For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

Plant Cell and Tissue Culture continues the high standards of Humana's Methods in Molecular Biology series. Its step-by-step approach (a hallmark of the series) is applied to a wide range of basic laboratory techniques and culture conditions appropriate to plant cells. Because of the diversity of cell types species and culture methods much of this volume is devoted to the culture of particular cell types and to the regeneration of these cells into whole plants. Special attention is also given to the genetic modification of plants as well as to the economic significance of plant products. Chapters cover a wide range of topics and techniques including: tissue culture media and selection cryopreservation callus culture techniques organ culture embryogenesis batch culture large-scale culture hormonal control fertilization techniques gene transfer cell immobilization production systems cell product purification DNA expression electrofusion of plant cells mutant selection mutagenesis techniques automation transfer of nuclei protoplast culture media analysis micropropagation. A detailed appendix lists the formulas for the most commonly employed plant cell media.Comprehensive easy to follow and a pleasure to use Pollard and Walker's Plant Cell and Tissue Culture is an essential tool for everyone--at all levels of proficiency and experience--involved in plant culture.

For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

Plant Cell and Tissue Culture continues the high standards of Humana's Methods in Molecular Biology series. Its step-by-step approach (a hallmark of the series) is applied to a wide range of basic laboratory techniques and culture conditions appropriate to plant cells. Because of the diversity of cell types, species, and culture methods, much of this volume is devoted to the culture of particular cell types and to the regeneration of these cells into whole plants. Special attention is also given to the genetic modification of plants, as well as to the economic significance of plant products. Chapters cover a wide range of topics and techniques, including:* tissue culture media and selection * cryopreservation * callus culture techniques * organ culture * embryogenesis * batch culture * large-scale culture * hormonal control * fertilization techniques * gene transfer * cell immobilization * production systems * cell product purification * DNA expression * electrofusion of plant cells * mutant selection * mutagenesis techniques * automation * transfer of nuclei * protoplast culture * media analysis * micropropagation.A detailed appendix lists the formulas for the most commonly employed plant cell media. Comprehensive, easy to follow, and a pleasure to use, Pollard and Walker's Plant Cell and Tissue Culture is an essential tool for everyone--at all levels of proficiency and experience--involved in plant culture.

For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

It is now seven years since the original Animal Cell Culture volume was published. During this time, techniques have advanced dramatically. The- fore, we have completely revised this edition, including many new chapters and updating old chapters. There has also been a proliferation of methods books covering specific areas of tissue culture. Thus, in this revised edition, we have left out chapters on hybridoma technology because of the many books dealing specifically with this subject. Instead, we have concentrated on the original purpose of this volume now entitled Basic Cell Culture Protocols- which was to provide easy-to-follow protocols for animal cell culture, that hopefully, will be useful to experts and beginners alike. We would like again to acknowledge Dr. Cliff Stanners who taught me (JWP) cell culture, as well as members of the Toronto Somatic Cell Genetics Group for their advice and encouragement, in particular, Drs. L. Siminovitch and V. Ling. Elyse Rizzo''s valuable secretarial assistance in compiling this new volume is greatly appreciated. Jeffrey Pollard John M. Walker W. Contents Preface ............................................................................................................. v Contributors ..................................................................................................... xi 1 Basic Cell Culture Jeffrey Pollard .................................................................................. 1 2 Establishment, Maintenance, and Cloning of Human Dermal Fibroblasts Gareth Jones and Clare J. Wise ..................................................

For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

Plant Cell and Tissue Culture continues the high standards of Humana's Methods in Molecular Biology series. Its step-by-step approach (a hallmark of the series) is applied to a wide range of basic laboratory techniques and culture conditions appropriate to plant cells. Because of the diversity of cell types species and culture methods much of this volume is devoted to the culture of particular cell types and to the regeneration of these cells into whole plants. Special attention is also given to the genetic modification of plants as well as to the economic significance of plant products. Chapters cover a wide range of topics and techniques including: tissue culture media and selection cryopreservation callus culture techniques organ culture embryogenesis batch culture large-scale culture hormonal control fertilization techniques gene transfer cell immobilization production systems cell product purification DNA expression electrofusion of plant cells mutant selection mutagenesis techniques automation transfer of nuclei protoplast culture media analysis micropropagation. A detailed appendix lists the formulas for the most commonly employed plant cell media.Comprehensive easy to follow and a pleasure to use Pollard and Walker's Plant Cell and Tissue Culture is an essential tool for everyone--at all levels of proficiency and experience--involved in plant culture.

For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

Plant Cell and Tissue Culture continues the high standards of Humana's Methods in Molecular Biology series. Its step-by-step approach (a hallmark of the series) is applied to a wide range of basic laboratory techniques and culture conditions appropriate to plant cells. Because of the diversity of cell types, species, and culture methods, much of this volume is devoted to the culture of particular cell types and to the regeneration of these cells into whole plants. Special attention is also given to the genetic modification of plants, as well as to the economic significance of plant products. Chapters cover a wide range of topics and techniques, including:* tissue culture media and selection * cryopreservation * callus culture techniques * organ culture * embryogenesis * batch culture * large-scale culture * hormonal control * fertilization techniques * gene transfer * cell immobilization * production systems * cell product purification * DNA expression * electrofusion of plant cells * mutant selection * mutagenesis techniques * automation * transfer of nuclei * protoplast culture * media analysis * micropropagation.A detailed appendix lists the formulas for the most commonly employed plant cell media. Comprehensive, easy to follow, and a pleasure to use, Pollard and Walker's Plant Cell and Tissue Culture is an essential tool for everyone--at all levels of proficiency and experience--involved in plant culture.

For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

It is now seven years since the original Animal Cell Culture volume was published. During this time, techniques have advanced dramatically. The- fore, we have completely revised this edition, including many new chapters and updating old chapters. There has also been a proliferation of methods books covering specific areas of tissue culture. Thus, in this revised edition, we have left out chapters on hybridoma technology because of the many books dealing specifically with this subject. Instead, we have concentrated on the original purpose of this volume now entitled Basic Cell Culture Protocols- which was to provide easy-to-follow protocols for animal cell culture, that hopefully, will be useful to experts and beginners alike. We would like again to acknowledge Dr. Cliff Stanners who taught me (JWP) cell culture, as well as members of the Toronto Somatic Cell Genetics Group for their advice and encouragement, in particular, Drs. L. Siminovitch and V. Ling. Elyse Rizzo''s valuable secretarial assistance in compiling this new volume is greatly appreciated. Jeffrey Pollard John M. Walker W. Contents Preface ............................................................................................................. v Contributors ..................................................................................................... xi 1 Basic Cell Culture Jeffrey Pollard .................................................................................. 1 2 Establishment, Maintenance, and Cloning of Human Dermal Fibroblasts Gareth Jones and Clare J. Wise ..................................................