Ascites Production in Mice
BU IACUCInstitutional Animal Care and Use Committee IACUC oversee... Approved October 2008; Revised January 2014, Revised May 2019
1. Avoiding pain and discomfort
In an attempt to avoid pain, discomfort, or distress in animals from the production of monoclonal antibodies (mAb) using mouse ascites, the NIHNational Institutes of Health in 1999 issued directives encouraging the use of alternative methods to produce monoclonal antibodies in vitro without compromising the aims of the study.
2. In vivo production
Institutional Animal Care & Use Committees critically evaluate all protocols which propose using the mouse ascites method, and only allow in vivo production on the basis of strong scientific justification.
3. Ascites method
There are legitimate circumstances for the use of the ascites method (for example, in vitro methods fail to generate sufficient quantities of mAb for use). However, since practical in vitro methods for large-scale production are available, the use of the ascites method should be the exception.
- Scientific justification for the proposed use of the ascites method.
- Why in vitro methods have been found to be unsuitable.
- A description of the methods that will be used in order to prevent or minimize pain and distress.
If in vivo production is sufficiently justified and approved by the IACUC, the following standards for monoclonal antibodies must be followed.
1. Volume of priming agent
The volume of the priming agent must be reduced to as small a volume as necessary to elicit the growth of ascetic tumors and at the same time reduce the potential for distress caused by the irritant properties of the priming agent. Although 0.5ml pristane has been considered standard for adult mice, the lower dose of 0.1–0.2ml has been shown to be effective for many hybridomas.
Although the time interval between priming and inoculation of hybridoma cells as well as the number of cells in the inoculum are determined empirically, inocula usually range from 10^5–10^7 cells in volumes of 0.1–0.5ml and are usually administered 10–14 days after priming. Generally, very high cell numbers are associated with greater mortality and 1 x 10^5 cells may elicit fewer ascetic tumors. Cell suspensions must be prepared under sterile conditions in physiological solutions.
3. Pathogen screening of biological materials used for ascites generation
Imported hybridomas or other materials of biological origin must be screened for adventitious murine pathogens before introduction into the animal host to prevent potential transmission of infectious agents from infected cell lines or products into facility mouse colonies and possibly to humans handling the animals. Please see the link below for more information on this topic as well as instructions on testing of these materials.
Pathogen Screening of Biological Materials Introduced into Rodents
Animals must be monitored at least once daily, seven days a week, by personnel familiar with clinical signs associated with ascites and circulatory shock. Bloating can result in severe respiratory distress and hypovolemia with associated decreased blood pressure and shock.
5. Relieving pressure
Ascites pressure must be relieved before abdominal distension is great enough to cause discomfort or interfere with normal activity. Manual restraint or anesthesia may be used for tapping. The tap must be performed by trained personnel using proper aseptic techniques. The smallest possible needle that allows for good flow must be used (18–22 gauge or smaller).
6. Withdrawing ascites
Wipe the abdomen with Betadine followed by 70% isopropyl alcohol. Withdraw ascites fluid through as small a needle as possible attached to a syringe. Massaging the abdomen gently will facilitate fluid removal.
7. After the tap
Animals must be monitored frequently over several hours following the tap to observe possible signs of shock due to fluid withdrawal. Pale eyes, ears, and muzzle and respiratory distress are indicative of circulatory shock. Shock may be prevented or treated with 1mL SQ and 1 mL IP of warm saline or LRS.
8. Limiting taps
The number of taps must be limited, based on good body condition of the animal. A maximum of three survival taps (the fourth being terminal) is allowed.
9. Euthanization if signs of distress
Mice should be euthanized appropriately before the final tap or promptly if there is evidence of debilitation, pain, or distress. Signs of distress include hunched posture, rough hair coat, reduced food consumption, emaciation, inactivity, difficulty in ambulation, respiratory problems, and solid tumor growth.
Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. ILAR. NRC. 1999. http://grants.nih.gov/grants/policy/antibodies.pdf
Guidelines for Ascites Production in Mice. NIH Animal Research Advisory Committee (ARAC) 2016. http://oacu.od.nih.gov/ARAC/documents/Ascites.pdf
Peterson, N.C. Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal Antibody Production. Comparative Medicine 50(5):516-526, 2000.
ILAR Journal 37(3): 141-152, 1995.