5 Insecta (Lepidoptera)
53
Procedure
1. Select larvae with slipped heads.
Sterilization
2. Surface sterilize the larvae by consecutive immersion, for approximately 2 min each, in baths of 20% Septisol, 0.1 % p-hydroxybenzoic
acid methyl ester and 0.1 % sodium hypochlorite.
3. Rinse sterilized larvae with sterile distilled water.
1. Fix the larvae on a sterile dissecting tray with pins.
Dissection
2. Make a lengthwise midline incision through the dorsal integument,
exposing the midgut.
3. Remove the Malpighian tubules and also the peritrophic membrane
containing the gut contents through a lengthwise incision through the
midgut, taking care not to contaminate the medium with gut contents.
4. Excise the posterior two-thirds of the midgut, and place it in a petri
dish containing Ringer's solution.
5. Rinse twice in sterile Ringer's solution containing 0.5% gentamicin
and 0.01 % antibiotic-antimycotic.
6. Cut the midgut into pieces approximately 1 mm 2 and transfer the pieces
to the culture medium.
1. Transfer the cut fragments of the midgut, with 3 ml culture medium, Culture
into the well of a culture plate. The culture medium must contain fat set-up
body extract.
2. Maintain the culture at 25°C by renewing one-third of the medium
with the same amount of fresh medium containing fat body extract
once a week.
Results
According to Sadrud-Din et al. {1994, 1996} and Loeb and Hakim {1996},
agranular round cells, mature columnar cells, and goblet cells appear in
the primary culture. The cultured cells proliferate without attaching to
the substrate. The addition of fat body extract to the medium promotes
the proliferation of stem cells, which are the source of differentiated cell
types. Isolated stem cells were only able to differentiate to mature goblet
and columnar cells when cell-free conditioned medium, taken from midgut cell cultures containing mature cells as well as stem cells and differentiating forms, or one of the midgut differentiating peptide foctors isolated from the conditioned medium or hemolymph, was introduced into
the culture medium (Fig. 4). Four peptides responsible for stem cell activation have been isolated and identified (Loeb et al., 1999 and personal
communication). When a stem cell differentiates, one of the divided sister cells remains as a proliferating stem cell, while the other sister cell is
committed to differentiate, as in the case of the differentiation of nerve
cells from neuroblasts.
53
Procedure
1. Select larvae with slipped heads.
Sterilization
2. Surface sterilize the larvae by consecutive immersion, for approximately 2 min each, in baths of 20% Septisol, 0.1 % p-hydroxybenzoic
acid methyl ester and 0.1 % sodium hypochlorite.
3. Rinse sterilized larvae with sterile distilled water.
1. Fix the larvae on a sterile dissecting tray with pins.
Dissection
2. Make a lengthwise midline incision through the dorsal integument,
exposing the midgut.
3. Remove the Malpighian tubules and also the peritrophic membrane
containing the gut contents through a lengthwise incision through the
midgut, taking care not to contaminate the medium with gut contents.
4. Excise the posterior two-thirds of the midgut, and place it in a petri
dish containing Ringer's solution.
5. Rinse twice in sterile Ringer's solution containing 0.5% gentamicin
and 0.01 % antibiotic-antimycotic.
6. Cut the midgut into pieces approximately 1 mm 2 and transfer the pieces
to the culture medium.
1. Transfer the cut fragments of the midgut, with 3 ml culture medium, Culture
into the well of a culture plate. The culture medium must contain fat set-up
body extract.
2. Maintain the culture at 25°C by renewing one-third of the medium
with the same amount of fresh medium containing fat body extract
once a week.
Results
According to Sadrud-Din et al. {1994, 1996} and Loeb and Hakim {1996},
agranular round cells, mature columnar cells, and goblet cells appear in
the primary culture. The cultured cells proliferate without attaching to
the substrate. The addition of fat body extract to the medium promotes
the proliferation of stem cells, which are the source of differentiated cell
types. Isolated stem cells were only able to differentiate to mature goblet
and columnar cells when cell-free conditioned medium, taken from midgut cell cultures containing mature cells as well as stem cells and differentiating forms, or one of the midgut differentiating peptide foctors isolated from the conditioned medium or hemolymph, was introduced into
the culture medium (Fig. 4). Four peptides responsible for stem cell activation have been isolated and identified (Loeb et al., 1999 and personal
communication). When a stem cell differentiates, one of the divided sister cells remains as a proliferating stem cell, while the other sister cell is
committed to differentiate, as in the case of the differentiation of nerve
cells from neuroblasts.