Hydrogel arrays formed via differential wettability patterning enable combinatorial screening of stem cell behavior

Abstract

Here, we have developed a novel method for forming hydrogel arrays using surfaces patterned with differential wettability. Our method for benchtop array formation is suitable for enhanced-throughput, combinatorial screening of biochemical and biophysical cues from chemically defined cell culture substrates. We demonstrated the ability to generate these arrays without the need for liquid handling systems and screened the combinatorial effects of substrate stiffness and immobilized cell adhesion peptide concentration on human mesenchymal stem cell (hMSC) behavior during short-term 2-dimensional cell culture. Regardless of substrate stiffness, hMSC initial cell attachment, spreading, and proliferation were linearly correlated with immobilized CRGDS peptide concentration. Increasing substrate stiffness also resulted in increased hMSC initial cell attachment, spreading, and proliferation; however, examination of the combinatorial effects of CRGDS peptide concentration and substrate stiffness revealed potential interplay between these distinct substrate signals. Maximal hMSC proliferation seen on substrates with either high stiffness or high CRGDS peptide concentration suggests that some baseline level of cytoskeletal tension was required for hMSC proliferation on hydrogel substrates and that multiple substrate signals could be engineered to work in synergy to promote mechanosensing and regulate cell behavior.

Statement of significance: Our novel array formation method using surfaces patterned with differential wettability offers the advantages of benchtop array formation for 2-dimensional cell cultures and enhanced-throughput screening without the need for liquid handling systems. Hydrogel arrays formed via our method are suitable for screening the influence of chemical (e.g. cell adhesive ligands) and physical (stiffness, size, shape, and thickness) substrate properties on stem cell behavior. The arrays are also fully compatible with commercially available micro-array add-on systems, which allows for simultaneous control of the insoluble and soluble cell culture environment. This study used hydrogel arrays to demonstrate that synergy between cell adhesion and mechanosensing can be used to regulate hMSC behavior.

Keywords: Cell adhesion; Human mesenchymal stem cell; Mechanosensing; Poly(ethylene glycol); RGD peptide; Thiol-ene.

Figure 1

Hydrogel array formation procedure and outputs. a–b) Hydrogels arrays were formed on gold-coated glass slides patterned with SAMs with differential wettability. c–e) Hydrogel precursor solutions deposited onto the hydrophilic SAM regions of the patterned slide were crosslinked via UV-initiated photopolymerization to form hydrogel spots. F) The resulting array is composed of hydrogel spots immobilized on a glass slide. g–h) The array formation procedure allows for the formation of hydrogel spots of various size, shape, and height.

Figure 2

Characterization of surface roughness of hydrogels formed using the array formation procedure. (a) Representative AFM height image of hydrogel (shown: 20 wt.%, 45% crosslinking) in liquid to determine the RMS roughness. (b) The calculated RMS roughness values (6.6–15.1 nm) for hydrogels with the lowest and highest stiffness values formed using this array formation procedure. Dotted line denotes minimum height used in previous studies investigating the influence of nanotopography of compliant surface on hMSC behavior. Sample size: n = 3 (b).

Figure 3

Characterization of the chemical and mechanical properties of hydrogel spots in the array. a) Schematic representation of array with hydrogel spots formed using thiolene chemistry. Array with hydrogel spots containing varying concentrations of immobilized CRGDS peptide (b, c) and encapsulated microspheres (b, d). Hydrogel spots with stiffness varied by changing concentration of PEG-NB or crosslinker density in the hydrogel precursor solution (e). Sample size: n = 4 (c–d) and n = 3 (e). Statistical significance was determined by two-factor ANOVA followed by Tukey HSD test, whereby * denotes statistical significant with p<0.05 and “NS” denotes no statistical significance.

Figure 4

Characterization of hydrogel physical properties and network structure. a) Hydrogel network mesh size predicted using calculations based on Flory-Rehner theory. b) Mass equilibrium swelling ratio of bulk hydrogels formed with varying PEG-NB (wt. %) and crosslinker density. Sample size: n = 3 (a–b). Statistical significance was determined by two-factor ANOVA followed by Tukey HSD test, whereby * denotes statistical significant with p<0.05 and “NS” denotes no statistical significance.

Figure 5

Demonstration of hMSC encapsulation in hydrogels formed using differential wettability patterning. All hydrogels presented CRGDS and were crosslinked with a MMP-degradable peptide. ad) Viability and density of hMSCs encapsulated in hydrogel spots presenting 2 mM CRGDS and formed using precursor solutions with varying concentrations of hMSC. a) Maximum intensity projection created by stacking images of the hydrogel acquired at 3 different focal planes in the hydrogel. c) Correlation of encapsulated hMSC density with hMSC concentration in precursor solution. e) Viability of ~500,000 hMSCs encapsulated in hydrogels spots containing varying concentrations of immobilized CRGDS peptide. Sample size: n ≥ 5 (b, d, e), n=3 (c). Statistical significance was determined by ANOVA followed by Tukey-Kramer test, whereby * denotes p<0.05 and “NS” denotes no statistical significance.

Figure 6

Effects of hydrogel spot stiffness and immobilized CRGDS concentration on hMSC behavior. a) hMSC culture on hydrogel spots presenting 4 mM CRGDS over the course of 8 days. hMSC b) cell attachment one day after cell seeding, c) cell spreading four days after cell seeding, and d) cell proliferation (indicated by normalized cell number from day 4 compared to day 1, C₄/C₁ > 1) after four days of culture Sample size: n = 4 (b–d).

Figure 7

Effects of hydrogel spot stiffness and immobilized CRGDS concentration on hMSC cytoskeletal structure. a) Focal adhesion (vinculin, green), stress fiber (F-actin, red), and nuclear (DAPI, blue) stain of hMSCs after 8 days of culture on hydrogel spots with varying stiffness and presenting 4 mM CRGDS. b) Correlation of hMSC focal adhesion density with immobilized CRGDS concentration (calculated by averaging all hydrogel spots with the same CRGDS concentration, regardless of stiffness). c) Average hMSC focal adhesion length on hydrogels of different stiffness values (calculated by averaging all hydrogel spots with the same stiffness values, regardless of CRGDS concentration). Sample size: n ≥ 25 (b–c). Asterisks denote statistical significance as determined by single factor ANOVA followed by Tukey-Kramer test, whereby ** p<0.01.

Figure 8

Effects of immobilized peptide identity on hMSC behavior. a) hMSC attachment on hydrogels presenting linear CRGDS or cyclic (RGDfC) after 3 days of culture. b) hMSC cell attachment one day after seeding and c) cell spreading after 3 days culture on hydrogel spots presenting 4 mM CRGDS or cyclic (RGDfC). Sample size: n ≥6 (b, c).

Figure 9

Demonstration of hydrogel array setup for soluble media screening. a) Hydrogel array assembly with commercially-available micro-array add-on for ability to introduce different soluble factors to b) each individual spot or c) group of spots in the array. b–c) Hydrogels stained with Trypan blue and media contains phenol red for enhanced contrast and visibility.