196 EFFECT OF MITROMYCIN-C ON VIABILITY AND CELL PROLIFERATION OF DOMESTIC CAT AND MOUSE EMBRYONIC FIBROBLASTS

Abstract

Nondomestic cat cloned embryos created by intergeneric nuclear transfer (ig-NT) have lower rate of blastocyst development than that observed in cloned embryos produced by inter-species NT. One promising technique for increasing the efficiency of ig-NT is to use embryonic stem cells (ESC) derived from the pluripotent inner cell mass (ICM) of pre-implantation blastocysts. Mouse embryonic fibroblasts (MEF) are commonly used as feeder cells to support the growth of human and mouse ESC, but they do not always maintain human ESC in an undifferentiated state and carry the risk of transmitting mouse retroviral virus and other pathogens. Thus, it is important to determine whether domestic cat embryonic fibroblasts (DCEF) are able to support cat ESC growth. Proliferation of MEF cells is inhibited before their use as feeder layers by exposure to gamma irradiation or Mitomycin-C. Therefore, in the present study, we determined what concentration of Mitomycin-C was required to inhibit proliferation of DCEF and MEF cells without affecting viability. Embryonic fibroblasts were generated from fetuses collected from a single pregnant domestic cat and mouse at 30 and 14 days of gestation, respectively. Tissue was minced and cultured in DMEM for 7 to 10 days before freezing and storage at −196°C. After thawed DCEF and MEF cells reached 80 to 100% confluence, proliferation was inhibited by exposure to 10, 30, or 40 µg of Mitomycin-C for 2.5 or 5 h. Cells were then washed and labeled with BrdU to measure DNA synthesis as a specific marker for replication. For cytotoxicity, cells were labeled with a dual fluorescent stain to evaluate membrane integrity and esterase activity. The amounts of DNA-labeled BrdU as well as calcein and ethidium homodimer-1 incorporated into the cells were quantified by flow cytometry (see Table 1). Proliferation of DCEF and MEF cells was affected similarly by both concentration and exposure time to Mitomycin-C, with the highest inhibition of DCEF cells occurring after treatment with 40 μg for 5 h (92% not replicating). Cell viability was influenced by both concentration and exposure time to Mitomycin-C, with the highest survival of DCEF cells occurring after exposure to 30 μg for 5 h (90%) and in MEF cells after exposure to 40 μg for 2.5 h (88% viable). In summary, these results indicate that cell proliferation of DCEF can be inhibited by Mitomycin-C, but a higher dosage and longer exposure time should be considered for use compared to the dosage commonly employed for inhibiting cell proliferation of MEF. High concentrations of Mitomycin-C did not significantly increase cytotoxicity of cells in either species.

Table 1.