In today’s global setting, Mammalian cell culture is an indispensable boon in the field of life sciences. It acts as a foundation brick for a variety of disciplines such as regulation, propagation, and differentiation as well as to achieve genetic manipulation.
Many biotechnology goods including viral vaccines are essentially reliant on mass culturing of animal cell lines; with the ongoing Covid-19 pandemic, it would have been impossible to manufacture viral vaccines if not for animal cell lines. Thus, one has an idea about how vital it is to sustain the purity of cell cultures and avoid contamination. Thus, if you want your Mammalian cell culture to prosper, here are 5 tips on how to do just that!
- Cell Analysis and Quality Check
Quality is crucial in all aspects of animal cell culture. The quality of cells used is bound to affect the quality of the cultures and the consequent scientific data and products derived from them. Thus, before going forward, the cells must be analyzed and checked for contamination. Few short- sure ways include:
- Conduct Viability Analysis- Trypan blue exclusion assay or Propidium Iodide assay
- Checking for Microbial Contamination- Fungal and Bacterial contamination can be accessed via the naked eye or by doing a brief test under a phase-contrast light microscope. Other methods include- DAPI Staining(Mycoplasma) or Gram staining.
If stem cells are to be analyzed endotoxic testing and HLA Testing can be conducted. Also, CD Markers can be used for their characterization using flow cytometry.
- Cell Line Authentication and Preventing Mixed Population
Animal cell culture is a vital tool in numerous laboratories for biomedical and clinical research purposes. Cell line authentication is required because countless cell lines are mistaken or contaminated via other cells, leading to annulling of published data and wasting time and laboratory resources. When two or more flasks, with different cell types, are kept in the same hood, there is a high chance of cross-contamination that in turn can affect the respective cell line’s authenticity and also risk a mixed population scenario. To avoid this,
- Avoid keeping more than one cell type in the same hood.
- If it is necessary to keep more than one cell type, make separate racks for separate cell types so that, no mixing can occur.
- Cellular Morphology Is Key
Examining the morphology of the cells in the culture (i.e., their shape and appearance), on a regular basis is crucial for successful experimentations. Confirming the healthy status of the cells can aid in the detection of any signs of early contamination and control it before it spreads to other laboratory cultures.
- Evaluation of overall cell form, cytoplasmic construction, flatness, and outline characteristics on a good phase-contrast light microscope additionally facilitates acquaintance with the morphology of the cell line one is operating, even if a few mammalian cell strains have comparable morphologies, being acquainted with those morphologies is essential.
- Conducting Trypan blue and Propidium Iodide assay can help check if the cell is in its integral form or not.
- TEM(transmission electron microscope), SEM (scanning electron microscope ), and AFM(atomic force microscopy) may also be conducted.
- Rapid Freeze/Thaw
Every so often cells are not cultured instantly, thus it is essential to preserve and store them for future experimentations. Freezing is the best way to store cells. However, freezing affects various factors such as proliferation, cytokine secretion, protein production, and mRNA expression. Additional penalties of the freeze-thaw process are mechanical damages caused by crystal formation, alteration of physical characteristics, and shape of cellular assemblies. To avoid these certain measures can be followed, such as:
- Incorporation of cryoprotectant constituents in the freezing media can avert crystal development and osmotic injury. For example- dymethylsulphoxide (DMSO), glycerol, ethylene glycol, or hydroxyethyl.
- Caspase inhibitors can be added to the freezing media to avert apoptosis of the cells as a consequence of stress.
- Using special vessels that have alcohol (i.e Isopropanol) that surround the sample tubes can help maintain the temperature gradient and avoidance of rupturing of cells.
- Developing New Cell Lines
Developing cell lines is important for a variety of applications including biologics manufacture, drug screening, and gene functional studies. The main problem related to cell lines is contamination via additional cell lines and mycoplasma. To avoid any kind of contamination some basic steps can be followed, including:
- Immediately after the first sub-culturing or passage, some cell vials should be cryopreserved as a backup.
- Cells that are to be isolated for sub-culturing, must be isolated when the media reaches 80% confluency because if the plates reach maximum confluence, the cells will stop growing due to contact inhibition that in turn can affect the cell’s growth rate and morphology.
If your lab is presently involved in biomedical research with animal cell culture, acquire quality-characterized, tissue-specific primary cells and stem cells from KOSHEEKA. Connect with us at email@example.com
Verma, A., Verma, M., & Singh, A. (2020). Animal tissue culture principles and applications. Animal Biotechnology, 269–293.
Aslantürk, O.S. (2018). In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages, InTech.
MacDonald, C. (1990).Development of new cell lines for animal cell biotechnology, Critical Reviews in Biotechnology,10(2):155-78