This paper reports work undertaken in the context of the project &#34;Advanced surface treatment for implantable medical devices&#34; funded by Innovation Fund Denmark. The author gratefully acknowledge the support from European Innovative Training Network MICROMAN &#34;Process Fingerprint for Zero-defect Net-shape MICROMANufacturing&#34; funded by the Horizon 2020 Framework Programme for Research and Innovation of the European Union.

1. Insert Fabrication by Additive Manufacturing (AM)
<ol>
	<li>Design the needed cavities by computer-aided design software (see Supplemental Materials for files used in this work). 
	NOTE: Manufacture the inserts directly on the building stage without support to avoid warping the inserts during curing. The tolerance is 0.05 mm.</li>
	<li>Choose a resin for photo polymerization, in particular one that can withstand pressure and heat during injection molding. 
	NOTE: The resin we chose (see Table of Materials) has a tensile strength of 56 MPa, elongation at break of 3.5%, a flexural strength of 115 MPa, a flexural modulus of 3350 MPa, and a heat deflection temperature of 140 &#176;C18.</li>
	<li>Mix the compounded photopolymer blend (proprietary methyl methacrylate and acrylamide blend, with a TiO2 based photo initiator) thoroughly for a minimum of 30 minutes by running the material canister on a wet-laboratory bottle roller, flask shaker or similar mixing device.</li>
	<li>Prepare the photo polymerization machine (voxel-size of 16 &#181;m):
	<ol>
		<li>For bottom-up AM machines, check that the vat coating is undamaged and thoroughly cleaned.</li>
		<li>For top-down machines, inspect the wiping system for any defects.</li>
		<li>Inspect the build stage and adjust the build plate using a 100 &#181;m gauge foil to correctly offset the build plate to the vat.</li>
		<li>Pre-ignite the light source of the machine tool if possible to reach steady-state temperature before progressing. This temperature should be 65 &#176;C in the light engine and 30 &#176;C inside the vat.</li>
	</ol>
	</li>
	<li>Import the geometry to the job preparation software. Follow the guidelines from the machine manufacturer19.
	<ol>
		<li>Assign appropriate machine parameters to match the chosen photopolymer (light intensity and layer thickness for the utilized machine).</li>
		<li>Run the build at the highest vertical machine resolution (25 &#181;m in this research).</li>
	</ol>
	</li>
	<li>Clean the printed inserts.
	<ol>
		<li>Rinse them in isopropanol exposed to ultrasonic bath for 3 min. Rinse thoroughly three times, and ensure that no photopolymer residue is left on the surface of the printed inserts.</li>
		<li>Allow the solvent to evaporate completely at room temperature and set the inserts in a desiccator to dry overnight at room temperature.</li>
	</ol>
	</li>
	<li>Post cure the dried inserts using UV light flashes twice, each time by 2000 flashes with a frequency of 10 flashes per second (see Table of Materials).</li>
</ol>
2. Injection Molding (IM)
<ol>
	<li>Install the micro injection module on a conventional injection molding machine (see Table of Materials). 
	NOTE: In our case, the injection module had a screw mounted at an angle of 45&#176;, the screw diameter was 8 mm, and the clamping force was 100 kN.</li>
	<li>Mount the inserts on mold plates.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57335/57335fig1.jpg" /> 
Figure 1: Injection machine with molds. (a) The injection side is assembled with the plates. The insert is highlighted by a red circle. (b) The movable side is assembled with the plates with ejection pin. The insert is highlighted by a red circle. <a href="https://www.jove.com/files/ftp_upload/57335/57335fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>To account for variations during the printing process, manually make any slight modifications to the inserts necessary in order to fit the mold.</li>
	<li>Screw the mold plates on the machine.</li>
	<li>Load PE granules onto the hopper.</li>
	<li>Set the machine parameter TMold at 60 &#176;C, TBarrel at 175 &#176;C, and Vinjection at 65 mm/s. Allow sufficient cooling time for the part to be demolded.</li>
	<li>Heat the screw at 31 &#176;C, 155 &#176;C, 165 &#176;C, 170 &#176;C, and 175 &#176;C from the 1st to the 5th session. When the melt temperature is 175 &#176;C, start accurate injection molding. Once the cavity is filled, maintain the packing pressure of 300 bar for 5 seconds to compensate for PE shrinkage.</li>
	<li>Open the mold and allow the ejection pin to push the PE part out of the moveable side of insert. If the PE part is clamped on the insert, take the part out by hand.</li>
	<li>Stop the machine at 100 cycles of injection molding (a cycle is defined from when the mold is closed until the part is ejected).</li>
</ol>
3. Quality Assessment
<ol>
	<li>Measure the diameter and depth of the holes on the inserts before injection molding by a laser scanning digital microscope20 . Measure on both inserts; on each insert, measure four holes on two areas with different distance to the gate &#34;near the gate&#34; and &#34;far from the gate&#34;.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57335/57335fig2.jpg" /> 
Figure 2: Measured areas highlighted on the CAD image of the inserts. (a) The movable side insert correspond to the backsides of the replica; (b) the injection side corresponds to the top sides of the replica. The injection gate locates at the center. Two areas with difference distance to the gate, &#34;Far from the gate&#34; and &#34;Near the gate&#34; are compared in the research. (This figure has been modified from Zhang et al. 201722) <a href="https://www.jove.com/files/ftp_upload/57335/57335fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Measure diameter and the height of the pillars on the tracked tines obtained from injection molding. 
	NOTE: Group the obtained PE parts into batches of 10 pieces; i.e., 1-10 is the 1st batch, 11-20 is the 2nd batch; until the last piece. Use one random sample in each batch and do the measurements on the top and back sides; on each side, measure four pillars on two areas with different distance to the gate (&#34;near the gate&#34; and &#34;far from the gate&#34;).</li>
	<li>Analyze data with the assistance of image processing software21 in order to obtain the dimensions.</li>
</ol>

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Software Manual, 2010 Available from: <a target="_blank" href="https://digfablab.wikispaces.com/file/view/Getting+started.pdf">https://digfablab.wikispaces.com/file/view/Getting+started.pdf</a> (2010)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Olympus LEXT OLS4100 laser scanning digital microscope Manual. , (2018).</li><li>. SPIP&#8482; - Analytical Software for Microscopy Available from: <a target="_blank" href="https://www.imagemet.com/products/spip/">https://www.imagemet.com/products/spip/</a> (2017)</li><li>Zhang, Y., Pedersen, D. B., G&#248;tje, A. 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The authors have nothing to disclose.

This method is suitable for fabrication of inserts for polymer parts with complex shapes. It replaces the tool steel mold cavity in the injection molding machine with a set of soft tool inserts made from polymers. the machining time and cost is reduced compared to conventional metal machining; therefore, the cycle of production is shortened. This process chain is suitable for products in intermediate scale (from 1000 - 10,000 cycles by injection molding or similar) production, but high variation in design. Moreover, for 3D printed inserts, no special mold plates are required. Standard commercial injection molding plates were purchased and machined to fit the inserts.
At the current technology state, soft tooling fails by a distinct mechanism17. The failure mechanism has been identified to be linked the heat deflection temperature of the soft tooling insert and the decomposition has been identified to be due to outgassing. Therefore, the most critical step is to choose the right resin for the inserts produced by additive manufacturing. The thermal and mechanical property determines the life of the inserts, i.e., how many cycles it can withstand during injection molding. It also determines the range of molded polymer; the mold temperature of the molded polymer should not be higher than the deflection temperature of the insert material.
The second critical step in the protocol is the design of the insert. General rules of mold design should be followed and mechanically weak parts should be avoided on a 3D printed polymer insert; otherwise, the tool life will be affected due to failures like cracks.
The surface quality of the obtained injection-molded parts is limited by the applied additive manufacturing process. Post processing such as chemical etching is a possible solution to improve the surface quality. Precision of the surface features is another issue due to the additive manufacturing process.
This method will create the possibility for creating micro features on real free-form surface on a new production platform meeting industrial demands. Therefore, this method has the potential to be applied and investigated for applications in connection with next generation medical devices that require micro features on complex shapes, for example in connection with medical devices or implanting devices14. This method will facilitate value creation through significantly reduced expenses for small-to-medium volume series productions, and positively affect the timescale for production in the gap between 1 - 10 individual devices and real mass production. It will open up new high-value products and design solutions.

The presented method aims at manufacturing complex surface with micro features by a soft-tooling process, i.e., to use polymer additive manufacturing to produce inserts for polymer injection molding. In other words, polymer parts with functional surfaces are injection molded by polymer inserts.
Surface functionalities can be realized by micro features; for instance, Doan et al.1 and Luchetta et al.2 demonstrate functional surfaces in the field of cell biology, and Hu et al.3 shows an example of optical elements, etc. One type of surface feature, micro pillars, has been investigated intensively for promoting cell proliferation. They are able to enhance the bonding between the proliferated tissues and the surface if the micro pillars are patterned in certain ways4,5.
Polymer replications of micro features have been intensively studied, and precise molding can be achieved by many processes6. For instance, Metwally et al. has reported high fidelity between the molded parts and mold for replication of micro and sub-micro features on flat surfaces7.
There are many protocols for manufacturing micro pillars or features; however, most of them can only be applied on flat surfaces or surfaces with constant curvature. For instance, Nian et al.8 showed that micro features could be achieved by hot embossing on a curved surface. Those protocols are not suitable for complex shapes with three-dimensional surfaces, which are required by most real-life devices. Apparently, the fabrication of a three dimensional cavity with micro features on the surface challenges current protocols; meanwhile, ejection of the parts may fail for high aspect ratio pillars on a complex surface if they are non-perpendicular to the demolding direction. Bissacco et al.9 used 3D mold inserts and obtained sub-micro features by injection molding; in their study, specific sub-micro features with a low aspect ratio were generated by an aluminum anodizing process and successfully replicated by polymer on a complex component.
Researchers have attempted to involve additive manufacturing in the process chains for polymer replication in order to achieve the designed surface texture. Lantada et al. described a process chain that starts from AM prototypes, and then employs coating techniques for obtaining metallic mold inserts for injection molding10. Polymer parts produced by AM have been proved to be directly applicable as mold inserts11,12, which is the soft tooling process presented in this protocol.
In our previous work, we demonstrated that &#216;4 &#181;m PEEK pillars (2 &#181;m high) were successfully demolded on a vertical wall by injection molding13. In this protocol, the investigated product is a ring with four tines, each characterized by having an angle of 60&#176;. This product has been studied by Zhang et al.14, where micro features were introduced by implementing pre-fabricated nickel plates, and micro pillars (&#216;4 &#181;m) with aspect ratio of 0.5 were obtained by silicone rubber injection molding on the tines.
In the presented method, micro features can be obtained on complex surfaces created by a soft tooling process chain. Steel mold cavities are replaced by a set of inserts made by vat photo-polymerization-based AM. Compared to metal AM, photopolymer based AM technology is able to achieve higher precision15. Moreover, the machining time and cost is reduced significantly compared to conventional tooling processes based on CNC machining. According to a recent case study16, by using soft-tooling for thermoforming, the cost is reduced by 91%, while the consumed time is reduced by 93%. This protocol is suitable for products requiring a high design flexibility and intermediate production volume. It has been proved that inserts manufactured from carbon fiber reinforced photopolymers can withstand up to 2500 injection molding cycles for polyethylene before noticeable mold deterioration17. The choice of injection-molded material is limited by the thermal properties of the chosen photopolymer for inserts. Polymers with high melting temperatures may not be applied in a photopolymer cavity. In this study, polyethylene (PE) was chosen to conduct the injection molding test.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Photopolymer resin&#160;</td>
			<td style="width:121px;">EnvisionTec</td>
			<td style="width:119px;">HTM140 V2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin mixing device</td>
			<td style="width:121px;">IKA</td>
			<td>Vortex Genius 3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3d printer</td>
			<td>Envisiontec</td>
			<td>Perfactory 3&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV light flash unit</td>
			<td style="width:121px;">EnvisionTec</td>
			<td style="width:119px;">Otoflash unit</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:24px;width:216px;">Polyethylene&#160;</td>
			<td style="width:121px;">lyondellbasell</td>
			<td style="width:119px;">PE Purell 1840</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injection moulding machine</td>
			<td>Arburg</td>
			<td>Allrounder 370A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Image processing</td>
			<td style="width:121px;">SPIP</td>
			<td style="width:119px;">6.2.8</td>
			<td></td>
		</tr>
	</tbody>
</table>

a soft tooling process chain for injection molding of a 3d component with micro pillars

The purpose of this paper is to present the method of a soft tooling process chain employing Additive Manufacturing (AM) for fabrication of injection molding inserts with micro surface features. The Soft Tooling inserts are manufactured by Digital Light Processing (vat photo polymerization) using a photopolymer that can withstand relatively high temperaturea. The part manufactured here has four tines with an angle of 60&#176;. Micro pillars (&#216;200 &#181;m, aspect ratio of 1) are arranged on the surfaces by two rows. Polyethylene (PE) injection molding with the soft tooling inserts is used to fabricate the final parts. This method demonstrates that it is feasible to obtain injection-molded parts with microstructures on complex geometry by additive manufactured inserts. The machining time and cost is reduced significantly compared to conventional tooling processes based on computer numerical control (CNC) machining. The dimensions of the micro features are influenced by the applied additive manufacturing process. The lifetime of the inserts determines that this process is more suitable for pilot production. The precision of the inserts production is limited by the additive manufacturing process as well.

Figure 3 shows the inserts fabricated by AM and the PE parts produced by injection molding. Two rows of circular holes were vertical to the surface on the tines (Figure 3 (b)). The nominal dimension of the holes on the inserts is &#216;200 &#181;m and 200 &#181;m in depth, with a center-to-center distance 400 &#181;m. The layer structures due to the AM process are clearly visible. On the bottom left line there is a visible folding mark, which took place unintentionally during the preparation for SEM.
The demolding process is illustrated in Figure 4. Pillars are not parallel to the demolding direction. No pillar breaking was observed in all the studied samples (Figure 5). In this sense, the demolding was successful. It is already visible that the pillars on position of far-from-the-gate are notably lower than on the top side due the pressure drop. Regarding the diameter, the difference from positions are not as prominent as for the height.
Figure 6 and Figure 7 illustrate how the replication propagated along the 100 cycles of injection molding. The degree of replication is defined by the ratio between the dimension of the pillars and the dimension of the inserts. Along the 100 cycles, stable process was achieved at each position regarding both vertical and lateral replication. The dimension of the holes on the inserts were unchanged after injection molding, which confirmed this conclusion. The height of the pillars positioned far from the gate were lower than those placed near the gate, because polymer flow increases its viscosity when the flow length increases.
The injection molding parameters were varied within the range allowed by the material datasheet. In the explored process window, most of the runs reached more than 80 cycles, confirming that the tool life of the AM inserts was not affected in the explored process window. However, cracking occurred to the inserts for the injection side in some tests when the injection pressure was high. This is due to the center part of the insert being much thinner.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57335/57335fig3.jpg" /> 
Figure 3: Inserts and replicas. (a) Inserts produced by additive manufacturing process form the mold cavity; one polyethylene (PE) replica produced by injection molding is displayed. Markings on the reference ruler are millimeters; numbers mark centimeters. (b) Scanning electron microscope (SEM) image shows the surface of the insert; (c) SEM image shows the surface of the PE parts made by injection molding. ((a) and (b) have been modified from from Zhang et al. 201722) <a href="https://www.jove.com/files/ftp_upload/57335/57335fig3large.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/57335/57335fig4.jpg" /> 
Figure 4: Successful demolding. The demolding was successful even though pillars are not parallel to the demolding direction. (a) Molds close and (b) molds open for demolding. (This figure has been modified from Zhang et al. 201722) <a href="https://www.jove.com/files/ftp_upload/57335/57335fig4large.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/57335/57335fig5.jpg" /> 
Figure 5: SEM image of the side view of one random tine. The investigated four positions are labeled on the image. (This figure has been modified from Zhang et al. 201722) <a href="https://www.jove.com/files/ftp_upload/57335/57335fig5large.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/57335/57335fig6.jpg" /> 
Figure 6: Pillar height replication degree. The replication degree of the pillar height is defined by height of the pillar/depth of the hole. The mean value of the replication degree at the four positions was displayed in each batch. One randomly selected sample was measured in each batch. The four positions are &#34;Back-far&#34; for the position far from the gate at the back side, &#34;Back-near&#34; for the position near the gate at the back side, &#34;Top-far&#34; for the position far from the gate at the topside, and &#34;Top-near&#34; for the position near the gate on the top side. (This figure has been modified from Zhang et al. 2017 22) <a href="https://www.jove.com/files/ftp_upload/57335/57335fig6large.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/57335/57335fig7.jpg" /> 
Figure 7: Pillar diameter replication degree. The replication degree of the pillars diameter is defined by diameter of the pillar/diameter of the hole. The mean value of the replication degree at the four positions was displayed in each batch. One randomly selected sample was measured in each batch. The four positions are &#34;Back-far&#34; for the position far from the gate at the back side, &#34;Back-near&#34; for the position near the gate at the back side, &#34;Top-far&#34; for the position far from the gate at the topside, and &#34;Top-near&#34; for position near the gate on the top side. (This figure has been modified from Zhang et al. 2017 22). <a href="https://www.jove.com/files/ftp_upload/57335/57335fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Soft Tooling Process Chain for Injection Molding of a 3D Component with Micro Pillars at JoVE.com

A Soft Tooling Process Chain for Injection Molding of a 3D Component with Micro Pillars

A protocol for fabricating injection molding inserts for complex geometry with micro features on surfaces employing Additive Manufacturing (AM) is presented.

The purpose of this paper is to present the method of a soft tooling process chain employing Additive Manufacturing (AM) for fabrication of injection ...

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Research

JoVE Journal

Engineering

12.5K Views.  Technical University of Denmark. A protocol for fabricating injection molding inserts for complex geometry with micro features on surfaces employing Additive Manufacturing (AM) is presented.

Video: A Soft Tooling Process Chain for Injection Molding of a 3D Component with Micro Pillars

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

Chemistry

Micro-masonry for 3D Additive Micromanufacturing

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called &#8216;inks&#8217;) from a substrate where they are generated to a different substrate by utilizing elastomeric stamps. Transfer printing enables the integration of heterogeneous materials to fabricate unexampled structures or functional systems that are found in recent advanced devices such as flexible and stretchable solar cells and LED arrays. While transfer printing exhibits unique features in material assembly capability, the use of adhesive layers or the surface modification such as deposition of self-assembled monolayer (SAM) on substrates for enhancing printing processes hinders its wide adaptation in microassembly of microelectromechanical system (MEMS) structures and devices. To overcome this shortcoming, we developed an advanced mode of transfer printing which deterministically assembles individual microscale objects solely through controlling surface contact area without any surface alteration. The absence of an adhesive layer or other modification and the subsequent material bonding processes ensure not only mechanical bonding, but also thermal and electrical connection between assembled materials, which further opens various applications in adaptation in building unusual MEMS devices.

This paper introduces a 3D additive micromanufacturing strategy (termed &#8216;micro-masonry&#8217;) for the flexible fabrication of microelectromechanical system (MEMS) structures and devices. This approach involves transfer printing-based assembly of micro/nanoscale materials in conjunction with rapid thermal annealing-enabled material bonding techniques.

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called &#8216;inks&#8217;) from a substrate where they are generated to ...

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

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Bioengineering

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid samples, such as chemical reagents and cell cultures, is predominantly accomplished through steady flows that are typically driven by syringe pumps, gravity, or capillary forces. The use of complementary oscillatory flows is seldom considered in applications despite its numerous advantages as recently demonstrated in the literature. The significant technical barrier to the implementation of oscillatory flows in microchannels is likely responsible for the lack of its widespread adoption. Advanced commercial syringe pumps that can produce oscillatory flow, are often more expensive and only work for frequencies less than 1 Hz. Here, the assembly and operation of a low-cost, plug-and-play type speaker-based apparatus that generates oscillatory flow in microchannels is demonstrated. High-fidelity harmonic oscillatory flows with frequencies ranging from 10-1000 Hz can be achieved along with independent amplitude control. Amplitudes ranging from 10-600 &#181;m can be achieved throughout the entire range of operation, including amplitudes &#62; 1 mm at the resonant frequency, in a typical microchannel. Although the oscillation frequency is determined by the speaker, we illustrate that the oscillation amplitude is sensitive to fluid properties and channel geometry. Specifically, the oscillation amplitude decreases with increasing channel circuit length and liquid viscosity, and in contrast, the amplitude increases with increasing speaker tube thickness and length. Additionally, the apparatus requires no prior features to be designed on the microchannel and is easily detachable. It can be used simultaneously with a steady flow created by a syringe pump to generate pulsatile flows.

The protocol demonstrates a convenient method to produce harmonic oscillatory flow from 10-1000 Hz in microchannels. This is performed by interfacing a computer-controlled speaker diaphragm to the microchannel in a modular manner.

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...