Growth of distal fetal rat lung epithelial cells in a defined serum-free medium

Abstract

Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-D-lysine substratum under serum-free culture conditions in Dulbecco's modified minimal essential medium with insulin (50 micrograms/ml), endothelial cell growth supplement (20 micrograms/ml), bovine pituitary extract (100 micrograms/ml), bovine serum albumin (50 micrograms/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 micrograms/ml), transferrin (5 micrograms/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 micrograms/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E1 (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung.

Changes in Expression of Platelet-Derived Growth Factor and Its Receptors in the Lungs of Newborn Rats Exposed to Air or 60% O2

“…We have also shown the presence of PDGF and its receptors (17) on type II pneumocytes in vivo, and shown increased expression of their mRNAs when fetal rat lung epithelial cells are exposed to 50% O 2 in vitro (42). PDGF-BB stimulates DNA synthesis in both fetal rat lung epithelial cells (18) and microvascular epithelial cells (43). Taken together, these studies formed the basis for investigating the expression of PDGF and its receptors in the normal growth of postnatal rat lung and in the neonatal rat pup model of hyperoxic lung injury.…”

Modulation of Sodium Transport in Alveolar Epithelial Cells by Estradiol and Progesterone

“…FDLE cells were isolated as described previously (8,20,21 6 cells per insert. The medium was changed daily, serum-free complete medium (Cellgro; Mediatech, Herndon, VA), supplemented with E2 and P (Sigma Chemical Co., Germany) as shown in Table 1, was used from the second day onward.…”

“…Measurement of bioelectric properties of FDLE monolayers. Experiments were performed on the fourth or fifth day of culture, which was ϳ72-96 h after seeding and 48 -72 h after applying E2 and P. The procedures of measurement are reported elsewhere (8,20,21). Ussing chamber experiments were included in the analysis only when the transepithelial resistance (R te ) exceeded 300 ⍀ ⅐ cm 2 .…”

Antioxidants as Therapy in the Newborn: Some Words of Caution

“…The growth promoting properties of O 2 -· at low concentrations, and cytotoxic properties at high concentrations, are illustrated in Figure 1. Primary cultures of rat fetal lung epithelial cells were exposed to increasing concentrations of either paraquat, an O 2 -· generator, or TEMPO, an O 2 -· scavenger, and assayed for DNA synthesis or cytotoxicity as previously described (94,95). Low concentrations of O 2 -· enhance cell DNA synthesis and protect against basal levels of cytotoxicity, whereas higher concentrations are cytotoxic and inhibit DNA synthesis.…”