Serum-free cryopreservation of bovine mammary fragments-Effects of growth factors

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1. It is considered that milk yield is depend on the number of mammary epithelial cells. However, the mechanism of proliferation and differentiation on bovine mammary epithelial cell has not been clarified. There is an economical limitation for using a cow as an experimental animal. In the slaughter house, bovine udders are usually thrown into the discard, therefore, we are able to get a lot of bovine mammary cells for these udders. It is beneficial that we cryopreserve the bovine mammary cells when we get a lot of fresh samples. Only a few reports have appeared about the culture of cryopreserved bovine mammary cells. All these reports have used serum in cryopreservation medium, and there is a possibility of undefined effect from serum to mammary cells.The objectives were to: 1) evaluate the proliferative response of mammary fragments to serum-free cryopreservation medium, methylcellulose-demethylsulfoxide-medium 199 (MC-DMSO-M 199), 2) compare with fresh and cryopreserved mammary grow … More th response to synthetic insulinlike growth factor-1 (IGF-1) or epidermal growth factor (EGF) in vitro. Tritium (^3H) thymidine incorporation and morphological aspects were monitored to determine the proliferation of mammary cells in embedded collagen gel culture.2.1) Pregnant 6 mice, lactating Holstein 3 cows, 6 steers and hormone treated (E/P) 4 ewes were used. 2) Mammary gland was digested with collagenase/hyaluronidase and mammary fragments were collected. One portion of fragments was used for fresh culture and other remaining fragments were frozen in serum-free or serum added cryopreservation medium. 3) After 6 and 12 months' cryopreservation, mammary cells were thawed immediately in 37C water and cultured in collagen gel.3.1) Pregnant mouse mammary fragments grew well in collagen gel after 5 months serum-free cryopreservation and there is no growth difference between 0.1%MC cryopreserved cells and 0.05% MC cryopreserved cells. Therefore, 0.1% concentration of MC is used. 2) In fresh culture, lactating mammary fragments did not proliferated and ^3H-TdR incorporation was also very low. On the other hand, steer and E/P treated ewe mammary fragments grew rapidly and they also showed high ^3H-TdR incorporation response to IGF-1 and EGF. 3) After 6 and 12 months' serum-free cryopreservation, steer and E/P ewe mammary fragments proliferated well and showed the same morphological change as the one of fresh culture. ^3H-TdR after cryopreservation also showed the high response to the growth factors. Less