The resolution of mixtures of viable mammalian cells into homogeneous fractions by zonal centrifugation.
Abstract: Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.
Publication Date: 1968-02-01 PubMed ID: 5638886PubMed Central: PMC2107352DOI: 10.1083/jcb.36.2.369Google Scholar: Lookup
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- Journal Article
Summary
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The research article focuses on the successful separation of mixtures of mammalian cells into homogeneous fractions using A-1X zonal centrifuge rotor and density gradients. The researchers found that cells remained viable post separation, and they document the potential this technique holds for large-scale fractionation of solid tissues, especially tumor cells.
Methodology Used
- The researchers used an A-1X zonal centrifuge rotor and density gradients comprising of Ficoll in modified Eagle’s MEM suspension-culture medium. Ficoll is a high molecular weight, sucrose-based polymer used for separating cells in biochemistry and cell biology. MEM or minimum essential medium, on the other hand, is a type of cell culture medium.
- They tested cell viability post separation by plating efficiency or by observing motility with time-lapse photography. Plating efficiency is the number of colonies originating from one cell, and time-lapse photography allows researchers to periodically record changes that happen slowly over time.
Results and Observations
- The researchers were able to separate rabbit thymocyte and HeLa cell mixtures with 99% and 89% purity, respectively. Thymocytes are immature T cells found in the thymus and HeLa cells are an immortal cell line utilized in scientific research.
- The researchers also separated mixtures of thymocytes and human acute leukemia cells (Roswell Park strain LKID) with 93% and 91% purity respectively.
- HeLa cells were separated from a mixture with horse leukocytes with 92% purity. Leukocytes are white blood cells that help the body fight off infections.
Additional Contributions
- The researchers also created a book of charts detailing the sedimentation position and velocity versus time of cells under standard conditions of gradient composition, angular velocity, and temperature.
- These charts can be utilized to determine the necessary centrifugation time for achieving maximum cell separation.
Conclusion and Future Possibilities
- The high-degree of success in separating mixtures of cells encourages the potential of this technique in large-scale fractionation of solid tissues, particularly tumor tissues.
- This technique could enable the preparation of viable cells of a single type from tumor tissues, which could significantly advance cancer research and treatment.
Cite This Article
APA
Boone CW, Harell GS, Bond HE.
(1968).
The resolution of mixtures of viable mammalian cells into homogeneous fractions by zonal centrifugation.
J Cell Biol, 36(2), 369-378.
https://doi.org/10.1083/jcb.36.2.369 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Biometry
- Centrifugation, Density Gradient
- Centrifugation, Zonal
- Computers
- Cricetinae
- Densitometry
- HeLa Cells
- Horses
- Humans
- Leukemia
- Leukocytes
- Models, Theoretical
- Rabbits
- Thymus Gland
- Viscosity
References
This article includes 1 references
- Anderson NG, Harris WW, Barber AA, Rankin CT Jr, Candler EL. Separation of subcellular components and viruses by combined rate- and isopycnic-zonal centrifugation.. Natl Cancer Inst Monogr 1966 Jun;21:253-83.
Citations
This article has been cited 16 times.- Selby PJ, Raghavan D. Role of laboratory chemosensitivity testing in the selection of cancer chemotherapy for individual patients.. J Clin Pathol 1981 May;34(5):455-63.
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- Cooper AJ, Bain AG. A demonstration of sub-population of rosetting cells in lymph nodes from sensitized guinea-pigs by zonal centrifugation.. Immunology 1971 Nov;21(5):781-94.
- Ball FR, Medzon EL. Sedimentation changes of L cells in a density gradient early after infection with vaccinia virus.. J Virol 1973 Sep;12(3):588-93.
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- Egan PM, Garrett JV, Gilbert CW. Separation of abnormal cells in the peripheral blood by means of the zonal centrifuge.. J Clin Pathol 1974 Sep;27(9):741-50.
- Pretlow TG 2nd, Glick MR, Reddy WJ. Separation of beating cardiac myocytes from suspensions of heart cells.. Am J Pathol 1972 May;67(2):215-26.
- Mills DM, Zucker-Franklin D. Electron microscopic study of isolated Kupffer cells.. Am J Pathol 1969 Feb;54(2):147-66.
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- Tse H, Dutton RW. Separation of helper and suppressor T lymphocytes on a ficoll velocity sedimentation gradient.. J Exp Med 1976 May 1;143(5):1199-210.
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