The authors thank Sungwon Lim for intellectual contributions to device and mold design as well as Jambu Jambulingam and Rebecca Grossman-Kahn for creating intravaginal silicone probes using this process. This work is supported by the Bill and Melinda Gates Foundation, the Vodafone Americas Foundation, and the FDA (2P50FD003793).

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None of the authors have any competing financial interests with respect to the work detailed in this article.

Of all the steps described, careful mold design is the most critical to success. The mold master should be created as a solid body with external geometries equal to the final device. These geometries should be adjusted to account for any material shrinkage due to the chosen elastomer as well as 3D printer resolution and tolerances. Placement of mold parting lines and through-holes for threaded rods and screws are dependent on one another. Adding parting lines increases the number of linear and rotational degrees of freed...

Liquid injection molding (LIM) (also known as reaction injection molding) is often used to manufacture elastomeric devices from thermosetting elastomers, but high tooling and equipment costs require a great deal of up-front capital investment1. Furthermore, LIM can be technically challenging and expensive to implement in cases with complex geometry and requirements for overmolding. As a result, it is typically impractical to use traditional LIM in ultra-low volumes or with early-stage device designs that often incur iterative revisions.
The typical procedure for injection molding elastomeric materials involves injecting liquid monomers at pressures around 150 psi into a mold using specialized molding machinery2. Temperatures and pressures are controlled to ensure laminar flow and prevent air being trapped in the mold3. Raw materials are typically two-part cure systems, such as platinum cure silicone, that are kept in separate and temperature controlled chambers prior to injection. Both components of the raw material are pumped into a high-pressure mixing chamber that subsequently feeds into the mold cavity. Curing is achieved by the presence of a catalyst as well as temperatures around 150-200 &#176;C4. Molds are typically machined from steel or aluminum to precise tolerances to create a good seal around parting edges3,5. Unfortunately, this process is generally more suited to larger scale manufacturing given high mold tooling costs as well as the requirement for specialized injection and feedback control systems.
For rapid prototyping of polyurethane (PU) parts, it is possible to use stereolithography (SLA) to create a mold master and produce a silicone rubber mold6,7. However, this technique is not suitable for overmolding since it is difficult to achieve precise alignment of overmolded components, as the silicone is, by design, not a rigid structure. Furthermore, production of devices with complex geometries, such as invaginations or hollowed out sections, is difficult or impossible. The requirement for complex or precise mold parting lines and rigid thin elements are more often than not, incompatible with the liquid rubber molding process.
The aforementioned production-scale or late-stage prototyping processes are often impractical for early-stage medical device development in which a few devices need to be produced for proof-of-concept and feasibility in human studies, as is often the case in academic laboratory and start-up company environments. The lack of alternatives often means that even early-stage development would incur high costs, requiring many device developers to limit device functionality or put development on hold while additional funds are raised. This contributes to a dramatic slowing of the development process since a large fraction of medical devices require implementation of complex features. It is also difficult to fund the costly development of such devices since proof-of-concept data is often not yet established. We encountered this roadblock in a recent project within this lab, which involved the development of a silicone intravaginal probe with overmolded electrical and optical sensors that required a cup-like tip to conform to specified cervical geometries. The process described in this article documents our attempt to circumvent this vicious cycle and rapidly reach proof-of-concept for LIM medical devices.
The technique shown in Figure 1 deconstructs the LIM process into 5 main activities: (1) mold design &#38; production, (2) mold assembly (3) elastomer mixing, (4) elastomer injection, and (5) elastomer curing &#38; demolding.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51745/51745fig1highres.jpg" width="600" /> 
Figure 1. Protocol Overview. Overview of the protocol, which involves: (1a) creating a mold using computer-aided design tools, (1b) 3D printing the mold pieces, (2) assembling the mold pieces using threaded rods and screws, (3) mixing liquid elastomer and loading it in a syringe, (4) injecting the liquid elastomer into the mold using a modified desiccator, (5a) curing the elastomer in a temperature-controlled oven, and (5b) demolding the cured elastomer device from the mold pieces.
Mold design involves development of a mold master in computer-aided design (CAD) software, subtraction of the mold master from a solid block and definition of mold parting lines. Mold pieces are created and then assembled using screws, rods, and nuts with overmolded components positioned in the mold cavity. Elastomer mixing involves combining parts A and B of raw material and degassing to remove potential void spaces in the material. Next, elastomer injection involves pressure-driven filling of the mold cavity, followed by elastomer curing in a temperature-controlled oven to ensure chemical crosslinking of the polymer chains.
Breaking down the injection molding process into these steps enables us to forego traditional LIM equipment in favor of low cost alternatives. For example, instead of machining a metal mold or casting a silicone rubber mold from a mold master, the molds created from the protocol described in this manuscript were created from acrylonitrile butadiene styrene (ABS) plastic using a fused-deposition modeling (FDM) 3D printer8,9. Compared to building metal molds or SLA molds, FDM is generally a cheaper and faster process. Fairly complex molds can be printed quickly on an in-house 3D printer, or cheaply produced by one of the many contract 3D printing services available. For example, a complex eight-piece 3D printed mold was used to cast the demonstrated intravaginal probe in the representative results section and shown in Figures 14&#160;and 15. All parts for this mold can be printed in approximately 1.5 days on an in-house 3D printer. Turnaround times for simpler molds can be a few hours. The overall length of time necessary to prototype a device using FDM 3D printers to create molds is similar to the time required to cast a mold out of silicone rubber and create a polyurethane prototype. However, using FDM 3D printers to create molds allows for several things that cannot easily be accomplished using a silicone mold: (1) many thermosetting elastomers can be used provided the 3D-printed mold can tolerate the required curing temperatures, (2) complex geometries can be created with the use of many different mold pieces and parting lines, and (3) use of rigid mold pieces allows precise and reproducible alignment of overmolded components within the mold cavity.
Instead of using a traditional LIM machine, which combines mixing, injection, and curing, it is possible to use a laboratory mixer to ensure homogenous mixing, a modified desiccator for injection, and a standard temperature-controlled oven for curing. The injection system was created using off-the-shelf components and involves the addition of a positive pressure supply line into the desiccator that connects to a syringe filled with mixed elastomer. Chamber pressurization in bench top desiccators is typically controlled by a three-way valve between the chambers, a vacuum supply line, and the atmosphere. The modified desiccator adds a positive pressure supply line feeding to the back of a syringe plunger. This enables the creation of a 40-50 psi pressure differential that is sufficient for liquid material injection into the mold cavity.
This technique allowed us to produce silicone intravaginal probes with overmolded electrical and optical sensors to collect proof-of-concept data for a Phase I clinical trial. Silicone was selected because of the need for biological inertness as well as the ability to sterilize with a variety of methods10,11. Furthermore, the device required a complex and unconventional cup-like geometry at the tip of the probe where sensors are located to interface with the cervix. Without the use of the described technique, it would have been a much more costly and lengthy process to produce these devices. This adaptation of the LIM process reduces cost and equipment requirements when compared to the traditional LIM process, making it practical to adopt a rapid and iterative approach to designing elastomeric devices.

<table border="0" cellpadding="0" cellspacing="0" width="1434">
	<tbody>
		<tr>
			<td>ABS Model Material</td>
			<td>Stratasys</td>
			<td>P430</td>
			<td>Model Material for uPrint Plus SE (Step: Mold Design &#38; Production)</td>
		</tr>
		<tr>
			<td>Soluble Support Material</td>
			<td>Stratasys</td>
			<td>SR-30</td>
			<td>Support Material for uPrint Plus SE (Step: Mold Design &#38; Production)</td>
		</tr>
		<tr>
			<td>Underwater Silicone Sealant, 2.8 Oz Tube, Clear</td>
			<td>McMaster-Carr Supply Company</td>
			<td>7327A21</td>
			<td>Silicone RTV for sealing gaps at mold parting lines (Step: Mold Assembly)</td>
		</tr>
		<tr>
			<td>Tubing, 1/8&#34; ID, 1/4&#34; OD, 1/16&#34; Wall Thickness, Ultra-chemical-resistant Tygon PVC, Clear</td>
			<td>McMaster-Carr Supply Company</td>
			<td>5046K11</td>
			<td>Forms runner/sprue adapter between mold and syringe with elastomer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Coupling, Adapter, Straight, Male Quick-turn (Luer lock) X 1/8&#34; Tube Barb, Nylon</td>
			<td>McMaster-Carr Supply Company</td>
			<td>51525K123</td>
			<td>Connect runner/sprue between mold and syringe with elastomer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Coupling, Adapter, Staight, Female Quick-turn (Luer lock) X 1/8&#34; Tube Barb, Nylon</td>
			<td>McMaster-Carr Supply Company</td>
			<td>51525K213</td>
			<td>Connect runner/sprue between mold and syringe with elastomer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Cap, Female Quick-turn (Luer lock), Nylon</td>
			<td>McMaster-Carr Supply Company</td>
			<td>51525K315</td>
			<td>Cap to prevent silicone from leaking out of mold after injection (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Liquid Silicone Rubber (LSR) 30 - 10:1, Implant Grade</td>
			<td>Applied Silicone Corporation</td>
			<td>PN40029</td>
			<td>Substitute with the elastomer of your choice.&#160; This is the one used for the intravaginal probe (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Syringes (BD), 1mL Slip-Tip, non-sterile clean, bulk</td>
			<td>Cole-Parmer</td>
			<td>WU-07945-00</td>
			<td>Syringes for transfering elastomer material (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Syringes (BD), 1mL Slip-Tip, non-sterile clean, bulk</td>
			<td>Cole-Parmer</td>
			<td>WU-07945-04</td>
			<td>Syringes for transfering elastomer material (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Syringe, 20mL, Open Bore, Solid Ring Plunger and Grip</td>
			<td>Qosina Corporation</td>
			<td>C1200</td>
			<td>Syringes for transfering elastomer material.&#160; Open bore is used for very viscous elastomers. (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Needle (BD), Non-sterile Clean with Shields, 18 gauge X 1.5&#34; Lg., Stainless Steel, BD Bulk&#160;</td>
			<td>Cole-Parmer</td>
			<td>WU-07945-76</td>
			<td>Used for removing air column between syringe plunger and elastomer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Plastic Cups, 12 Oz., Clear</td>
			<td>Safeway</td>
			<td>N/A</td>
			<td>Used for mixing silicone in THINKY Mixer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Polyethylene Bag, Open-Top, Flat, 5&#34; Width X 6&#34; Height, 2-MIL Thk.</td>
			<td>McMaster-Carr Supply Company</td>
			<td>1928T68</td>
			<td>Used for mixing silicone in THINKY Mixer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Rubber Band, Latex Free, Orange, Size 64, 3-1/2&#34; L X 1/4&#34; W</td>
			<td>McMaster-Carr Supply Company</td>
			<td>12205T96</td>
			<td>Used for mixing silicone in THINKY Mixer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Parafilm Wrap, 4&#34;W</td>
			<td>Cole-Parmer</td>
			<td>EW-06720-40</td>
			<td>Used for mixing silicone in THINKY Mixer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Syringe Barrels with Stoppers, Luer Lock, Air Operated,&#160; 50mL</td>
			<td>EWD Solutions</td>
			<td>JEN-JG50A-15</td>
			<td>Smaller syringes can be used if less elastomer is required, but make sure it is compatible with Air Operated Syringe Adapter in injection chamber (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Sealant Tape, Pipe Thread, 50&#39;Lg X 1/4&#34; W, .0028&#34; Thk, 0.5 G/CC Specific Gravity&#160;</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4591K11</td>
			<td>Teflon Tape for air-tight seals around at threads (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Scalpel Blades, Disposable, No. 22</td>
			<td>VWR</td>
			<td>21909-646</td>
			<td>Used for cutting tubing and demolding (Step: Curing &#38; Demolding)</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>VWR</td>
			<td>21903-005</td>
			<td>&#160;(Step: Curing &#38; Demolding)</td>
		</tr>
		<tr>
			<td>2-Propanol, J. T. Baker</td>
			<td>VWR</td>
			<td>JT9334-3</td>
			<td>&#160;(Step: Curing &#38; Demolding)</td>
		</tr>
		<tr>
			<td>uPrint Plus SE 3D Printer</td>
			<td>Stratasys</td>
			<td>uPrint Plus SE</td>
			<td>Other 3D printers can be used (Step: Mold Design &#38; Production)</td>
		</tr>
		<tr>
			<td>Screw, Cap, Hex Head,&#160; 1/4&#34;-28 , 2-1/2&#34; Lg, 18-8 Stainless Steel</td>
			<td>McMaster-Carr Supply Company</td>
			<td>92198A115</td>
			<td>Screws used with nuts to compress mold (Step: Mold Assembly)</td>
		</tr>
		<tr>
			<td>Nut, Hex, 1/4&#34;-28, 7/16&#34; Wd, 7/32&#34; Height, 18-8 Stainless Steel&#160;</td>
			<td>McMaster-Carr Supply Company</td>
			<td>91845A105</td>
			<td>Screws used with nuts to compress mold (Step: Mold Assembly)</td>
		</tr>
		<tr>
			<td>Stud, Fully Threaded, 1/4&#34;-28, 1&#34; Lg, 18-8 Stainless Steel&#160;</td>
			<td>McMaster-Carr Supply Company</td>
			<td>95412A567</td>
			<td>Threaded-rods can be cut to desired length and are used with nutes to compress mold (Step: Mold Assembly)</td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>THINKY USA Inc.</td>
			<td>ARE-310</td>
			<td>Mixers are strongly recommended for fine mixing and to reduce degassing time, but hand mixing is fine (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Laboratory Weigh Scale</td>
			<td>Mettler-Toledo International Inc.</td>
			<td>EL602</td>
			<td>&#160;(Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Desiccant Vacuum Canister, Reusable,&#160; 10-3/4&#34; OD</td>
			<td>McMaster-Carr Supply Company</td>
			<td>2204K7</td>
			<td>This desiccator is used for degassing the elastomer (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Custom 3D-Printed Mixer-to-Cup Adapter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Modeled in Solidworks CAD and 3D printed (Step: Elastomer Mixing)</td>
		</tr>
		<tr>
			<td>Tubing, Smooth Bore, 1/4&#34; ID, 1/2&#34; OD, 1/8&#34; Wall Thickness, High Purity Tygon PVC, Clear</td>
			<td>McMaster-Carr Supply Company</td>
			<td>5624K51</td>
			<td>Tubing outside of Desiccator (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Tubing, Smooth Bore, 3/8&#34; ID, 5/8&#34; OD, 1/8&#34; Wall Thickness, High Purity Tygon PVC, Clear</td>
			<td>McMaster-Carr Supply Company</td>
			<td>5624K52</td>
			<td>Tubing to adapt to Air/Vacuum Supply (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Reducer, Straight, Vacuum Barb 3/8&#34; Tube ID X Vacuum Barb 1/4&#34; Tube ID, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>44555K188</td>
			<td>Adapt Tubing outside Desiccator to Tubing leading to Air/Vacuum Supply (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Clamp, Hose &#38; Tube, Worm-Drive, for 7/32&#34; to 5/8&#34; OD tube, 5/16&#34; Wd., 316 SS</td>
			<td>McMaster-Carr Supply Company</td>
			<td>5011T141</td>
			<td>Used on tubing to create Air/Vacuum-tight seal at junctions (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Clamp, Hose, Smooth-Band Worm-Drive, for 1/2&#34; to 3/4&#34; OD tube, 3/8&#34; Wd., 304 SS</td>
			<td>McMaster-Carr Supply Company</td>
			<td>5574K13</td>
			<td>Used on tubing to create Air/Vacuum-tight seal at junctions (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Tee, Vacuum Barb 1/4&#34; Tube ID, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>44555K138</td>
			<td>Tee Junction between Vacuum, Three-way T-valve on Desiccator, and Three-way L-valve (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Tee, 1/4 NPT Female X Female X Male, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>50785K222</td>
			<td>Tee Junction between Pressure Gauge, Chamber, and Three-way L-valve (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Valve, Ball, Straight, T-Handle, 1/4 NPT Female X Male, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4082T42</td>
			<td>Three-way L-valve (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Adapter, Straight, Vacuum Barb 1/4&#34; ID Tube X 1/4 NPT Male, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>44555K132</td>
			<td>Adapter for Three-way L-valve-to-Tubing (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Saw, Hole, Bimetal. 1-3/8&#34; OD, 1-1/2&#34; Cutting Depth</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4066A25</td>
			<td>Used to cut holes in Desiccator for throughwall fittings (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Arbor, 9/16&#34; to 1-3/16&#34; Saw, 1/4&#34; Hex</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4066A76</td>
			<td>Used to cut holes in Desiccator for throughwall fittings (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Arbor Adapter for 1-1/4&#34; Thru 6&#34; Dia Hole Saws</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4066A77</td>
			<td>Used to cut holes in Desiccator for throughwall fittings (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Straight, Through-Wall, 1/2 NPT Female, Polypropylene</td>
			<td>McMaster-Carr Supply Company</td>
			<td>36895K141</td>
			<td>Throughwall fittings leading to Pressure/Vacuum Gauges (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Adapter, Straight, Reducing,&#160; Bushing, Hex, 1/2 NPT Male X 1/4 NPT Female, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4429K422</td>
			<td>Reducing tube diameter inside the Desiccator to adapt to Air-operated Syringe System (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Adapter, Straight, Reducing, Bushing, Hex, 1/4 NPT Male X 1/8 NPT Female, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4757T91</td>
			<td>Reducing tube diameter inside the Desiccator to adapt to Air-operated Syringe System (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Coupling, Adapter, Straight, Vacuum Barb 1/4&#34; ID Tube X 1/8 NPT Female, Brass</td>
			<td>McMaster-Carr Supply Company</td>
			<td>44555K124</td>
			<td>Reducing tube diameter inside the Desiccator to adapt to Air-operated Syringe System (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Syringe Adapters, Air Operated, 30/50mL</td>
			<td>EWD Solutions</td>
			<td>JEN-JG30A-X6</td>
			<td>Air operated syringe adapter on the inside of the Desiccator; must be compatible with syringes used to hold elastomer (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Gauge, Dual-Scale Vacuum, 2-1/2&#34; Dial, 1/4 NPT Male, Bottom Connector, 30&#34; Hg-0, Steel Case</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4002K11</td>
			<td>Vacuum Gauge (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Gauge, Dual-Scale Vacuum and Compound, 3-1/2&#34; Dial, 1/4 NPT Male, Center Back, 30&#34; Hg-0, 100 PSI, Steel Case</td>
			<td>McMaster-Carr Supply Company</td>
			<td>4004K616</td>
			<td>Pressure Gauge leading to Air-operated Syringe System (Step: Elastomer Injection)</td>
		</tr>
		<tr>
			<td>Oven, Vacuum, Isotemp, Economy&#160;</td>
			<td>Fisher Scientific</td>
			<td>280A</td>
			<td>Standard non-vacuum oven can be used (Step: Curing &#38; Demolding)</td>
		</tr>
		<tr>
			<td>Solidworks CAD</td>
			<td>Dassault Syst&#232;mes</td>
			<td>Solidworks Research Subscription</td>
			<td>Other CAD Software can be used for mold master and mold design (Step: Mold Design &#38; Production)</td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51745/51745fig2highres.jpg" width="500" /> 
Figure 2. 2D CAD Sketches. A)&#160;2D Sketch that can be radially revolved about the Y-axis to produce a cup-like feature similar to the one on the intravaginal probe device. B)&#160;Teardrop-shaped 2D sketch that can be extruded out of the plane into a prism-like structure that forms the handle of the intravaginal probe device. ...

Watch this Scientific Journal Video about Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding at JoVE.com

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

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 ...

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Research

JoVE Journal

Bioengineering

19.7K Views.  University of California, San Francisco. 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.

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

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing

Dipstick urinalysis provides quick and affordable estimations of multiple physiological conditions but requires good technique and training to use accurately. Manual performance of dipstick urinalysis relies on good human color vision, proper lighting control, and error-prone, time-sensitive comparisons to chart colors. By automating the key steps in the dipstick urinalysis test, potential sources of error can be eliminated, allowing self-testing at home. We describe the steps necessary to create a customizable device to perform automated urinalysis testing in any environment. The device is cheap to manufacture and simple to assemble. We describe the key steps involved in customizing it for the dipstick of choice and for customizing a mobile phone app to analyze the results. We demonstrate its use to perform urinalysis and discuss the critical measurements and fabrication steps necessary to ensure robust operation. We then compare the proposed method to the dip-and-wipe method, the gold standard technique for dipstick urinalysis.

Dipstick urinalysis is a quick and affordable method of assessing one&#8217;s personal state of health. We present a method to perform accurate, low-cost dipstick urinalysis that removes the primary sources of error associated with traditional dip-and-wipe protocols and is simple enough to be performed by lay users at home.