Arthropod Cell Culture Systems

Karl Maramorosch

• Publisher: CRC Press
• Year: 2018
• Print ISBN:
  9781315890807
• eBook ISBN:
  9781351086806
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Invertebrate cell culture is increasingly being used in various areas of biological research. Research in cellular biology and pathology that previously depended primarily on in vitro investigations of vertebrate animal cell systems is now being conducted using invertebrate cells. Specialists and pioneers from the United States, Japan, Switzerland, Slovakia, and China have presented original contributions to create a well-balanced cross-section of current developments. Topics discussed include the preparation of cell culture media; cultivation of mosquito, lepidopteran, grasshopper, and tick cells; the application of such cells to mammalian and plant virus research; and diverse applications in medicine, biology, and agriculture. A special chapter devoted to the work of Japanese cell culture pioneers is also featured. All chapters are supported by tables, photographs, and up-to-date bibliographies.

Chapter 1

INSECT CELL CULTURE MEDIA

Jun Mitsuhashi

Table of Contents

• I. Introduction
• II. History of Medium Development
• III. Medium Components
• IV. Preparation of Media
  • A. Preparation of Stock Solutions
    • 1. Stock Solutions of Inorganic Salts
    • 2. Stock Solutions of Amino Acids, Sugars, and Organic Acids
    • 3. Stock Solutions of Vitamins
  • B. Preparation of Ready to Use Medium
• V. Preparation of Medium Additives
  • A. Preparation of Insect Hemolymph
  • B. Preparation of Water-Insoluble Additives
• VI. Sterilization
• VII. Test for Contamination
• VIII. Storage
• IX. Medium Improvement
  • A. Serum-Free Media
  • B. Chemically Defined Media
  • C. Low-Cost Media
• References

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Jun Mitsuhashi, D.Agr., is Professor of Applied Entomology at Tokyo University of Agriculture and Technology. He received his Bachelor of Agriculture in applied entomology in 1955 and Doctor of Agriculture in 1965 at The University of Tokyo. 1955–1984, National Institute of Agricultural Sciences; 1984–1988. Forest Research Institute; 1988-present, Tokyo University of Agriculture and Technology; 1962–1964, Boyce Tompson Institute for Plant Research; 1968–1969, Division of Entomology, CSIRO; 1993–1994. President of Japanese Society of Applied Entomology and Zoology. Insect cell culture; insect endocrinology; artificial rearing of insects, insect endosymbiotes; insect pathology. Address: Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan.

I. Introduction

Since Grace¹ established the first insect cell line, very many cell lines have been established during the last 3 decades from various insects. In order to maintain these cell lines, various culture media have been used, because there is no single culture medium that could maintain the growth of every insect cell. Some cell lines can be maintained in relatively nutrient-poor media but others cannot. Formulation of widely usable media is one of the tasks in developing new culture media.

II. History of Medium Development

When the first trial to culture insect tissues in vitro was made, insect hemolymph itself was used as the culture medium by Goldschmidt.² At present, we know that hemolymph itself is not suitable for culture media, because it soon turns dark due to the formation of melanin by the action of phenol oxidase, and the resulting melanin is toxic to the cultured tissues or cells. Goldschmidt did not mention how he could overcome this sort of trouble. In the early stage of insect tissue cultures, most researchers used insect hemolymph or simple saline solutions as culture media, and the results were always short survival of tissues or cells. Trager³ formulated a culture medium by combining inorganic salts, egg albumin digest, maltose, and insect hemolymph (Table 1). This medium was the first medium formulated specifically for insect cell growth and it supported growth for a considerable time. After Trager’s work, several investigators examined the ability of vertebrate cell culture media to support insect cell growth although none of them gave promising results. Wyatt⁴ formulated a synthetic culture medium by combining 6 inorganic salts, 21 amino acids, 4 other organic acids, and 3 sugars (Table 2). Her formulation was based on the chemical analyses of hemolymph from several insect species.⁵ Although the medium could not support cell growth by itself, it supported the cell growth well when it was fortified by the addition of heat-treated insect hemolymph. This medium actually gave a clue to the formulation of insect cell culture media, and even at present we are using various modifications of her medium. Wyatt’s medium was improved by Grace,¹ and with this medium, he established the first insect continuous cell lines. His modification was the addition of ten water-soluble vitamins, change of balance of inorganic salts, and increment of sucrose (Table 3). Grace’s medium is well known as the medium which can support growth of various insect tissues, when supplemented with insect hemolymph or vertebrate sera, especially fetal bovine serum (FBS), which can be purchased commercially at present. Another type of culture medium is a chemically undefined medium whose major components are natural substances. Mitsuhashi and Maramorosch⁶ formulated a simple medium presently called the MM medium. This medium contained lactalbumin hydrolysate, TC-yeastolate, and FBS as natural substances (Table 4). Originally this medium was developed for culturing leafhopper cells. It turned out later that this medium was well suited for mosquito cell cultures and also for culturing cells from various other insects, belonging to many orders. Furthermore, this medium is now known to support growth of many cell lines without sera supplements.⁷

In recent years, serum-free culture media were developed by many investigators. Now many insect cell lines can be cultured in serum-free media, but usually their growth rates are inferior in serum-free media, compared to serum-containing media. Some media sold commercially are said to maintain cell growth at the same rate as serum-containing media. However, these media do not necessarily support cell growth of every cell line. Most serum-free media contain more or less chemically undefined substances. It is, therefore, required to formulate completely chemically defined media. Once such a chemically defined medium was formulated,⁸ however, this medium was rather difficult to prepare and it could support only limited cell line species (Table 5). Some media sold by industries are said to be almost chemically defined, but they cannot be used for biochemical studies of cultured cells because the formulation has not been disclosed.

TABLE 1
Trager’s Medium

TABLE 2
Wyatt’s Medium

Recent development of biotechnology using insect cell cultures requires large-scale cultures of insect cells. Developments of low cost media are desired for large-scale cultures of insect cells especially for the purpose of production of viral insecticides. By the development of serum-free media, the cost of media has decreased considerably and now we can purchase fairly low-cost media, but still further cost reduction is needed.

III. Medium Components

In general, the main ingredients of culture media consist of inorganic salts, proteins, sugars as an energy source, and vitamins.

TABLE 3
Grace’s Medium

TABLE 4
Mitsuhashi and Maramorosch’s MM Medium

Insect cells are said to be insensitive to ion balance and change of pH. Most media contain potassium chloride, sodium phosphate, sodium bicarbonate, magnesium chloride, and calcium chloride. Some media also contain sodium chloride or magnesium sulfate. Sodium chloride is not important for insect cells probably because insect hemolymph contains only a little sodium chloride. Since most insect cells are tolerant to a wide range of pH, incorporation of buffer to inorganic salt composition is not common, although some media contain some buffers, such as tris buffer,⁹ glycine buffer,¹⁰ and so on. There are two types of inorganic salt compositions, one that contains about 0.8% sodium chloride and is similar to vertebrate Ringer solution, the other that does not contain sodium chloride and instead contains potassium chloride or magnesium chloride as main components. Many cell lines can be cultured in media based on either type of inorganic salt composition.

TABLE 5
Wilkie et al.’s CDM (Chemically Defined Medium)

As a protein source, usually combinations of free amino acids are used. The amino acid mixture consists of 20 amino acids, which are constituents of protein, and β-alanine. In vertebrate cell culture, β-alanine has never been a component of culture media. In insect cell cultures, it has been incorporated since Wyatt’s medium,³ because insect hemolymph reportedly contains considerable amounts of β-alanine. It turned out later, however, that β-alanine was not necessary or was even detrimental for the growth of insect cells.¹¹ Based on this finding, some recently developed media omitted β-alanine from the amino acid mixture. Among 20 amino acids, arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, pheny...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Preface
• The Editors
• Contributors
• Table of Contents
• Chapter 1 Insect Cell Culture Media
• Chapter 2 Mosquito Cell Lines
• Chapter 3 Lepidopteran Cell Cultures
• Chapter 4 Grasshopper Cell Culture
• Chapter 5 Tick Cell Culture: Applications in Biology
• Chapter 6 Leafhopper Cell Culture for Virus Research
• Chapter 7 Invertebrate Cells for Mammalian Virus Research
• Chapter 8 Japanese Invertebrate Cell Culture Pioneers
• Index