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://jove.unicartagenaproxy.elogim.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><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>631-1575</td><td>Dimensions: 24&amp;times;60 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Polystyrene spheres</td><td>Fisher Scientific</td><td>TSS#4314A</td><td>Diameter = 140 &amp;micro;m; 3x10&lt;sup&gt;4&lt;/sup&gt; 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 &amp;micro;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>M&lt;sub&gt;w&lt;/sub&gt;= 4 kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with M&lt;sub&gt;w&lt;/sub&gt; = 4.6&amp;nbsp;kDa</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</td><td>Laysan Bio</td><td>ACRL-PEG-SVA-3400-1g</td><td>M&lt;sub&gt;w&lt;/sub&gt; = 3.4 kDa</td></tr><tr><td>2-hydroxy-4&#039;-(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</td><td>Parafilm M&amp;nbsp;</td><td>#PM996</td><td>Kept at 9&quot; with allows intensity of 10.84&amp;nbsp;mW/cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Bluewave 200 UV spotlight</td><td>Blaze Technology&amp;nbsp;</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></td></tr><tr><td>Collagen Type I</td><td>Sigma Aldrich</td><td>C3867-1VL</td><td>From rat. 1x, w/o CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;amp; MgCl&lt;sub&gt;2&lt;/sub&gt;; pH = 7.2</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>20012-027</td><td>16% W/V AQ. 10 x 10&amp;nbsp;ml</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)&amp;nbsp;</td><td>Bio-Rev PTE LTD</td><td>3820-024</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium(DMEM)&lt;br/&gt; 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>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&amp;nbsp;&amp;deg;C, 5% Humidity</td></tr><tr><td>Forma Steri-Cycle CO&lt;sub&gt;2&lt;/sub&gt; 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 for mammalian cells</td><td>Life Technologies</td><td>L3224&amp;nbsp;</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&amp;nbsp;</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 labeled 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&amp;nbsp;</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&amp;nbsp;</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

Universidad de Cartagena UDEC

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

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

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

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

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

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 ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...