Spatially controlled cell adhesion on three-dimensional substrates
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The microenvironment of cells in vivo is defined by spatiotemporal patterns of chemical and biophysical cues. Therefore, one important goal of tissue engineering is the generation of scaffolds with defined biofunctionalization in order to control processes like cell adhesion and differentiation. Mimicking extrinsic factors like integrin ligands presented by the extracellular matrix is one of the key elements to study cellular adhesion on biocompatible scaffolds. By using special thermoformable polymer films with anchored biomolecules micro structured scaffolds, e.g. curved and µ-patterned substrates, can be fabricated. Here, we present a novel strategy for the fabrication of µ-patterned scaffolds based on the “Substrate Modification and Replication by Thermoforming” (SMART) technology: The surface of a poly lactic acid membrane, having a low forming temperature of 60°C and being initially very cell attractive, was coated with a photopatterned layer of poly(L-lysine) (PLL) and hyaluronic acid (VAHyal) to gain spatial control over cell adhesion. Subsequently, this modified polymer membrane was thermoformed to create an array of spherical microcavities with diameters of 300 µm for 3D cell culture. Human hepatoma cells (HepG2) and mouse fibroblasts (L929) were used to demonstrate guided cell adhesion. HepG2 cells adhered and aggregated exclusively within these cavities without attaching to the passivated surfaces between the cavities. Also L929 cells adhering very strongly on the pristine substrate polymer were effectively patterned by the cell repellent properties of the hyaluronic acid based hydrogel. This is the first time cell adhesion was controlled by patterned functionalization of a polymeric substrate with UV curable PLL-VAHyal in thermoformed 3D microstructures.
KeywordsThree-dimensional cell culture Biopolymer Patterning Microthermoforming Tissue engineering
Two-dimensional (2D) cell cultures are routinely used as models to study the principles of cell biology. However, the commonly applied tissue culture plastic (TCP) substrates do rarely provide environments that resemble the in vivo conditions and thus are not accurate models of the natural situation (Prestwich 2007). Major drawbacks of these 2D cell culture systems are the lack of structural architecture, the limited material variety and no adequate control of many biochemical cues. Cells are forced to adapt to the flat, rigid substrate which may result in altered metabolism and reduced functionality (Beigel et al. 2008). Therefore, the simplified 2D technology does not allow for tissue engineering in which the maintenance of differentiation and cell type specific functions are playing a pivotal role (Cukierman et al. 2001).
In natural tissues, the extracellular matrix (ECM) surrounds nearly all cells by providing a complex three-dimensional meshwork of fibers combined with many biochemical and physical cues. This microenvironment is specific for each cell type and controls cell viability, i.e. proliferation, differentiation and metabolism (Streuli 1999). The ECM is a highly hydrated, gel-like material composed of collagen-fibers, proteoglycans and glycoproteins (Lodish et al. 2000) with unique mechanical characteristics like stiffness, porosity and permeability.
It is well known that cells respond in vitro to chemically and topographically patterned surfaces, for a review see: (Lim and Donahue 2007). To mimic a natural three-dimensional (3D) environment, advanced cell culture systems have been developed. These matrices or scaffolds are often porous substrates like sponges, fiber meshworks or microcapsules that can support cell growth, organization and differentiation on or within their structure (Barbucci et al. 2004; Ju et al. 2008; Lee et al. 2008). Another technique to culture cells three-dimensionally is the use of microwell arrays made of polymers (Bhatia and Chen 1999; Giselbrecht et al. 2006; Wu et al. 2006). In this context, microfabrication tools such as photolithography, plasma etching, soft lithography and microthermoforming are employed to produce microarrays and cell culture chips with characteristic and reproducible geometries to create organotypic structures (Borenstein et al. 2007). However, depending on the polymer material and its surface characteristics, cell adhesion inside of microcavities may only be controlled globally if no further cell confinement process is applied. As a consequence, cells statistically seeded from suspension onto a microwell array will usually also adhere to all exposed surfaces leading to a mixed population of a cell monolayer between the wells and a 3D cell culture situation inside the microcavities (Altmann 2008). This might lead to misinterpretations of some observations.
Several approaches to restrict cell adhesion to microcavities have been proposed. Mohr et al. coated a moulded polyurethane substrate with a protein resistant self-assembled monolayer of triethylene glycol-terminated alkanethiols between the wells and a Matrigel™ adsorbate inside the wells. This device was capable of maintaining undifferentiated human embryonic stem cells in culture for weeks without passaging (Mohr et al. 2006). Ochsner et al. passivated the plateau of a polydimethylsiloxane microstructure by inverted microcontact printing of poly(L-lysine)-graft-poly(ethylene glycol). Subsequent incubation with a fibronectin solution selectively coated the wells with this cell-attractive protein to control the shape of single cells (Ochsner et al. 2007). A method called ‘reactive microcontact printing’ to control topography and localized presentation of extracellular matrix and transmembrane hematopoietic stem cell regulatory proteins was described by Lutolf (Lutolf et al. 2009). Our approach of fabricating microstructured cell culture substrates with a site-specific functionalization is based on the SMART-technology (Substrate Modification And Replication by Thermoforming) (Giselbrecht et al. 2006; Truckenmuller et al. 2008). SMART combines microtechnical thermoforming with a pre-modification of a planar polymer film with a high spatial resolution. Because the forming of the film occurs in its softened and not in its molten state pre-modifications are preserved. To guide cell adhesion locally we used a patterned hyaluronic acid (Hyal) layer as pre-modification. Hyaluronic acid, a major component of the natural ECM, is a water soluble polysaccharide. Hyal or its derivatives are usually deposited onto a surface by a sol-to-gel phase transformation. To obtain cell-inoculated scaffolds or drug-immobilized matrices, various chemical and physical cross linking and immobilization techniques of Hyal have been developed (Allison and Grande-Allen 2006; Ifkovits and Burdick 2007; Kogan et al. 2007). It was observed that most Hyal derivatives are cell and even protein repellent (Barbucci et al. 2003) and must therefore be modified to render them adhesive. A common approach to promote cell adhesion is to use artificial polymers like polylysine (Blau et al. 2001; Quirk et al. 2001). Picart et al. demonstrated that a polyelectrolyte layer made from polycationic poly(L-lysine) (PLL) and polyanionic Hyal acts as a ECM-like environment (Picart et al. 2001).
In this report, we modified this approach to generate thermomechanically processable films. The cell adhesion mediator, poly(L-lysine), was mixed with a photocurable Hyal derivative. This mixture was immobilized onto biocompatible PLA films (Desmet et al. 2009) by masked UV irradiation. The pre-patterned thin PLA film was then processed by microtechnical thermoforming to yield microwell arrays with an aligned pattern of cell repellent and attractive sites. The preservation of the cell adhesion pattern is possible due to the permanent material cohesion during the solid state forming process and, moreover, the surface patterns can be easily aligned to the obtained microwell topography of the thermoformed polymer membrane, to control cell adhesion with high precision even locally inside of each well.
2 Materials and methods
2.1 Synthesis of vinylated hyaluronic acid (VAHyal)
The coupling of 4-vinyl aniline to sodium hyaluronate, was described by Matsuda et al. (Matsuda and Magoshi 2002). Hyaluronic acid (200 mg, MW 1.2–2.6 × 106 Da, Lifecore) was dissolved in 160 ml Ca2+/Mg2+-free phosphate-buffered saline (PBS, Gibco). After the addition of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (191 mg, 1 mmol, Sigma), and stirring for 15 min, the solution was adjusted to pH = 3 with 1 N HCl (Roth). 4-Vinylaniline (240 µL, 2 mmol, Aldrich) was added and the reaction mixture was stirred at 4°C for 24 h. Purification of the vinyl derivatized biomacromolecule (VAHyal) was carried out using a dialysis membrane (cut off MW: 12 × 103 Da, Sigma). Dialysis against distilled water for 3 days resulted in the complete removal of any non-transformed vinyl aniline and soluble side products from the solution. After freeze drying, the product was stored in a cool and dark place.
The identity and purity of VAHyal, a yellowish powder, was confirmed by 1H-NMR spectroscopy (Bruker, Avance, 250 MHz). The yield of vinylation, the number of styryl groups incorporated into hyaluronan, was calculated based on the UV absorbance of VA: ε260 nm = 2.1 × 104 l mol-1 cm-1.
2.2 Machinery and PLA-membrane processing
2.2.1 Thermoforming mould insert
Microstructured brass plates (RECORD Metall-Folien) of 300 µm thickness with a mechanically milled array of 25 × 25 circular through-holes of 350 µm diameter were used as mould inserts. The thickness of these inserts corresponds to the maximum depth of the microcavities to be thermoformed. These thin mould inserts, shown in Fig. 1 (yellow), allowed the alignment of through-holes to the circle pattern of a corresponding photo mask, Fig. 1 (black/white), for a site-specific immobilization of VAHyal before thermoforming (see chapter 2.2.2).
2.2.2 Preprocessing and immobilization of VAHyal on PLA membranes
A PLA membrane, Fig. 1 (red), was fixed on the mould insert of the thermoforming machine, Fig. 1 (yellow). The fixed PLA film on the mould insert was plasma treated in a radio frequency plasma generator (Oxford Plasma Technologies) with capacitive coupling (water cooled electrodes of 250 mm diameter and 35 mm distance) using the following parameters: N2 atmosphere, flow: 50 standard cm3/min, pressure: 356 Pa, radio frequency: 13.56 MHz, power: 150 W, duration: 3 min.
The photo polymerization process to graft either pure VAHyal or the mixture of VAHyal and ε-poly-L-lysine (0.1% w/v solution in water, Sigma) on PLA films was initiated by Irgacure 2959 (Ic), (Ciba), and N-Vinylpyrrolidone (Vp), (Merck) as photoinitiators. The cytocompatibility of Irgacure 2929 was shown by Bryant et al. (Bryant et al. 2000). Leach et al. did show cross linking of a similar Hyal derivative (Leach et al. 2003).
15 mg/mL VAHyal, 1 mg/mL Vp, and 1 mg/mL Ic, in aqueous EtOH;
0.1 mg/mL PLL, 1 mg/mL Vp, and 1 mg/mL Ic, in aqueous EtOH.
To produce mixed VAHyal/PLL coatings, a working solution (c) was mixed from the VAHyal stock solution, (a), and the PLL stock solution, (b), in a ratio of 2:1. Otherwise, pure (a) was used. (a) or (c) was spin-coated onto the plasma treated PLA membrane. The following spin-coating protocol was applied: 10 s at 200 rpm, followed by 60 s at 3000 rpm, followed by 5 s at 4,000 rpm. Subsequently, this layer, Fig. 1 (green), was irradiated by polychromatic UV light (HBO high pressure mercury lamp, Osram) to yield a water-insoluble photocrosslinked VAHyal, (pclVAHyal). Irradiation dose: 11 J cm-2 measured at λ 365 nm. In case of masked irradiations, the transparency of the modified PLA film allowed the alignment of irradiation masks with the through holes of the mould insert. Finally, the pattern was developed by washing the samples with demineralised H2O. This process is schematically presented in Fig. 1.
2.3 Surface analysis
Contact angle measurements were performed on PLA samples with the sessile drop method using a goniometer (OCA10, dataphysics). To visualize the photocrosslinked VAHyal layers, staining with crystal violet (0.5% in 20:80 MeOH:H2O, 30 min) was used. Thicker pclVAHyal layers stain violet, thin physisorbed layers can not be detected by this technique. In addition, X-ray photoelectron spectroscopy (XPS) was performed on a VG Scientific ESCALAB 5 with Alpha 110 analyser (Thermo Fisher Scientific), excitation by non-monochromatic MgKα-radiation, power 200 W, pass-energy 20 eV, electron emission angle 60° to the surface normal. Data analysis was performed according to R. Hesse et al. (Hesse et al. 2007)
2.4 Cell culture
HepG2 cells (ATCC, strain: HB-8065) were cultured in Minimum Essential Medium, (MEM), (Gibco) supplemented with 10% foetal calf serum (PAA), 1% non-essential amino acids (PAA), 1% Na-pyruvate (PAA), 1% Glutamax (Gibco), 1% penicillin/streptomycin (PAA) and 0.1% phenol red (Sigma). L929 cells (ATCC, strain: C3H/an, designation: NCTC clone 929) were cultured in MEM supplemented with 10% foetal horse serum (ATCC) and 1% penicillin/streptomycin. Both cell lines were cultured in tissue culture flasks (BD Falcon) under culture conditions of 37°C, >95% relative humidity, 5% CO2.
The modified PLA-substrates were used in cell culture experiments after sterilization with 70% isopropanol and subsequent washing with PBS. The scaffolds were pre-incubated overnight with the correspondent cell culture medium. For cell seeding into the microstructured scaffolds, 300 µL cell suspension with 5 × 105 cells were placed on the thermoformed area of the PLA film. Spreading of the suspension was avoided by a silicone ring placed on the PLA film. After three hours of pre-incubation with the cell suspension, cell culture medium was added to cover the PLA scaffold completely.
Cell adhesion and proliferation was monitored by staining with May-Grünwald’s eosin methylene blue (Merck). Fluorescence micrographs were recorded on a confocal laser scanning microscope (Leica LSM TCS SP5) after rinsing the scaffold with PBS and cell staining with 0.5% Syto16® (Invitrogen) and 0.1% propidium iodide (Fluka) in MEM (30 min).
3 Results and discussion
3.1 Chemical modifications
The vinylated hyaluronic acid product was isolated as yellowish powder. The successful coupling of VA to Hyal was verified by NMR and UV/VIS spectroscopy. 1H-NMR (Bruker, 250 MHz, D2O, 25°C) σ = 7.49 (m, 4H, aromatic), 6.75 (m, 1H, vinyl-CH-), 5.22 (m, 1H, vinyl-CH2-), 4.56 (m, 2H, OH-CH2-), 3.63–3.41 (m, 10H, H-polysaccharide), 1.93 (s, 3H, CH3-). The number of derivatized carboxylic groups was calculated from UV absorption data to be 2,5 per molecule. Successful UV initiated immobilization and cross linking of this derivative on PLA was observed.
3.2 Surface modifications
Contact angles of a 1:1 ethanol:water mixture on native and plasma treated PLA
PLA, N2 -plasma treated
50° ± 2°
26° ± 2°
less than 10°, fully wettable
Measured atomic concentrations from XP spectra for native PLA, nitrogen plasma treated PLA, UV irradiated PLA, plasma and UV treated PLA, photoinitiators Ic+Vp on plasma treated PLA with UV treatment and VAHyal/Ic/Vp on plasma treated PLA with UV treatment
Measured atomic concentrations [%] for:
PLA + N2
PLA + UV
PLA + N2 + UV
PLA + N2 + Ic + Vp + UV
PLA + N2 + VAHyal/Ic/Vp + UV
In the case of immobilized VAHyal with Ic and Vp from solution (a) nitrogen was detected on PLA. Therewith, successful photocrosslinking of VAHyal was confirmed. Further, it was verified that the photoinitiators Irgacure and Vinylpyrrolidone did not modify the PLA surface, which otherwise may result in an altered cell adhesion: No indications were found in XP spectra (data not shown) and in the elemental ratios, see Table 2.
We observed no nitrogen containing functionalities on N2-plasma treated PLA. It can be concluded, that the N2 plasma treatment did not insert any nitrogen into the PLA film, while it notably hydrophilized the surface, thereby facilitating the subsequent spin-coating process with the VAHyal-solution (Table 1).
3.3 Thermoforming process
During the thermoforming process the PLA film was heated up to 60°C and had to resist a pressure of 2 to 3 MPa. We investigated the behavior of a uniform and a patterned VAHyal layer on the PLA film after stretching in a thermoforming process at 60°C and 3 MPa (according to Fig. 1).
To rule out the possibility that pclVAHyal would decompose during the thermoforming process, the melting temperature of pclVAHyal was determined. At 180°C a slow decomposition of the polymer was observed; thus it can be assumed that thermoforming conditions for PLA membranes are compatible with pclVAHyal layers.
3.4 Cell culture
HepG2 hepatoma cells were cultured on VAHyal patterned PLA to validate it as a suitable cell culture substrate. L929 mouse fibroblasts, a cell line with high adhesiveness on many cell culture substrates like polystyrene, PLA, and even PDMS, were used to demonstrate the effectiveness of cell patterning. On PLA films that were uniformly covered with a pure pclVAHyal layer, neither HepG2 nor L929 cell adhesion was observed. Surprisingly, we found also no cell adhesion in any region of a patterned flat pclVAHyal film on PLA, comparable to the sample shown in Fig. 3, irrespective of local UV doses. In the non-irradiated areas no cross linking of the biopolymer should have occurred, leaving VAHyal fully water soluble and therefore those PLA regions should have remained cell-attractive. In these areas, an adsorbate of VAHyal on non-irradiated PLA surfaces could not be visualized by crystal violet staining, but the presence of a nitrogen peak in XP spectra (data not shown) clearly indicated the presence of a thin VAHyal layer.
Having successfully patterned 2D PLA films, pclVAHyal/PLL pre-treated PLA films were subjected to the thermoforming process and tested with HepG2 and L929 cell cultures to demonstrate the more sophisticated manufacturing of patterned three-dimensional substrates.
Figure 7a shows a part of a three dimensional PLA scaffold which was plasma treated and exposed to UV applying an aligned circle mask (UV light blocked for membrane areas which become reshaped to microcontainers). Irrespective of local UV exposure HepG2 cell adhesion and spreading was observed inside and between the thermoformed cavities.
To prepare the sample shown in Fig. 7b, VAHyal (solution (a)) was spin-coated onto plasma treated PLA, exposed to UV using the aligned circle mask and subsequently thermoformed (according to Fig. 1). In contrast to a flat PLA film where no cell adhesion was monitored at all for pclVAHyal patterns, the stretched areas of the PLA film inside the microcavities provided a cell attractive surface. As shown in Fig. 7b, cells were able to adhere to the PLA substrate within the ruptured pclVAHyal layer that had been generated by strong stress during the thermoforming process. The generated noncoherent VAHyal structures allowed for cell adhesion between the VAHyal fragments, (compare to Fig. 5). This side-effect of stretching, which was found for thermoformed patterned pclVAHyal substrates (Fig. 7b) and also for uniform, flood exposed, pclVAHyal layers (Fig. 5) opens an easy way to restrict cell adhesion to the microcavities.
If plasma treated PLA was spin-coated with VAHyal/PLL, irradiated with UV through the aligned circle mask and then thermoformed, HepG2 cells exclusively adhered inside the cavities (see Fig. 7c). This agrees with the cell adhesion observed for HepG2 on flat PLA patterned with pclVAHyal/PLL (see Fig. 6). On elevated surfaces between cavities cells were completely absent (Fig. 7c).
It was observed that cells initially spread between the pclVAHyal/PLL lines (Figs. 9a and b) and later grew across the pclVAHyal/PLL lines, until they finally filled the entire cavity as three dimensional aggregates. In contrast to other techniques (Lutolf et al. 2009; Mohr et al. 2006; Ochsner et al. 2007), the presented method based on the SMART-technology allows spatially controlled cell adhesion inside of microcavities.
It is generally accepted that three dimensional multicellular organoids represent a more realistic tissue model as compared to isolated single cells lacking cell/cell contacts or conventional cell monolayers in 2D configurations (Prestwich 2007, Beigel et al. 2008). Especially, in case of highly metabolizing primary liver cells, but even in case of conventionally hepatoma cells like HepG2, three dimensional cell culture configurations often outperform their classical 2D counterparts (Altmann 2008), e.g. we have shown recently that in HepG2 cytochrome oxidase II and III genes are upregulated more than fivefold in 3D vs. 2D culture (Gottwald et al. 2007). We presented an advancement of the SMART process by including a patterned biofunctionalization of the substrate preceding its forming. This allowed us to precisely define cell-adhesive and non-adhesive regions that can be independently controlled and partially separated from the mechanical alteration of surface topography. Thus, by combining the coating of a plasma-activated PLA substrate with a mixture of VAHyal and PLL, its 2D UV patterning by photolithography followed by a final thermoforming process, a biochemically functionalized substrate for three-dimensional cell culture was obtained. The concept had been validated by a HepG2 cell culture model. HepG2 and L929 cells exclusively aggregated inside the non-irradiated yet VAHyal/PLL-coated, thermoformed microcavities, while unwanted adhesion and spreading between the cavities was prevented by photochemically crosslinked VAHyal/PLL coatings.
Evaluation of substrate and coatings displayed by symbols with regard to cell adhesion (+) or no cell adhesion (-)
Substrate and coating:
Flat, non stretched areas
Curved thermoformed areas, areal draw ratio <2a
PLA without coating
PLA + VAHyal (non irradiated)
PLA + pclVAHyal
PLA + VAHal/PLL (non irradiated)
PLA + pclVAHyal/PLL
The authors would like to thank Michael Bruns for his help with XPS measurements, Alex Gerwald for contributing ”SMART processed” membranes, and Sebastian Bartels for his assistance with cell culture and staining.
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