Primary cell cultures represent the physiological state of the tissue of origin. However, they have a finite lifespan undergo senescence after a certain number of passages or subcultures. While lower passages of the cells usually contain a mixed cell population, high passages can lead to loss of the differentiated properties of the cell type. Continuous culturing of primary cells can lead to accumulation of genetic and phenotypic changes in cells thus losing their original properties. There are multiple factors that can drive these changes including selective pressure of the culture media and growth conditions. Moreover, multiple passages can increase susceptibility of the primary culture to microbial contamination. Generally for primary cultures, passages between 2 to 5 are suitable for maintaining the genetic and phenotypic properties.
How to avoid increasing passage numbers
To access your primary culture at low passages, it is recommended to freeze down multiple vials of your stock cultures. This ensures that your experiments can be repeated with reproducibility at similar passages. Kosheeka offers high quality and pure population of primary cells at very low passages for your effective research. Our cells have undergone strict quality control and microbiological testing to ensure high reliability. In general, it is a healthy practice to passage a minimum number of times as per your experimental requirements and freeze down your working culture vials for future requirements. Seeding density is also important to prevent slow or overgrowth of cells. A very low density can often be an additional stress to the primary cells while a high density will make passaging very frequent. Finally, one must record the passage number correctly and label the flasks after every passage to avoid confusion. There is no way one can determine the passage number of cells experimentally. However, for freezing the cells, the passage number must not be increased.
How do you know if a passage is no longer needed?
Primary cells from different patients respond differently to cell culture. Therefore, it is essential to monitor the morphology routinely and observe for any abnormalities or changes under an inverted microscope. It also recommended to characterize the cells at various passages and determine their growth curves to establish population doubling time.
Is passage number the definite guide to the age of cells?
Passage number does not tell us the actual number of population doublings. Depending on the seeding density of the cells, the number of cell divisions will vary. If the split ratio is 1:2, cells will undergo fewer doublings as compared to flasks with a split ratio of 1:5. So one must not rely solely on the passage number to assess other properties of cells. Passage number must only be used for reference purpose only.