How to Reform Poor Cell Conditions: Causes and Solutions

News 17 7 月, 2024

In the world of cell culture, researchers often encounter poor cell conditions. Common signs include unclear cell edges, deflated cells, poor refractivity, dirty background, many black dots, numerous cell debris (apoptotic cell fragments), vacuoles in individual cells (apoptosis) with many vacuoles, and aging cells that appear flattened, with slow proliferation, enlarged nuclei, and increased cell processes. These phenomena collectively refer to poor cell conditions. The root causes of deteriorating cell conditions typically include:

– Inadequate or unsuitable cell nutrition

– Cell contamination

– Cell aging

– Cell recovery issues

Analyzing the Causes of Poor Cell Conditions

Let’s delve into the underlying reasons affecting cell conditions.

1. Inadequate or Unsuitable Cell Nutrition

When cells lack proper nutrition, it is necessary to consider the culture medium.

– Fetal Bovine Serum (FBS): FBS, derived from animals, contains essential nutrients (plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances, etc.) needed for cell culture. When switching serum, cells need an adaptation period. Choose the appropriate serum based on your cell type.

– Basic Culture Medium: Besides serum, cells need a basic culture medium to form a complete culture medium. The basic medium, with defined components such as amino acids, inorganic salts, vitamins, carbon-containing substances, proteins, and other nutrients, supplements what is missing in serum. Different cell lines require different nutrient content, so choose the medium (e.g., DMEM, RPMI-1640, M-199, MEM) accordingly. When preparing your own basic medium, ensure the addition of necessary nutrients (e.g., sodium bicarbonate) and adjust the pH. Inappropriate media can alter cell characteristics. The pH indicator phenol red in the medium has weak estrogen-like effects, stimulating cells with estrogen receptors.

Recommendations: Use the most suitable culture medium. If conditions do not allow, gradually increase the ratio of new medium during medium change (1/4, 1/2, 3/4, then full) and passage several times to help cells adapt. Maintain appropriate conditions in the cell culture incubator (optimal temperature: 37°C, CO2 concentration: 5%, pH: 7.2-7.4, osmotic pressure, etc.).

2. Cell Contamination

Contamination usually comes from the following sources:

– Fungal Contamination: Resembling cotton-like floating substances, fungal contamination can be identified under a microscope by observing mycelia. Early contamination does not yellow the medium.

– Yeast Contamination: Yeast appears as oval-shaped, beaded structures under a microscope, often forming connected structures during budding. Severe contamination can change the pH of the medium and is hard to eliminate.

– Bacterial Contamination: The medium turns yellow or white and turbid within a short period, cells stop growing or die, and some cells exhibit abnormal morphology (multinucleated or polygonal), detachment, and a background filled with black dots under a microscope.

– Mycoplasma Contamination: Mycoplasma, common in human skin, respiratory tract, reproductive tract, and digestive tract, as well as animal-derived serum and trypsin, can pass through standard 0.22μm filters. Mycoplasma contamination is difficult to detect early but can alter cell morphology, proliferation, gene expression, and experimental results.

– Black Particulate Contamination: Often referred to as unknown particulate phenomena or biological contamination, black particulate contamination shows various shapes (ovoid, spherical, rod-shaped) and movement under high magnification, potentially causing cell growth inhibition or apoptosis.

– Viral Contamination: Viral infections can come from host animal cells or patients and are cell-specific. Under a microscope, infected cells show more fragments, swelling, shrinkage, and detachment. Severe cases show local damage plaques in monolayer cells.

– Cell Cross-Contamination: Approximately 15-20% of cell lines in use are not what scientists believe them to be. Cross-contamination often occurs due to experimental errors (shared culture media, unchanging pipette tips, mislabeled cell lines), leading to inaccurate data collection. STR analysis, isoenzyme analysis, chromosome typing, and AFLP are common methods for cell line identity verification.

3. Cell Aging

Cell aging is common in both primary cells and cell lines, often overlooked by operators. Aging cells exhibit slower proliferation, abnormal nuclear size and multinucleation, increased surface secretions, enlarged cell size, flattened appearance, and vacuoles in the cytoplasm. Aging cells do not die immediately but show altered gene expression.

Solution: There is no reversible solution; replace with new cells promptly.

4. Cell Recovery Issues

Cell recovery mainly concerns freezing issues, as recovery problems are rare due to simple and rigid procedures. Potential issues include:

– Overly Long Digestion Time During Freezing: Long digestion times can lead to cells losing adhesion ability upon recovery, causing initial adhesion followed by death as the cells enter an irreversible apoptosis program.

– Quality of Cryoprotectant: Whether using glycerol or DMSO, quality is crucial to prevent cell death.

– Amount of Cryoprotectant: ATCC recommends no more than 7% DMSO, ideally above 5%. Excessive amounts are detrimental.

– Mixing DMSO Properly: Proper mixing of DMSO has some impact, though not substantial.

– Strict Temperature Gradient During Freezing: Adhering to proper temperature gradients is crucial during the freezing process.

– Contamination Detection: Contamination should be quickly observable; eliminating centrifugation may be beneficial.

– Cell Density Before Freezing: Ensure cells are not overly dense before freezing.

Conclusion

Understanding and addressing these factors can help improve cell conditions and ensure successful cell culture experiments. By analyzing and tackling these issues, researchers can maintain healthier cell cultures and achieve more reliable experimental results.