Aqueous 2-Phase System for the Separation and Recovery of Mammalian Cells from Contaminated Cultures
Tufts researchers have developed a method to separate cultured mammalian cells from contaminants based on density using aqueous two-phase systems (ATPS) which allows recovered cells to be cultured after decontamination without the need for antibiotic treatments.
The in vitro culture of mammalian cells is a crucial part of biomedical research. Aseptic technique is necessary to ensure that only the cells of interest are growing in the culture. Contamination, however, is still a relatively common occurrence in cell culture laboratories. Typical contaminants include bacteria, yeast, and mold. These microorganisms affect the way cells behave in culture (e.g., change morphology, growth, and viability) and alter the culture conditions (e.g., by changing pH or by competing for resources in the medium). Nonviable cells (i.e., cells present in culture that lack membrane integrity as demonstrated by the inability to exclude dyes), cell fragments, and debris can also be considered contaminants because their presence can bias seeding densities or skew the results of functional assays. Cultures that contain a large fraction of nonviable cells (e.g., cultures that are overgrown) may not be usable unless the viable cells can be isolated from the dead cells.
Antibiotics, such as penicillin and streptomycin, are used to treat contamination or prevent it prophylactically. However, these compounds may adversely interfere with normal processes in cultured cells, exacerbate contamination issues by masking poor aseptic technique, or promote antibiotic resistance and the development of resistant microorganisms. To prevent the spread of microorganisms to other cultures and to avoid the negative effects of antibiotic use, contaminated cultures are typically destroyed with bleach. Although this approach is an effective way to contain and eliminate contaminants, it also results in the disposal of the cultured cells. This is, at a minimum, a waste of time and effort for the researchers and money for the lab. Moreover, if the cells are rare or valuable (e.g., primary or engineered), their disposal may not be acceptable. The capability to decontaminate and recover the desired population of cells would be far more appropriate in these cases. Additionally nonviable cells in cultures can bias cell counts and affect the results of assays. Cultures that contain a large fraction on nonviable cells (e.g., cultures that are overgrown) may not be usable unless the viable cells can be isolated from the dead cells.
Use aqueous two-phase systems (ATPS) for density-based separations of biomolecules and cells. ATPSs form when solutions of water-soluble polymers are mixed above a threshold concentration and molecular weight. These systems produce two thermodynamically stable phases with different densities separated by a liquid-liquid interface. The inherent biocompatibility associated with using water as a common solvent makes ATPS particularly well suited for density-based separations. Cells can be partitioned based on affinity into either phase or can be isolated based on density at the liquid-liquid and liquid-container interfaces. Microorganisms that may contaminate cell cultures such as bacteria (e.g., E. coli) and yeast have higher buoyant densities than cultured mammalian epithelial cells (e.g., HeLa). Under these conditions, contaminated cultures will separate upon centrifugation with the mammalian cells coalescing to the liquid-liquid interface and the denser contaminates forming a pellet at the bottom of the container. After centrifugation, the desired cells can be recovered from the interface by pipette, washed, and transferred to fresh medium for further culture. The contaminants can be bleached and disposed of.
The concentration of polymers and buffer are carefully selected to generate systems with a pH and osmolality close to physiological conditions and a density step above the density of mammalian cells but below the density of microorganisms. This biocompatible system allows for the cells to be separated from contaminants by density and recovered without a loss of viability. Tests showed that cell recovery was an average of 74.4 ± 2.7%. Recovered cells had comparable viability and growth rates to control populations that were not introduced to ATPS.
Viability enrichment in overgrown cell cultures
•Simple, gentle, and nondestructive process.
•Cells can be recovered without treatments with antimicrobial compounds.
•Offers a more complete separation. The sharp density steps in ATPS allow cells and contaminants to be isolated at different interfaces with significant phase volume between them. The use of ATPS is also preferable because cells with slightly different densities become concentrated at an interface rather than in a disperse band like in density gradients.
• Isolation at the interface and separation in distance allows for more complete recovery of the cells without recovery of the contaminants.
•Density-based separation of cell populations allows non-viable and lysed cells to be removed, enriching the cell population for viable cells with similar densities.
IP STATUS US Provisional Patent filed.