Capillary Force Lithography for Cardiac Tissue Engineering

Summary

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Abstract

Cardiovascular disease remains the leading cause of death worldwide¹. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart’s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale². Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering^3-5.

A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling². Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart^6-9.

Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)⁸ and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function^10-14.

Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively^15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (λ = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 °C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS².

Introduction

Cardiovascular disease is the leading cause of morbidity and mortality in the world and present a weighty socio-economic burden on an already strained global health system^1,17. Cardiac tissue engineering has two distinct goals: (1) to regenerate damaged myocardium after ischemic disease or cardiomyopathy or (2) to create a high fidelity model of the heart for in vitro drug screening or disease modeling.

The heart is a complex organ that must work constantly to supply blood to the body. Densely packed laminar structures of cardiomyocytes and supportive tissues are arranged in helical patterns throughout the heart wall^18,19. The heart is also electromechanically coupled²⁰ in a highly coordinated fashion to efficiently eject blood to the body²¹. Several major hurdles remain to be addressed, however, before nature’s intricate design can reliably be recapitulated in vitro. First, although robust cardiomyocyte differentiation methods continue to be developed²², hPSC-CMs still exhibit rather immature phenotypes. Their electromechanical properties and morphology most closely match fetal levels²³. Second, when kept in traditional culture conditions, both stem cell-derived and primary cardiomyocytes fail to assemble into native, tissue-like structures. Rather, cells become randomly oriented and do not exhibit the banded rod-shaped appearance of adult myocardium²⁴.

The extracellular matrix (ECM) environment with which cells interact plays a significant role in numerous cellular processes^11,13,25. The ECM consists of complex, well-defined molecular and topographical cues that significantly influence the structure and function of cells^6,26. Within the heart, cellular alignment closely follows the underlying nanometer scale ECM fibers². The impact of these nanotopographical cues on cell and tissue function, however, is far from completely understood. Preliminary studies of nanometer scale cell-biomaterial interaction indicate the potential importance and impact of sub-micron topographical cues for cell signaling²⁷, adhesion^28-30, growth³¹, and differentiation^32,33. However, due to the difficulty in developing reproducible and scalable nanofabricated substrates, such studies could not reproduce the multi-scale cellular effects of the complex in vivo ECM environment. In this protocol, a straightforward and cost-effective nanofabrication technique to produce cell culture scaffolds mimicking native cardiac ECM fiber alignment is described, allowing for a wide range of novel investigations of cardiomyocyte-biomaterial interactions. Understanding how cardiomyocytes interact with the nanoscale ECM environment could allow for the ability to control cellular behavior to more closely mimic native tissue function. Furthermore, cell monolayers are a simplified experimental system compared to 3D structures but still exhibit complex multi-cellular behavior for insightful investigations and functional screening^2,34-36. Finally, such scaffolds could be used to improve cellular graft function when implanted into the heart for regenerative purposes³⁷.

Protocol

All procedures are conducted at room temperature (~23 °C) unless otherwise noted.

1. Fabrication of Silicon Master

1. Clean silicon wafer with 100% ethanol or xylene and dry under O₂/N₂ gas.
2. Place silicon wafer in spin-coater at rotation speeds of 2,000-4,000 rpm to produce a 0.3-0.5 µm thick film.
3. Pattern the photoresist film with the correct dimensions by using a photolithography system
4. Fully immerse the patterned photoresist-coated silicon wafers in an appropriate volume of photoresist developing solution.
5. Rinse the developed photoresist-coated silicon wafers with deionized water.
6. To form arrays of sub-micron scale ridges with near vertical side walls, deep reactive ion etch the exposed silicon using an etching system.
7. Remove the remaining photoresist by placing the silicon wafer in a plasma asher system.
8. Cut the silicon wafers with a diamond-tipped cutter into the appropriately sized silicon masters for subsequent replica molding.

2. Fabrication of PUA Mold from Silicon Master

NOTE: Volume to be added to silicon master for nanofabrication will vary depending on the area of the nanopatterned master to be replicated as well as the viscosity of the polyurethane acrylate (PUA) solution.

1. Clean silicon master surface with 100% ethanol or xylene and dry under O₂/N₂ gas.
2. Place silicon master pattern side up in a Petri dish.
3. For a silicon master with a 2 cm x 2 cm surface pattern, pipette 40 µl of PUA to the pattern surface.
4. Place a sheet of 4 cm x 4 cm transparent polyester (PET) film over the dispensed PUA.
5. Press down on the PET sheet and spread the PUA underneath the sheet across the pattern face using a roller or flat edged surface (such as a card) so that the entire pattern is covered by the PUA prepolymer.
6. Place silicon master, prepolymer, and PET approximately 10 cm below a 20 Watt (115 V) UV light (λ = 365 nm) for 50 sec. To be effective, the UV light wavelength can be anywhere between 310-400 nm. The intensity of the light is 10-15 mW/cm² at the surface of the substrate.
7. After curing, remove PET film slowly with forceps. PUA should attach to the PET film with a negative of the silicon master nanopattern.
8. Cure PUA/PET nanopatterns under UV for at least 12 hr prior to use. Overexposure is not an issue.
9. To clean silicon masters, place another film of PET on top of the master without the addition of PUA and expose to UV light (λ = 365 nm) for 50 sec and remove PET film. This will remove any unreacted monomers.
10. Rinse silicon master with 100% ethanol or xylene and dry under O₂/N₂ gas.

3a. Nanopatterning Polyurethane Polymer

1. Prepare 25 mm diameter circular glass slides by placing in an ozone treatment chamber for 10 min.
2. Place ozone-treated glass slides onto small PDMS block for easy handling.
3. Apply thin layer of surface adhesion promoter with paintbrush to glass slides. Air dry glass slides for 30 min.
4. Place the glass slide on a piece of printer paper.
5. Drop dispense 10 μl of polyurethane (PU) pre-polymer (NOA 76) to center of glass slide. Make sure no bubbles are present after addition.
6. Place PUA mold, pattern face down, onto the glass slide. Disperse the PU uniformly across the surface of the glass slide by rolling a rubber cylinder roller along the PUA mold. Printer paper will absorb polymer overflow.
7. Watt UV lamp. Polymerization time of the PU is dependent on power of UV source.
8. Remove the sample from UV light source and carefully peel the PUA mold from PU coated glass slide. Polymerization of the PU is considered complete when the PUA mold peels cleanly away from the sample and the PU glass slide has an iridescent appearance.
9. Place finished samples in desiccator for storage for as long as a month.

3b. Nanopatterning Poly(Lactide-Co-Glycolide) Hydrogel

1. Create a flat PDMS mold by vigorously mixing silicone elastomer base and silicone elastomer curing agent in a 10:1 ratio.
2. Pour mixed PDMS precursor solution into a Petri dish so that the PDMS precursor reaches 5 mm up the edge of the dish (i.e. so that the PDMS is 5 mm thick).
3. Place Petri dish and PDMS precursor in a desiccator for 1 hr to degas.
4. Move Petri dish and PDMS precursor into a 65 °C oven for at least 2 hr to cure.
5. After PDMS is cured, use a razor to cut the flat PDMS into 3 cm x 3 cm square sections. These will be the flat PDMS molds used later in the patterning process.
6. Clean 25 mm diameter circular glass slides by placing in isopropyl alcohol for 30 min in a water sonicator.
7. Dry cleaned glass slides under O₂/N₂ gas.
8. Drop dispense 100 μl of PLGA solution (15% w/v PLGA in chloroform) onto glass slide.
9. Place a flat PDMS mold on top of the dispensed PLGA to absorb the solvent and obtain a flat PLGA surface. Apply a light pressure (~10 kPa) by placing a 200 g weight on top of the PDMS for 5 min.
10. Slowly peel away flat PDMS mold and place the cover glass on a preheated plate (120 °C) for 5 min to remove residual solvent and increase adhesion between the PLGA and the cover glass.
11. Place the NP PUA mold on top of the flat PLGA and apply constant pressure (~100 kPa) and heat (120 °C) by placing a 1 kg weight on top of the PUA mold while on the heating plate for 15 min.
12. Remove the weight from PLGA cover slide and allow the substrata to cool to room temperature. Do not remove PUA mold until the substrata have been cooled.
13. Once the substrata have sufficiently cooled, carefully peel away the NP PUA mold, revealing the NP PLGA substratum. The NP PLGA substratum should have an iridescent appearance.
14. Place finished samples in desiccator for storage for as long as a month.

4. Cell Seeding and Culture

NOTE: This protocol describes the culture of neonatal rat ventricular myocytes (NRVMs) and H7 human embryonic stem cell-derived cardiomyocytes (hESC-CMs) but other cell sources may be used.

1. Attach the ANFS coverslips (PU or PLGA) to a 35 mm tissue culture polystyrene dish. Pipette 20 µl of Norland Optical Adhesive (NOA83H) to the bottom of the dish and gently place the ANFS coverslip on top of the NOA. Allow glue to spread out and cover entire coverslip bottom. Cure NOA by exposing dish to UV for 10 min.
2. Sterilize ANFS by rinsing with 2 ml of 70% aqueous ethanol solution for 5 min, twice. Remove ethanol by aspiration. Allow ANFS to completely air dry for ~1 hr under the UV sterilization lamp (λ = 200-290 nm) in the biological safety cabinet.
3. Cellular adhesion is enhanced by coating the ANFS in fibronectin overnight. Dilute fibronectin in DI water to 5 µg/ml. Pipette 2 ml of fibronectin solution into dish. Place in incubator at 37 °C and 5% CO₂ overnight (at least 6 hr).
4. Obtain NRVMs, hESC-CMs, or other cardiac cells of interest according to previous protocols²².
5. Centrifuge cell sample at 1,000 rpm for 3 min to pellet the cells.
6. Carefully remove the supernatant by aspiration. Make sure not to disturb the pellet.
7. Resuspend cells in appropriate culture media to a concentration of 4.6 x 10⁶ cells/ml.
8. Carefully pipette 200 µl of cell suspension onto sterilized ANFS. Make sure cell suspension remains on the coverslip.
9. Place cells in incubator at 37 °C and 5% CO₂ for 4 hr to allow cells to attach to ANFS.
10. Add 2 ml of warm culture media to dish and replace cells in incubator under the same conditions.
11. After 24 hr, remove media and wash with 2 ml of DPBS twice to remove excess cells.
12. Add 2 ml of warm culture media to dish and replace cells in incubator under the same conditions. Culture the cells to confluence. Replace media every other day.

Results

Figure 1 is a schematic overview of the production process for the two fabrication methods. Due to the diffraction of light caused by the nanoscale topography, nanopatterning should result in an iridescent surface to the ANFS. Figure 2 depicts this iridescent surface on a well-patterned 25 mm NP-PU coverslip (Figure 2A) with 800 nm ridge and groove width (Figure 2B). The iridescent appearance of the ANFS will vary slightly depending on the ridge and groo...

Discussion

Functionally mature cardiac tissues are lacking for both in vivo and in vitro applications of cardiac tissue engineering. The CFL nanofabrication methods described here are robust techniques for achieving cellular alignment and influencing macroscopic tissue function due to the scalability of the system. Large areas can easily be patterned and used for cell culture. Macroscopic cellular alignment is essential in cardiac tissue engineering in order to create biomimetic, functional tissue as it influences...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fibronectin	BD Biosciences	354008
NOA 76	Norland Products, Inc.	7606B
Surface Adhesion Promotor (Glass Primer)	Minuta Tech
PUA	Minuta Tech	MINS-311RM
Soft Rubber Roller	Speedball
Silicon Wafers	NOVA Electronic Materials	FA01-9900
Photoresist	Shipley	SPRT510
Photoresist Developer	Shipley	MF320
Electron-Beam Lithography System	JEOL	JBX-9300FS
Etching System	Surface Technology Systems	NP10 8UJ
Plasma Asher System	BMR Technology Co.	DSF-200
Ozone Cure System	Minuta Tech	MT-UV-O- 08
Fusion Cure System	Minuta Tech	MT-UV-A 11
NOA 83H	Norland Products, Inc.	8301
Spin Coater	Laurel Technology	WS-400-6NPP
Skyrol PET Film	SKC Co., Ltd.	23038-59-9
25 mm Glass Slides	Corning	2948
Sylgard 184 Silicone Elastomer Kit	Dow Corning	6/5/2553
Poly(D,L-lactide-co-glycolide)	Sigma-Aldrich	P2191-1G
Chloroform	Sigma-Aldrich	372978-1L
500 g Weights	Global Insustrial	T9FB503120
Isopropyl Alcohol	EMD Millipore	PX1835-2
Hot Plate	Corning	PC-420D
Sonicator	Branson	B2510MTH