9.12: Chapter Summary
- Page ID
- 223069
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Cryopreservation preserves cells for later use. Ice formation, water movement, concentrated solutes, and cryoprotectant exposure can affect survival. Select a suitable source culture and follow its approved formulation, cooling method, and storage instructions. Plan viable cells per vial and fill volume together. Extra preparation volume needs enough cells to maintain the target concentration. An intermediate freezer step differs from long-term cryogenic storage. Cooling records, transfer times, and safe cryogenic handling matter throughout the process. Cell-bank labels and inventory must connect every vial to its source, status, and storage location. Post-thaw viability measures a fraction of recovered cells. Viable recovery compares recovered viable cells with the number loaded. Later attachment and growth provide further evidence about the usefulness of the bank.
Key Terms
|
Cryopreservation |
Cell bank |
Extracellular ice |
intracellular ice |
|
Cryoprotectant |
DMSO |
Bank acceptance criteria |
Freezing batch |
|
Cryovial |
headspace |
Cooling rate |
Intermediate storage |
|
Temperature excursion |
Traceability |
Master cell bank |
working cell bank |
|
Quarantine |
bank release |
Post-thaw viability |
viable-cell recovery |
Review Questions
1. Explain how cooling too quickly and cooling too slowly can create different kinds of freezing stress.
2. Why can a contaminated culture remain a problem after it has been frozen?
3. A culture has a large total cell count but low viability and unusually slow growth. What evidence would you review before deciding whether to bank it?
4. A suspension contains 2.4 × 10⁶ viable cells/mL in 5.0 mL. The target is 2.0 × 10⁶ viable cells in each 0.8 mL vial. Calculate the total viable cells, complete vial number, final concentration, and packaged volume.
5. A plan calls for five 1.0 mL vials at 3.0 × 10⁶ viable cells/mL plus 0.4 mL of extra suspension at that concentration. How many viable cells are needed? Explain why an extra liquid allowance alone is insufficient.
6. Equal volumes of a cell suspension and a cell-free freezing solution are mixed. What happens to cell concentration and total cell number? What further information is needed to calculate final DMSO concentration?
7. Explain why a −80 °C freezer setting is not evidence that vials cooled at 1 °C per minute.
8. Design a label and inventory entry for one vial. Include stable identification, culture history, storage coordinates, and current status.
9. Why might a laboratory keep a protected master bank and a separate working bank? How should records connect them?
10. A vial loaded with 2.0 million viable cells yields 1.2 million viable and 0.3 million dead cells immediately after thawing. Calculate post-thaw viability and immediate viable-cell recovery. Explain the different denominators.
11. A thawed culture has high initial viability but poor attachment and little growth. Propose three possible explanations and identify records or measurements that could help evaluate them.
12. An oxygen alarm sounds while a colleague is preparing to retrieve a vial. State the appropriate immediate response and explain why sample recovery cannot take priority.
Apply Your Understanding
You have 12 million viable cells and need 2 million per vial at 0.8 mL per vial. Calculate the complete vial number, final concentration, and packaged volume before allowing for losses.
A frozen vial was loaded with 2.0 million viable cells. After thawing, 1.2 million viable and 0.3 million dead cells are recovered. Calculate viability and viable recovery, then explain their difference.
The cooling record is complete, but the long-term transfer time is missing. Identify the uncertainty and the records that could help investigate it.
A banked culture has high initial post-thaw viability but little later growth. Propose two possible causes and one relevant check for each.