22
General Methods
increased. This is useful for the improvement of culture media, but difficult to apply to primary cultured cells, because growth or active metabolism of cells in a culture medium is a prerequisite for reliable information to be obtained regarding nutrient consumption.
Empirical formulation of culture media is also possible. For this, much
trial and error is necessary. The strategy the present author has adopted
is to use many substances that are considered favorable for cell maintenance or growth, at several concentrations, and when favorable changes
are observed in cultured tissues or cells, a reduction or increase in some
of the components of the medium is made, and the results reviewed. In
this case, a favorable change can be noticed by observing the culture frequently, and some experience is required. Various combinations of components and their concentrations should be tested. This requires the
preparation of many media. Even so, this method is successful when no
information on formulation or improvement of culture media is available.
3 Primary Culture
The in vitro culture of cells or tissues taken from living animals is called
the primary culture until the first subculture is made. In order to set up
a primary culture, various techniques have been developed depending
on the size of the materials, the type of tissues or organs, and the culture
vessels to be used. These techniques are mentioned in Part II of this book.
Primary culture can be started with free cells or with small fragments of
tissues. The former is called a free cell culture and the latter an explant
culture.Animals whose cells or tissues are to be cultured are preferably
sterile. If animals to be used can be raised aseptically, these sterile animals should be used as donors of tissues. Otherwise, animals should be
surface sterilized. Commonly, treatment with 70% ethanol for 5-10 min
is used. Sodium hypochlorite, benzalkonium chloride, or mercuric chloride may be used instead of ethanol.
3.1 Free Cell Culture
Free cell culture is used for the culture of originally free cells, such as
hemocytes, or free cells obtained by dissociating tissues. In free cell cultures, the primary culture can be made by collecting free cells by centrifugation and by dispersing them in culture media. However, to obtain
free cells from rigid tissues, dissociation of the tissues is necessary. This
is accomplished by moderate digestion of tissues by enzymes (see chapter 4, subprotocols 1-3). Under favorable conditions, these cells will proliferate.
General Methods
increased. This is useful for the improvement of culture media, but difficult to apply to primary cultured cells, because growth or active metabolism of cells in a culture medium is a prerequisite for reliable information to be obtained regarding nutrient consumption.
Empirical formulation of culture media is also possible. For this, much
trial and error is necessary. The strategy the present author has adopted
is to use many substances that are considered favorable for cell maintenance or growth, at several concentrations, and when favorable changes
are observed in cultured tissues or cells, a reduction or increase in some
of the components of the medium is made, and the results reviewed. In
this case, a favorable change can be noticed by observing the culture frequently, and some experience is required. Various combinations of components and their concentrations should be tested. This requires the
preparation of many media. Even so, this method is successful when no
information on formulation or improvement of culture media is available.
3 Primary Culture
The in vitro culture of cells or tissues taken from living animals is called
the primary culture until the first subculture is made. In order to set up
a primary culture, various techniques have been developed depending
on the size of the materials, the type of tissues or organs, and the culture
vessels to be used. These techniques are mentioned in Part II of this book.
Primary culture can be started with free cells or with small fragments of
tissues. The former is called a free cell culture and the latter an explant
culture.Animals whose cells or tissues are to be cultured are preferably
sterile. If animals to be used can be raised aseptically, these sterile animals should be used as donors of tissues. Otherwise, animals should be
surface sterilized. Commonly, treatment with 70% ethanol for 5-10 min
is used. Sodium hypochlorite, benzalkonium chloride, or mercuric chloride may be used instead of ethanol.
3.1 Free Cell Culture
Free cell culture is used for the culture of originally free cells, such as
hemocytes, or free cells obtained by dissociating tissues. In free cell cultures, the primary culture can be made by collecting free cells by centrifugation and by dispersing them in culture media. However, to obtain
free cells from rigid tissues, dissociation of the tissues is necessary. This
is accomplished by moderate digestion of tissues by enzymes (see chapter 4, subprotocols 1-3). Under favorable conditions, these cells will proliferate.