Cell Co-culture Patterning Using Aqueous Two-phase Systems

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Phase System Characterization: Determining Thresholds for Phase Separation

1. 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 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dextran 500,000 kDa	Pharmacosmos, Denmark
Polyethylene Glycol 35,000 kDa	Sigma-Aldrich, St. Louis, MO
Hela	ATCC, Manassas, VA
HepG2 C3A	ATCC, Manassas, VA
NIH 3T3	ATCC, Manassas, VA
Cell Tracker	Invitrogen, Carlsbad, CA
DMEM	Gibco, Carlsbad, CA
RPMI	Gibco, Carlsbad, CA
F12	Gibco, Carlsbad, CA
Fetal Bovine Serum	Gibco, Carlsbad, CA