Cell Culture Protocol
This protocol describes standard procedures for maintaining mammalian cell lines in culture, covering thawing from cryostorage, routine subculturing (passaging), cell counting, cryopreservation, and aseptic technique. The protocol is applicable to common adherent and suspension cell lines and uses Solarbio cell culture reagents.
Principle
Mammalian cell culture maintains cells in an artificial environment that mimics physiological conditions (37°C, 5% CO₂, humidified atmosphere, nutrient-rich medium supplemented with growth factors from fetal bovine serum). Adherent cells attach to the culture vessel surface and must be detached enzymatically (trypsinization) or mechanically for passage. Suspension cells grow freely in medium and are subcultured by dilution. Both cell types require sterile technique to prevent microbial contamination and defined media formulations to support growth.
Materials
| Reagent |
Recommended Solarbio Product |
SKU |
| D-PBS (Ca²⁺/Mg²⁺-free) |
Dulbecco's PBS without calcium or magnesium |
D1040 |
| Trypsin-EDTA 0.25% |
Trypsin-EDTA (0.25%), phenol red in D-PBS |
T1300 |
| Fetal Bovine Serum |
FBS (characterized, heat-inactivated) |
S1810 |
| Cell Culture Medium |
DMEM (high glucose), RPMI 1640, MEM, or DMEM/F12 |
Various |
| Penicillin-Streptomycin (100×) |
Penicillin (10,000 U/mL) + Streptomycin (10,000 μg/mL) |
P1400 |
| DMSO (sterile, cell culture grade) |
Dimethyl sulfoxide, ≥99.7%, sterile-filtered |
D8060 |
Equipment Checklist
| Item |
Specification |
Purpose |
| Biological safety cabinet (BSC) |
Class II, Type A2 (laminar flow) |
All sterile manipulations |
| CO₂ incubator |
37°C, 5% CO₂, 95% humidity |
Cell growth |
| Inverted microscope |
Phase contrast, 10× and 20× objectives |
Cell morphology and confluency checks |
| Water bath |
37°C, clean and sanitized |
Medium and reagent warming |
| Refrigerated centrifuge |
Swing-bucket rotor, 200×g capability |
Cell pelleting |
| Hemocytometer or automated cell counter |
Standard or trypan blue exclusion |
Cell counting and viability |
| Cryogenic storage |
Liquid nitrogen (−196°C) or −80°C (short term) |
Long-term cell storage |
| Controlled-rate freezer |
Isopropanol freezing container |
Controlled −1°C/min cooling |
Detailed Protocol
Section A: Thawing Cryopreserved Cells
| Step |
Detail |
Time |
Notes |
| Warm complete medium |
37°C water bath, 15 min |
15 min |
Complete medium = basal medium + 10% FBS + 1% P/S |
| Prepare culture vessel |
Label with cell line, passage number, date |
— |
10 cm dish or T25 flask for standard thaw |
| Thaw cryovial |
37°C water bath with gentle swirling |
~1 min |
Keep the cap out of the water to prevent contamination |
| Decontaminate vial |
Spray with 70% ethanol |
30 s |
Wipe dry before opening in BSC |
| Transfer cells |
1 mL cell suspension into 9 mL pre-warmed complete medium |
1 min |
Use a sterile serological pipette |
| Centrifuge |
200×g, 5 min at RT |
5 min |
Brake on for most cell lines (off for fragile primary cells) |
| Remove supernatant |
Aspirate carefully without disturbing cell pellet |
— |
Use a sterile Pasteur pipette or serological pipette |
| Resuspend pellet |
1–2 mL fresh complete medium by gentle pipetting |
30 s |
Avoid foaming |
| Seed |
Transfer to culture vessel containing 5–10 mL complete medium |
1 min |
Rock gently to distribute cells evenly |
| Incubate |
37°C, 5% CO₂ |
Overnight |
Check attachment and morphology the next day |
Section B: Subculturing (Passaging) — Adherent Cells
Perform subculturing when cells reach 70–90% confluency, typically every 2–4 days depending on cell line.
| Step |
Detail |
Time |
Notes |
| Remove spent medium |
Aspirate from the side of the vessel |
— |
Do not disturb the cell monolayer |
| Wash cells |
Add 5 mL (T25) or 10 mL (T75) D-PBS (D1040) |
1 min |
Add D-PBS gently to the side; rock to cover; remove completely |
| Add trypsin |
0.5 mL (T25) or 1–2 mL (T75) of 0.25% Trypsin-EDTA (T1300) |
1 min |
Ensure entire monolayer is covered |
| Incubate at 37°C |
Place vessel in incubator |
2–3 min |
Observe under microscope periodically; cells should round up |
| Tap vessel |
Gently tap the side of the flask to dislodge cells |
10 s |
Do not over-tap |
| Neutralize trypsin |
Add 3–5 mL complete medium (containing serum) |
30 s |
Serum contains trypsin inhibitors; mix gently |
| Collect cells |
Pipette cell suspension to a conical tube |
30 s |
Rinse the surface once more |
| Centrifuge |
200×g, 5 min at RT |
5 min |
Brake on |
| Remove supernatant |
Aspirate carefully |
— |
Avoid pellet disruption |
| Resuspend in fresh medium |
1–5 mL complete medium depending on pellet size |
30 s |
Count cells if a specific seeding density is required |
| Seed at desired ratio |
Typical split ratios: 1:3 to 1:10 |
— |
Add appropriate volume of cell suspension to fresh medium in new vessel |
| Incubate |
37°C, 5% CO₂ |
2–4 days |
Until next passage |
Important: Over-trypsinization damages cells. If cells are still adherent after 5 minutes at 37°C, wash the cells with D-PBS again and add fresh trypsin. Do not scrape — this causes membrane damage.
Section C: Subculturing — Suspension Cells
| Step |
Detail |
Notes |
| Mix culture gently |
Swirl or rock flask to resuspend settled cells |
Do not vortex or shake vigorously |
| Collect sample |
Transfer aliquot to a sterile tube |
For counting and passaging |
| Count cells |
Trypan blue exclusion on hemocytometer |
Record viable cell density |
| Calculate dilution |
Seed at 1–3 × 10⁵ cells/mL in fresh medium |
Split ratio typically 1:2–1:5 |
| Transfer to new vessel |
Add calculated volume to pre-warmed fresh medium |
Vent cap slightly for gas exchange |
| Incubate |
37°C, 5% CO₂ |
Passage every 2–3 days |
Section D: Cell Counting and Viability
- Mix 10 μL cell suspension with 10 μL 0.4% trypan blue in a microcentrifuge tube.
- Load 10 μL of the mixture onto a hemocytometer.
- Count viable (clear/unstained) and dead (blue) cells in the four corner squares.
- Calculate:
Cell density (cells/mL) = (Average viable cells per square) × 2 (dilution factor) × 10⁴
Viability (%) = (Viable cells / Total cells) × 100
| Quality Standard |
Acceptable Range |
| Viability after thaw |
≥85% |
| Viability at passage |
≥90% |
| Cell density at confluency (adherent) |
1–5 × 10⁴ cells/cm² |
| Doubling time (typical immortalized line) |
18–30 h |
Section E: Cryopreservation
| Component |
Volume |
Final Concentration |
| Complete culture medium |
70% |
70% v/v |
| Fetal Bovine Serum (S1810) |
20% |
20% v/v |
| DMSO (D8060) |
10% |
10% v/v |
| Cell density |
— |
1–5 × 10⁶ cells/mL |
| Step |
Detail |
| Prepare freezing medium |
Mix components fresh; keep on ice |
| Harvest cells |
Trypsinize adherent cells, count, centrifuge at 200×g for 5 min |
| Resuspend pellet |
In ice-cold freezing medium at the target cell density |
| Aliquot |
1 mL per cryovial; label with cell line, passage number, date, and initials |
| Cool at −1°C/min |
Place in isopropanol freezing container at −80°C for 24 h |
| Transfer to LN₂ |
Move to liquid nitrogen storage (−196°C) within 24–48 h |
| Record location |
Document the storage location (rack, box, position) |
Aseptic Technique Checklist
| Practice |
Frequency |
Purpose |
| Clean BSC with 70% ethanol |
Before and after each use |
Surface decontamination |
| UV sterilization of BSC |
30 min before use |
Air and surface decontamination |
| Warm media in sealed water bath |
Before each use |
Prevents thermal shock to cells |
| Spray all items with 70% ethanol before placing in BSC |
Every item |
Reduces contamination risk from packaging |
| Check media for turbidity or pH change |
Each use |
Detects microbial contamination |
| Mycoplasma testing of culture supernatant |
Monthly |
Detects the most common cell culture contaminant |
| Check incubator CO₂ level and water pan |
Weekly |
Ensures stable environment and humidity |
| Replace HEPA filter in BSC per manufacturer schedule |
Per schedule |
Protects from contamination |
Contamination Response
| Contamination Type |
Signs |
Immediate Action |
Long-Term Prevention |
| Bacterial |
Cloudy medium, pH drop (yellow), visible turbidity |
Discard culture immediately; bleach flask; test all shared media |
Use fresh antibiotics (P1400); inspect all reagents |
| Fungal |
Mycelia or yeast on medium surface |
Discard culture; BSC decontamination with 70% ethanol; change HEPA filter |
Use antifungal agents for valuable cultures |
| Mycoplasma |
Subtle pH change, no visible growth |
Test using PCR-based mycoplasma detection kit; discard positive cultures |
Monthly mycoplasma testing; quarantine new lines |
| Cross-contamination |
Mixed cell morphology, unexpected growth |
Verify by STR profiling; discard mixed lines |
Separate handling of different cell lines in BSC |
Troubleshooting Table
| Problem |
Cause |
Solution |
Prevention |
| Cells not attaching |
Trypsin over-treatment |
Reduce trypsin incubation to 2 min; check cells under scope every 30 s |
Use fresh trypsin at correct concentration |
|
Vessel coating required |
Use poly-L-lysine or collagen-coated flasks for difficult lines |
Pre-coat vessels before seeding |
|
Mycoplasma contamination |
Test using mycoplasma detection kit |
Test all new cultures before co-culturing |
| Slow growth |
Medium nutrient depletion |
Replace with fresh complete medium; increase FBS to 15% |
Passage before medium becomes acidic |
|
CO₂ levels incorrect |
Verify CO₂ at 5% |
Calibrate CO₂ sensor quarterly |
|
Contamination |
Check for mycoplasma |
Monthly mycoplasma screening |
| Cell clumping after trypsinization |
Incomplete trypsin neutralization |
Add more complete medium; DNase I treatment (50 μg/mL) |
Use serum-containing medium for neutralization |
|
High passage number |
Thaw earlier passage |
Record passage number; discard after P20–P30 |
| Viability low after thaw (<50%) |
Freezing medium without FBS |
Always use 90% FBS + 10% DMSO (or 20% FBS + 70% medium + 10% DMSO) |
Prepare freezing medium fresh; keep cold |
|
Slow thawing |
Thaw in 37°C water bath with constant agitation |
Thaw quickly but aseptically |
|
Cells stored at −80°C for >1 month |
Transfer to LN₂ within 48 h |
Use −80°C only for short-term storage (<1 month) |
| Cells detaching spontaneously |
Over-confluent |
Passage before 90% confluency |
Count cells; seed at consistent density |
|
Temperature fluctuations |
Check incubator temperature |
Monitor temperature daily |
Optimization Notes
- For primary cells: Use lower trypsin concentration (0.05% Trypsin-EDTA, T1350), shorter incubation (1–2 min at 37°C), and coat vessels with 0.1% gelatin or collagen. Primary cells have higher sensitivity to enzymatic treatment and require gentler handling.
- For serum-free culture: Use defined serum-free medium and pre-coated culture vessels. Supplement with specific growth factors as required by the cell type. Remove serum gradually (10% → 5% → 2% → 0% over 2 weeks) rather than abruptly.
- For 96-well plate assays: Trypsinize cells from a T75 flask, count, and prepare a cell suspension at the desired density (e.g., 5 × 10⁴ cells/mL for 5,000 cells/well in 100 μL). Seed using a multichannel pipette. Avoid edge wells — fill them with sterile D-PBS to reduce evaporation effects.
- For scaling up: T75 → T175 flasks (2.5× surface area) or 10-layer CellSTACK (10,000 cm²). Adjust trypsin and medium volumes proportionally. For large-scale production, use microcarrier beads in spinner flasks or bioreactors.
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