Name: creating transient cell membrane pores using a standard inkjet printer
Uploaded: 2012-03-16T12:00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer at JoVE.com

The authors would like to acknowledge Dr. Thomas Boland for the idea of using the HP DeskJet printers for cell printing. Funding for this project from NSF RII-EPS 0903795 and NIH K25 HL0922280.

1. Converting the HP DeskJet 500
It should be noted that this technique should work with many commercially inkjet printers. However, older printers tend to work better as they use ink cartridges with larger diameter nozzles, which do not clog as easily. In addition, older printers tend to use mechanical paper feed sensors that are easier to bypass. Printers with optical sensors can be tricked but using a small strip of paper on the far edge of the printer during each cycle, but is a bit more difficult than the mechanical system to "trick". The currently commercially available printers that work the best have low resolution (DPI).
Higher resolution printers tend to clog more easily. The resolution of the HP Deskjet 500 is 300 DPI. There are many commercially available printers (HP Deskjet series and others) that have a resolution of 600 DPI. This type of printer can be used with only a small increase in nozzle clogging issues that can be alleviated using careful cleaning (section 3).
<ol>
	<li>Remove the top plastic case of the printer by unlocking several plastic clips from the bottom base of the printer and slowly lifting the top off.</li>
	<li>Unscrew button/display light panel from top of printer, leaving it connected to the printer's motherboard.</li>
	<li>Clean the inside of the printer, especially the areas where the ink cartridge rests and where printing occurs.</li>
	<li>Locate the cables supplying power to the paper feed mechanisms and unplug them from the motherboard.
	<ol>
		<li>In the HP DeskJet 500, these are found just below the paper tray towards the left of the front.</li>
	</ol>
	</li>
	<li>Locate paper detection mechanism. Bypass the mechanism by affixing a string or wire loop to serve as a manual pull handle.
	<ol>
		<li>In the HP DeskJet 500, the paper detection mechanism is a gray plastic lever found above and behind the printing/paper feed mechanism.</li>
	</ol>
	</li>
	<li>Create a stage in front of the paper feed mechanism (where the paper would be fed from and deposited) in order to bring the desired printing slides to a level just below the cartridge print head.
	<ol>
		<li>For our experiments, the foam shipping holder for 15 mL centrifuge tubes was used with several microscope slides taped in place in the printing region to bring the final level of the slides to the desired height.</li>
	</ol>
	</li>
	<li>To maintain aseptic technique, the printer can be placed inside a standard biohazard cabinet or tabletop laminar flow hood.</li>
	<li>It is important to note that modifying a commercially available ink-jet printer will usually void the printer's manufacturer warranty.</li>
</ol>
2. Converting Stock HP Ink Cartridges (HP 26 Black ink cartridge)
<ol>
	<li>Remove the cartridge from its packaging, leaving the protective tape covering the printer contacts and print head for the time being.</li>
	<li>Stabilize the body of the cartridge (black portion) either with a clamp or vise (simply firmly gripping the cartridge by hand usually worked as well), leaving the green top clear of any obstacle.</li>
	<li>Using pliers or an adjustable wrench, grasp the green top of the cartridge and twist back and forth several times until it breaks free.
	<ol>
		<li>This should not take much force, but the top is no longer needed, so it is okay if it breaks when removing it.</li>
	</ol>
	</li>
	<li>Using screwdrivers, pry off the clear plastic piece now exposed.
	<ol>
		<li>Again, this should not take much force, but it will not be needed, so it is okay if it breaks during removal.</li>
	</ol>
	</li>
	<li>Empty any remaining from the reservoir.</li>
	<li>Remove the plastic protective tape covering the printer contacts and print head.</li>
	<li>Thoroughly flush the reservoirs with water.
	<ol>
		<li>Use a pipette or syringe to push water through the channels.</li>
		<li>Water will likely leak from the print head during this process; this is acceptable, and does not cause any damage to the functionality of the cartridge.</li>
	</ol>
	</li>
	<li>When the water runs clear, allow the cartridge to dry.</li>
</ol>
3. Cleaning Ink Cartridge
<ol>
	<li>In order to maintain a clean printing environment, the ink cartridge should be cleaned before and after use.
	<ol>
		<li>This also helps with avoiding crystallization of salts and other biological material in the cartridge that could cause blockages.</li>
	</ol>
	</li>
	<li>Fully submerge the cartridge in a beaker full of de-ionized water, and sonicate for 15 minutes before and after printing.</li>
	<li>After sonication, remove the cartridge from the water, and shake out excess water.</li>
	<li>Spray 70% ethanol into the cartridge to create a more aseptic environment.
	<ol>
		<li>Ensure that the ethanol has dried before adding the bioink printing solution.</li>
	</ol>
	</li>
</ol>
4. Making Cell Suspension - "Bioink"
<ol>
	<li>Culture cells until ready to passage.
	<ol>
		<li>For 3T3 fibroblasts: Seed cells on T-75 flasks at approximately 1.3x104 cells/cm2 in Dulbeccos Modified Eagles Medium (DMEM) with 10% Fetal Bovine Serum (FBS). Culture cells for two days in an incubator at 37 &deg;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Make fluorescent g-actin stock solution at concentration 50 &mu;g/ml in Phosphate Buffer Solution (PBS).</li>
	<li>Passage cells.
	<ol>
		<li>For 3T3 fibroblasts: Remove media from flask and rinse twice with PBS. Cover cells with 5 ml 0.25% Trypsin+EDTA and incubate for 5 mins.</li>
		<li>Add 5 ml of fresh medium and pipette the cell suspension into a 15 ml conical centrifuge tube and centrifuge at 1000 rpm for 5 min. Aspirate spent medium.</li>
	</ol>
	</li>
	<li>Resuspend the cells in PBS with fluorescent g-actin stock solution to create bioink.
	<ol>
		<li>Bioink should have a final concentration of g-actin of 10 &mu;g/ml and a cell concentration of 1x105 cell/ml. This concentration has been optimized to limit amount of cells per drop and clogging of the printer head.8</li>
		<li>Note that 250 &mu;l of bioink prints three cover slips with the pattern shown in Figure 2.</li>
	</ol>
	</li>
</ol>
5. Bioprinting
<ol>
	<li>Power on and let the printer warm up.</li>
	<li>Place desired printing surface (here, we use 22 mm x 22 mm coverslips) on the center of the stage prepared in step 1.6.</li>
	<li>Create a printing pattern file.
	<ol>
		<li>Open Microsoft Word (or any other drawing software), and draw the desired pattern.</li>
		<li>Pick a desired print pattern for cell printing in the premade file.</li>
	</ol>
	</li>
	<li>Load the prepared cartridge with desired cell suspension.
	<ol>
		<li>Pipette suspension into the small circular well at the bottom of cartridge compartment. Use approximately 100-120 &mu;l of solution. The printed drop size is around 130 picoliters.8</li>
	</ol>
	</li>
	<li>Print the file with the HP Deskjet 500 Printer.
	<ol>
		<li>For best results, print smaller patterns multiple times (5), by changing the amount of copies desired in the word processing program.</li>
	</ol>
	</li>
	<li>Printer will warm up again, then cartridge will move to the "ready position".</li>
	<li>When cartridge moves into the ready position (slightly to the left of the ink drip well below and stays there), pull up on the paper feed mechanism wire, and printing should commence.
	<ol>
		<li>For multiple copies of the printing, the paper feed mechanism will have to be released after every cycle or page printed. The cartridge will then return to the "ready" position, and the paper feed mechanism wire should be raised again.</li>
	</ol>
	</li>
	<li>The printer will print onto the coverslip placed on the center of the printing stage in step 5.2 (see Figure 2).</li>
</ol>
6. Representative Results
The representative results of the entire conversion process of a standard, off-the-shelf HP Deskjet 500, standard HP 26 series cartridges will create a printer with the capability of printing cell solutions for many types of analysis as stated before. The completed printer after conversion is shown in Figure 3, with a printing stage to place the microscope cover slip onto. The printer can be useful in analysis in many fields, including but not limited to: single cell mechanics, tissue engineering, gene transfection, biosensor micropatterning, and direct cell therapies. 1-10, 16-22
In this example, an HP Deskjet 500 printer and HP 26 series ink cartridges were modified for bioprinting. Using this printer setup with a bioink consisting of a fibroblast cell suspension in a g-actin monomer solution, cells were printed onto glass microscope coverslips. Figure 1 illustrates representative results of printed fibroblast cells, which show incorporated fluorescent actin monomers. The results were obtained in a controlled aseptic environment in which the cells were printed into customizable patterns.
The pattern used in this example was created in Microsoft Word (Figure 2). This pattern created a continuous line of the printing solution across the majority of the microscope slide. Figure 4 shows the straight line in which the bioink and cells are mutually deposited. It should be noted that immediately after printing, there is an increase of background fluorescence because the bioink solution, in which the cells are suspended, contains free excess fluorescent actin monomers. This background fluorescence significantly decreases after the addition of growth media on the cells (Figure 1), which washes excess monomer from the substrate.
<img alt="Figure 1." src="/files/ftp_upload/3681/3681fig1.jpg" /> 
Figure 1. Representative images of 3T3 fibroblasts 3 hours after printing using the modified inkjet printer. The interior of the cells show incorporated fluorescently tagged actin monomers. Scale bars represent 50 &mu;m.
<img alt="Figure 2." src="/files/ftp_upload/3681/3681fig2.jpg" /> 
Figure 2. Design used to print bioink.
<img alt="Figure 3." src="/files/ftp_upload/3681/3681fig3.jpg" /> 
Figure 3. Printer after conversion with Styrofoam stage. The orange wire in the middle bypasses the paper feed lever sensor.
<img alt="Figure 4." src="/files/ftp_upload/3681/3681fig4.jpg" /> 
Figure 4. Representative image of 3T3 fibroblasts printed in a localized printing zone. Microscopy image taken 5 minutes after printing at 20x magnification.
<img alt="Figure 5." src="/files/ftp_upload/3681/3681fig5.jpg" /> 
Figure 5. Fluorescence image (40x magnification) taken 3 hours after printing showing a fibroblast with internalized fluorescent actin monomers. Scale bar represents 50 &mu;m.
<img alt="Figure 6." src="/files/ftp_upload/3681/3681fig6.jpg" /> 
Figure 6. Fluorescent microscopy (20x magnification) of a cell taken 15 minutes after printing. The image shows both g-actin (green) and the nucleus (blue). From left to right: g-actin with Alexa Fluor 488, nucleus with DAPI, and an overlay of both.
<img alt="Figure 7." src="/files/ftp_upload/3681/3681fig7.jpg" /> 
Figure 7. Fluorescent microscopy showing two fibroblasts in a line pattern 3 hours after printing. Image on left shows fluorescently labeled actin monomers inside cells. Image on right is an overlay of the fluorescent channel with the background image to show that although there may be some debris on the slide (bottom left corner), it does not fluoresce. Scale bars represent 50 &mu;m if size not indicated the far right is a Control group of non-printed cells incubated for 3 hours with fluorescently tagged monomers. This control is to show that monomers could not penetrate the cell membrane without membrane permeabilization by cell printing.

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Part A. 16, 1749-1759 (2010).</li><li>Deitch, S., Kunkle, C., Cui, X., Boland, T., Dean, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+matrix+alignment+using+inkjet+printer+technology.">Collagen matrix alignment using inkjet printer technology.</a> Proc. 1094 (DD. , 7-16 (2008).</li><li>Ilkhanizadeh, S., Teixeira, A., Hermanson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+printing+of+macromolecules+on+hydrogels+to+steer+neural+stem+cell+differentiation.">Inkjet printing of macromolecules on hydrogels to steer neural stem cell differentiation.</a> Biomaterials. 28, 3936-3943 (2007).</li><li>Ringeisen, B. R., Orthon, C. M., Barron, J. A., Young, D., Sparago, B. 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We have nothing to disclose.

The process to convert a standard inkjet desktop printer for bioprinting is not particularly difficult. The most challenging step is determining how to bypass the paper feed mechanism, which is dependent on the brand and model of printer used. However, this is relatively simple when the paper feed sensor is mechanical as described here. For models with optical feed sensors, other techniques may need to be employed to trick the printer into thinking it is using paper; for instance, one can run a small piece of paper through the printer while it prints on the microscope slide. Bypassing the paper feed mechanism is likely to be the most difficult step in applying these procedures to different printer models.
When constructing a stage to hold the coverslips for printing, it is important to ensure proper alignment and height. The stage should allow for the coverslip to be placed in the middle of the printing area. In addition, it should place the coverslip at an adequate height to allow clearance for the print cartridge to pass over the slide without disrupting it. The exact height of the stage will depend on the printer model.
To ensure that printed cells do not get washed away, the slides were placed in an incubator immediately after printing for approximately 30 minutes to allow for cell attachment before additional cell growth media was added. Because the cells were printed in a solution that includes large amounts of PBS the cells did not dry out and remained viable. Cells were imaged using fluorescence microscopy to visualize the distribution of the tagged g-actin monomers inside the cell (Figures 1, 5-7). When printing with 3T3 fibroblasts, Figure 5 shows a representative result, with the actin causing much of the cell to fluoresce, but showcasing lines of increased brightness. The incorporation of fluorescently tagged g-actin into the cytoskeleton is useful for the study of cytoskeletal dynamics and cellular mechanics.12-15 A limitation of this technique is that it is only applicable for molecules and proteins that have diameters smaller than approximately 10 nm.
To ensure that cells were actually being processed by the converted printer, DAPI (1:5000 in PBS) was added to the bioink in place of half of the volume of pure PBS. The fluorescent stain DAPI binds to amino acid rich portions of DNA located in the nucleus. Therefore DAPI is a useful stain in fluorescently imaging nuclei of the printed cells. Figure 6 shows a representative image of a cell displaying both Alexa Fluor 488 (actin) and DAPI (nucleus) fluorescence with an overlay image of both. Figure 7 also illustrates how the cells will fluoresce once the g-actin monomers have been incorporated while non-biological material that has been deposited into the sample will not fluoresce because of the absence of g-actin monomers.
One important consideration for bioprinting is the aqueous media being used for the bioink. It was found that using standard cell growth media with serum yielded inconsistent results. This is most likely due to clogging of the print-head nozzles by serum proteins. The use of PBS increased the consistency of printed patterns and the number of cells deposited. One drawback to using PBS is that cells should not be left in the suspension for prolonged periods of time. However, fibroblasts in these examples were able to tolerate the bioink conditions for at least an hour with no change in cell viability. This is consistent with several prior findings, which report that cells printed via thermal inkjet mechanisms have been shown to have high viability rates.2-8
This modified printer setup can be used for applications other than cell printing. 16-22 Matrix proteins, such as collagen or fibronectin, can be easily printed onto substrates with this technique, which can be useful for cell patterning. For instance, collagen type I printed into line patterns will result in aligned collagen substrates that can be used for in vitro cell culture studies.17 In addition to matrix proteins, other molecules, including growth factors, can be reliably localized on substrates for cell studies and potential therapeutic applications.18
A major limitation of the design described in the above steps is that this printer is not capable of printing in more than one dimension. This limits the potential for use in patterned applications such as scaffold printing. To allow for 3D printing, a specialized stage needs to be used. The stage needs to have incremental height adjustments for layer-by-layer deposition of bioink.
Bioprinting has shown promise as an efficient and cost-effective method for tissue engineering, gene transfection, micropatterning and microarray fabrication.1-12, 16-22 The future applications of this type of device are numerous, including: creation of controlled and patterned cellular microenvironments, incorporation of macromolecules into cell cytoplasm, and deposition of cells onto scaffolds and structures that do not occur naturally or efficiently.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>HP DeskJet 500</td>
			<td>Hewlett-Packard</td>
			<td>C2106A</td>
			<td>Discontinued from manufacturer. Purchased refurbished DEC Trader.</td>
		</tr>
		<tr>
			<td>HP 26 Black Ink Cartridge</td>
			<td>Hewlett-Packard</td>
			<td>51626A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Actin from rabbit muscle, Alexa Fluor 488 conjugate, 200 &mu;g</td>
			<td>Invitrogen</td>
			<td>A12373</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>MP Biomedicals</td>
			<td>ICN1860454</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbeccos Modified Eagles Medium (DMEM)</td>
			<td>Thermo Scientific</td>
			<td>SH3002201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td>Used at 0.5% in cell culture media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>Used at 0.5% in cell culture media</td>
		</tr>
		<tr>
			<td>Unidirectional Flow Clean Bench</td>
			<td>Envirco</td>
			<td>VLF 797</td>
			<td>Optional housing for keeping printer aseptic</td>
		</tr>
	</tbody>
</table>

creating transient cell membrane pores using a standard inkjet printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

Watch this Scientific Journal Video about Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer at JoVE.com

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

creating-transient-cell-membrane-pores-using-a-standard-inkjet-printer

Research

JoVE Journal

Bioengineering

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.