Subculture: After the primary cell culture is successful, it needs to be separated and cultured, otherwise the cells will have insufficient living space or excessive density, and nutritional disorders, which will affect cell growth. The process in which cells are separated and diluted from the original culture flask and transferred to a new culture flask is called subculture. Passage cells allow the expansion of cultured cells (to form cell strains), they can be cloned, and are easy to preserve, but may lose some special cell and differentiation characteristics. The greatest advantage of subcultured cells to form cell lines is to provide a large amount of long-lasting experimental materials to facilitate experiments.

Equipment required for subculture experiment

AlphaClean1300 Clean Bench, inverted microscope, HF180 CO2 Incubator, culture bottle, straw, waste liquid tank, etc.

AlphaClean1300 Clean Bench
HF180 CO2 Incubator

Reagents needed for experiment: 0.25% pancreatin, culture medium (containing 10% calf serum)
Cells needed for experiment: Adherent cell line

Subculture Experiment Operating Steps

(1) Wash your hands with soap before entering the sterile room, and wipe and disinfect your hands with 75% alcohol.

(2) Observe the cell morphology under an inverted microscope to determine whether the cells need to be passaged (passage criteria: 1. Grasp the criteria for whether the cells are healthy: healthy cells have full morphology, good refractive index, and can be passaged when they are densely grown. 2. Wait for cells cover the bottom surface of the bottle and dish, and it can be used. It can also be subcultured and expanded or replaced with a maintenance solution.) and the number of times the cells need to be diluted. Preheat the culture solution at 37°C.

(3) Wipe the AlphaClean1300 Clean Bench with 75% alcohol or 0.1% bromogeramine solution
(4) Turn on the UV lamp of the Clean Bench to irradiate the countertop for about 30 minutes, turn off the UV lamp of the Clean Bench, turn on the exhaust fan to clean the air and remove ozone. It is recommended to use Heal Force AlphaClean1300 Clean Bench, which has a UV lamp reservation function. According to the need, you can set the time period for disinfection in advance and run automatically, saving operation steps.

AlphaClean1300 Clean Bench

(5) Use an infrared sterilizer or light an alcohol lamp; take out the sterile test tube, Bast straws and graduated straws; install a rubber tip; after the alcohol lamp flame is slightly burned, insert it into the sterile test tube.
(6) Sterilize the mouth of the culture liquid bottle with 75% alcohol, and place it diagonally on the shelf next to the alcohol lamp after passing the flame of the alcohol lamp.
(7) Aspirate or discard the old culture fluid in the culture flask.
(8) Add a small amount of trypsin solution and EDTA mixture into the bottle. It is better to cover the bottom of the culture flask.
(9) Place the digestion in a 37°C incubator or room temperature (25°C). After 2 to 5 minutes, place the culture flask under an inverted microscope for observation. When the cytoplasm is found to shrink and the intercellular space increases, stop immediately Digestion.
(10) Aspirate the digestion solution, add a small amount of Hanks’ solution into the bottle, turn the culture bottle gently to wash away the residual digestion solution, and then add the culture solution. If only trypsin digestion is used, after aspirating the trypsin solution, a small amount of serum-containing culture solution can be directly added to terminate the digestion.
(11) Use an elbow pipette to suck the culture medium in the bottle, and gently pipette the cells on the bottle wall in sequence to separate them from the bottle wall to form a cell suspension. When pipetting, the action should be gentle to prevent excessive force from damaging the cells.
(12) After counting with a counting plate, inoculate them into new culture flasks and place them in HF180 CO2 Incubator or HF90 CO2 Incubator for culture.

HF90 CO2 Incubator
HF180 CO2 Incubator

(13) The cell culture medium exchange time should be determined according to the state of cell growth and experimental requirements. Generally, the growth medium should be changed once after 2 to 3 days. After the cells are covered with the bottom surface of the vessel, it can be used; it can also be continued to be subcultured and expanded or replaced with a maintenance solution.

Subculture Operation precautions

  1. Master the time of cell digestion. When the digestion time is too short, the cells should not fall off the bottle wall. Excessive digestion will cause the cells to fall off and damage;
  2. Master the digestion concentration. When the digestion solution concentration is too high, the digestion time should be shortened, and when the digestion solution concentration is too low, the cell digestion time will be relatively prolonged.

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