Research was supported by seed funds from The Agricultural Research Organization to MK.

1. Growing the plants and root preparation
<ol>
	<li>Option 1: Prepare adventitious roots from stem.
	<ol>
		<li>Take a rooting tray for growing plants.</li>
		<li>Fill the tray with soil.</li>
		<li>Add one seed of M82 tomato cultivar to each cell in the tray.</li>
		<li>Cover the seeds with a little soil.</li>
		<li>Water the tray from the bottom with a dropper as the water fills the bottom of the tray and the soil absorbs water.</li>
		<li>Add 2 mL of fertilizer per 1 L of water to the bottom of the tray once a week.</li>
		<li>Grow in a growing chamber at 25 &#176;C.</li>
		<li>Use lighting conditions of 9 h light (7:00-16:00) alternated with 15 h of darkness.</li>
		<li>After 3 weeks remove the plant from the soil.</li>
		<li>Cut the root system from the plant at the point of interaction with the stem.</li>
		<li>Put the rootless plant in a beaker filled with water.</li>
		<li>After a few days, cut the adventitious roots that emerge from the stem and use them for replication.</li>
	</ol>
	</li>
	<li>Option 2: Prepare seed germinating roots.
	<ol>
		<li>Wet a Petri dish sized filter paper with water.</li>
		<li>Put several M82 seeds (no more than 10) on the paper, inside a Petri dish.</li>
		<li>Incubate the plate at 25 &#176;C.</li>
		<li>Hydrate the paper every day.</li>
		<li>After germinated roots are long enough (approximately 5 days), remove the seeds and use the roots for replication.</li>
	</ol>
	</li>
</ol>
2. Preparation of the root negative replica from polyurethane
<ol>
	<li>To generate negative replica solution, add 9.49 g of diurethane dimethacrylate to a 20 mL vial.
	<ol>
		<li>Add 1.45 mL of ethyl methacrylate to the vial.</li>
		<li>Stir at room temperature (RT) until the solution looks clear and becomes homogeneous. 
		NOTE: Approximately 2 h is sufficient to reach a homogeneous solution.</li>
		<li>Add 3 mL of the plasticizer, diethyl phthalate, and stir for 1 h at RT. 
		NOTE: Diethyl phthalate is miscible in acrylate monomer.</li>
		<li>Add 300 &#181;L of the photo initiator, 2-hydroxy-2-methylpropiophenone, and stir overnight at RT. Continue stirring until all bubbles are removed. 
		NOTE: The protocol can be paused here. The solution can be kept at RT.</li>
	</ol>
	</li>
	<li>To generate the negative replica of the root, take a clean glass slide and pour 1 mL of the negative replica solution on it.
	<ol>
		<li>Place 2&#8210;3 roots over the solution. Do not allow the roots to be fully covered by the solution.</li>
		<li>Keep the slide under 8 W ultra violet (UV) lamp for 8&#8210;10 min. Do not keep the solution under UV light for too long. 
		NOTE: It is important not to keep the solution under the UV light for too long as it makes the polyurethane too hard, making it impossible to remove the root.</li>
		<li>Switch off the UV lamp, remove the replica from the glass slide and put it in a Petri dish filled with ethanol, to remove unreacted monomer.</li>
		<li>To obtain the negative replica, remove the root from the replica very slowly using forceps.</li>
	</ol>
	</li>
</ol>
3. Prepare the root positive replica from PDMS.
<ol>
	<li>To generate the mixture for the positive replica, place 10 g of dimethyl siloxane in a paper cup.
	<ol>
		<li>Add 1 g of curing agent and mix thoroughly.</li>
		<li>Keep the mixture in a desiccator under vacuum for 2 h to remove air bubbles.</li>
	</ol>
	</li>
	<li>To generate the positive replica, place the polyurethane negative replica in a Petri dish.
	<ol>
		<li>Pour the PDMS mixture on top of the negative replica.</li>
		<li>Apply vacuum for 2 h to assure coverage of the microstructure.</li>
		<li>Keep the Petri dish overnight at RT.</li>
		<li>Separate the cured positive replica from the negative replica by hand.</li>
	</ol>
	</li>
</ol>
4. Prepare the root positive replica from ethyl cellulose.
<ol>
	<li>To generate the ethyl cellulose solution,&#160;put 1.32 ml of diethyl pthalate as a plasticizer in a 100 ml cup.
	<ol>
		<li>Add 20 mL of ethanol and stir at RT for 2 h.</li>
		<li>Add 3.3 g&#160;of ethyl cellulose and stir overnight.</li>
	</ol>
	</li>
	<li>To generate the positive replica, place the polyurethane negative replica in a Petri dish.
	<ol>
		<li>Pour the ethyl cellulose solution on top of the negative replica.</li>
		<li>Keep the Petri dish overnight at RT under the hood.</li>
		<li>Remove the positive replica from the negative replica very slowly by forceps.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We present a novel method for the replication of root surface microstructure. This method relies on existing methods of leaf surface microstructure replication4. In order to develop this method, we had to tweak the existing method for leaves. We realized that the problematic step in copying the leaf replication method into roots involves the first step of the root molding. This is the most sensitive part of the method as it involves the biological tissue. As a result, we wanted to choose a polymer that would demand relatively gentle conditions for curing and hence causing minimal damage to the biological tissue. We chose polyurethane because it can be polymerized quickly (within 10 min) under UV light29. Additionally, it is very hard once polymerized30 and we hoped that this property would allow for the relatively easy removal of the root from the polyurethane mold.
The presented method is a two-step approach in which the negative image (negative replica) is formed in the first step and the replication is formed in the second step, based on the negative replica. This extends the range of materials we can work with. Leaf surface microstructure replication was mainly performed on PDMS or epoxy materials11,31. Some work was done with other materials, specifically materials supporting microorganism growth13,32. This is because in recent years this method has been used to study microorganism-surface interactions in the context of leaf surface structure. However, no cellulose-like materials have been used in this method in the context of leaves. We suggest the use of a polyurethane negative replica as a mold and a variety of materials for the positive replica. In other words, making the positive replica, from a variety of materials, is relatively easy once a good negative replica is made. We currently use cellulose derivatives, but are exploring the possibilities of using more relevant materials to root surface such as pectin and lignin33,34 in combination with cellulose derivatives.
The method also expands upon the existing method of leaf surface microstructure replication since the leaf is a 2D surface while the root surface is curved and hence is a 3D surface. Our method does not enable the replication of the whole surface since embedding the whole root in the polyurethane solution does not allow for its release. Therefore, one side of the root has to be chosen when replicating the root surface microstructure. The generated synthetic surface is curved and represents roughly half the surface, but not all of it. Our assumption is that the structural features of the root surface are mostly symmetrical about the axis along the root length. However, in studies where such symmetry is not assumed, one should be careful to choose the appropriate side root to replicate.
We present two options for roots to be used as molds. The first is the option of adventitious roots grown from the stem and the second is the option of germinated roots on paper. The first option is mostly meant to assist researchers in practicing the method as these roots are more robust and easier to work with. The second option represents the genetic differences that can be found between roots of different cultivars, regardless of the environmental conditions. These surfaces can be used as important research tools, however, one should be aware that the environment can have a strong influence on the root surface structure, specifically the soil in which the roots are grown35,36. Due to the mechanical stress inflicted by the soil, some morphological changes are bound to happen, in addition to wounds accruing on the surface as the root penetrates the soil37. Removal of roots from soil, as well as cleaning them, without damaging their structure is a very difficult task. Hence, we are not optimistic as to the ability to use this method to reliably mimic the root surface microstructure of roots grown in soil. However, for research that focuses on genetic differences or environmental differences where the change in microstructure is noticeably clear, this method can be used as a tool to study the influence of root surface microstructure.
Our method produces an inert surface mimicking of only the microstructural properties of the root surface. While this method is designed to separate the structural effects in root-environment interactions from all other effects, we cannot ignore the chemical compounds in those interactions. Some microorganisms may not survive or function on the surface without the addition of compounds, specifically nutrients. The next step in the development of this platform will be the controlled addition of chemical compounds to study their effects on the different interactions when combined with structure.
This method was developed as a first step in the development of a synthetic platform to study root-microorganism interactions. Here we mimic the microstructure of the root surface and this initial platform can be used to study the influence of surface microstructure on microorganism behavior. However, this platform is limited since it lacks many other elements from the natural system. This platform should be further developed with the use of the right materials to generate the surface and with the addition of other, critical, chemicals into the system. In a more advanced platform, we can also imagine spatial distribution of the chemicals. However, since currently no other method exists to isolate structural effects in root-microorganism interactions, we hope researchers could use this initial platform to ask structure-specific questions in those interactions.

Replication of leaf surface microstructure is a known method in the biomimetics research field1,2,3,4. The earliest replications of the leaf surface microstructure were performed using nail polish and rubber materials applied on the leaf surface for better visualization of microstructure, specifically stomata5,6,7,8,9,10. The method was then improved, and advanced polymers were used to mimic leaf surface microstructure using soft lithography, especially in the context of biomimetics of super hydrophobic surfaces2,3,4,11,12. In recent years, this method was proven as a useful tool in the study of the interaction between the leaf surface and microorganisms residing on the surface whether they are pathogenic13,14 or beneficial, as part of the natural leaf phyllosphere15. Simplification of the natural system was proven extremely useful in the study of surface-microorganism interactions even when purely synthetic systems were used as surfaces15,16,17,18.
While replication of leaf surface microstructure was shown to be a useful tool for studying the interaction occurring on the surface of the leaf with different microorganisms, no such tool exists for plant roots. Plant roots are harder to study since they reside below the ground and all interactions occur within the soil. Similar to leaves, root surface microstructure is likely to play a role in root-microorganism interactions. However, currently no method exists to isolate the specific role of root surface microstructure in the complex root-microorganism interactions. The most studied root surface microstructural feature is the root hairs19,20,21. Root hairs have an important role in increasing the surface area and by that allowing more efficient intake of nutrients and water22, however their involvement as a structural feature in root-microorganism interactions has never been tested.
The most widely used polymer for soft lithography in leaves is polydimethyl siloxane (PDMS). PDMS properties resemble those of the leaf cuticle15,23. However, in plant roots, the most abundant material is cellulose24,25 which has different properties than those of PDMS26,27,28. Using PDMS to build a synthetic platform for studying the surface microstructure effects in root-environment interactions is, hence, less than ideal.
The protocol presented here enables the formation of synthetic root surface microstructure replica from various materials. Like the method for leaf surface microstructure replication this is a two-step process. The first step uses the biological tissue (root) as a source for molding into a polyurethane mold (a negative replica). The polyurethane mold, which represents the negative image of the root surface microstructure, can then be used as a base to generate the positive replication of the root surface microstructure from a variety of materials, including PDMS and cellulose derivatives. This root surface replication can later be used as a platform to understand the surface structure role in root-microorganism interactions.

<table><tbody><tr><td>2-hydroxy-2-methylpropiophenone</td><td>Sigma</td><td>405655</td><td></td></tr><tr><td>Diethyl phthalate</td><td>Across</td><td>114520010</td><td></td></tr><tr><td>Diurethane dimetharylate</td><td>Sigma</td><td>436909</td><td></td></tr><tr><td>Ethyl cellulose</td><td>Across</td><td>232705000</td><td></td></tr><tr><td>Ethyl methacrylate</td><td>Sigma</td><td>234893</td><td></td></tr><tr><td>Shaphir Solution</td><td>GAT fertilizer</td><td>6-2-4</td><td></td></tr><tr><td>Sylgard 184 kit</td><td>Polymer-G</td><td>510018400500</td><td></td></tr></tbody></table>

biomimetic replication of root surface microstructure using alteration of soft lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

To form the root surface microstructure replication, a root must be chosen for molding. We grow tomato plants in soil, making the use of the natural root from the root system extremely challenging. Removal of soil from the root system can be difficult and additionally, the root system roots are fragile and can break upon molding attempts. We therefore suggest to first use more rigid roots, to establish the protocol in the lab. The formation of such roots is described in Figure 1A. The plant root system is removed after the plant was grown for 3 weeks and the rootless plant is placed in water for about a week until adventitious roots emerge from the stem. Those roots can be used for replication during the protocol establishment. Once the protocol has been well established, a more realistic root surface structure is desired. Here we suggest avoiding roots grown in soil as the full removal of soil in extremely challenging. Instead we suggest the use of germinating roots, supplying valuable information on the root surface microstructure of a genetically specific plant. The growth of such roots is described in Figure 1B. The seeds are placed on a wet filter paper and incubated at 25 &#176;C. After approximately 5 days, during which the filter paper is kept moist, the germinated roots are long enough for replication. Those roots are more fragile than the previously suggested roots and require more delicate care.
The production of the root surface microstructure replica is a two-step process. In the first step the natural root is being molded into a polyurethane based mold (the negative replica). The advantage of this step is that all materials for the polyurethane mold are being prepared and the root is placed on top of the prepared solution at the very end for a 10 min exposure to UV. As a result, the biological tissue is not exposed to harsh conditions for too long and can be gently handled at the end of the process. If all protocol steps are followed, a good negative replica is generated. This replica will show the cell structure of the root surface as well as holes representing the location of the root hairs (Figure 2A). If some critical steps in the protocol are not being followed, the procedure will fail. One such step is the placement of the root on the polyurethane solution prior to curing. The root must be placed very gently to avoid the submergence of it in the polyurethane solution. Such submergence, of any part of the root, will cause the entrapment of the root in the hard polymer with no ability to remove it. If such an event occurs, the root will remain within the negative replica after it is cured (Figure 2B). Another crucial step is regarding the curing time by UV light. The recommended curing time is 8&#8210;10 min. Going past 10 min will result in an extremely hard polyurethane mold, making it impossible to remove the root without breaking it within the polyurethane mold. The breakage of the root can sometimes be visible to the naked eye, e.g., when a large piece is broken (Figure 2C, top, marked with purple arrows). However, sometimes small root pieces are left in the material which are difficult to spot by the naked eye and a microscope has to be used (Figure 2C, bottom, marked with purple arrows). We recommend carefully examining the polyurethane negative replica with a microscope prior to the continuation of the protocol to make sure no residual root is present.
Once the polyurethane negative replica is prepared; many materials can be used for the preparation of the positive replica. The preparation of the positive replica, using the polyurethane negative replica as a mold, is straight forward and depends completely on the quality of the polyurethane negative replica. To generate the positive replica we have used both PDMS&#8212;as it is well known in the field of soft lithography (Figure 3A)&#8212;and ethyl cellulose as a material that better mimics the properties of the root surface which is mostly composed of cellulose (Figure 3B). The SEM image of the PDMS replica shows the root hairs very clearly. The hairs are in the elongation zone, where they begin to emerge. Hence, the length of root hairs varies along the root surface as they become longer, much like in the natural root (Figure 3A). Ethyl cellulose generates harder and less flexible film than PDMS. Hence, the removal of it from the negative mold requires more care. However, some hairs and the surface microstructure are visible under the light microscope (Figure 3B). We used those two materials to generate the positive replica, however, any material that can form a film will be a good candidate for the positive replica, using the polyurethane negative replica.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61437/61437fig01.jpg" /> 
Figure 1: Tomato plant roots for replication.&#160;(A) A tomato (M82) plant is grown at 25 &#176;C with 9 h of light and 15 h darkness. After 3 weeks, the plant is removed from the soil and the root system is cut off. The rootless plant is put in water until adventitious roots emerge from the stem after about a week. These roots do not show the exact structure as the roots from the root system, but they represent a good model. Those roots are less fragile than the root system roots and hence are preferred to work with when establishing the technique in the lab. (B) Tomato (M82) seeds are put on a wet filter paper in a Petri dish and incubated at 25 &#176;C. The paper is hydrated every day and the seeds are germinating. The roots are growing and after approximately 5 days are long enough to be used for replication. These roots are gentler and should be used once the method is well established. <a href="https://www.jove.com/files/ftp_upload/61437/61437fig01large.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/61437/61437fig2v2.jpg" /> 
Figure 2: Microscopy images of polyurethane negative replica.&#160;(A) SEM image of polyurethane negative replica made according to a protocol following all the steps. Cell structure is clearly visible. Yellow arrows point at holes formed by the hairs in the root. (B) Light microscopy images of polyurethane negative replica with a root inside of it as it was fully covered with the solution and the removal of it was impossible. The polyurethane negative was cured with the root inside. The root is visible by eye and using light microscopy. It is impossible to remove this root from the cured replica. (C) Light microscopy images of polyurethane negative replica that was kept under UV light for too long. As a result, root could not be fully removed from the polymer with either large particles visible by eye (top image, marked with purple arrows) or small fractions visible only by microscope (lower image, marked with purple arrows). <a href="https://www.jove.com/files/ftp_upload/61437/61437fig2v2large.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/61437/61437fig03.jpg" /> 
Figure 3: Microscope images of positive replica.&#160;(A) SEM micrograph of a positive replica made from PDMS. Enlargement shows root hairs. (B) Light microscopy images of a positive replica made from ethyl cellulose. Hairs are shown in the images on the right while surface texture is visible in the image on the left. <a href="https://www.jove.com/files/ftp_upload/61437/61437fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography at JoVE.com

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

biomimetic-replication-root-surface-microstructure-using-alteration

Research

JoVE Journal

Bioengineering

5.6K Views.  Agricultural Research Organization (Volcani Center). Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Video: Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Developmental Biology

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

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.

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 ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

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Rozdziały w tym wideo

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Microfluidic Model to Mimic Initial Event of Neovascularization

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

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

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices