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Cell Subculture

Cell subculturing is the basis of most cell biology experiments. It is one of the most common use methods for tissue culture. The cells are grown in culture container and needs to transfer to other containers for further growth which is called subculture.

Cell culture system can be divided into adherent cell culture and suspension cell culture.

Simply, adherent cells are grown by attaching to the surface of the culture container, such as normal cells, tumor cells, etc.,. Suspension cells do not depend on the surface of culture container, and they grow in culture medium by suspension, such as lymphocytes.

Subculture method of two kinds of cells are different, here is the culture steps for your reference:

Consumables and Instruments:

●cell culture flask/dishes

●Pasteur Pipettes

●Centrifuge Tubes

●Serological Pipettes

●CO₂Incubator（37℃）

●37℃ water bath

●Micro Pipettes

●Clean Bench

●Centrifuge

●Microscope

Reagent：

●Cell culture medium or 90％DMEM

●10％ FBS

●0.25% Pancreatic enzymes

●PBS Buffer

●Iodine

●75% Alcohol

Adherent Cells

Tips

Trypsin is the most common used enzyme in cell subculture which can disrupt connection between cells and between cells and flasks.

Adherent cells treated with trypsin can be dispersed into single cells by external force (such as pipetting), and then diluted and inoculated to provide sufficient nutrients and space for cell growth.

Steps:

1.Take out cell culture flasks/dishes from incubator and observe cell conditions under microscope to check whether the cell density that can be passaged.

2.Autoclave the clean bench and required consumables and waterbath enzyme, buffer and cell culture medium.

3.Pipette used cell culture medium and add PBS buffer. Avoid disrupting the cell layer by shaking the dishes/flasks gently and rinse the cells.

4.Add enzyme by micro pipettes and shake the dishes/flasks and rinse the cells.

5.Put dishes/flasks into 37℃ incubator and digest for around 2 minutes and then take out. Observe the cells under microscope. When the cells become round and fall off, terminate the digestion. Use 75% alcohol to spray the cell culture dishes/flasks and then transfer to clean bench.

6.Add serum medium by micro pipettes and stop digestion. Blow cells to detach from the bottom and observe suspensions with single cells.

7.Fill cell suspensions into centrifuge tubes and centrifuge it under ambient temperature, set speed at 1000rpm for 2-5 minutes. (speed and time depend on experiments)

8.After centrifugation, spray centrifuge tube with 75% alcohol and transfer to clean bench. Open the cap, dispose supernatant, and mix cells with culture medium gently for re-suspension.

9.Fill cell suspensions into 2 or more new cell culture dish/flask, add cell culture medium and put on the lid.

10.Observe the cell density to make sure cell quantity.

11.Mark new cell culture dishes/flasks with subculture time, cell type, operator and put into incubator, waiting for cell growth.

Suspension Cells：

Suspension cell subculture is much more simple than adherent cells. It does not need enzyme to digest, here are 2 methods:

Direct Subculture

1.Put flasks into clean bench and wait for 5-10 minutes. When suspension cells go to the bottom of flasks/dishes, pipette 1/2~!2/3 medium solution inside.

2.Transfer cell suspensions into 2 or more cell culture dishes/flasks.

3.Mark subculture time, cell type , operator and then put into incubator.

Subculture by Centrifuge

1. Take out of the cell suspensions into centrifuge tube and then put on the clean bench.

2. Set speed at 1000rpm for 3-5 minutes and dispose supernatant.

3. Add serum medium for re-suspension.

4. Fill cell suspensions into new cell culture dish/flask, add cell culture medium and mix gently.

5. Mark new cell culture dishes/flasks with subculture time, cell type, operator and put into incubator.

Operation Tips

1. All subculture procedures must be sterile and all consumables must be sprayed by 75%

alcohol before putting on the clean beanch.

2. Pipette tips must be disposable without any cross contamination.

Welso can provide all the consumables and instrument during the whole cell culture process. Please contact us for any inquiry or download our catalogue from website.