Influence of supramammary lymph node extract on in vitro cell proliferation

Abstract

Objectives: Experiments were conducted to evaluate whether or not bovine supramammary lymph node extract (LNE) could support cell proliferation when it was substituted for bovine growth serum (BGS) in cell culture media.

Materials and methods: Two different preparations of LNE were tested. The first yielded protein concentration of 3 mg/mL and the second contained 27 mg/mL protein. Three cell lines (MDA-MB-435, MAC-T and 1C6) were used in serum starvation assays to evaluate LNE. Cell proliferation assays were used to determine growth stimulation in the presence of LNE, and short-term or rapid adaptation cultures were evaluated for LNE effects on cell survival.

Results: Heat-inactivated preparation 1 supported cell proliferation as well as or better (12-39%) than BGS following 2 days of serum starvation in culture. The second lymph node preparation provided a stimulatory effect (263-702% greater than BGS across all cell lines) following serum starvation at 2.7 and 5.4 mg/mL protein supplementation. A gradual adaptation process with lymph node supplementation into media maintained cell population growth on a short-term basis. However, once cells were trypsinized or scraped and re-seeded into 2.7 mg/mL LNE protein containing media, cells were unable to re-adhere, leaving them detached, and eventually appearing to be dead.

Conclusions: Substitution of BGS with LNE protein dramatically stimulated cells to proliferate, but did not allow for rapid cell population growth adaptation in vitro.

Figure 1

Proliferation indices (PI) of MDA‐MB‐435 human breast cancer cells in a serum starvation assay with lymph node extract (LNE) of preparation 1 (a) and preparation 2 (b). Following 2‐day starvation, cells were cultured in media containing non‐heat‐inactivated LNE (striped bars), heat‐inactivated LNE (white bars), and bovine growth serum (black bars) at different protein concentrations of supplementation. Data are expressed as means ± SEM. Means are significantly different (P = 0.05) from respective heat‐inactivated means within protein concentrations. Dashed line is the PI (1.0) of cells cultured with 0 mg/mL bovine growth serum or LNE protein.

Figure 2

Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of bovine growth serum (BGS) and lymph node extract (LNE) from lymph node preparation 2. Lane 1, protein standards; lane 2, BGS; and lane 3, LNE.

Figure 3

Proliferation indices (PI) of MDA‐MB‐435 human breast cancer (a), MAC‐T bovine mammary epithelial (b), and 1C6 hybridoma B (c) cells in a serum starvation assay with lymph node extract (LNE) of preparation 2. Following 2‐day starvation, cells were cultured in media containing heat‐inactivated LNE (white bars), and bovine growth serum (black bars) at different protein concentrations of supplementation. Data are expressed as means ± SEM. Means are significantly different (P ≤ 0.05) from respective heat‐inactivated means within protein concentrations. Dashed line is the PI (1.0) of cells cultured with 0 mg/mL bovine growth serum or LNE protein.

Figure 4

Photomicrographs of cells subjected to rapid adaptation assay with lymph node extract (LNE) following initial adherence for 24 h with bovine growth serum (BGS) containing media. MDA‐MB‐435 human breast cancer cells (a–e), MAC‐T bovine mammary epithelial cells (f–j), and 1C6 hybridoma B‐cells (k–o) were provided with increasing concentrations of LNE in place of BGS, daily. Day 0 (a, f, k): 0 mg/mL LNE; day 1 (b, g, l): 0.675 mg/mL LNE, day 2 (c, h, m): 1.35 mg/mL LNE, day 3 (d, i, n): 2.025 mg/mL LNE, and day 4 (e, j, o): 2.7 mg/mL LNE. Bar = 20 µm.