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

Summary

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.

Abstract

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' 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.

Introduction

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 ....

Protocol

1. ICC Scaffold Fabrication (Figure 1)

1. Prepare the polystyrene (PS) lattices (diameter = 6 mm; 8-13 layers of beads).
   1. To prepare the mold, cut the tips off from 0.2 ml boil-proof microcentrifuge tubes at the 40 µl level. Adhere the top of the cut-tubes to 24 x 60 mm² microscope cover glass slips with water-proof glue.
   2. Put the PS spheres (diameter = 140 µm) 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 severa....

Results

The representative results for the structural characterization of the ICC scaffold and the comparison of each ICC scaffold condition's efficacy in culturing hepatocytes are shown and explained below. The ICC scaffold conditions used in these results are collagen coatings of 0 µg/ml (Bare), 20 µg/ml (Collagen 20), 200 µg/ml (Collagen 200), and 400 µg/ml (Collagen 400) and the initial Huh-7.5 cell seeding number is 1x10⁶.

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
0.2 mL PCR tube	Axygen Scientific	PCR-02D-C	Boil-proof
Gorilla Glue	Gorilla Glue, Inc.	Depends on vendor. This was purchased from a local store.
Glass slides	VWR	631-1575	Dimensions: 24×60 mm
Polystyrene spheres	Fisher Scientific	TSS#4314A	Diameter = 140 um; 3x10^4 particles per milliliter and 1.4% size distribution
Ethanol	Merck	1.00983.1011	absolute for analysis EMSURE; Dilute to 70% with Milli-Q water
Ultrasonic Bath	Elma	S10H	Equiment
Furnace	Nabertherm	N7/H	Equipment
200 µL pipette tip	Axygen Scientific	T-210-Y-R-S
Rocking shaker	VWR	444-0142
Polyethylene Glycol (PEG)	Merck	1.09727.0100	Mw= 4kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with Mw = 4.6kDa
Centrifuge	Beckman Coulter	392932	Equipment
Acrylate-Poly (Ethylene Glycol) - Succinimidyl Valerate	Laysan Bio	ACRL-PEG-SVA-3400-1g	Mw = 3.4 kDa
2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone	Sigma Aldrich	410896
Vortex	VWR	58816-123	Equipment
Microcentrifuge	Eppendorf	5404 000.413
Paraffin Film	Parafilm M	#PM996	Kept at 9" with allows intensity of 10.84 mW/cm^2
Bluewave 200 UV spotlight	Blaze Technology	120008, 122300
Tetrahydrofuran (THF)	Merck	107025
Orbital shaker	Heidolph	543-123120-00-5	From rat
Collagen Type I	Sigma Aldrich	C3867-1VL	1X, w/o CaCl & MgCl; Ph = 7.2
Phosphate Buffered Saline (PBS)	Gibco	20012-027	16% W/V AQ. 10x10ml
Paraformaldehyde	VWR	43368.9M	Equipment
Freezone 4.5 freeze drier	Labconco	7750020	Equipment
Sputter coater	Jeol Ltd.	JFC-1600	Equipment
Scanning Electron Microscope	Jeol Ltd.	JSM 5310
Anti-mouse primary antibodies against Collagen type I	Abcam	ab6308
Anti-mouse secondary antibody conjugated with Alexa Fluor 488	Life Technologies	A21121
Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon)	Bio-Rev PTE LTD	3820-024
Dulbecco's Modified Eagle's Medium(DMEM) 2.5 g/L Glucose w/ L-Gln	Lonza	12-604F
Fetal Bovine Serum (FBS)	Gibco	A15-151
Penicillin-Streptomycin (P/S)	Life Tchnologies	15140-122 E
APC49‐Huh ‐7.5 Cell Line	Apath
100 mm Corning non-treated culture dishes	Sigma Aldrich	CLS430591
0.25% Trypsin-EDTA	Gibco	25200-056	Equipment; 37°C, 5% Humidity
Forma Steri-Cycle CO2 Incubators	Thermofisher Scientific	371
Hausser Bright-Line Phase Hemacytometer	Thermofisher Scientific	02-671-6
Live/Dead Viability/Cytotoxicity Kit 'for mammalian cells	Life Technologies	L3224
CCK-8 Assay	Dojindo Laboratories	CK04-11	Monosodium-salt reagent (MSR)
Infinite 200 PRO microplate reader	Tecan
Albumin Human ELISA kit	Abcam	ab108788
Triton X-100	Bio-Rad	#1610407
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A2153-50G
Anti-mouse primary antibodies (against CYP3A4, albumin)	Santa Cruz Biotechnology	sc-53850; sc-271605
DAPI	Life Technologies	D3571
Alexa Fluor 555 labelled Phalloidin	Life Technologies	A34055
Trizol	Life Technologies	15596-026
Chloroform	VWR	22706.326
Isopropanol	Fisher Scientific	67-63-0
DPEC water	Thermofisher Scientific	AM9916
Nanodrop 2000c Spectrophotometer	Thermofisher Scientific	ND-2000
iScript Reverse Transcription Supermix	Bio-Rad Laboratories	1708840
SYBR select Master Mix for CFX	Life Technology	4472937
Primers (to be chosen)
CFX96 Real-Time System, C-1000 Touch Thermal Cycler	Bio Rad Laboratories	SOFT-CFX-31-PATCH