**4.17 Challenges during passage of cell**

During cell culture, laboratory personnel and researcher should follow up the cell routinely. Splitting of the cell depends on cell doubling number, cell type, pH level, media, and so many cell culture-related issues. There is a lot of challenge situation faced by laboratory workers. The most common problems that cause major issues in the laboratory may also ruin the running works are misidentification of cell line, Contamination of culture and media, Rough handling, Poor cell growth, Poor cell attachment, Improper trypsinization during harvesting, Improper cell count, Improper split ratio during the passage, Clumping of cell, Cell death. Incubation time, temperature, and CO2 level also have a great impact on cell culture as well as on subculture.

## **4.18 Maintenance of cell**

The routine follow-up of cell morphology is necessary. To maintain a good cell line routinely change of the medium is essential for the both proliferating or nonproliferating cells. The culture medium should be changed repeatedly in the case of proliferating cells compared to the non-proliferating one. The rate of cell growth, cell morphology and metabolism of cell indicates the urgency and time interval of medium change. For example, HeLa cells are rapidly growing transformed cells, in the case of HeLa cell the culture medium should be changed twice within 7 days, whereas for slowly growing non-transformed cells (like IMR-90 cells) the culture medium may be changed once in a week. Continuous cell lines, Chicken embryo fibroblast cell (CEFC), Embryonic cells and transformed cells develop quickly that's why these cells need rapid sub-culture and altering the culture medium. While normal cells are grow slowly. Generally altering the medium depends on pH level. Immediately change whole medium when the pH level appeared 7.0, cells are stop proliferating at pH 6.5, and the cells may lose their durability and viability when the pH level drop in between 6.5 to 6.0. The drop rate of pH is commonly estimated for each and individual cell line with a selected culture medium. If the drop rate of pH is less than 0.1 units/day, that indicates no harm and no need to hurry to change the culture medium immediately. When the drop rate of pH is 0.4 units/day, that indicates the culture medium need to be changed immediately [13]. A laboratory person or a researcher can easily maintain cells by maintaining SOP of cell handling and culture procedure, by counting passaging time because 10–30% density of cell is standard but at 80–90% density cell should be split as well as cell count may be helpful in this regard. Must pay attention to media quality, color, clarity, foul smell results from infection of the cell. Cell health and cell concentration, appearance observed regular interval by bright field microscope with 20–60x magnification may help to maintain cells and eradicate clumping, detachment, apoptosis. Quality control (QC) documents, Certificate of Analysis (COA), Cell transportation SOP are crucial to maintaining cells. Logbook entry for all the daily activities like pH level daily basis, media condition, temperature, CO2 level, assigned peoples information, cell condition, passage number, all the information about the cell very much essential to maintain cell for either small or large scale work.

#### **4.19 Mycoplasma: a issue**

Mycoplasma contamination is a serious and widespread problem in cell culture. Mycoplasma is often passed from culture to culture and from lab to lab. Mycoplasma can ruin whole research if data collected from mycoplasma-infected cells or cultures. Among all the contaminants (biological) in the laboratory mycoplasma have the capability to spread rapidly and causes detrimental effect on cells because of their detection rate is very low as well as their serious impact on cell lines. In spite of the fact that mycoplasmas are actually microscopic organisms (like bacteria) but they have some particular characteristics that make them identical. Mycoplasmas can easily survive and multiply at high densities without producing any noticeable signs. They are very harmful to any cell culture. Mycoplasmas can easily alter the host cells' metabolism and morphology, cause chromosomal aberrations and damage of cell that provoke cytopathic effects. Mycoplasma ought to be tested at least once a month is recommended in laboratory and research work. Two different testing methods, such as DAPI staining and PCR are helpful but a commercially available mycoplasma kit is also recommended for the detection [37]. A routine screening process might be helpful to eradicate mycoplasma contamination from the lab. The following points are essential to prevent mycoplasma issues [38]. a). Wearing personal protective equipment (PPE) during cell culture that includes a dedicated, clean lab coat and gloves b). Checking the COA, QC pass of cells' origins c). Ensure proper sterilization d). Always clean the working area e). To avoid cross-contamination work with only one cell at a time f). Always ensure covering the media bottles and CCF. Do not use the hood for storage and work always within biosafety cabinet g). Should be cautious in the use of antibiotics because it is reported that antibiotics have no impact on mycoplasmas h). Maintain logs for record-keeping that help to identify possible contamination sources if needed i). SOP develop for routine mycoplasma screening.
