I. What is the Difference Between Primary Cells and Cell Lines?

Primary cells are derived directly from human or animal tissues and typically have a limited lifespan. Compared to cell lines, primary cells retain many characteristics and markers of the donor, making them suitable for establishing more accurate disease models.

Cell lines, on the other hand, have undergone prolonged passaging and are usually genetically transformed. A cell line can generally be passaged 30–80 times. Unlike primary cells, cell lines lose many of the authentic features of the original tissue from which they were isolated. However, due to their ease of handling and extensive published literature supporting their use, researchers often prefer cell lines for convenience.

II. How to Choose the Right Culture Medium?

MEM is generally preferred for adherent cell culture, while RPMI-1640 is commonly used for suspension culture. However, selecting the appropriate medium for primary culture is a critical consideration. In theory, the choice should be based on cell type, nutritional requirements, the physiological conditions of the cell's native microenvironment, and the availability of optimized formulations.In practice, primary culture often requires testing different types of media, and in some cases, even specific variations of a medium—such as DMEM supplemented with non-essential amino acids (NEAA) and glutamine, or DMEM without L-glutamine but containing NEAA. The optimal formulation depends entirely on the specific nutritional needs of the cell type and its original microenvironment. Sometimes, specialized media like HAM’s F-12 or additional growth factors (e.g., HGF for hepatocytes or retinoic acid [RA] for neuronal cells) may be necessary.

Downstream applications should also be considered. For instance, if a cell line is used for fluorescence assays, phenol red should be excluded from the medium due to its high autofluorescence, which can elevate background signals.

III. Why is Serum Required in Cell Culture?

Serum is commonly supplemented into culture media at concentrations ranging from 2% to 20%, serving as a source of comprehensive nutrients, hormones, growth factors, and attachment factors for cells. Additionally, serum acts as a buffer in the culture system, protecting cells from detrimental factors such as pH fluctuations, proteolytic activity, heavy metals, and endotoxins, which could otherwise inhibit growth or induce toxicity.

Despite its advantages, serum use presents certain challenges, including batch-to-batch variability, undefined composition, and potential interference with downstream purification processes. While serum-free culture systems are increasingly adopted in many applications, serum remains indispensable in certain cell culture contexts due to its irreplaceable benefits.

IV. Why is Bovine Serum Classified into Fetal Bovine, Newborn Calf, and Calf Serum? How to Choose?

Bovine serum is mostly a by-product of the livestock industry. The differences between fetal bovine serum (FBS), newborn calf serum, and calf serum stem from the varying collection times—or, in other words, the age of the cattle.

Fetal bovine serum is obtained via cardiac puncture from unborn calves (3-8 months of gestation), while newborn calf serum is collected through venous blood draw within 12-24 hours after birth. Calf serum, on the other hand, is acquired via arterial blood collection from calves aged 16-22 weeks. Although the age classification for newborn or young calves may vary, it is certain that the younger the cattle, the higher the proportion of growth-promoting factors, attachment factors, hormones, and other bioactive components in the serum—and the lower the antibody levels.

Due to its superior biochemical composition, FBS is considered the gold standard for serum and is consequently the most expensive. Serum selection is typically based on a combination of cell type and cultivation costs. For delicate and difficult-to-culture cells, fetal bovine serum is the only option, whereas easier-to-culture tumor cell lines and conventional cell lines may be suitable for newborn calf or calf serum.

V. Are Black Spots in Cell Culture Contamination? How to Prevent and Handle Them?

First, visually inspect whether the culture medium appears cloudy. If it is cloudy, contamination can generally be confirmed. If not, observe the black spots under a microscope to assess their size, shape, and movement patterns—check whether they are uniform, whether they are moving, whether they exhibit Brownian motion, or if they are moving rapidly in straight lines.If the black spots are irregular in size and display Brownian motion, they may be cell debris (indicating poor cell condition or over-digestion). Protein precipitates from repeatedly freeze-thawed serum or cellular metabolites can also form black spots. However, if the black spots are uniform in size and move rapidly, bacterial contamination is likely.

Preventive Measures:

1）Time cell passaging appropriately—perform it at the optimal stage rather than when cells are senescent.

2）Control digestion time to prevent excessive breakdown of cell fragments.

3）Minimize repeated freeze-thaw cycles of serum and other reagents, and adjust the medium’s pH to the optimal level.

4）Strictly maintain cleanliness of water and labware.

Handling Method：If the black spots are confirmed to be contaminants, promptly treat and discard the cells. If microscopic observation shows that the cells are in good growth condition with no changes compared to before the appearance of the black spots, no action is required. Alternatively, the following methods may be used for treatment.

1）Suspension Cells：Collect the cell supernatant by slow centrifugation (500-600 rpm/min, 5-6 min) and transfer the cells to a new culture flask.

2）Adherent Cells:Wash the cells 2-3 times with PBS. During washing, gently tap the culture flask to remove loosely attached debris and particles.Discard the PBS and add a low concentration of trypsin (e.g., 0.05%, for 1 minute) to digest and dislodge particles and debris between the cells.Remove the low-concentration trypsin, then digest the cells as usual.Centrifuge the collected cell suspension at a low speed (500-600 rpm/min, 5-6 min) and transfer the cells to a new culture flask.Increase the serum concentration appropriately during subsequent culturing.

VI. What are the symptoms of bacterial contamination? How should it be handled?

Bacterial contamination is the most common type of contamination in cell culture, primarily caused by improper handling or the use of incompletely sterilized consumables and reagents. In most cases, bacterial contamination is detected visually by observing turbidity in the culture medium. Aerobic bacteria multiply rapidly and can make the medium cloudy within 24 hours of infection, whereas anaerobic bacteria grow slowly under standard cell culture conditions, requiring a longer time before turbidity becomes noticeable.

The most common contaminants include Bacillus subtilis, Escherichia coli, Pseudomonas, and Staphylococcus albus. Under the microscope, these bacteria may appear as long rods, short rods, or granular particles. In cases of rod-shaped bacterial contamination, if the bacterial load is low, the medium may remain clear, but microscopic examination often reveals fast-moving rod-shaped bacteria along their axis. If the contamination is granular, the medium typically becomes cloudy or develops white, sand-like precipitates.

Contamination Handling：Unless the culture is extremely valuable, it is generally recommended to discard the contaminated cells and restart the culture from a frozen stock. If the cells remain viable, one may attempt rescue by washing them with PBS and then treating them with antibiotics. However, due to the widespread overuse of antibiotics in cell culture in China, adding penicillin-streptomycin (double-antibiotic) is usually ineffective. For rod-shaped bacterial contamination, treatment with 100 μg/ml gentamicin for one week can be effective. For granular bacterial contamination, 25 μg/ml ciprofloxacin for one week may yield relatively good results.

VII. How to Handle Fungal Contamination?

The most common fungal contaminants in cell culture include Aspergillus fumigatus, Aspergillus niger, Mucor, Candida albicans, and yeast. Mold contamination is often visible to the naked eye as hyphal growth, while yeast contamination can be identified microscopically by its distinct budding morphology.

Contamination Handling:Unless the culture is extremely valuable, it is recommended to discard the contaminated cells as soon as possible and thoroughly disinfect the incubator and related equipment. If only a single well or a few wells in a culture plate are contaminated, carefully aspirate the medium and add a high concentration of sodium hydroxide to seal the well, preventing the spread of contamination to neighboring wells or flasks.If attempting to salvage the cells, amphotericin B can be used for treatment. However, note that amphotericin B is highly toxic and should only be applied when the cells maintain at least 50% viability and show no significant decline in health.

KMD Biosciences has established a comprehensive mammalian expression system, including but not limited to FreeStyle 293-F, Expi 293-F, Expi-CHO-K1, and Expi CHO-S cell lines. Combined with KMD-designed high-expression vectors (featuring full-length CMV promoters and optimized secretion signal peptide sequences), we are capable of providing clients with high-yield recombinant protein expression and production in mammalian cells.

The mammalian protein expression system is ideal for scientists requiring recombinant expression of proteins with native conformations, such as those needing glycosylation or phosphorylation modifications. Leveraging high-density fermentation technology, KMD Biosciences' scientists can achieve recombinant antibody expression levels as high as 6.3 g/L, making this system particularly suitable for high-level production of recombinant monoclonal antibody drugs.

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