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Primary Cell Culture: What, Why, and How?

Primary cells have become indispensable tools for in vitro research. They have provided valuable insights owing to their phenotypic and genotypic similarity to tissues. The primary cell culture has led to a deeper understanding of cells, disease pathology, genetic profile, cell signaling, and drug mechanisms. Therefore, these cells have found applications in pre-clinical studies for screening drugs for their efficacy, metabolism, absorption, toxicity, etc. The 3D culture of these cells have constructed tissue models that are improved representations of the in vivo environment. Initially, primary cell culture was a troublesome task, but at present, medium composition and protocols have been optimized. The commercial availability of these cells has facilitated primary cell research.

What Are Primary Cells?

Primary cells (Fig 1) are extracted directly from the disaggregation of the tissue. The following characteristics distinguish them from other cells:

Life span: The key characteristic of these cells is their limited life span; that is, they can proliferate up to a few cell divisions before undergoing senescence.

Anchorage-Dependent: Ignoring a few exceptions, all primary cells need to adhere to a substrate, named as anchorage dependence. Due to this requirement, they can only grow as a single layer, or say, a monolayer above a substrate. This feature has been particularly useful for 3D cell culture, where cells are directed by an extracellular matrix to migrate in different directions and form the initial structure of a tissue. These tissues mimic the structure and function of the original tissues, rendering them as better alternatives to cells.

Contact Inhibition: Primary cells tend to inhibit cell proliferation when they are in contact with other cells. It is a regulatory mechanism to prevent unwanted growth. The absence of this mechanism in cancer cells leads to unlimited growth.

Types of Primary cells | Kosheeka
Fig 1. Images of primary cells. a. Human primary fibroblast cells from skin (100X), b. Human umbilical vein endothelial cells (100X), and c. Human hepatocytes (200X) (Source: From left to right-https://doi.org/10.5812/jssc.69080, Angio-Proteome, 10.5487/TR.2015.31.2.137)

Why Primary Cells?

Cervical cancer cell extraction from a patient stunned the scientific community due to their unlimited life span. Thus, they were named as immortalized cell lines, and scientists have created numerous cell lines since then. The unlimited life span enables easy handling of cell lines. The immortalization modifies the basic characteristics of the cell line, rendering them different from the tissue they belong to. However, primary cells retain all the features of the tissue from which they originated. Studies have proved that the genome and proteome of these cells are more similar to the tissue than the immortalized cell lines. Therefore, these cells are better representatives of a tissue, and the research on these cells can be translated to tissue level. Thus, culturing them is an important endeavor in research.

When to Culture Primary Cells?

Gradually, cells in a culture deplete the nutrients of the media and accumulate waste products, including even dead cells. Cells also exhaust the area of the culture dish. Hence, they require transfer to a new culture vessel containing fresh media. This subsequent culturing process is termed as subculture or passaging. The passage number holds special importance for primary cells for it monitors their life span. Cells should be subcultured in their log phase of growth when they are going through proliferation. Log phase is determined by the cell confluence. Cell confluence is the percentage of the culture vessel area that cells cover. Log phase is said to be achieved when cells reach 80% confluence. But it is important to note that media depletion occurs before the cells are due for subculture. Hence, instead of subculture, replace the media and with fresh media regularly to promote cell growth.

Preparation before Subculture

Ensure necessary preparations prior to the subculture process. To maintain an aseptic environment, wash your hands properly before entering the cell culture lab. Incubate the required cell culture reagents at room temperature (Fig. 2). Sterilize the working surface inside the biosafety cabinet (BSC class I or II) or laminar hood by wiping with 70% ethanol followed by exposure to UV light for at least 15 minutes. Remember to minimize the exposure to harmful UV radiation. Wear gloves before commencing subculture and disinfect them with 70% ethanol. Before placing anything inside the biosafety cabinet (BSC class I or II), wipe it with disinfectant, such as bottles of media, buffer, and trypsin-EDTA solution; packs of culture vessels (plates or flasks) and centrifuge tubes; autoclaved tips; discard box, etc.

How to Subculture?

Place the cells in the biosafety cabinet (BSC class I or II) and remove the media gently (Fig. 2). Remove debris from the vessel by washing the cell layer with phosphate buffer saline at least twice. Proceed with detaching the cells from the culture vessel by addition of trypsin-EDTA in quantity sufficient to cover the cell layer. After incubation for a few minutes, inspect the cells under a microscope for detachment. Afterwards, deactivate trypsin by adding culture medium. Follow with transferring the cells to a tube and centrifuging them at the given speed and time. Dissolve the pellet in the medium after removal of the supernatant and measure the cell count on a hemocytometer. Pipette the required amount of cells into a new culture vessel containing fresh media. Observe the cells once more in the microscope and store them in the incubator.

Process of sub culturing primary cells | Kosheeka
Fig 2. Process of subculturing primary cells. (Source: Pelobiotech.com)

What to Keep in Mind?

One of the challenges of primary cell culture is contamination. The thumb rule of a cell culture lab is to reduce the sources of contamination as much as possible. Several practices such as washing hands before entering the culture lab, using separate footwear for the culture lab, avoiding talking and eating, etc., are followed to maintain an aseptic environment. Additionally, avoid opening cell culture vessels and reagents outside the sterile conditions of a biosafety cabinet or hood. To prevent any further risk of contamination, maintain clean surfaces of all equipment in the culture lab and perform fumigation on a regular basis. It is recommended to keep the working space in the cabinet or hood decluttered for adequate working space. During subculture, keep the bottles and vessels covered as much as possible. Avoid pipetting the cells harshly to prevent any cellular damage. These precautions will ensure a contamination-free primary cell culture. 

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Conclusion

Primary cells are the key to the pursuit of scientific discipline. But complications arise from their definite life span and limited cell count. They quickly overshadow these constraints due to their resemblance in terms of genome, behavior, and function to that of the tissue. Thus, safety and efficacy studies are preferable on these cells to estimate the tissue response before proceeding to clinical trials. Their culture process is a quite simple step-by-step protocol, with the risk of contamination being the usual obstacle. Following standard protocols and maintaining stringent aseptic conditions, our team at Kosheeka assures the quality of the primary cells. We deliver a wide range of primary cells derived from different tissues like liver, skin, blood vessels, etc. after performing rigorous quality checks so that your research can continue without any hindrance.

FAQs

What is primary cell culture?

Culture of primary cells in a lab is denoted as primary cell culture. Primary cells are obtained from the tissue directly and are accurate representatives of the tissue of origin. Therefore, these cells are employed in investigational studies.

What does passaging mean?

Sub culturing is also named as passaging. Primary cells have a limited life span. To monitor their life span, researchers count the number of times cells have been subculture or passaged. Scientists have observed that cells accumulate mutations the longer they stay in culture. Thus, primary cells with low passage numbers are preferred for research.

What is the purpose of Trypsin-EDTA solution?

Trypsin-EDTA solution is used to dissociate the adhered cells in the culture. Cells use surface proteins like adhesion molecules to attach to a substrate. Trypsin is a serine protease that breaks peptide bonds of a protein at the C-terminal of Lysine and Arginine amino acids. EDTA sequesters calcium ions that are required for adhesion. Together, trypsin and EDTA dislodge the cells from the plate.

Dr.Swati Chitrangi (PhD)

Dr. Swati Chitrangi, PhD, Head of Production at Advancells Group, will be leading the session. With over 15 years of experience in regenerative medicine, stem cell therapy, and organoid research, Dr. Swati has contributed significantly to the advancement of disease modelling and drug discovery using organoids. Her deep expertise in precision medicine and patient-specific organoid development will provide valuable insights into the transformative potential of these advanced models.
Dr. Swati holds a PhD in Bioengineering and an MBA in Strategic Management from the Indian Institute of Management (IIML-2025), providing her with a unique blend of scientific and business acumen. She has been involved in several pioneering research projects and has authored publications on patient-derived organoids for precision oncology, the derivation of human iPSC lines, and engineered 3D in vitro models for drug toxicity studies. Her work emphasizes the translation of cutting-edge stem cell technology into practical applications for patient care and drug development.

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