Name: tracking hypoxic signaling within encapsulated cell aggregates
Uploaded: 2011-12-16T13:00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Thanks to the Kristi Anseth lab of the University of Colorado, Boulder for generously supplying MIN6 cells. Funding for this project has been provided by the NSF.

1. PEGDM synthesis and photoactive PEGDM macromer solution preparation
<ol>
	<li>Weigh 2g of PEG (linear, 10,000Da) into a 40mL glass vial with a hard plastic cap.</li>
	<li>Add approximately 308&mu;L of methacrylic anhydride and loosely cap the vial.</li>
	<li>Microwave the vial on high for 2 minutes in a standard domestic microwave. Wearing heat resistant gloves, thoroughly vortex the vial, then microwave on high for an additional 5 minutes.</li>
	<li>Briefly allow the vial to cool enough to be handled with gloves. Uncap it and slowly add methylene chloride while swirling the vial. Add enough so that the solution appears clear and homogeneous, vortexing the sample as needed. The total volume of methylene chloride used should be 1.5 - 2 mL.</li>
	<li>Precipitate PEGDM in 200 mL of cold, stirred diethyl ether by adding the solution dropwise.</li>
	<li>Collect the PEGDM by vacuum filtration and allow it to dry.</li>
	<li>Dissolve the PEGDM in an adequate amount of deionized water (typically 100 -150 mL).</li>
	<li>Dialyze the solution against deionized water for 5 days in dialysis tubing with a molecular weight cut-off of 1,000 Da. Replace the water once daily.</li>
	<li>Aliquot the solution into appropriate containers and freeze at -80&deg;C overnight. Lyophilize the solution for 4 days to yield a purified white PEGDM powder. Store the powder at 4&deg;C when not in use.</li>
	<li>To prepare a photoactive PEGDM solution, first prepare a 0.5 weight percent photoinitiator stock solution by dissolving Irgacure 2959 in deionized water. Vortex the solution and store it in the dark.</li>
	<li>Prepare a 10 wt% PEGDM and 0.025 wt% Irgacure 2959 solution in Hank's Balanced Salt Solution (HBSS). Vortex thoroughly to ensure dissolution of the PEGDM. We typically prepare 1mL of this solution at a time.</li>
	<li>Sterilize the solution by filtering it through a 0.2 &mu;m syringe filter into a 1.8 mL microcentrifuge tube. Wrap the tube in aluminum foil and store at 4&deg;C</li>
</ol>
2. Culture, infection, and aggregation of MIN6 cells
<ol>
	<li>MIN6 cells are cultured in RPMI 1640 medium supplemented with 10% FBS, 7mM glucose, 100 units/mL penicillin/streptomycin, and 0.5 &mu;g/mL amphotercin B in a humidified environment at 37&deg;C and 5% CO2.</li>
	<li>To release the cells from the treated culture flask surface, aspirate the medium, rinse the cells with 37&deg;C calcium/magnesium-free HBSS, aspirate the HBSS, then add 2 mL of 37&deg;C trypsin-EDTA solution and allow the cells to incubate for 3 minutes.</li>
	<li>Firmly jar the side of the flask to knock the cells free, then add 8 mL of 37&deg;C medium and vigorously pipette the cell suspension up and down taking care not to introduce bubbles.</li>
	<li>Pipette the cells into a sterile 15 mL centrifuge tube and perform a cell count using a hemocytometer or other cell counting procedure.</li>
	<li>In preparing to infect the cells with the maker virus, first determine the total number of cells to be infected and calculate the volume of virus suspension required to achieve the desired multiplicity of infection (MOI) (50 to 100 infectious particles per cell).</li>
	<li>Dilute the virus in HBSS by carefully pipeting the proper amount of virus suspension into a pre-aliquoted volume of HBSS in a 1.8 mL microcentrifuge tube. 50 &mu;L of HBSS per well of aggregating cells is appropriate.</li>
	<li>Disperse the cells by pipeting them up and down in their tube, then pipet the necessary volume to achieve 400,000 cells per well into the wells of a 6-well suspension culture plate.</li>
	<li>Add the appropriate volume of diluted virus to each well to achieve the desired MOI.</li>
	<li>Add medium to each well to bring the total volume up to 1.7 mL.</li>
	<li>Place the plate on an orbital shaker inside the incubator. Set the shaker on a low setting so that the medium gently washes around the wells (&tilde;100 rpm). Allow the cells to aggregate for 24-36 hours. Aggregates with an approximate diameter of 75-175 &mu;m should be formed.</li>
</ol>
3. Encapsulation of cells in PEGDM
<ol>
	<li>Cells may be encapsulated as a dispersion (step 2.4) or following aggregation (step 2.10). Since the hydrogel precursor PEGDM solution is a liquid, cells may tend to settle prior to crosslinking and hydrogel formation. If settling is expected to occur rapidly, such as with larger aggregates, it may be beneficial to produce the hydrogel in two steps so that the cells settle to a central plane of the gel. A one-step hydrogel synthesis procedure appropriate for dispersed cells is shown (Figure 2) and the two-step procedure is shown as well (Figure 3) and detailed below (3.2-3.9).</li>
	<li>Create a vessel in which the hydrogel will be formed by cutting the tapered tip off of a 1 mL plastic syringe and placing it open end up in a rack.</li>
	<li>Pipet 20 &mu;L of photoactive PEGDM solution into the open end of the syringe onto the plunger making sure the volume covers the entire plunger surface. Adjust the plunger in small increments to achieve a flat fluid surface.</li>
	<li>Place the pipet in the rack and position the rack underneath a UV lamp (365nm, &tilde;7 mW/cm2) for approximately 8 minutes to allow for hydrogel crosslinking. During this time, preparation of the cells may be begun</li>
	<li>Pipet a volume containing the desired number of cells/aggregates into a 1.8 mL microcentrifuge tube, cap, and centrifuge the tube at 130 g for 5 minutes.</li>
	<li>Using a micropipettor, carefully remove the media without disturbing the cell pellet at the tip of the tube. As the media head approaches the pellet, it may be helpful to use a finer, 10 &mu;L pipet tip.</li>
	<li>Gently resuspend the cell pellet in 20 &mu;L of photoactive PEGDM solution (use a wide-mouthed pipet tip if working with aggregates) and add the cell suspension to the syringe on top of the previously crosslinked hydrogel portion.</li>
	<li>Expose the syringe to an additional 8 minutes of UV light to complete construction of the hydrogel. When finished, the cells should be fully encapsulated within the cylindrical hydrogel (Figure 3).</li>
	<li>Eject the hydrogel from the syringe and into 1 mL of HBSS in the well of a 24-well plate to briefly wash the gel. Transfer the gel to 1 mL of media in the well of a 24-well plate and culture under the desired conditions.</li>
</ol>
4. Hypoxia tracking
<ol>
	<li>At any point during the culture, image the cells using fluorescent microscopy to track the initiation of hypoxic signaling. Depending on what is being imaged you can image in the multi-well plate or transfer the gel to a microscope slide prior to placement on the microscope stage. Peak excitation of Ds Red is achieved at 556nm and peak emission occurs at 586nm. Emission is fairly intense and thus photobleaching has not been observed to be problematic. Lag time between initiation of protein production and first signal detection is approximately 12 hours. Signal is specific enough to identify individual signaling cells, but resolution may be inadequate for determining intracellular signal localization. In signaling cells, signal is typically observed uniformly throughout the cytoplasm. Signal intensity can also increase with extended exposure to hypoxia, though we have not yet fully determined if this is due to increased protein expression, overall protein accumulation, or delayed protein maturation.</li>
</ol>
5. Representative results
A representative example of MIN6 aggregates encapsulated in a PEG hydrogel is shown in Figure 4. The crosslinked gel will be solid throughout, taking the shape of the vessel in which the reaction was performed. A gel with smooth outer surfaces is preferable for implantation to aid in prevention of a foreign body response. Within the gel, cell aggregates should be fully enclosed in the matrix and homogenously distributed to allow for better nutrient transport.
Representative images of hypoxic signaling in MIN6 aggregates are pictured in Figure 5. Identical MIN6 aggregates infected with the marker virus were cultured in either 20% O2 (5a) or 1% O2 (5b) for 44 hours before image capture.
<img alt="Figure 1" src="/files/ftp_upload/3521/3521fig1.jpg" /> 
Figure 1. Schematic of the activity of our hypoxia marker system. Adenoviral insertion of the Ds Red DR gene and upstream HRE promoter allows for hypoxia-induced production of the fluorescent protein under control of HIF-1.
<img alt="Figure 2" src="/files/ftp_upload/3521/3521fig2.jpg" /> 
Figure 2. Procedural flow chart for the single-step encapsulation of dispersed MIN6 cells in a crosslinked PEGDM hydrogel. For each gel, the dispersed cells are suspended in 40&mu;L of the photoactive PEGDM macromer solution. This is placed in a decapitated 1mL syringe and the hydrogel is formed under 365nm UV light after 10-12 minutes. Upon completion, the hydrogel disk is removed from the syringe, washed and placed in a medium-filled well plate for incubation.
<img alt="Figure 3" src="/files/ftp_upload/3521/3521fig3.jpg" /> 
Figure 3. Procedural flow chart for the dual-step encapsulation of aggregated MIN6 cells in a crosslinked PEGDM hydrogel. For each gel, a half-gel is first formed by UV crosslinking of 20&mu;L of the photoactive PEGDM macromer solution for 8 minutes. MIN6 aggregates are carefully suspended in an additional 20&mu;L of photoactive macromer solution which is added on top of the pre-formed half-gel. The full gel is formed by an additional 8 minutes of UV exposure with aggregates fully encapsulated in the medial plane of the gel.
<img alt="Figure 4" src="/files/ftp_upload/3521/3521fig4.jpg" /> 
Figure 4. Image of a 40&mu;L hydrogel under 20X magnification. MIN6 aggregates (~400,000 total cells) are clearly seen within the gel. Hydrogel diameter is approximately 6mm (bar = 1mm).
<img alt="Figure 5" src="/files/ftp_upload/3521/3521fig5.jpg" /> 
Figure 5. Fluorescent hypoxia signaling in aggregated MIN6 cells in a PEGDM hydrogel. Cells that were encapsulated and then placed in incubation at 20% O2 for 44 hours do not display hypoxia signaling (a) while cells that were encapsulated then incubated in 2% O2 for 44 hours display clear, ubiquitous signal. (bar = 100&mu;m) (b).

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Biotechnol. 10, 87-99 (1984).</li><li>Iwata, H., Amemiva, H., Matsuda, T., Takano, H., Hayashi, R., Akutsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microencapsulated+islets+in+agarose+gel+as+bioartificial+pancreas+by+studies+of+hormone+secretion+in+culture+and+by+xenotransplantation.">Evaluation of microencapsulated islets in agarose gel as bioartificial pancreas by studies of hormone secretion in culture and by xenotransplantation.</a> Diabetes. 39, 224-225 (1989).</li><li>Zhang, W. J., Laue, C., Hyder, A., Schrezenmeir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purity+of+alginate+affects+the+viability+and+fibrotic+overgrowth+of+encapsulated+porcine+iselt+xenografts.">Purity of alginate affects the viability and fibrotic overgrowth of encapsulated porcine iselt xenografts.</a> Transplant Proc. 33, 3517-3519 (2001).</li><li>Lin-Gibson, S., Bencherif, S., Cooper, J. A., Wetzel, S. J., Antonucci, J. M., Vogel, B. M., Horkay, F., Washburn, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+PEG+dimethacrylates+and+their+hydrogels.">Synthesis and characterization of PEG dimethacrylates and their hydrogels.</a> Biomacromolecules. 5, 1280-1287 (2004).</li><li>Weber, L. M., He, J., Bradley, B., Haskins, K., Anseth, K. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+properties+influence+ECM+production+by+chondrocyte+photoencapsulated+in+poly(ethylene+glycol)+hydrogels.">Hydrogel properties influence ECM production by chondrocyte photoencapsulated in poly(ethylene glycol) hydrogels.</a> J. Biomed. Mater. Res. 59, 63-72 (2001).</li><li>Bryant, S. J., Nuttelman, C. R., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytocompatibility+of+UV+and+visible+light+photoinitiating+systems+on+cultured+NIH/3T3+fibroblasts+in+vitro.">Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro.</a> J. Biomater. Sci. Polymer Edn. 11, 439-457 (2000).</li><li>Dionne, K. E., Colton, C. K., Yarmush, M. 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No conflicts of interest declared.

The method presented here offers a quick and simple technique for cell encapsulation in a PEG hydrogel with minimal use of non-physiological conditions. PEG represents a very useful encapsulation material for its biocompatibility and ease of modification. Simple variation of PEG percentage in the photoactive solution, for instance, may be used to adjust mechanical properties, such as compressive modulus, and transport properties through pore size. Also, PEG is easily modified by the addition of side chains. PEG hydrogels, therefore, represent both a promising clinical device and a flexible platform for in vitro research
A method for tracking hypoxia in PEG-encapsulated cells has also been presented. This method is useful for the simplicity of hypoxia detection and for avoiding the need to sacrifice the cells of interest. The technique may be applied to a variety of types of cells in a variety of conditions making its usefulness broad. For instance, hypoxia as a cue for stem cell differentiation may be tracked in stem cell micromass cultures. However, this method can only be applied to disperse cell systems or system in which dispersed cells are later aggregated. Also, detection of the fluorescent signal may be difficult in larger or denser tissues.

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments (optional</td>
</tr>
<tr>
<td>PEG</td>
<td>Sigma-Aldrich</td>
<td>309028-500G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Methacrylic Anhydride</td>
<td>Sigma-Aldrich</td>
<td>276685-100ML</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Microwave</td>
<td>Emerson</td>
<td>MW8784SB</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vortexer</td>
<td>Scientific Industries</td>
<td>SI-A236</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Methylene Chloride</td>
<td>Sigma-Aldrich</td>
<td>D65100-1L</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Diethyl Ether</td>
<td>Sigma-Aldrich</td>
<td>346136-1L</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dialysis Tubing</td>
<td>Spectrum </td>
<td>132640</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Freezer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Lyophilizer</td>
<td>Labconco</td>
<td>7670521</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vacuum pump</td>
<td>Welch</td>
<td>8917Z-01</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Irgacure 2959</td>
<td>Ciba-Geigy</td>
<td>029891301PS04</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>HBSS</td>
<td>Mediatech</td>
<td>21-022-CV</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Syringe Filter</td>
<td>VWR</td>
<td>28145-477</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Mediatech</td>
<td>*</td>
<td>*custom formulation</td>
</tr>
<tr>
<td>FBS</td>
<td>PAA Laboratories</td>
<td>A15-351</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Mediatech</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Amphotericin B</td>
<td>Mediatech</td>
<td>30-003-CF</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Incubator</td>
<td>Thermo Scientific</td>
<td>3597</td>
<td>Napco Series 8000 WJ w/ O2 suppression</td>
</tr>
<tr>
<td>Trypsin EDTA</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Orbital Shaker</td>
<td>VWR</td>
<td>12620-926</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>UV Lamp</td>
<td>Sanyo Denri</td>
<td>FLR40SBLB/M</td>
<td>Holds two 40W, 365nm blacklight blue UV bulbs</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Eppendorf</td>
<td>5811 000.010*</td>
<td>*order number.&nbsp; 
Model 5810 R</td>
</tr>
<tr>
<td>Microscope</td>
<td>Nikon</td>
<td>TI-ND6-PFS</td>
<td>With filterset for 556nm excitation/ 586nm emission</td>
</tr>
</table>

tracking hypoxic signaling within encapsulated cell aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

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Bioengineering

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...