BLS Cell culture Assignment

COLLEGE OF VETERINARY MEDICINE AND BIOMEDICAL SCIENCES

DEPARTMENT OF MICROBIOLOGY, PARASITOLOGY AND BIOTECHNOLOGY

BLS 201: TISSUE AND CELL CULTURE TECHNIQUES

BLS Y2

GROUP ASSIGNMENT – MEDIA AND CELL CULTURE 2024

Instructions

1. Each group must attempt all questions
2. Each group will be given one or two questions to present during class hours
3. The presenter will be selected randomly during the day of presentation
4. The presentation will be scored

 

Question 1: Cell culture media

Discuss the importance of cell culture media in supporting the growth and maintenance of different cell types. Explain the key components and considerations for designing and selecting appropriate cell culture media for specific cell lines or research objectives. Provide examples of how variations in media composition can impact cellular behavior and experimental outcomes.

Question 2: Cell culture techniques

Compare and contrast adherent cell culture methods with suspension cell culture techniques, and highlight the specialized equipment and vessels used for each. Discuss the advantages and challenges associated with these two approaches.

Question 3: Cell Count

HELA cells attached to a cell culture flask at 80% confluency were split using trypsin and versene in 50 ml cell culture medium. Trypsinized cells were centrifuged at 1500 rpm and the cell pellet was diluted in 10 ml of RPMI-1640 containing fetal bovine serum to obtain a stock cell suspension. 100 µl of the stock cell suspension was then added onto 1900 µl of RPMI-1640. From the cell-RPMI-1640 mixture, 300 µl were added onto 100 µl of trypan blue and cells were then added onto a haemocytometer. The picture below as seen under the light microscope.

• • a) Explain the function (s) of trypsin, versene, trypan blue and fetal bovine serum used during cell splitting and counting.
  • b) Calculate the total number of cells in the stock cell suspension
  • c) Calculate the volume (in µl) of the stock cell suspension required to make 20 ml of cell suspension at 5 x 10⁵ cells/ml

 

Question 4: Total Cell Count

A researcher performed a cell count using a hemocytometer after trypsinizing and resuspending a cell culture. The researcher diluted the cell suspension by a factor of 1:10 and loaded it into the hemocytometer chamber. The cell counts in four large squares of the hemocytometer were recorded as follows:

• Square 1: 100 cells
• Square 2: 105 cells
• Square 3: 95 cells
• Square 4: 110 cells

Given that the volume of each large square is 0.1 mm³, calculate the total number of cells per milliliter in the original, undiluted cell suspension.

Question 5: Cell Viability

During a cell culture experiment, a student-stained cells with Trypan Blue and counted the following:

• Total cells (both live and dead) counted: 200
• Non-stained (viable) cells: 160

Calculate the percentage of cell viability. If the initial cell suspension volume was 5mL and the concentration of viable cells was found to be 2×10⁶ cells/mL, calculate the total number of viable cells in the culture.

Question 6: Cell Seeding Density

You are planning to seed cells into a 96-well plate to perform a drug assay. You want to seed 10,000 cells/well in a final volume of 200 μL per well.

If the concentration of your cell suspension is 2.5×10⁶ cells/mL, calculate the volume of the cell suspension required to seed one well. Additionally, calculate the total volume of cell suspension needed to seed all 96 wells.

Question 7: Dilution Factor and Cell Count

A cell suspension was diluted 1:5 (i.e., 1 part cell suspension + 4 parts medium) before counting cells using a hemocytometer. The average cell count in the counting chamber (considering four large squares) was 80 cells.

Calculate the concentration of cells per milliliter in the original, undiluted sample.

 

Question 8: Splitting and passaging of cells

You have a cell culture with a concentration of 8×10⁵cells/mL in a 10mL volume. You plan to split this culture at a 1:4 ratio into a new flask.

Calculate:

• a) The number of cells you will transfer into the new flask.
• b) The volume of fresh medium needed to achieve the desired 1:4 split.

Question 9: Calculating Cell Concentration After Trypsinization

You trypsinize a confluent cell culture in a T-75 flask, resuspend the cells in a 10 mL of medium, and count them using a hemocytometer. You find an average of 50 cells in each of the four large squares (volume of each large square is 0.1 mm³).

Calculate:

• a) The concentration of cells per milliliter.
• b) The total number of cells harvested from the T-75 flask.

Question 10: Multiplicity of Infection (MOI)

A researcher wants to infect a culture of 2×10⁶ cells with a virus at a multiplicity of infection (MOI) of 0.5.

• a) How many viral particles are required to achieve this MOI?
• b) If the stock concentration of the virus is 1×108 virions/mL, what volume of the virus stock should be added to the cell culture?

Question 11: Adjusting Multiplicity of Infection (MOI) for Different Cell Numbers

You are working with two cell cultures:

• • Culture A has 5×10⁵ cells.
  • Culture B has 2×10⁶ cells.

You want to infect both cultures with a virus at an MOI of 2 using a viral stock concentration of 1×10⁹ virions/mL.

• a) Calculate the number of viral particles needed for each culture.
• b) Determine the volume of virus stock required to infect each culture at the desired MOI.
• c) Explain the differences between high MOI and low MOI infections in cell culture experiments. Provide examples of when each type of infection might be used in virological research.

Question 12: Determining Viral Titer After Infection

You have infected a culture of 1×10⁶ cells with a virus at a Multiplicity of infection (MOI) of 1. After a 24-hour incubation, you harvested the supernatant and performed a plaque assay. The assay showed 4×10⁷ plaque-forming units (PFU)/mL.

• a) What is the total viral yield in the supernatant if the harvested volume was 5 mL?
• b) Explain how an increase in MOI would affect the viral yield, assuming the cells are not limited by factors such as nutrient availability or cell viability.

Question 13: Calculating Multiplicity of Infection (MOI) from Viral Stock and Cell Concentration

A lab technician has a viral stock with a concentration of 5×10⁷ PFU/mL and a cell culture containing 4×10⁶ cells. The technician adds 0.2 mL of the viral stock to the culture.

• a) What is the MOI used for this infection?
• b) If the technician wanted an MOI of 5, what volume of the viral stock should have been added?

Question 14: Virus Dilution and Infection

You are planning to infect 1×10⁷ cells with a virus at an MOI of 0.1. The virus stock has a concentration of 2×10⁹ virions/mL.

• a) Calculate the number of viral particles required.
• b) If the virus needs to be diluted 1:10 before use, what volume of the original stock should be taken and how much diluent should be added to prepare enough virus for the infection?

Question 15: Determining Cell Concentration for a Given MOI

A virologist wants to achieve an infection at an MOI of 0.1 using a viral stock with a concentration of 3×10⁸virions/mL. The virologist plans to use 0.5 mL of the viral stock for the infection.

• a) How many cells should the virologist have in the culture to achieve the desired MOI?
• b) If the virologist only has 2×10⁶ cells available, what adjustment should be made to the volume of virus stock used?

Question 16: Viral Concentration from a Plaque Assay

After performing a virus infection in a cell culture, you collect the supernatant and perform a plaque assay on it. You plate 0.1 mL of a 10⁻⁴ dilution of the supernatant and observe 50 plaques.

• a) Calculate the concentration of the virus in the original supernatant in PFU/mL.
• b) If you want to infect a culture at an MOI of 1 using this virus, how many cells can be infected with 1 mL of the supernatant?

Question 17: Virus Release Kinetics

You are conducting an experiment to study the release kinetics of a virus. You infect 1×10⁶ cells at an MOI of 0.1 and collect supernatants at 6-, 12-, 24-, and 48-hours post-infection. The viral concentrations measured (in PFU/mL) are as follows:

• • hours: 1×10⁶
  • hours: 5×10⁶
  • hours: 2×10⁷
  • hours: 1×10⁸
• a) Plot a graph of viral concentration vs. time.
• b) Describe the trend observed and discuss possible factors influencing the release kinetics of the virus.

Question 18: Design of Cell Culture Laboratory

How does the design of a cell culture laboratory impact the efficiency, reproducibility, and overall success of cellular experiments? Discuss key considerations in laboratory layout, equipment selection, and workflow optimization, highlighting the importance of creating an environment that fosters cell viability, experimental consistency, and researcher safety.

Question 19: Emerging Cell Culture Technologies

Explore the emerging technologies and advancements in cell and tissue culture, such as 3D cell culture, organoids, and organ-on-a-chip microfluidic systems. How are these technologies changing the landscape of cell and tissue culture research?

Question 20: Quality Control in Cell Culture

Explain the importance of quality control and characterization in cell culture. What are the key parameters that should be monitored to ensure the reliability and reproducibility of experimental results?

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