Cryopreservation of Biologically Functional Submandibular Gland Rudiments from Fetal Mice

Abstract

Background/aim: Cryopreservation of cell lines has been widely used in the laboratory; however, cryopreservation of organs is still considered to be difficult. The submandibular gland (SMG) of fetal mice is one of the best-characterized organs. We investigated the conditions for cryopreserving SMG rudiments.

Materials and methods: Embryonic day 13 SMG rudiments were cryopreserved with or without a cryoprotectant. They were thawed and incubated in DMEM/F12 medium. Moreover, the influence of EGF stimulation on the signaling cascade after frozen-thawing the rudiments was analyzed by Western blotting.

Results: When SMG rudiments were cryopreserved without a cryoprotectant, all cells in the rudiments died. However, the SMG rudiments that had been preserved in a cryoprotectant showed branching morphogenesis. Additionally, the responsiveness of signaling cascades to EGF did not differ between frozen with a cryoprotectant and non-frozen rudiments.

Conclusion: Cryopreservation might be a useful technology for preserving tissues from small organs, such as fetal SMG rudiments.

Keywords: AKT; Cryopreservation; EGF; ERK1/2; branching morphogenesis; submandibular gland.

Figure 1. Morphological changes in cultured SMG rudiments. (A) E13 non-frozen control SMG rudiments floating in DMEM/F12 medium. (B-E) SMG rudiments cryopreserved at –80˚C for 24 h either in a freezing tube box (Quick) or a Mr. Frosty (Slow) with (B) DMEM/F12, (C) DMEM/F12 medium containing 10% DMSO, (D) TC only, and (E) CB only. All SMG rudiments were cultured for 0-72 h. Some of cryoprotective agents (CPA) were protected the cells of SMG rudiments although the protective effects were different from each other. The black arrow indicates a hypertrophylike structure. Scale bar=200 μm. TC: TC Protector; CB: CELLBNKER1.

Figure 2. Influence of cryoprotective agents on branching morphogenesis of cultured SMG rudiments. The number of endpieces (A) and the area of the epithelium (B) of E13 cultured SMG rudiments held under the different conditions were determined. All SMG rudiments were cultured for 72 h. Values represent the mean±SD of four independent experiments. N.D.: not detected; Quick: quick freezing (using a freezing tube box); Slow: slow freezing (using a Mr. Frosty). *p<0.05, **p<0.01.

Figure 3. Western blot analysis of ERK1/2 phosphorylation by EGF in cultured SMG rudiments with or without cryopreservation. E13 SMG rudiments were collected and separated into two groups. The control group was not frozen but was held at 4˚C for 24 h, and the other group was cryopreserved in Mr. Frosty at –80˚C for 24 h in CB as a CPA. SMG rudiments were cultured and stimulated with EGF (20 ng/ml) for 0, 10, and 30 min. The SMG rudiments were homogenized, and the resulting supernatants were analyzed by Western blotting. (A) Upper panel: Phosphorylated ERK1/2 (p-ERK1/2); lower panel: Total ERK1/2 (t-ERK1/2). (B) The intensities of the Western blot bands were analyzed using a CS analyzer and expressed as a ratio (pERK1/2 per t-ERK1/2 at each incubation time). Values represent the mean±SD of three independent experiments. ANOVA compared with 0 min. *p<0.05.

Figure 4. Western blot analysis of AKT phosphorylation by EGF in cultured SMG rudiments with or without cryopreservation. E13 SMG rudiments were collected and separated into two groups. The control group was held at 4ºC for 24 h, and the other group was cryopreserved in a Mr. Frosty at –80˚C for 24 h in CB as a CPA. The SMG rudiments were cultured and stimulated with EGF (20 ng/ml) for 0, 10 and 30 min. The SMG rudiments were homogenized, and the resulting supernatants were analyzed by Western blotting. (A) Upper panel: Phosphorylated AKT (p-AKT); lower panel: Total AKT (t-AKT). (B) The intensities of the Western blot bands were analyzed using a CS analyzer and expressed as a ratio (p-AKT per t-AKT at each incubation time). Values reprsent the mean±SD of three independent experiments. ANOVA compared with 0 min, *p<0.05.