Methods of Cell Separation PDF
Preview Methods of Cell Separation
Methods of Cell Separation Volume I BIOLOGICAL SEPARATIONS Series Editor: Nicholas Catsimpoolas Massachusetts Institute of Technology Cambridge, Massachusetts Methods of Protein Separation, Volume 1 Edited by Nicholas Catsimpoolas Methods of Protein Separation, Volume 2 Edited by Nicholas Catsimpoolas Biological and Biomedical Applications of Isoelectric Focusing Edited by Nicholas Catsimpoolas and James Drysdale Methods of Cell Separation, Vol. 1 Edited by Nicholas Catsimpoolas • A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher. Methods of Cell Separation Volume 1 Edited by Nicholas Catsimpoolas Massachusetts Institute of Technology Plenum Press· New York and London Library of Congress Cataloging in Publication Data Main entry under title: Methods of cell separation. (Biological separations) Includes bibliographies and index. 1. Cell separation. I. Catsimpoolas, Nicholas. II. Series. [DNLM: 1. Cell separa- tion-Methods. WH25 M592] QH585.M49 574.8'7'0724 77-11018 ISBN-13: 978-1-4684-0822-5 e-ISBN- 13: 978-1-4684-0820-1 DOl: 10.1007/978-1-4684-0820-1 © 1977 Plenum Press, New York Sof'tcover reprint of the hardcover 1st edition 1977 A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N.Y. 10011 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microftlming, recording, or otherwise, without written permission from the Publisher Contributors Nicholas Catsimpoolas, Biophysics Laboratory, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cam bridge, Massachusetts 02139 Gerald M. Edelman, The Rockefeller University, New York, New York 10021 Ann L. Griffith, Biophysics Laboratory, Department ofN utrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massa chusetts 02139 Torvard C. Laurent, Institute of Medical and Physiological Chemistry, Biomedical Center, University of Uppsala, S-75123 Uppsala, Sweden Hilkan Pertoft, Institute of Medical and Physiological Chemistry, Biomedi cal Center, University of Uppsala, S-75123 Uppsala, Sweden Herbert A. Pohl, Department of Physics, Oklahoma State University, Stillwater, Oklahoma 74074 Theresa P. Pretlow, Departments of Pathology and Engineering Biophys ics, University of Alabama Medical Center, Birmingham, Alabama 35294 Thomas G. Pretlow II, Departments of Pathology and Engineering Bio physics, University of Alabama Medical Center, Birmingham, Ala bama 35294 Urs S. Rutishauser, The Rockefeller University, New York, New York 10021 Ken Shortman, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia J. A. Steinkamp, Biophysics and Instrumentation Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87545 Harry Waiter, Laboratory of Chemical Biology, Veterans Administration Hospital, Long Beach, California 90822, and Department of Physiol ogy, College of Medicine, University of California, Irvine, California 92717 v Preface 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. It is hoped that new workers with fresh ideas )Vill join us in the near future to achieve further and much needed progress in this important area of biological research. Nicholas Catsimpoolas Cambridge, Massachusetts vii Contents Chapter 1 Preparative Density Gradient Electrophoresis and Velocity Sedimentation at Unit Gravity of Mammalian Cells .............. . Nicholas Catsimpooias and Ann L. Griffith I. Density Gradient Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . 1 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 B. Apparatus and Procedures ........................... 2 C. Velocity of Cell Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 D. Applications.... .. .. . . .. .. .. .. .. .. .. .. . . .. . . .. . . .. .. 9 II. Velocity Sedimentation at Unit Gravity. . . . . . . . . . . . . . . . . . . . 13 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 B. Experimental Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 C. Considerations and Interpretations .................... 20 D. Separation of Human Blood Cells. . . . . . . . . . . . . . . . . . . . . 21 III. Conclusions............................................ 23 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Chapter 2 Isopycnic Separation of Cells and Cell Organelles by Centrifugation in Modified Colloidal Silica Gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Hakan Pertoft and Torvard C. Laurent I. Introduction ........................................... 25 II. Preparations of Colloidal Silica . . . . . . . . . . . . . . . . . . . . . . . . . .. 26 III. Methodology........................................... 27 ix x CONTENTS A. Formation of Gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 B. Selection of Centrifuge and Rotors .................... 31 C. Running Conditions ................................. 32 D. Fractionation of Gradients .... " ...... " " .. .. .. .. .. .. 32 E. Data Analysis ...................................... 32 F. Removal of Gradient Material ........................ 37 IV. Centrifugation of Cells .................................. 43 A. Separation of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 B. Buoyant Densities of Cells ........................... 48 C. Properties of Isolated Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . 48 V. Centrifugation of Subcellular Particles. . . . . . . . . . . . . . . . . . . . . 53 A. Buoyant Densities of Subcellular Particles. . . . . . . . . . . . . . 53 B. Size Limit for Banding in Silica Gradients. . . . . . . . . . . . . . 57 C. Purification of Various Subcellular Particles ............ 57 VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 61 Chapter 3 Dielectrophoresis: Applications to the Characterization and Separation of Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Herbert A. Pohl I. Introduction ........................................... 67 II. Mechanism of Nonuniform Field Effects and Dielectrophoresis 68 III. Polarization Mechanisms in Biological Materials. . . . . . . . . . . . 71 A. Bulk Polarization Processes .......................... 71 B. Interfacial and Space Charge Polarization Processes. . . . . 72 IV. Experimental Collection and Separation of Cells. . . . . . . . . . . . 75 A. Methods and Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 B. Observations on Yeast, Saccharomyces cerevisiae ...... 85 C. Experiments on Canine Blood Platelets ................ 104 D. Experiments on Red Blood Cells ..................... 115 E. Experiments on Chloroplasts. . . . . . . . . . . . . . . . . . . . . . . .. 124 F. Mitochondria Experiments ........................... 130 G. Observations on Bacteria ............................ 136 H. The Construction of Oriented Living Cell Masses ....... 149 1. Single Cell Dielectrophoresis ......................... 153 J. Continuous Separations of Cells by Dielectrophoresis . . .. 161 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 165 CONTENTS xi Chapter 4 Separation of Viable Cells by Velocity Sedimentation in an Isokinetic Gradient of Ficoll in Tissue Culture Medium .................... 171 Thomas G. Pretlow II and Theresa P. Pretlow I. Introduction ........................................... 171 II. Historical Development of Technique ..................... 172 III. Isopycnic Sedimentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 173 IV. Velocity (Including Isokinetic) Sedimentation in Ficoll Gradients .......... " ................ " .. .. .. .. .. .. .. .. 175 A. Velocity Sedimentation Prior to the Development of the Isokinetic Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 175 B. Isokinetic Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 177 V. Selected Theoretical Considerations. . . . . . . . . . . . . . . . . . . . . .. 179 A. Medium for Velocity Sedimentation ................... 179 B. Properties That Determine Rate of Sedimentation. . . . . .. 180 C. Misuse of Velocity Sedimentation to Determine Size .... 180 VI. Critical Analysis of Data from Experiments in Cell Separation 183 A. Characterization of Starting Sample ................... 184 B. Recovery .......................................... 186 C. Expression of Purification. . . . . . . . . . . . . . . . . . . . . . . . . . .. 186 D. Morphology ................. . . . . . . . . . . . . . . . . . . . . . .. 187 References. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. 188 Chapter 5 Fractionation and Manipulation of Cells with Chemically Modified Fibers and Surfaces .......................................... 193 Urs S. Rutishauser and Gerald M. Edelman I. Introduction ........................................... 193 II. Affinity Fractionation of Cells. . . . . . . . . . . . . . . . . . . . . . . . . . .. 193 III. Fiber Fractionation of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 194 IV. Manipulation of Cells and the Study of Localized Perturbations at the Cell Surface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 196 V. Procedures ............................................ 197 A. Preparation of Chemically Modified Fibers and Surfaces. 197 B. Preparation of Cell Suspensions . . . . . . . . . . . . . . . . . . . . . .. 202 C. Binding of Cells to Fibers .......................... " 203 xii CONTENTS D. Observation and Quantitation of Bound Cells .. . . . . . . . .. 205 E. Removal of Cells from the Fiber. . . . . . . . . . . . . . . . . . . . .. 206 VI. Applications ........................................... 207 A. Fractionation of Lymphoid Cell Populations . . . . . . . . . . .. 208 B. Binding of Cells to Antibody- or Lectin-Coated Fibers. .. 216 C. Studies on Fiber-Cell Interactions .................... 218 D. Cell Agglutination Induced by Lectins ................. 220 E. Studies of Adhesion among Neural Cells of the Chick Embryo ............................................ 223 F. Isolation of Membrane Fragments and Receptors. . . . . . .. 223 VII. Prospective Applications ................................ 226 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 227 Chapter 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 ................................ 229 Ken Shortman I. Introduction 229 II. Separation of B from T Lymphocytes .. . . . . . . . . . . . . . . . . . .. 230 A. Electrophoretic and Adherence Separation ............. 230 B. Sedimentation and Buoyant Density Separation. .. .. .. .. 232 III. Separation of Functionally Distinct B-Lymphocyte Subsets .. 233 A. The General Approach Leading to a Model of B-Cell Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 233 B. Electrophoretic and Adherence Column Characterization of Adult Mouse Virgin and Memory AFC Progenitors and of AFC ............................................ 236 C. Sedimentation Rate Analysis of Adult Mouse Virgin and Memory AFC Progenitors. . . . . . . . . . . . . . . . . . . . . . . . . . .. 238 D. Density Distribution Analysis of Adult Mouse Spleen Virgin and Memory AFC Progenitors . . . . . . . . . . . . . . . . .. 240 E. The Characteristics of Newbom, Unstimulated Virgin AFC Progenitor B Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 241 IV. General Conclusions ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 242 V. Appendix: Technical Aspects of the Separation Procedures.. 244 A. General Points, Preliminary Cell Preparation, and Damaged Cell Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 244
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