Abstract The use of preclinical studies with animal models in biomedical research provides a critical step in the assessment of novel therapeutic approaches. In vivo studies using small animals play a pivotal role in generating and improving methods for therapeutic treatment of cancer including radiotherapy (RT). Rodents, and in particular mice, have led to extensive development in cancer research. Currently, there are two commercially available radiation devices for small animal irradiation: The Small Animal Radiation Research Platform. Both deliver targeted radiation to preclinical animal models with an accuracy equivalent to clinical RT using a rotating gantry and implement high-resolution cone beam-CT and 3D bioluminescent tomographic imaging. However, the complexity of these systems makes them rather expensive, which is often a hindering factor for hospitals or research institutions to acquire them. The Gammacell 40 Exactor is a low-dose research irradiator widely available, commonly used for radioimmunology studies and is suitable to treat murine models. The device implements two 137Cs sources; each one has a nominal activity of 55.5 TBq and together they can produce a central dose rate of 1.0 Gy/minute. In this study two collimators were designed capable of precisely collimating a 1 cm radiation beam for focal irradiation, accommodated within a Gammacell 40 Exactor irradiator. This machine allows to insert in the main chamber collimator designs with a maximum dimension of 312 mm in diameter and 106 mm in height. The one-chamber collimator was designed cylindrical with an internal space for the mouse of 100 mm in diameter and a height of 50 mm. This chamber was surrounded on its sides by a cylindrical wall of steel and lead, and two 25mm-thick lead lids at the top and bottom with central apertures of 10mm in diameter. This geometry conforms the beam of the two Cs137 sources to a 10mm beam. In the wall that surrounds the inner chamber, four ventilation apertures were designed, which allow the mice to breath while they are been irradiated, in an anesthetized state. The four-chamber collimator was designed to be capable of irradiating up to 8 mice at a time. For this collimator the apertures were angled to 13 degrees to cope with the divergence of the two sources of Cs137. They conform an angled 10mm beam. The dimensions of the inner chambers were 25mm thick lead for the lids and internal height 50mm, also with breathing apertures for the mice. Monte Carlo (MC) simulations were executed as a verification method. These simulations were implemented using EGSnrc in particular DOSxyznrc and BEAMnrc to simulate the sources, collimators and phantom. Two simulations were run in DOSXYZnrc for the two radiation sources of the Gammacell exactor at 0 and 180 degrees, and the results were added to find the total radiation at mid-point of the internal chamber. These simulations were run in a phase-space option, which allows to modify the angle of the sources, to 0 and 180 degrees in the source type option. For dosimetric verification, radiochromic films and gel dosimetry are projected to be used. Radiochromic films are composed of layers that experience coloration upon receiving radiation, with no need of further chemical, optical or thermal development. One of the main advantages of gel dosimeters is the possibility of storing 3D distribution of the radiation dose, as they are made of radiosentitive chemicals that polymerize upon radiation. The simulations showed that the design of the collimators was able to correctly create well-collimated beams with a diameter of 1cm and to reduce out-of-field dose in both collimator designs. In the four-chamber collimator the simulations proved that the angled hole copes with the divergence of the two radiation sources. The results will also be verified against dosimetric measurements with Gafchromic films and dosimetric gels. Citation Format: Jessica Benitez, Davide Fontanarosa, Robert Mazzieri, Davide Moi. Collimator design and simulation for preclinical radiotherapy applications using a clinical cell irradiator [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B204.