When receiving a cell line in the lab for the first time, several pieces of information related to the cell line should be organized and recorded. These details are crucial for ensuring successful cell propagation, expansion, cryopreservation, and storage. It is strongly recommended to document the following information before starting cell expansion: (1) Background information (2) Subculturing (3) Cryopreservation (4) Cell line storage.

Monitoring and recording the morphology and behavior of cells is crucial. It is recommended to routinely and carefully examine cultured cells before subculturing to assess their status and health. Observations of cell morphology can help researchers determine:

(1) Cell morphology: Are the cells healthy or deteriorating (such as cellular senescence or necrosis)?

(2) Is there any evidence of contamination?

(3) Differentiation of cell types and assessment of cell density.

Most cell cultures grow in either suspension or adherent conditions. However, in some cases, a mixed population of suspension and adherent cells may be observed.

Using a T25 culture flask as an example. First, check the color of the culture medium and inspect for any leakage. Then, observe the cell condition under an inverted microscope and capture images at different magnifications to rule out contamination or poor cell condition. After confirming the cells are in good condition, disinfect the outer surface of the cell flask and place it in the incubator for a few hours (depending on cell density) to stabilize the cell state.

Adherent cells: When the cell growth density exceeds 80%, passaging can be performed as needed. If the confluence is less than 80%, perform a partial medium change by removing half of the original medium and adding an equal amount of fresh complete medium. Continue culturing until the cell density exceeds 80%, then proceed with passaging.

Suspension cells: Transfer the liquid from the culture flask to a centrifuge tube and centrifuge at 500g for 5 minutes. Gently discard the supernatant. Resuspend the cell pellet at the bottom of the tube in 10 mL of complete medium by gentle pipetting. Transfer the suspension to a new culture flask and incubate overnight. Perform subculturing based on cell density and growth status.

Cell counting: Dilute the cell suspension to 200-2000 cells per milliliter in a serum-free medium. Add an equal volume of 0.4% trypan blue solution to 10-100 µl of the cell suspension. Mix gently and count the cells using a hemocytometer. Living cells can reject trypan blue and remain translucent, while dead cells will be stained blue. It is also more convenient to directly use a cell counter for counting.

Selecting the appropriate culture medium is crucial for the success of cell culture. Different types of cells have varying growth requirements and specific culture conditions. First, the nutrients in the culture medium are essential for maintaining cell growth and metabolism. Different types of cells require various amino acids, sugars, vitamins, inorganic salts, lipids, and other nutrients to meet their physiological needs. If the culture medium lacks necessary nutrients, cells may be unable to grow and divide normally, and this can even lead to cell death^[3][4].

When selecting the appropriate culture medium, prioritize the culture conditions provided by the cell source company. If the source is uncertain, you can refer to the recommended medium for the corresponding cell type from ATCC.

Secondly, growth factors and hormones in the culture medium play an important role in regulating cell growth and differentiation. These growth factors can promote cell proliferation, development, and differentiation, and play a key role in cell signaling pathways. Different types of cells require specific growth factors and hormones to maintain their particular functions and characteristics. For example, neurotrophic factors are often added to the culture medium for neural cells to promote growth and synapse formation; fetal bovine serum is commonly added to the medium to provide the growth factors, proteins, and other essential components needed by the cells.

In addition, antibiotics can be added when necessary to prevent contamination and protect the cells. For example, double antibiotics (penicillin/streptomycin) or even triple antibiotics (penicillin, streptomycin, amphotericin B) can be used to inhibit the growth of microorganisms such as bacteria and fungi. Penicillin-streptomycin is effective in inhibiting the growth of most Gram-positive and Gram-negative bacteria, while amphotericin B can be used to prevent fungal and yeast contamination.

Over time, the number of cells will increase, but space and resources are limited. Therefore, when cells reach a certain density, it's time to provide them with a larger living space to continue proliferating.

Using a T25 culture flask as an example, the common steps for cell passaging are as follows:

(1)Remove the cells from the incubator and observe them under a microscope. If the confluence exceeds 80%, proceed with passaging.

(2)Preparation: Prewarm the culture medium and PBS in a 37°C water bath. Place the consumables needed for passaging (e.g., pipettes, pipette tips, T25 culture flasks) in a biosafety cabinet, sterilize them with UV light for 30 minutes, and ensure proper ventilation. Disinfect the trypsin, prewarmed medium, and PBS with 75% ethanol before placing them in the biosafety cabinet.

(3)Aspirate the old culture medium from the flask, rinse the cells with 5 mL of PBS, and then aspirate the PBS.

(4)Add 1 mL of trypsin, gently shake the flask to ensure the trypsin fully covers the cells, and incubate it in a 37°C incubator for 30 seconds to 2 minutes (the actual incubation time may vary depending on the cell line used).

(5)Observe under a microscope, and when ≥90% of the cells have detached, add a volume of complete medium containing serum equal to twice the volume of the trypsin to stop the digestion. Pipette the surface of the cell layer several times to disperse the cells into a single-cell suspension.

(6)Centrifuge at 500 g for 3~5 minutes, then resuspend the cell pellet in complete medium containing serum.

(7)Distribute the cell suspension into culture flasks according to the desired passaging ratio, add fresh complete medium, gently shake the flask to evenly distribute the cells, and label it accordingly.

(8)Observe the cell density and condition under a microscope, then return the cells to the incubator. When using trypsin for passaging animal cells, several factors need to be considered. Trypsin has proteolytic activity that can affect various physiological and metabolic functions of the cells.