Isolation, subculture, cryopreservation and characterization of cells MCQs With Answer

Introduction: This quiz collection focuses on Isolation, Subculture, Cryopreservation, and Characterization of cells — core practical competencies for M.Pharm students specializing in cellular and molecular pharmacology. The questions probe critical steps of primary cell isolation, establishment and maintenance of cultures, aseptic passaging strategies, and principles of cryogenic storage. Emphasis is placed on factors influencing cell viability, contamination control, authentication and phenotypic/genotypic characterization methods used in research and biopharmaceutical development. Each MCQ is designed to test conceptual understanding and procedural reasoning, preparing you for laboratory decision-making, troubleshooting and ensuring reproducible cell-based studies.

Q1. Which method is most appropriate for initial isolation of adherent primary cells from soft tissue biopsy when preserving cell-surface proteins is critical?

• Mechanical mincing followed by overnight culture
• Enzymatic digestion with trypsin-EDTA for prolonged periods
• Gentle enzymatic digestion using collagenase and dispase
• Direct explant culture without enzymes

Correct Answer: Gentle enzymatic digestion using collagenase and dispase

Q2. What is the primary reason for using a 1:3 or 1:4 split ratio during subculture of rapidly growing mammalian cell lines?

• To reduce nutrient consumption and slow growth deliberately
• To maintain cells below confluence and preserve exponential growth phase
• To increase differentiation by forcing cell–cell contact
• To dilute accumulated antibiotics and selection agents

Correct Answer: To maintain cells below confluence and preserve exponential growth phase

Q3. Which component is most commonly used as a cryoprotectant to prevent intracellular ice formation during freezing of mammalian cells?

• Glyceraldehyde
• 10% Dimethyl sulfoxide (DMSO)
• Polyethylene glycol 4000
• Sodium chloride solution

Correct Answer: 10% Dimethyl sulfoxide (DMSO)

Q4. What is the recommended controlled-rate freezing step for most mammalian cells prior to liquid nitrogen storage?

• Rapid cooling at −10°C per minute until −80°C
• Controlled cooling at approximately −1°C per minute to −80°C
• Slow cooling at +1°C per minute to 0°C, then plunge to −196°C
• No controlled rate; direct transfer to liquid nitrogen is preferred

Correct Answer: Controlled cooling at approximately −1°C per minute to −80°C

Q5. Which of the following best describes the purpose of using serum in cryopreservation media?

• Serum supplies cryoprotectant activity equivalent to DMSO
• Serum provides proteins that reduce osmotic shock and act as extracellular cryoprotectants
• Serum accelerates ice nucleation to protect cells
• Serum eliminates the need for controlled-rate freezing

Correct Answer: Serum provides proteins that reduce osmotic shock and act as extracellular cryoprotectants

Q6. After thawing frozen cells, what immediate step minimizes DMSO toxicity and improves post-thaw viability?

• Incubate cells at room temperature with high antibiotic concentration
• Rapidly dilute and remove DMSO by adding warm culture medium and centrifugation
• Plate cells directly without changing medium to retain osmolarity
• Expose cells to a 4°C cold shock to slow metabolism

Correct Answer: Rapidly dilute and remove DMSO by adding warm culture medium and centrifugation

Q7. Which assay is most appropriate for quantitatively measuring viability immediately after thawing?

• Crystal violet staining
• Trypan blue exclusion with automated counting
• Alkaline phosphatase staining
• Phase-contrast microscopy observation only

Correct Answer: Trypan blue exclusion with automated counting

Q8. Which characterization method is best for confirming the presence of a specific cell-surface marker on cultured cells?

• Mycoplasma culture
• Flow cytometry using fluorescently labeled antibodies
• Gram staining
• Electron microscopy of whole cells

Correct Answer: Flow cytometry using fluorescently labeled antibodies

Q9. During subculture, what is the primary function of EDTA when used in combination with trypsin?

• EDTA inhibits trypsin activity to protect cells
• EDTA chelates calcium and magnesium to disrupt cell–cell and cell–matrix adhesion
• EDTA acts as an energy source for cells during detachment
• EDTA sterilizes the culture by acting as an antimicrobial

Correct Answer: EDTA chelates calcium and magnesium to disrupt cell–cell and cell–matrix adhesion

Q10. What is the most reliable molecular approach for authenticating a human cell line to rule out cross-contamination?

• PCR for common bacterial contamination
• Short tandem repeat (STR) profiling
• Observation of morphology under light microscope
• Growth rate comparison only

Correct Answer: Short tandem repeat (STR) profiling

Q11. Which marker assay is commonly used to detect cellular senescence in cultured cells?

• SA-β-galactosidase staining at pH 6.0
• MTT metabolic assay
• Propidium iodide staining for cell cycle
• Bromodeoxyuridine incorporation for proliferation

Correct Answer: SA-β-galactosidase staining at pH 6.0

Q12. Why is it recommended to minimize the number of passages for primary cells used in experiments?

• Higher passages always increase proliferation indefinitely
• Genetic drift and phenotypic changes accumulate with successive passages
• Passaging increases contamination risk but does not affect cell phenotype
• Primary cells require high passage to adapt to culture conditions

Correct Answer: Genetic drift and phenotypic changes accumulate with successive passages

Q13. Which technique would you choose to evaluate chromosomal stability in a cultured cell line prior to regulatory studies?

• Flow cytometric immunophenotyping
• Karyotyping or G-banding analysis
• Mycoplasma PCR assay
• Live/dead staining

Correct Answer: Karyotyping or G-banding analysis

Q14. For long-term storage of cells, what is the typical storage temperature of liquid nitrogen vapor phase to ensure viability and safety?

• −80°C in mechanical freezers
• Approximately −150°C to −190°C in vapor phase
• Room temperature in desiccators
• −20°C in standard freezers

Correct Answer: Approximately −150°C to −190°C in vapor phase

Q15. Which step is most important to reduce the risk of mycoplasma contamination in cell culture?

• Regular use of broad-spectrum antibiotics in all cultures
• Routine screening by PCR and strict aseptic technique
• Daily medium changes with unfiltered reagents
• Incubation at lower temperature to inhibit microbes

Correct Answer: Routine screening by PCR and strict aseptic technique

Q16. When establishing a primary cell culture from tissue explants, what is the role of a feeder layer?

• To provide enzymatic digestion of the tissue
• To supply growth factors and extracellular matrix support for fragile primary cells
• To increase shear stress for cell differentiation
• To act as a physical barrier preventing contamination

Correct Answer: To supply growth factors and extracellular matrix support for fragile primary cells

Q17. Which parameter calculated from growth curve data best reflects the intrinsic proliferation rate of a cell line?

• Confluence at a single time point
• Doubling time in the exponential growth phase
• Total culture volume
• Number of passages performed

Correct Answer: Doubling time in the exponential growth phase

Q18. Which microscopic feature indicates healthy adherent epithelial cells in culture?

• Rounded, refractile cells floating in medium
• Flat, tightly packed cobblestone morphology with clear cell borders
• Large vacuolated cells and detachment
• Granular debris covering the monolayer

Correct Answer: Flat, tightly packed cobblestone morphology with clear cell borders

Q19. For phenotypic characterization of a differentiated cell type, which combined approach gives both spatial and quantitative information?

• Brightfield microscopy alone
• Immunocytochemistry for marker localization plus flow cytometry for quantification
• Mycoplasma culture and Gram stain
• pH measurement of culture medium

Correct Answer: Immunocytochemistry for marker localization plus flow cytometry for quantification

Q20. Which practice is considered best for maintaining reproducibility and traceability of cell-based experiments in a lab?

• Maintaining detailed cell bank records with passage numbers, freeze/thaw logs, and authentication data
• Relying solely on morphology to identify cell lines
• Using antibiotics to mask contamination without documentation
• Pooling cells from multiple donors without records

Correct Answer: Maintaining detailed cell bank records with passage numbers, freeze/thaw logs, and authentication data

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