Light-Induced Cell Alignment and Harvest for Anisotropic Cell Sheet

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Light-induced cell alignment and harvest for anisotropic cell sheet technology Chao Liu, Ying Zhou, Miao Sun, Qi Li, Lingqing Dong, Liang Ma, Kui Cheng, Wen-Jian Weng, Mengfei Yu, and Huiming Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07202 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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ACS Applied Materials & Interfaces

Light-induced cell alignment and harvest for anisotropic cell sheet technology †

†

Chao Liu1, , Ying Zhou1, , Miao Sun1, Qi Li1, Lingqing Dong3, *, Liang Ma4, Kui Cheng3, Wenjian Weng3, Mengfei Yu1,2, * & Huiming Wang1,2, * 1

The Affiliated Stomatologic Hospital, Zhejiang University, Hangzhou 310003, China.

2

The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou

310003, China. 3

School of Materials Science and Engineering, State Key Laboratory of Silicon

Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China. 4

The State Key Laboratory of Fluid Power Transmission and Control, Zhejiang

University, Hangzhou 310027, China. †

These authors contributed equally to this work.

*

Correspondence and requests for materials should be addressed to:

[email protected] (M. Yu), [email protected] (L. Dong) and [email protected] (H. Wang).

KEYWORDS:

light-induced, cell alignment, anisotropic cell sheet, TiO2 nanodots

film, tissue engineering.

ACS Paragon Plus Environment


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ABSTRACT Well-organized orientation of cells and anisotropic extracellular matrix (ECM) are crucial in engineering biomimetic tissues, such as muscles, arteries and nervous system, etc. This strategy, however, is only beginning to be explored. Here, we demonstrated a light-induced cell alignment and harvest for anisotropic cell sheets (ACS) technology using light-responsive TiO2 nanodots film (TNF) and photocrosslinkable gelatin methacrylate (GelMA). Cell initial behaviors on TNF might be controlled by micropatterns of light-induced distinct surface hydroxyl features, owing to a sensing mechanism of myosin II-driven retraction of lamellipodia. Further light treatment allowed ACS detachment from TNF surface while simultaneously solidified the GelMA, realizing the automatic transference of ACS. Moreover, two detached ACS were successfully stacked into a 3D bilayer construct with controllable orientation of individual layer and maintained cell alignment for more than 7 days. Interestingly, the anisotropic HFF-1 cell sheets could further induce the HUVECs to form anisotropic capillary-like networks via upregulating VEGFA and ANGPT1 and producing anisotropic ECM. This developed integrated-functional ACS technology, therefore, provides a novel route to produce complex tissue constructs with well-defined orientations and may have a profound impact on regenerative medicine.


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INTRODUCTION Tissue engineering aims to construct functional living tissues in vitro to replace damaged tissues or organs in regenerative medicine application.1 One of the challenges inherent is replicating the complex three-dimensional (3D) structures of most tissues which are critical to their function, such as skeletal muscle and heart tissues, bone, tendons and ligaments, arteries and nervous system, etc.2 In fact, there is growing evidence that morphogenesis and homeostasis of tissues are regulated by extracellular microenvironment. And various material cues, including topography, chemical properties and biological cues, have been proved as potent regulators of cell function, lineage commitment and epigenetic status.3-4 The further understanding of material-induced cell responses has accelerated the development of smart biomaterials and provided more techniques and tools for tissue engineering.5 Among these, the combination of cell sheet (CS) engineering and surface patterning seems a promising approach to replicate complex structures. CS engineering makes it possible to fabricate a sheet full of cells along with their natural extracellular matrix (ECM), which can be transferred and stacked to construct a multi-layer scaffold-free tissue.6-7 Compared to other tissue engineering techniques for creating 3D tissue constructs, the absence of scaffold in CS engineering avoided the problems came with the biodegradation process of the scaffold, such as biodegradation and potential cytotoxicity.8 Recently, T. Okano and co-workers have made progress by



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combining the thermoresponsive cell sheet technology with topological patterning, micro-contact printing or polymers grafting, which generated effective biophysical cues the temperature-sensitive substrates without interrupting the detachment of cell sheets.9 Furthermore, the possibility of using submicrometer-grooved10 or nanostructured PNIPAAm substrates11-12 for constructing anisotropic cell sheets (ACS) was also While CS engineering emphasizes the importance of biocompatibility for harvesting confluent cells, the surface engineering aims to precisely control cell adhesion and spreading, utilizing the so-called non-fouling materials that are intrinsically impeditive cells and proteins adsorption.13 Therefore, a major challenge for ACS engineering is to integrate opposite intrinsic property of materials used in surface engineering with the requirements of CS engineering. We have recently demonstrated a light-induced cell detachment approach by using a TiO2 nanodots film (TNF).14-15 However, these previous efforts have only reported the detachment of isotropic cell sheets, lacking the integrated functions of transferring and manipulation of detached cell sheets as well. Recently, the light patterning has been demonstrated

as

a

way to

fabricate

the

hydrophilic/hydrophobic

patterns,16

polymer brushes17 and small-molecule organic nanowire18 on TiO2 film. A novel method for fabricating cell microarrays based on photocatalysis of cell-repellent coating on TiO2 film

was

also

reported.19-22

In

fact,

one

property

of

TiO2,

known

as

showed the great improvements in the attachment and proliferation of osteogenic cells on


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ultraviolet (UV) light pre-irradiated TiO2 film.23-25 Surface hydroxyl group features on TiO2 controlled by photofunctionalization initiate a hierarchical signal processing thus guide the selective protein adsorption as well as the subsequent cell behaviors.15, 26-27 It implied the possibility of cell patterning by UV light on TiO2 film without the coating and the combination with light-induced cell detachment.

Meanwhile, the newly

developed photopolymerizable gelatin methacrylate (GelMA), synthesized by adding methacrylate groups to amine-containing side-groups of gelatin,28-29 might be an ideal photocrosslinkable hydrogel to integrate detachment and stack of the light-induced CS. In this work, we designed a technology of integrated functions of cell alignment and harvest ACS using light-responsive TNF and photocrosslinkable GelMA. The mechanism underlying the effects of micropatterned cues originated in light-induced distinct hydroxyl on cell behaviors was discussed. Moreover, the human foreskin fibroblasts (HFF-1) ACS were successfully transferred and stacked assisted by GelMA. Also, the orientation features and “anisotropy duplicate” of transferred HFF-1 ACS to induce anisotropic capillary-like networks were evaluated.

RESULTS AND DISCUSSION Light-induced Cell Alignment and ACS Harvesting. To obtain anisotropy in cell sheet engineering, a suitable micropatterning approach should be applied to the substrates without interrupting cell detachment. In this work, the micropatterned cues were



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simply by adding a photomask-assisted 254 nm UV light (UV254) pretreatment (Figure 1A). The TNF was prepared on quartz substrates as reported previously.30-31 After 1-hour pre-irradiation treatment (supporting information, Figure S1), the HFF-1 cells labelled with CellTrackerTM Green were immediately seeded on the patterned TNF, and they lined up parallel to the patterns in photomasks. During the following 5-day incubation, the aligned HFF-1 cells proliferated and reached to be confluent while maintaining the orientation. In contrast, HFF-1 cells (stained with CellTrackerTM Red) adhered randomly on the nonpatterned TNF and arranged in a vortex way after being confluent. Figure 1B illustrated the process of UV-induced ACS detachment. After 20-minute 365nm UV light (UV365) irradiation from the bottom of the TNF, confluent cells were peeled off from the surfaces spontaneously as an intact monolayer. The general morphology of detached cell sheets was recorded under stereomicroscope. A distinctive shrink ratio was inevitable during observation as reported previously.32 In comparison to isotropic cell sheets shrinking equally in two dimensions, the ACS were observed in asymmetric shrinkage between the vertical and parallel sides. The live-dead staining of detached cell sheets and cells remained on the substrates was examined immediately after light-induced (Figure S2), showing few cells remained on substrate and the survival of ACS was not compromised by the detachment process. Micropatterning32-35 or nanopatterning11-12 approaches have been used in CS engineering. Generally speaking, nanopatterning controlled adhesion formation at a


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integrin dimer level and thus was adequate for most kinds of cells. However, it usually required highly delicate devices and complex fabricating processes.3 Micropatterning can confine cells thus dictated their shape, but it might require a two-step cell culture with serum-free and serum-containing media, or needed to be more selective in choosing the appropriate cell types.9 The light patterning method used in this work was a kind of micropatterning approach, with high degree-of-freedom in pattern fabrication and relative ease of access to equipment.19 An anisotropic cell sheet could be obtained simply via one-pot cell seeding, meanwhile maintained the high-efficiency and safety of cell detachment technology. Therefore, we believed it provided a simple tool for ACS fabrication and greatly broadened the range of applications of light-induced cell sheet technology.



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Figure 1. Overview of light-induced cell alignment and anisotropic cell sheets (ACS) detachment on TiO2 nanodots film (TNF). (A) Photomask-assisted UV254 illumination used to pattern TNF, while the nonpatterned ones were exposed to UV254 without photomask. HFF-1 cells stained with CellTrackerTM Green or CellTrackerTM Red were seeded on patterned or nonpatterned TNF, respectively. Fluorescence images showed the adhesion of fibroblasts at 12 hours and confluence at 5 days after cell seeding. (B) illumination from underneath was used to harvest ACS. Phase contrast microscopic images showed the HFF-1 monolayers underwent spontaneous detachment after UV365


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illumination on both patterned and non-patterned TNF. White arrows indicated the detaching edges and directions of cell sheets. The stereomicroscopic images showed the general morphology of detached anisotropic and isotropic cell sheets.

Photocrosslinkable GelMA Hydrogel Casting Method for Light-induced ACS Detachment, Transferring and Stacking. We now have achieved the results that cells were patterned and harvested from the TNF simply by a two-step UV illumination. However, the ACS showed a general shrinkage after the light-induced spontaneous detachment, and the delicate cell sheets were unsustainable to be manipulated further. In order to maintain the sizes and shapes of ACS and facilitate the transferring and stacking process, the photocrosslinkable GelMA hydrogel was introduced, as illustrated in Figure 2A. Briefly, the melt GelMA prepolymer solution was added to the confluent cells on patterned TNF, and then proceeded to the UV illumination. As UV light solidified the gel, the intact cell sheet was simultaneously detached from the TNF surface and then to the bottom of photocrosslinked GelMA. The delicate cell sheets could be easily manipulated using forceps with the assistance of GelMA. Trypan blue staining was used visualize the process that intact cell sheet was transferred from patterned TNF onto the crosslinked GelMA (Figure 2B). Sequentially the detached cell sheet could be stacked another cell sheet to fabricate 3D tissues. Because cell sheets were harvested with associated ECM, we speculated that it was easy for them to connect with each other



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through layering and co-incubation. The resultant bilayer tissue was detached and transferred in the same manner. Furthermore, multilayer complex tissue could be constructed by repeating this stacking process. The ACS were stained with CellTrackerTM Green or Red to discern individual layers during the stacking. And the stacked ACS maintained their previous alignment at least 3 days, indicating a successful organized 3D layered cell sheet (Figure 2C). Although the gelatin-coated plunger or stamp had been widely used for harvesting and manipulating cell sheets,36 the gelatin was not optimal for other cell sheet strategies due to additional procedure of lowering temperature and relative poor mechanical and stability. By adding methacrylate groups to the amine-containing side-groups of gelatin, GelMA was a functionalized hydrogel sharing the advantages of both natural and synthetic hydrogels.28 A desirable mechanical robustness could be obtained by just modifying

the

degree

of

methacrylation

without

compromising

the

cellular

biocompatibility. Also, this hydrogel’s stability at 37 °C facilitated long-term cell viability.29 These properties contained in the photocrosslinkable GelMA might facilitated the integration of light-induced detachment, transfer and stack of ACS. What’s more, cells or/and proteins could be encapsulated into GelMA before photopolymerization, which had been demonstrated to promote the osteogenesis and microvascularization of engineered implantation.37-38 This light-induced ACS technology of integrated functions


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seems to provide a promising strategy to combine the cell sheet technology and hydrogel system to construct vascularized large-volume engineered tissues in future.

Figure 2. The photocrosslinkable GelMA hydrogel casting method to maintain the morphology of ACS and facilitate the stacking process. (A) Schematic illustration of the UV-induced detachment, transferring and stacking of ACS assisted by GelMA hydrogel. (B) The ACS was transferred from the surface of TNF substrate to GelMA without



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distortion. ACS was fixed and stained with trypan blue to enhance visibility. Scale bar, 1 cm. (C) Confocal images of z-stack showed two membrane-stained ACS were stacked to construct a bilayer tissue using photocrosslinkable GelMA casting method. Individual ACS was stained with CellTrackerTM Green and CellTrackerTM Red before detachment. The arrows indicated the orientation of cell alignment of individual layers. Scale bar, 100 µm.

Micropatterned Cues on TNF Are Generated by Photomask-assisted UV Illumination. Though cell patterning on TiO2 film have been reported previously,19-22 we here to described another novel method to generate micropatterned cues directly on TiO2 surface without the cell-repellent coating. We observed the effects of UV pretreatment at aspects of wettability, protein adsorption and cell attachment to confirm the generation of micropatterned cues, as shown in Figure 3. Figure 3A showed the surface topography of TNF. The homogeneous distribution of TiO2 nanodots assured the high efficiency of UV light absorption, which was reflected in the instant changes of water contact angle (WCA) of TNF under UV illumination (Figure S3). The water drop condensation and fluorescein-conjugated BSA adsorption on the three groups (Figure 3B) showed the wettability increased dramatically after UV illumination, as well as the protein adsorption. And water columns and protein patterns with the width of 50 µm were forming on the patterned TNF. The quantitative analysis of BSA adsorption (Figure S4)


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and cell attachment and proliferation (Figure 3C) further demonstrated the moderately enhanced bioactivity of UV patterned TNF. The confocal images (top images in Figure 3D) pictured the well-organized cell alignment on patterned TNF, whereas cells adhered and spread randomly on both control groups. High magnification images vividly revealed that cells were much larger with their processes spreading on the +UV and patterned TNF surfaces, whereas cells remained small and round with little cytoskeletal development on -UV surface (bottom images in Figure 3D). The cell morphology on +UV and patterned TNF surfaces was also significantly different. Cells on the +UV surfaces were still in round-shape with process in random directions, and vinculin diffused in the cytoplasm and assembled at the termini of actin fibers. In contrast, elongated shape oriented along the pattern direction in patterned group, with vinculin predominantly localized at the top of the ridges. Cytomorphometric evaluations of the area, perimeter and Feret’s diameter further demonstrated a considerate upregulation in cell spread and cytoskeletal development after UV pretreatment and an effective control of cell morphology by photomask-assisted UV pretreatment. The upregulation of protein adsorption and cell attachment, proliferation, differentiation, and mineralization of osteogenic cells on titanium or TiO2 surface after UV illumination has been reported and termed as photofunctionalization. And these effects were further enhanced on titanium surface modified with nanoscale topology.39-40 The photofunctionalization has always been reported to enhance bone-implant integration



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before, yet it is the first time to be related with the light patterning. With the assistance of photomask-assisted irradiation, the effect of photofunctionalization was limited to microscale level. The possible mechanism underlying photofunctionalization was reported to involve photohydrophilicity,23 photocatalysis of hydrocarbon25 and electrostatic properties41 of UV-treated TiO2 surface. Previously, the upregulated proportion of terminal hydroxyl group (TiOHT) was observed on titanium surface after UV preirradiation and reported to be an intrinsic factor which had strong electrostatic interactions with the amino group (NH3+) of proteins in media26 and regulated the conformation of the initial protein adsorption and subsequent cellular behaviors.42 The similar changes of TiOHT were observed on the pre-irradiated TNF (Figure S5) and the generation of micropatterned cues based on altered surface hydroxyl groups was schematically illustrated in Figure S6. Meanwhile, as the light patterning method used here was possibly based on the photofunctionalization effects, it was speculated that the stem cell sheets or osteoblast sheets obtained by light-induced ACS technology might exhibit better osteogenesis differentiation properties. The potential application of this technology in bone tissue engineering remains to be explored.


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Figure 3. The generation of light-induced micropatterned cues on TNF. (A) The SEM image of TNF. Scale bar, 300 nm. (B) The images of water drop condensation and fluorescein-conjugated BSA adsorption on -UV, +UV and Patterned TNF. Scale bar, 200 µm. (C) The cell attachment (6h) and proliferation (1d, 3d, 5d) on -UV, +UV and Patterned TNF. The data are presented as the mean ± SD, n=3, *: p < 0.05, **: p < 0.01. (D) Confocal images of initial spread and arrangement of HFF-1 6h after seeding on -UV, +UV and Patterned TNF. Vinculin was stained by AlexaFluor488 (green), F-actin stained by Rhodamine (red) and nuclei stained by DAPI (blue). Scale bar, 50 µm. (E) Cytomorphometric evaluations performed using confocal images were shown as scatter dot plot (n=50).

Lamellipodia Are Involved in Sensing Micropatterned Cues on TNF during Light-induced Cell Alignment. We further studied the mechanism underlying cell alignment on TNF after photomask-assisted UV illumination. Since cells adhered and spread limited within fluorescein-protein stripes at initial stage and were confluent in a uniform orientation coincided with the orientation of protein and patterns in photomask (Figure S7), it was reasonable to speculate that micropatterns play a vital role in cell alignment. Cells seeded on patterns of 30 or 50 µm showed well-defined orientation both in the early stage of adhesion and in the later confluent stage (Figure 4A). However, with the width reduced to 15 µm, cells adhered randomly as the nonpatterned groups.


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Similarly, though cells could adhere and form stripes on the surface with the width increased to 100 µm, they extended randomly in the stripes, further immigrated and proliferated randomly on the unirradiated parts. For further quantitative analysis of cell alignment, we converted the phase contrast microscopic images into frequency distribution through two dimensional Fast Fourier transform method (2D FFT, Figure S8). We analyzed cell orientation distribution when cells adhered on surface (12 hours) and first became confluent (5 days). Cell orientation distribution peaks of 30 and 50 µm group were higher and narrower compared with the other groups in both stages (Figure 4B). “Alignment index” was defined and used as a comparable indicator of the anisotropy in cell and cell sheet. As expected, the Alignment indexes of 30 µm and 50 µm groups were significantly upregulated, compared to those of others both in 12h and 5d (Figure 4C). It seemed that the width of 30 to 50 µm was optimum to induce HFF-1 cell alignment, which might be attributed to the actual diameter of cells. In consistent with previous studies, width of 30-50 um was suitable for many types of cells derived from human, such as vascular smooth muscle cells,35 mesenchymal stem cell,43 fibroblasts,32, 34 myoblasts44 and so on. However, it seemed too large for cell lines derived from mouse. As the length of long axis and short axis of HFF-1 cells were measured as 100 ± 30µm and 25 ± 5µm, respectively, we speculated that it’s necessary to ensure the width of patterns were just between the length of cellular long axis and short axis for achieving well-defined cell alignment.



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It’s reported that cells might probe the extracellular environment and sense substrate topography before spreading by protruding cellular protrusions.45-46 The observations of SEM results showed the well-patterned cells (30µm) did not extend any lamellipodia in perpendicular direction to patterns, but formed thick lamellum with active lamellipodia at the leading edge along the direction of the patterns. On the contrary, the weakly polarized morphology of nonpatterned fibroblasts was typically characterized by multiple lamellipodia, which exhibited intermittent protrusion with random direction, contacting with one another (white arrows, Figure 4D). It indicated extensions onto unirradiated zone tended to release and retract, whereas extensions onto irradiated area were stable to allow continuous spreading. As the protrusion rate was the difference between the actin polymerization rates and retrograde flow rates,47-48 some researchers reported the fibroblasts probed substrate rigidity49 or nanotopographies50 with filopodia extensions and actomyosin-dependent traction forces were crucial for sensing.51 Myosin II was an actin-binding protein that had actin crosslinking and contractile properties, and believed to take central stage in cell adhesion and migration.46 Inhibition of myosin II activity with blebbistatin greatly decreased the rate of actin retrograde flow in the lamellum and increased protrusiveness.52-53 Therefore, a myosin II-driven, filopodia-based probing mechanism has gain lots of attention.45 Consistent with the previous research, inhibition of myosin II or Rho-associated coiled coil-containing kinase (ROCK), a upstream regulator of myosin II,52 also caused cells lost their anisotropy and arranged randomly on


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the micropatterned TNF (Figure S9). These results indicated that actin-containing lamellipodia in front of the leading edge might be responsible for micropattened cues sensing and the retraction of lamellipodia, and eventually the cellular shape and orientation (Figure 4E).



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Figure 4. Cells sensing the micropatterned cues by lamellipodia during light-induced alignment. (A) Cell alignment on patterned TNF of varied widths. The representative


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images (groups of 15µm, 30µm, 50µm, 100µm and nonpatterned) were taken at 12h and 5d after cell seeding by a phase contrast microscope. (B) The frequency distribution of cell alignment converted by the representative images through 2D FFT. (C) The comparison of “Alignment index” among varied widths of patterns after cell seeding 12h and 5d. All the data are presented as the mean ± SD, n=3, n.s.: p>0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001. (D) The SEM images of cells on patterned and nonpatterned TNF. Cells were fixed and pictured at 6h after cell seeding. The lamellipodia were pointed by white arrows. (E) Schematic illustration of the hypothesis of cell sensing micropatterned cues and alignment on varied patterns. a,d) Actin-containing lamellipodia extended in front of the lamellum at the leading edge to probe the substrates and b, e) established nascent adhesions at a distance in front of the leading edge. c) The nascent focal complex could mature into focal adhesions and mediate cell extension on the UV-treated patterns (bright area), while f) the nascent adhesions disassembled and protrusions retracted on the UV-untreated areas (dark area).

The Formation of Capillary-like Networks and “Anisotropy Duplicate” on ACS. The detached ACS was transferred by GelMA casting method onto a tissue culture polystyrene (TCPS) surface as shown in Figure 5A. It showed that the orientation of the cytoskeleton of cell sheets could maintain for at least 7 days. Furthermore, the transferred ACS produced anisotropic extracellular collagen fibers after cultured in media containing



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L-ascorbic acid, while they were deposited randomly in control group. It indicated the alignment of fibroblasts provided anisotropy to deposited collagen. Although multilayer tissue could be fabricated effectively using the GelMA casting method, the lack of sufficient oxygen and essential nutrients supply could induce necrosis in this cell-dense tissue fabrication.54 Therefore, the vascularization inside multilayer tissues has gained extensive attention. Human umbilical vein endothelial cells (HUVECs) were seeded on the reattached HFF-1 sheets to explore the angiogenetic effects of ACS without any exogenous addition. Different with the previous studies,55 our HUVECs could self-assemble and form anisotropic capillary-like branching networks even on a single HFF-1 cell sheet, as shown in Figure 5B. Because HFF-1 cells were usually used as a feeder layer for supporting and nourishing other cells, it might be the reason that HFF-1 cell sheets showed better biological effects for angiogenesis. Quantification of the formation of capillary-like networks revealed statistically significant differences between anisotropic and isotropic cell sheets. The networks on anisotropic fibroblast sheet showed higher total length, average branch length, number of branch points and number of branches (Figure 5C), which indicated that the ACS provided a better niche for angiogenesis. Furthermore, the angiogenesis-related genes (vascular endothelial growth factor A, VEGFA and Angiopoietin 1, ANGPT1) and ECM secretion related genes (transforming growth factor- β, TGF-β and collagen I, COL-I) were examined using real-time polymerase chain reaction (RT-PCR) (Figure 5D). The expression of VEGFA,


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ANGPT1 and COL-I was obviously upregulated while TGF-β of aligned HFF-1 was similar with that from randomly adhering cells. VEGF may act in a juxtacrine/paracrine manner as a survival and/or stabilizing factor for endothelial cells, and ANGPT1 binds the endothelial-specific receptor tyrosine kinase Tie-2 (also known as TEK) and plays a critical role in endothelial sprouting and vessel wall remodeling.56 TGF-β is a multifunctional cytokine produced by perivascular cells and endothelial cells and is involved in multiple processes, including ECM production and mesenchymal cell differentiation into mural cells, with both pro- and anti-angiogenic properties depending on concentration and local microenvironment.56 Fibroblasts play an essential role in the angiogenesis process through their production of extracellular matrix molecules and other essential growth factors. And it seemed that the alignment of fibroblasts enhanced their pro-angiogenesis property. Similar results had been reported, for example, parallel microgroove could enhance the directed differentiation of cardiac progenitors into cardiomyocyte-like cells57 or help to reprogram somatic cells into pluripotent stem cells,58 suggesting that biophysical signals could act as potent regulators of stem cell function, lineage commitment, and epigenetic status.4 All these results indicated the control of cell arrangement could be an effective approach to influence individual cell function. In addition, the anisotropic myoblast sheet placed on top of other cell sheets in fabricating thick tissue was able to change the cell orientation in several layered cell



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sheets underneath.44 HUVECs co-cultured on the ACSs in this study also showed well-defined alignment while maintaining the branching structure. The degree of anisotropy of capillary-like networks was quantified in the same manner described above, showing the orientation distribution peaks of networks on ACS and isotropic HFF-1 cell sheets were distinct (Figure 5E). Because the orientation of HUVECs was originated from the fibroblast cell sheets, we could nominate this phenomenon as “anisotropy duplicate”. Since fibroblast-derived ECM scaffold was directly used and reported to promote directional tubulogenesis,59 we considered that the anisotropic HFF-1 cell sheets might control the orientation of co-cultured cells via secreting well-aligned ECM (Figure 5A), which served as a biophysical cue to promote individual cell self-assembly. In natural development, embryogenesis and wound healing heavily rely on self-organized activities, such as cell migration and aggregation.60 Inducing tissue formation via self-organized activities is a natural way to reconstitute tissue architectural features in microenvironments, which can’t be replaced by artificial or mechanical fabrication method, especially for vascularization and neurotization. The “anisotropy duplicate” therefore provides us an effective approach to obtain anisotropy of certain cells.


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Figure 5. The formation and analysis of capillary-like networks on transferred ACS using GelMA casting method. (A) The orientation of cell alignment maintained after cell sheets transferring. The cell sheets were transferred onto TCPS using GelMA casting method and cultured for 7 days with L-ascorbic acid. Fluorescence images showed the alignments of cytoskeleton and the secretion of type I collagen in transferred cell sheets. F-actin was stain by Rhodamine (red), nuclei stained by DAPI (blue) and type I collagen



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stained by Fluorescein (green). Scale bar, 200 µm. (B) The fluorescence photographs of the formation of capillary-like networks on transferred cell sheets. The fluorescence images were taken at 7 days after co-culture, and HUVECs were stained by Fluorescein (green) and cytoskeleton of HUVECs and HFF-1 cell sheets were stained by Rhodamine (Red). Scale bar, 200 µm. (C) Quantitative analysis of the extent of capillary-like network formation on HFF-1 cell sheets was carried out by measuring the total length and average length of capillaries, the number of branch points per unit of area and the number of branches per unit of area. (D) The expression of angiogenesis related genes (VEGFA, ANGPT1) and ECM secretion related genes (TGF-β, COL-I) of anisotropic and isotropic HFF-1 cell sheets using RT-PCR relative to the level of GAPDH mRNA expression. All the data are presented as the mean ± SD. n=3, *: p < 0.05, **: p < 0.01, ***: p < 0.001. (E) The histogram of frequency distribution of capillary-like networks forming on the anisotropic and isotropic cell sheets.

CONCLUSION In summary, we designed a technology of integrated functions of cell alignment and harvest anisotropic cell sheets (ACS) using light-responsive TiO2 nanodots film (TNF) and photocrosslinkable GelMA. This integrated functional technology was based on the hierarchical signal processing originated from the micropatterns of distinct surface hydroxyl feature triggered by photomask-assisted photofunctionalization to initiate


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protein adsorption as well as subsequent cell behaviors. The pattern widths of 30 and 50 µm were verified as the optimum width to induce HFF-1 cell alignment, which might be attributed to comparative size between the patterns with the cultured cells and the finitude of myosin II-driven lamellipodia-based sensing mechanism. Furthermore, the light-induced solidification of photocrosslinkable GelMA and simultaneously cell detachment from TNF surface were conducive to ACS harvest and further transfer. Moreover, the detached HFF-1 ACS with alignment ECM and upregulated VEGFA and ANGPT1 could further induce the formation of anisotropic capillary-like networks of HUVECs. These results suggested that the cellular orientation with distinct mechanical and biological ECM feature of ACS could be a promising strategy to produce complex tissue constructs with well-defined orientations and may have a profound impact on regenerative medicine.

MATERIALS AND METHODS Preparation and Characterization of TNF. TNF was prepared on substrate through a phase separation-induced self-assembly method.30-31 Briefly, a precursor sol containing titanium tetrabutoxide (TBOT, Sinopharm Chemical Reagent), acetylacetone (AcAc, Lingfeng Chemical Reagent) and polyvinyl pyrrolidone (PvP, K30, Sinopharm Chemical Reagent), was spin-coated on the quartz surface and further heat treated at 500 °C for 1 hour. The dots were polycrystalline and characterized by scanning electron



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microscopy (SEM, Hitachi Model M-1000). The water contact angle (WCA) was determined using a sessile drop method with a contact angle meter (Dataphysics OCA20), and the process of water condensation on TNF surfaces was recorded with a CCD camera installed on an optical microscope. A UV light source (254 nm, 300 µW/cm2) was used for micropatterning through a photomask which was made from quartz and formed Cr lines with varied widths (line/space ratio of 1:1), and another UV light source (365 nm, 2 mW/cm2) was used for cell sheet detachment as described previously.14 Cell Culture and Seeding on TNF. Human foreskin fibroblasts (HFF-1, SCRC-1041) were obtained from American Type Culture Collection (ATCC) cultured in Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 15% fetal bovine serum (FBS, Sciencell). The cells were seeded onto TNF at different densities according to each experiment: 2×104 cells/cm2 for observation of the cellular morphology and behaviors on different TNF surfaces, or 5×104 cells/cm2 for construction of cell sheets. The general morphology of HFF-1 cells cultured on varied substrates were observed everyday by a phase contrast microscope (Olympus). To visualize cell alignment or individual layers, cells were labeled with either 1 µM CellTrackerTM Green CMFDA (Invitrogen) or 1 µM CellTrackerTM Red CMTPX (Invitrogen) for 30 min before seeding or detachment. Inhibitors were added to cell culture media at the following concentrations: Blebbistatin (10 µM, Selleck) or Y-27632 (10 µM, Miltenyi Biotec).


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Human umbilical vein endothelial cells (HUVECs, PCS-100-010, ATCC) were cultured in vascular cells basal medium supplemented with Endothelial Cell Growth Kit-VEGF (ECGM, ATCC), and 1×104 cells/cm2 of HUVECs for co-culture with ACS in a 1:1 mixture of DMEM/ECGM media. HUVECs within 6th passage were used. All the cells were incubated at 37°C in an atmosphere of 5% CO2, replaced fresh media every 2–3 days and harvested using 0.25% trypsin-1mM EDTA (Gibco) at 80% confluence. Assessment of Cell Sheet Viability. The morphology of detached cell sheets was observed and recorded using a StereoMicroscope (Olympus). The cell sheet viability was assessed using a live/dead viability/cytotoxicity assay kit (Invitrogen): calcein-AM as a live cell reporting dye for intracellular esterase activity and ethidium homodimer-1 as a dead cell reporting dye for plasma membrane integrity. Briefly, detached cell sheets were incubated with a mixture of 1mM calcein-AM and 4 mM ethidium homodimer-1 for 30 min and examined using a fluorescence microscope (Zeiss Ax10). Preparation of GelMA prepolymer solution. GelMA was synthesized as described previously.29 Briefly, type-A porcine skin gelatin (Sigma–Aldrich) was dissolved at 10% (w/v) in Dulbecco’s phosphate buffered saline (DPBS) (GIBCO) at 60 °C. Methacrylic anhydride (MA) (Sigma–Aldrich) was added to the gelatin solution until the final concentration of 10% (v/v) at a rate of 0.5 mL/min under stirring conditions and then reacted for 3 h at 50 °C. Following a 5-times dilution of pre-warm DPBS to stop the reaction, the GelMA solution was dialyzed against deionized water using 12–14 kDa



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cut-off dialysis tubes for 7 days at 50 °C to remove unreacted MA and additional by-products. The solutions were lyophilized to be a white porous foam and stored at room temperature. Before use, the freeze-dried GelMA (6 w/v% final) was mixed with the photoinitiator (Irgacure 2959) (0.5 w/v%, CIBA Chemicals) in DPBS at 80 °C until fully dissolved. GelMA Casting Method. The GelMA casting method was developed to assist the detachment, transfer and stacking of ACS. Briefly, the melt GelMA prepolymer solution (37°C) was added to the confluent cells on patterned TNF, and then proceeded the illumination from the bottom of the culture plate (365nm, 2mw/cm2). After 20min illumination, the solidified GelMA was then removed from the TNF surface with blades and forceps and the cell sheet adhered to the bottom surface of this GelMA cast, which could be further transferred to a new surface and allowed to culture on the GelMA cast surface for at least 7 days. To enhance the visibility, the confluent cells on patterned TNF were fixed with ethyl alcohol and stained with 0.04% trypan blue before detachment. To create multilayered tissues, the GelMA cast which adhered the first cell sheet was transferred and carefully placed on top of the second cell sheet on TNF with a desired angle, and incubated for 1 hour to reattach. After the incubation, the same UV illumination was carried out for the detachment of the bilayer tissue. The fabricated tissue could be easily handled and transplanted reproducibly. And by repeating stacking cell sheets, the 3-D multilayer tissue could be fabricated.


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Protein Adsorption on TNF. Fluorescein-conjugated bovine serum albumin (BSA, 3.0 µg/mL in PBS, Molecular Probes) was incubated with patterned TNF and control group at 37 °C for 0.5 h. After being washed with PBS thoroughly, the BSA adsorption was observed through a fluorescence microscope (Zeiss Ax10). For quantitative analysis of protein adsorption, 300 µL of BSA solution (100 µg/mL in PBS)) was firstly pipetted onto samples. After incubation for 24 h at 37 °C, nonadherent proteins were rinsed and the rinse solution was collected and mixed with bicinchoninic acid (Pierce Biotechnology) at 37 °C for 60 min. The amount of the removed and inoculated BSA was quantified using a microplate reader (Molecular Devices SpectraMax) at 562 nm, and the remained BSA could be acquired by subtracting the two values above. Cell Adhesion and Proliferation. Initial cell attachment was evaluated by measuring the amount of cells attached to sample surfaces after 6 and 24 h incubation. Further cell proliferation was quantified in terms of cell density for 3, 5 and 7 culture days. Cell attachment and proliferation was assessed using the alamarBlue Cell Viability Reagent (Invitrogen). Referring to manufacturer’s instructions, cells were incubated in complete media containing 10% alamarBlue dye for 3 h, followed by fluorescence quantification using a microplate reader (Molecular Devices SpectraMax) with excitation and emission wavelengths of 540 and 590 nm respectively. Scanning Electron Microscope. The protrusions of cells on TNF were observed by SEM. After 12 h of cell culture, cells were fixed in 2.5% glutaraldehyde overnight at 4



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◦

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C. Then the samples were washed with PBS for three times, postfixed with 1% osmium

tetroxide for 2 h, and dehydrated in ethanol of ascending concentrations (30, 50, 70, 80, 90, 95, 100 (v/v)) for 15 min at each step. In the end, the samples were dehydrated in Hitachi Model HCP-2 critical point dryer with liquid CO2. The dehydrated sample was coated with gold-palladium in Hitachi Model E-1010 ion sputter for 5 min and observed in Hitachi Model TM-1000 SEM. Immunofluorescent Staining and Imaging. For immunofluorescence, cells were permeablized in 0.4% Triton X-100 in PBS, blocked in 2% FBS containing 2% BSA in PBS. Primary antibody labeling for mouse anti-vinculin antibody (Abcam) or rabbit anti-type I collagen antibody (Abcam) was performed in 2% BSA in PBS for 8-16 h at 4 °C. Secondary antibody labeling was performed in the same procedure with AlexaFluor488

labeled

anti-mouse

IgG

antibody

(Invotrogen)

or

fluorescein

isothiocyanate labeled anti-rabbit IgG antibody (santa Cruz) for 1 h at room temperature. To observe the capillary-like networks, HUVECs were visualized by the green fluorescence of Fluorescein-conjugated Ulex Europeus Agglutinin I (UEA-I, Vector Laboratories). Rhodamine Phalloidin (Cytoskeleton) was used for cytoskeleton staining and DAPI (Molecular Probe) for nuclei staining. Immunofluorescence microscopy was conducted using a Nikon A1Ti confocal laser scanning microscope or a Zeiss Ax10 inverted fluorescent microscope.


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Quantitative analysis. To quantitatively analyze the anisotropy of patterned cells and cell sheets, two dimensional Fast Fourier transform (2D FFT) techniques had been used to turn spatial information of images into frequency information.43 The phase contrast and fluorescent images were conducted 2D FFT and the orientation information was collected by ImageJ software (NIH). These data were then normalized such that the total sum was unity, and were plotted as a representation of frequency distribution of cell orientation (Figure S3). To directly compare the alignment of cells and cell sheets among different samples, the “Alignment Index” was defined as distribution values summed from ± 5˚ around the maximum peak and converted to a percentage. Cytomorphometric evaluations of area, perimeter and Feret’s diameter and the quantitative analysis of the formation of capillary-like networks on cell sheets (the total length of capillary-like network, the average length of capillary-like network, the number of capillary-like branches and the number of branches points) were quantified on fluorescent images using Image J software (NIH) referred to previous reports.24, 28 Real-time polymerase chain reaction (RT-PCR) analysis. The RT-PCR was used to detect the expression of several functional genes of fibroblasts (COL-I, TGF-β, VEGFA and ANGPT1) in the harvested ACS and isotropic HFF-1 cell sheets. Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA concentration was determined using NanoDrop 2000c. First-stranded complementary DNAs (cDNAs), synthesized from 1 mg of the isolated



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RNA by oligo (deoxythymidine) (oligo (dT)) using PrimeScriptTM RT Reagent Kit (TaKaRa), were applied as templates for real-time PCR using SYBR Premix Ex Taq (TaKaRa). The primer sequences were listed in Table S1. Forty cycles were used to amplify all gene sequences and the comparative expression level was obtained by transforming the logarithmic values into absolute values using 2 -△△CT, with GAPDH as the housekeeping gene for normalization. Statistical analysis. All values were expressed as means ± standard deviation (SD). Statistical analyses were carried out by a one-way analysis of variance (one-way ANOVA) and Turkey test for multiple comparison tests or using Student’s t-test, at a significance level of P < 0.05 in GraphPad Prism 6.0.

ASSOCIATED CONTENT Supporting Information This supporting information is available free of charge via the Internet at http://pubs.acs.org. Additional characterization data (Figures S1−S9 and Tables S1) (PDF) AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected]


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* E-mail: [email protected] Author Contributions † These authors contributed equally to this work. Notes The authors declare no competing financial interests.

ACKNOWLEDGES This work was financially supported by National Natural Science Foundation of China (81600838, 81670972, 81371120, 51502262, 51472216, 51372217), Medical Technology and Education of Zhejiang Province of China (2016KYB178) and Zhejiang Province Key Science and Technology Innovation Team (Grant No. 2013TD02).

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Light-Induced Cell Alignment and Harvest for Anisotropic Cell Sheet

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