Mastering the Challenges of Primary Cell Culture: Unlocking True In Vivo Insights

Primary cells are terminally differentiated cells that are directly isolated from the tissue or organ of interest. Primary cells are a heterogeneous population of cells, representing in vivo tissue microenvironment. Primary cells provide more accurate insights of in vivo conditions as compared to immortalized cell lines. It’s a more valuable tool to study physiological and pathological mechanisms.  Primary cells closely mimic the in vivo physiological conditions of the cell. Primary cells maintain in vivo functions for a short duration. Many primary cells are difficult to isolate and culture in vitro, as they often fail to adhere and proliferate.

During primary cell culture, Fibroblast-like cells generally grow rapidly and dominate growing primary cell types. Cells isolated and cultured in vitro divide definitely and finally enter senescence. Limited lifespan is a major drawback of using primary cells. Viral transfection and the use of small molecules can prevent senescence and induce proliferation. For example, Human kidney-2 (HK-2) cells are the most commonly used immortalized normal adult kidney cell lines. The HK-2 cell line was established by immortalizing proximal tubule cells by transduction with human papillomavirus 16 (HPV-16) E6/E7 genes. HK-2 cells maintain characteristics of proximal tubule phenotype such as a parathyroid hormone-stimulated

1. Isolation and Initial Viability: Isolation of primary cells from source tissue and procurement of tissue from biopsy or cadaver is the first challenge working while working with primary cells. Isolation of cells needs advanced cell culture expertise to minimize damage and contamination. Different tissues require specific techniques for cell extraction, generally involving mechanical disaggregation or enzymatic digestion, achieving high cell viability after cell isolation is crucial. Primary cells are prone to get damaged and stressed during isolation, which leads to low yields and compromised cell health.
2. Heterogeneity and Purity: Primary cells are heterogeneous populations as they are directly isolated from tissue, so they contain all cell types from source tissue. Cell population varies depending on isolation and passaging methods. Sorting of cell of interest by fluorescence-activated (FACS) or magnetic-activated cell sorting (MACS) is often used but these methods are time-consuming and may compromise cell health. Heterogeneity in the cell population leads to inconsistent experimental outcomes. 
3. Culture Conditions: Establishing optimal culture conditions for primary cells is another significant challenge as they are prone to dedifferentiate. Unlike immortalized cell lines which proliferate in standard culture conditions, primary cells require complex and precisely tailored culturing methods. This includes the addition of specific growth supplements, signaling molecules, extracellular matrix etc. For example, Primary neural cells require specialized culture media with specific growth supplements like B27, N2, Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Glial-Derived Neurotrophic Factor (GDNF), signaling molecules like neurotrophins and extracellular matrices like poly-L-lysine, Laminin, etc, to support cell proliferation. 
4. Limited Lifespan and Proliferation Capacity: Primary cells have limited lifespan in vitro culture conditions, reflecting lifespan constraints of normal cells in the body. It restricts their usage for an extended period. Unlike immortalized cell lines which can be passaged indefinitely, primary cells can be passaged for only a few passages (~5 to 6) before undergoing senescence. This demands frequent isolation of cells from fresh tissues, which makes it a tedious and costly process, this also leads to inconsistent experimental outcomes. Primary cells are also sensitive to passaging, and excessive passaging causes phenotypic changes and loss of cellular functionality. 
5. Senescence and Genetic Stability: Primary cells frequently undergo senescence due to developmental signals, different kinds of stress (oxidative or genotoxic), nutrient deprivation, inflammation, various intrinsic or extrinsic stimuli, telomere shortening, mitochondrial dysfunction, changes in telomere structure, mitogenic signals, epigenetic changes etc. Senescence can be caused by suboptimal culture conditions and repeated passing. Senescence is characterized by permanent cessation of cell division, alterations in gene expression and functions. Many times, multiple genetic mutations are also observed in primary cells, which causes genetic instability as well. 
6. Cost and Resource Intensiveness: Culturing Primary cells are generally costly and labor-intensive as compared to immortalized cell lines. Primary cells require costly specialized media, growth factors, small molecules, and extracellular matrix components. Additionally, the need for frequent isolation of fresh cells and the complexity of maintaining optimal culture conditions is a cost and labor-intensive process. This also limits the scalability and accessibility of primary cells.
7. Contamination Risk: Primary cells are particularly more prone to contamination by microorganisms such as bacteria, fungi and mycoplasma, as tissue or organ samples are sourced directly from operation theatre. Tissue sample handling procedure decides the contamination risks. Even a minor aseptic handling compromises the whole cell culture process. This leads to loss of valuable samples and experimental setbacks. Strict aseptic techniques and regular monitoring are essential to mitigate this risk.
8. Cell Recovery: When we pass cells it gets separated from other cells and extracellular matrix (ECM), which adversely affects the primary cells, which leads to anoikis, a type of programmed cell death when cells detach from ECM. To avoid anoikis, sensitive cells are incubated at 40 C with shaking for an hour in a recovery media containing supplements like serum, sugar, antioxidants, anti-apoptotic agents (e.g. Pan-caspase inhibitors) etc. Primary cells that do not proliferate in vitro, for example hepatocytes, are generally cryopreserved in small aliquots immediately after thawing, leaving aside the required cell number. Non-proliferating primary cells are generally cryopreserved with extra caution, to avoid any cell loss during thawing. Cryopreservatives such as Dimethyl Sulfoxide (DMSO) or galactose are generally used during cryopreservation to prevent the formation of ice crystals. Controlled rate freezers (CFR) are generally preferred to freeze primary cells, which rapidly drop the required temperature in appropriate timing to minimize ice crystal formation. Most cryopreserved cells are recommended to ship and store in liquid nitrogen.

Conclusion: Primary cells are a valuable tool in biomedical research, offering more physiologically relevant models as compared to immortalized cell lines. At Kosheeka, a branch of Advancells Group, we have successfully overcome these challenges associated with primary cells to provide high-quality primary cells for research and therapeutic applications.  By leveraging our optimized isolation techniques, stringent aseptic techniques, and quality check protocols, we provide highly viable and pure populations of primary cells.

Our dedicated scientific team tailor-made culture conditions for different cell types, preventing cellular loss and maintaining cellular functionality. We mitigate the limited proliferation capacity of primary cells by using senescence inhibitors and regular isolation of cells from fresh tissues. By closely monitoring genetic stability and maintaining optimal culture conditions, we mitigate the risks of senescence and mutations. We follow robust quality checks for primary cells like flow cytometry analysis of specific markers, mycoplasma, bacterial/fungal testing etc. We follow rigorous aseptic techniques to mitigate contamination risks. Our expertise in cryopreservation and recovery techniques ensures cell viability and functionality for extended periods.

Following these techniques, Kosheeka provides high-quality primary cells that closely mimic in vivo conditions, facilitating ground-breaking research and advanced therapeutic applications. We are committed to delivering reliable primary cells for biomedical research.