8.13: Chapter Summary
- Page ID
- 223056
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Choose feeding, dilution, passaging, or plating according to the culture’s condition and the goal. Adherent cells may need release from a surface before passage. Suspension cells can often be diluted, while full medium replacement requires a way to retain them.
Calculate transfers from measured viable concentration. A split ratio describes a fraction of the source, but final density also depends on culture volume or growth area. For multiwell plates, plan cells per well, final volumes, dispensing allowances, controls, and well identities before starting.
After transfer, check the cells rather than assuming that a correct calculation ensured recovery. A patchy NHDF layer may be unsuitable for a scratch assay. A THP-1 suspension needs appropriate viable concentration and condition. Keep passage history, actual handling details, and follow-up observations linked to each vessel.
Key Terms
|
Culture maintenance |
Medium change |
Passaging |
Plating |
|
Culture dilution |
Dissociation |
Trypsin |
EDTA |
|
Enzyme neutralization |
Split ratio |
Plate map |
Vehicle control |
|
Edge effect |
Conditioned medium |
Passage number |
Population doubling |
|
Senescence |
Review Questions
1. Explain why a medium change may be appropriate for one NHDF culture while another needs passaging, even if both have consumed nutrients.
2. Why should THP-1 medium not be discarded during routine maintenance without a plan to retain the cells?
3. Explain what trypsin and EDTA contribute to dissociation. Why must the stopping method match the reagent and medium?
4. A source suspension contains 1.2 × 10⁶ viable cells/mL. Calculate the source and fresh-medium volumes needed for 18.0 mL at 0.30 × 10⁶ viable cells/mL. Assume no other additions or cell losses.
5. In Question 4, the operator actually transfers 5.0 mL of source and adds 13.5 mL of medium. Calculate the resulting starting concentration and explain what must be recorded.
6. A recovered adherent-cell suspension contains 3.2 × 10⁶ viable cells in 8.0 mL. Calculate the viable cells and suspension volume in one quarter. Explain why its seeding density depends on the destination area.
7. An experiment uses 150 µL per well at 3.0 × 10⁵ viable cells/mL. Calculate viable cells per well and stock volume per well from a stock of 1.5 × 10⁶ viable cells/mL.
8. For 16 wells at 150 µL each, calculate the exact total volume and the volume to prepare with a stated 10% dispensing allowance. Explain why the allowance does not change the volume placed in each well.
9. A student dispenses a THP-1 suspension without remixing it. Why might the first and last wells receive different cell numbers despite equal volumes?
10. An NHDF plate reaches the scheduled scratch time but has patchy growth. What evidence should guide the decision to proceed, delay, or repeat?
11. A treatment well has more secreted protein per milliliter than its control but also contains more viable cells. Identify information needed before interpreting this as increased secretion per cell.
12. A passage 5 source is divided into four maintenance vessels. Explain how to label their passage and source relationships under a one-transfer-one-passage convention.
13. Describe two reasons a count taken the next day may differ from a calculated starting concentration. Which records help distinguish them?
14. Explain why twelve wells from one cell preparation do not represent twelve cell cultures obtained independently.
Apply Your Understanding
A student removes all spent liquid from a THP-1 culture as if it were an NHDF flask. Explain what happens to the cells and propose an appropriate way to renew the medium.
A stock contains 1.2 million viable cells/mL. Calculate the stock and medium volumes for 18.0 mL at 0.30 million viable cells/mL, assuming no other additions or losses.
Sixteen wells require 150 µL each. Calculate the preparation volume with a specified 10% dispensing allowance. Explain why each well still receives 150 µL.
An NHDF plate has patchy growth at the planned scratch time. Explain how this affects interpretation and identify the evidence needed to proceed, delay, or repeat.