This work was supported by the Coulter Foundation, Beyster Foundation, the Undergraduate Research Opportunity (UROP) summer program for ATA and a National Science Foundation Graduate Student Research Fellowship (Grant no. DGE 0718128; ID: 2010101926) for JBW.

1. Phase System Characterization: Determining Thresholds for Phase Separation
<ol>
	<li>Prepare solutions containing PEG and DEX in the desired buffer or cell culture medium as shown in Figure 1 (purple dots) in 15 ml or 50 ml conical tubes. Hereafter, PEG and DEX will refer to 35 kDa PEG and 500 kDa DEX; however, critical concentrations will change depending on the two polymers used. Record the mass of PEG and DEX in each solution. High-concentration polymer solutions may take several hours to dissolve. Vortexing can be used for serum-free solutions. For media containing proteins or serum, place the tubes on a rocking stage until both polymers are fully dissolved. Record the weight of the media used to dissolve the polymers and take note of the initial concentrations.</li>
	<li>Once the polymers are fully dissolved, the solutions should appear cloudy. This is the first indication that phase separation has occurred. To confirm this, allow the polymer solutions to rest in a vertical position at room temperature for 20 min. Centrifugation at 1,000 x g can be used to accelerate the phase separation process. The denser bottom phase will be DEX-rich and the top phase will be PEG-rich.</li>
	<li>Slowly add additional buffer or media to the tubes. Small increments should be used so as not to overshoot the phase separation point.</li>
	<li>When the solution becomes clear and no longer phase-separates after centrifugation, the threshold for phase separation has been reached. Record the final weight of the tube.</li>
	<li>Using the previous recorded weights for the polymers, along with the final weight after adding media, determine the % wt/wt of each the two polymers at which phase separation no longer occurs.</li>
	<li>Plot these values as % wt/wt PEG on the y-axis and % wt/wt DEX on the x-axis. This plot, known as the binodal curve, can be used to determine the threshold concentration for phase separation for different concentrations of PEG/DEX in a specific cell culture medium.</li>
</ol>
2. Configuration 1: Exclusion Patterning (96-well Plate Format)
<ol>
	<li>Prepare separate solutions of 5.0% wt/wt PEG and 12.8% wt/wt DEX in cell culture medium. Use ATPS solutions of at least twice the critical point to ensure that an ATPS remains after the polymers equilibrate with respect to each other. There is a small amount of flux of DEX into the PEG-rich phase and vice versa, therefore, working too near the critical concentration can result in loss of the phase system as concentrations drop below critical point. Similarly, transferring the cell culture dish to a humidity- and temperature-controlled incubator can alter the two-phase properties and make the two solutions miscible. Note: Some cells may perform better with other ATPS formulations. Acceptable formulations may be selected based on the binodal curves determined from Part 1.</li>
	<li>Harvest the cells to be used for exclusion patterning. Determine the total number/concentration of cells available. Optional: label the cells with CellTracker or other non-cytotoxic labels for fluorescence microscopy.</li>
	<li>Pellet the cells and resuspend the pellet in an appropriate volume of 5.0% PEG to achieve the desired number of cells for exclusion. For example, one well of a 96-well plate requires 37,500 fibroblast cells to be resuspended in 200 &mu;l of PEG to produce confluence the following day. Scale these numbers as appropriate for other cell types and culture substrate sizes.</li>
	<li>Using a micropipettor, dispense 0.5 &mu;l droplets of DEX onto a dry cell culture substrate. Larger volume droplets produce larger exclusion zones. Droplets ranging in size from 0.1 to 1 &mu;l are recommended for exclusion micropatterning. Optional: DEX droplets can be deposited 24 hr ahead of time and allowed to dehydrate at room temperature. This can produce cleaner patterns.</li>
	<li>Dispense 200 &mu;l of PEG cell suspension into the well to cover the DEX droplets.</li>
	<li>Place in a humidified incubator for 12 hr at 37 &deg;C, 5% CO2. Make sure that the dish is not tilted during handling and that it is placed on a level incubator shelf to avoid disrupting the patterns.</li>
	<li>Remove the PEG solution and wash three times with 200 &mu;l of culture medium.</li>
	<li>Add fresh culture medium and return to the incubator.</li>
	<li>Monitor the cultures periodically to observe cell movement into the exclusion zone.</li>
</ol>
3. Configuration 2: Island Patterning (96-well Plate Format)
<ol>
	<li>Prepare solutions of 5.0% wt/wt PEG and 12.8% wt/wt DEX in cell culture medium, as above.</li>
	<li>Harvest the cells to be used for island patterning. Determine the total number/ concentration of cells available.</li>
	<li>Pellet and resuspend the cells in an appropriate volume of 12.8% DEX to achieve the desired concentration of cells for island patterning. Concentrations of 5,000 cells/&mu;l or less are recommended for strongly adherent cell types. For cells that have difficulty attaching or cells that loosely adhere, concentrations of up to 10,000 cells/&mu;l may be considered.</li>
	<li>Working quickly to avoid drying, pipette 0.5 &mu;l droplets of DEX onto a dry cell culture substrate, as described above. Do not allow droplets to dry. Optional: 200 &mu;l of PEG solution can be added to the well ahead of time. DEX droplets can then be deposited into the PEG solution where they will sink to the bottom and contact the culture surface. This can produce cleaner island patterns.</li>
	<li>Cover the DEX droplets with 200 &mu;l of PEG.</li>
	<li>Place in a humidified incubator for 12 hr at 37 &deg;C, 5% CO2. Make sure that the dish is not tilted during handling and that it is placed on a level incubator shelf to avoid disrupting the patterns.</li>
	<li>Remove the PEG solution and wash three times with 200 &mu;l of culture medium.</li>
	<li>Add fresh culture medium and return to the incubator.</li>
	<li>Monitor the cultures periodically to observe cell movement and proliferation outwards from the islands.</li>
</ol>
4. Configuration 3: Exclusion Co-cultures (96-well Plate Format)
<ol>
	<li>Prepare solutions of 5.0% wt/wt PEG and 12.8% wt/wt DEX in cell culture medium, as above.</li>
	<li>Harvest the cells to be used for exclusion and island patterning. Determine the total number/concentration of cells available for each cell type. Optional: Some cell pairings may display dramatically different proliferation indices. To prevent the excluded cells from overpopulating the island patterned cells (especially for long term cultures), treat the cells used for exclusion with mitomycin C for 2 hr or irradiate them before harvesting. This will prevent proliferation. Fluorescent CellTracker dyes can be used to distinguish the two cell populations if necessary.</li>
	<li>Pellet the cells and resuspend the pellet for exclusion patterning in an appropriate volume of 5.0% PEG to achieve the desired number of cells, as above. Resuspend the pellet for island patterning in an appropriate volume of 12.8% DEX to achieve the desired concentration of cells, as above.</li>
	<li>Using a micropipettor, dispense 0.5 &mu;l droplets of DEX cell suspension onto a dry cell culture substrate. Do not allow droplets to dry.</li>
	<li>Cover the DEX droplets with 200 &mu;l of PEG cell suspension.</li>
	<li>Place in a humidified incubator for 12 hr at 37 &deg;C, 5% CO2. Make sure that the dish is not tilted during handling and that it is placed on a level incubator shelf to avoid disrupting the patterns.</li>
	<li>Remove the PEG solution and wash three times in 200 &mu;l of culture medium.</li>
	<li>Add fresh culture medium and return to the incubator.</li>
	<li>Monitor the co-cultures to observe interaction between cell populations. Optional: Controls can be prepared by performing exclusion or island patterning individually, by co-culture cells that do not interact or by blocking pathways of interest in one or both cell populations before or after patterning.</li>
</ol>

<ol>
	<li>Hatti-Kaul, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+two-phase+systems+:+methods+and+protocols.">Aqueous two-phase systems : methods and protocols.</a> Methods in biotechnology. xiii, 440 (2000).</li><li>Albertsson, P. A. k. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Partition of cell particles and macromolecules: separation and purification of biomolecules, cell organelles, membranes, and cells in aqueous polymer two-phase systems and their use in biochemical analysis and biotechnology. , 346 (1986).</li><li>Yamada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cell+partitioning+using+an+aqueous+two-phase+flow+system+in+microfluidic+devices.">Continuous cell partitioning using an aqueous two-phase flow system in microfluidic devices.</a> Biotechnol. Bioeng. 88 (4), 489-494 (2004).</li><li>Soohoo, J. R., Walker, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+aqueous+two+phase+system+for+leukocyte+concentration+from+whole+blood.">Microfluidic aqueous two phase system for leukocyte concentration from whole blood.</a> Biomed. Microdevices. 11 (2), 323-329 (2009).</li><li>Hahn, T., Hardt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+size+separation+of+DNA+samples+at+liquid-liquid+interfaces.">Concentration and size separation of DNA samples at liquid-liquid interfaces.</a> Anal. Chem. 83 (14), 5476-5479 (2011).</li><li>Hatti-Kaul, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+two-phase+systems.+A+general+overview.">Aqueous two-phase systems. A general overview.</a> Mol. Biotechnol. 19 (3), 269-277 (2001).</li><li>Hustedt, H., Kroner, K. H., Menge, U., Kula, M. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+recovery+using+two-phase+systems.">Protein recovery using two-phase systems.</a> Trends in Biotechnology. 3 (6), 139-144 (1985).</li><li>Keating, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+Phase+Separation+as+a+Possible+Route+to+Compartmentalization+of+Biological+Molecules.">Aqueous Phase Separation as a Possible Route to Compartmentalization of Biological Molecules.</a> Acc Chem. Res. 45 (12), 2114-2124 (2012).</li><li>Helfrich, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+and+assembly+of+metal+particles+and+their+bioconjugates+in+aqueous+two-phase+systems.">Partitioning and assembly of metal particles and their bioconjugates in aqueous two-phase systems.</a> Langmuir. 21 (18), 8478-8486 (2005).</li><li>Diamond, A. D., Hsu, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prote.+Partitioning+in+PEG/Dextran+Aqueous+Two-Phase+Systems.">Prote. Partitioning in PEG/Dextran Aqueous Two-Phase Systems.</a> AIChE Journal. 36 (7), 1017-1024 (1990).</li><li>Y-T, Z. -. Q., Zhu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+interfacial+tension+of+aqueous+two-phase+systems.">Modeling of interfacial tension of aqueous two-phase systems.</a> Chemical Engineering Science. 54 (4), 433-440 (1999).</li><li>Liu, Y., Lipowsky, R., Dimova, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+dependence+of+the+interfacial+tension+for+aqueous+two-phase+polymer+solutions+of+dextran+and+polyethylene+glycol.">Concentration dependence of the interfacial tension for aqueous two-phase polymer solutions of dextran and polyethylene glycol.</a> Langmuir. 28 (8), 3831-3839 (2012).</li><li>Rha, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Tension+of+Polyethylene+Glycol/Potassium+Phosphate+Aqueous+Two-Phase+Systems.">Interfacial Tension of Polyethylene Glycol/Potassium Phosphate Aqueous Two-Phase Systems.</a> Physics and Chemistry of Liquids: An International Journal. 38 (1), 25-34 (2000).</li><li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Generation+of+Multiplexed+Cell+Cocultures+Using+Acoustic+Droplet+Ejection+Followed+by+Aqueous+Two-Phase+Exclusion+Patterning.">Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning.</a> Tissue Eng. Part C. Methods. 18 (9), 647-657 (2012).</li><li>Tavana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolitre+liquid+patterning+in+aqueous+environments+for+spatially+defined+reagent+delivery+to+mammalian+cells.">Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells.</a> Nat. Mater. 8 (9), 736-741 (2009).</li><li>Tavana, H., Mosadegh, B., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+aqueous+biphasic+systems+for+non-contact+cell+printing+on+cells:+engineering+heterocellular+embryonic+stem+cell+niches.">Polymeric aqueous biphasic systems for non-contact cell printing on cells: engineering heterocellular embryonic stem cell niches.</a> Adv. Mater. 22 (24), 2628-2631 (2010).</li><li>Tavana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microprinted+feeder+cells+guide+embryonic+stem+cell+fate.">Microprinted feeder cells guide embryonic stem cell fate.</a> Biotechnol. Bioeng. , (2011).</li><li>Tavana, H., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+biphasic+microprinting+approach+to+tissue+engineering.">Aqueous biphasic microprinting approach to tissue engineering.</a> Biomicrofluidics. 5 (1), 13404 (2011).</li><li>Frampton, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precisely+targeted+delivery+of+cells+and+biomolecules+within+microchannels+using+aqueous+two-phase+systems.">Precisely targeted delivery of cells and biomolecules within microchannels using aqueous two-phase systems.</a> Biomed. Microdevices. 13 (6), 1043-1051 (2011).</li><li>Hossein Tavana, K. K., Bersano-Begey, T., Luker, K. E., Luker, G. D., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehydration+of+Polymeric,+Aqueous,+Biphasic+System+Facilitates+High+Throughput+Cell+Exclusion+Patterning+for+Cell+Migration+Studies.">Rehydration of Polymeric, Aqueous, Biphasic System Facilitates High Throughput Cell Exclusion Patterning for Cell Migration Studies.</a> Advanced Functional Materials. 21 (15), 2920-2926 (2011).</li><li>Falconnet, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+engineering+approaches+to+micropattern+surfaces+for+cell-based+assays.">Surface engineering approaches to micropattern surfaces for cell-based assays.</a> Biomaterials. 27 (16), 3044-3063 (2006).</li><li>Lim, J. Y., Donahue, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sensing+and+response+to+micro-+and+nanostructured+surfaces+produced+by+chemical+and+topographic+patterning.">Cell sensing and response to micro- and nanostructured surfaces produced by chemical and topographic patterning.</a> Tissue Eng. 13 (8), 1879-1891 (2007).</li><li>Ringeisen, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jet-based+methods+to+print+living+cells.">Jet-based methods to print living cells.</a> Biotechnol. J. 1 (9), 930-948 (2006).</li><li>Wright, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+static+and+dynamic+patterned+co-cultures+using+microfabricated+parylene-C+stencils.">Generation of static and dynamic patterned co-cultures using microfabricated parylene-C stencils.</a> Lab Chip. 7 (10), 1272-1279 (2007).</li><li>Takayama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+cells+and+their+environments+using+multiple+laminar+fluid+flows+in+capillary+networks.">Patterning cells and their environments using multiple laminar fluid flows in capillary networks.</a> Proc. Natl. Acad. Sci. U.S.A. 96 (10), 5545-5548 (1999).</li><li>Berthier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pipette-friendly+laminar+flow+patterning+for+cell-based+assays.">Pipette-friendly laminar flow patterning for cell-based assays.</a> Lab Chip. 11 (12), 2060-2065 (2011).</li><li>Davidson, R. L., O'Malley, K. A., Wheeler, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-induced+mammalian+cell+hybridization:+effect+of+polyethylene+glycol+molecular+weight+and+concentration.">Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration.</a> Somatic Cell Genet. 2 (3), 271-280 (1976).</li><li>Johnson, D. M., LaFranzo, N. A., Maurer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Two-Dimensional+Patterned+Substrates+for+Protein+and+Cell+Confinement.">Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement.</a> J. Vis. Exp. (55), e3164 (2011).</li><li>Moon, S., Lin, P., Keles, H. O., Yoo, S., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Title+Cell+Encapsulation+by+Droplets.">Title Cell Encapsulation by Droplets.</a> J. Vis. Exp. (8), e316 (2007).</li></ol>

The authors have no competing financial interests.

The ATPS cell micropatterning method requires very little expertise beyond proficiency in cell culture techniques and can be quickly mastered. The advantages of this approach are that it is inexpensive, rapid and compatible with a variety of cell types and culture formats. For these reasons, our protocol should be easily adopted by life scientists, particularly those who study cell proliferation, migration and chemotaxis, and the influence of juxtacrine and paracrine interactions among cell populations. The assays...

Aqueous two-phase systems (ATPSs) form when solutions of two incompatible polymers are mixed together at high enough concentrations. Phase separation is influenced by a variety of factors that include the molecular weight and polarity of the polymers, temperature of the solutions, pH and ionic content of the aqueous solvent 1, 2. The point at which the two polymer solutions separate is determined by the physiochemical properties of the chosen phase system, but generally occurs at low polymer concentrations (less than 20% wt/wt) under non-denaturing conditions, allowing ATPSs to be used for biotechnology applications 3-9.
By far the most extensively studied ATPS is the polyethylene glycol (PEG)/dextran (DEX) system. The ATPS formed by these inexpensive and biocompatible polymers was originally described for the purification of biomolecules by way of molecular partitioning 2, 10. Partitioning occurs when additional molecules or particles that do not contribute to the phase system are mixed with PEG and DEX. Based on their relative affinities for either DEX or PEG, the molecules or particles will preferentially reside within one of the two phases or at the interface. Another property of the PEG/DEX ATPS is the existence of interfacial tension between the two polymer phases. ATPSs formed by PEG and DEX generally display interfacial tensions that are much lower than other liquid-liquid two-phase systems such as oil and water; however, the interfacial tension forces still exert effects on small particles such as viruses, cells and protein aggregates 2, 11-13. Finally, since higher molecular weight PEG and DEX separate at low concentrations (less than 5% wt/wt for high molecular weight polymers varieties) in the presence of physiological concentrations of salts, there are few if any deleterious effects on mammalian cells incorporated within these systems 14-16.
Recently, the interfacial properties and partitioning effects of ATPSs have been applied by our lab for cell patterning 14, 16-20. This was accomplished by micropatterning a denser DEX solution on cell culture substrates in the presence of PEG. When cells are incorporated into the PEG phase, they are excluded from entering the DEX droplets due to PEG/DEX interfacial tension 20. When cells are patterned in the DEX phase, they are retained at the surface of the cell culture substrate by interfacial tension and partitioning 16, 17, 19.
In contrast to other methods for cell patterning, ATPS cell patterning is easy to learn and only requires rudimentary knowledge about the polymers themselves, and the ability to perform cell culture and use a micropipettor. Other methods for cell patterning often involve specialized equipment and training that are not easily translated to the life sciences. For example, some methods (microcontact printing or inkjet printing) pattern cells indirectly by applying patterns of cell adhesive biomolecules to a culture substrate that subsequently serve as sites for cell attachment 21, 22. Although indirect approaches are useful for some cell types, they require a high degree of user skill and specialized equipment to fabricate the patterning tool, and can lack specificity depending on the particular cell type/biomolecule pattern. Alternatively, cells can be deposited with high pattern specificity by way of direct patterning approaches that include laminar flow patterning, stenciling and inkjet printing 23-26. However, these techniques also require user expertise and specialized equipment, and may damage cells during the printing process. Although these approaches generally produce precise patterns of cells, for cell patterning to be a useful tool in the life sciences, it must be cost effective and simple to implement.
Here we report a detailed protocol for generating patterned cell cultures using the ATPSs described in our previously-published applications. Using only micropipettors, users can generate cell exclusion zones or cell islands for migration assays. This is achieved by way of PEG/DEX interfacial tension that either retains cells in the DEX phase or excludes cells deposited in the PEG phase from DEX. By combing these two fundamental patterning techniques, it is possible to rapidly generate co-cultures of cells such as liver-fibroblast cell co-cultures. Patterning methods, ATPS parameters and expected results are described in detail.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Manufacturer</td>
 </tr>
 <tr>
 <td>Dextran 500,000 kDa</td>
 <td>Pharmacosmos, Denmark</td>
 </tr>
 <tr>
 <td>Polyethylene Glycol 35,000 kDa</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 </tr>
 <tr>
 <td>Hela</td>
 <td>ATCC, Manassas, VA</td>
 </tr>
 <tr>
 <td>HepG2 C3A</td>
 <td>ATCC, Manassas, VA</td>
 </tr>
 <tr>
 <td>NIH 3T3</td>
 <td>ATCC, Manassas, VA</td>
 </tr>
 <tr>
 <td>Cell Tracker</td>
 <td>Invitrogen, Carlsbad, CA</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco, Carlsbad, CA</td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Gibco, Carlsbad, CA</td>
 </tr>
 <tr>
 <td>F12</td>
 <td>Gibco, Carlsbad, CA</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Gibco, Carlsbad, CA</td>
 </tr>
 </tbody>
</table>

cell co-culture patterning using aqueous two-phase systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

To select an appropriate combination of PEG and DEX for cell patterning it is important to determine the binodal curve. This curve delineates the points at which an ATPS can form and can vary for a given set of polymers based on temperature, pH and ionic content. For culturing cells that require customized medium formulations it may be necessary to experimentally determine the binodal curve. This is accomplished by generating a series of ATPSs that are far from the binodal and varying in their PEG and DEX contents...

Watch this Scientific Journal Video about Cell Co-culture Patterning Using Aqueous Two-phase Systems at JoVE.com

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

cell-co-culture-patterning-using-aqueous-two-phase-systems

Research

JoVE Journal

Bioengineering

18.3K Views.  University of Michigan. Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Video: Cell Co-culture Patterning Using Aqueous Two-phase Systems

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

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

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

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

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...