CRYOPRESERVATION OF MAMMALIAN CELLS

1. SUMMARY

1.1. Purpose

This SOP details the method of cryopreservation used for cell lines used in the Research Laboratory at CMEM. The goal is to create a uniform and reproducible protocol to cryo-preserve and identify all cell lines stored in the Liquid Nitrogen (LN2) storage tanks.

1.2. Scope

This procedure applies to all CMEM research laboratory personnel freezing mammalian cell lines for research, storage and distribution.

1.3. Responsibility

It is the responsibility of the Laboratory Operations Manager to ensure all required personnel are properly trained and understand this Trash.MitowikiSOP.

2. REQUIRED MATERIALS

2.1. Required Equipment
* Sterile biosafety cabinet
* -80°C Ultra low freezer
* Stereo-microscope, inverted
* Centrifuge
* Incubator
* Pipette-Aid
* Ice bucket
* Liquid nitrogen storage tank
* Cryogloves, Eye protection
* Isopropanol freezing containers
* Cell Counter or Hemocytometer
* Black extra-fine alcohol proof pen

2.2. Required Supplies
* 1, 5, 10 ml sterile serological pipets
* 50ml Stericup filter system, 0.22µm membrane
* 15 or 50ml sterile centrifuge tubes
* 1.5ml Cryovials (Nunc)
* Cryoflex tubing (Nunc)

2.3. Reagents
* PBS without CaCl and MgCl
* DMEM Invitrogen
* Certified FBS or BCS
* Dimethyl Sulfoxide (DMSO)
* 0.05% Trypsin-EDTA
* Ethanol
* Isopropanol

2.4. Reagent Preparation

2.4.1. Cryopreservation Media

The following media has to be made fresh right before beginning the freezing procedure and should be kept on Ice at all times. The total volume should be determined dependent on the number of samples to be frozen, +10%.


1. Added to a Stericup filter system are:

* 20% Certified FBS (e.g. 2ml)

* 60% Culture medium without supplements (e.g. 6ml).

2. The media is then filter-sterilized using the 0.22µm Stericup filter.

3. Finally, 20% of sterile DMSO (2ml) from sterile 5ml ampoules or aliquots in glass vials is added.

4. This freezing medium has to be kept sterile and on ice.

3. PROCEDURE

3.1. Preparation of cells for freezing

3.1.1. The flasks have been checked the day before freezing and fed with fresh medium, assuring that cells will be still in their exponential growth phase when harvested.

3.2. Generation of an electronic record for the procedure

3.2.1.

A researcher planning on storing cells in Liquid Nitrogen, has to consult the supervisor or lab manager for allocation of storage space in the LN tanks with information regarding the number of slots needed for storage and preferences regarding their location in the tanks.

3.2.2. A record will be generated in form of an electronic spreadsheet listing all the cell lines that are to be frozen. The template for this electronic spreadsheet can be found on the CMEM server and will contain the following information: * A " Unique Sample Identifier", consisting of eight characters. Starting with the researcher initials (no middle initial being the letter "U"), a number (starting at 1 and increasing for researchers sharing the same initials), and a four digit number unique number, in chronological succession of the researchers freezing log (0001 to 0006, 0007 to 0011 etc.) assigned to EACH cryovial:

AUF

1

XXXX

Researcher Initials
(three letters, with the letter "U" serving as placeholder)

Numerator

Sample ID number.
A consecutive series of numbers 0001 to 9999, increasing with sample number.

* "TYPE" of the cell line (the template spreadsheet will contain a drop-down menu for cell types)
* "GENOTYPE" of the cell line (e.g. Rho0; ANT -/+; etc.) to be filled in by the researcher.
* Passage number "P01"
* " DATE " of freezing
* "Freezing history" "Unique Identifier of sample originally thawed (e.g. "CPE10099")
* " NOTES " regarding specific sample properties or origin

IMPORTANT:

NO "HIPAA" RELEVANT INFORMATION (Patient/Donor name and other personal information) SHOULD BE KEPT IN THIS SPREADSHEET UNDER ANY CIRCUMSTANCES!

If the researcher is unfamiliar with "HIPAA" procedures and needs to work with human cell lines he should contact the lab-manager or your supervisor for further instructions.

NOTE: It is admissible to temporary carry this information in a spreadsheet, however this information should be entered into the secured database as soon as possible and/or be kept "secure" according to HIPAA regulations.


3.3. Labeling of sterile cryovials

3.3.1.

Before beginning to freeze cells, the researcher has to transfer the following information from the electronic record generated under section 3.2. to the cryovial(s):

* The " Unique Identifier" as defined under 3.2.1., consisting of the researchers initials, his identifier and the four digit sample number from the spreadsheet (e.g. AUF10001, AUF10002...).
* The " Date " of freezing (e.g. 01/20/09)

Optional field entries on the tube are:
* "TYPE" of the cell line (same nomenclature as on the spreadsheet)
* "GENOTYPE" of the cell line (e.g. Rho0; ANT -/+; etc.)
* " P01 " Passage number

AGAIN:

NO "HIPAA" RELEVANT INFORMATION ON THE VIAL!


3.4. Cell Harvesting

3.4.1.

Transfer of all flasks to be frozen in the sterile biosafety cabinet and aspiration of the growth media using sterile serological pipettes.

3.4.2.

Washing of cells with sufficient PBS to remove all growth media.

3.4.2.a,

For adherent cells: Addition of just enough Trypsin-EDTA solution to each flask to cover cells, followed by incubation for 5 minutes at 37°C.

3.4.2.b,

Recapping the flasks tightly and dislodging the cells from the flask surface by gently tapping the side of the flask with the heel of the hand.

3.4.2.c,

Addition of enough Cell Culture Medium to each flask to neutralize the Trypsin-EDTA (in general at least 10 volumes).

3.4.2.d,

Dispersion of cells with a sterile, disposable pipette to break up cell chunks.

3.4.3.

Transfer of suspension from the flasks into 15/50ml conical tubes, using equal volumes for all tubes to balance them in the centrifuge.

3.4.4.

Centrifugation of the suspension at 200 x g for 5 minutes.

3.4.5.

Aspiration of the medium from each tube, and careful resuspension of the pellet in PBS to remove residual medium.

3.4.6.

Repetition of steps 3.2.4. and 3.2.5. as needed.

3.5. Freezing of Cells

3.5.1.

Resuspension of the cell pellet in a suitable amount of PBS and subsequent removal of an aliquot to count the number of cells in the cell counter. NOTE: The aliquot may require dilution.

3.5.2.

Repetition of step 3.2.8., followed by resuspension of cells in a suitable amount of freezing medium. NOTE: Cells should be frozen at a density of 10x10E6cells/vial.

3.5.3. Quick, but careful addition of 0.5-1ml cell suspension containing 1X10E6 cells into each cryovial using a sterile 1ml pipette. NOTE: The top of the vial (including threads) should not come in contact with medium, otherwise tubes will not properly seal when placed in LN2!

3.5.4.

Tightening of the screw cap to hand-tight (resulting in slight pressure to the rubber seal).

3.5.5.

Optional:

CryoflexTM tubing can be used to seal the tubes stored in the liquid nitrogen phase. After cutting the tubing to size (allowing for ca. 1 inch overhang at each end of the vial), the vial is inserted and the tubing ends are shrunk by careful and slow exposure to a heat source. The heated ends can then be crimped by using forceps. The excess tubing can then be trimmed away. CryoflexTM tubing can be removed by cutting around the screw cap at the rubber seal.

3.5.6.

Placement of vials into the isopropanol freezing container and immediate transfer to a -80°C freezer.

3.5.7.

Overnight storage to freeze cells at -1°C to -3°C per minute. NOTE: Extended storage of cells at -80°C will result in loss of viability of cells!

3.5.7.

Transfer of samples from the -80°C freezer to the Liquid Nitrogen (LN) Storage Tanks THE NEXT DAY to the freezer, rack, box and slot(s) allocated to the researcher by the supervisor or lab-manager.

AGAIN:

Transfer of samples ONLY into the LN freezer, rack, box and slots allocated to the researcher by the supervisor and/or lab-manager!

It is imperative to follow all safety and handling procedures applicable to working with Liquid Nitrogen. This includes, but is not limited to the Personnel Protection Equipment (e.g. closed toe shoes / laboratory coat / cryoprotection gloves / eye protection!)!

4. RESULTS REPORTING

4.1. All mammalian cell lines stored and kept in liquid nitrogen tanks have to be recorded in the central database.

4.1.1.

When storing human cell lines, all patient/donor information (generated or transferred while in compliance with HIPAA regulations), should be kept in the Central Database managed and updated by the supervisor and/or lab manager.

4.1.2.

Requests for HIPAA protected cell lines or HIPAA-protected information should be addressed to the relevant supervisor and/or lab manager.

Again: If you have never heard of "HIPAA" and need to work with human samples you should ask the lab-Manager or your supervisor for information).

5. PROCEDURE NOTES

6. REFERENCES

HIPAA guidelines can be found at:

http://www.policies.uci.edu/quickviews/privacy.html#medical

7. CHANGE HISTORY

First version: Adopted: 01/21/2009

Topic revision: r1 - 12 Feb 2016, MartyBrandon

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