Bovine mammary explant versus primary cell cultures: Effect of bovine somatotropin and insulinlike growth factor-I on DNA content and protein synthesis

Abstract

Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 +/- 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulin-like growth factor-I, and somatotropin + insulin-like growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G0G1 phase was increased by: (a) insulin-like growth factor-I in explant cells; (b) somatotropin, insulin-like growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulin-like growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G0G1 or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more beta-casein than primary cells but secretion trends for beta-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.

Transcriptomics Comparisons of Mac-T cells Versus Mammary Tissue during Late Pregnancy and Peak Lactation

“…Even though it has been reported that Mac-T cells can express and synthetize caseins [13,38] the amount produced is often below the limit of detection [2]; certainly not at the level of mammary tissue. Therefore, contrary to previous reports [19,34], the qPCR data in the present study indicated that the usefulness of Mac-T cells for studying milk protein synthesis, particularly if expression of milk proteins is considered, is limited.…”

“…A direct comparison between mammary explants and primary cells from the same animals indicated a greater quantity and secretion of β-casein in explants vs. expanding primary cells in response to somatotropin and insulin like growth factor I (IGF-I); however, the difference decreased when cells reached confluence [34]. The authors concluded that "primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion".…”

A Proteomic Analysis of Differentiation in the Mammary Epithelium

“…As a model, the explant is an excellent way to observe the interactions between all of the cell types within the gland that may depend on structural relationships to function similarly to in vivo (Keys, 1997). In addition, an effect observed in vivo can be validated in culture when that tissue is separated from circulating ligands and systemic signals (Telang, 1989).…”

“…In addition, an effect observed in vivo can be validated in culture when that tissue is separated from circulating ligands and systemic signals (Telang, 1989). Some drawbacks to explant culture (excluding xenografts) are: (a) the short life of the tissue in culture as they are only viable for a few day at most (Keys, 1997); (b) the mix of cell types within an explant, making study of the epithelium exclusively impossible, and knowing the proportion of epithelial cells in the explant is a challenge; (c) explants cannot be frozen cryogenically, so some experiments are not repeatable over time with samples from a single isolation event. While explants from rodent mammary glands have been used in experiments for over 50 years, conducting successful explant studies with ruminant mammary tissue has presented a much greater challenge, and is more narrowly studied (Matitashvili, 1997).…”

“…Other advantages to using primary cell culture are: (a) the ability to study a single cell type; (b) primary cells secrete proteins and lipids directly into media which can then be measured quantitatively; (c) primary cells can proliferate for a greater volume of samples and can be kept in culture for extended periods provided the right combination of growth factors and nutrients; (d) primary cells can be cryogenically frozen and used for future research or to repeat an experiment at a later time, even with cells isolated from the same animal (Keys, 1997). A few drawbacks are: (a) the necessity for validation that the intended cell type was indeed the one isolated and cultured and the likelihood of a small percentage of contaminating mesenchymal cells (Matitashvili, 1997); (b) an environment unlike the in vivo environment, cryopreservation and extended passaging of cells may cause a change in the cells morphology and function (Keys, 1997). Morphology and secretory activity may also be affected by whether primary cells are plated on plastic culture dishes, artificial extracellular matrices, or attached collagen gel (Matitashvili, 1997).…”