol>
		</li>
		<li>Pipette 50 &#181;l of the secondary antibody stock solution on the scaffold. Cover the scaffold with paraffin film, wrap the dish with aluminum foil and store at RT for 2 hr.</li>
		<li>Wash 3x with 2 ml PBS.</li>
		<li>Pipette 200 &#181;l of 0.2% 4&#39;-6-diamidino-2-phenylindole (DAPI; a nucleus stain) solution on the scaffold and keep at RT for 2-3 min. Cover the plate with aluminum foil.</li>
		<li>Wash 2x with 2 ml PBS.</li>
		<li>Using a dropper, place a drop of mounting media on the substrate.</li>
		<li>Carefully put the scaffold on a glass slide and image using CLSM47.</li>
	</ol>
	</li>
	<li>Assess the gene expression by real-time polymerase chain reaction (qPCR). Use standard kits for reverse transcriptase PCR (RT-PCR)53 and qPCR54 as per manufacturer&#39;s instructions. Extract the RNA from the cells as described below.
	<ol>
		<li>Place the scaffold (from step 3.7) in a 1.5 ml microcentrifuge tube.</li>
		<li>Pipette 1 ml of an RNA extraction solution into the tube and keep in a sonicator for 5 min at RT.</li>
		<li>Pipette 200 &#181;l of chloroform into each microcentrifuge tube and shake the tube vigorously in hand for 15-20 sec. Keep the tubes at RT for ~3 min until the phases separate.</li>
		<li>Centrifuge the sample at 13,000 x g, 4 &#176;C for 15 min and remove the tubes carefully so that phases do not mix.</li>
		<li>Carefully pipette 500-600 &#181;l of the upper aqueous phase from the first tube into a second microcentrifuge tube.</li>
		<li>Add an equivalent volume (500-600 &#181;l) of isopropanol to this second tube.</li>
		<li>Invert the tube 3-5 times and leave the tube standing at RT for 10 min.</li>
		<li>Centrifuge the sample at 13,000 x g, 4 &#176;C for 15 min.
		<ol>
			<li>If a pellet is not visible at the bottom of the tube, centrifuge again for 5 min. 
			Note: If there is still no visible pellet, the amount of RNA may be insufficient.</li>
		</ol>
		</li>
		<li>Invert tubes with the cap open to discard supernatant and pipette in 1 ml 70% ethanol diluted in DEPC water into the tube.</li>
		<li>Slightly vortex the tube so the pellet detaches from the wall of the tube and then let the tube air dry.</li>
		<li>Add 50 &#181;l of DPEC water to resuspend the pellet.</li>
		<li>Keep pipetting until the pellet dissolves.</li>
		<li>Keep for 10 min at 55 &#176;C in order to denature the double-stranded RNA into single-stranded RNA.</li>
		<li>Lightly drum fingers on the tube bottom and then centrifuge the tubes briefly (7,500 x g, 4 min, 4 &#176;C).</li>
		<li>Keep the tubes in ice until performing reverse transcriptase55 and real-time PCR as described in 56.</li>
	</ol>
	</li>
</ol>

The authors have no competing financial interests to disclose.

Tissue engineering scaffolds are rapidly evolving to provide all the physical and biochemical cues necessary to regenerate, maintain, or repair tissues for the application of organ replacement, studying disease, developing drugs, and many others57. In liver tissue engineering, primary human hepatocytes rapidly lose their metabolic functions once isolated from the body, creating a great need for engineering scaffolds and developing platforms to maintain the hepatic function. The current in vitro hepato...

The liver is a highly vascularized organ with a multitude of functions, including detoxification of the blood, metabolism of xenobiotics, and the production of serum proteins. Liver tissue has a complex three-dimensional (3D) microstructure, comprising of multiple cell types, bile canaliculi, sinusoids, and zones of different biomatrix composition and different oxygen concentrations. Given this elaborate structure, it has been difficult to create a proper liver model in vitro1. However, there is a rising demand for functional in vitro models hosting human hepatocytes as platforms for testing drug toxicity2 and studying diseases associated with the liver3.
Current liver tissue engineering platforms have simplified the complexity of the liver by isolating one, or focusing on a few, of the liver&#39;s parameters, namely co-culture of cells4, biochemical composition of the zonal microenvironments5, flow dynamics6,7 and the configuration of the biomatrix8. Configuration of the biomatrix can be broken into parameters such as scaffold materials, composition of extracellular matrix (ECM) proteins, matrix stiffness as well as the design and structure of the scaffold. There has been a rise in tissue engineering studies using synthetic hydrogels, especially poly(ethylene glycol) (PEG) hydrogels9, given the ability to tune the hydrogel&#39;s mechanical properties, bioactivity, and degradation rate. Regarding liver-related research, the biocompatible hydrogel was applied for virus infection study of liver disease3. As a hepatocyte platform design, numerous studies have utilized hepatocyte sandwich cultures10,11 and cell encapsulation within a hydrogel12,13 to provide the 3D environment and cell-ECM and cell-cell interaction which are essential to mimic in vivo microenvironment. However, these platforms do not possess a high degree of control and spatial organization, leading to non-uniform properties through the scaffold14.
The inverted crystal colloidal (ICC)14 scaffold is a highly organized 3D scaffold for cell culture that was first introduced in the early 2000s. The scaffold&#39;s unique structure can be attributed to the simple fabrication process using a colloidal crystal, an ordered lattice of colloidal particles of variable diameter. Briefly, to summarize the process, particles are neatly arranged and annealed using heat to form a lattice. The leaching of this lattice, by an organic solvent, in a polymerized hydrogel results in hexagonally packed spherical cavities15 with high surface area. This highly ordered scaffold has been previously made with both synthetic and natural materials, including but not limited to poly(acrylamide)16-21, poly(lactic-co-glycolic acid)15,22-30, poly(ethylene glycol)31,32, poly (2-hydroxyethyl methacrylate)21,33-35, and chitosan36-39. ICC scaffolds made of non-fouling materials tend to promote cellular spheroids within the cavities14,23,40. Multiple cell types have been shown to successfully proliferate, differentiate and function within this configuration, including chondrocytes41, bone marrow stromal cells42, and stem cells43,44. Regarding hepatocyte, studies have been conducted with ICC scaffolds made of Na2SiO3 and poly(acrylamide), but not PEG. With simple bioconjugation strategies (i.e., amine coupling through EDC/NHS), ECM proteins-conjugated PEG-based scaffolds can be fabricated, that can prove more cell binding sites to be a more in vivo like environment and enhance hepatic function.
In this manuscript and the associated video, we detail the fabrication of the ICC scaffold using poly(ethylene glycol) diacrylate (PEGDA) hydrogel and a polystyrene microsphere lattice, optimized for hepatocarcinoma (Huh-7.5) culture. We demonstrate the differences between the generally nonadhesive bare PEGDA ICC scaffolds and the collagen-coated PEGDA ICC scaffold in terms of scaffold topology and cell performance. Cell viability and function are measured qualitatively and quantitatively to assess Huh-7.5 cell behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tube</td>
			<td>Axygen Scientific</td>
			<td>PCR-02D-C</td>
			<td>Boil-proof</td>
		</tr>
		<tr>
			<td>Gorilla Glue</td>
			<td>Gorilla Glue, Inc.</td>
			<td>Depends on vendor. This was purchased from a local store.</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>VWR</td>
			<td>&#160;631-1575</td>
			<td>Dimensions: 24&#215;60 mm</td>
		</tr>
		<tr>
			<td>Polystyrene spheres&#160;</td>
			<td>Fisher Scientific</td>
			<td>TSS#4314A</td>
			<td>Diameter = 140 um; 3x10^4 particles per milliliter and 1.4% size distribution</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>1.00983.1011</td>
			<td>absolute for analysis EMSURE; Dilute to 70% with Milli-Q water</td>
		</tr>
		<tr>
			<td>Ultrasonic Bath</td>
			<td>Elma</td>
			<td>S10H</td>
			<td>Equiment</td>
		</tr>
		<tr>
			<td>Furnace</td>
			<td>Nabertherm</td>
			<td>N7/H</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tip</td>
			<td>Axygen Scientific</td>
			<td>T-210-Y-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking shaker</td>
			<td>VWR</td>
			<td>444-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol (PEG)</td>
			<td>Merck</td>
			<td>1.09727.0100</td>
			<td>Mw= 4kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with Mw = 4.6kDa</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>392932</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Acrylate-Poly (Ethylene Glycol) - Succinimidyl Valerate&#160;</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-SVA-3400-1g</td>
			<td>Mw = 3.4 kDa</td>
		</tr>
		<tr>
			<td>2-hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenone</td>
			<td>Sigma Aldrich</td>
			<td>410896</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>58816-123</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5404 000.413</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Film&#160;</td>
			<td>Parafilm M&#160;</td>
			<td>#PM996</td>
			<td>Kept at 9&#34; with allows intensity of 10.84 mW/cm^2</td>
		</tr>
		<tr>
			<td>Bluewave 200 UV spotlight</td>
			<td>Blaze Technology&#160;</td>
			<td>120008, 122300</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF)</td>
			<td>Merck</td>
			<td>107025</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Heidolph</td>
			<td>543-123120-00-5</td>
			<td>From rat</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Sigma Aldrich</td>
			<td>C3867-1VL</td>
			<td>1X, w/o CaCl &#38; MgCl; Ph = 7.2</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)&#160;</td>
			<td>Gibco</td>
			<td>20012-027</td>
			<td>16% W/V AQ. 10x10ml</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>43368.9M</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Freezone 4.5 freeze drier</td>
			<td>Labconco</td>
			<td>7750020</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Jeol Ltd.</td>
			<td>JFC-1600</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Jeol Ltd.</td>
			<td>JSM 5310</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse primary antibodies against Collagen type I</td>
			<td>Abcam</td>
			<td>ab6308</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse secondary antibody conjugated with Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon)&#160;</td>
			<td>Bio-Rev PTE LTD</td>
			<td>3820-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium(DMEM) 
			2.5 g/L Glucose w/ L-Gln</td>
			<td>Lonza</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Life Tchnologies</td>
			<td>15140-122 E</td>
			<td></td>
		</tr>
		<tr>
			<td>APC49&#8208;Huh &#8208;7.5 Cell Line</td>
			<td>Apath</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Corning non-treated culture dishes</td>
			<td>Sigma Aldrich</td>
			<td>CLS430591</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Equipment; 37&#176;C, 5% Humidity</td>
		</tr>
		<tr>
			<td>Forma Steri-Cycle CO2 Incubators</td>
			<td>Thermofisher Scientific</td>
			<td>371</td>
			<td></td>
		</tr>
		<tr>
			<td>Hausser Bright-Line Phase Hemacytometer</td>
			<td>Thermofisher Scientific</td>
			<td>02-671-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Viability/Cytotoxicity Kit &#39;for mammalian cells</td>
			<td>Life Technologies</td>
			<td>L3224&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 Assay</td>
			<td>Dojindo Laboratories</td>
			<td>CK04-11</td>
			<td>Monosodium-salt reagent (MSR)</td>
		</tr>
		<tr>
			<td>Infinite 200 PRO microplate reader&#160;</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Human ELISA kit</td>
			<td>Abcam</td>
			<td>ab108788</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bio-Rad</td>
			<td>#1610407</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse primary antibodies (against CYP3A4, albumin)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53850; sc-271605</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 labelled Phalloidin</td>
			<td>Life Technologies</td>
			<td>A34055</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>22706.326</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>DPEC water</td>
			<td>Thermofisher Scientific</td>
			<td>AM9916</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000c Spectrophotometer</td>
			<td>Thermofisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript Reverse Transcription Supermix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>1708840</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR select Master Mix for CFX</td>
			<td>Life Technology</td>
			<td>4472937</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers (to be chosen)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Real-Time System, C-1000 Touch Thermal Cycler</td>
			<td>Bio Rad Laboratories</td>
			<td>SOFT-CFX-31-PATCH&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The representative results for the structural characterization of the ICC scaffold and the comparison of each ICC scaffold condition&#39;s efficacy in culturing hepatocytes are shown and explained below. The ICC scaffold conditions used in these results are collagen coatings of 0 &#181;g/ml (Bare), 20 &#181;g/ml (Collagen 20), 200 &#181;g/ml (Collagen 200), and 400 &#181;g/ml (Collagen 400) and the initial Huh-7.5 cell seeding number is 1x106.
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Watch this Scientific Journal Video about Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering at JoVE.com

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.

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 ...

fabrication-inverted-colloidal-crystal-poly-ethylene-glycol-scaffold

Research

JoVE Journal

Bioengineering

9.8K Views.  Nanyang Technological University. 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.

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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

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

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

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

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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

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

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

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

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...