Protein patterning on hydrogels by direct microcontact printing: application to cardiac differentiation

Abstract

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.

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Hydrogels are networked materials with high water content, which allows easy diffusion of soluble factors and oxygen. Due to their biocompatibility and swelling properties, hydrogels are widely used for mammalian cell cultures. Because of their mechanical and chemical properties, hydrogels mimic extracellular matrix architecture and have become trendy materials to resemble soft tissues.^1–3 Protein patterning of biocompatible surfaces, like hydrogels, plays an important role mimicking chemical heterogeneity of native tissues where cells collectively arrange in complex functional structures.^4–7 In particular, cardiac microenvironment has been replicated through fibronectin patterns to obtain more realistic cardiac cell cultures.^8–11

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 strategies^15–17 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.

Fig. 1 Schematics of hydrogel micropatterning process. (a) After the gelation process, the hydrogels were freeze-dried (b) to enable the application of the microcontact printing technique (c). Then, the hydrogel were reconstituted (d) and cells were cultured.

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

Fig. 2 Fluorescence microscopy images of hydrogels patterned with streptavidin Texas Red® conjugate. 5 μm wide lines printed on freeze-dried Matrigel (a) before and (b) after reconstitution. (Scale bar: 30 μm.) 20 μm wide lines printed on freeze-dried gelatin (c) before and (d) after reconstitution. (Scale bar: 60 μm.) (e) Fluorescence intensity profile corresponding to dash line of (d), showing the uniformity of the printing.

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).

Fig. 3 Cell alignment on micropatterned gelatin and application in cardiac differentiation. Fluorescence microscopy pictures of NIH-3T3 mouse embryonic fibroblasts cultured on (a) non-patterned and (c) patterned gelatin (fibronectin lines 20 μm in width) hydrogels after 4 hours of culture. Cell nuclei are stained in blue, actin cytoskeleton in green and fibronectin in red. Scale bar: 100 μm. Normalized histograms (bin = 10°) depicting the distribution of the angles between cell cytoskeleton fibers (in green) and cell nuclei (in blue) with the pattern direction on (b) non-patterned (d) and patterned gelatin hydrogels. Human embryonic stem cells (hESCs) cultured on micropatterned gelatin with cardiac differentiation medium. (e) Elongated hESC cells following the pattern direction after 24 hours. Scale bar: 125 μm. (f) Beating colony from hESC on patterned gelatin at day 30, expressing GATA4 (stained in blue) and ASA (stained in red) cardiac markers. Scale bar: 200 μm. (g) Plot of the number of beating foci as a function of the culture time on patterned (red) and non-patterned (black) gelatin substrates (mean ± SEM).

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 combinations^33,34 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.

Conclusions

In conclusion, we have developed a simple, straightforward procedure to create protein micropatterns applicable to hydrogels. This procedure employs the robust, well-proven microcontact printing technique with PDMS stamps and requires minor pre-processing of the hydrogels by freeze-drying. Matrigel™ and gelatin hydrogels have been successfully micropatterned using this methodology. We found that NIH-3T3 cells adhere well on patterned hydrogels, and aligned and elongated following the patterns. Moreover, in a preliminar study, we obtained hESC-derived cardiac lineage cells at earlier time points on patterned hydrogels than on non-patterned hydrogels. The described extended microcontact printing technique will be useful in cell culture platforms such as stem cells, where the use Matrigel™ or gelatin hydrogels is required.

Acknowledgements

Authors acknowledge the financial support of CIBER-BBN (Instituto de Salud Carlos III) with assistance from the European Regional Development Fund, the Commission for the Universities and Research of the Generalitat de Catalunya (2009 SGR 505) and the “Fundación Botín” (Santander, Spain). This work was also financially supported by the CARDIO-STEM Project (PLE2009-0147), funded by the MICINN under the National Program for the internationalization of R&D, TERCEL-ISCIII-MINECO, Cardiocel and Fundación Cellex.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, Fig. S1–S4 and Video 1. See DOI: 10.1039/c4ra03374d

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