Controlling major cellular processes of human mesenchymal stem cells using microwell structures.

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Xun Xu, Weiwei Wang, Karl Kratz, Liang Fang, Zhengdong Li, Andreas Kurtz, Nan Ma,* and Andreas Lendlein*

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Controlling Major Cellular Processes of Human Mesenchymal Stem Cells using Microwell Structures

clinically relevant scale without major loss of potency. An increasing number of preclinical and clinical evidences have demonstrated their established safety record and therapeutic efficacy.[1–3] MSCs can be found in almost all postnatal organs and tissues. Among these, adipose tissue is a particularly practical cell source as it is abundant and readily accessible with minimal risk to the patient.[4–6] MSCs contribute to tissue regeneration and maintenance by orchestrating a variety of spatially and temporally controlled functions. For example, MSCs can migrate through the physical barriers and home to the injury site.[7] Here, MSCs can remodel the extracellular matrix (ECM) and facilitate tissue formation by modulating the immune response and stimulating endogenous stem/progenitor cells.[3,8] Ideally, the MSCs themselves should be able to proliferate to a meaningful amount and be directed by intrinsic signals towards desired differentiation pathways at the site of injury. Because of these important functions, MSC-based therapy has been intensively explored. However, their efficacy is often hampered by dysregulated migration, insufficient expansion rate, differentiation into undesired lineages, and malignant transformation.[9–13] These limitations remain major obstacles for fully realizing the endogenous regeneration capacity of MSCs.

Directing stem cells towards a desired location and function by utilizing the structural cues of biomaterials is a promising approach for inducing effective tissue regeneration. Here, the cellular response of human adipose-derived mesenchymal stem cells (hADSCs) to structural signals from microstructured substrates comprising arrays of square-shaped or round-shaped microwells is explored as a transitional model between 2D and 3D systems. Microwells with a side length/diameter of 50 µm show advantages over 10 µm and 25 µm microwells for accommodating hADSCs within single microwells rather than in the inter-microwell area. The cell morphologies are threedimensionally modulated by the microwell structure due to differences in focal adhesion and consequent alterations of the cytoskeleton. In contrast to the substrate with 50 µm round-shaped microwells, the substrate with 50 µm square-shaped microwells promotes the proliferation and osteogenic differentiation potential of hADSCs but reduces the cell migration velocity and distance. Such microwell shape-dependent modulatory effects are highly associated with Rho/ROCK signaling. Following ROCK inhibition, the differences in migration, proliferation, and osteogenesis between cells on different substrates are diminished. These results highlight the possibility to control stem cell functions through the use of structured microwells combined with the manipulation of Rho/ROCK signaling.

1. Introduction Mesenchymal stem cells (MSCs) have emerged as an attractive cell source in regenerative medicine. They are multipotent stem cells that can be expanded in vitro or ex vivo to a X. Xu,[+] Dr. W. Wang,[+] Dr. K. Kratz, Dr. L. Fang, Z. Li, Prof. N. Ma, Prof. A. Lendlein Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies Helmholtz-Zentrum Geesthacht Kantstraße 55 14513, Teltow, Germany E-mail: [email protected]; [email protected] X. Xu, Z. Li, Prof. N. Ma, Prof. A. Lendlein Institute of Chemistry and Biochemistry Freie Universität Berlin Takustraße 3 14195, Berlin, Germany Dr. K. Kratz, Prof. N. Ma, Prof. A. Lendlein Helmholtz Virtual Institute −Multifunctional Materials in Medicine Berlin and Teltow Kantstraße 55 14513, Teltow, Germany

Prof. A. Kurtz Berlin-Brandenburg Center for Regenerative Therapies Charité – University Medicine Berlin Augustenburger Platz 1 13353, Berlin, Germany Prof. A. Kurtz College of Veterinary Medicine and Research Institute for Veterinary Science Seoul National University Gwangk-ro 1, Gwanak-gu, Seoul 151–747, Republic of Korea [+]X.X.

and W.W. contributed equally to this work.

DOI: 10.1002/adhm.201400415

Adv. Healthcare Mater. 2014, 3, 1991–2003

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MSC behavior is tightly regulated by both intrinsic programs and extrinsic factors, including physical, chemical, and biochemical cues provided by their surrounding environment.[14,15] These multiple cues are integrated to exert a combinatorial influence on the fate and function of MSCs. Among these signals, structural cues presented by biomaterials have shown potential to direct MSC behavior as a complement to traditionally applied biochemical methods. Numerous approaches and models were developed to examine the influence of structural cues on stem cell functions. In this context, the most widely used model is the “cell-adhesive micropatterns” system, where micropatterns with specific size, shape, and distribution were created by immobilizing cell-adhesive molecules on smooth substrates, which are nonadhesive for cells.[16,17] Anchoragedependent cells were physically constrained to specific sizes and shapes on the microscale within the micropatterns so as to analyze their subsequent response to the geometric cues of the micropatterns. At the single cell level, the lineage commitment of MSCs can be modulated by the shape and size of the celladhesive micropatterns independent of biochemical cues such as soluble factors. It was found that larger-sized micropatterns created using entities with cell-adhesive properties like peptides (e.g., arginine-glycine-aspartic acid (RGD)) or proteins (e.g., fibronectin) facilitated osteogenic differentiation but inhibited adipogenic differentiation of MSCs.[18,19] In addition, individual MSCs confined to the micropatterns preferentially underwent adipogenic differentiation on flower-shaped but osteogenic differentiation on star-shaped micropatterns.[20] Although this “cell-adhesive micropatterns” system is simple to apply and favors cell attachment following predefined micropatterns, it still has several drawbacks. First, it might restrict the overall cell movement and limits the cell–cell adhesion and communication especially for single cell micropatterns. In addition, such 2D systems could not replicate the 3D structural aspects of intact tissues.[21,22] Cells growing on 2D surface are forced to adopt unnatural characteristics such as an aberrant flattened morphology, whereas 3D culture may enhance the proliferation and differentiation of stem cells.[23,24] There is a need to reduce the gap between flat-surface cell culture systems and culture conditions resembling a tissue environment. To this end, we created polystyrene (PS)- based microstructured substrates as a transitional model between 2D and 3D systems. Given the sensitivity of MSCs to structural cues, we hypothesized that MSCs may be able to respond to the structural aspects offered by our microstructured substrates. These microstructured substrates comprising arrays of microwells in either square or round shape were used to probe the cellular response of MSCs to structural signals. The microstructured substrates were designed to allow MSCs to freely migrate on the substrates without confinement by the surface structures (microwells or inter-microwell area) and distribution of cell-adhesive molecules, and the microwells were sized to accommodate at least one individual cell. It was shown that the diameters of suspended and adherent hADSCs are about 15–45 µm and 20–80 µm respectively,[25] and the thickness of the fully spreading hADSCs is generally below 5 µm. Therefore, to accommodate the resulting spatial requirement, the microwells were designed with a side length/diameter of 10, 25, or 50 µm and a uniform depth of 10 µm. The distance


between the microwells was designed as twice the side length/ diameter of the microwells, which provided support for MSC spreading and migration on the inter-microwell area. For the substrates with 50 µm microwells, the inter-microwell spacing (100 µm) could prevent individual cells from spreading on several microwells and consequently exclude the influence of multiple microwells on a single cell. Further, by injection molding, the microstructured PS substrates were embedded into PS inserts sized to fit the standard 24-well tissue culture plate (TCP). This insert design restricts cell interactions to the microstructured substrates, excluding the influence of other materials such as those utilized to support or fix the test materials. Prior to cell seeding, the microstructured substrates (both the microwells and the inter-microwell area) were coated with human vitronectin. As a glycoprotein of the ECM, vitronectin is essential for overcoming the poor cell adhesion properties of pure PS,[26] thereby facilitating cell adhesion and spreading as well as unconstrained movement on the substrates. After cell seeding, the following sequential events in cell behavior were explored: i) initial interaction with microwells (t = 2 h); ii) cell distribution, morphology, gene profile, and migration (t = 2 d); iii) cell phenotype, proliferation, and differentiation (t > 2 d). The mechanism of the regulatory effects of microstructured substrates on MSCs associated with ROCK signaling was investigated.

2. Results 2.1. Design, Creation, and Characterization of Microstructured Substrates For studying the influence of the microwell structures on the behavior of hADSCs, a series of inserts with microstructured substrates comprising arrays of uniform square-shaped or round-shaped microwells was prepared. In order to obtain microwells with high precision, the soft-lithography technique was applied to achieve microstructures from PS. The injection molding was utilized for embedding such substrates into inserts fabricated from the identical polymer to ensure that the hADSCs are only in contact with one material during the experiments (Figure 1A,B, Figure S1, Supporting Information). As reference materials, PS inserts with a smooth culture surface were prepared using the identical processing techniques. Digital microscopy and optical profilometry analysis of the micron-scaled arrays revealed the successful replication of the side length/diameter (Table S1, Supporting Information) and depth (10.0 ± 0.5 µm) of the templates. X-ray photoelectron spectroscopy (XPS) analysis confirmed negligible contamination due to elements other than carbon and hydrogen (for details see Method S4, Supporting Information). The prepared PS substrates exhibited similar wettability with water represented by advancing contact angles (θadv) in the range of 103° to 115° (Table S1, Supporting Information). After coating the culture surfaces with vitronectin, there was no remarkable change in θadv but a slight increase in the hysteresis, which could be attributed to the higher chemical inhomogeneity of the coated surface in comparison with the original PS substrates.




FULL PAPER Figure 1. Creation and characterization of microstructured substrates. A) Microstructured PS substrates comprising square-shaped (Square-50) or round-shaped (Round-50) microwells with a side length/diameter of 50 µm and a depth of 10 µm created via soft-lithography (bar = 100 µm). B) PS insert prepared via injection molding. The microstructured PS substrates are integrated as cell culture surfaces at the bottom. C) AFM images representing the roughness of the vitronectin-coated microstructures, which were taken inside the microwells and in the inter-microwell area. D) Quantification of human vitronectin adsorbed onto the bottom of the PS inserts (n = 4). E) Surface density of vitronectin inside the microwells examined by analyzing the fluorescent intensity in different areas of the microwell bottom (illustrated as gray) (n = 4). F) Representative laser scanning microscopic images of the microwells coated with human vitronectin. Vitronectin was stained with protein labeling dye (green) (bar = 10 µm).

A more detailed surface analysis including micromechanical properties, nanoscale surface roughness and surface density of vitronectin was performed for the substrates with 50 µm square-shaped (Square-50) and round-shaped (Round50) microwells. Atomic force microscopy (AFM) based indentation revealed similar micromechanical properties (reduced Young’s modulus (Er) of 7–12 GPa) inside the microwells, on the inter-microwell area as well as on the smooth PS substrate (Table S2, Supporting Information). The nanoscale surface roughness was investigated by scanning electron microscopy (SEM) and AFM on different parts of the substrates. The rootmean-square roughness (Rrms) varied from 3 to 4 nm in the inter-microwell area, and from 23 to 44 nm inside the microwells (Table S2, Supporting Information). SEM and AFM images (Figure S2,S3, Supporting Information) demonstrated the higher surface roughness in the microwells, which may be related to the differences in the microstructured Si-wafer templates. After coating the substrates with human vitronectin, the difference in nanoscale roughness between the microwells and the inter-microwell area was maintained, whereby a slight increase in Rrms values of the inter-microwell area and a minor decrease in the nanoscale roughness inside the microwells were observed. The Rrms varied from 4 to 8 nm in the intermicrowell area, and from 17 to 22 nm inside the microwells for the coated microstructures (Table S2, Supporting Information). The representative AFM images of the vitronectincoated microstructures are shown in Figure 1C. The adsorption and density of human vitronectin were quantified via laser


scanning microscopy and enzyme-linked immunosorbent assay (ELISA). Similar vitronectin adsorption was observed on coated Square-50 and Round-50 substrates, and the vitronectin surface density was at the same level in the different areas in the microwells (Figure 1D–F).

2.2. Influences of Vitronectin Coating on Cell Adhesion and Microwell Size on Cell–Microwell Interaction The influence of human vitronectin on hADSC adhesion was studied by comparing the cell attachment on vitronectin coated and uncoated substrates at 24 h after cell seeding. hADSCs attached and spread efficiently on vitronectin-coated substrates with typical spindle-shaped MSC morphology. However, highly limited cell adherence with incomplete spreading was observed on the uncoated substrates (Figure S4, Supporting Information). To further examine the influence of microwell size on cell attachment, hADSCs cultured on different microstructured substrates were stained for F-actin, vinculin, and nuclei at 2 d after cell seeding. The cells on microstructured substrates with 10 µm square-shaped (Square-10) or round-shaped (Round-10) microwells exhibited spreading over several microwells. The cell bodies were only partially accommodated in the 25 µm square-shaped (Square-25) and round-shaped (Round25) microwells, whereas they were completely accommodated in the 50 µm square-shaped (Square-50) and round-shaped (Round-50) microwells (Figure S5, Supporting Information).



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higher than that outside the microwells (Figure 2B; Square-50: (7.9 ± 2.1) × 104 vs (0.4 ± 0.2) × 104 cells cm−2; Round-50: (7.8 ± 1.0) × 104 vs (1.1 ± 0.2) × 104 cells cm−2).

2.4. Cell Morphology, Focal Adhesion, and Cytoskeletal Structure To evaluate the influence of microwells on cell morphology, focal adhesion, and cytoskeletal structure, the cells were stained for F-actin, vinculin, and nuclei 2 d after cell seeding. 3D images (Figure 3) were reconstructed from multilayer images acquired by confocal laser scanning microscopy (Figure S6, Supporting Information). The cells on the smooth surface (vitronectin-coated PS) displayed a flat and fibroblast-like morphology. Their focal adhesions were mostly located at the outer edge of the cells and were connected by well-oriented strong stress fibers. However, the shape of the cells in the microwells was highly modulated by the structures of the microwells. The cells in the microwells displayed a more three-dimensional shape and cytoskeletal orientation, which were different from that of cells on the smooth surface. Interestingly, cells in the Square-50 and Round-50 microwells showed very different distributions of focal adhesion and cytoskeletal stress fibers. The focal adhesions of the cells in the Square-50 microwells were formed mainly at the corners and were connected by the stronger F-actin stress fibers. In contrast, the cells in the Round-50 microwells exhibited more homogeneously distributed focal adhesions, which were dispersed throughout the cells as well as the weaker stress fibers. Following ROCK inhibition (Y-27632 treatment), the formation of stress fibers and focal adhesion was found to be remarkably inhibited on both of the microstructured substrates (Figure S7, Supporting Information). Figure 2. Initial hADSC interaction with microwells and cell distribution on microstructured substrates. A) Representative cell images at t = 2 h after seeding on microstructured substrates. The cell nucleus was stained with Hoechst 33342 (bar = 50 µm). B) Density of cells distributed inside/ outside of the microwells on microstructured substrates with 50 µm square-shaped (Square-50) or round-shaped (Round-50) microwells at t = 2 d (n = 4; *Sig < 0.05).

To focus on the cellular characteristics of hADSCs within the individual microwells, we selected Square-50 and Round-50 substrates for further evaluation.

2.3. Cells Distribution on Microstructured Substrates Two hours after cell seeding, the migration of non-attached hADSCs on microstructured substrates was recorded with timelapse microscopy. The rolling of cells into the 50 µm microwells occurred before they formed firm adhesions, as shown in the example of a square-shaped microwell (Figure 2A). Two days after cell seeding, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and the number of cells inside and outside the microwells was manually counted to examine the cell distribution on the microstructured substrates. The results showed that the cells were more likely to migrate into the 50 µm microwells instead of adopting a random distribution. Consequently, the density of the cells inside the microwells was

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2.5. Cell Motility In order to examine the motility of cells on microstructured substrates, random cell movement was recorded by time-lapse microscopy 2 d after cell seeding. It was observed that hADSCs moved freely on the microstructured substrates without physical constraints (Figure 4 and Movie S1, Supporting Information). For example, the cells initially located inside the microwells moved out and some of them re-entered the microwells and vice versa. In spite of the free cell migration, the cells showed different migration velocities and distances on different microstructured substrates (Figure 5A). We observed that compared to cells on the Square-50 substrate, cells on the Round-50 substrate showed remarkably higher total and straight migration distances (1154.59 ± 44.43 vs 724.23 ± 25.23 µm and 295.89 ± 28.56 vs 197.50 ± 19.27 µm). The velocity (total distance/migration time) of the migrating cells was dependent on the microwell structure with higher values observed on the Round-50 substrate than on the Square-50 substrate (0.82 ± 0.03 vs 0.51 ± 0.02 µm min−1). There was no significant influence of microwell structures on the migration tortuosity (total distance/straight distance). Following ROCK inhibition, the total distance and velocity of cell migration were significantly reduced on the Round-50 substrate but not on the Square-50




FULL PAPER Figure 3. Morphology, focal adhesion, and cytoskeletal structure of hADSCs in 50 µm square-shaped (Square-50) and round-shaped (Round-50) microwells and on a smooth surface. Results were presented as 3D reconstruction images (green: vinculin; red: F-actin; blue: nuclei). White dash lines indicate the outlines of the microwells and the interface of the smooth surface (bar = 50 µm).

substrate. Compared to untreated cells, the treated cells showed a reduction in velocity of approximately 32% and 4% in the Round-50 and Square-50 groups, respectively. The differences in migration parameters such as total distance, straight distance, and velocity between the Square-50 and Round-50 groups were clearly diminished after ROCK inhibition. The migration tortuosity was not affected by ROCK inhibition (Figure 5B). These data indicated a strong involvement of ROCK in cellular motility in response to structural features.

2.6. Gene Profile and ROCK Expression To identify differentially expressed genes regulating focal adhesion and migration, we performed real-time PCR array analysis. The heat map demonstrated the changes of the expression levels of the selected genes (Figure 6A). Notably, cell migrationpromoting genes (Cdc42, ITGB1, ILK, and PAK1) were downregulated whereas migration-inhibiting genes (AKT3, ZYX, and PTEN) were upregulated in cells on the Square-50 substrate compared to the Round-50 substrate. There was a strong upregulation of genes associated with the components of focal adhesion and cytoskeletal structure (PLEC, TNS1, ZYX, ACTN1, ACTN4, PARVA, and PTK2) in the Square-50 group compared to the Round-50 group. Moreover, the genes responsible for the remodeling of pre-existing actin filaments and actin polymerization (RhoA, ROCK1, and DIAPH1) were remarkably upregulated in cells on the Square-50 substrate in comparison with the Round-50 substrate. Based on this finding, we analyzed the protein level of ROCK1 via Western blot (Figure 6B,C). It was observed that hADSCs on the Round-50 substrate expressed the ROCK1 protein at a similar level to that on the smooth surface (TCP), whereas ROCK1 was upregulated in cells on the Square-50


substrate, indicating a close association of ROCK1 with the microwell-mediated cellular response.

2.7. Cell Viability, Proliferation, and Cell Cycle To examine the potential microstructure-dependent effects on cell viability, live/dead staining was performed. Eight days after cell seeding, only a few dead cells were observed on both microstructured substrates, indicating that the microstructures did not affect cell survival (data not shown). The efficient proliferation of stem cells is critical to fully realize their regeneration capacity. Here, the effect of microstructured substrates on hADSC proliferation was firstly determined by exploring the expression of the nuclear protein Ki67 (Figure 7A,B). We found that the total percentage of Ki67+ cells on the Square-50 substrate (75.6 ± 4.5%) was higher than that on the Round-50 substrate (62.3 ± 4.7%) and the smooth surface (vitronectin coated PS, 54.9 ± 6.3%). The cells inside and outside the microwells were separately counted to further examine the exact effect of the microstructures on cell proliferation. For the cells outside the microwells in both groups, there was no significant difference in the percentage of Ki67+ cells. On the Round-50 substrate, the cells inside and outside the microwells showed a similar percentage of Ki67+ cells (53.5 ± 9.2% vs 64.5 ± 7.3%). However, on the Square-50 substrate, the cells inside the microwells presented a much higher Ki67+ percentage than those outside the microwells (84.5 ± 5.7% vs 55.3 ± 10.0%). These results indicated that the square-shaped microwells might promote the hADSC proliferation potential in comparison with the round-shaped microwells and the inter-microwell area. In order to clarify whether the square-shaped microwells facilitate cell proliferation over extended periods of time,



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Figure 4. Dynamic movement of hADSCs on microstructured substrates with 50 µm square-shaped (A) or round-shaped (B) microwells. Two days after cell seeding, the cells were monitored up to 24 h. Cells moving into or out of the microwells are identified by blue and green arrows respectively; the red arrows indicate the cells dividing inside the microwells. (bar = 50 µm)

hADSCs were cultured up to 10 d and the cell number at the indicated time points was determined using the Cell Counting Kit-8. After 10 d of culture, the number of cells on the Square-50 substrate ((2.3 ± 0.1) × 105 cells/insert) was higher than that on the Round-50 substrate ((1.7 ± 0.2) × 105 cells/ insert) and the smooth surface ((1.3 ± 0.2) × 105 cells/insert). When treated with ROCK inhibitor (Y-27632), the proliferation of cells on all substrates was strongly inhibited. The proliferation advantage of the Square-50 substrate over the Round-50 substrate was eliminated by exposure to Y-27632. After 10 d of culture, the number of cells in the Square-50, Round-50, and smooth groups was (3.9 ± 1.8) × 104, (4.2 ± 0.5) × 104 and (2.0 ± 0.5) × 104 cells/insert, respectively (Figure 7C). Finally, we examined the effect of microstructured substrates on the percentage of hADSCs in different phases of the cell cycle (Figure 7D). In presynchronized cells, no obvious difference in cell cycle distribution was observed between the Square-50, Round-50, and smooth groups. After Y-27632 treatment, the percentage of cells in G2/M phase increased for all groups, indicating that ROCK inhibition could induce G2/M phase arrest. Interestingly, there was a higher percentage of cells in G2/M phase on the Square-50 substrate (7.76 ± 1.66%) as compared with the Round-50 substrate (3.48 ± 0.73%) and 1996


smooth surface (4.44 ± 0.96%), suggesting a higher sensitivity of cells on the Square-50 substrate to late cell cycle arrest/delay upon ROCK inhibition.

2.8. Phenotype and Osteogenic Differentiation To examine the effects of microwells on cell phenotype, we compared the phenotypic markers of hADSCs before and after 10 d of culture on the microstructured substrates. Flow cytometry results demonstrated that the main surface markers of MSCs (CD90, CD73 and CD105) were not influenced by the microwells (Figure S8, Supporting Information). To evaluate the influence of microstructured substrates on the osteogenic differentiation of hADSCs, the alkaline phosphatase (ALP), an osteoblast marker was stained after 14 d of culture and the cellular mineralization was examined by staining the cells with Alizarin Red S (ARS) after 21 d of culture. It was found that hADSCs cultured in normal growth medium without induction did not undergo spontaneous differentiation (data not shown). In contrast, under the inductive conditions, the cells differentiated into the osteogenic lineage, as indicated by the positive ALP and ARS staining




FULL PAPER Figure 5. Migration of hADSCs on microstructured substrates with 50 µm square-shaped (Square-50) or round-shaped (Round-50) microwells. The cell nuclei were stained with Hoechst 33342 and were tracked up to 24 h to generate the trajectories (A) and quantify the parameters (B; mean ± standard error of the mean (SEM); n = 44; *Sig < 0.05) of cell migration with and without Y-27632 treatment.

(Figure 8A,C). Semiquantitative analysis of the ALP expression (ALP positive area) and the calcium content (ARS covered area) revealed that the cells had the higher osteogenic differentiation capacity on the Square-50 substrate than on the Round-50 substrate. When treated with Y-27632, the ALP expression and the calcium deposition of the cells on the Square-50 substrate were strongly suppressed, while the osteogenic differentiation capacity of cells on the Round-50 substrate and the smooth surface (TCP) was not affected (Figure 8B,D). This result suggested that the differentiation of hADSCs towards the osteogenic lineage on the Square-50 substrate was dependent on Rho/ROCK signaling.


3. Discussion The microwell systems have demonstrated their importance in regulating the cellular behavior. They have been used to modulate the growth of pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells to generate homogeneous cell colonies with defined sizes and shapes (e.g., embryoid bodies).[27–30] However, there is limited understanding on the effect of microwell geometry on the behavior of MSCs. Regulating and controlling the major cellular behavior of MSCs using the microwells is of great clinical value as the traditional applied surface with smooth polystyrene could not



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Figure 6. PCR array and ROCK1 expression of hADSCs on microstructured substrates comprising 50 µm square-shaped (Square-50) or round-shaped (Round-50) microwells and smooth surface. A) PCR array heat map showing the fold change of differentially expressed genes involved in cell migration and focal adhesion (left: Square-50/Smooth; middle: Square-50/Smooth; right: Square-50/Round-50). B) The expression level of ROCK1 protein normalized to GAPDH in cells on microstructured substrates and smooth surface (set as 1) (n = 3; *Sig < 0.05). C) The representative image of Western blot analysis of ROCK1 and GAPDH proteins.

fully satisfy the clinical requirement of MSC expansion.[13] Hence, there is a great need to identify and develop microwell culture system to support MSCs growth and maintain their multipotent state for therapeutic applications. In this study, PS inserts with microstructured substrates containing arrays of equidistantly placed microwells were successfully prepared via soft-lithography and injection molding, as a transitional model between 2D and 3D systems in order to investigate the modulatory effects of microwells on hADSCs. Previous studies using 2D micropatterns revealed that the cellular behavior could be strongly regulated by multiple geometric factors including the size, aspect ratio, corners sharpness, local edge curvature, and anisotropic features.[16,20,31–34] The cells are very sensitive to subtle geometric cues, particularly the corners and curvature, preferentially forming their cytoskeletal structures at the corners or convex curves. For example, the cells growing on the 2D square micropatterns extended the lamellipodia, filopodia, and fascin microspikes from the corners, but on the round micropatterns, these cell processes occurred at random points along the circumference.[31] Therefore, the microwells with square or round shape were fabricated on the substrates. The surface characteristics of the PS substrates such as wettability, local mechanics, nano topography, and the depth of the microwells were kept constant, while the side length/diameter and the structure of the microwells were systematically varied. Coating the substrates with human vitronectin remarkably facilitated cell adhesion and spreading. This model could fulfill the following requirements: i) hADSCs interacted with homogeneous substrates without unwanted contact with other

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materials; ii) hADSCs could efficiently attach and freely migrate on the substrates. However, the size requirement, whereby at least one cell could be entirely accommodated in a single microwell, could only be fulfilled by the substrates comprising 50 µm microwells. Therefore, further studies on the cellular effects of hADSCs within single microwells were focused on substrates with 50 µm microwells. Based on previous studies of hADSC adhesion and doubling time,[35] we examined the early cell attachment to the microwells before spreading at 2 h after cell seeding, complete spreading and migration at 2 d and later cell replication and differentiation after 2 d. We observed that hADSCs were preferentially distributed inside the microwells rather than in the inter-microwell area, especially at the early culture stage (t = 2 d) when the cell confluence was at a low level. The mechanism for this difference in cell distribution is not clear, but may be attributed to the different spatial presentation of biochemical and physical signals inside and outside the microwells. Previous studies using 2D models demonstrated that the cell shape could strongly influence the cell adhesion plaques and cytoskeletal stress fibers with regard to their distribution and intensity.[20,31,36] In consistence with these findings, our result showed that the cell shape, focal adhesion distribution, and cytoskeletal organization were strongly affected by the structure of the microwells. Importantly, the cells in the microwells presented a more 3D morphology than those growing on the smooth surface, indicating the potential of these microwells to affect intracellular spatial arrangements. After attachment, the cells exhibited unconstrained and dynamic migration on both microstructured substrates




FULL PAPER Figure 7. Proliferation and cell cycle analysis of hADSCs on microstructured substrates comprising 50 µm square-shaped (Square-50) or round-shaped (Round-50) microwells and on substrates with smooth surface. A) Percentage of Ki67+ cells on the whole substrates, inside and outside the microwells after 2 d of culture (n = 4; *Sig < 0.05). B) Representative images of stained hADSCs on microstructured substrates (bar = 50 µm). C) The proliferation rate of hADSCs on microstructured substrates and smooth surface (n = 4; *Sig < 0.05 for Square-50, Y-27632 (−) vs Square-50, Y-27632 (+); #Sig < 0.05 for Round-50, Y-27632 (−) vs Round-50, Y-27632 (+); ψSig < 0.05 for Smooth, Y-27632 (−) vs Smooth, Y-27632 (+); &Sig < 0.05 for Square-50, Y-27632 (−) vs Round-50, Y-27632 (−); δSig < 0.05 for Square-50, Y-27632 (−) vs Smooth, Y-27632 (−); ζSig < 0.05 for Round-50, Y-27632 (−) vs Smooth, Y-27632 (−)).D) Percentage of cells in different phases of the cell cycle after 2 d of culture (n = 3; *Sig < 0.05) with and without Y-27632 treatment.

with different velocity and distance. Rho family of GTPases including Cdc42, Rac, and Rho plays a key role in coordinating the cellular responses relevant to cell migration. The formation of filopodia and lamellipodia is regulated by Cdc42 and Rac, respectively, while Rho regulates the formation of focal adhesion and the assembly of stress fibers, and is therefore responsible for cell contractility, cell body contraction, and retraction of the rear end.[37–39] Our gene array analysis indicated that the expression level of Cdc42 was upregulated, and the expression levels of RhoA and Rac2 were downregulated in cells on the substrate with round-shaped microwells when compared to cells on the substrate with square-shaped microwells. This result is in agreement with previous findings that the cell migration could be regulated positively by Cdc42 and negatively by Rho.[40,41] In addition, the positive regulators for cell migration such as PAK1,[42] ITGB1[43] and ILK[44] were found to be downregulated in cells on the substrate with square-shaped microwells, whereas the inhibitors such as AKT3,[45] ZYX,[46] and PTEN[47] were upregulated, suggesting that the structure of the microwells could regulate the gene expression and consequently influence the cell migration. ROCK, the downstream effector of Rho, contributes to physiological processes by controlling the actin-cytoskeleton assembly and cell contractility, and regulates several cellular functions such as cell migration, apoptosis, mitosis,


cytokinesis, and cell cycle progress.[48,49] Based on our initial findings, we speculated that the Rho/ROCK pathway might be critical for the response of hADSCs to the structural cues. To verify our hypothesis, we analyzed the protein level of ROCK1, and we found that the cells on the substrate with square-shaped microwells expressed more ROCK1 than those on the substrate comprising round-shaped microwells. Chen’s group has suggested the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling. The activation of ROCK by GTP-Rho was improved by the cell adhesion and the associated changes in cell shape.[50] In agreement with their findings, our results demonstrated that the square-shaped microwells, which allowed the formation of stronger stress fibers and denser focal adhesions, led to the upregulation of ROCK1 protein levels. As a central player, ROCK may participate in cell migration through the regulation of cellular contraction, stress fiber assembly, and thus cell-substratum contact.[51,52] However, the precise function of ROCK in regulating cell migration is still intriguing. On one hand, Rho/ROCK signaling mediates retraction of the trailing edge of cells and is implicated in adhesion disassembly during cell detachment.[53] The contractile nature of stress fibers could provide the driving force for cell body contraction and promote cell migration. Inhibition of RhoA induced an elongated cell morphology with impaired rear-end



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Figure 8. Osteogenic differentiation of hADSCs on two types of microstructured substrates comprising either 50 µm square-shaped (Square-50) or round-shaped (Round-50) microwells and on substrate with smooth surface. The hADSCs in osteogenic induction medium with and without Y-27632 treatment were stained by ALP staining kit after 14 d of culture (A; bar = 100 µm) and by ARS after 21 d of culture (C; bar = 2 mm). The osteogenic differentiation levels of hADSCs were expressed as the percentage of the ALP-positive area per unit total area (B; mean ± SEM; n = 6; *Sig < 0.05) and the percentage of the ARS covered area per unit total area (D; mean ± SEM; n ≥ 6 for microstructured substrates and n ≥ 3 for smooth surface; *Sig < 0.05).

detachment and migration.[54] On the other hand, Rho/ROCK signaling might inhibit cell migration since the reorganization of strong actin bundles and focal adhesions was a relatively slow process under many conditions.[51,52] As a result, depending on the cell type and environments, the bidirectional regulatory influence of ROCK on cell migration has been reported, either as a promoter[55] or as an inhibitor.[40,56] Here, the slower migration of hADSCs on the substrate with square-shaped microwells might be due to the inhibiting effect of the stronger stress fibers on cell motility. Following Y-27632 treatment, the stress fibers and focal adhesion were remarkably inhibited on both microstructured substrates. The decrease of cell motility on the substrate with round-shaped microwells could be attributed to the inhibited actin-cytoskeleton assembly and cell contractility by ROCK blockage. Interestingly, the ROCK blockage did not noticeably affect the migration activity of the cells in the “square” group, which might be explained by the bidirectional regulatory influence of ROCK on cell migration: the movement of the untreated cells was limited by the strong stress fiber, whereas the contractility for cell migration decreased in the treated cells. This bidirectional effect of ROCK is consistent with a previous study in which the human bronchial epithelial (16HBE) cells treated with high concentration (10 and 25 μ M) of Y-27632 displayed a similar motility as the untreated cells. The highest motility was achieved at 0.5 µ M Y-27632.[57] In

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addition, different subtypes of actin stress fibers play distinct roles in cell migration. For instance, the dorsal stress fibers promote cell migration and the ventral stress fibers maintain stable adhesions at the trailing edge.[58] And these subtypes might have different responses to ROCK blockage. Notably, we found a higher percentage of Ki67+ cells inside square-shaped microwells when compared to round-shaped microwells and the inter-microwell area of both substrates. Ki67 is a nuclear cell proliferation marker that is present during all the active phases of the cell cycle (G1, S, G2/M), but is absent in resting cells (G0).[59] To our knowledge, this is the first time that the effect of microwell shape on MSC proliferation activity has been demonstrated. Our results indicated that squareshaped microwells led to a higher cell proliferation activity than round-shaped microwells or the inter-microwell area, which might be due to the higher expression of ROCK by the cells in the square-shaped microwells. Previous studies have shown that Rho/ROCK pathway participates in the cell proliferation process and the low expression of ROCK may result in a low cell proliferation.[60,61] This was in agreement with our finding that the proliferation of hADSCs on both substrates decreased to a similar level following ROCK blockage. Given the fact that the cells proliferated faster on the substrate with squareshaped microwells, our results indicated that the proliferation of hADSCs was more dependent on Rho/ROCK signaling on




4. Conclusion Microstructured substrates comprising arrays of square-shaped or round-shaped microwells were developed as a transitional model between 2D and 3D systems in order to explore the cellular response of hADSCs. The differently structured microwells exerted highly distinct effects on major cellular process


including cell morphology, migration, gene expression, proliferation, and differentiation. The substrate with 50 µm square-shaped microwells promoted in situ proliferation and differentiation of cells towards osteogenic lineage under induction. The velocity and distance of random cell migration were much higher on the substrate comprising 50 µm round-shaped microwells. All of these cellular influences from structural cues are closely related to the intracellular Rho/ROCK signaling pathway. These findings allow a better understanding of MSC response to structural cues and highlight the possibility to control the fate and functions of MSCs through structured features via manipulation of Rho/ROCK signaling. This knowledge might help to direct cells/stem cells towards desired locations and functions through utilization of biomaterial structures. This approach of induced and controlled endogenous regeneration using a polymeric biomaterial may also be applicable to other stem/progenitor cells and to various types of implants, such as microstructured substrates of orthopedic implants, where the integration into relevant tissues could be potentially improved.

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the substrate with square-shaped microwells than on that with round-shaped microwells. Moreover, ROCK blockage led to the retardation of cell division in G2/M phase, which could be explained by the deficiency of contractility. However, a remarkable enrichment of cells in G2/M phase was observed on the substrate with square-shaped microwells, suggesting the higher dependency of cell cycle progression on ROCK signaling in the “square” group. The lineage commitment of MSCs can be regulated by related factors including cell shape, cytoskeletal tension, and Rho/ ROCK signaling.[18] In addition, the actomyosin contractility, which can be modulated by geometric cues, is of great importance to direct the differentiation of MSCs. It was found that MSCs growing on star-shaped micropatterns preferred to differentiate into osteoblasts, but those growing on flower-shaped micropatterns showed higher potential for differentiation into adipocytes. By using pharmacological agents to manipulate the cytoskeleton, the lineage commitment of MSCs was controlled on both geometric patterns.[20] In consistence with these findings in a 2D system, our study demonstrated that the structure of microwells could regulate the differentiation of hADSCs. The higher osteogenic differentiation on the substrate with square-shaped microwells could be attributed to the cell shapes and particularly the stronger stress fibers in these cells that produced higher contractility. Interestingly, hADSCs were found to be more responsive to ROCK inhibitor in the “square” group than in the “round” group: the blockage of ROCK, impairing the cell contractility, suppressed the osteogenic differentiation of cells on the substrate comprising square-shaped microwells, but did not influence the osteogenic differentiation of cells on the substrate with round-shaped microwells and on the smooth surface. These results suggested that the microwells might direct the cell differentiation by modulating the cell contractility and regulating the ROCK responsiveness. We hypothesize that the microstructured substrates could regulate the cellular behavior of hADSCs via the following mechanism. When the cell shape is manipulated by the microwells, the cells sense the different structural cues via integrins on the cell surface and further determine the cell behavior. Compared with the round-shaped microwells, the squareshaped microwells may generate a higher local cytoskeleton tension at the corners which is mediated by integrins,[31,62] and such an increase of cytoskeleton tension promotes the local assembly of focal adhesion and stress fibers.[63] Intracellularly, the cytoskeletal tension is required to activate ROCK, and the feedback signal could be subsequently generated to remodel the stress fibers and focal adhesion.[50] Furthermore, the induced alternation in cytoskeleton and Rho/ROCK activity by the change of cell shape could contribute to multiple cellular processes.

5. Experimental Section Preparation and Characterization of Microstructured Inserts: Microstructured PS inserts with a suitable size to fit the standard 24-well TCP were prepared by a two-step processing procedure. First, the films of PS substrates were microstructured by soft-lithography according to the reference.[64] Then, the microstructured PS films were embedded into PS inserts via injection molding. The PS inserts were gas sterilized (gas phase: 10 wt% ethylene oxide, 54 °C, 65% relative humidity, 0.17 MPa, 3 h gas exposure time, and 21 h aeration phase). The insert bottom was coated with human vitronectin prior to seeding cells. The vitronectin adsorption amount and distribution were characterized by enzyme-linked immunosorbent assay (ELISA) and fluorescent staining, respectively. The microstructured substrates were characterized by digital microscopy and optical profilometry for the well size and by SEM, AFM, contact angle measurements as well as XPS for the surface properties. (for details see Supporting Method S1-S4, Supporting Information) Cell Culture: In our study, hADSCs were isolated from human adipose tissue obtained by abdominal liposuction from a female donor after informed consent (No.: EA2/127/07; Ethics Committee of the Charité – Universitätsmedizin Berlin, approval from 17.10.2008; for details see Method S5, Supporting Information). The cells were cultured in Dulbecco’s modified eagle medium (Life Technologies, Darmstadt, Germany) supplemented with 10 vol% fetal bovine serum (FBS, Biochrom, Berlin, Germany), 2 mM L-glutamine and 100 IU mL−1 penicillin plus 100 µg mL−1 streptomycin, and were used from passage three for all experiments. ROCK Inhibition: Y-27632 (Sigma–Aldrich, St. Louis, MO, USA) was used as a selective inhibitor of ROCK and the medium containing Y-27632 (10 μ M) was applied to cells 24 h after cell seeding. In cell cycle analysis, the blocking was performed after the synchronization of the cells in the G0/G1 phase. Time-Lapse Microscopy: Cell migration on microstructured substrates was tracked using a phase contrast time-lapse imaging microscope (IX81 motorized inverted microscope, Olympus, Hamburg, Germany). A bold line cage incubator provided the humidified atmosphere (37 °C, 5% CO2) for cell growth. Suspended cells were added into the inserts at a density of 1.0 × 104 cells cm−2. Cells were observed at 2 h after adding of the cell suspension to monitor the migration of the non-attached cells and 2 d after cell seeding when all of the cells were attached and well spread. The images were processed using ImageJ software (National Institutes of Health, USA) combined with the software plug-ins



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www.MaterialsViews.com “manual tracking” and “chemotaxis and migration tool” (ibidi GmbH, Martinsried, Germany) to calculate the migration parameters. Real-Time PCR and Immunoblotting: The cells were cultured on microstructured substrates for 2 d. Thereafter, the gene profile was studied by real-time PCR using a Human Focal Adhesions RT2 Profiler PCR Array microplate (QIAGEN GmbH, Germany) and a StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The ROCK1 protein expression level was investigated by immunoblotting. (for details see Method S8,S9, Supporting Information) Cell Proliferation Assay: The cells were seeded at a density of 1.0 × 104 cells cm−2 and the cell culture medium was changed every 3 d. The number of cells at the indicated time points was determined using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Munich, Germany). In brief, the old medium was carefully aspirated and 500 µL of fresh medium was added into each insert, followed by adding 50 µL CCK-8 solution. After 2.5 h of incubation at 37 °C, 100 µL medium/CCK-8 mixture was transferred from each insert into a transparent 96-well TCP, and the absorbance of the mixture was measured at a wavelength of 450 nm and a reference wavelength of 650 nm using a microplate reader (Infinite 200 PRO, Tecan Group Ltd., Männedorf, Switzerland). The cell number was calculated via a standard curve, which was generated by measuring a series of samples with known cell numbers. Cell Differentiation Assay: Cells were seeded at a density of 0.5 × 104 cells cm−2 and cultured in normal growth medium and osteogenic induction medium (StemPro Osteogenesis differentiation kit, Life Technologies GmbH, Germany). The medium was replaced with fresh medium every 3 d. After 14 d culture, the cells were fixed with 4% (w/v) formaldehyde solution and the ALP was stained using the Stemgent ALP Staining Kit II (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) to evaluate the early stage osteogenic differentiation. After 21 d of culture, the cells were fixed and stained with 2% (w/v) ARS solution (Sigma–Aldrich, St. Louis, MO, USA) at pH 4.2 to identify the calcium deposits. The differentiation levels were described as the percentage of ALP positive area per unit total area and the percentage of ARS covered area per unit total area, which were calculated using ImageJ software (National Institutes of Health, USA). Statistics: Statistical analysis was performed using the two-tailed independent-samples t-test, and a significance level (Sig.) < 0.05 was considered to be statistically significant. Data are presented as mean ± standard deviation if not indicated otherwise.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements The authors acknowledge Dr. Manfred Gossen and Dr. Nilay Lakhkar for their input on the manuscript revision as well as Dr. Thomas Weigel, Mrs. Manuela Keller, and Mr. Mario Rettschlag for technical support. This work was financially supported by the Helmholtz Association of German Research Centers (including Helmholtz-Portfolio Topic “Technology and Medicine”) and the German Federal Ministry of Education and Research (BMBF project number 0315696A “Poly4Bio BB”). Received: July 15, 2014 Revised: September 2, 2014 Published online: October 14, 2014 [1] B. Parekkadan, J. M. Milwid, Annu. Rev. Biomed. Eng. 2010, 12, 87. [2] A. I. Caplan, J. Cell. Physiol. 2007, 213, 341. [3] N. K. Satija, V. K. Singh, Y. K. Verma, P. Gupta, S. Sharma, F. Afrin, M. Sharma, P. Sharma, R. P. Tripathi, G. U. Gurudutta, J. Cell. Mol. Med. 2009, 13, 4385.

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Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

Discriminating cellular heterogeneity using microwell-based RNA cytometry.

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Modulation of the stemness and osteogenic differentiation of human mesenchymal stem cells by controlling RGD concentrations of poly(carboxybetaine) hydrogel.

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Treatment of Collagen-Induced Arthritis Using Immune Modulatory Properties of Human Mesenchymal Stem Cells.

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Guiding osteogenesis of mesenchymal stem cells using carbon-based nanomaterials.

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Generation of a biomimetic human artificial cornea model using Wharton's jelly mesenchymal stem cells.

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

Generation of induced pluripotent stem cells from human mesenchymal stem cells of parotid gland origin.