ANIMAL CELL CULTURE

SUBCULTURING Transfer of Cells from spent up media to fresh media. when primary culture is first subcultured it gives rise to secondary culture the secondary to a tertiary, and so on.

Primary culture P1 Selection Secondary culture(Cell line) Characterization P2 Tertiary culture P3 Cell Strain P4 P5

Most subcultivation methods involve a disruption of intercellular and cell-to-substrate connections. Trypsin is the most common cell and tissue dissociating agent used. After the cell dissociation, cells in suspension are diluted in fresh media and transferred into an appropriate sterile vessel. Cells will re-attach and grow in this vessel after a period of time, depending on the cell line.

Subculture of Monolayer

TERMINOLOGY cell line: Once a primary culture is subcultured (or passaged), it becomes known as a cell line. Several cell lineages of either similar or distinct phenotypes is present. cell strain: A characterized cell line derived by selection or cloning. Passage Number: It is the number of times that the culture has been subcultured, Generation Number: It is the number of doublings that the cell population has undergone

Split ratio: The divisor of the dilution ratio of a cell culture at subculture (e.g., one flask divided into four, or 100 mL up to 400 mL, would be a split ratio of 4). Plating efficiency: The percentage of cells seeded at subculture that gives rise to colonies. Seeding efficiency: The percentage of the inoculum that attaches to the substrate within a stated period of time (implying viability, or survival, but not necessarily proliferative capacity). Cell concentration: Number of cells per mL of medium. Cell density: Number of cells per cm2 of substrate

Serial Subculture

FACTORS INDICATES THE NEED FOR SUBCULTURING Drop in pH, Cell concentration, Cell type, Morphological deterioration,

Drop in pH Growing Cell Releases Acidic Metabolites in Medium Reduces pH of Media Affect the Cell Growth Hence,the cells needs to be subcultured

Optimum pH for Growth Cells Optimum pH Fibroblast cells 7.4-7.7 Transformed cells 7.0-7.4 Epidermal cells 5.5 When pH reduces from pH7.0 to pH6.5---cell stop growing, If pH drops further from pH6.5 to pH6.0—cells start losing their viability.

Phenol RED Commonly used Ph Indicator. Red in color at pH7.4 Change in Ph results in change in color. pH of Medium Corresponding color 7.8 Purple 7.6 Pink 7.4 Red 7.0 Orange 6.5 Yellow <6.5 Lemon Yellow

Rate of change of pH If the rate of change of pH is 0.1unit/day: the subculturing can be delayed for few days but if the change in pH id 0.4 unit/day: then it needs to be subculture immediately

Cell Concentration High cell concentrations exhaust the medium faster than low concentrations. Hence needs to be subculture sooner.

Cell Type Normal cells: Transformed cell: Deplete media slowly once the confluence is reached they stop growing cells arrest in the G1 phase of the cell cycle Deteriorate very little, So,subculturing can be delayed Transformed cell: Continue to grow confluent monolayer and beyond saturation density Hence they need to be subcultured within few days.

Hence need to be subcultured soon The continuous cell lines and embryonic cells also deplete media at much faster rate. Hence need to be subcultured soon IMR 90 Cell line 3T3-A31 Cell line

Morphological Deterioration Significance of Cell Morphology: The culture be examined carefully to confirm the absence of contamination. The cells should also be checked for any signs of deterioration,such as: Granularity around the nucleus. Increased cytoplasmic vaculation. Rounding of cells.

Vacuolation and granulation in bronchial epithelial cells Such signs may imply that the culture requires a medium change, or may indicate a more serious problem, e.g., inadequate/toxic medium,microbial contamination, or senescence of the cell line

Time Since Last Subculture If cells have not reached a high-enough density (i.e., they are not confluent) by the appropriate time, then increase the seeding density, or if they reach confluence too soon, then reduce the seeding density.

Subculture of Cells Growing in Suspension Almost same as subculture of monolayers. Replacement of the medium (feeding) is not usually carried out with suspension cultures, and instead, the culture is either diluted, or the bulk of the cell suspension is withdrawn and the residue is diluted back to an appropriate seeding concentration Trypsin treatment is not required, Subculture is quicker, Less traumatic for the cells, Scale-up is easier

USEFUL NOTE When handling a cell line for the first time, or when using an early-passage culture with which you have little experience, It is good practice to subculture the cell line to a split ratio of 2 or 4 at the first attempt, As you gain experience and the cell line seems established in the laboratory, it may be possible to increase the split ratio—i.e., to reduce the cell concentration after subculture