Is Slow Cell Growth an Abnormal Condition? How to Adjust it Properly?

In cell culture, many researchers may wonder: why are my cells growing so slowly? Is it contamination? Are the culture conditions unsuitable? Or have I made an error in the process? In reality, recognizing and adjusting the conditions affecting cell growth is a critical aspect of successful cell culture.

In this article, we explore five common causes of slow cell growth and provide practical recommendations to restore cells to their optimal growth state. These guidelines aim to support your experiments and help you confidently manage and adjust cell culture conditions.

1. Intrinsic Characteristics of Cells

Some cell types naturally grow at slower rates due to their inherent biological properties, such as Caco-2 cells (human colorectal adenocarcinoma cells) and LNCaP clone FGC cells (human prostate cancer cells). These cell lines have prolonged passage cycles, making the culture process more complex and time-consuming.

Take LNCaP clone FGC cells as an example: these cells not only grow slowly but also cause the culture medium to acidify quickly. Additionally, they do not form a uniform monolayer but tend to grow as colonies. Therefore, it is essential not to disturb the culture within the first 48 hours after passaging. During medium changes, handle the cells carefully to avoid disturbing the colonies, ensuring stable colony formation and normal cell growth.

In summary, when working with slow-growing cells, it is crucial to closely monitor their growth state and adjust culture conditions as needed to provide an optimal environment for their growth.

2. Impact of Cell Growth Density

Cell density is a key factor influencing cell growth. When density is high, cells compete for limited nutrients and space, leading to slower growth. When density is low, cell signaling, metabolic activity, and colony effects are diminished, negatively affecting growth.

For example, HL-60 cells (human promyelocytic leukemia cells) grow better at high densities. If HL-60 cells show slow growth and poor condition at low densities, you can try vertically placing the culture flask to increase local cell density. As the density increases, cell growth typically improves.

RAW264.7 cells (mouse monocyte/macrophage leukemia cells), on the other hand, are an exception. These cells grow rapidly but begin to differentiate if not passaged within three days, resulting in altered morphology, functionality, and reduced proliferation rates.

To ensure normal cell proliferation and maintain optimal growth conditions, regularly observe cell growth, perform timely medium changes or passaging, and adjust culture parameters as needed.

3.Selection and Adaptation of Culture Medium

The choice of culture medium is critical for cell growth.

For cell lines like C127 (mouse mammary tumor cells), 293T (human embryonic kidney cells), and COS-7 (African green monkey kidney cells transformed by SV40), DMEM high-glucose medium (4500 mg/L glucose) is generally recommended. Low-glucose medium can limit cell growth and even cause cell death.

For specific cell types such as mesenchymal stem cells, general complete media may lack essential growth factors (e.g., TGF-β, FGF2), which can restrict proliferation[1]. For these cells, it is advisable to use medium specifically formulated to meet their growth needs.

Serum, as a vital source of nutrients, must also be carefully selected to ensure culture stability and reliability. Additionally, regular medium changes (every 2-3 days) help maintain adequate nutrients and prevent the accumulation of metabolic waste, thereby protecting cells from damage.

The pH of the culture medium is another critical factor influencing cell growth. Most cells thrive at a pH range of 7.2–7.4. However, sensitivity to pH varies. Primary cells are more sensitive to pH fluctuations. Established cell lines are generally more adaptable. To monitor pH changes, phenol red is commonly added to the culture medium as a pH indicator. If the medium turns yellow, indicating acidification, a medium change should be performed to ensure normal cell growth.

It is important to note that once a culture medium is opened, it should be used promptly to avoid degradation or contamination. If storage is required, aliquot the medium to prevent repeated warming. If you notice significant color changes compared to unopened medium, check for microbial contamination. If contamination is ruled out, the change may be due to pH buffer system shifts, which can be rebalanced by placing the medium in the incubator for a short period before use.

To ensure proper cell growth, choose a culture medium appropriate for the cell type and follow the cell culture instructions closely.

4. Influence of Experimental Procedures

Several factors during experimental procedures can affect cell growth. Key considerations include:

Enzymatic Digestion:

The digestion time for adherent cells must be carefully controlled. Over-digestion damages cells, compromising their viability. Insufficient digestion leads to incomplete separation, resulting in aggregation and poor adhesion or proliferation.

Optimize digestion time by closely observing the cells under a microscope to minimize damage and ensure proper growth.

Cryopreservation and Thawing

Cells can sustain damage during freezing or thawing due to factors such as inappropriate cryopreservation solutions, insufficient cell density during freezing, improper storage or prolonged handling during thawing. Following standardized protocols can minimize cell damage. When freezing a new cell type, conduct a preliminary test to ensure cells can recover and grow normally after thawing.

Suspension Culture in Shakers

For suspension cultures, shaker amplitude and speed are critical. Adjust parameters based on the cell type to ensure cells remain in a healthy suspended state, allowing for normal growth.

Sterile Technique

Strict sterile techniques are essential to prevent microbial contamination. Use sterile reagents and culture media, and regularly disinfect the culture environment. If contamination occurs, immediately perform a thorough cleaning and decontamination.

5. Influence of Culture Environment

Environmental factors such as temperature, carbon dioxide levels, and oxygen availability play a critical role in cell growth. Most cells are cultured at 37°C, and deviations from this temperature can directly impact their growth and viability. Carbon dioxide concentration is essential for maintaining the pH balance of the culture medium; levels that are either too low or too high can disrupt this balance and hinder cell growth. Similarly, adequate dissolved oxygen in the medium is crucial for cell metabolism, as insufficient oxygen can significantly impair cell growth and survival. To ensure optimal conditions for cell culture, it is important to regularly monitor and maintain the incubator’s temperature and gas control systems.

To ensure optimal conditions for cell culture, it is important to regularly monitor and maintain the incubator’s temperature and gas control systems.

When cells exhibit slow growth, it is important to systematically investigate the underlying causes. Consider factors such as the intrinsic properties of the cells, cell density, culture medium, and procedural details. By analyzing these elements and making appropriate adjustments, you can restore cells to their optimal growth state and ensure reliable experimental results.

Reference：

[1] Chu DT, Phuong TNT, Tien NLB, et al. An Update on the Progress of Isolation, Culture, Storage, and Clinical Application of Human Bone Marrow Mesenchymal Stem/Stromal Cells. International Journal of Molecular Sciences. 2020 Jan 21;21(3):708. doi: 10.3390/ijms21030708. PMID: 31973182; PMCID: PMC7037097.