Name: improved visualization and quantitative analysis of drug effects using micropatterned cells
Uploaded: 2010-12-02T18:00:00
Duration: 15 min 41 s
Description: Watch this Scientific Journal Video about Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells at JoVE.com

1. 	Cell Preparation and Seeding on Micropatterns
<ol>
<li>Place 2 CYTOOchips in 2 independent wells of a 6 well plate ensuring that you read "CYTOO" in the lower right corner of the CYTOOchip.
Micropatterns can be fluorescently labeled with Cy3 or Cy5 dyes. CYTOOChips used for this experiment have Cy3 fluorescently labeled micropatterns. 
CAUTION: Keep chips in the dark.
</li>
<li>Collect HeLa cells (~80% confluent) by trypsinization. Check under a microscope that cells are properly dispersed by the trypsin treatment. Collect and centrifuge cells at 300g for 4 min, then gently resuspend the cell pellet. Count cells and dilute to a concentration of 15,000 cells/ mL in completed DMEM/F12 culture media.</li>
<li>Dispense 4 mL (60,000 cells) into each well containing a CYTOOchip. 
CAUTION: The plate should be moved as little as possible to avoid inducing any rotating movements in the medium as this will tend to concentrate cells at the center.</li>
<li>Let the cells sediment for 10 min under the hood then move them to the cell incubator. After 10-20 min, cells will start to adhere to the micropatterns. After 10 min, check regularly under the microscope.</li>
<li>As soon as cells have attached, change the cell medium and gently flush the coverslip surface using the following procedure:</li>
<li>Keeping the plate flat, gently aspirate the medium with a pipette from the side of the well. 
CAUTION: Never let the chip dry out, this means never aspirate off all the liquid but leave enough to cover the CYTOOchip at all times.</li>
<li>Add 4 mL PBS and aspirate again first starting at the center of the chip then moving to the side of the well. Aspirate off the PBS as described without exposing the chip to air. Repeat 3-4 times.</li>
<li>Check under the microscope for floating cells:
<ul>
<li>If a large number of floating cells remain, repeat the washing procedure.</li>
<li>If you see very few cells, aspirate off part of the PBS and replace it with 2 mL fresh medium. Aspirate and add 4 mL of fresh medium twice.</li>
</ul>
</li>
<li>Place the plate in a tissue culture incubator at 37&deg;C with 5% CO2 for a minimum of three hours to allow cells to achieve full spreading. 
NOTE: Using this method between 10-30% (2000-6500) micropatterns will be occupied by a single or multiple cells. 
CAUTION: The time needed for cells to adhere before the washing step and the time needed for full spreading of cells on micropatterns will be specific to each cell line.</li>
</ol>
2. 	Blebbistatin Treatment
<ol>
<li>Dissolve the blebbistatin in 100% DMSO to obtain a stock solution of 17 mM. Dilute the stock solution further to 5 mM with 100% DMSO. Make a final dilution in culture medium to obtain a final concentration of 5 &mu;M blebbistatin in 0.1% DMSO.</li>
<li>For control conditions, prepare 4 mL of medium containing 0.1% DMSO only.</li>
<li>Aspirate the media on cells and replace it with the 4 mL medium prepared for treated and control conditions.</li>
<li>Incubate cells for 1hr at 37&deg;C.</li>
</ol>
3. 	Cell Fixation and Staining of Actin
<ol>
<li>Solution preparation for cell fixation 
Please see reagent section for the preparation of cytoskeleton buffer (CB) and PFA 5%. CB is particularly recommended for fluorescence labeling of actin filaments and microtubules. 
<ul>
<li>Prepare 1xCB-sucrose: add 1 g of sucrose to 2 mL of CB. 
Inclusion of sucrose helps preserve internal cell structures.</li>
<li>Prepare 4% PFA-CB-sucrose: Add 1 mL of 1xCB-sucrose to 4 mL of warm PFA 5%. 
CAUTION: This solution can be kept at 4&deg;C for a few days only.</li>
</ul>
</li>
<li>PFA fixation
<ul>
<li>Use forceps to pick up the CYTOOchip on the CYTOO logo and place it (without washing) in a well of a 6-well plate containing 2 mL of 4% PFA-CB-sucrose solution</li>
<li>Incubate 10 min at RT</li>
<li>Remove PFA-CB-sucrose and wash once with PBS</li>
<li>Do a second wash with 2 mL of 100 mM NH4Cl for 10 min in order to quench residual PFA crosslinking activity 
NOTE: PFA fixed slides can be stored in PBS for several days at 4&deg;C before organelle staining for fluorescent imaging.</li>
</ul>
</li>
<li>Permeabilization of cell membrane with Triton X-100 
Permeabilization of cells with detergent allows antibodies or other probes to penetrate the cell membrane and eliminates some of the cytosolic pool of cytoskeleton proteins that can otherwise reduce the contrast of cytoskeletal polymers making it difficult to analyse their localization. 
<ul>
<li>Remove NH4Cl and add 2 mL/well of 0.1% Triton X-100-CB for 3 min at RT</li>
<li>Wash twice with 2 mL PBS</li>
</ul>
</li>
<li>Actin staining 
Fluorescent-phalloidin which binds to native actin only is used to stain actin filaments.
<ul>
<li>Add 2 mL of FITC-conjugated phalloidin diluted 1:2000 in PBS with 1.5% BSA</li>
<li>Incubate 1 h at RT</li>
<li>Wash once with 2 mL PBS</li>
</ul>
</li>
<li>Nuclei staining
<ul>
<li>Add 2 mL of Hoechst (stain with 1&mu;g/ mL in PBS)</li>
<li>Incubate 3 min at RT</li>
<li>Wash 2 times with 2 mL PBS</li>
</ul>
</li>
<li>Mounting of slide
<ul>
<li>Pick up the CYTOOchips on the CYTOO logo and mount CYTOOchip onto a standard microscope slide using 25-30 &mu;L of Mowiol or any other mounting solution.</li>
</ul>
</li>
</ol>
4. 	Microscope Setup and Automated Image Acquisition
Numerous imaging software packages exist that control the microscope for fast automated image acquisition and display. This example uses Metamorph imaging software and automatic acquisitions are performed on a Nikon Eclipse Ti microscope equipped with a CCD Hamamatsu camera and an Intensilight mercury-fiber illuminator. Images are acquired at 20x magnification in three wavelengths corresponding to DAPI (nuclei), Cy-3 (micropatterns) and FITC-Phalloidin (actin). The number of micropatterns visualized per field will vary depending on the camera and objective setups. CYTOO micropatterns are positioned at defined intervals from each other (100 or 130 &mu;m) across the chip greatly facilitating acquisition.
<ol>
<li>Select 20x magnification</li>
<li>Open Multidimensional Acquisition (in the Menu bar: Apps/Multidimensional Acquisition) and set up the different parameters of the experiment:
<ul>
<li>Number of wavelengths: 3 </li>
<li>Wavelength types: DAPI, FITC, Cy3</li>
<li>Set exposure time for each wavelength by first focusing on a field of cells</li>
<li>Autofocus can be performed on each wavelength</li>
<li>Select where to save data</li>
</ul>
</li>
<li>Select Cy3 wavelength and position the camera so that 12 micropatterns are visualized and centered in the camera field (4 columns and 3 rows of patterns).</li>
<li>Create a journal running MultiDimensional Acquisition. The procedure for creating a journal is described in Figure 1.</li>
<li>Open Scan stage function (in the Menu bar: Devices/Stage/Scan Stage). To acquire 24 images each containing 12 micropatterns at 20x magnification, enter 6 columns and 4 rows with -400 &mu;m X step size and 300 &mu;m Y step size. In Set Journal to Execute, upload the journal MDA.JNL. Then press Scan to start the automated acquisition of the whole block in the three wavelengths. 
NOTE: The X and Y step sizes indicated here will vary according to the number of micropatterns present in a camera field. Depending on your stage set up, direction in X and/or Y may require negative values for movement in the correct direction.</li>
</ol>
5. 	Automated Image Processing and Analysis
Many image processing and analysis software packages exist. The procedures described below outline image processing and analysis steps performed by 2 custom-made macros written for the open source program ImageJ. The first macro CellRef creates a Reference Cell, the second Hypotenuse measures rigidity/collapse of the cell membrane. Both are available upon request through <a href="http://www.cytoo.com">www.cytoo.com</a>.
<ol>
<li>Create image stacks for all three channels centered on micropatterns (macro CellRef). 
Use of micropatterns avoids cell clustering and clumping, greatly simplifying the first step in automated image processing which is localization and segmentation of individual cells. The fluorescently labeled micropattern images are used to define and delimit regions centered on the micropatterns. These regions are then transposed onto the images acquired in other channels and cropped. Corresponding cropped images are then assembled into stacks for each wavelength. A RGB-stack is also created from the merge of the three channels (Figure 2).</li>
<li>Apply single cell selection filter (macro CellRef) 
As indicated above, 10-30% (2000-6500) of the micropatterns are occupied by a single cell or multiple cells, while the remaining micropatterns are empty. To select images that show micropatterns occupied by just a single cell, the macro detects the number of nuclei per image and eliminates from all stacks (RGB and different wavelengths) those containing more than one nucleus or no nuclei (Figure 3).</li>
<li>Eliminate non-normalized and cells that are not fully stretched across the micropattern using cell area cut-off filter (macro CellRef) 
To remove dividing cells that would have escaped the nuclei filter, another filter using the FITC channel for actin is applied. Cell area is calculated and those below a certain cut off (cell areas about 2 times less than interphase cell area) are removed from all stacks. The cell envelope area is determined by the size of the pattern. </li>
<li>Align cell images (macro CellRef) 
From the previous steps, images containing single micropatterned cells were selected. Even though these images are centered on micropatterns, they are never all perfectly aligned to each other. To conclude the Image processing, an ImageJ plugin (MultiStackReg) is applied to realign all collected images and all channels based on the micropattern position. This step generates aligned stacks of individual images that can now be used for single cell analysis or creation of a Reference Cell (Figure 4)</li>
<li>Building a Reference Cell (macro CellRef) 
Growing cells on micropatterns means that all the cells have similar shapes. This normalization permits a precise alignment and overlay of many cell images. Summing up the signal in each image over the whole stack allows one to visualize and measure an "average drug effect". The final overall image or Reference Cell is a unique and useful tool for representation and image analysis and can only be obtained with micropatterned cells (Figure 4). In ImageJ, the Reference Cell is constructed by making a projection of the filtered and aligned images from a stack (Image>Stacks>Z-project). Several projection methods can be applied such as average or maximum intensity but usually a median projection is chosen which eliminates main outliers of the image stack. To facilitate examination of the results, a LUT (Look Up Tables) converting the monocolor image into a color-coded frequency map is applied.</li>
<li>Measuring and quantifying parameters of interest (macro Hypotenuse) 
In this video article, L-micropatterns are used because the form organizes the actin cytoskeleton into a single major stress fiber. By highlighting actin in this way, blebbistatin's impact on the cytoskeleton can be determined in many ways: measuring changes in the curvature of the actin stress fiber, staining intensity, length or thickness of the stress fiber, changes in morphometric features of the actin filaments or cell shape etc. These parameters can be measured either on individual cell images or on the final Reference Cell itself. 
Here, the impact of 5 &mu;M blebbistatin was characterized by counting the number of collapsed cells using a second ImageJ macro named Hypotenuse (Figure 7). Briefly, thresholded images of the L-micropattern are used to define a theoretical hypotenuse of the cell triangle shape. Next, thresholding of the actin stained images is performed in order to approximate actual cell shape. The area between the theoretical hypotenuse of the cell triangle and the actual concave cell membrane is then measured. When the cell has collapsed, an area underneath the theoretical hypotenuse appears. The percentage of collapsed cells is used to compare control and treated conditions.</li>
</ol>
6. Representative Results
Examples of what cells should look like on micropatterns immediately and several hours after the critical washing steps described in 1.6-1.8, are shown in Figure 5. After 15 min on CYTOOchips, round HeLa cells are observed at equal distances indicating that they have adhered to the fibronectin micropatterns (Figure 5a). Adhesion is complete when cells remain immobilized despite gentle shaking of the culture vessel. To minimize the number of micropatterns occupied by multiple cells, chips are then flushed with PBS to remove cells floating over the cytophobic areas. After several hours, cells that are fully stretched over the micropatterns will look like those shown in Figure 5b.
Typical single cell images obtained after incubation of cells with blebbistatin, fixation and staining of actin and nuclei are presented in Figure 6. After automated acquisition of multiple images and image processing using the ImageJ CellRef macro, a color-coded frequency map is generated that depicts the intensity of actin averaged over all cell images (Figure 6c and 6f). The comparison of the Reference Cell for nontreated and treated conditions shows the potential of this approach for easy observation of the phenotype under study and is especially useful for exploring cellular drug effects.
An example of one possible analysis of the effect of blebbistatin on cells is presented in Figure 7. The number of collapsed cells (ie. cells with a blank area existing under the theoretical hypotenuse) detected in 50 control cells and 50 cells treated with 5 &mu;M blebbistatin as detected by the ImageJ Hypotenuse macro is shown. The increased number of collapsed cells in the blebbistatin treated population reflects relaxation of the stress fibers and hence tension at the membrane due to blebbistatin inhibition of actinomyosin contractile forces within the cell.

<img src="/files/ftp_upload/2514/2514fig1.jpg" alt="Figure 1" /> 
Figure 1. Procedure to create a new journal with Metamorph.
<img src="/files/ftp_upload/2514/2514fig2.jpg" alt="Figure 2" /> 
Figure 2. Flow chart of the automatic image processing procedure.
<img src="/files/ftp_upload/2514/2514fig3.jpg" alt="Figure 3" /> 
Figure 3. Filtering out of single cells.
<img src="/files/ftp_upload/2514/2514fig4.jpg" alt="Figure 4" /> 
Figure 4. Building a Reference Cell.
<img src="/files/ftp_upload/2514/2514fig5.jpg" alt="Figure 5" /> 
Figure 5. Cell seeding. A) Hela cells adhered to micropatterns after the flushing step (4x magnification) B) HeLa cells after full spreading on L micropatterns (10x magnification).
<img src="/files/ftp_upload/2514/2514fig6.jpg" alt="Figure 6" /> 
Figure 6. Comparison of nonpatterned and micropatterned cells in control and drug treated conditions. HeLa cells were seeded for 3 hours and treated with 5 &mu;M blebbistatin for 1 hour (lower panel) or left untreated (upper panel). In control nonpatterned cells (a), actin assembles into multiple fibers which disassemble imperceptibly upon treatment with 5 &mu;M blebbistatin (d). On L-micropatterns, control cells adopt a triangular shape (b) and develop a major actin fiber (stress fiber) between the 2 apices of the L. Compared to nonpatterned cells (d), blebbistatin induces a major phenotypic change on L-micropatterned cells (e). Direct visualization of drug effects and the comparison of control and treated conditions can be visualized rapidly with the Reference Cell presentation built from 20 cells. Similar to individual cells, a clear and intense stress fiber is present on the control Reference Cell (c), while blebbistatin treated cells collapse and the major stress fiber disappears (f).
<img src="/files/ftp_upload/2514/2514fig7.jpg" alt="Figure 7" /> 
Figure 7. Example of an analysis of the effects of blebbistatin on cells using the macro Hypothenuse. While 25% of control cells were collapsed, this increased 3 fold to 75% in the 5 &mu;M blebbistatin treated cells reflecting the weakened actinomyosin contractile network (n=50 cells).

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All the authors except Anne B&eacute;ghin are employees of CYTOO S.A, which produces reagents and instrumentation featured in this article.

Choice of micropattern
While the effects of 5 &mu;M blebbistatin are barely detectable on standard culture supports, efficient quantification is possible on micropatterns that concentrate actin into a single major stress fiber. This enhances visualization of the actin cytoskeleton aiding accurate quantification of blebbistatin effects on the cell. The choice of micropattern shape is critical in the design of the assay and is designed according to the phenotypic change to be highlighted in the study.
Conclusions
Micropatterns facilitate the visualization and quantification of drug effects, especially at low concentrations. The replacement of homogenous planar substrates with adhesive micropatterns for cell-based assays improves the accuracy, sensitivity and quality of data produced. In consequence, fewer cells are needed for analysis in order to achieve robust statistical outcomes 3. Adhesive micropatterns offer a real opportunity for improving functional studies and exploring phenotypic changes at lower drug concentrations in order to avoid inducing toxic or indirect drug effects. If adequate screening platforms are used, micropatterns can meet the demands of pharmaceutical companies for improving gene/drug screening applications. Some additional examples of recent publications that have exploited micropatterns for analyzing and quantifying cellular phenomenon are cited below 3-13.

<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td colspan="5" align="center">Cell culture</td>
 </tr>
 <tr>
 <td colspan="2">CYTOOchips 20x20 L-M-FN</td>
 <td>CYTOO</td>
 <td>10-114</td>
 <td>CYTOOchips are available for adsorption of the protein of your choice</td>
 </tr>
 <tr>
 <td colspan="2">PBS 1x</td>
 <td>Gibco</td>
 <td>14040-091</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Counting chamber (Kova slide)</td>
 <td>Prolabo</td>
 <td>71287.01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">DMEM/F-12 + Glutamax-I</td>
 <td>Gibco-Invitrogen</td>
 <td>25300-054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">FBS (f tal bovine serum)</td>
 <td>PAA</td>
 <td>31331-028</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Penicillin &amp; Streptomycin</td>
 <td>PAA</td>
 <td>A15-101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">6 wells-plate</td>
 <td>Dutscher</td>
 <td>353046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">centrifuge</td>
 <td>Fisher Scientific</td>
 <td>M65838</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Microscope</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5" align="center">Drugs, fixation and immunostaining</td>
 </tr>
 <tr>
 <td colspan="2" >Cytoskeleton Buffer 1X (CB)</td>
 <td >Sigma</td>
 <td >MES&nbsp;: M8250 
 KCl&nbsp;: I2636 
 MgCl&nbsp;: M2393 
 EGTA : E3889</td>
 <td >10 mM MES pH 6.1, 138mM KCl, 
 3mM MgCl, 
 2mM EGTA</td>
 </tr>
 <tr>
 <td colspan="2" >Sucrose</td>
 <td >Sigma</td>
 <td >S2395</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">PFA 32 %</td>
 <td>Electron Microscopy Sciences</td>
 <td>15714</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">PFA 5%</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Mix 10 mL of PFA 32% with 54 mL of CB 1.185X</td>
 </tr>
 <tr>
 <td colspan="2">Triton-X100</td>
 <td>Sigma</td>
 <td>T9284</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">PBS tablets</td>
 <td>Gibco-Invitrogen</td>
 <td>18912-014</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Bovin serum albumin (BSA)</td>
 <td>Sigma</td>
 <td>A7806</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Phallodin-FITC</td>
 <td>Sigma</td>
 <td>P5282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Hoescht</td>
 <td>Invitrogen</td>
 <td>H3570</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">Blebbistatin</td>
 <td>Euromedex</td>
 <td>BN0640</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">DMSO</td>
 <td>Sigma</td>
 <td>D8418</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">NH4Cl 0.1M</td>
 <td>Sigma</td>
 <td>M11209</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5" align="center">Image Acquisition</td>
 </tr>
 <tr>
 <td colspan="2">Epifluorescent microscope </td>
 <td>Nikon</td>
 <td>Eclipse Ti</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">CCD Camera</td>
 <td>TBC</td>
 <td>TBC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">ImageJ Software</td>
 <td><a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a></td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

improved visualization and quantitative analysis of drug effects using micropatterned cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

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Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

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Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

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

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

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

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

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...