Methods of Cell Separation

Presently, the need for methods involving separation, identification, and characterization of different kinds of cells is amply realized among immu­ nologists, hematologists, cell biologists, clinical pathologists, and cancer researchers. Unless cells exhibiting different functions and stages of differ­ entiation are separated from one another, it will be exceedingly difficult to study some of the molecular mechanisms involved in cell recognition, specialization, interactions, cytotoxicity, and transformation. Clinical diag­ nosis of diseased states and use of isolated cells for therapeutic (e. g. , immunotherapy) or survival (e. g. , transfusion) purposes are some of the pressing areas where immediate practical benefits can be obtained by applying cell separation techniques. However. the development of such useful methods is still in its infancy. A number of good techniques exist based either on the physical or biological properties of the cells, and these have produced some valuable results. Still others are to be discovered. Therefore, the purpose of this open-end treatise is to acquaint the reader with some of the basic principles, instrumentation, and procedures pres­ ently in practice at various laboratories around the world and to present some typical applications of each technique to particular biological prob­ lems. To this end, I was fortunate to obtain the contribution of certain leading scientists in the field of cell separation, people who in their pioneer­ ing work have struggled with the particular problems involved in separating living cells and in some way have won.

Contenu

1 Preparative Density Gradient Electrophoresis and Velocity Sedimentation at Unit Gravity of Mammalian Cells.- I. Density Gradient Electrophoresis.- A. Introduction.- B. Apparatus and Procedures.- C. Velocity of Cell Transport.- D. Applications.- II. Velocity Sedimentation at Unit Gravity.- A. Introduction.- B. Experimental Procedures.- C. Considerations and Interpretations.- D. Separation of Human Blood Cells.- III. Conclusions.- References.- 2 Isopycnic Separation of Cells and Cell Organelles by Centrifugation in Modified Colloidal Silica Gradients.- I. Introduction.- II. Preparations of Colloidal Silica.- III. Methodology.- A. Formation of Gradients.- B. Selection of Centrifuge and Rotors.- C. Running Conditions.- D. Fractionation of Gradients.- E. Data Analysis.- F. Removal of Gradient Material.- IV. Centrifugation of Cells.- A. Separation of Cells.- B. Buoyant Densities of Cells.- C. Properties of Isolated Cells.- V. Centrifugation of Subcellular Particles.- A. Buoyant Densities of Subcellular Particles.- B. Size Limit for Banding in Silica Gradients.- C. Purification of Various Subcellular Particles.- VI. Concluding Remarks.- References.- 3 Dielectrophoresis: Applications to the Characterization and Separation of Cells.- I. Introduction.- II. Mechanism of Nonuniform Field Effects and Dielectrophoresis.- III. Polarization Mechanisms in Biological Materials.- A. Bulk Polarization Processes.- B. Interfacial and Space Charge Polarization Processes.- IV. Experimental Collection and Separation of Cells.- A. Methods and Procedures.- B. Observations on Yeast, Saccharomycescerevisiae.- C. Experiments on Canine Blood Platelets.- D. Experiments on Red Blood Cells.- E. Experiments on Chloroplasts.- F. Mitochondria Experiments.- G. Observations on Bacteria.- H. The Construction of Oriented Living Cell Masses.- I. Single Cell Dielectrophoresis.- J. Continuous Separations of Cells by Dielectrophoresis.- References.- 4 Separation of Viable Cells by Velocity Sedimentation in an Isokinetic Gradient of Ficoll in Tissue Culture Medium.- I. Introduction.- II. Historical Development of Technique.- III. Isopycnic Sedimentation.- IV. Velocity (Including Isokinetic) Sedimentation in Ficoll Gradients.- A. Velocity Sedimentation Prior to the Development of the Isokinetic Gradient.- B. Isokinetic Gradient.- V. Selected Theoretical Considerations.- A. Medium for Velocity Sedimentation.- B. Properties That Determine Rate of Sedimentation.- C. Misuse of Velocity Sedimentation to Determine Size.- VI. Critical Analysis of Data from Experiments in Cell Separation.- A. Characterization of Starting Sample.- B. Recovery.- C. Expression of Purification.- D. Morphology.- References.- 5 Fractionation and Manipulation of Cells with Chemically Modified Fibers and Surfaces.- I. Introduction.- II. Affinity Fractionation of Cells.- III. Fiber Fractionation of Cells.- IV. Manipulation of Cells and the Study of Localized Perturbations at the Cell Surface.- V. Procedures.- A. Preparation of Chemically Modified Fibers and Surfaces.- B. Preparation of Cell Suspensions.- C. Binding of Cells to Fibers.- D. Observation and Quantitation of Bound Cells.- E. Removal of Cells from the Fiber.- VI. Applications.- A. Fractionation of Lymphoid Cell Populations.- B. Binding of Cells to Antibody- or Lectin-Coated Fibers.- C. Studies on Fiber-Cell Interactions.- D. Cell Agglutination Induced by Lectins.- E. Studies of Adhesion among Neural Cells of the Chick Embryo.- F. Isolation of Membrane Fragments and Receptors.- VII. Prospective Applications.- References.- 6 The Separation of Lymphoid Cells on the Basis of Physical Parameters: Separation of B- and T-Cell Subsets and Characterization of B-Cell Differentiation Stages.- I. Introduction.- II. Separation of B from T Lymphocytes.- A. Electrophoretic and Adherence Separation.- B. Sedimentation and Buoyant Density Separation.- III. Separation of Functionally Distinct B-Lymphocyte Subsets.- A. The General Approach Leading to a Model of B-Cell Development.- B. Electrophoretic and Adherence Column Characterization of Adult Mouse Virgin and Memory AFC Progenitors and of AFC.- C. Sedimentation Rate Analysis of Adult Mouse Virgin and Memory AFC Progenitors.- D. Density Distribution Analysis of Adult Mouse Spleen Virgin and Memory AFC Progenitors.- E. The Characteristics of Newborn, Unstimulated Virgin AFC Progenitor B Cells.- IV. General Conclusions.- V. Appendix: Technical Aspects of the Separation Procedures.- A. General Points, Preliminary Cell Preparation, and Damaged Cell Removal.- B. Electrophoretic Separation.- C. Density Separation.- D. Sedimentation-Velocity Separation.- E. Adherence Separation.- References.- 7 Multiparameter Analysis and Sorting of Mammalian Cells.- I. Introduction.- A. Historical Background of Instrumentation Development.- B. Recent Applications of Flow Analysis and Sorting.- II. Description of the LASL Multiparameter Cell Separator.- A. Principle of Analysis and Separation.- B. Flow Chamber.- C. Laser-Beam-Shaping Optics and Wavelength Considerations.- D. Cell Sensing Methods.- E. Signal Processing.- F. Cell Separation Logic.- III. Examples of Multiparameter Cell Analysis and Sorting Applications.- A. Separation of [3H] Thymidine-Labeled Cells Based on DNA Measurements.- B. Analysis and Separation of Tumor Cells Based on DNA and Cell Volume Measurements.- C. DNA and Cell Volume Analysis of Drug-Treated Cultured Cells.- D. Analysis of the Cell-Surface Density of Lectin Binding to Cultured Cells.- E. Differential Analysis and Separation of Mammalian Leukocytes.- F. Measurement of Nuclear and Cytoplasmic Diameter in Single Cells.- G. Cell Characterization by Multiangle Light Scatter.- IV. Summary and Conclusions.- References.- 8 Partition of Cells in Two-Polymer Aqueous Phases: A Surface Affinity Method for Cell Separation.- I. Introduction.- A. Usefulness.- B. Background.- C. Properties of the Phases.- D. Partition of Cells.- E. Properties of the Cells Measured by Partition.- II. Methods.- A. Preparation of Cells for Partition.- B. Stock Solutions.- C. Phase Systems.- D. Partition.- E. Phase Selection.- F. Countercurrent Distribution.- III. Results.- A. Separation of Cells.- B. Subfractionation of Cell Populations and Tracing Membrane Changes That Occur as a Function of Normal or Abnormal inVivo Processes.- C. Membrane Alterations Due to…