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 &#34;Materials Driven Regeneration&#34;. The authors would like to thank Alv&#233;ole for their correspondence, help, and troubleshooting.

The authors have no conflicts of interest to disclose.

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 &#181;m. 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 &#181;m 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.

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 &#181;m), while cells have been reported to respond to geometrical protein patterns as small as 0.1 &#181;m2,42. Sevcik&#160;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 &#181;m 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 &#312; = 1/2500 to &#312; = 1/125 &#181;m-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 &#181;m44. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-vinculin antibody, mouse monoclonal IgG1</td>
			<td>Sigma</td>
			<td>V9131</td>
			<td>Dilution: 1/600</td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, Fraction V</td>
			<td>Roche</td>
			<td>10735086001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 + GlutaMAX (1x)</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMi8 epifluorescent microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Biosolve</td>
			<td>0005250210BS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Serana</td>
			<td>758093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji/ImageJ, version v1.53k</td>
			<td>www.imageJ.nih.gov</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent highlighter</td>
			<td>Stabilo</td>
			<td>4006381333627</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescin-labeled gelatin</td>
			<td>Invitrogen</td>
			<td>G13187</td>
			<td>Concentration: 0.01%</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Merck</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 24 x 60 mm, #1</td>
			<td>VWR</td>
			<td>631-1575</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips, &#248; = 32 mm, #1</td>
			<td>Menzel-Gl&#228;ser</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCX PL fluotar L 20X/0.40na microscope objective</td>
			<td>Leica</td>
			<td>11506242</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human dermal fibroblasts</td>
			<td>Lonza</td>
			<td>CC-2511</td>
			<td></td>
		</tr>
		<tr>
			<td>Human primary keratocytes</td>
			<td>MERLN Institute for Technology-Inspired Regenerative Medicine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illustrator, Version 26.0.1</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory oven</td>
			<td>Carbolite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X software, version 3.5.7.23225</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leonardo software, version 4.16</td>
			<td>Alv&#233;ole</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-manager, version 1.4.23</td>
			<td>Open imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>mPEG-succinimidyl valerate MW 5,000 Da</td>
			<td>Laysan Bio</td>
			<td>MPEG-SVA-5000</td>
			<td>Concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>Negative glass mold</td>
			<td>FEMTOprint</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Live Readyprobes Reagent (Hoechst 33342)</td>
			<td>Invitrogen</td>
			<td>R37605</td>
			<td>2 drops/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (&#248;=100 mm)</td>
			<td>Greiner Bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin Atto 647N</td>
			<td>Sigma</td>
			<td>65906</td>
			<td>Dilution: 1/250</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma asher</td>
			<td>Emitech</td>
			<td>K1050X</td>
			<td></td>
		</tr>
		<tr>
			<td>PLPP (photoinitiator)</td>
			<td>Alv&#233;ole</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
			<td>Concentration: 0.01%</td>
		</tr>
		<tr>
			<td>PRIMO</td>
			<td>Alv&#233;ole</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine-labeled fibronectin</td>
			<td>Cytoskeletn, Inc.</td>
			<td>FNR01</td>
			<td>Concentration: 10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Secondary antibody with Alexa 488, Goat anti-mouse IgG1 (H)</td>
			<td>Molecular Probes</td>
			<td>A21121</td>
			<td>Dilution: 1/300</td>
		</tr>
		<tr>
			<td>Secondary antibody with Alexa 555, Goat anti-mouse IgG1 (H)</td>
			<td>Molecular Probes</td>
			<td>A21127</td>
			<td>Dilution: 1/300</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Leurell Technologies Corporation</td>
			<td>model WS-650MZ-23NPPB</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>DOW</td>
			<td>1673921</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP8X confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tridecafluoro(1,1,2,2-tetrahydrooctyl)trichlorosilane</td>
			<td>ABCR</td>
			<td>AB111444</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x), no phenol red</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of multicue cellular microenvironments by uv-photopatterning of three-dimensional cell culture substrates

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.

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 (&#312; = 1/250 &#181;m-1, &#312; = 1/1,000 &#181;m-1, and &#312; = 1/3,750 &#181;m-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 &#312; = 1/250 &#181;m-1 was patterned using the single focal plane method (one pattern), whereas the pits with &#312; = 1/1,000 &#181;m-1 and &#312; = 1/3,750 &#181;m-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 &#181;m wide, and 5 &#181;m 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63988/63988fig01.jpg" /> 
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 &#176;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. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63988/63988fig02.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63988/63988fig03.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63988/63988fig04.jpg" /> 
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 (&#312; = 1/1,000 &#181;m-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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63988/63988fig05.jpg" /> 
Figure 5: Brightfield images after seeding. Primary keratocytes (left) and dermal fibroblasts (right) 4 h after seeding on 3D geometrical features (concave pit of &#312; = 1/1,000 &#181;m-1 and semicylinders of &#312; = 1/500, 1/375, 1/250, 1/175, and 1/125 &#181;m-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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63988/63988fig06.jpg" /> 
Figure 6: Characterization of circular patterns on concave pits. (A) The maximum-intensity projection (XY) and orthogonal view (XZ) of aconcave pit (&#312; = 1/250 &#181;m-1) patterned using LIMAP (linewidth: 20 &#181;m, gap width: 20 &#181;m) 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 &#312; = 1/1,000 &#181;m-1 and &#312; = 1/3750 &#181;m-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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63988/63988fig07.jpg" /> 
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 &#181;m wide lines (rhodamine-fibronectin, red) and 10 &#181;m wide gaps. Scale bar = 5 &#181;m. (B) Dermal fibroblasts stained for F-actin (green) cultured on concave semicylinder patterned with 20 &#181;m wide lines (rhodamine-fibronectin, red) and 20 &#181;m wide gaps. Scale bar = 5 &#181;m. (C) Saddle surface patterned with 20 &#181;m wide lines (rhodamine-fibronectin, red) and 20 &#181;m wide gaps. Scale bar = 5 &#181;m. (D) Concave pit patterned with concentric circles of 20 &#181;m wide lines (gelatin-fluorescein, green) and 20 &#181;m wide gaps. The F-actin cytoskeleton of the human keratocytes is stained using phalloidin and visualized in red. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63988/63988fig08.jpg" /> 
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 &#181;m wide, and 5 &#181;m gaps) concave semicylinder. Cells are stained for F-actin (magenta), vinculin (green), and nuclei (blue). Scale bar = 100 &#181;m. (B) Zoom-in of a cell adhering to the multicue environment. Scale bars = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63988/63988fig09.jpg" /> 
Figure 9: Brightfield timelapse images of human dermal fibroblasts on a patterned, concave cylinder. The concave semicylinder (&#312; = 1/250 &#181;m-1) was patterned with parallel lines (5 &#181;m wide and 5 &#181;m 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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63988/63988fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Pattern example on a 3D cylindrical substrate. 3D representation of a convex cylinder patterned with rhodamine-fibronectin (red). <a href="https://www.jove.com/files/ftp_upload/63988/Video 1.zip" target="_blank">Please click here to download this Video.</a>
Video 2: 3D representation of dermal fibroblasts cultured on a patterned, 3D cylindrical substrate (&#312; = 1/500 &#181;m-1). Dermal fibroblasts cultured for 24 h on a patterned (fibronectin lines, red, 10 &#181;m wide, and 10 &#181;m gaps) convex semicylinder. Cells are stained for F-actin (magenta), vinculin (green), and nuclei (blue). <a href="https://www.jove.com/files/ftp_upload/63988/Video 2.zip" target="_blank">Please click here to download this Video.</a>
Video 3: 3D representation of human keratocytes cultured on a patterned, 3D pit (&#312; = 1/3,750 &#181;m-1). 3D representation of human keratocytes cultured for 24 h in a concave, patterned pit (gelatin circles, green, 20 &#181;m wide, and 20 &#181;m gaps). Cells are stained for F-actin (red). <a href="https://www.jove.com/files/ftp_upload/63988/Video 3.zip" target="_blank">Please click here to download this Video.</a>
Video 4: Brightfield timelapse imaging of human dermal fibroblasts on a patterned, concave cylinder. The concave semicylinder (&#312; = 1/250 &#181;m-1) was patterned with parallel lines (5 &#181;m wide and 5 &#181;m 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. <a href="https://www.jove.com/files/ftp_upload/63988/Video 4.zip" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates at JoVE.com

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

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 ...

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Bioengineering

2.3K Views.  Eindhoven University of Technology. 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.

Video: Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...