Biology Assignment on Cell Culture & Cell Cytotoxicity

Question

Task: This is a lab report on biology assignmentcontaining 2 parts
1)introduction to cell culture
Objective:
a. observes pre cultured CHO cells using phase contrast microscope.
b.aseptically sub culture CHO cells in a laminar hood.
c. Calculate the number of cells/ml and viability of cell using hemocytometer. introduction should talk about principle of cell culture

Introduction Questions
Why is it necessary for a bio pharmaceutical scientist to have an understanding of cell culture techniques
Describe the potential use of cytotoxic assays in drug discovery, development and manufacturing industries.

Result section Questions:
What indication of a cell culture contaminants should you watch out for
Why are cells pre washed in PBS prior to trypsination
What could go wrong in trypsination step
Why is it important to triturate the cells after media re-suspension post trypsin

2) Cell cytotoxicity (alamar blue)
objective
A.observe pre cultured HepG2 cells using phase contrast microscope.
B.Aseptically passage hepg2 cells in a laminar hood
C. To calculate the number of cells/ml and viability of a cell using hemocytometer.
D.to create a cytotoxicity profile of dieldrin on hepG2 cells using alamar blue assay

Introduction should answer this question:
What is dieldrin
Origin, use and culture of HepG2 cells
MTT assay- potential application, disadvantages associated with use

Answer

Part: I
Introduction to cell culture
Principle of cell culturedefined in the biology assignment:
The principle of plant and animal tissue and cell culture illustrates thein vitromaintenance and propagation of cells and tissues of organs in an artificial environment that is appropriate for their (cells and tissues) growth. For in vitro maintenance and propagation of cells, the culture condition may or may not mimic the in vivo conditions. The cell culture conditions – which may or may not mimic in vivo condition - include pH, carbon di-oxide concentration, oxygen supply, osmoregulation, and definitely appropriate nutrient media.

The culture media play the most important part while carrying out cell and/or tissue culture. However, the nutrient media is not same for all kinds of tissue or cells – not only they differ in case of plant and animal tissues, but also in case of different animal or plant cells and tissues. In addition to this, cell culture principle includes the necessity of maintaining a sterile and aseptic environment and cell culture media where continuous supply of nutrient media becomes a mandate in order to ensure growth of cells and tissues in a sophisticated incubatory environment.

Introduction Questions:
Why is it necessary for a bio pharmaceutical scientist to have an understanding of cell culture techniques
There are several necessities for a bio pharmaceutical scientist to have an understanding of cell culture techniques. They are mentioned below:
1. Cell cultures are required for the application of scientific research and to investigate the application and effectiveness of drugs(Tekarslanahin et al., 2017).
2. Cytotoxicity study is an important part of biopharmaceutical science. Different animal cells are required to investigate the permeability and cytotoxicity of drug molecules. Since 3D cells often time mimic the in vivo condition, the cell and tissue culture becomes an essential tool to understand the drug delivery system.
3. Bio pharmaceutical deals with embryonic studies. Therefore, it is necessary for a bio pharmaceutical scientist to understand the side effects of various drugs that the expecting mother consumes while she is bearing a child in her womb(Tekarslanahin et al., 2017).
4. Cancer study is an important part of bio pharmaceutical science. Various cancer cell lines are required as a research model for cancer study and anti-cancer drug study. It is important to mention that during the time of cancer therapy, as well as during trial (done by pharmaceutical companies), anti-cancer drugs are tested through different cell lines. Therefore, it is necessary to know the basic principles of cell and tissue culture(Tekarslanahin et al., 2017).
5. There are several cancer cells line are employed during the application of anti-cancer drugs, they are as follows: HeLa (human cervix adenocarcinoma), Caco-2 (Human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), A549 (Human Lung Carcinoma), U87MG (human Glioblastoma- astrocytoma), HT-29 (human colon adenocarcinoma), HEP - G2 (human hepatocellular carcinoma), K- 562 (human chronic myeloid leukaemia), CHO (chinese hamster ovary cells), etc (Ferreira et al., 2013).
6. The knowledge of cell and tissue culture is important for in vivo toxicity testing.
7. In order to carry cell viability assays, the bio pharmaceutical scientists need to know cell and tissue culture in artificial environment that may or may not mimic the in vitro condition. It is necessary to note that cell viability assays are employed for in vitro drug application and formulation of studies that involves toxicology ("Cell Viability and Proliferation Assays", 2021).

The potential use of cytotoxic assays in drug discovery, development and manufacturing industries:
Cytotoxic assays in drug discovery and development ensures pharmaceutical safety. The preliminary tests of drug formulation and development should include a vast array of screening for toxicity of the applied ingredients. The canonical drug toxicity tests show significant chances for the pharmaceutical developers, because not only it reduces the chance of associated loss of human life, but as well it wanes the chance of great fiscal loss of investment. The potential uses of cytotoxic assays (Bácskay et al., 2018) in different domains are discussed below:
• Cell lines can be isolated primarily from the tissues or organs. The pharmaceutical scientists, as well as drug development and manufacturing companies can easily study their detailed structure, the pattern of protein expressions, cell metabolism, and genetic code(Bácskay et al., 2018).
• Cytotoxic assays are important in early drug development(Bácskay et al., 2018).
• Cytotoxic assays are great substitute of animal study. Animal experiments are cost effective, and laborious. At the same time, animal experiments need a huge space, continuous supply of food, and most importantly individuals who have animal handling experience(Bácskay et al., 2018).
• It helps to understand the biological activity of the cells chosen(Bácskay et al., 2018).

Result section Questions:
What indication of a cell culture contaminants should you watch out for Cell cultures are usually contaminated by bacteria, fungi, virus, and mycoplasma. However, they can also be contaminated by other cell lines, molecules, as well as unwanted endogenous agents.
Usually fungal and bacterial contamination occurs because of flawed techniques, and contaminated and impure source material. Usually, viral and mycoplasma contaminations are very difficult to detect. In addition to this, they are highly contagious. They usually viral and mycoplasmalcontamination cannot be detected by standard light microscope("Cell Culture Contamination Troubleshooting", 2021).
Detection of fungal and bacterial contamination:
• When the cell culture media, along with the cell cultures becomes contaminated by fungi and/or bacteria, they look turbid in naked eye. Usually, the turbidity increases with time, as the fungal cell starts to multiply in number and starts disseminating throughout the culture("Cell Culture Contamination Troubleshooting", 2021).

• At the same time, colour change might also be noticed alongside of increased turbidity once the fungi and/or bacteria contaminate the media("Cell Culture Contamination Troubleshooting", 2021).
• Standard light microscope also reveals the presence of fungi and bacteria in a tissue culture("Cell Culture Contamination Troubleshooting", 2021).

Detection of mycoplasma contamination:
Mycoplasma are belongs to a genus of bacteria. Since they do not contain cell wall, like other bacteria, they cannot be killed even if multiple antibiotics are applied in the cell culture media. Here are some signs of mycoplasma contamination:
• Unlike other genus of bacteria, mycoplasma cannot be detected by casual observation. They are difficult to detect under standard light microscopes because of their exclusive morphology and significantly small size ("Cell Culture Contamination Troubleshooting", 2021).
• Usually they do not change the turbidity of the cell culture or the nutrient media even after contaminating them. However, they do not obliterate mammalian cells even after infecting them ("Cell Culture Contamination Troubleshooting", 2021).
• They cause dramatic shift to the result of an experiment by changing the metabolism of animal cells, accentuating cell growth, intruding the nature of intra and extra-cellular cell attachment, and even by causing chromosomal aberration ("Cell Culture Contamination Troubleshooting", 2021).

Why are cells pre washed in PBS prior to trypsination
During animal cell culture, the animal cells are pre-washed in PBS in order to remove the serum of media from the cell surface. Removing of the serum from the cells, ensures the detachment of the animal cells from the petri plates. Trypsin plays an important role in detachments of cells from the petri plate. If the serum is not removed, it will inactivate the trypsin. This is why animal cells are pre-washed with PBS before trypsination step.

In addition, PBS is non-toxic to most cells and it maintains a neutral pH.
What could go wrong in trypsination step
A wrong thing about trypsinization is over-trypsinizing the cells which might occur during the primary cell passaging. One might use high concentration of trypsin or trypsin concentration which is higher than the recommended trypsin concentration. It might damage the entire animal cell.

The second thing that might go wrong is, if the cells are not being observed closely under the microscope during trypsinization. Also, after trypsinization, it is necessary to completely neutralize the cell, in order to avoid cell damage.

why is it important to triturate the cells after media re-suspension post trypsin.
After trypsinization, it is necessary to completely neutralize the cell, in order to avoid cell damage.

Part II
Cell Cytotoxicity (alamar blue)
What is Dieldrin
Dieldrin (C12H8Cl6O), an organo-chlroide, was usually used as insecticides back in 1948. It is chemically closely related to aldrin. Aldrin further reacts to form dieldrin. It is also a sterio-isomer of endrin. It was named after Diels-Alder reaction. It had been developed as a DDT alternative. Dieldrin is toxic to human, it inhibits GABA and exhibits cytotoxicity and neurotoxicity(Honeycutt & Shirley, 2014).

Origin, use and culture of HepG2 cells
HePG2 is originated from “human liver carcinoma cells”, obtained from a Caucasian male who was fifteen years old. That man had hepatocellular carcinoma which was in well-defined state.
HepG2 They are immortalized cell lines. They are commonly used in cancer research which are close adhered to hepatotoxicity and metabolism of drugs by the cancer cells.

Culture of HepG2 Cells:
Requirement:
1. Eagle’s Minimum Essential Medium (Aka EMEM), supplemented with FBS (10% Concentration)("HepG2 liver hepatocellular carcinoma cells transfection", 2021).
2. DMEM and RPMI1640 can also be used instead of EMEM.

Method of Culture:
1. It requires to add fresh culture medium every two to three days.
2. The doubling time of HepG2 takes around 48 hours.
3. To passage the cells, it is necessary to since the monolayer of HepG2 cells with 1X PBS for two times.
4. Now pre-warmed (37 degree Celsius) 0.05% Trypsin-EDTA Solution (Concentration 0.05%) should be added in order to cover the bottom of the glass conical flask.
5. It should be incubating for five to seven minutes.
6. Now the cells will start to detach. It is time to neutralize the cells with trypsin. Therefore, 4x volume of trypsine will be added alongside of 10% FBS. Now the cells have to be gently re-suspended in the liquid by pipetting.
7. Clumping of cells should be avoided, and therefore the flask must not be agitated while waiting for detachment,
8. The appropriate incubation temperature of this cell is 37 degree Celsius, in a humidified atmosphere where the carbon di-oxide concentration is 5% ("HepG2 liver hepatocellular carcinoma cells transfection", 2021).

MTT assay- potential application, disadvantages associated with use

MTT or 3 - (4 , 5 - dimethylthiazol- 2 - yl)-2 , 5- diphenyl - 2H - tetrazolium bromide is required to determine the cell viability, as well as cell proliferation. This assay determines the viability of cells in terms of activities that are reductive in nature. The reductive activities include enzymatic conversion as well. It is necessary for high through output cell remuneration technology (van Tonder et al., 2015).

Disadvantages of MTT assay:
• Linear range of the MTT or 3 - (4 , 5 - dimethylthiazol- 2 - yl)-2 , 5- diphenyl - 2H - tetrazolium bromide assay have shown highest variability. It suggests an accuracy which is however compromised.
• In addition it shows a vast diversification in the calculated concentration of IC50.
• MTT assay shows its best result only when all the test parameters are considered, however, which is hard to maintain at times (van Tonder et al., 2015).
• The pH of the solution used for solubilisation should be set to acidic range. It converts the red phenol into yellow which further avoids intrusion with recording absorbance.
• The volatile substances present in the MTT solution starts to evaporate, which is one of the primary concern of MTT assay("Is Your MTT Assay the Right Choice", 2021).
• It remains stable only for a few hours in the room temperature.
• The MTT assay lacks sensitivity. It has less sensitiviry as compared to luminescent and fluorescent methods of cell assay. Not only that the detection sensitivity of MTT assay varies a lot among cell to cel("Is Your MTT Assay the Right Choice", 2021)l.
• The variety compounds used in MTT assay often interferes during the experiment("Is Your MTT Assay the Right Choice", 2021).
• When MTT is exposed for a long time the reagent becomes alkaline and which further produce formazan. It in turn increases the background absorbance ("Is Your MTT Assay the Right Choice", 2021).

• MTT reagents displays effects that are cytotoxic in nature ("Is Your MTT Assay the Right Choice", 2021).
• The tetrazolium reduction affects the cell metabolism during MTT assay ("Is Your MTT Assay the Right Choice", 2021).

References
Bácskay, I., Nemes, D., Fenyvesi, F., Váradi, J., Vasvári, G., &Fehér, P. et al. (2018). Role of Cytotoxicity Experiments in Pharmaceutical Development. Cytotoxicity. https://doi.org/10.5772/intechopen.72539
Cell Culture Contamination Troubleshooting. Sigmaaldrich.com. (2021). Retrieved 20 December 2021, from https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-culture-troubleshooting-contamination.
Cell Viability and Proliferation Assays. Sigmaaldrich.com. (2021). Retrieved 20 December 2021, from https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/imaging-analysis-and-live-cell-imaging/cell-viability-and-proliferation.
Ferreira, D., Adega, F., &Chaves, R. (2013). Oncogenomics and Cancer Proteomics - Novel Approaches in Biomarkers Discovery and Therapeutic Targets in Cancer. https://doi.org/10.5772/1745

HepG2 liver hepatocellular carcinoma cells transfection. HepG2 (Liver Hepatocellular Carcinoma) Cell Line - Cell Culture and Transfection Protocol. (2021). Retrieved 20 December 2021, from https://www.hepg2.com/. Honeycutt, M., & Shirley, S. (2014). Dieldrin. Biology assignmentEncyclopedia Of Toxicology, 107-110. https://doi.org/10.1016/b978-0-12-386454-3.00132-9 Is Your MTT Assay the Right Choice. Promega.in. (2021). Retrieved 20 December 2021, from https://www.promega.in/resources/pubhub/is-your-mtt-assay-really-the-best-choice/.

Tekarslanahin, ., Mesut, B., &Özsoy, Y. (2017). Applications of cell culture studies in pharmaceutical technology. ACTA PharmaceuticaSciencia, 55(3), 63. https://doi.org/10.23893/1307-2080.aps.05519 van Tonder, A., Joubert, A., & Cromarty, A. (2015). Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Research Notes, 8(1). https://doi.org/10.1186/s13104-015-1000-8