Chips and Tips

Development of a cell culture microdevice with a detachable channel for clear observation

21 Feb 2018

By Sian Carrington, Editorial Assistant.

Eriko Kamata, Momoko Maeda, Kanako Yanagisawa, Kae Sato*

Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, Bunkyo, Tokyo 112-8681, Japan

*E-mail: satouk@fc.jwu.ac.jp

Why is this useful?

There have been many reports on microfluidic devices for cell culture having upper and lower microchannels separated by a thin PDMS membrane. In these devices, the lower channel often interferes with the microscopic observation of cells cultured in the upper channel. To avoid interference, a microdevice with a detachable lower channel was developed.

What do I need?

Materials:

PDMS (SILPOT 184w/c, Dow Corning Toray)

Hexane

PMMA sheet (56 × 76 × 2 mm)

Glass slides (52 × 76 mm and 26 × 76 mm)

Cover slip (24 × 60 mm)

1 kg weight

PTFE tubing (1 × 2 mm and 0.46 × 0.92 mm)

Tygon tubing (1.59 × 3.18 mm)

Biopsy Punches (1 mm and 2 mm, Kai corporation)

Equipment:

Vacuum desiccator

Oven (65 ˚C and 100˚C)

Spin coater

Plasma generator

Vacuum pump

What do I do?

PDMS molding

Mix the elastomer and curing agent at a 10:1 mass ratio. De-gas the mixture under vacuum until no bubbles remain (20 min). Pour the degassed PDMS mixture onto a master, which has an upper channel (1 × 1 × 10 mm) or a lower channel (0.5 × 2 × 15 mm) structure, and then place it in an oven at 65˚C for 1 h. Peel off the PDMS replica from the master and adhere it to a glass slide (26 × 76 mm). Place it in an oven at 100˚C for 1 h (Fig.1).

Preparation of a thin PDMS membrane

Spin coat 600 µL of the PDMS prepolymer (at a 10:1 mass ratio) on a PMMA sheet at 500 rpm for 20 s followed by 2,400 rpm for 600 s. Bake it at 65˚C for 1.5 h.

Fig.1 PDMS sheet with the upper channel, that with the lower channel, PDMS membrane, and tubing.

Permanent bonding of the PDMS membrane and the sheet with the upper channel

Punch the inlet and outlet holes at both the ends of the upper channel with a 2-mm biopsy punch. Expose both the bonding surfaces of the PDMS membrane and the PDMS sheet with the upper channel (upper sheet) to plasma at 100 W, 35 s (Fig.2a and 2b). Laminate and bake them at 65˚C for 1 h (Fig.2c). Remove the PMMA sheet, and punch a hole to connect the sheet with the lower channel by using a 1-mm biopsy punch from the membrane side.

Fig.2 (a) Schematic diagram of bonding the PDMS membrane and the upper sheet. (b) Plasma treatment. (c) Bonded PDMS membrane and sheet.

Detachable bonding of the PDMS membrane and the PDMS sheet with the lower channel

The PDMS sheet with the lower channel (lower sheet) is bonded to the PDMS membrane by using a PDMS prepolymer diluted with hexane (a dilution ratio of 1:3) as a glue¹ (Fig.3a). Spin coat the diluted PDMS prepolymer (600 µL) at 2,000 rpm for 30 s on the surface of a glass slide (52 × 76 mm) to cover the slide with a thin layer of the glue and incubate it for 10 min to dry the solvent (Fig.3b). Place the lower sheet on the coated glass slide (Fig.3c). Apply glue at the four corners of the PDMS membrane bonded with the upper sheet (Fig.3d). Peel off the lower sheet from the glass slide and place the glue-coated surface of the sheet on the PDMS membrane (Fig.3e). After 30 min of incubation, bond the lower sheet to a cover slip by plasma bonding. Place a 1-kg weight and a glass slide on the device and bake it at 100˚C for 1 h (Fig.3f).

Fig.3 (a) Schematic diagram of bonding of the PDMS membrane and lower sheet. (b) Spin coat the diluted PDMS prepolymer (glue). (c) Lower sheet is placed on the thin film of the diluted PDMS prepolymer. (d) The diluted PDMS prepolymer is applied on the four corners of the PDMS membrane. (e)The glue-coated surface of the lower sheet is placed on the PDMS membrane. (f) A weight and a glass slide are placed on the device to bake it at 100˚C.

Tubing

Connect polytetrafluoroethylene (PTFE) tubes (1 × 2 × 100 mm) with Tygon tubes (1.59 × 3.18 × 10 mm) to the holes present at both ends of the upper microchannel. Connect a PTFE tube (46 × 0.92 × 150 mm) to the hole of the lower channel. Apply PDMS prepolymer at the root of the tubes, and then bake it at 100˚C for 1 h for firm connection (Fig.4a and b).

Cell culture

Introduce a cell suspension into the upper microchannel, which is manually precoated with 0.1 mg/mL of fibronectin. Incubate the device at 37˚C with 5% CO₂ for 16 h to allow cells to adhere to the bottom of the upper channel (surface of the PDMS membrane).

Detachment of the lower sheet for cell observation

Remove the lower sheet from the device carefully (Fig.4c and d). Place the rest of the device on a cover slip for observation with an inverted microscope (Fig.4 f and h).

Fig.4 (a) The complete microdevice. (b) Side view of the microdevice. The cell culture channel (upper) is filled with water containing a red food color, while the lower channel is filled with water containing a blue food color. (c) and (d) The lower sheet is peeled off from the microdevice carefully. Phase contrast images of cells (e) before and (f) after detachment of the lower sheet. Fluorescent images of cells stained with CellTracker Red CMTPX (g) before and (h) after detachment.

Conclusion

We developed a microfluidic device with a detachable lower microchannel. It is important that different bonding techniques be used for each side of the PDMS membrane. If the lower channel is filled with air and the device is incubated in a CO₂ incubator, dew condensation is often observed in the lower channel when the device is taken out from the incubator. The condensation in the lower channel makes observation difficult (Fig.4e and g). This problem was solved with the detachable device.

Acknowledgment

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP16H04170.

Reference

Chueh, B.H., Huh, D., Kyrtsos, C.R., Houssin, T., Futai, N., Takayama, S. (2007). Leakage-free bonding of porous membranes into layered microfluidic array systems. Analytical Chemistry 79 (9), 3504–3508.

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A solvent-based method to fabricate PMMA microfluidic devices

01 Nov 2017

By Harriet Riley, Development Editor.

Mustafa Al-Adhami; Abhay Andar; Elizabeth Tan; Govind Rao; Yordan Kostov*

Center for Advanced Sensor Technology, University of Maryland, Baltimore County, 1000 Hilltop Circle, TRC 252, Baltimore, Maryland, 21250.

*Email: kostov@umbc.edu

Why is this useful?

The demand for microfluidics has steadily increased, due in part to the growing popularity of point-of-care devices [1]. Often, microfluidic chips are fabricated in thermoplastics [1]. Thermoplastics are synthetic polymers that have gained popularity due to their ability to be molded into complex structures [3, 4]. They are often used as a safer and cheaper alternative to glass [3, 4]. However, proper sealing of these devices proves challenging, especially in the field of medical testing, where the demand for reliable devices is high. For example, pressure-sensitive adhesives, common sealants, can limit the size of microfluidic channels; some adhesive can exhibit reactive groups that interfere with analytical processes that run on the chip [5]. Hence, a method of sealing that is free from the aforementioned limitations is needed.

Here, a solvent-based method is presented. Polymethylmethacrylate (PMMA), a thermoplastic, exhibits softening at temperatures above its glass transition temperature (T_g) returning to its original state when cooled. This transition introduces several direct bonding options [6]. However, T_gof PMMA is 115 °C. The pressure required for bonding even at this temperature is fairly high. This can lead to imperfections in the channel dimensions, as the bulk of the material softens. The application of a weak solvent decreases T_g only for the surface of the plastic, thus reducing the required temperature and pressure for the process. The decreased pressure reduces the possibility of channel deformation. Furthermore, as the solvent-induced softening is limited only to the surface (the first few microns), the deeper channel structures are not affected. Hence, a direct solvent bonding method allows for an adhesive-free bonding and avoids a temperature-induced deformation. As a bonus, the mechanical properties of the bond are greatly enhanced [7]. It is worth noting that this approach is valid for microfluidic devices with channel depths greater than 100 microns, typically for devices produced by a direct laser etching.

Another advantage of this technique is that it results in the production of sterile devices when the weak solvent is ethanol. They can be manufactured quickly using basic equipment found in any laboratory [7]. To demonstrate this method, PMMA was used along with 90% ethanol as a solvent to bond to other sheet materials such as:

PMMA sheets,, to manufacture microfluidic mixers and other microfluidic devices for sample processing (Figure 1, a, d) [7];
ePTFE membranes, to make microfluidic de-bubblers (Figure 1, c); and,
Cellulose Acetate membranes, to make custom made dialysis devices (Figure 1, b).

Figure 1. A) Dialysis device B) Redox assay enclosure C) Microfluidic debubbler D) Microfluidic mixer

What do I need?

Materials:

PMMA sheet, thickness of 1.5 mm (Astra Products)
PMMA sheet, thickness of 0.2 mm (Astra Products)
10K, 20K MWCO Cellulose Acetate membranes (Thermo Fisher Scientific)
ePTFE membrane (Sterlitech,product number: PTU021350)
Metal vise (McMaster, product number: 5226A3)
1000 grit Sandpaper (McMaster)
Silicone pad (McMaster)
KIM-WIPES (Kimberley Clark)
2 rectangular metal sheets (McMaster)
Deionized water
90% Ethanol (Decon Labratories, Fisher Scientific, Reagent grade)

Equipment:

CO2 Laser cutter (Universal laser systems)
Conventional toaster oven
Computer aided design (CAD) software (CorelDRAW X4)

What do I do?

Design devise
1. Draw the desired devise design using any available computer aided design (CAD) software.
2. Export the CAD file into .dxf file format that is compatible with the laser cutter software.
Laser cut design
1. Place the PMMA sheet of 1.5 mm in thickness onto the bed of the laser cutter.
2. Calibrate laser cutter according to sheet thickness.
3. Select the correct laser setting in the laser cutter software. It should be height-aligned according to the material and thickness of the sheet.
4. Laser cut the core pathway of the design onto the 1.5 mm thickness of PMMA.
5. Next, place the other PMMA sheet of 0.2 mm in thickness onto the bed of the laser cutter and repeat points b-c
Roughen the PMMA sheets
1. After laser cutting, there should be a total of three pieces: core channel design (1) and cover sheets (2).
2. Rinse the PMMA cutouts with deionized water and wet the sandpaper with tap water.
3. Sand the wetted PMMA cutouts in a figure eight-like motion onto the wetted sandpaper until it is milky white. This is to ensure flat conformity on the PMMA sheets to increase bondage.
4. Rinse PMMA cutouts with deionized water and dry using “KIM WIPES”.
Bond the PMMA sheets
1. Preheat the temperature-controlled oven to 55 °C. Place the metal vise and the rectangular metal sheets in the oven to preheat as well. Thermal gloves should be used for safety.
2. Place a clean sheet of silicone onto a rectangular metal (aluminum) plate.
3. Next, place the base cover of the microfluidic cassette on top of the silicone pad in a sandwich manner.
4. Spray the 90% ethanol to the base cover sheet until all area is wetted.
5. Place the core channel sheet of the PMMA cutout.
6. Spray the 90% ethanol to the core channel sheet until the complete area, to be bonded, is wetted.
7. Place the final cover sheet of the microfluidic cassette.
8. Place the other silicone pad and the other rectangular metal sheet following it.
9. Place the sandwiched PMMA sheet, silicone pad, and rectangular metal sheet in the metal vise carefully so it does not misalign the cassette (Figure 2.)
10. Tighten the vise until can’t turn the lever anymore.
11. Place the vise with the microfluidic cassette sandwich into the preheated oven for five minutes. This has to happen before the vise is cooled down.
12. Allow to cool to room temperature and remove from metal vise and silicone pad.
13. Add inlet and outlet fittings, either glue them on or place them depending on the method of preference
Bonding PMMA to ePTFE(or cellulose acetate):
1. Preheat the temperature-controlled oven to 85°C. Place the metal vise and the rectangular metal sheets in the oven to preheat as well. Thermal gloves should be used for safety.
2. Place a clean sheet of silicone onto a rectangular metal (aluminum) plate.
3. Next, place the membrane of the microfluidic cassette on top of the silicone pad in a sandwich manner.
4. Spray the 90% ethanol on the membrane sheet until all area is wetted.
5. Place the core channel sheet of the PMMA cutout.
6. Spray the 90% ethanol to the core channel sheet until the complete area, to be bonded, is wetted.
7. Place the final PMMA cover sheet of the microfluidic cassette.
8. Place the other silicone pad and the other rectangular metal sheet following it.
9. Place the sandwiched device, silicone pad, and rectangular metal sheet in the metal vise carefully so it does not misalign the cassette (Figure 2.)
  Figure 2. Bonding setup
10. Tighten the vise until can’t turn the lever anymore.
11. Place the vise with the microfluidic cassette sandwich into the preheated oven for five minutes. This has to happen before the vise is cooled down.
12. Allow to cool to room temperature and remove from metal vise and silicone pad and remove the excess membrane if any with scissors.
13. Add inlet and outlet fittings, either glue them on or place them depending on the method of preference

What else do I need to know?

In order to align the sheets on top of each other, three methods were used:

For PMMA-PMMA devices it is enough to eyeball the alignment. The adhesion forces between the PMMA sheets with ethanol in between are enough to keep them fixed in place.
An alignment manifold was also fabricated where the machined sheets are placed in. The manifold will prevent the movement of the sheets (Figure 3a.).
Where the membrane is thick enough to drill a hole through, it is better to use three alignment pins. The pin holes are pre-fabricated when the device is machines. Toothpicks have been used as alignment pins (Figure 3b.).
Figure 3. A) Alignment manifold B) 3 wooden pins are used to keep the layers from moving
For thinner membranes, the machined PMMA is bonded on a slightly bigger membrane. The excess membrane is then removed with scissors.

Acknowledgements

This work was funded by DARPA, Biologically-derived Medicines on Demand (Bio-Mod) Project Grant (N66001-13-C-4023) for financial support.

References

Sia, S. K., & Kricka, L. J. (2008). Microfluidics and point-of-care testing. Lab on a Chip, 8(12), 1982. doi:10.1039/b817915h
Materials Used in Microfluidic Devices. (n.d.). SpringerReference. doi: 10.1007 /springerreference_67093
Liu, K., & Fan, Z. H. (2011). Thermoplastic microfluidic devices and their applications in protein and DNA analysis. The Analyst, 136(7), 1288. doi:10.1039/c0an00969
Tsao, C., & DeVoe, D. L. (2008). Bonding of thermoplastic polymer microfluidics. Microfluidics and Nanofluidics,6(1), 1-16.doi:10.1007/s10404-008-0361-x
Hong, T., Ju, W., Wu, M., Tai, C., Tsai, C., & Fu, L. (2010). Rapid prototyping of PMMA microfluidic chips utilizing a CO2 laser. Microfluidics and Nanofluidics, 9(6), 1125-1133. doi:10.1007/s10404-010-0633-0
Visakh, P. M., & Thomas, S. (2011). Engineering and Specialty Thermoplastics: Nylons: State of Art, New Challenges and Opportunities. Handbook of Engineering and Specialty Thermoplastics, 1-9. doi:10.1002/9781118229064.ch1
Al-Adhami, M., Tilahun, D., Gurramkonda, C., Rao, G., Kostov, Y (2016) Rapid detection of microbial contamination using a microfluidic devise. In: Biosensors and Biodetection: Methods and Protocols, Second Edition. Ed. A. Rasooly and B. Prickril. Springer.

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Manual Razor Patterned Tape Based Prototyping for Droplet Microfluidics

19 Oct 2017

By Harriet Riley, Development Editor.

Saifullah Lone^ab* I. W. Cheong^b and S. T. Thoroddsen^a

^aDivision of physical Sciences and Engineering, King Abdullah University of Science & Technology, (KAUST), Thuwal, 23955-6900, Saudi Arabia.
^bInstitute of Advanced Energy Technology, Kyungpook National University, Daegu, South Korea,
Phone: +821053165673, Office: +82-53-950-7590, FAX: +82-53-950-6594. Email: saifullah.lone@gmail.com, inwoocheong@gmail.com, and sigurdur.thoroddsen@kaust.edu.sa

Why is it Useful?

The subject of droplet microfluidics has grown in importance among researchers in chemistry, physics and biology, hence it has found applications in drug delivery, encapsulation, single-cell analysis, pickering-emulsion and phase-separation. For generating monodisperse droplets, various methods have been employed in constructing microfluidic devices. Emulsions with a coefficient of variation ≤ 5% have been previously reported in T-junction, flow focusing, co-axial, as well as other types of microfluidic devices. Microdroplets with ≤100 µm size offer attractive applications in industry and biology. Small channel-diameters attained by clean-room soft lithography is the most precise technique for fabricating microfluidic devices.^{1, 2} This technique is widely used to make master molds for PDMS-based devices.³ However, regarding the cost and complexity, it is difficult to install clean-room soft lithography in financially challenged countries and laboratories. Therefore, the cost and special clean-room training restricts its wide-spread application. To develop low cost robust technologies; inkjet printing, controlled numerical machining, xurography or razor-writing, printed circuit technology, print-and-peel (PAP) microfabrication and 3-D printing have been tested to fabricate microfluidic devices without clean-room technology. However, creating droplets under 100 µm size ranges by non-cleanroom technologies is challenging and open for upgradation. Recently, a rapid prototyping technique for microfluidics has been reported by employing laser-patterned tape⁴ This technique relies on computer-controlled CO₂ laser beam. This work was further simplified by manual razor patterned tape-based prototyping for patterning mammalian cells.⁵ Building on this prototyping concept, we extended the idea to produce monodisperse droplets under 100 µm size rages by overlapping the razor patterned tape strips (at right angles) on a flat glass surface. The production of monodisperse emulsion under 100 µm size ranges are greatly useful in pharmaceutical and cosmetic industries. Hence, our approach may well serve as one of the simplest approaches to fabricate droplet microfluidic generators.

What do I need?

One-sided adhesive tape (Temflex 1500 electrical, thickness 150 µm)
Flat glass slides, such as a microscope slide
30cm stainless steel metal ruler
Sharp razor blade
Uncured mixture of PDMS base and curing agent (10:1 w/w)
Oxygen plasma
Oven or hot plate
A microfluidic PDMS puncher for drilling holes
Deionized water (D.I. water) and 20 cSt and 10 cSt silicone oil

What do I do?

Figure 1 outlines the prototyping procedure. Prototyping begins by attaching adhesive tape on a flat glass substrate. With a sharp razor-blade, the tape is cut into fine parallel strips. The thickness of the tape (150 µm) determines the depth of the microchannel, but this can be increased by attaching multiple layers of tape on top of each other. Next the tape is removed from the regions outside the fine strips.

To construct a cross-junction, one strip of the tape is lifted and horizontally placed on top of another at an angle of 90ᴼ. The junction is pressed gently to ensure the strips are well attached. These adhering strips of tape serve as a master for PDMS-based replica casting.

A mixture of PDMS silicone elastomer base and a curing agent (in 10:1 ratio) is poured on top of the master within a plastic petri dish. The mixture is degassed under vacuum for 1 h and cured for 4 hrs at 65°C. Cured PDMS replica is then cut and peeled-off from the master. The master can be used repeatedly to fabricate multiple copies of the PDMS replica by following the afore-mentioned steps. Inlet and outlet holes are drilled through PDMS replica, which is then bonded on a glass substrate, after both replica and glass has been exposed to oxygen plasma. Figure 1(g) shows the resulting PDMS-device for generating monodisperse water-in-oil (W/O) emulsion. The technique is easily extended to fabricate T-junction or double T-junction prototypes (Figure 1h and i).

Figure 1. (a) Manual Razor Patterned Tape Based Prototyping for Droplet Microfluidics (b) Strips of adhesive tape on flat glass-substrate cut by a sharp razor-blade and a ruler, (c) The PDMS-casting, by pouring a mixture of PDMS silicone elastomer base and curing agent on top of the master in a plastic container. (d) Cut and peeled-off replica after curing. (e) Final assembled cross-junction microfluidic PDMS device. (f) Microfluidic device connected to syringe pumps under an optical microscope. (g) Video frame showing water-in-oil droplet formation in a flow-focusing prototype. Panels (h) and (i) show razor patterned tape-based T-junction and double T-junction prototypes, respectively.

Figure 2. (a) Image sequence from a video recorded at 10 kfps showing water droplet formation at a cross-junction. Time between subsequent frames is 200 µs. (b) Droplet size as a function of capillary number based on the viscosity and flow-rate of the continuous-phase (20 cSt silicone oil), for the channel in panels (c) for 300 µm wide channel above the horizontal line and (d) for 150 µm channel below the horizontal line with 10 cSt silicone oil. (c) Images extracted from video, showing flow regimes and droplet sizes as a function of flow-rate, with 300 µm channel width and 150 µm channel depth. (d) Video frame from smaller square channel with 150 µm width and depth.

Figure 2(a), demonstrates the droplet formation at a cross junction in our tape-based microfluidic device, with a channel width and depth of 300 µm and 150 µm respectively. In Figure 2(c), we kept the flow rate of aqueous phase fixed at 25 µl/min, while systematically increasing the flow-rate of outer continuous oil-phase (20 cSt silicone oil). As the outer flow-rate is increased, the regime is found to shift from dripping at lower flow-rate to jetting at higher flow-rate (Figure 2(c)). For lowest flow-rate, the aqueous-phase breaks into elongated plugs, while at higher flow-rates regular drops are pinched off. Various factors affect the size of the droplets, but it is primarily determined by competition between viscous stress in the continuous phase F_v~ µU/d which tends to rip off the drop and the interfacial tension F_s ~ s/d which try to keep the drop attached. Here µ is the dynamic viscosity of the outer phase and U is its velocity; while s is the interfacial tension between the water and the oil, s=0.040 N/m. For small channels, the characteristic length-scale d is the same for the two forces and it therefore drops out of the balance and when F_v~ F_s then the non-dimensional capillary number Ca=µU/s characterizes their relative strength. Figure 2b shows the droplet-size as a function of Ca. When the flow-rate of oil-phase reaches 65 µl/min, the size of the droplets reaches ~100 µm and the droplet breakup occurs at a large distance from the cross-junction. Figure 2(d) shows drop formation with a channel width of 150 µm and a channel depth of 150 µm. In this case, the droplet size reaches down to ~73 µm, when the oil-phase (10 cSt) is flowing at 25 µl/min and aqueous-phase at 10 µl/min.

Acknowledgement: This work was jointly funded by King Abdullah university of Science & Technology (KAUST), Thuwal, Saudi Arabia , and the Ministry of Trade, Industry and Energy, Korea (Grants No. 10067082 and 10070241).

Reference

[1] Qin, D.; Xia, Y.; Whitesides, G. M. Rapid prototyping of complex structures with feature sizes larger than 20 μm. Adv. Mater. 1996, 8, 917-919.

[2] Xia, Y.; Whitesides, G. M. Soft Lithography. Angew. Chem. Int. Ed. 1998, 37 550- 575.

[3] Duffy, D. C.; McDonald, J. C.; Schueller, O. J.; Whitesides, G. M. Rapid Prototyping of Microfluidic Systems in Poly (dimethyl siloxane). Anal. Chem., 1998, 70, 4974–4984.

[4] Luo, L. W.; Teo, C. Y T.; Ong, W. L.; Tang, K. C.; Cheow, L. F.; Yobas, L. Rapid prototyping of microfluidic systems using a laser-patterned tape J. Micromech. Microeng. 2007, 17 N107–N111

[5] Anil, B. S.; Ali, H.; Cheul, H. C.; and Raquel, P-C. Adhesive-tape soft lithography for patterning mammalian cells: application to wound-healing assays. BioTechniques, 2012, 53 315–318.

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Rapid Inoculation and Recovery of Microbes in a Microfluidic Device

04 Oct 2017

By Harriet Riley, Development Editor.

Greiner, A.^1,2, Tekwa, E.W.^1,3, Gonzalez, A.¹, Nguyen, D.⁴

¹Department of Biology, McGill University, 1205 Dr. Penfield, Montreal, QC, H3A 1B1, Canada.
²Department of Ecology and Evolution, University of Toronto, 25 Willcocks Street, Toronto, ON, M5S 3B2, Canada
³Department of Ecology, Evolution, and Natural Resources, Rutgers University, 14 College Farm Road, New Brunswick, NJ, 08901, USA.
⁴Meakins Christie Laboratories, Research Institute of the McGill University Health Centre, and Department of Medicine, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada.

Why is this useful?

Microfluidic devices are used for many different types of experiments across the medical, ecological and evolutionary disciplines (Park et al., 2003; Keymer et al., 2008; Connell et al., 2013; Hol & Dekker, 2014). For example, microfluidic devices for microbial experiments require inoculation into smaller chambers that simulate natural microbial environments such as porous soils (Or et al., 2007) and biological hosts (Folkesson et al., 2012). These devices often involve complicated pump setups and irreversible seals. We developed a technique that requires only common lab equipment and makes the device reusable while also allowing the microbes to grow undisturbed (based on Tekwa et al., 2015; Tekwa et al., in review). Here, we provide a detailed guide for the assembly and the previously undocumented non-destructive disassembly of polydimethylsiloxane (PDMS) experimental devices to recover microbes in situ, which can then be plated for relative counts and further molecular analyses of population changes. This is complemented by videos for each step.

Figure 1: Microfluidic device containing 14 habitats on an elastomer (PDMS) layer pressed onto a 60mm x 24mm glass cover slip. The habitats are 10 or 20µm in depth, range from 1400 µm to 2670 µm in diameter and take the shape of a ring or network of patches. This device is used to test the effects of habitat patchiness on microbe dynamics. Habitats were dyed blue for visualization. For more information see Tekwa et al. (2015).

What do I need?

Single-layer PDMS devices with habitats on one side
Pipette + sterile pipette tips
Sterile petri dishes (1/device)
Sterile tweezers
Inoculum
Filtered water
Kimwipes
Autoclavable plastic container
Ethanol
Tinfoil
Sterile 1μL inoculating loop (1/habitat)
Sterile eppendorfs
Phosphate-buffered saline (PBS)
Biological safety cabinet (BSC)

How do I do it?

Clean the PDMS devices: The PDMS device should be pre-treated once with 0.01N HCl for one hour and plasma-treated to keep it hydrophilic and amenable to bonding to glass or plastic substrates (Cho et al., 2007; Tekwa et al., 2015). Fill autoclavable plastic container 1/3rd full of 70% ethanol and PDMS devices and then cover with tinfoil, let them sit in this for >30 minutes before carefully disposing of the ethanol down the sink. Fill and empty the container with water 10 times in order to rinse the devices. Lastly, fill the container with filtered water, seal with tin foil and autoclave in order to sterilize the devices.
Inoculating the devices (perform in a Biological Safety Cabinet, BSC): Set the devices to dry in the BSC for 30 minutes. Place the devices with features facing up on the lid of a petri dish and place a small amount (i.e. 0.7 µL) of inoculum onto each (of 14 in sample device Fig. 1) habitat. The amount of liquid must be sufficient to fill the habitats, but not too much as to prevent bonding between PDMS and the cover glass/petri dish (Fig. 2). Using sterile tweezers, pick up the device and place face down into the centre of a petri dish or cover glass, sealing it to the surface by pushing on the back with gloved fingers repeatedly, using a kimwipe to wick excess liquid away from the side. Then surround, but not touch, the device with kimwipes soaked in filtered water (Fig. 3) to ensure that the device does not dry out in the incubator, before closing the petri dish. Place upright in incubator for the desired amount of time. The experiment can now proceed untouched for up to 24 hours (see Supplementary video).

Figure 2. Device with bacteria droplets, 1 droplet per habitat.

Figure 3. An ‘upright’ petri dish + kimwipes + device + coverslip ready to be incubated.

Recovering from the devices (perform in a BSC): Open petri dish, carefully remove and discard kimwipes and then use sterile tweezers to gently unseal the device and place face up in the lid of the petri dish. By around 12 hours, the spaces between the habitats will be void of liquid from PDMS absorption, preventing microbes from being mixed across chambers during disassembly. Habitats that have dried out will appear white (Fig. 4) and cannot be used. Dip sterile inoculating loop into an eppendorf with PBS, then use that loop to scrape one of the habitats (Fig. 5). Dip that inoculating loop back into the eppendorf media again, which can then be grown overnight for further analyses such as plating for relative cell count (if there are different strains) and other molecular analyses. Repeat for the rest of the habitats that you are interested in, using new inoculating loops. The PDMS device can now be cleaned as in Step 1 and reused again.

Figure 4. View of an inoculated and incubated device, looking through the bottom of a petri dish.

Figure 5. Recovering bacteria from a habitat in the disassembled PDMS device.

What else should I know?

The recovery technique can be used to estimate relative proportions of different types of microbes (e.g. morph frequencies) which is useful when performing competition assays and evolutionary experiments. Unlike in Tekwa et al. (2015), this technique forgoes the use of a confocal microscope; assessment of the contents of the device is instead performed through direct microbe recovery and standard plating procedures.

Links to Videos

These videos go through the specific procedure that we used to perform experiments on competition and cooperation in Pseudomonas aeruginosa and may be useful in determining specific amounts of media, growth times, etc. that may be utilized for experiments with similar PDMS microfluidic devices.

Part 1 – Intro + Washing the Devices https://youtu.be/Bne3ZN3wU4Q

Part 2 – Inoculating the Devices https://youtu.be/p-UYIRreYyM

Part 3 – Recovering from the Devices https://youtu.be/YLnmGxqMYgE

Supplementary – Fluorescent Bacteria Experiment https://youtu.be/lgMtryS62pA

Acknowledgements

AGr was supported by an NSERC Undergraduate Student Research Award and by an NSERC Discovery Grant. EWT was supported by the Fonds Québécois de la Recherche sur la Nature et les Technologies and the Québec Centre for Biodiversity Science. AGo was supported by the Canada Research Chair program and NSERC Discovery grants. DN was supported by a CFI Leaders Opportunity Fund (25636), a Burroughs Wellcome Fund CAMS award (1006827.01) and a CIHR salary award.

References

Cho, H., Jönsson, H., Campbell, K., Melke, P., Williams, J. W., Jedynak, B., … & Levchenko, A. (2007). Self-organization in high-density bacterial colonies: efficient crowd control. PLoS biology, 5(11), e302.

Connell, J. L., Ritschdorff, E. T., Whiteley, M., & Shear, J. B. (2013). 3D printing of microscopic bacterial communities. Proceedings of the National Academy of Sciences, 110(46), 18380-18385.

Folkesson, A., Jelsbak, L., Yang, L., Johansen, H. K., Ciofu, O., Høiby, N., & Molin, S. (2012). Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nature reviews. Microbiology, 10(12), 841.

Hol, F. J., & Dekker, C. (2014). Zooming in to see the bigger picture: Microfluidic and nanofabrication tools to study bacteria. Science, 346(6208), 1251821.

Keymer, J. E., Galajda, P., Lambert, G., Liao, D., & Austin, R. H. (2008). Computation of mutual fitness by competing bacteria. Proceedings of the National Academy of Sciences, 105(51), 20269-20273.

Or, D., Smets, B. F., Wraith, J. M., Dechesne, A., Friedman, S. P. (2007). Physical constraints affecting bacterial habitats and activity in unsaturated porous media – a review. Advances in Water Resources, 30(6), 1505-1527.

Park, S., Wolanin, P. M., Yuzbashyan, E. A., Silberzan, P., Stock, J. B., & Austin, R. H. (2003). Motion to form a quorum. Science, 301(5630), 188-188.

Tekwa, E. W., Nguyen, D., Juncker, D., Loreau, M., & Gonzalez, A. (2015). Patchiness in a microhabitat chip affects evolutionary dynamics of bacterial cooperation. Lab on a Chip, 15(18), 3723-3729.

Tekwa, E.W., Nguyen, D., Loreau, M., Gonzalez, A. Defector clustering is linked to cooperation in a pathogenic bacterium. In review.

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Rapid and easy fabrication of glass-bottom culture dishes for long-term live cell imaging

25 Jul 2017

By RSC Internet Services.

Ayako Yamada¹²³, Jean-Louis Viovy¹²³, Catherine Villard¹²³ and Stéphanie Descroix¹²³

¹ Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, Paris, France.

² Sorbonne Universités, UMPC Univ. Paris 06, Paris, France

³ Institut Pierre-Gilles de Gennes, Paris, France

Email: ayako.yamada@curie.fr

Why is this useful?

Glass is a versatile surface for chemical treatments, and it still is by far the most used substrate for surface engineering (e.g. micropatterning, surface chemistry) or plasma-bonding of PDMS microfluidic devices. For cell culture on such substrates, glass-bottom culture dishes are desired to keep over the cells well defined medium volumes, and to protect the cells from contamination and medium evaporation. Moreover, they are optically better suited for microscopy observation than polystyrene dishes routinely used for cell culture. Although glass-bottom culture dishes are commercially available (e.g. Fluorodish from WPI), the presence of plastic walls limits the treatments that can be performed onto the glass bottom surface and those are more expensive ( 5 € per dish; φ 50 mm) than polystyrene dishes (e.g. φ 40 mm dish from TPP, 0.5 € per dish). In this Tip, we describe an easier way than a previous Tip¹ to transform a polystyrene culture dish into a glass-bottom one, while preserving the possibility to apply to the glass any treatment before its assembly into a dish. Note that in this method, the body of the culture dish will be upside down and the lid is thus no longer lifted above the dish opening by lid stoppers. However, the gas exchange through the gap between the body and its lid seems to be enough to culture cells healthily in this dish. In summary, this Tip provides a low-cost and rapid solution for cell culture in a microfluidic device or on an engineered surface directly in a culture dish, suited for a long-term live cell imaging.

What do I need?

φ 40 mm polystyrene tissue culture dish (e.g. TPP #93040)
φ 40 mm cover slide (e.g. Thermo Fisher Scientific #11757065, ∼0.2 € per slide)
Large screw driver (or a similar tool)
Uncured mixture of PDMS base and curing agent (10:1 w/w)
Oven or hotplate
Ferromagnetic metal plate (e.g. lid of a PDMS container, optional)
Cylindrical magnets (optional)

What do I do?

Place a polystyrene culture dish upside down on a surface and hit a few times the center of the dish bottom with the grip of a large screw driver (Fig. 1a) until the dish bottom falls apart from the dish wall (Fig. 1b). The bottom should fall easily with the success rate around 9 over 10. Avoid breaking the dish wall by hitting the bottom too strongly.
Spread uncured PDMS mixture on a flat substrate (e.g. a larger plastic Petri dish) and coat the edge of the dish (broken part up) with PDMS (Fig. 1c).
Place the dish (broken part up) on a cover glass slide (Fig. 1d) and cure PDMS in an oven or on a hotplate e.g. at 80 °C for 10 min (Fig. 1e).
Surface treatment (e.g. micro-contact printing) or plasma-bonding of a PDMS chip to the glass surface can be performed after or before the dish assembly (Fig. 1f).

To keep humidity for on-chip cell culture, the dish can be filled with e.g. phosphate buffered saline (Fig. 2a). Dishes with chips or micropatterns loaded with cells can be placed in a CO₂ incubator with or without further protection (Fig. 2b).
Long-term live cell imaging can be performed using a stage top incubator (Fig. 2c).

What else should I know?

Depending on the support type of microscopes, it might be necessary to well align the contours of the dish and the glass slide. This can be done using cylindrical magnets (3 per dish) and a ferromagnetic metal plate (Fig. 3a) during PDMS curing in an oven or on a hotplate (Fig. 3b).

Acknowledgement

This work is supported by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” program (reference: ANR 10-NANO 0207) and ERC Advanced Grant CellO (FP7-IDEAS-ERC-321107).

Reference

[1] Caballero D, Samitier J, Different strategies for the fabrication of cell culture chambers for live-cell imaging studies. Chips and Tips, 02 Dec 2014 (https://blogs.rsc.org/chipsandtips/2014/12/02/different-strategies-for-the-fabrication-of-cell-culture-chambers-for-live-cell-imaging-studies/)

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Multilayer photolithography with manual photomask alignment

05 Jun 2017

By RSC Internet Services.

Frank Benesch-Lee, Jose M. Lazaro Guevara, and Dirk R. Albrecht

Worcester Polytechnic Institute, Worcester, MA 01609 USA

Why is it useful?

Modern microfluidic devices can incorporate channels of different heights to fulfill their designed function. Examples include hydrodynamic focusing [1], cell traps [2], and chambers that isolate cellular components [3]. These devices are fabricated from a multilayer SU-8 photoresist master mold. Each layer height requires a separate set of photolithographic steps, including photoresist spin, photomask alignment, exposure, and bakes, followed by a development step at the end to reveal the 3D resist pattern.

Mask aligners have microscopes and stage micrometers for precise, micron-scale alignment of each layer’s photomask with visible marks on the substrate wafer. They are indispensable tools for creating multilayer patterns with accurate registration, but while available in cleanrooms at many research universities, their substantial expense may place them out of reach of teaching institutions and individual laboratories.

In contrast, single-layer microfluidics can be prepared using an inexpensive UV light source, or even a self-made one [4]. In principle, manual photomask alignment could be made under a microscope, then brought to the UV source, yet this poses several complications. First, alignment features can be very difficult to see using inexpensive microscopes or stereoscopes, especially in thin SU8 layers, due to poor contrast between exposed and unexposed regions before development. Second, misalignment can occur during movement to the exposure system.

Here we present a manual photomask positioning method that yields a 50 µm accuracy, without the aid of a mask aligner.

What do I need?

Equipment and supplies for photolithography:
- Spin coater, and UV exposure system
- Substrate wafer and SU-8 photoresist
Small microscope (e.g. USB) or stereo microscope
Photomask transparencies for each layer
Scotch tape
Fine-tip permanent marker
Straight razor blade
Cutting mat
4 small (3/4”) or mini (1/2”) binder clips
Glass plate, approx. 4 x 5”, compatible with exposure system

What do I do?

Cut the photomasks from the transparency sheet, leaving 4 corner tabs. Align the two masks relative to each other under the microscope (Figure 1a) and clip them together with a binder clip. Ensure correct mask orientation and check alignment accuracy at multiple alignment marks across the mask. (Note that horizontal alignment accuracy with a stereomicroscope is low, because each eye’s optical path is angled 5 – 8 degrees, whereas vertical alignment is unaffected. Align in the vertical direction first then rotate the masks 90 degrees to ensure accurate alignment in both horizontal and vertical directions.) Add binder clips to each corner (Figure 1b), and verify alignment. Next, remove one binder clip at a time and use a straight razor blade to cut a sharp V-notch into each tab, through both masks. Press the blade straight down to avoid shifting the alignment. Replace the binder clip, and proceed to the next corner until all 4 notches are cut (Figure 1c).

Spin the first layer of SU-8 onto the wafer to the desired thickness and prebake. Attach 4 pieces of scotch tape onto the bottom of the wafer so that the sticky side faces up (Figure 2a). Position the first mask on the wafer, pressing gently to adhere it to the tape tabs. Use a fine-tip marker to trace the alignment notches (Figure 2b) onto the scotch tape (Figure 2c). Transfer to the UV exposure system and expose. Carefully remove the mask without detaching the scotch tape from the wafer and postbake. Scotch tape is compatible with 95 °C baking. Apply an additional piece of tape to cover the sticky tape tabs to protect the marker from smearing and allow smooth alignment of the next mask.

Spin coat the next photoresist layer and prebake (Figure 3a). Mount the wafer onto a glass plate with a loop of scotch tape to keep it in place. Position the second mask onto the wafer, ensuring that alignment “V” markings are centered within each alignment notch and across all 4 corners (Figure 3b). Affix the mask to the glass plate with thin (2-3 mm wide) pieces of tape, and adjust alignment as necessary. Carefully transfer the glass plate with wafer and aligned photomask for exposure (Figure 3c).

Repeat step 3 for any additional layers. Remove the tape tabs and develop the photoresist. Evaluate alignment accuracy under a microscope (Figure 4).

Conclusions:

In this tip, we present a method for manual alignment of multiple transparency photomasks. We achieved repeatable accuracy of <100 µm and as good as 50 µm (Figure 4a). These accuracies are within required tolerances of many multilayer designs (Figure 4b). In many cases, minor design alternatives can relax alignment tolerances, such as in a trap design containing a thin horizontal channel that allows fluid bypass but captures larger objects (Figure 4c). In this example, a 100 µm wide bypass channel only partially covered the trap indentations, whereas widening the bypass channel to 400 µm enabled a functional device despite slight misalignment. Overall, this simple method allows fabrication of microfluidic device molds containing multiple layer heights, without expensive mask alignment equipment, to an accuracy of at least 50 µm. Furthermore, after alignment marks are cut, no microscope is needed at all during the photolithography process, speeding the fabrication of multiple masters.

Acknowledgments:
Funding provided by NSF IGERT DGE 1144804 (FBL), Fulbright LASPAU (JMLG), University of San Carlos of Guatemala (JMLG), NSF CBET 1605679 (DRA), NIH R01DC016058 (DRA), and Burroughs Wellcome CASI (DRA).Acknowledgments:

References:

Chih-Chang, C., H. Zhi-Xiong, and Y. Ruey-Jen, Three-dimensional hydrodynamic focusing in two-layer polydimethylsiloxane (PDMS) microchannels. Journal of Micromechanics and Microengineering, 2007. 17(8): p. 1479.
Erickson, J., et al., Caged neuron MEA: A system for long-term investigation of cultured neural network connectivity. Journal of Neuroscience Methods, 2008. 175(1): p. 1-16.
Taylor, A. M., et al., A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods, 2005. 2(8): p. 599-605.
Erickstad, M., E. Gutierrez, and A. Groisman, A low-cost low-maintenance ultraviolet lithography light source based on light-emitting diodes. Lab on a Chip, 2015. 15(1): p. 57-61.

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A simple, bubble-free cell loading technique for culturing mammalian cells on lab-on-a-chip devices

28 Feb 2017

By RSC Internet Services.

Sahl Sadeghi^1* and Meltem Elitas¹

¹ Faculty of Engineering and Natural Sciences, Sabanci University, 34956, Istanbul, Turkey

^*Sahl Sadeghi wrote the paper.

Purpose

Lab-on-a-chip (LOC) devices significantly contribute different disciplines of science. Polydimethylsiloxane (PDMS) is one of the main materials, which is widely used for the fabrication of biological LOCs, due to its biocompatibility and ease of use. However, PDMS and some other polymeric materials are intrinsically water repellant (or hydrophobic), which results in difficulties in loading and operating LOCs. The eminent consequence of hydrophobicity in LOCs for biological systems is the entrapment of air bubbles in microfluidic channels. Although the oxygen plasma treatment of PDMS reduces the surface hydrophobicity for a certain period of time, the hydrophilic property of PDMS vanishes over time¹. The persistent problem of bubbles in the microfluidics led to several studies conducted to overcome it. Some of these solutions suggested implementing bubble traps²^,³, surface treatment of LOCs through hydrophilic coatings⁴, and using actively controlled bubble removal systems ⁵^,⁶.

Although the aforementioned design complexities are introduced to LOCs in order to reduce the clogging problem caused by the bubbles, these modifications also result in higher production cost, complex operation, and long device preparation time. In many single-cell experiments without losing or damaging the rare cells, these cells needs to be introduce into the LOCs. Here, we present a simple method that enables loading a small number of cells without introducing bubbles in the microfluidics channels.

Materials

· PDMS	(Dow Corning Sylgard 184 Silicon Elastomer Kit)
· Pipette tips	(20-200 ul, Eppendorf, # 3120000917)
· Pipetman	(Gilson, P200, #69989-5)
· Aqueous ethanol 70%	(ZAG Chemistry)
· Cell culture medium	(DMEM, PAN Biotech, #P04-01548)
· Mammalian cells	(MCF7, ATCC-HTB-22)
· Sterile syringe	(BD 10 ml Syringe, Luer-Lok Tip, #300912)
· Sterile Hamilton syringe	(Hamilton, 100 ul SYR, #84884)

Procedure

Step 1: Insert two 200-ul pipet tips at the inlet and outlet ports of the PDMS device as illustrated in Figure 1.

Step 2: Introduce a 70% aqueous ethanol into the inlet-pipet tip using a pipetman. Thus, the inner surface of microfluidic channels will be disinfected and the fluid flow will be tested within the micro channels as it is applied in many other protocols for LOCs⁷^,⁸.

Step 3: Gently apply pressure pressing the pipetman to force ethanol solution flow through the micro channels and cavities of the PDMS device. Take care to avoid applying negative pressure from the outlet-pipet tip, which might create air leakage through the pipet connections. Besides, applying a negative pressure will directly affect the amount of a gas dissolved in the liquid according to Henry’s law⁹ that might contribute formation of more bubbles inside the micro-channels. The positive pressure will facilitate removal of the air bubbles via dissolving them.

Step 4: After flushing the chip with ethanol solution, inspect the chip to ensure bubble removal. In case of air bubbles, repeat the steps 2 and 3.

Step 5: Fill the syringe (10 ml) with medium or phosphate buffered saline (PBS). Take care to remove the air bubbles inside the syringe, mount and lock the needle on the syringe. Then, flow medium through the needle too make sure the needle is full of medium without any bubble. Insert the needle in the inlet-pipet tip; gently apply positive pressure to replace the ethanol with medium. Next, collect the excess medium from the outlet-pipet tip.

Step 6: Fill the inlet pipet with fresh medium in such a way that due to certain height (h) between the levels of the medium in the inlet and outlet pipet tips, very slow medium flow will be established inside the micro-channels.

Step 7: Load your cells into the Hamilton syringe and take care to ensure that there is no air bubble inside its needle and syringe. Insert the needle of the Hamilton syringe into the inlet-pipet tip as explained in Step 5, Figure 1. Introduce the cells via applying gentle positive pressure to the syringe. Established flow streams in the PDMS chip will deliver the released cells to the desired positions in the chip. Flow rate can be arranged adjusting the applied positive pressure and amount of medium collected in the inlet and outlet pipet tips. The excess supernatant from the outlet-pipet tip can be collected, and fresh medium can be supplied through the inlet-pipet tip during the experiment.

References

Tan, S.H., N.T. Nguyen, Y.C. Chua, and T.G. Kang,. Biomicrofluidics, 2010. 4(3).
Zheng, W.F., Z. Wang, W. Zhang, and X.Y. Jiang,. Lab on a Chip, 2010. 10(21): p. 2906-2910.
Wang, Y., D. Lee, L.S. Zhang, H. Jeon, J.E. Mendoza-Elias, T.A. Harvat, S.Z. Hassan, A. Zhou, D.T. Eddington, and J. Oberholzer, . Biomedical Microdevices, 2012. 14(2): p. 419-426.
Wang, Y.L., C.E. Sims, and N.L. Allbritton,. Lab on a Chip, 2012. 12(17): p. 3036-3039.
Karlsson, J.M., M. Gazin, S. Laakso, T. Haraldsson, S. Malhotra-Kumar, M. Maki, H. Goossens, and W. van der Wijngaart,. Lab on a Chip, 2013. 13(22): p. 4366-4373.
Cortes, D.F., T.-X. Tang, D.G.S. Capelluto, and I.M. Lazar,. Sensors and Actuators B: Chemical, 2017. 243: p. 650-657.
Benavente-Babace, A., D. Gallego-Perez, D.J. Hansford, S. Arana, E. Perez-Lorenzo, and M. Mujika,. Biosensors & Bioelectronics, 2014. 61: p. 298-305.
Yesilkoy, F., R. Ueno, B.X.E. Desbiolles, M. Grisi, Y. Sakai, B.J. Kim, and J. Brugger,. Biomicrofluidics, 2016. 10(1).
Henry, W., Phil. Trans. R. Soc. Lond., 1803. 93: 29–274.

Figure 1 – Schematic of cell loading procedure in a microfluidic PDMS device.

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A novel low cost method to prepare a cross-linked gelatin membrane for potential biological applications

01 Feb 2017

By RSC Internet Services.

Gabriele Pitingolo¹and Valerie Taly¹

¹ INSERM UMRS1147, CNRS SNC 5014, Université Paris Descartes, Equipe labellisée Ligue Nationale contre le cancer 2016. Paris, France.

email: gabriele.pitingolo@parisdescartes.fr

Why is this useful?

In recent years, several gelatin types (i.e. GelMA, A or B) have been used in pharmaceutical formulation and tissue engineering due to their excellent biocompatibility, propensity to cell differentiation and availability at low cost.¹ The use of gelatin as biomaterial is also advantageous for the possibility to tune the mechanical properties of the substrate, changing the concentration in water and the degree of cross-linking. However, Transwell^® is the most used permeable support with microporous membranes and is a standard method for culturing cells.² This commercial type of support has been widely used to study the molecular secretion by different cell types and also to reproduce several in vitro physiological barriers (i.e. blood brain barrier).³ Transwell^® cell culture inserts are convenient, because they are sterile and easy-to-use, but they are very expensive (~ 300 $ for a 12 pack) and possess a limited range of biomaterial properties because they are made from polyester or polycarbonate. Furthermore, as shown by Falanga and colleagues, these porous membranes are often integrated onto microfluidic chip for permeability studies.⁴

Recently, Yong X. Chen et al. proposed an alternative method to prepare a suspended hydrogel membrane platform for cell culture, designing a complicated protocol to synthesize the GelMA and to fabricate an open-grid structure made of polylactic acid (PLA) polymer using a commercial printer.⁵ Our tip shows a novel low-cost method for preparing a cross-liked gelatin membrane as a permeable support useful for potential biological applications. In addition, the proposed protocol doesn’t require the use of sophisticated fabrication technologies or expensive materials. It uses gelatin from porcine skin and the formaldehyde vapor technique to cross-link the gelatin membrane. As proof of concept, we integrate the gelatin membrane into a microfluidic chip, to show the possibility to develop a platform for comparable studies in static and dynamic conditions.

Furthermore, to demonstrate the resistance to culture temperature (around 37° C) of the prepared gelatin membrane, we tested the mechanical properties (Young’s modulus) before and after the incubation time (2 days). Finally, we observed the preservation of the mechanical properties and structural integrity that makes the membrane usable for studies with cell culture.

What do I need?

Transwell insert
Porcine gelatin type A
Formaldehyde solution
Scalpel
PMMA milled chamber or similar

What do I do?

Remove the porous membrane from the Transwell^® insert (Fig. 1a-1c) or alternatively use a similar homemade support. To facilitate this step is convenient to use a scalpel to incise the membrane along the entire diameter.

After the removal of the membrane, the Transwell® support is ready to use. Position the structure at the center of the PMMA chamber (depth 2 mm at least) (Fig 2a) and pour liquid 10% w/v gelatin, without bubbles, onto the PMMA chamber and inside the Transwell^® support (Fig 2b). After 2 min of stabilization, put the system in the fridge, for at least 10 minutes.

After the gelation time (10 minutes at 4°C) it is possible to remove the formed gelatin membrane from the PMMA chamber, with the aid of a scalpel to facilitate the detachment (Fig 3a-3b). As shown in Figure 3c the gelatin membrane it appears very flat, an ideal characteristic for cell cultivation (Fig 3c). To guarantee the preservation of the mechanical properties during the cell culture step, cross-link the gelatin membrane using the classical protocol “cells-biocompatible”, such as glyceraldehyde⁶, formaldehyde⁷ and glutaraldehyde⁸ methods or natural products such as genipin.⁹

In this case, we used the vapor formaldehyde method to cross-link the prepared gelatin membrane and to obtain a system with lower aqueous solubility, higher mechanical strength and stability against enzymatic degradation. We exposed the gelatin membrane to formaldehyde vapors for 1 day. In Figure 4a we show the difference, after 48 h of culture conditions, between a sample cross-linked (left) and not (right). The final depth of the cross-linked gelatin membrane is around 1 mm, however, it is possible to change the depth tuning the liquid gelatin amount. Finally, we calculated, before and after the incubation time, the young’s modulus of the cross-linked gelatin membrane, observing a similar value of 40 kPa (compression test by using hydraulic testing system Instron DX).

Integration of the gelatin membrane into a microchip. In this section, we detail the integration of the gelatin membrane into a microfluidic chip. As example, we used the same geometry proposed in our previous work⁴, to make a device for a permeability experiment (Figure 5a). As shown in Figure 5b, just pour the liquid gelatin into the smaller drilled microchannel, using a pipette to form a thin uniform layer of gelatin (Figure 5c). After gelation at 4° C, cross-link the formed gelatin membrane using the previously described method, the result is shown in Figure 5d. To bond the different PMMA-PDMS substrates we propose here a magnetic approach recently developed by our group¹⁰, to preserve the gelatin membrane by physical-chemical stress as in the case of solvent evaporation and plasma bonding. Figure 5e shows the final chip.

Conclusions: In this tip, a novel biocompatible gelatin permeable support was obtained by using a simple and low cost fabrication method. Vapor formaldehyde method or other chemical crosslinking approach can be applied to crosslink the integrated gelatin membrane for the use as potential scaffolds for cell culture. Furthermore, the Young’s modulus and the thickness of the permeable membrane can be adjusted by changing the initial concentration of the gelatin, the degree of cross linking and the amount of liquid gelatin. Finally, we showed the integration of the gelatin membrane into a modular microchip. Therefore, we propose an easy and low cost method to prepare a permeable gelatin membrane for cell biology and for other applications.

Acknowledgements

This work was carried out with the support of the Pierre-Gilles de Gennes Institute equipment (“Investissements d’Avenir” program, reference: ANR 10-NANO 0207).

References

Geckil, Hikmet, et al. “Engineering hydrogels as extracellular matrix mimics.” Nanomedicine 5.3 (2010): 469-484.
https://www.corning.com/worldwide/en/products/life-sciences/products/permeable-supports/transwell-guidelines.html
Guarnieri, Daniela, et al. “Shuttle‐Mediated Nanoparticle Delivery to the Blood–Brain Barrier.” Small 9.6 (2013): 853-862.
Falanga, A. P., Pitingolo, G., Celentano, M., Cosentino, A., Melone, P., Vecchione, R. & Netti, P. A. (2016). Shuttle‐mediated nanoparticle transport across an in vitro brain endothelium under flow conditions. Biotechnology and Bioengineering.
Chen, Yong X., et al. “A Novel Suspended Hydrogel Membrane Platform for Cell Culture.” Journal of Nanotechnology in Engineering and Medicine 6.2 (2015): 021002.
Sisson, Kristin, et al. “Evaluation of cross-linking methods for electrospun gelatin on cell growth and viability.” Biomacromolecules 10.7 (2009): 1675-1680.
Usta, M., et al. “Behavior and properties of neat and filled gelatins.” Biomaterials 24.1 (2003): 165-172.
Talebian, A., et al. “The effect of glutaraldehyde on the properties of gelatin films.” Kemija u industriji 56.11 (2007): 537-541.
Bigi, A., et al. “Stabilization of gelatin films by crosslinking with genipin.” Biomaterials 23.24 (2002): 4827-4832.
Pitingolo Gabriele, et al. “Fabrication of a modular hybrid chip to mimic endothelial-lined microvessels in flow conditions.” Journal of Micromechanics and Microengineering” (Accepted manuscript)

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Electric drill/driver as centrifuge with 3D-printed custom holders for non-conventional containers

17 Oct 2016

By Liz Bowley.

Minkyu Kim^a, Guanya Shi^b, Ming Pan^c, Lucas R. Blauch^a, and Sindy K.Y. Tang^a*

^aDepartment of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA

^bUndergradute Visiting Research Program, School of Engineering, Stanford University, Stanford, CA 94305, USA

^cDepartment of Materials Science and Engineering, Stanford University, Stanford, CA 94305, USA

*sindy@stanford.edu

Why Is This Useful?

Many processes in biological and chemical preparation require centrifugation steps. The transfer of samples between the original sample container and tubes required by commercial centrifuges increases the risk of sample contamination, and often leads to the loss of samples. Commercial centrifuges are also not readily available outside laboratory settings. Here we show the design of a simple 3D-printed holder for attaching to the chuck of an electric drill/driver which we use as a centrifuge. The advantages of this method include: 1) The holder can be designed to hold non-conventional containers (e.g., syringes, glass vials, capillaries). 2) Electric drill/drivers are more widely available than centrifuges. We show that a variety of samples (e.g., water-in-oil emulsions, cell suspensions, food and drinks, wet soil) in various containers can be centrifuged with our method. This method should be useful for field work outside of the laboratory, and for the wider DIY community interested in home-based applications that require centrifugation, such as blood separation and related diagnostics, separation of interstitial water from wet soil for pollution detection, extraction and identification of allergens in food samples, and fluid clarification (e.g., olive oil, wine) by accelerating sedimentation.

What do I need?

Figure 1. Photo of components needed.

Electric drill/driver (DeWalt DC742KA Cordless Compact Drill/Driver Kit [1]).
3D-printed custom holder.
3D-printed custom support for the drill/driver.
Four 1 mL-syringes (NORM-JECT, Part No.: 4010-200V0) as an example of non-conventional sample containers.
One long bolt (Pan Head Machine Screw, Zinc, #8 x 1-1/4”) and one matching nut (Hex Nut, Zinc, #8-32). The dimensions of the bolt should match the chuck of the drill/driver.
Four short bolts (Pan Head Machine Screw, Zinc, #6 x 1/2”) to secure the 1 mL-syringes to the 3D-printed custom holder.

What do I do?

Design a custom holder using Solidworks or other CAD software.
1. Measure the outer diameter (w₁= 6.5 mm) of the 1 mL-syringe (Fig. 2a). We included a 0.5 mm-tolerance in deciding the width of the slot (w₂ = 7 mm) into which the 1 mL-syringe will be secured (Fig. 2b).
2. Decide the angle (q = 60°) at which the 1 mL-syringe will be tilted relative to the plane of rotation.
3. Decide the length (w₃ = 80 mm) and the thickness (w₄ = 15 mm) of the holder.
4. Measure or identify the outer diameters of the short and long bolts, and use these values for f3 and f5 respectively.
Design a custom support for the drill/driver (Fig. 2c). The dimensions of this support are not critical so long as the drill/driveris stable and does not topple during operation.
3D-print the holder and the support. We used a 3D-printer by ROBO 3D [2]. The resolution of the 3D-printer in xyz direction is 100 mm. The material we used was polylactic acid (PLA).
Assemble the centrifuge (Fig. 2d).
1. Put one long bolt through the center of the holder and tighten with the nut.
2. Insert the bolt into the chuck of the drill/driver and tighten the bolt by pushing the trigger of the drill/driver a few times.
3. Make sure the bolt is fixed in the chuck and aligned to the drill/driver.
4. Place the drill/driver in the 3D-printed support.
5. Secure four 1 mL-syringes to the holder using the four short bolts.
Start the centrifuge by pushing the trigger of the drill/driver for 5-10 minutes. The plane of rotation should be parallel to the floor.
Unscrew the short bolts to remove the syringes.
If desired, measure the rotational speed of the drill/driver before inserting the real samples. We used the SLO-MO mode in iPhone to calibrate the rotational speed of the drill/driver [3] (Fig. 2e).
Results (Fig. 3).
1. Water-in-oil emulsion: Micron-sized uniform water-in-oil droplets were collected in a 1 mL-syringe. After a needle was connected to the syringe, the syringe was secured to the 3D-printed holder with needle pointing up. The holder was balanced before centrifugation by adding another syringe containing an equal weight of fluids to the opposite side of the holder. We centrifuged the sample at a speed of 374 rpm for 10 minutes. Droplets were then injected into a microchannel to measure the change of volume fraction before and after centrifugation. The volume fraction was defined as the ratio of the total volume of water droplets to the total volume of fluids filling up the channel. After centrifugation, the volume fraction of the emulsion increased from 60% to 86% without a change in the size of the droplets. Neither break-up nor coalescence of the droplets was observed.
2. Stentor coeruleus: To demonstrate the concentration of cell suspensions, we used Stentor coeruleus (www.carolina.com) as a model. We filled a 3 mL-syringe with about 20 Stentor cells suspended in 2 mL of aqueous culture media (concentration ~ 10 cells/mL). A needle was connected to the syringe which was then secured in the 3D-printed holder with the needle pointing down. The cell suspension was centrifuged at a speed of 374 rpm for 5 min. The cells were concentrated at the bottom, close to the entrance into the needle. It was then possible to inject this concentrated cell suspension through the needle into a polyethylene tubing. Fig. 2-i) shows the microscopic image of the cells in about 15 mL of aqueous culture media in the tubing (concentration ~ 400 cells/mL).
3. Korean rice wine (Makgeolli): A separate holder was designed to hold a 20 mL-glass vial. The vial was filled with 12 mL of Korean rice wine and centrifuged at a speed of 1252 rpm for 10 minutes. The sediment was clearly observed after centrifugation. On the other hand, the sediment was not observed for more than 20 minutes without centrifugation.
4. Wet soil: 10 mL of wet soil in a 20 mL-glass vial was centrifuged at a speed of 1252 rpm for 10 minutes. Interstitial water was separated from the soil.

Figure 2. a) A 1-mL syringe as non-conventional container. b) Drawing of 3D-printed holder generated by SolidWorks. c) Drawing of 3D-printed support generated by SolidWorks. d) Photograph of experimental setup. The 3D-printed holder was connected to the drill/driver placed on the 3D-printed support. Four 1 mL-syringes were then tightened using the short bolts. e) Calibration plot of the rotational speed versus the trigger levels on different modes of the drill/driver. Mode 1 and Mode 2 indicate different gear settings in the transmission of the drill/driver. The numbers 1 to 3 in each mode indicate user-defined trigger levels. The rotational speed ranges from 120 rpm to 1252 rpm. The rotational speeds were measured using SLO-MO function in iPhone 6.

Figure 3. a) Photographs of emulsion in 1 mL-syringe and microscopic images of emulsion injected into a microchannel before and after centrifugation. The scale bar in the photographs is 5 mm. b) Photographs of Stentor cells in 3 mL-syringe before and after centrifugation. The red arrows indicate individual cells. The scale bar is 5 mm. i) Microscopic image of 6 Stentor cells in a polyethylene tubing after centrifugation. The scale bar is 300 mm. c) Photographs of Korean rice wine in 20 mL-glass vial before and after centrifugation. The red box indicates sediments. The scale bar is 10 mm. d) Photographs of wet soil in 20 mL-glass vial before and after centrifugation. The red box shows interstitial water separated from wet soil. The scale bar is 10 mm.

What else should i know?

The centrifugal force can be increased by lengthening the arms (w₃) holding the containers.
The rotational speed can be measured using a high-speed camera or a smart phone with slow-motion videotaping capability, so long the frame rate is sufficient for the rotational speed used.
The load in the centrifuge should be balanced.
For safety purposes, safety goggles should be worn. The centrifuge should also be placed inside a safety barrier (e.g., a sturdy laundry basket). The safety instructions for the drill/driver should also be observed.
After centrifugation for 10 minutes, we found that the drill/driver started to heat up. If centrifugation time longer than 10 minutes is needed, it should be possible to perform multiple rounds of 10-min centrifugation steps with breaks in between to cool down the drill/driver.

Conclusion
In this work, we demonstrated that 3D-printed holders attached to an electric drill/driver can be used for the centrifugation of samples in non-conventional containers. As 3D-printers and hand drills are easily accessible, we expect this tip to find immediate use in settings outside laboratories for field work, and also at home for DIY users.

Acknowledgements

We acknowledge support from the Stanford Woods Institute for the Environment and the National Science Foundation (Award #1454542 and #1517089).

References

1. http://www.dewalt.com/products/power-tools/drills/drills-and-hammer-drills/12v–38-10mm-cordless-compact-drilldriver-kit/dc742ka

2. http://store.robo3d.com/collections/all/products/r1-plus-3d-printer?variant=6274616835

3. http://www.imore.com/how-to-record-video-iphone-ipad

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Simple and Low-cost Contact Angle Measurements Using a Smartphone with a PDMS-Lens

19 Jul 2016

By Catherine McKenna.

Jonas M. Ribe^a, Nils R. Skov^b, Ole-Andreas K. Kavli^a, Armend G. Håti^a, Henrik Bruus^b and Bjørn T. Stokke^a

^a Department of Physics, Norwegian University of Science and Technology, NO–7491 Trondheim, Norway

^b Department of Physics, Technical University of Denmark, DK–2800 Kongens Lyngby, Denmark

jonas.ribe@ntnu.no

Why Is This Useful?

Contact angle measurements are important for characterizing the wettability of a liquid to a solid surface. In microfluidics they are of special interest as they provide insight into the intermolecular interactions between the sample liquid and the microchannel surface. Contact angle measurements are also important when assembling polydimethylsiloxane (PDMS) devices using oxygen plasma bonding. For optimal bond strength the water contact angle of plasma treated PDMS should be minimized as shown by Bhattacharya et al. [1] A current hurdle in measuring contact angles is the requirement of a setup that is expensive and non-portable. Here we show a method for measuring contact angles using materials and equipment found in a typical microfluidics lab.

What do I need?

Consumables:

Equipment:

Smartphone
Digital scale
Desiccator with vacuum pump
Oven
Syringe pump (optional)
Light source

For measurements:

Pipette (0.5–3μL)
Sample (e.g. deionized (DI) water or other liquid sample)

What do I do?

Prepare PDMS:

Weigh 10:1 PDMS (Sylgard 184) in a plastic cup on the digital scale
Mix the PDMS by hand using a plastic spoon
Degas the PDMS in a desiccator to remove the bubbles

Make PDMS-lens:

Use the tip of the plastic spoon handle (or a pipette) to place a small droplet of uncured PDMS in the center of a glass cover slip. Repeat with various amounts of PDMS to obtain lenses with varying magnification.
Mount the cover slips upside down (e.g. between two glass slides) and cure the PDMS hanging at 70 °C for 15 min. Longer curing times might be necessary, if the drop is relatively large.
Center the PDMS-lens over the camera of your smartphone and fixate it using tape.
Test the focus of your camera. For our camera setup the best images were captured with lenses that focus around 2 cm.

Contact angle measurements:

Smartphone contact-angle setup: (A) Focus test of a PDMS lens. (B-C) The smartphone mounted on a syringe pump. The PDMS-lens is mounted on the front facing camera of an iPhone 6S and the sample is centered in front of the lens. The sample is mounted on the pusher block of a syringe pump which can be moved to adjust the focus.

Make a sample stage preferably using a syringe pump or some other system that you can move. We mounted the smartphone on the syringe holder block with the camera pointing towards the pusher block. Make a sample holder on the pusher block using glass slides or other consumables found in the lab. Align the center of the stage with the center of the camera. Tip: aligning is easier if done using the sample that you want to measure. Put the sample on the block and move it into focus by releasing the pusher block and sliding it away/towards the camera. Increase the height of the stage until the top of the sample is centered in the camera.
Place the light source behind the sample and illuminate the stage evenly. Tip: put the sample stage in front of a white wall and light up the wall for a homogenous background and optimal contrast.
Place a small drop (0.5–3 μL) of DI water on top of the sample using a pipette. Place the drop near the sample edge closest to the camera.
Move the sample edge into focus. Block out ambient light in the room.
Measure the contact angle of the drop in the image e.g. using ImageJ [2] software with a plugin for contact angle measurements [3] or get a rough estimate using an app on your smartphone.[4]

Contact angle measurements of water on PDMS: (A) Raw image from iPhone 6S front-facing camera with PDMS-lens. (B) Direct measurement using app on smartphone (based on θ/2 calculation) (C-E) ImageJ measurements using DropSnake plugin. Unmodified PDMS (C) and PDMS treated with oxygen plasma with increasing intensity (D-E).

What else should I know?

The focal length of the PDMS-lens is determined by the volume of PDMS used as described by Lee et al. [5]. However, it is difficult to control the volume of PDMS using a pipette due to the high viscosity of PDMS. We recommend making a range of lens sizes and testing them on your smartphone camera to see which gives the right focal length. If your digital scale has milligram precision you can measure the amount of PDMS used for each lens. The mass of each PDMS-lens is typically less than 10 mg. You can decrease the focal length further by adding PDMS to an already cured lens. Modern smartphones have both a rear-facing and a front-facing camera and in our experience the drop focusing was easier when using the front facing camera. The images taken here were captured with an iPhone 6S from Apple using the front-facing camera with a 5MP sensor. The weight of the cured PDMS lens was 7 mg.

Tip: you can also remove the PDMS-lens from the cover slip and place it directly on your camera. Although, it might be more difficult to center.

Calculating contact angles from images of sessile drops can be done using a range of techniques.[6] If the drop volume is small and the contact angles are not extreme, we can generally neglect droplet distortion due to gravitational effects. Extrand and Moon [7] calculated that gravitational effects can be neglected for a water droplet sitting on a hydrophilic surface (θ=5°) if its volume is less than 5 μL and less than 2.7 μL on a hydrophobic surface (θ=160°). If we assume the drop to be spherical, the contact angle can be estimated by multiplying the angle between the base and the height of the droplet by 2. This is referred to as the θ/2-method and is implemented by e.g. the Contact Angle Measurement app [4] for iOS. Sessile drop measurements are generally limited by the experimental setup and operator error, but typically has a precision of ±3°.[8] Image-processing algorithms relying on curve fitting of the droplet outline can enhance reproducibility. ImageJ [2] with DropSnake-plugin [3] uses active contours (energy minimization) to track the outline of the drop and calculate contact angles. This increases precision, but is slower and currently requires analysis on a separate computer.

Acknowledgements

The Research Council of Norway is acknowledged for the support to the Norwegian Micro- and Nanofabrication Facility, NorFab (197411/V30).

References

S. Bhattacharya, A. Datta, J. M. Berg and S. Gangopadhyay, J. Microelectromech. S., 2005 14, 590–597
ImageJ software
DropSnake ImageJ-plugin for contact angle measurements
Contact Angle Measurement iOS app (Japanese)
W. M. Lee, A. Upadhya, P. J. Reece, and T. G. Phan, Biomed. Opt. Express, 2014, 5, 1626–1635
Y. Yuan and T. R. Lee, Surface Science Techniques, Springer, Berlin/Heidelberg, 2013, 51, 3–34.
C. W. Extrand and S. I. Moon, Langmuir, 2010, 26, 11815–11822.
A.F. Stalder, G. Kulik, D. Sage, L. Barbieri and P. Hoffmann, Colloids and Surfaces A: Physicochem. Eng. Aspects, 2006, 286, 92–103.

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Electric drill/driver as centrifuge with 3D-printed custom holders for non-conventional containers

Simple and Low-cost Contact Angle Measurements Using a Smartphone with a PDMS-Lens

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