General Guidelines for Cell Culture

 

 

Licia Miller   Product Manager

 

Cells cultured in the laboratory can be roughly divided into the following three categories according to cell type.

 

(1) Adherent cells: cells adhere to the culture container (such as tissue culture plastic).

(2) Suspension cells: All cells grow in suspension in the growth medium and do not grow attached to the culture container.

(3) Semi-adherent cells: Some cells are loosely adhered to the culture vessel, while others may be suspended in the growth medium.

 

Cells can also be classified according to their growth characteristics:

 

Finite Proliferation: Proliferates and maintains viability for only a limited number of population doublings. Common examples are primary cells isolated directly from tissue, or cell lines that cease to grow (senesce) after a certain number of passages.

 

Continuous: Cells that have the capacity to divide indefinitely (immortalized). Often produced by transformation to acquire a cancer-like phenotype (e.g., loss of contact inhibition, unlimited growth).

 

Here we will focus on the most common cell types: adherent cells and suspension cells. Please note, All procedures involving manipulation of cultured cells should be performed using aseptic techniques and appropriate controls.

 

Phase 1   Cryopreservation

 

Genetic instability accumulates in cells that are continuously cultured. Therefore, cell lines should be frozen and stored as soon as possible after receipt. This ensures that the cell bank is as genetically close to the source material as possible and reduces the risk of contamination. The freezing process should freeze the cells in a controlled manner (ideally at a rate of 1°C per minute) in the presence of a cryoprotectant such as DMSO to prevent ice crystals from forming within the cells and causing loss of culture viability.

 

Experimental Steps

 

1. Observe the cells under a microscope to check their overall appearance and ensure that there is no sign of microbial contamination. At the same time, ensure that the cells are in the logarithmic growth phase, the cell density should not exceed the guidance range of the cell line, and the viability should generally be greater than 90%.

 

2. Collect and count cells according to standard subculture methods. Cryopreserve suspension cells at a density of 2-5×106 cells/ml, and cryopreserve adherent cells at a density of 1-2×106 cells/ml.

 

3. Select the appropriate cryoprotectant based on the characteristics of the cell line, calculate the required freezing solution based on the number of cells, and prepare the freezing solution according to the formula.

 

Note that if DMSO is added to the freezing solution, try to prepare it in advance because DMSO will release heat when added.

 

4. Centrifuge the cell suspension at 200 × g for 5 minutes, remove the supernatant, and resuspend the cell pellet in PBS.

 

5. Centrifuge at 200 × g for 5 minutes and remove the supernatant.

 

6. Resuspend the cell pellet in the prepared freezing solution, mix thoroughly, transfer to an appropriate number of cryovials, and label important information such as date, name, cell number, passage number, and cell type.

 

7. Place the cryovials in a cryo box and place at -80°C overnight. This will control the temperature drop.

 

8. Transfer to a liquid nitrogen tank for long-term storage.

 

Phase 2   Cell Revival

 

When cells are needed, they should be thawed as quickly as possible to minimize adverse effects on cell viability. It is recommended that the cryopreservative be removed from the culture medium by centrifugation during thawing.

 

Experimental Steps

 

1. Remove the cryovials from liquid nitrogen storage and place in a 37°C water bath. Gently shake the tube to accelerate thawing. Monitor the vials carefully. When the vials are thawed to the size of soybeans, the cryovials can be removed from the water bath.

 

2. Transfer the cells (1 ml) from the cryopreserved tube to a 15 ml centrifuge tube containing pre-warmed culture medium (4 ml) and centrifuge at 200-250 × g for 5 minutes.

 

Note: The centrifugation speed and time are for reference only and can be adjusted according to specific experimental requirements.

 

3. Remove the supernatant and resuspend the cell pellet with an appropriate amount of pre-warmed culture medium, select a suitable inoculation density, and inoculate the cells into the corresponding culture container for culture.

 

4. According to the difference of the cell type, add the required growth factors and other ingredients.

 

Phase 3   Observation

 

Cells should be regularly inspected microscopically and visually for signs of microbial contamination. Microscopic examination should also be used to determine the overall health of the cells and to determine if subculturing is necessary.

 

Experimental Steps

 

1. Observe the status of cell culture with the naked eye to eliminate contamination.

 

For adherent cells, the growth medium should be clear. If it is not, this may be a sign of contamination, or that the cells have passed confluence and are dying or detaching from the culture surface.

 

For suspension cells, the medium will be more turbid due to the cells being suspended in the solution. If the turbidity is higher than expected, coupled with an acidic medium, it may indicate contamination or high cell density and the need to subculture.

 

Temperature levels, carbon dioxide, and the metabolism of ingredients can all affect the pH of the growth medium.

 

2. Observe the growth of cells and whether they are contaminated under an optical microscope through the following aspects.

 

Cell attachment. Ensure that cells are adhered to the culture vessel or are in suspension as expected.

Cell morphology. Inspect the culture to confirm that the cell morphology is as expected. Different morphologies may be a sign of contamination or differentiation.

Confluency (for adherent cells). This is the percentage of the cell culture surface that is covered by cells. 100% means that the cells completely cover the surface of the culture vessel. The percentage at which cells need to be subcultured depends on the cell line, but 70% is the most common.

Cell density. Monitor the health and growth characteristics of suspension cells by performing a cell count. Collect a sample of cells and perform a cell count using a hemacytometer or automated cell counter. The density at which the cell line needs to be subcultured depends on the cell line.

 

Be sure to test cells regularly to exclude mycoplasma contamination, and monitor cells continuously during the culture process for signs of bacterial, fungal, and yeast contamination. Different cell lines may also contaminate the culture.

 

Stage 4   Cell Maintenance and Subculture

 

If the cells are healthy and have reached the desired confluence, they can be subcultured and/or plated for experimental use.

If cells have not yet reached the desired confluence and 2-3 days have passed since the last subculture, change the medium and continue until the desired confluence is reached.

If cells show signs of contamination, they should be discarded.

 

● Exchange the medium

 

When culturing cells, it is necessary to change the culture medium regularly to prevent the accumulation of toxic metabolites (such as lactate) and to ensure a continuous supply of growth medium components. The accumulation of cellular metabolites is often monitored by pH indicators (such as phenol red) to determine the appropriate time to complete the cell culture medium change.

 

Experimental Steps

 

1. Aspirate the growth medium.

 

For suspension cells, first transfer the culture to a centrifuge tube and centrifuge at 200 - 250 × g for 5 minutes to remove the culture medium.

For adherent cells, tilt the culture vessel slightly and aspirate the culture medium directly, being careful not to aspirate it completely.

 

2. Refill with fresh growth medium.

 

For suspension cells, resuspend the cell pellet in an appropriate amount of fresh growth medium, mix well, and transfer to a new culture container.

For adherent cells, add an appropriate amount of fresh growth medium directly along the wall.

 

3. Place the culture container in the incubator for cultivation in time and continue to monitor cell growth and signs of contamination.

 

● Subculture

 

If the cells grow to the desired confluence or density, they should be subcultured. Subculture, also known as passaging, is the process of transferring cells from a previous culture to a new culture vessel and adding fresh culture medium for continued growth.

 

Experimental Steps

 

1. Wash and collect cells.

 

For suspension cells, transfer the culture to a centrifuge tube and centrifuge at 200-250 × g for 5 minutes. The culture medium was removed and the cell pellet was suspended in an appropriate volume of PBS, washed once, and the PBS was removed by centrifugation.

Resuspend the cell pellet in fresh growth medium.

 

For adherent cells:

(1) Tilt the culture vessel slightly, aspirate the culture medium directly, add an appropriate volume of PBS to wash , make sure the cell culture is covered, aspirate and discard the PBS.

(2) Add an appropriate digestion enzyme, such as trypsin, and incubate at 37°C until the cells are detached.

(3) Add fresh culture medium and transfer the cell suspension to a centrifuge tube.

(4) Centrifuge at 200-250 × g for 5 minutes.

(5) Remove the culture medium and resuspend the cell pellet in an appropriate volume of PBS. Mix well and wash thoroughly.

(6) Centrifuge at 200-250 × g for 5 minutes.

(7) Aspirate and discard the PBS and resuspend the cell pellet in fresh growth medium.

 

Note: Any coating of the culture vessel required should be performed prior to cell isolation.

 

2. Select the appropriate dilution ratio for subculturing, inoculate an appropriate amount of cells into a new culture container, and add growth medium to the required volume.

 

3. Label the culture container with key information, including cell type, passage number, seeding density/splitting ratio, date, and operator, etc. and incubate as required.

 

4. Continue to monitor cells daily for growth and signs of contamination.

 

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/