In the case of wall culture and suspension culture, the criteria for judging whether to be subgrade are roughly the same.
However, there are some differences between mammalian and insect cell lines.
When we can start the generation, we can look at the cell density and the level of the medium.
Cell density
(1) mammalian cell: it should be carried out in the long term when the cell wall culture cells enter the logarithmic growth period.
When normal cells reach the confluence state, they cease to grow (contact inhibition), and it takes a long time to recover after revaccination.
Conversion cells can continue to proliferate even when they reach confluence, but usually deteriorate after about two doubling times.
Similarly, the cells in suspension culture should be passed on to the logarithmic growth period, not to meet the state.
When the confluence state is reached, the cells of the suspension culture will aggregate into a mass, and the culture medium will become cloudy when rotating the bottle.
(2)Insect cells: the insect cell enters the logarithm of the logarithm of the logarithm of the log, and does not reach the confluence state.
Firmly attached insect cells to achieve convergence condition can also be represented, while cells more easily from the incubators turned from down, but if repeated insect cells in density than extend the convergence conditions, the multiplication of cells number will decrease, vitality will reduce, stick a wall ability will decline.
On the other hand, the insect cells that have been cultured in the pasted wall need a great mechanical force to achieve the confluence status, so that the cells can be separated from the single-cell layer.
Repeatedly in the prequel to the confluence state, the number of cells is reduced, the vitality is decreased, and the health is poor.
The medium is depleted
(1)Mammalian cells: the pH value of the growth medium is decreased, and the lactic acid is usually expressed as a byproduct of cell metabolism.
Lactic acid has cytotoxicity, and lower pH is also a negative factor for cell growth.
The speed of pH change is usually determined by the concentration of cells in the culture system, the higher the concentration of cells, the faster the medium is depleted.
If the pH value decreases rapidly (> 0.1-0.2 pH), and the cell concentration increases, then the cell can be passed on.
(2)Insect cells: growth medium used for insect cell culture is usually more acidic than mammalian cell culture medium
For example, the pH of tnm-fa and Grace media for Sf9 cell culture was 6.2.
Different from mammalian cell culture, the pH of the culture medium in the growth of insect cells will gradually increase, but generally not more than 6.4.
But, like mammalian cells, the pH of the growth medium, which is more dense, begins to decline.
Transfer time arrangements
According to the predetermined time, cell transmission can ensure the stable biological behavior of the cell and facilitate the monitoring of its health.
The inoculation density was gradually adjusted from one inoculation density until it reached the stable growth rate and yield suitable for the cell.
Cells growth pattern is usually clear the deviation so poor health (for example, metamorphism, pollution) or dysfunction of cultivation system of a certain component (for example: not the best temperature, medium too old).
We strongly recommend that you keep detailed records of cell culture, records and extend the time of feeding medium type, dissociation method used, minute rate, morphological observation, inoculation concentration, production and usage of antibiotics.
It is best to conduct experiments and other unconventional operations (such as changing medium type) in accordance with the time of transmission.
If the arrangement of your experiment does not coincide with the conventional transfer time arrangement, it should be ensured that when the cell is still in the delayed period or has reached the confluence state, when the growth is stopped, no transmission is carried out.
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