Finite lifespan pre-stasis HMEC from specimens 184 (batch D), 48R (batch T), and 240L (batch B), and post-selection HMEC 184 (batch B, agonescence at ~passage 15), 48R (batch S, agonescence at ~passage 23), and 240L (agonescence at ~passage 18) were obtained from reduction mammoplasty tissue of women aged 21,16, and 19 respectively. Cells were initiated as organoids in primary culture in either serum-free MCDB170 medium (MEGM, Lonza, Walkersville, MD) plus supplements (2 (link)), or serum-containing media MM (1 (link)) or M85, and subjected to multiple partial trypsinizations, as described (18 ). Post-selection HMEC were cultured in MCDB170 as described (2 (link),18 ,19 ). M85 medium is composed of 50% MM medium and 50% bicarbonate-free supplemented MCDB170; M87 medium is composed of 50% MM4 (MM without the conditioned media)(1 (link)) and 50% supplemented MCDB170. Cholera toxin was added to M85 and M87 at a final concentration of 0.5 ng/ml and oxytocin (Bachem) at 0.1 nM. M85A and M87A media were supplemented with 0.1% AlbuMAX I (Invitrogen). Fibroblasts from specimens 184, 48, and 240L were obtained by growing primary reduction mammoplasty cells in DME/F12 with 10% FBS and 10 μg/ml insulin. 250MK cells were obtained from aspirated milk fluids1 .
Total PD level for each culture was calculated beginning at passage 2 using the formula PD=log2(Nfinal/Ninitial) where Ninitial is the number of cells seeded in a dish at each passage and Nfinal is the number of cells recovered from the dish. No corrections were made for plating efficiency.
Total PD level for each culture was calculated beginning at passage 2 using the formula PD=log2(Nfinal/Ninitial) where Ninitial is the number of cells seeded in a dish at each passage and Nfinal is the number of cells recovered from the dish. No corrections were made for plating efficiency.
