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FAQ: How to Enhance The Success of Your Cell Culture

Quality, consistency, and reproducibility are all hallmarks of successful cell culture that help researchers derive insights into cellular behavior that advance our understanding of biological development to enable the investigation of disease mechanisms, aid in the development of new drugs, and more.

In this December edition of the Lifeline Cell Technology (LCT) blog, we present the top five most frequently asked cell culture questions. Whether you’re a seasoned researcher or a newcomer to the world of cell biology, we want to help set you up for cell culture success to grow ‘happy cells’ for all your experimental needs.

Q1: What’s the difference between primary and secondary cell culture?

A primary cell culture refers to cells that have been isolated directly from tissues or organs and established in an in vitro culture environment. These cultures more accurately recapitulate the physiological responses observed in vivo. However, primary cell cultures have a finite lifespan and provide a limited number of cells for experimentation.

In contrast, secondary cell cultures, commonly referred to as cell lines, have been genetically transformed and can divide indefinitely. While secondary cultures are easier to maintain and are highly uniform, they are vulnerable to genetic drift, which can cause their responses to differ from the cells in the original host tissue. Researchers should carefully consider these distinctions when selecting the most suitable cell culture approach for their specific research objectives.

Characteristic Primary Cell Culture Secondary Cell Culture (cell line)
Source Derived directly from tissues or organs. Derived from primary cultures and are generally to generate established cell lines
Lifespan Limited lifespan due to finite replicative capacity. These cells have been immortalized, allowing for continuous replication over multiple passages.
Genetic Stability These cells maintain the genetic characteristics of the tissue/organ of origin. These cells may undergo genetic drifting, changes or mutations acquired during repeated passaging and culture maintenance.
Complexity Represents the heterogeneity and complexity of cells in their in vivo environment. Less complex and more uniform, as cells have been adapted to grow long-term in a laboratory setting.


Q2: What are the advantages/disadvantages of serum in cell culture media formulations?

Serum has a long history of use as a cell culture media additive, serving as a rich source of nutrients, growth factors, hormones, attachment factors, and other essential components that support cell growth and proliferation. Fetal bovine serum (FBS) is commonly used in cell culture media because it can support a wide variety of different cell types. While using serum can keep your cells healthy and happy, there are some significant disadvantages including:

Batch-to-Batch Variability: Serum is undefined, and its composition can vary considerably between batches, leading to inconsistencies in cell culture conditions and experimental results.

Risk of Contaminants: Serum contains undefined or undefined factors, such as viruses, prions, or mycoplasma, posing a risk of introducing contaminants to cell cultures or could trigger an immune response in cultured cells.

Ethical and Cost Considerations: Serum is an animal-derived product, a characteristic that raises ethical considerations. Additionally, FBS can be expensive, which can increase the overall expense of the cell culture media.

Advances in cell culture technology have led to the development of serum-free—and even chemically defined media—as alternatives to serum that allow for more precisely defined culture conditions, including many of the cell culture media products available at LCT. Researchers can have better control over the composition of the media, creating a highly controlled environment for experimental reproducibility and consistency using their cell type of interest.

For researchers interested in transitioning away from serum-containing culture media to LCT serum-free formulations, it is not necessary to wean cells even if they have been cultured in media containing 20% serum, because LCT media formulations are optimized to support the growth requirements of each specific cell type.

Q3: How can specialized culture media formulations enhance the viability and functionality of primary cells in my research?

Specialized culture media formulations, such as those offered by LCT, can significantly enhance the viability and functionality of primary cells in research. Each formulation is designed and optimized to provide an environment that closely resembles in vivo conditions for the cell type of interest. The nutrient composition, and precise levels of growth factors and cytokines are tailored to the cell-specific nutritional requirements to maintain cellular phenotype, viability, and functionality, which enhances the relevance and translatability of cell culture research. Additionally, LCT media products do not contain antimicrobials and are phenol red-free to minimize confounding, off-target effects to ensure reproducible and consistent experimental results.

Q4: How do I establish a culture from cryopreserved cells?

NOTE: This protocol provided is for use with Lifeline Cell Technology cells.

Storage of Cryopreserved Cells:

  • Primary cells should be stored in the liquid phase of liquid nitrogen (-196 ⁰C).
  • Cells may degrade if stored at -80°C for more than 24 hours, with a significant increase in cell death after 72 hours.

Preparation Before Thawing:

  • Calculate the seeding density based on the information provided in the Certificate of Analysis.
  • Determine the seeding rate at 3,000 to 5,000 viable cells/cm2.

Media Preparation:

  • Aliquot the required volume of complete medium into the final tissue culture flask.
    • Use 1 mL of medium per 5 cm2 of the flask’s surface area.
  • Pre-warm the complete medium to room temperature.

Thawing Process:

  • Check the integrity of the cryovial seal before placing it in a water bath.
  • Place the closed vial containing cells in water bath for ~60 seconds and remove when there are still some small ice crystals remaining in the vial.
    • DO NOT submerge the vial more than half-way during thawing.
  • Dry the vial and then douse in 70% Ethanol (EtOH) before placing in BSC.

Cell Resuspension:

  • Gently resuspend cells in the cryovial tube.

Culturing Process:

  • Based on the calculations, aliquot an appropriate volume of the cell suspension into the culture flask containing pre-warmed complete medium. Avoid letting the media touch the mouth of the culture flask.
  • Gently rock the flask to ensure even distribution of cells. It is not recommended to spin the cells before adding them to the culture flask.
  • Place cell culture flask in the 37⁰C, 5% CO2 incubator.

Post-Thawing Steps:

  • It is recommended to change the media within 24 hours to remove the residual dimethyl sulfoxide (DMSO).
    • Observe cell attachment before changing the media.

Q5: When subculturing primary cells, what are the most important considerations to keep in mind?

Subculturing, also known as passaging, is the process of transferring a small portion of cells from an existing culture to a new culture vessel with fresh growth medium to maintain and propagate the cell population, allowing for continuous cell growth and expansion. For primary cells, this process is known as population doubling. It’s a routine part of most research labs but there are some key considerations that can set you up for success.

Timing is Key:

Cells should be checked microscopically daily to monitor health, grow rates and confluency (% surface area covered with cell monolayer). The optimal phase for passaging cells is typically when they reach the logarithmic or exponential growth phase.

This phase is characterized by rapid cell division when the cells are actively proliferating. In adherent cell cultures, this often corresponds to a confluence level of about 80–90%, while in suspension cultures, it’s when cells are actively dividing but have not yet reached high density or confluence. Cells in this phase are more likely to recover well after subculturing, helping to ensure a healthy, viable cell population with the desired biological characteristics. Passaging cells too late can lead to overcrowding, apoptosis and senescence.


The temperature of reagents such as trypsin and fresh growth medium should closely match the incubator temperature where the cells are cultured. Avoiding fluctuations in temperature helps to minimize cellular shock or stress during the subculturing process, ensuring higher cell viability. Additionally, enzymes like trypsin, commonly used for cell detachment, have optimal activity at physiological temperatures. Maintaining consistent temperatures throughout passaging helps creates a stable environment that minimizes cell culture variability.

Quality Control Checks:

Routine testing for sterility helps ensures the absence of bacterial or fungal contaminants. In particular, the presence of mycoplasma can alter cell behavior and metabolism and have adverse effects on cells, compromising experimental results.

As part of quality control checks in cell culture, maintaining good aseptic technique is paramount. This includes practicing proper sterile procedures during all stages of cell handling, such as subculturing and media changes, to prevent contamination.

At Lifeline Cell Technology, we offer a diverse and specialized portfolio of complete cell culture media kits. These come complete with basal media, supplements, and growth factors tailored to support the growth and maintenance of a wide range of human primary cell types, including endothelial cells, epithelial cells, and stem cells.

Whether you’re interested into vascular biology, epithelial studies, hematopoietic or stem cell research, our cell culture media kits provide the optimal environment for cultivating ‘happy cells’ to power your experiments with precision and reliability.

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