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 dissolv...

<ol>
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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, ...

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18.3K visualizações.  University of Michigan. De duas fases aquosas sistemas foram usados ​​para as populações em simultâneo múltiplos padrões de células. Este método fácil e rápido para a padronização de células tira vantagem da separação de fases de soluções aquosas de dextrano e polietileno glicol e a tensão interfacial que existe entre as duas soluções poliméricas.