Methods: Rat submandibular salivary-gland integrin a6b1 positive cells were secluded, cultured and limiting diluted. Thereafter cells were grown on growth-factors enriched or reduced Matrigel (basement membrane extra cellular matrix) to form clusters. We have tested various conditions leading to morphological differentiation, including seeding level, matrigel concentrations and growth-media supplements (FCS, BPE, and HGF). Cultures of the human neoplastic submandibular-gland intercalated duct cell-line (HSG) served as a control model for differentiation process in all growth conditions.
Results: HSG cells formed acinar and reticular structures when grown on both types of Matrigel for one or two days in high or low seeding densities (respectively). a6b1 cells formed reticular appearances after one day in culture when seeded in high density under all media supplements. However, in low seeding density those cells formed reticular structure after 18 days, only when grown on not diluted enriched Matrigel and unsuplemented media. a6b1 cells grown on reduced matrigel in low seeding density with growth media and 0.5% BPE, formed granule-splinter like structures after three days in culture, in all Matrigel concentrations tested.
Conclusions:
1. When seeded in low density, the appearance of the reticular structures created by á6â1 cells was much later than those created by HSG.
2. Cells seeding concentration may have crucial impact on morphological differentiation.