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

1.认为产奶量取决于乳腺上皮细胞的数量。然而，牛乳腺上皮细胞的增殖分化机制尚不清楚。用母牛作实验动物有经济上的限制.在屠宰场，牛的乳房通常被丢弃，因此，我们能够从这些乳房中获得大量的牛乳腺细胞。在获得大量新鲜样本的情况下，对牛乳腺细胞进行冷冻保存是非常有益的。牛乳腺细胞冷冻培养的研究报道较少。所有这些报道都在冷冻保存培养基中使用了血清，血清对乳腺细胞的影响可能不确定。目的是：1）评价乳腺碎片对无血清冷冻保存培养基甲基纤维素-脱甲基亚砜-培养基199（MC-DMSO-M 199）的增殖反应，2）与新鲜和冷冻保存的乳腺生长进行比较 ...更多信息 在体外对合成胰岛素样生长因子-1（IGF-1）或表皮生长因子（EGF）的反应。监测氚（^3H）胸苷掺入和形态学方面以确定乳腺细胞在包埋胶原凝胶培养物中的增殖。2.1）使用妊娠小鼠6只、泌乳荷斯坦奶牛3头、阉牛6头和激素处理（E/P）的母羊4只。2)用胶原酶/透明质酸酶消化乳腺，收集乳腺碎片。一部分片段用于新鲜培养，其余片段在无血清或添加血清的冷冻保存培养基中冷冻。3)3.1）无血清冻存5个月后，妊娠小鼠乳腺组织块在胶原凝胶中生长良好，0.1%MC冻存细胞与0.05%MC冻存细胞生长无差异。因此，使用0.1%浓度的MC。2)在新鲜培养物中，泌乳期的乳腺碎片不能增殖，并且^3 H-TdR掺入也很低。另一方面，阉牛和E/P处理的母羊乳房碎片生长迅速，它们也显示出对IGF-1和EGF的高^3 H-TdR掺入反应。3)无血清冻存6个月和12个月后，阉牛和E/P母羊的乳房组织块增殖良好，形态学变化与新鲜培养物相同。~ 3 H-TdR对生长因子的反应也较强。少

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