Cell Handling Procedures

● Cryopreserved Cell Handling Procedure (Cell Recovery)

1. Preparation Before Operation

Before starting the procedure, irradiate the biosafety cabinet with UV light for 30 minutes. Ensure that the water bath is clean and preheated to 37°C.

2. Thawing and Initial Cell Recovery

Rapidly thaw the cryovial containing 1 mL of cell suspension in a 37°C water bath with gentle shaking. The cells should be completely thawed within 1–2 minutes whenever possible.

Immediately transfer the thawed cell suspension into a centrifuge tube containing 9 mL of pre-warmed complete culture medium, and gently pipette to mix.

3. Centrifugation and Cell Resuspension

Centrifuge the cell suspension at 1000 rpm for 3 minutes.

Remove the supernatant and gently resuspend the cells in 1 mL of complete culture medium.

4. Cell Seeding and Culture

Transfer the cell suspension into a T25 culture flask containing 4 mL of complete culture medium.

Mix gently and place the flask in a 37°C, 5% CO₂ incubator for culture.

(For cells with specific requirements, please follow the recommended culture conditions provided in the corresponding cell datasheet.)

5. Cell Passaging

When cells reach the appropriate confluency for passaging, perform cell passage according to the passaging procedure described in the recovery flask cell handling procedure.

● Recovery Flask Cell Handling Procedure (Processing and Passaging)

(I) Handling After Receiving Cells

1. Initial Inspection and Stabilization

After receiving the recovery flask containing cells:

• Wipe the surface of the culture flask with 75% ethanol.

• Observe cell morphology and status under a microscope.

• Place the cells in a cell culture incubator and allow them to stabilize for 2–4 hours.

2. Cell Observation and Documentation

Carefully read the cell datasheet to understand relevant cell information.

After stabilization, remove the culture flask and:

• Observe the cells under a microscope.

• Take photographs.

• Record the cell status.

3. Determining Whether to Passage

If the cell confluency is above 80%, cells can be passaged normally.

For the first passage, a recommended split ratio is 1:2–1:3.

If the confluency is below 80%:

• Remove the culture medium from the flask.

• Leave approximately 6 mL of original culture medium in the flask.

• Continue culturing until the cells reach approximately 80% confluency before passaging.

During this process, loosen the flask cap or replace it with a vented cap to ensure proper gas exchange.

(II) Cell Passaging Procedure (For Adherent Cells)

1. Washing Cells

Remove the culture medium.

Wash the cells gently with 2–3 mL PBS once or twice.

2. Cell Digestion

Remove PBS.

Add 1–2 mL of 0.25% trypsin-EDTA to a T25 flask (or a 10 cm culture dish).

Gently shake the flask/dish to ensure complete coverage of the surface.

3. Cell Detachment

Place the flask/dish in a 37°C incubator for 1–2 minutes for digestion.

For difficult-to-digest cells, digestion time may be appropriately extended.

Observe the cells under a microscope. When cells become rounded or begin moving in a sand-like pattern but have not completely detached, add 2–3 mL of complete culture medium to terminate digestion.

Gently pipette to generate a uniform cell suspension.

4. Centrifugation and Resuspension

Centrifuge the cell suspension at 1000 rpm for 3 minutes.

Remove the supernatant and gently resuspend the cells in fresh complete culture medium.

5. Cell Seeding

According to the cell datasheet and the specific cell condition, distribute the cell suspension into new culture flasks or dishes containing complete culture medium at an appropriate split ratio.

6. Cell Culture

After gently mixing the cell suspension, place the culture flask or dish into a 37°C, 5% CO₂ incubator.

(For cells with special requirements, culture according to the recommended conditions provided in the corresponding cell datasheet.)

● Precautions

1. Maintain Sterile Conditions

All operations should be performed under sterile conditions to avoid contamination.

2. Culture Medium Requirements

The culture medium supplied during transportation should not be used for cell culture.

Please prepare and use culture medium according to the recommended specifications for cell growth.

3. Biosafety Requirements

All animal-derived cells should be considered as having potential biological hazards.

Experimental operations must be performed in a Class II biosafety cabinet.

4. Personal Protection During Cell Recovery

When recovering cryopreserved cells, it is recommended to wear:

• Protective gloves

• Protective clothing

• Face protection

These precautions help prevent potential injuries caused by cryovial breakage or cap release during thawing.

Conclusion

Proper cell handling is essential for maintaining cell viability, stability, and experimental reproducibility.

By following standardized procedures for cryopreserved cell recovery, cell culture, passaging, and biosafety practices, researchers can improve cell survival rates and ensure reliable experimental outcomes.

MiaoLingBio is committed to providing high-quality biological resources and professional technical support to help researchers achieve efficient and reproducible cell culture results.