Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.
The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.
In vivo, cells are subjected to a wide variety of environmental cues that can be of mechanical, physical, and biochemical nature that originate from the extracellular matrix (ECM). Numerous environmental cues have been identified to play vital roles in the regulation of cell behavior, such as proliferation, differentiation, and migration1,2,3,4,5. One of the most widely investigated phenomena is contact guidance, describing adhesion-mediated cell alignment along anisotropic biochemical or topographical patterns present on the extracellular substrate6,7,8,9,10,11. Beyond directing the alignment of cells, contact guidance cues have also been shown to influence other cell properties such as cell migration, organization of intracellular proteins, cell shape, and cell fate12,13,14,15. Additionally, the geometrical architecture of the 3D cellular environment has also been acknowledged for its regulatory influence on cell behavior16,17. In the human body, cells are exposed to a range of curved geometries, ranging from microscale collagen fibers, capillaries, and glomeruli, up to mesoscale alveoli and arteries18,19. Interestingly, recent in vitro studies have shown that cells can sense and respond to such physical cues, from the nano- to mesoscale20,21,22,23.
To date, most studies investigating cell response to environmental cues have been largely performed using experimental setups that isolate single cues. While this approach has allowed tremendous progress in understanding the basic mechanisms behind cellular sensing of environmental cues, it poorly recapitulates the in vivo environment that simultaneously presents multiple cues. To bridge this gap, it is useful to develop culture platforms whereby multiple environmental cues can be independently and simultaneously controlled. This concept has gained increasing traction lately24,25, with studies combining matrix stiffness and ligand density26,27,28,29, substrate stiffness and porosity30, substrate stiffness and 3D microniche volume31, surface topography and contact-guidance cues32,33,34, and nanoscale contact-guidance cues with mesoscale curvature guidance cues23. However, it remains challenging to combine contact-guidance cues with a variety of 3D geometries in a controlled and high-throughput way.
This research protocol addresses this challenge and introduces a method to create cell culture substrates with a controlled combination of patterned adhesive areas of ECM proteins (contact-guidance cues) and substrate curvature (geometrical cues). This approach allows for the dissection of cell response in a biomimetic multicue environment in a systematic and high-throughput manner. The knowledge acquired can aid in further understanding of cell behavior in complex environments and can be used to design instructive materials with properties that steer cell responses into a desired outcome.
3D protein photopatterning
Creation of adhesive areas of ECM proteins (contact-guidance cues) on cell culture materials can be accomplished using a variety of techniques, for example, by deep-ultraviolet (deep-UV) patterning or microcontact printing35,36. Deep-UV patterning makes use of UV-light that is projected through a mask on a polymeric material to degrade passivation polymers at specific locations on the cell culture substrate. The patterned substrate is then incubated with a ligand of interest, resulting in adhesive areas that support cell attachment and culture on predefined locations12,37,38. An alternative way to introduce protein patterns is by means of microcontact printing, where elastomeric stamps containing a desired shape are coated with a protein of choice and are pressed on a cell culture substrate, thereby transferring the protein coating to which cells can adhere35,37,39,40. Unfortunately, since both techniques rely on mask preparation and soft lithography methods, the experiments are time-consuming and labor-intensive, as well as limited in terms of pattern flexibility. In addition, both deep-UV patterning and microcontact printing are most suited for planar materials and are technically difficult, if not impossible, for patterning ligands in a 3D environment.
To improve on these conventional methods, Waterkotte et al. combined maskless lithography, chemical vapor deposition, and thermoforming to generate micropatterned 3D polymeric substrates41. However, this technique relies on the use of thermoformable polymer films and offers low protein-pattern resolution (7.5 µm), while cells have been reported to respond to geometrical protein patterns as small as 0.1 µm2,42. Sevcik et al. described another promising method to nanopattern ECM ligands on substrates containing nano- and micrometer topographies43. Using microcontact printing, ECM proteins were transferred from polydimethylsiloxane (PDMS) stamps to a thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) substrate. Subsequently, the thermoresponsive property of the pNIPAM network allowed them to transfer the two-dimensional (2D) protein pattern to a topographic PDMS substrate (10-100 µm deep grooves), thereby controlling the localization of adhesion sites on topographical features. However, not all possible microtopographies can be patterned since decreased wettability issues make it more difficult to pattern deeper topographic substrates. Trenches with a depth-to-width aspect ratio of 2.4 have been reported to be the ultimate limit to successfully transfer the pattern to the topographic substrate43. Additionally, the flexibility of varying patterns and the resolution of the generated patterns are poor due to the requirement of microcontact printing.
This paper describes a method that overcomes the abovementioned bottlenecks and offers a flexible and high-throughput method to create multicue substrates that can be used for cell culture (see Figure 1). Physiologically relevant geometries (cylinders, domes, ellipses, and saddle surfaces) with curvatures ranging from ĸ = 1/2500 to ĸ = 1/125 µm-1 are predesigned and microfabricated in PDMS chips. Subsequently, contact-guidance cues are created on top of the 3D geometries using a variety of digital pattern designs by employing a photopatterning technique with a resolution as small as 1.5 µm44. To this end, the PDMS chips are initially passivated to prevent cells and proteins from adhering; this passivation layer can then be removed by combination of the photoinitiator 4-benzoylbenzyl-trimethylammonium chloride (PLPP) and UV-light exposure45. A digital mask is designed to specify the locations of UV-exposure, and, thus, the area where the passivation layer is removed. Proteins can subsequently adhere to these areas, enabling cell attachment. Since the patterning is performed using a digital (rather than a physical) mask, a variety of patterns can be created quickly without the hassle and cost associated with designing and fabricating additional photomasks. In addition, a diverse range of ECM proteins (e.g., collagen type I, gelatin, and fibronectin) can be patterned on the substrate. Although this protocol is performed using cell culture chips made of PDMS, the principle can be applied to any other material of interest46.
In the studies described in this protocol, primary human keratocytes were used. This research was performed in compliance with the tenets of the Declaration of Helsinki. Primary keratocytes were isolated from leftover human cadaveric corneoscleral tissues from Descemet Membrane Endothelial Keratoplasty surgery, which were obtained from the Cornea Department of the ETB-BISLIFE Multi-Tissue Center (Beverwijk, the Netherlands) after obtaining consent from the next of kin of all deceased donors.
NOTE: See the Table of Materials for details about all materials, reagents, equipment, and software used in this protocol.
1. Fabrication of 3D cell culture substrates
2. Fabrication of flat PDMS samples (control samples)
3. Substrate passivation of 3D cell culture substrates
4. Storage of patterned cell culture substrates
NOTE: 3D cell culture substrates can be stored during different steps in the process.
5. Design of digital masks used for photopatterning
NOTE: Patterning of 3D substrates can be performed using a single or multiple focal planes (see Figure 3). A single focal plane can be used on features that are not larger than one digital mirror device (DMD, approximately 300 µm x 500 µm) and that are not too tall (50-100 µm). In that case, design a digital pattern using the TIFF-mode. For features that exceed the dimensions of one DMD and are relatively tall, divide the patterning of the substrate into multiple steps. In this case, multiple patterns are designed using the PDF-mode that all individually focus on single focal planes.
6. UV-photopatterning of 3D cell culture substrates
7. Protein incubation
NOTE: It is advised to use freshly protein-incubated substrates for cell culture. Only proceed to this part of the protocol if the cell seeding (step 8) is done directly afterwards.
8. Cell seeding
NOTE: This protocol uses human primary keratocytes and human dermal fibroblasts. The keratocytes were harvested from human corneal tissue from patients, in line with Dutch guidelines for secondary use of materials, and previously characterized as keratocytes47. These cells are cultured in DMEM supplemented with 5% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin (P/S), and 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (vitamin C) at 37 °C for a maximum of four passages. Human dermal fibroblasts were purchased and cultured in DMEM supplemented with 10% FBS and 1% P/S at 37 °C for a maximum of 15 passages. For seeding of both keratocytes and dermal fibroblasts on the photopatterned cell culture chip, 20,000 cells per chip were used.
9. Staining, image acquisition, and analysis
By means of the described protocol, 3D PDMS cell culture substrates can be UV-photopatterned to create precise and high-throughput adhesive areas suitable for cell attachment. In this way, cells are subjected to both relevant substrate geometries and adhesive ligand patterns simultaneously. Cell properties such as orientation, cell area, and number of focal adhesions can easily be monitored and used to better understand cell behavior in complex, in vivo-like environments.
To verify the patterning events on the 3D PDMS substrates, atomic surface compositions of the material in different stages of the protocol have been measured using atomic X-ray photoelectron spectroscopy (XPS)48. In summary, the XPS measurements showed the presence of PEG chains with an increased carbon signal on passivated samples, which was reduced after photopatterning. Incubation with fibronectin resulted in an increase of carbon signal, again indicating successful protein adhesion on the surface of the cell culture chip. Next, pattern resolution and alignment on 3D features were characterized on a variety of circle-patterned, concave pits (ĸ = 1/250 µm-1, ĸ = 1/1,000 µm-1, and ĸ = 1/3,750 µm-1, see Figure 6). From the maximum-intensity projections, it can be concluded that the protein pattern was successfully patterned on all three 3D features. The intensity profile in Figure 6A shows high pattern resolution with sharp transitions between patterned and non-patterned areas. Additionally, consistent protein intensity across the complete pattern in the pit was obtained.
The concave pit with ĸ = 1/250 µm-1 was patterned using the single focal plane method (one pattern), whereas the pits with ĸ = 1/1,000 µm-1 and ĸ = 1/3,750 µm-1 were patterned using two and three focal planes (patterns), respectively. As can be seen in the maximum-intensity projections in Figure 6, both methods result in perfect alignment of the patterns on top of the features. No misaligned transitions between the two different focal planes and patterns can be observed.
Using the described protocol, a wide range of protein pattern designs can be applied to a variety of geometries (see Figure 7 and Video 1). To illustrate the versatility of this method, semicylinders (convex and concave), a saddle surface, and pit were patterned using lines and circles of various widths. The photopatterned materials can subsequently be used for cell culture (see Figure 7, Figure 8, Video 2, Video 3, and Video 4). An example of dermal fibroblasts cultured on a patterned (fibronectin lines, red, 5 µm wide, and 5 µm gaps) concave semicylinder is shown in Figure 8, Figure 9, and Video 4. During the experiment, cells sense and adhere to the multicue cell culture substrate and remain viable over time. As can be seen from the immunofluorescent staining in Figure 8, cells form focal adhesions (vinculin clusters) mainly on the fibronectin lines.
Another example study making use of these cell culture materials was recently published by our group48. In this study, human myofibroblasts and endothelial cells were subjected to the combination of contact guidance cues and geometrical topographies. In vivo, both types of cells experience curvature- and contact-guidance cues in native tissues such as in the human vasculature. By subjecting the cells in vitro to an environment that combines both environmental cues, the in vivo situation can be recapitulated, providing a deeper understanding of the role of the microenvironment on cell behavior. Human myofibroblasts were shown to align with contact guidance cues (parallel fibronectin lines) on concave cylindrical substrates48. However, on convex structures with increasing curvatures, the geometrical cues overruled the biochemical cues, suggesting that myofibroblasts can sense both the degree and sign of curvature. Interestingly, endothelial cells could only adhere to the concave multicue substrates and not to the convex PDMS substrates. On concave, protein-patterned substrates, the endothelial cells are oriented in the direction of the contact guidance cue. This fundamental in vitro knowledge has physiological relevance in the field of vascular tissue engineering and can eventually aid in the design of smart tissue engineering constructs.
Figure 1: The experimental timeline of applying contact guidance cues on 3D cell culture substrates. First, positive cell culture chips are produced from a negative PDMS mold containing a range of geometries. Uncured PDMS is poured into the mold and cured for 3 h at 65 °C. Subsequently, the PDMS is treated with O2-plasma and incubated with PLL and mPEG-SVA (blue, labeled) to passivate the surface of the cell culture substrate. After washing, the substrate is flipped upside-down in a droplet of photoinitiator (PLPP, green, labeled) and UV-photopatterned using the LIMAP approach. Here, a digital mask with a user-defined pattern is used to cleave the passivation layer at defined locations. Next, a protein solution (red, labeled) can be incubated and will only adhere to the locations where the passivation layer is removed. Cells seeded on the substrate are subjected to both geometry and protein patterns, which enables research into cell behavior in complex, in vivo-mimicking environments. Abbreviations: PDMS = polydimethylsiloxane; mPEG-SVA = methoxypolyethylene glycol-succinimidyl valerate; PLPP = 4-benzoylbenzyl-trimethylammonium chloride; LIMAP = Light-Induced Molecular Adsorption of Proteins. Please click here to view a larger version of this figure.
Figure 2: Different stages during the production and passivation of the 3D cell culture substrate. The negative glass mold (#1) is designed with computer assisted design software and produced using a femtosecond-laser direct-write technique. This mold is utilized to produce the intermediate positive PDMS chip (#2) and negative PDMS mold (#3), which are subsequently used to produce the final cell culture chip (#4). Abbreviation: PDMS = polydimethylsiloxane. Please click here to view a larger version of this figure.
Figure 3: Schematic illustration of the two patterning methods. Left: UV-photopatterning is performed on smaller features (approximately one DMD) using a single focal plane and pattern. As a result, the complete feature is patterned in one go. Right: When larger features are used (larger than one DMD), the patterning is divided over multiple focal planes and patterns. Abbreviation: DMD = digital mirror device. Please click here to view a larger version of this figure.
Figure 4: A typical example of a normal and dried-out protein-incubated substrate. Maximum-intensity projections (XY) and orthogonal views (XZ) of normal and dried-out protein-incubated substrates. When washing a patterned cell culture substrate after incubation with a protein solution, it is crucial to always keep the sample wet. Although the pattern is identical in all images on the features (ĸ = 1/1,000 µm-1), the gelatin-fluorescein (green) aggregated forming a major clump when the sample was left to dry for a few seconds. If the sample always remains wet, correct protein patterns can be observed. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 5: Brightfield images after seeding. Primary keratocytes (left) and dermal fibroblasts (right) 4 h after seeding on 3D geometrical features (concave pit of ĸ = 1/1,000 µm-1 and semicylinders of ĸ = 1/500, 1/375, 1/250, 1/175, and 1/125 µm-1). The top-left inserts represent the line pattern used for the patterning of the geometries. White arrows indicate spreading cells that already show alignment. Scale bars = 250 µm. Please click here to view a larger version of this figure.
Figure 6: Characterization of circular patterns on concave pits. (A) The maximum-intensity projection (XY) and orthogonal view (XZ) of aconcave pit (ĸ = 1/250 µm-1) patterned using LIMAP (linewidth: 20 µm, gap width: 20 µm) and incubated with gelatin-fluorescein (green). The intensity profile along the white line is plotted against the distance, showing a consistent pattern quality and resolution. (B) Additional patterning performed on concave pits with ĸ = 1/1,000 µm-1 and ĸ = 1/3750 µm-1, showing flexibility in terms of geometrical features that can be used for patterning. Again, both the maximum intensity projections (XY) and orthogonal views (XZ) are visualized. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 7: 3D microscopy data of patterned structures. Typical examples of 3D patterned cell culture materials after photopatterning and cell culture, visualized using 3D rendering software. (A) Convex semicylinder patterned with 10 µm wide lines (rhodamine-fibronectin, red) and 10 µm wide gaps. Scale bar = 5 µm. (B) Dermal fibroblasts stained for F-actin (green) cultured on concave semicylinder patterned with 20 µm wide lines (rhodamine-fibronectin, red) and 20 µm wide gaps. Scale bar = 5 µm. (C) Saddle surface patterned with 20 µm wide lines (rhodamine-fibronectin, red) and 20 µm wide gaps. Scale bar = 5 µm. (D) Concave pit patterned with concentric circles of 20 µm wide lines (gelatin-fluorescein, green) and 20 µm wide gaps. The F-actin cytoskeleton of the human keratocytes is stained using phalloidin and visualized in red. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 8: Immunofluorescent staining of human dermal fibroblasts on a photopatterned, concave semicylinder. (A) Maximum-intensity projection (XY) and orthogonal sections (XZ and YZ) of human dermal fibroblasts cultured for 24 h on a patterned (fibronectin lines, red, 5 µm wide, and 5 µm gaps) concave semicylinder. Cells are stained for F-actin (magenta), vinculin (green), and nuclei (blue). Scale bar = 100 µm. (B) Zoom-in of a cell adhering to the multicue environment. Scale bars = 50 µm. Please click here to view a larger version of this figure.
Figure 9: Brightfield timelapse images of human dermal fibroblasts on a patterned, concave cylinder. The concave semicylinder (ĸ = 1/250 µm-1) was patterned with parallel lines (5 µm wide and 5 µm gaps) and incubated with rhodamine-fibronectin before cell seeding. The timelapse imaging is started 1 h after initial cell seeding (left, 0 min), when cells are still rounded and non-adherent (arrows). After approximately 24 h (middle, 1,420 min), cells adhered to the multicue substrate and show an alignment response according to the contact guidance pattern. Both the alignment response and cell viability are maintained throughout the entire culture duration (right, 3,180 min). Scale bars = 200 µm. Please click here to view a larger version of this figure.
Video 1: Pattern example on a 3D cylindrical substrate. 3D representation of a convex cylinder patterned with rhodamine-fibronectin (red). Please click here to download this Video.
Video 2: 3D representation of dermal fibroblasts cultured on a patterned, 3D cylindrical substrate (ĸ = 1/500 µm-1). Dermal fibroblasts cultured for 24 h on a patterned (fibronectin lines, red, 10 µm wide, and 10 µm gaps) convex semicylinder. Cells are stained for F-actin (magenta), vinculin (green), and nuclei (blue). Please click here to download this Video.
Video 3: 3D representation of human keratocytes cultured on a patterned, 3D pit (ĸ = 1/3,750 µm-1). 3D representation of human keratocytes cultured for 24 h in a concave, patterned pit (gelatin circles, green, 20 µm wide, and 20 µm gaps). Cells are stained for F-actin (red). Please click here to download this Video.
Video 4: Brightfield timelapse imaging of human dermal fibroblasts on a patterned, concave cylinder. The concave semicylinder (ĸ = 1/250 µm-1) was patterned with parallel lines (5 µm wide and 5 µm gaps) and incubated with rhodamine-fibronectin before cell seeding. The timelapse imaging is started 1 h after initial cell seeding, when cells show initial adherence to the multicue environment. During the complete timelapse, cells predominantly orient along the contact guidance cues, while cell viability is maintained. Please click here to download this Video.
Nowadays, cell behavior is often studied on flat culture substrates that lack the complexity of the native cell microenvironment. 3D environments such as scaffolds and hydrogels are used as an alternative. Although these cell culture environments improve the in vivo relevance, both systematic studies of cell behavior and the feasibility of readout methods remain challenging. To systematically investigate cell behavior on representative culture substrates, consistent multicue substrates that allow for microscopic readout are needed. Therefore, in this protocol, we describe a method to create multicue cell culture substrates with physiologically relevant geometries and patterned ECM proteins. The main challenge in combining environmental cues such as tissue geometry and contact-guidance cues on in vitro platforms is largely of a technological nature. Conventional methods to apply contact-guidance cues (e.g., soft lithography, deep-UV patterning, and microcontact printing35,36) to cell culture materials have been optimized for planar substrates. The need for 3D cell culture materials combined with contact guidance cues highlighted several technological challenges, such as poor pattern alignment, resolution, and flexibility. To overcome these challenges, a high-throughput, maskless, light-based patterning method can be used45,49. Here, an optical microscope enables precise pattern alignment and a resolution in the order of micrometers (see Figure 6). Further, the use of a digital mask enables researchers to study cell behavior on a wide range of patterns without the need to fabricate labor-intensive physical masks.
The UV-photopatterning approach can be used in combination with a variety of 3D geometries (e.g., cylinders, saddles, domes, pits) produced from a range of materials48. The 3D cell culture substrates used in this study are made from PDMS; however, other materials can be used as well. This might require different steps to produce the final cell culture substrate containing the features of interest. Since cells have been shown to be sensitive to surface roughness of cell culture materials, it is important to create the cell culture chips with a smooth surface so that the observed cells’ response can be fully attributable to the 3D geometry and contact-guidance cues50,51. Measurement methods such as optical profilometry, scanning electron microscopy, or atomic force microscopy can be used to measure surface roughness. After fabrication of the cell culture material, one can select a patterning method based on one or multiple focal planes dependent on the specific dimensions of the feature of interest (see Figure 3). Typically, a single focal plane is used to pattern an area within a Z-range of approximately 50 µm. The pattern resolution was shown to be consistent using this rule of thumb (see Figure 6). However, a downside of this method is the increased patterning time with the introduction of multiple focal planes and patterns. In our hand, using multiple focal planes, 3D geometrical features up to 16 mm x 16 mm x 0.17 mm (X x Y x Z) have been successfully patterned with high pattern quality.
Additionally, it is important to mention that the height (Z-axis) of geometrical features that can be used in combination with this protocol is limited. Since both the UV-photopatterning and many cellular readouts rely on a microscopy setup, the working distance of the objectives determines the maximum height of a feature. In our hand, geometries exceeding a height of 300 µm can still be UV-photopatterned, and readouts have been performed using a confocal microscope with 40x objectives. Thus, mechanobiological research ranging from intracellular to cellular and tissue scales is possible using the described protocol.
Another factor that needs to be taken into account is the risk of samples drying out during or after the UV-photopatterning49. This is especially relevant when using 3D geometries, as convexities are often exposed outside cell culture materials. As shown in Figure 4, this might result in uneven patterns with protein aggregates forming on top of the feature of interest. The washing of the cell culture chips following protein incubation and during cell culture is critical for the proper coating of 3D geometries. Therefore, it is advised to always leave small volumes of a working solution (PBS, PLPP, protein solution, cell culture medium) on top of the cell culture chip.
So far, several protein coatings (fibronectin, collagen type I and IV, gelatin, FNC) and cell types (human bone marrow stromal cells, human myofibroblasts, human endothelial cells, human keratocytes, and dermal fibroblasts) have been used in combination with the described photopatterning approach on structured cell culture materials. As shown in a previous study48, the optimization of protein incubation parameters is key for the systematic investigation in new cell types. Therefore, before performing a new experiment with new proteins or cells, it is advised to test a range of protein concentrations, incubation temperatures, and incubation times. By comparing cell morphology after initial adhesion on homogeneous, patterned flat areas with cell morphology under ‘normal’ cell culture conditions, an optimized set of experimental parameters can be obtained. Additionally, each cell type might require a different amount of time after seeding to show a recognizable adhesion morphology on a specific multicue environment (see Figure 5). To this end, it is crucial to optimize the time required per cell type to present contact events on the patterned area during the washing in step 8.4. For example, we observed that on line patterns, human keratocytes show elongated morphologies within the first 30 min after seeding, while endothelial cells and dermal fibroblasts require multiple hours before showing a change in adhesion morphology. The experimental parameters required for protein incubation (step 7) and cell seeding (step 8) might thus be dependent on the protein and cell type of choice.
The presented approach to apply contact guidance cues on 3D geometries can aid in creating a deeper understanding of cell behavior in complex multicue environments. This can include investigations into intracellular components, such as focal adhesions and nuclei, and can also involve experiments performed on a larger, cell, or tissue scale using the proposed method. Eventually, it is anticipated that the knowledge gained can be used in the design of tissue engineering applications, where complex cellular environments are designed to steer cell behavior towards a desired outcome.
The authors have nothing to disclose.
We thank Dr. Nello Formisano (MERLN Institute for Technology-Inspired Regenerative Medicine) for providing human primary keratocytes. This work has been supported by the Chemelot InSciTe (project BM3.02); the European Research Council (grant 851960); and the Ministry of Education, Culture and Science for the Gravitation Program 024.003.013 "Materials Driven Regeneration". The authors would like to thank Alvéole for their correspondence, help, and troubleshooting.
Anti-vinculin antibody, mouse monoclonal IgG1 | Sigma | V9131 | Dilution: 1/600 |
Bovine Serum albumin, Fraction V | Roche | 10735086001 | |
DMEM, high glucose, pyruvate | Gibco | 41966029 | |
DMEM/F-12 + GlutaMAX (1x) | Gibco | 10565018 | |
DMi8 epifluorescent microscope | Leica Microsystems | ||
Ethanol | Biosolve | 0005250210BS | |
Fetal Bovine Serum | Serana | 758093 | |
Fiji/ImageJ, version v1.53k | www.imageJ.nih.gov | ||
Fluorescent highlighter | Stabilo | 4006381333627 | |
Fluorescin-labeled gelatin | Invitrogen | G13187 | Concentration: 0.01% |
Formaldehyde solution | Merck | F8775 | |
Glass coverslips 24 x 60 mm, #1 | VWR | 631-1575 | |
Glass coverslips, ø = 32 mm, #1 | Menzel-Gläser | ||
HCX PL fluotar L 20X/0.40na microscope objective | Leica | 11506242 | |
HEPES | Gibco | 15630080 | |
Human dermal fibroblasts | Lonza | CC-2511 | |
Human primary keratocytes | MERLN Institute for Technology-Inspired Regenerative Medicine | ||
Illustrator, Version 26.0.1 | Adobe | ||
Laboratory oven | Carbolite | ||
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | Sigma-Aldrich | A8960 | |
Leica Application Suite X software, version 3.5.7.23225 | Leica Microsystems | ||
Leonardo software, version 4.16 | Alvéole | ||
Micro-manager, version 1.4.23 | Open imaging | ||
Mowiol 4-88 | Sigma-Aldrich | 81381 | mounting medium |
mPEG-succinimidyl valerate MW 5,000 Da | Laysan Bio | MPEG-SVA-5000 | Concentration: 50 mg/mL |
Negative glass mold | FEMTOprint | ||
NucBlue Live Readyprobes Reagent (Hoechst 33342) | Invitrogen | R37605 | 2 drops/mL |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140163 | |
Petri dish (ø=100 mm) | Greiner Bio-one | 664160 | |
Phalloidin Atto 647N | Sigma | 65906 | Dilution: 1/250 |
Phosphate Buffered Saline | Sigma | P4417 | |
Plasma asher | Emitech | K1050X | |
PLPP (photoinitiator) | Alvéole | ||
Poly-L-lysine, sterile-filtered | Sigma-Aldrich | P4707 | Concentration: 0.01% |
PRIMO | Alvéole | ||
Rhodamine-labeled fibronectin | Cytoskeletn, Inc. | FNR01 | Concentration: 10 µg/mL |
Secondary antibody with Alexa 488, Goat anti-mouse IgG1 (H) | Molecular Probes | A21121 | Dilution: 1/300 |
Secondary antibody with Alexa 555, Goat anti-mouse IgG1 (H) | Molecular Probes | A21127 | Dilution: 1/300 |
Spin coater | Leurell Technologies Corporation | model WS-650MZ-23NPPB | |
SYLGARD 184 Silicone Elastomer Kit | DOW | 1673921 | |
TCS SP8X confocal microscope | Leica Microsystems | ||
tridecafluoro(1,1,2,2-tetrahydrooctyl)trichlorosilane | ABCR | AB111444 | |
TrypLE Express Enzyme (1x), no phenol red | Gibco | 12604013 | |
Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 |