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Suspension Cells Cryopreservation Protocol Comparison

David Ma, Shirley Pan

2018-06-28 00:49:45 in Protocols

Examples of cells that can be grown in suspension culture:

Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than in adherent conditions. Typical cells grown in suspension culture include PBMCs, CD4+ etc.

Part 1: Preparing Samples

Step	Common Protocol
1	Transfer the suspension cell with culture medium to a 50ml centrifuge tube and centrifuge at 1000 RPM and 4℃ for 3 minutes.
2	Carefully remove the upper clear supernatant with a pipette.
3	Add 5ml PBS and resuspend sample.
4	Take 10uL sample and count the viability of cells. The count of cells should be greater than 2 x 10⁶/ml.
5	Centrifuge the suspension again at 1000 RPM and 4℃ for 3 minutes.
6	Carefully remove the clear PBS supernatant with a pipette.

Part 2: Freezing Samples

Step	Conventional Protocol Using Freezing Containers	MetaLocker™ BioFlash Drive™ Optimized Protocol
1	Prepare cryoprotective solution by mixing 10% DMSO and 90% Fetal Bovin Serum (or selected culture medium, such as serum-free culture medium).	(No need)
2	Resuspend the cells with the cryoprotective solution. The concentration should be 1⁶/ml.	Resuspend the cells with Fetal Bovin Serum (or selected culture medium, such as serum-free culture medium). The concentration should be 1⁶/ml.
3	Transfer the cell solution to a regular cryogenic vial. (Mark the vial if needed).	Transfer the cell solution to a MetaLocker™ BioFlash Drive™ (Mark the vial if needed. You can also register the vial with our BioUSB APP)
4	Prepare a room temperature freezing container: 1) If an IPA-based freezing container is used, IPA needed to be replaced every 5 times of use. 2) If a foam-based freezing container is used, empty slots needs to be filled with dummy vials.	(No need)
5	Transfer the regular cryogenic vials to a freezing container.	(No need)
6	Put the freezing container into a -80℃ freezer for 12 hours.	Put the freezing container into a -80℃ freezer or directly to vapor phase nitrogen environment. -80℃ freezer is NOT recommended for storage longer than 6 months. * Do not immerse the sample into liquid nitrogen.
7	Transfer the cryogenic vials to a cryogenic storage cardboard box.	(No need)

Part 3: Thawing Samples

Step	Conventional Protocol Using Freezing Containers	MetaLocker™ BioFlash Drive™ Optimized Protocol
1	Warm culture medium to 37℃ with water bath.	Warm culture medium to 37℃ with water bath.
2	Put on safety measures such as low temperature gloves and goggles.	Put on safety measures such as low temperature gloves and goggles.
3	Take the selected samples out from freezers.	Take the selected samples out from freezers.
4	Put the regular cryogenic vials into 37℃ for about 5 minutes (until there is no visible ice).	Put the regular cryogenic vials into 37℃ for about 5 minutes.
5	Centrifuge the cryogenic vial 1000 RPM and 4℃ for 3 minutes.	(No need)
6	Carefully remove the clear supernatant.	(No need)
7	Resuspend the cell pellet with PBS	(No need)
8	Take 10uL of sample, calculate the viability by counting the number of cells.	Transfer the sample to a petri dish with 5 times culture medium (40% FBS and 60% culture medium, or any selected serum-free culture medium), and melt the remaining sample (if any) with medium from the petri dish.
9	Centrifuge the suspension again 1000 RPM and 4℃ for 3 minutes.	Take 10uL of sample, calculate the viability by counting the number of cells.
10	Resuspend the cell pellet with culture medium and start culturing based on need.	Culture the cell based on need. Washing is not necessary.

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