Subculture calculations based on split ratios and confluence.
3.1 Terminologies and principles
Key terminologies:
- Split Ratio: Ratio of cell suspension transferred to fresh medium (e.g., 1:3 means 1 part cells to 2 parts fresh medium).
- Passage Number: Number of times cells have been subcultured since primary culture.
- Confluence-Based Seeding: Calculating cell numbers based on percentage surface coverage.
- Subculturing: Transferring cells from one culture vessel to fresh medium in new vessels.
- Trypsinization: Use of proteolytic enzyme to detach adherent cells.
Principles:
- Growth Phase Optimization: Cells should be passaged during log phase growth, typically at 70-90% confluence.
- Split Ratio Determination: Based on cell doubling time and experimental timeline.
- Surface Area Correlation: Cell number directly relates to culture vessel surface area at specific confluence.
3.2 Split Ratio and Passaging Strategy
Split ratio defines how cells are redistributed during subculture.
Example: 1:3 split yields three flasks at one-third density.
Worked example:
A researcher needs to seed 6 T-75 flasks with 4.0 × 10⁵ cells per flask for an experiment. Currently, the researcher has a T-25 flask containing 2.0 × 10⁶ cells.
a) Calculate the total number of cells required to seed all 6 T-75 flasks.
b) Determine whether the current T-25 flask contains enough cells to seed all T-75 flasks directly.
c) If not, calculate a suitable split ratio and explain how many subculture steps are required to generate enough cells.
d) After performing a 1:3 split, calculate the total number of cells available. Will this be sufficient for the experiment?
Step-by-step calculations
Given
- Number of T-75 flasks = 6
- Desired seeding density per flask = 4.0 × 10⁵ cells/flask
- Cells available in T-25 flask = 2.0 × 10⁶ cells
a) Total number of cells required
Formula:
Total cells required = Number of flasks × Cells per flask
Substitute values:
Total cells = 6 × 4.0 × 10⁵ cells
Total cells = 2.4 × 10⁶ cells
b) Are the current cells sufficient?
Available cells = 2.0 × 10⁶ cells
Required cells = 2.4 × 10⁶ cells
Comparison:
2.0 × 10⁶ < 2.4 × 10⁶
Conclusion: Not enough cells are available. A subculture step is needed.
c) Determine a suitable split ratio
A split ratio divides a flask into multiple new flasks to expand cell number.
- Let’s choose 1:3 split, meaning 1 part cells seeded into 3 new flasks.
- After a 1:3 split, the number of cells increases by a factor of 3:
Cells after 1:3 split = Available cells × Split factor
Cells after 1:3 split = 2.0 × 10⁶ × 3
Cells after 1:3 split = 6.0 × 10⁶ cells
Subculture steps required: Minimum 1 subculture step is sufficient to generate enough cells.
d) Check if the split yields enough cells for the experiment
- Cells required = 2.4 × 10⁶
- Cells after 1:3 split = 6.0 × 10⁶
Since 6.0 × 10⁶ > 2.4 × 10⁶
Conclusion: Yes, the 1:3 split provides enough cells to seed 6 T-75 flasks.
Summary
| Step | Calculation / Result | Conclusion |
| Total cells required | 6 × 4.0 × 10⁵ = 2.4 × 10⁶ | Required for seeding 6 flasks |
| Cells available | 2.0 × 10⁶ | Not sufficient |
| Split strategy | 1:3 → 2.0 × 10⁶ × 3 = 6.0 × 10⁶ | Sufficient |
| Subculture steps | 1 | Minimum required |
| Outcome | 6.0 × 10⁶ > 2.4 × 10⁶ | Enough cells available |
3.3 Confluence-Based Seeding
Cell number is proportional to surface area and confluence percentage.
Worked example
A researcher plans to seed 6 T-25 flasks with cells at 30% confluence. The following information is available:
- A T-25 flask reaches 100% confluence at 6.0 × 10⁵ cells/flask.
- Available cell suspension concentration = 2.0 × 10⁶ cells/mL.
- a) Calculate the number of cells required per flask to achieve 30% confluence.
b) Calculate the total number of cells required for all 6 flasks.
c) Calculate the volume of cell suspension (in mL and µL) needed per flask.
d) Provide a general formula to calculate the volume of cell suspension required based on the target confluence (%), cells at 100% confluence, and cell suspension concentration.
Step-by-step calculations
Given
- Target confluence = 30%
- Cells at 100% confluence in T-25 flask = 6.0 × 10⁵ cells/flask
- Number of flasks = 6
- Cell suspension concentration = 2.0 × 10⁶ cells/mL
a) Cells required per flask
Formula:
Cells per flask = Target % × Cells at 100% confluence
Substitute values:
Cells per flask = 0.30 × 6.0 × 10⁵ cells
Cells per flask = 1.8 × 10⁵ cells/flask
b) Total cells required for 6 flasks
Total cells = Cells per flask × Number of flasks
Total cells = 1.8 × 10⁵ × 6
Total cells = 1.08 × 10⁶ cells
c) Volume of cell suspension per flask
Formula:
Volume (mL) = Cells required per flask / Cell suspension concentration
Substitute values:
Volume = (1.8 × 10⁵ cells) / (2.0 × 10⁶ cells/mL)
Volume = 0.09 mL
Convert to microlitres:
0.09 mL × 1000 µL/mL = 90 µL
Volume per flask = 0.09 mL = 90 µL
d) General formula
Where:
- V add = volume of cell suspension to add (mL)
- Target % confluence = desired confluence in percent (e.g., 30%)
- N100% = number of cells at 100% confluence in one flask
- C suspension = cell concentration in suspension (cells/mL)
Practice Questions
- Define passage number.
- Why passage at 80% confluence?
Answers
- Number of subcultures.
2. Cells are in log phase.