Invertebrate animals span a huge biological realm of form and function, and have evolved many unique characteristics. The wealth of variety within this diverse group not only contributes to the health of the ecosystem, but also provides a virtually unlimited potential for biological discovery. Models for basic research originally included a large number of invertebrates. However,over the past few decades, most research has focused on a limited number of systems, such as flies and worms, for which molecular genetic technology is easily available. Nevertheless, two things have recently changed this focus:the development of RNA interference (RNAi) techniques to perturb gene expression in diverse organisms and a renewed interest in the evolution of developmental mechanisms. One main caveat to being interested in diverse organisms is the comparable paucity of technical information regarding the handling of tissues and cells from these organisms. Knowing where to begin with any new animal system requires a bit of patience, a certain amount of stubbornness, and, now thanks to Mitsuhashi, a good road map to plan your way through this new unknown.
For scientists initiating tissue culture projects, Invertebrate Tissue Culture Methods starts with a detailed introduction into the necessary and suggested equipment needed to properly equip facilities for invertebrate culture research. General principles for establishing and maintaining cultures are discussed, with an emphasis upon those areas most likely to delay the progress of establishing a viable culture. Although media selection can partly depend upon empirical determination, several successful culture solutions are described in the text, which may serve as a template to customize media selection for animals of a specific phyla. In addition, several suggested media additives are described to maintain the proper nutrition and growth of the culture. Once a culture media has been selected, contamination of the culture must be avoided; to this end, frequent sources of contamination are outlined in this book, such as food and bacteria in the gut, as well as from the incompletely sterilized external surfaces of the animal. Avoiding an excess of unwanted blood, which will oxidize to toxic metabolites, including melanin, is another recurring theme in the initial establishment of a healthy invertebrate tissue culture.
Tissues of interest are usually best identified through careful dissection and consultation of available anatomical information. Invertebrate Tissue Culture Methods offers a comprehensive array of figures, many of which are focused on the proper identification and removal of a variety of specific tissues from a wide range of invertebrate phyla: Insecta, Prochordata,Echinodermata, Mollusca, Annelida, Nematoda, Platyhelminthes, Coelenterata,Porifera, and various non-insect arthropods. Although the emphasis of the text is on previously reported culture methods for representative members of these invertebrate phyla, one can also extrapolate these methods to new tissue types and organisms. An equally broad portion of the book is devoted to the establishment of whole-organ culture for selected tissues from the above phyla, and this section usefully focuses on those tissues that have been successfully maintained. Techniques for studying developmental processes in embryonic tissues, as well as in imaginal discs and gonadal tissue, are outlined for most phyla, although any given species may not be covered in great depth. Most major organ systems are represented, and are discussed from both a developmental and a functional perspective, including the nervous system, the circulatory system and hemocytes, intestinal tissues, and species-specific exocrine tissues (such as silk and pheromone glands). Once the appropriate tissues have been removed, there are a number of general methods in cell culture described that can be applied to fit individual experimental design, including protocols for cellular dissociation, subculture and the induction of cell growth.
The concluding portion of Invertebrate Tissue Culture Methodsfocuses on related techniques for the study of cells in culture. These instructive chapters offer general directions for the characterization and manipulation of viable tissue culture. Broadly, these chapters describe the genetic identification of cell lines, methods to evaluate and document cell growth and viability, techniques for optimal photography, and tips for creating large-scale invertebrate tissue cultures. This final section offers a concise compilation of several modern and traditional approaches to the characterization of cell culture, and is outlined in a protocol-driven style that is accessible and easily followed. An appendix is provided to the main text, listing the various formulations of media, as well as media suppliers,together with an anthology of previously reported continuously cultured cell lines.
There are a number of places where this book could have been improved. While an outline of basic cell culture and sterile techniques will prove useful to the uninitiated researcher, in reality, this introductory section could have been replaced with a reference to any of a number of standard cell culture handbooks. In addition, the section on organ culture could have been expanded to focus on the techniques that currently apply to flies, as the myriad of techniques developed for flies will equally apply for other organisms. Even a table of references for additional fly organ culture protocols would have been useful. Also, the book was short on descriptions of molecular technologies, and this aspect could have been developed to cover techniques such as in situ hybridization and, in particular, the use of RNAi. However, overall this book will prove to be an essential companion to any laboratory wishing to research non-standard invertebrates.
One of the most exciting features of this book is the new opportunity for discovery that will accompany the reading of this text, and while it could certainly be used as a teaching device in a formal laboratory setting, it is also suited for hands-on use in any invertebrate research lab. As scientific questions of developmental and evolutionary interest continue to probe invertebrate systems, researchers are likely to continue to require a broad source of species-relevant information, and, to this end, Invertebrate Tissue Culture Methods provides an excellent resource.
Invertebrate Tissue Culture Methods
By Jun Mitsuhashi
Springer-Verlag (2002) 446 pages