Guide to the method of adherent cell passage

Most cell lines and primary cells grow in a single layer (single layer of cells) or on the surface of glass or treated plastic objects. In order to maintain the healthy and active proliferation of cells, it is usually necessary to carry out regular passage of cells.

The most common method of passage is to use a protease such as trypsin or collagenase to disrupt the intercellular and intercellular connections between the cells and the culture medium. When the cells are dissociated into a single cell suspension, they are then diluted and dispensed into a new culture vessel. There, the cells can re-adhere to growth and division, and then after a period of incubation, the cells are once again full (close to confluence). At this point, the cells need to be passaged or used in downstream experiments.
Note: The following guidelines describe the basic principles of routine passage and maintenance of typical monolayer cells. In order to achieve consistent results, it is important to maintain a good culture record, which should include the date of passage and the number of passages.
Cellular examination: Good habits should be developed to routinely and carefully examine cells for their status and health.

First, visually inspect the culture vessel for visible microbial contamination, such as changes in the pH of the medium , turbidity, or particulate-like objects. Also pay attention to the presence of small fungal colonies because they are not easily found under the microscope. These colonies may float on the surface of the liquid, especially near the perimeter of the culture vessel.
Secondly, observe the normal morphology and growth state of the cells under an inverted microscope, and pay attention to the signs of microbial contamination visible under any microscope. In some cell lines, the cells floating in the culture fluid are usually dead cells. However, many cells become rounded when mitotic, forming a very refraction (very bright) sphere and floating after being disturbed. The difference between the two is that dead cells usually become rounded off but generally do not have strong refractive power.

In addition to these daily tests, periodic periodic fungal, bacterial, and mycoplasma tests are required on the cells.
Preparation of culture medium: Prepare a fresh culture solution recommended for use in a specific cell line at the time of purchase or in the literature, and mark information such as passage time and cell number on the culture container with a marker. Make sure to add supplemental ingredients (eg glutamine, growth factors, antibiotics, etc.) and pre-position the incubator for pH equilibration. A 75 cm ² flask requires approximately 12-15 ml of culture medium. How to use other sizes of culture vessels requires adjustment.

Collect cells:

Most cells undergo cell passage before complete growth (and some room for growth) to achieve optimal growth because the cells are in an active logarithmic growth phase. Any abnormalities observed in the experiment should be recorded on a specific record for each cell line.

The principle of optimal collection of cells is to destroy the connection between cells and culture vessels and cells in the most gentle manner. For most cells, this requires the use of a digestive solution containing chemicals or enzymes. Trypsin is the most commonly used digestive juice and is used in conventional amounts between 0.05% and 0.25% . The optimal working concentration of trypsin is usually the lowest concentration at which cells can be detached from the wall of the culture vessel to form a single cell suspension in a relatively short period of time ( 10-15 min ). Some cells are more difficult to digest than other cells. In this case, collagenase or a chelating agent such as EDTA is usually added to trypsin to improve digestion.
When the cells are grown in a culture medium containing mammalian serum, the cells need to be washed first to remove the serum, since the presence of serum inhibits the activity of the trypsin. Serum residue is a common cause of pancreatic digestion failure. Buffers that do not contain calcium and magnesium ions are required for cell digestion because these two ions play an important role in cell-to-cell and adsorption between cells and culture vessels.
Collection of monolayer cells:

1. Remove cell culture fluid
2. Add 5 ml of digestive juice to wash the cells once and then aspirate (the reagent amount for this method is for 75 cm ² culture flasks, if other culture vessels are used, it is necessary to increase or decrease the amount of reagents). If trypsin is included in the digestive juice, all serum residue needs to be removed because the serum contains trypsin inhibitor. (Note: You can also use DPBS to clean 1-2 times here, saving point of digestive juice)
3. Add 2-3 ml of digestive juice and observe the digestion of the cells under an inverted microscope every 5 minutes . For cells that are particularly difficult to digest, digestion can be performed at 37 degrees. Preheating the digestive juice can reduce the digestion time. To avoid cell agglomeration, do not tap or shake the flask while waiting for the cells to fall off.
4. Using a pipette, add 6-8 ml of growth medium to the cell suspension and pipette the bottom of the flask to collect all remaining cells. At this point, a simple observation under an inverted microscope should show that at least 95% of the cells in the cell suspension are single cells. If not, you may need to blow further.
5. Collect the cell suspension, perform cell counting or aliquot as needed, and dispense to the prepared culture vessel. For some cell lines or enzyme digests (collagenase), it is necessary to remove the enzyme digest by gentle centrifugation ( 125 g , 5 min ) prior to distribution .
Counting or aliquoting of cells:

In order to obtain the growth rate of the cells or to inoculate the cells at a known concentration, it is necessary to count the cell suspension, and we can use a hemocytometer or an electronic cell counter. The advantage of a hemocytometer is that it is relatively inexpensive and can simultaneously detect cell viability. The cell suspension was gently mixed, and 0.5 ml was taken out for cell counting. To this was added 0.5ml viable cell staining solution such as trypan blue (trypan blue) or erythrosine B (erythrosin B), mix well, remove the portion of the sample, carefully added to a clean hemacytometer.
The actual concentration of cells in the cell suspension (number of cells per ml of suspension) and the viability of the cells were calculated, and then the volume of suspension required to achieve a suitable passage density was calculated. In addition to cell counting, cell suspensions are often equally divided by the number of subculture flasks. For example, 1 : 2 passage means aliquoting the cell suspension obtained in one culture flask into two new culture flasks of equal culture area, which is suitable for conventional cell passage when the precise density of cells is not very important.
Cell inoculation:

Dispense the appropriate cell suspension into a well-prepared cell culture flask (adding a high concentration of cell suspension directly to an empty culture flask will result in uneven adherence and growth of the cells). For fast growing cells, the density of inoculation can be lower. However, some cells do not grow well below a certain seeding density. However, most cells can grow well at an initial density of 10 ³ -10 ⁴ cells per square centimeter or higher ( 7.5 x 10 ⁴ to 7.5 x 10 ⁵ cells per 75 cm ² flask ).

Cell culture:

The cells were returned to the incubator, most preferably mammalian cells grown at a constant temperature of 35 degrees to 37 degrees. In addition to maintaining a stable temperature, the incubator also needs to maintain high humidity and CO ₂ levels for non-closed culture vessels such as Dish or multiwell plates . The high humidity prevents the loss of volatilization of the liquid in the culture vessel, which in turn leads to a high osmotic pressure caused by concentration of the culture solution.
When the culture medium uses a buffer system containing an appropriate amount of NaHCO ₃ , increasing the level of CO ₂ (usually 5% to 10% ) allows the culture to be maintained at a suitable pH range ( 7.0-7.6 ). In order for this type of cushioning system to work properly, a non-sealed (capped loose cap) or gas permeable flask is required to ensure proper gas exchange. If no CO _{2 is} available , there is a buffer system that maintains the culture in a suitable pH range in a closed culture vessel .
The next day, the next observation was performed under the microscope to ensure that the cells had reattached and actively proliferated. The medium is changed as required during the culture. For most actively proliferating cells, it is usually necessary to change the fluid 2-3 times a week .

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