A. G. Castaño*a,
V. Hortigüelaab,
A. Lagunasbc,
C. Cortinae,
N. Montserratbf,
J. Samitierbcd and
E. Martínezabd
aBiomimetic systems for cell bioengineering, Institute for Bioengineering of Catalonia (IBEC), C/Baldiri Reixac 10-12, 08028 Barcelona, Spain. E-mail: agarciac@ibecbarcelona.eu
bCentro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain
cNanobioengineering, Institute for Bioengineering of Catalonia (IBEC), C/Baldiri Reixac 10-12, 08028 Barcelona, Spain
dDepartment of Electronics, University of Barcelona, C/Martí i Franquès 1, 08028 Barcelona, Spain
eOncology program, Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, 08028 Barcelona, Spain
fCenter of Regenerative Medicine in Barcelona, Barcelona Biomedical Research Park (PRBB), C/Doctor Aiguader, 88, 7a Planta, 08003 Barcelona, Spain
First published on 20th June 2014
An extended microcontact printing technique to chemically pattern hydrogels is reported. The procedure employs standard polydimethylsiloxane stamps and requires minor pre-processing of the hydrogels by freeze-drying. Micropatterned Matrigel™ and gelatin hydrogels induce NIH-3T3 cell alignment and elongation. Furthermore, human embryonic stem cells cultured on fibronectin-patterned hydrogels display beating foci earlier than those cultured on non-patterned substrates.
Protein patterning has been extensively applied to stiff substrates; however the patterning of soft, sticky materials such as hydrogels is not straightforward. Several works have been published reporting successful chemical patterning of hydrogels. Most of them rely on complex techniques such as jet printing,12 microfluidics,13,14 and photo-immobilization strategies15–17 to generate micropatterns of biomolecules on hydrogels. Microcontact printing and related techniques are the most widely used to create molecular patterns onto surfaces because of their high versatility, easy procedure and low cost.18 However, these straightforward methods could exhibit an inefficient application on very soft or tacky substrates such as hydrogels.19 Attempts to expand microcontact printing to hydrogels are based on the use of agarose hydrogel stamps,20,21 the use of intermediate poly(vinyl alcohol) layers,19 require sequential steps on glass slides,22 require substantial chemical modification of the hydrogels,23,24 or need complex combination with stereolithography methods.25
Herein we present a simple and versatile method to create protein micropatterns on soft hydrogels by direct microcontact printing without the need of hydrogel chemical modifications, intermediate layers, or multiple steps. As in the conventional method, we keep on using polydimethylsiloxane (PDMS) stamps. To make hydrogels able to withstand the stamping procedure and to avoid sticking or slip effects, hydrogels are freeze-dried prior to the printing procedure. To prove the success of this expanded technique we choose to print protein features onto Matrigel™ and gelatin substrates, which are hydrogels commonly used in stem cell culture.26,27 We achieved the protein printing on these hydrogels, the micropatterns remained after reconstitution and during cell culture, and they successfully guided cellular alignment. The applicability of the extended microcontact printing approach has been validated in promoting cardiac differentiation. In a preliminary study, human embryonic stem cells have been seeded on fibronectin micropatterned hydrogels until beating foci were observed. Micropatterned substrates displayed promising results, as beating foci were observed earlier than on nonpatterned substrates.
For the microcontact printing process, the hydrogels were freeze-dried (Fig. 1). First, Matrigel™ (at dilutions ranging from 1/40 to 1/10 v/v in DMEM) and gelatin (at dilutions ranging from 0.05% to 0.5% w/v in Milli-Q water) were poured on piranha-activated glass slides. After gelation (2 h at 37 °C), the excess of solution was removed and samples were rinsed with Milli-Q water to avoid the presence of salts. Subsequently, samples were freeze-dried by immersion in liquid nitrogen and vacuum dried for 24 hours at −50 °C and 0.06 mbar of pressure.
After checking the hydrogel surface by scanning electron microscopy (SEM), those dilutions leading to homogeneous distribution of the material on the surface were selected (Fig. S1 in the ESI†). They correspond to 0.1 w/v for gelatin and 1/40 v/v for Matrigel™. To ensure that cell-adhesive properties of the hydrogels were not modified by the freeze-drying process, substrates were reconstituted and NIH-3T3 cells were cultured. After 4 hours of culture, NIH-3T3 cells preserved the typical fibroblast morphology and no significant differences in cell adhesion percentages were found between the freeze-dried and the control hydrogels (Fig. S4 in the ESI†).
Microcontact printing process was then carried out on freeze-dried hydrogels (Fig. 2). PDMS stamps featuring lines of several width and pitch combinations, from 2 μm to 20 μm, were employed to pattern streptavidin, laminin, and fibronectin proteins (Fig. S2 in the ESI†). No chemical modification of the hydrogel surface was performed; the proteins were immobilized by physical adsorption. The success of the printing procedure (uniformity, reproducibility and stability) was checked by fluorescence microscopy before reconstitution and 24 hours after the addition of 10 mM PBS buffer (Fig. 2). Dehydration is accompanied by an increase in stiffness of hydrogel-like materials such as collagen fibrils, which ensures the successful transfer of the pattern.28,29
It was observed that the procedure did not alter the pattern dimensions when the hydrogels were reconstituted. Protein patterning was observed on the surface and showed limited diffusion within a region ∼8 μm thick into the bulk of the hydrogels (Fig. S3 in the ESI†). Once the printing was accomplished, fibronectin lines of 20 μm in width were printed onto freeze-dried Matrigel™ and gelatin hydrogels. Such size was selected in accordance to the results found in literature demonstrating successful cell alignment.5,30,31 Thereafter, NIH-3T3 cells were seeded at a cell density of 104 cells per cm2 under serum starvation conditions to highlight the effects of the micropattern. Unlike most of the works within this field, we have not passivated the non-patterned regions of the micropatterned surfaces, so cells were able to adhere freely to all over the sample surface in a way that mimics better the in vivo situation. After 4 hours of culture, both gelatin and Matrigel™ hydrogels showed significant alignment of NIH-3T3 cells on the patterned regions with respect to non-patterned substrates, used as controls (Fig. 3 and S5 in the ESI†). In particular, on patterned gelatin more than 60% of cells showed actin fibers and nuclei aligned with the pattern direction within an angle of 30°, while on patterned Matrigel™ the percentage increased up to 70% (Fig. 3d and S5d in the ESI†). Moreover, cells were elongated because of the pattern effects (Fig. 3e).
Once pattern effects on cell guidance were demonstrated on NIH-3T3 cells, the applicability of the technique was further extended to cardiac differentiation studies. For this purpose, disaggregated human embryonic stem cells (hESC) were seeded on fibronectin patterned hydrogels and non-patterned hydrogels, and their differentiation toward cardiac lineage cells was monitored for 30 days. During this differentiation assay, hESC cultured on micropatterned hydrogels displayed cell alignment at early culture stages (24 h) (Fig. 3e), expressed cardiac markers (GATA4 and alpha sarcomeric actin (ASA))(Fig. 3f), and beating foci (Video 1 in the ESI†) appeared at earlier time points than non-patterned substrates (day 18 versus day 30) (Fig. 3f and g). Through these results we demonstrate the beneficial effects of the anisotropy introduced on the substrate by the micropattern on the cardiac differentiation process.
One of the major advantages of human embryonic stem cells (hESCs) arises in their dual ability to self-renew and differentiate into all the cell types of the body. While differentiation of hESCs into cardiomyocytes has been well reported the process remains still inefficient.32–35 So far intense research has been documented in the use of specific media culture combinations33,34 or the use of specific cardiac-related genes35 promoting cardiac differentiation. Hydrogels have been previously reported as potential candidates for hESCs differentiation towards cardiomyocytes due to their tunable composition and physical properties.36 In that regard, we believe that the development of hydrogel-based strategies favoring cardiac differentiation will represent a major finding in the field of pluripotent stem cells differentiation and regenerative medicine. Because of its versatility, compatibility with standard cell culture methodology and minor modification of the hydrogel surface, we believe our patterning strategy to be advantageous for such applications.
Footnote |
† Electronic supplementary information (ESI) available: Experimental details, Fig. S1–S4 and Video 1. See DOI: 10.1039/c4ra03374d |
This journal is © The Royal Society of Chemistry 2014 |