基本的组织培养:细胞传代

Immortalized cell lines derive from multicellular organisms with mutations affecting cell cycle regulation, allowing indefinite proliferation. Stem cell lines are generated by isolating self-renewing, multipotent cells from adult or embryonic tissues. Both can be maintained indefinitely in culture under appropriate conditions, but stem cells retain pluripotency while immortalized cells do not.

Toxic metabolites accumulate in cell culture media over time, lowering pH and reducing nutrient availability. Fresh media maintains cell health and supports continued expansion. Media color indicators like phenol red signal when changes are needed—orange indicates fresh media, yellow indicates acidic conditions requiring immediate media change, and pink suggests pH is too basic.

Confluency refers to the percentage of culture container surface covered by cells. At 90% confluency, adherent cells cover most of the container bottom and must be passaged to prevent overgrowth and maintain cell health. Monitoring confluency ensures cells are split at the optimal time for expansion and prevents toxic metabolite accumulation.

Complete media requires serum such as heat-treated Fetal Bovine Serum for growth factors, antibiotics like penicillin and streptomycin to prevent contamination, and additional growth factors such as fibroblast growth factor to prolong expansion. Each cell line requires a specific growth supplement cocktail tailored to its needs. Store complete media at 4°C when not in use.

Trypsin is a digestive enzyme that cleaves proteins binding cells to plastic surfaces. Cells are first washed with calcium- and magnesium-free phosphate buffered saline, then trypsin is added and incubated at 37°C for approximately 5 minutes. Careful timing is critical to avoid damaging cell surface proteins. Fresh media is added to stop proteolysis before transferring cells to a conical tube for counting using hemacytometer methods and applications.

For cells sensitive to enzymatic digestion or extremely adherent to plates, a cell scraper can gently remove cells from the culture container bottom. Alternatively, a serological pipette can be used in a firm scraping motion while rinsing with media. Mechanical dissociation requires care, as delicate cells can be damaged by these methods.

Multilayer flasks expand cell cultures 3-5 fold compared to single-layer flasks, enabling faster and more reproducible scale-up of cell expansion. These specialized culture vessels provide increased surface area for cell growth, making them ideal for generating large numbers of cells efficiently while maintaining appropriate culture conditions.