9: Passaging Suspension Cells

BACKGROUND 

THP-1 cells (a human cell line commonly used to study immune cell behavior) normally grow as a suspension culture (cells dispersed in liquid medium rather than attached to the vessel). Passaging (transferring some cells to a new vessel with fresh medium so they can continue growing) helps keep cells within a suitable density range. Because routine THP-1 cells are not attached, they usually do not need a detachment enzyme. As cells divide, the number of living cells per milliliter rises, nutrients are consumed, and wastes accumulate. If a culture becomes too dense, cell growth or health may change. Transferring a measured portion into fresh medium gives the new culture an appropriate starting density.

Count the cells before deciding how much culture to transfer. A hemocytometer (a counting slide with a grid of known dimensions) and trypan blue (a dye that stains many dead cells blue while living cells remain clear) let you estimate the number of living cells per milliliter. Viability (the percentage of counted cells that are living) is also useful because two cultures with the same total cell count may contain different numbers of living cells. Use the allowable cell density (the number of cells per milliliter) and target starting density specified in the assigned cell product sheet or laboratory procedure. These limits can differ among cell stocks and experiments. If the culture looks clumped or contaminated, report that observation before proceeding.

For a routine dilution, select the target concentration and final volume from the assigned procedure. The dilution equation (a relationship used to calculate how much original culture is needed to reach a target concentration) is C₁V₁ = C₂V₂. Here C₁ is the measured living-cell concentration in the original culture; V₁ is the volume of that culture to transfer; C₂ is the desired living-cell concentration in the new vessel; and V₂ is its final volume. Rearrange to find V₁ = C₂V₂ ÷ C₁. Fresh-medium volume = V₂ − V₁. For example, with C₁ = 1.0 × 10⁶ living cells/mL, C₂ = 2.5 × 10⁵ living cells/mL, and V₂ = 20 mL, transfer 5 mL of original culture and add 15 mL of fresh medium. The units cancel to give milliliters. Check that enough culture is available and that the new vessel can safely hold the final volume.

A routine dilution usually leaves the cells in their existing liquid and does not require a centrifuge. If a separate procedure calls for complete medium replacement, centrifugation (spinning a sample to collect cells) may be needed before adding fresh medium. Use only its specified force and time because processing can stress cells or cause cell loss. PMA treatment (exposure to phorbol 12-myristate 13-acetate, a chemical that can change THP-1 behavior) may cause cells to attach to the vessel; those cells need a different handling procedure. Record what you actually transfer and later compare the new culture’s observed density with its calculated starting density.

MATERIALS

• THP-1 culture and its product sheet or laboratory SOP (standard operating procedure, an approved set of instructions)
• Appropriate complete growth medium and sterile receiving vessel
• Class II biosafety cabinet (a ventilated enclosure that helps protect cultures and workers) and required personal protective equipment
• Sterile serological pipettes (graduated pipettes for milliliter volumes), pipette aid, micropipette (a device for small microliter volumes), and sterile tips
• Hemocytometer, coverslip, microscope, 0.4% trypan blue, and sample tube
• Disinfectant, wipes, and appropriate liquid waste container
• Culture record and calculator
• Centrifuge and sterile tubes only if the assigned procedure requires them

METHODS

1. Review the assigned THP-1 product sheet or SOP for medium, allowed density range, target reseeding density (the intended living-cell concentration immediately after transfer), vessel, final volume, incubation conditions, and special handling. Label a new vessel with cell identity, date, passage number, and operator.

2. Inspect the culture under the microscope. Record appearance, clumping, medium condition, and possible contamination. Report suspected contamination before opening the vessel. Prepare the biosafety cabinet and bring in only required supplies.

3. Gently mix the closed culture to disperse settled cells without vigorous shaking. In the cabinet, remove a small aseptic sample (a sample collected while preventing contamination) sufficient for counting; do not withdraw one-tenth of the culture unless the procedure specifically requires it.

4. Count clear and blue cells using the hemocytometer and trypan blue method from the cell counting lab. Record each square’s counts, the dye dilution and any additional sample dilution, living cells/mL, and percent viability. If the grid is too crowded or sparse to count reliably, prepare and document a suitable new sample.

5. Choose the target living cells/mL and final volume from the assigned instructions. Calculate V₁ = C₂V₂ ÷ C₁ and fresh-medium volume = V₂ − V₁. Show your units and ask a second student to check the calculations. Consult the instructor if you would need more old culture than is available or more final volume than the vessel allows.

6. Add the calculated fresh complete medium to the receiving vessel. Gently remix the source culture immediately before withdrawing V₁. Transfer the calculated volume aseptically into the receiving vessel. Mix gently to distribute cells evenly; do not add cells to an empty vessel unless the cell-specific procedure directs it.

7. If complete medium replacement is explicitly required, follow that separate SOP for cell collection, centrifugation settings, supernatant (the liquid above collected cells) removal, and resuspension (mixing collected cells back into liquid). Record the settings and any cell loss. Do not substitute a generic 600 × g spin.

8. Return the vessel to its specified incubator. Record source and receiving vessel IDs, cell passage number, actual volumes transferred and added, medium lot, date and time, identities of both students and the steps each performed. Note the calculated starting density in the new vessel.

9. At the next scheduled observation, remix and recount a sample as directed. Record cell density, viability, appearance, and any unexpected growth or contamination. Clean the cabinet and dispose of waste according to laboratory procedures.

EXERCISE Passaging Suspension Cells                                                                                NAME____________________________

EXPECTATIONS

1. Predict what would happen to the new culture’s starting density if you transferred twice the calculated old-culture volume but kept the same final volume. Explain your prediction.

2. Predict how using the same transfer volume from a culture with lower viability could affect growth after passaging.

RESULTS

Record your measurements and observations below. Show the C₁V₁ = C₂V₂ calculation and the fresh-medium calculation with units. Add the follow-up count when it is available.

Cell identity, passage, source and new vessel IDs: ____________________________

Appearance and medium condition before passage: ____________________________

Clear and blue counts in four squares: ____________________________

Dye and any additional sample dilution: ____________________________

C₁ measured living cells/mL and percent viability: ____________________________

C₂ target living cells/mL and V₂ final volume: ____________________________

V₁ calculated transfer and fresh-medium volumes: ____________________________

Actual transfer and medium volumes; medium lot: ____________________________

Calculated starting density and follow-up count: ____________________________

Date, operator identities, and deviations: ____________________________

Record your measurements and observations below. Show the C₁V₁ = C₂V₂ calculation and the fresh-medium calculation with units. Add the follow-up count when it is available.

Date: __________ Student: __________________ Partner: __________________

Cell identity: __________ Source passage: ______ New passage: ______

Source vessel ID: __________ Receiving vessel type and ID: ________________

Assigned SOP or product sheet: __________ Allowed density (cells/mL): ______

Appearance clumping and medium condition before passage: __________________

________________________________________________________________________

Raw cell counts

Dye dilution factor: ______ Additional sample dilution factor: ______

Total counting dilution factor: ______ Counting problems or repeat reason: ______

Density and transfer plan

Calculations

Show V1 = C2 × V2 ÷ C1 and fresh medium = V2 − V1. Include units. Use actual volumes to calculate the actual starting density.

________________________________________________________________________

________________________________________________________________________

________________________________________________________________________

________________________________________________________________________

Calculation checked by: ____________________ Date and time: __________

Complete medium name and lot: __________________________________________

Transfer date and time: __________ Incubator ID and location: __________

Incubation conditions: __________________________________________________

Student work record

If complete medium replacement was required: procedure reference __________

Actual centrifugation force (× g): ______ Time (min): ______ Cell loss if observed: ______

Use N/A for steps not performed.

Follow up culture record

Deviations unexpected observations and actions taken

________________________________________________________________________

________________________________________________________________________

________________________________________________________________________

CONCLUSIONS

1. Did the measured culture density justify passaging under the assigned THP-1 instructions? Explain with your actual result and the specified limit.

2. Compare your calculated starting density with the measured density at the next observation. Explain a reason they could differ, including the time that passed between measurements.

3. How would failing to mix the source culture immediately before sampling and transferring affect the count and the new vessel’s starting density?

4. Under what circumstances would a complete medium replacement be needed instead of a routine dilution, and what additional measurements or records would that require?