156
- Measure subcutaneous tumor size (tumor length, L and width,
W) with digital caliper twice a week for 2–4 weeks (see Note
27 ). - Measure tumor volume (TV) with the formula of (W^2 XL)/2
[ 25 ]. Calculate relative tumor volume (RTV) with the follow-
ing formula: RTV = TVn/TV 0 , where TVn is the tumor volume
at the day of measurement and TV 0 is the tumor volume on the
first day of measurement [ 12 , 26 ]. - Euthanize all mice at the end of study.
- After euthanasia, examine the mouse for the extent of the
tumors and measure the tumor weight. - Harvest the tumors into three fractions and immerse two frac-
tions into liquid nitrogen and one fraction in 4% formaldehyde
for fixation followed by paraffin embedding. - Agitate the cell suspension in 50 ml Falcon tube to prevent the
cells from settling and draw up into the syringe (1 ml tubercu-
lin syringe) and needle (25 G × 5/8), the amount of cells in
serum free medium (1 ml, 10 × 105 to 10 × 106 cell per ml) to
be administered (see Note 28). - Gently remove the animal (SCID mouse) from the cage and
restrain appropriately in the head-down position. Identify ana-
tomical landmarks in order to inject into the appropriate area
of the abdomen (see Note 29). - Insert needle with the bevel facing up into the lower right
quadrant of the abdomen towards the head at 30–40° angle to
horizontal (see Note 30). - Pull back on the plunger to ensure negative pressure and if
there is negative pressure, depress the plunger until the medium
with the cells has been fully administered (see Note 31). - Pull the needle straight out and place the syringe/needle
directly into a biohazard sharp container. - Place the animal back into a clean cage and observe for
10–15 min for any complications. - Evaluate peritoneal tumor formation (Fig. 1 ) [ 12 ] and mouse
survival from the day of cancer cell injection until death. - Examine mouse daily for signs of distress or development of
jaundice and measure body weight once a week. - Euthanize mouse when they become moribund (see Note 32)
according to predefined criteria like rapid weight loss (>20%)
or weight gain (>20% due to ascites), loss of ability to ambu-
late, labored respiration, or inability to drink or feed to avoid
animal suffering [ 27 , 28 ] in line with the local animal care
committee protocol (Fig. 2 ) [ 12 ].
3.4 Preparation
of Mice
and Intraperitoneal
Injection of Cancer
Cells
3.5 Mice
Survival Study
Md Sazzad Hassan and Urs von Holzen