永生化猪乳腺上皮细胞、猪乳腺上皮细胞系

Primary hVICs (passage 2) were cultured to 50–60% confluence and infected with pGMLV-SV40T-puro lentivirus (NewgainBio, Wuxi, China) at a multiplicity of infection of 80 supplemented with 5 µg/mL polybrene (Sigma-Aldrich, Buchs, Switzerland).

Tissue was cultured until cells became visible around the tissue, and when the fusion reached 90% (FIGURE 1A) §ask ¦lled with the prepared culturing medium was sent to the company for further immortalisation. Cell immortalisation was done for cell stability and longer-term use. Immortalised cells were cultured with 10% FBS and 1% PS in the DMEM medium.  After the cells multiplied and merged, they were routinely passed and grown ( NEWGAINBIO Inc. Wuxi, Jiangsu, China) (FIGURE 1B-C).

Mouse primary cultured renal vascular ECs and VSMCs were obtained from Newgainbio company, which were tested by Factor VIII and α-smooth muscle actin (α-SMA), the marker of ECs and VSMCs. RNeasy Mini Kit was used for RNA extraction, and the above protocols were repeated.

Chicken intestinal epithelial cells were obtained from NEWGAINBIO company. Cells were cultured on 37℃, with 5% CO2, in the Ham’s F-12 Nutrient (DMEM/12) that contained the following supplementations: fetal bovine serum (5%), in-sulin (5 µg/mL), transferrin (5 µg/mL), selenium (5 ng/mL), epidermal growth factor (5 ng/mL) and penicillin-streptomycin (100–100 U/mL) for cell culturing (full DMEM/12). Experiments were performed with chicken intestinal epithelial cells and working solutions were prepared with plain DMEM/12 without supplementation. For the investigations, cells were seeded onto 96-well, 24-well or 6-well polystyrene cell culture plates.

Porcine primary colon epithelial cells (Newgainbio company, Wuxi,China) were cultured in Dulbecco's Modified Eagle's Medium (Solarbio, Beijing, China) containing 10 % fetal bovine serum (BioInd, Kiryat shmona, Lsrael) at 37 ◦C and 5 % CO2 humidity.