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 m...

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.
<p class="jove_content" fo:kee...

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 ...

The overall goal of this procedure is to fabricate PEG-based inverted colloidal crystal scaffolds with an extracellular matrix coding for liver tissue engineering applications. Liver tissue engineering is an exciting field for tissue engineering and regenerative medicine. We re creating 3D engineering scaffold that allow liver cell to maintain their function for a long time.The implications of this technique extends toward drug testing as liver cells cultured with this system may retain their function longer and better response to drug. Though this method can provide insight into tissue engineering, it can also be applied to other systems, such as those using stem cells. Demonstrating the procedure will be Hitomi, a graduate student from my laboratory.To prepare the micro-bead molds, first cut the tips from 0.2 milliliter boil-proof microcentrifuge tubes at the 40 microliter level. Use waterproof glue to adhere the tops of the cut tubes to glass microscope cover slips and then glue around the molds. Next, replace the water in a vial of polystyrene microspheres with 70%ethanol.Place the sphere solution in an ultrasonic bath and sonicate the mixture at 30 watts for three minutes to loosen the aggregated spheres. Once the microspheres have been prepared, pipette 100 microliters of ethanol into each of the molds. Then, cut four millimeters off of the top of a 200 microliter pipette tip and use it to pipette 25 microliters of the polystyrene microspheres into the mold twice.Place the molds on a rocking shaker and set it to 120 RPM. Let the samples sit on the shaker overnight. The next day, place the molds under a microscope and check that the spheres are ordered hexagonally.Let the ethanol evaporate at room temperature for two nights. At this time, carefully place the mold and bead complex into a 130 degrees Celsius oven for six hours to anneal the polystyrene beads and form the negative of the 3D scaffold. First, synthesize polyethylene glycol diacrylate macromers using established protocols as described in these two references.Next, prepare a 50%solution of PEGDA in deionized water and centrifuge the mixture until it is completely dissolved. For extracellular matrix conjugated scaffolds add an addition 10%of a krill oil PEG NHS into the 50%PEGDA solution. Then, prepare a 20%stock solution of the photo-initiator in 70%ethanol.Add 50 microliters of the photo-initiator solution for each milliliter of the PEGDA mixture. Then, vortex the mixture for one minute. Next, peel away the molds from the glass slides, push the lattices out carefully using a spatula and place them into separate 1.5 milliliter centrifuge tubes.Pipette 300 microliters of the PEGDA solution into each tube, and then centrifuge the tubes to allow the PEGDA solution to infiltrate into the polystyrene lattice. Next, remove the lattice from the tube using tweezers and carefully blot excess PEGDA solution from the surface. Place the lattice on paraffin film covered glass with the flat, circular surface facing up.Then, expose the scaffold to 365 nanometer ultraviolet light for five minutes using a UV spot lamp. Place around ten PEGDA polymerized crystal lattices into a new vial and add 20 milliliters of THF. Shake the vials on an orbital shaker at 300 RPM for six hours.Remove the vial every one to two hours and exchange the polymer-containing solvent with fresh THF. After the last rinse, add water to the used THF solution to check if the PS spheres have completely dissolved. If the solution is white, perform additional rinses until the solution runs transparent.To sterilize the scaffolds, prepare a 50 milliliter microcentrifuge tube with two milliliters of 70%ethanol per scaffolds and place the scaffolds in the tube using a spatula. Allow the scaffolds to soak in ethanol for one hour. After one hour, carefully pour the ethanol out and replace it with an equal volume of sterile PBS.Then, centrifuge the scaffolds to remove any bubbles. Store the centrifuge scaffolds at four degrees Celsius and remove them every one to two hours to replace the solution with fresh PBS. After a few changes in PBS, remove the scaffolds to be coated in collagen and place them in a tube containing one milliliter of collagen type one per scaffold to be coated.Next, centrifuge the tube as before, and then, shake the scaffolds at 400 RPM on an orbital shaker for 30 minutes. Store the scaffolds overnight at four degrees Celsius. Culture hepatocarcinoma cells in 100 millimeter cell culture dishes with 10 milliliters of culture medium.Change the medium every three days until the cell density reaches 75 to 80%of the culture surface. When the cells are ready, carefully place the scaffolds with the flat surface facing upward into a 24-well plate. Then, rinse each scaffold with two milliliters of PBS.Aspirate the PBS and add two milliliters of growth medium to the scaffolds. After 30 minutes, remove the medium and allow the scaffold to dry for one hour. Next, harvest the hepatocarcinoma cells using the trypsin digestion method.After pelleting the cells, remove the supernatant and re-suspend the pellet so that there are one million cells per 25 microliters. Slowly pipette 25 microliters of the cell suspension directly on top of each scaffold. Then, cover the plate and place it in the incubator overnight.Next, transfer the scaffolds carefully into a new 24-well plate and pipette two milliliters of medium into each well before returning the plate to the incubator. A scanning electron microscope of the scaffold is shown here. It is made up of a hexagonal closest packed arrangement of pores that average 100 to 105 microns in diameter which forms interconnects with the neighboring pores that are mostly between 35 and 40 microns in diameter.When seeded with hepatocarcinoma cells, the cells grown and proliferate on the scaffolds that are pre-coated in collagen type one but do not expand in number on the bare scaffolds. In addition, the hepatocarinoma cells on the collagen-coated scaffolds showed greater cell function in a concentration dependent manner. On day 14, the hepatocarcinoma cells cultured in the 400 microgram per milliliter coated ICC scaffolds secreted more than three times the amount of albumin as those cultured in the bare scaffold.While attempting this procedure, it is important to remember to make sure the polystyrene spheres in the lattice are arranged properly. Poor arrangement will lead to poor inter-connectivity which can adversely affect cell activity. After watching this video, you should have a good understanding of how to fabricate celciated PEG-based inverted colloidal crystal scaffolds with a coating.Don't forget that working with tetrahydrofuran can be hazardous and precautions such as operating in a fume hood and wearing gloves and goggles should always be taken while performing this procedure.

fabrication-inverted-colloidal-crystal-polyethylene-glycol-scaffold

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

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