Jun Mitsuhashi

Development of highly nutritive culture media

SummaryA highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.

Preparation of Media

Media consisting of a few chemicals maybe prepared by dissolving these chemicals in solution one after the other. Following is the preparation of MM medium (Mitsuhashi and Maramorosch, 1964) as an example of such media. (For the composition of the medium, see Appendix 1.)

A continuous cell line from the cupreous chafer, Anomala cuprea hope (insecta, coleoptera, scarabaeidae)

SummaryA continuous cell line was obtained from the culture of embryonic cells of the cupreous chafer, Anomala cuprea Hope. The cells showed substrate-dependent growth and formed loose networks. Population doubling time was about 4.5 d. The mode of chromosome number was about 32 (4n). The cell line was designated FRI-AnCu-35.

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata

The prothoracic glands (PGs) taken from the last instar of the common armyworm, Pseudaletia separata, were cultured in various media for the purpose of finding a suitable medium for relatively long-term culture of PGs. Among the tested culture media, MGM-450 medium without serum was the best to maintain PG cells viable for relatively long periods, and to continue to secrete ecdysteroids. Secretion of ecdysteroid by the PG in vitro became marked when the PG was taken from last instar larvae older than 2 days after the last molt. PGs cultured in any of the media secreted ecdysteroid only within the first 2 h after placing them in culture, however, in the MGM-450 medium, the PGs secreted ecdysteroid even after 5 days of culture.

General Cell Culture Methods

Trypsin is the most widely used enzyme in mammalian cell culture. However, it cannot be used for the culture of some insect cells because of their sensitivity to it. The action of trypsin is inhibited by the presence of Mg2+ and Ca2+. Therefore, trypsin should be dissolved in a physiological solution free of Mg2+ and Ca2+. Rinaldini’s solution (Rinaldini, 1959; see Appendix 1) is often used as one such solution. Balanced salt solution (BSS) or phosphate-buffered saline (PBS) from which Mg2+ and Ca2+ have been omitted can also be used. The optimum pH for trypsin is 8–9. Trypsin is often used with ethylenediamine tetraacetic acid (EDTA).

Facilities and Equipment

Tissue culture should be done free of microbial contamination. This can be realized by keeping the room as free of microorganisms as possible, and by using sterile techniques. If possible, three separate rooms, i.e., a sterile room, an observation and maintenance room, and a washing and sterilizing room, are desirable (Fig. 1). However, this requires a large space. After the development of laminar flow cabinets (hoods), most investigators now use laminar flow cabinets rather than a sterile room. Therefore, setting up one room with laminar flow cabinets, and another room with equipment for observation and maintenance, has become common practice (Fig. 2). Washing and sterilization may be performed in a regular laboratory, because modern sterilizing equipment does not liberate much water vapor and heat. However, if strict sterility is required, it may be better to place a laminar flow cabinet in a sterile room (Fig. 3).

Large-Scale Cell Culture

The large-scale culture of cells requires the optimization of various factors, such as cell density, cell viability, growth stage, cell nutrition, composition of media, dissolved oxygen concentration (DO), temperature, pH, and osmotic pressure of the media. In addition, the cost of the media is important for industrialization, and in this context, whether the media can be sterilized by heating is important.

Basic Information and Overview

It is not necessary to spray alcohol in the cabinet before use. Turn the UV light off and turn the fan and fluorescent light on. The gas burner cannot be lit before the fan is operational in most laminar flow cabinets. While working, keep the front glass shutter as low as possible, leaving only enough space to insert your arms under the shutter.

Identification of Cell Lines

Discrimination between and identification of cell lines are important in order to avoid confusion due to mislabeling and accidental cellular contamination. Cell morphology is not a reliable criterion for the identification of cells, because the shape of a cell changes easily with different culture conditions, and a cell population usually consists of various-shaped cells, even in a cloned cell line. Chromosome analysis can be used to discriminate between and identify cell lines; however, lepidopteran cell lines have numerous small chromosomes and this makes determination of the karyotype difficult in these cases. Isozyme pattern analysis is also useful in the identification of cell lines, but isozyme pattern analysis does not clearly distinguish cell lines derived from taxonomically close species, or cell lines derived from the same species. A reliable method for discriminating cell lines is the recently developed restriction fragment length polymorphism (RFLP). However, this method has some drawbacks: radioisotopes, a large number of cells, and southern hybridization are needed in RFLP, and the procedure itself is not simple. More recently, a polymerase chain reaction (PCR) method for the study of phylogeny came into use. This technique is a good tool for the discrimination of cell lines. Furthermore, a method of random amplified polymorphic DNA (RAPD)-PCR has been developed. Because gene analyses of insect cell lines are scarce, a method of RAPD-PCR using appropriate short single primers seems to be most practical.

Examination of Drug Effects

Cultured invertebrate cells may be useful in drug toxicity tests, because most invertebrate cell lines are easy to maintain. Unlike mammalian cells, invertebrate cells can be cultured at lower and over a relatively wider range of temperatures, and do not require a CO2 incubator. Most insect cell lines do not adhere to a substrate, and subculture is very easy. These characteristics of invertebrate cell culture give some advantage to the use of invertebrate cells over mammalian cells in drug toxicity tests.