Cell Banking

1.    Scope

·       This SOP explains how to freeze the cells slowly for the preparation of Cell culture stocks.

2.    Definition

·       DMSO             -           Dimethyl Sulphoxide

·       LN₂                             -                  Liquid Nitrogen

·       PBS                 -           Phosphate Buffer Saline

3.    Material

·       PBS (Without Ca & Mg)

·       10% GM with 10% DMSO

·       Nunc Cryo-Vial

·       Pipette

·       Incubator

·       Freeze-80ºĊ

·       Gauge-piece

·       Liquid nitrogen

·       Microscope

·       Trypsin-EDTA  

4.    .  Procedure

• Low passaged cells of RD and L20B cell line are received from Enterovirus research centre, Mumbail.
• 2 flasks of 25 cm ² of each of L20B #21 and RD # 230 were received in the lab in the month of August 2008.
• On arrival , the flask is checked Macroscopically for the following:

o   Breakage in the flask

o   Loosening of the caps

o   Leakage in the flask

o   Flask labels – these are checked for the values mentioned in the form accompanying the flask- Cell line, #, Date of preparation

o   Turbidity and fungal colonies

o   Colour changes in the media( yellow or deep red)

o   Date of preparation of flask

·       Flask is examined microscopically for the following:

o   Monolayer – the sheet is observed for the monolayer and the percentage is filled in the flask register. It should be 80-90% complete.

o   Detachments of cells if any

o   Floating debries

o   Bacterial and fungal contamination

Note: If the cells are contaminated, they are discarded and a new shipment of cells is requested.

·       Document accompanying the form is checked for the following:

o       Cell line

o       # no

o       Date of preparation

o       Seeding count

o       Mycoplasma testing

o       Growth medium

o       Condition of cells

o       Absolute passage number

·       Document of cell line history is kept in cell line history file no PF-03

·       The details mentioned in the form are checked and marked with comments on the form. The passage number received is treated as absolute passage number.

Adaptation of newly received cells:

·       The external surface of flask is decontaminated with spirit swab before transferring to cell culture area.

·       Aseptically remove the medium and add 10 ml of 10 % GM in the 25 cm ²   flask. Remove the medium from inside of the neck or cap area.

·       Incubate the culture at 36°C for overnight to acclimatize the cells.

·       If the sheet is confluent, the flask is subcultured, if not, then it is incubated and monolayer is allowed to complete to 80-90%. Flask is observed everyday for monolayer and the percentage of sheet is entered in flask register.

Freezing of cells:

·       Cryo vial are labeled with type of cell line, absolute passage no and date of freezing with an adhesive tape and also write with marker on vial.

·       One person discards the growth medium from flask.

·       Add (3ml for 25cm² flask and 5ml for 75 cm² flask) PBS (Without Ca & Mg), and wash the cells for two times.

·       Add 3ml Trypsin EDTA for 25 cm² flask and 5ml for 75 cm² and leave it until streaks appears and then remove it with sterile 5ml pipette.

·       Tap gently the flask so that cell sheet will get detached.

·       To make cell suspension add 3ml 10% growth medium in 25cm² flask and 5ml 10% growth medium in 75 cm²  flask.

·       Do cell count as referred in SOP no.6.8

·       Transfer cell suspension in to centrifuge tube.

·       Before centrifugation balance properly.

·       Centrifugation is done at 1000rpm for 10 min at 4° C.

·       After centrifugation discard the supernatant carefully with sterile 5ml pipette. Leave the pallet in centrifuge tube.

·       Prepare 10% growth medium with 10% DMSO.

·       For preservation of cells, the cell count should be 2.0 × 10⁶ cell/ml for 25 cm² and   4.0 × 10⁶ cell/ml for 75 cm².

·       Prepare dilution of concentrated cell with growth medium containing 10% FBS and 10% DMSO.

·       Transfer the cells (1ml) in to each vial which is previously labeled, tighten the cap and seal with sticking plaster.

·       Put vials in the nalgene container having Isopropyl alcohol.

·       Place container into-80°Ċ deep freeze over night so that temperature drops -1⁰C/minute.

·       On second day place the vial in special holding cane with marking and tie the vial with rubber band to the cane.

·       Place cane in canister and place canister in to Liquid Nitrogen container.

1.    Safety conditions  

• DMSO is powerful solvent so avoid it to come it contact with skin when preparing or using DMSO containing freezing medium.
• When working with LN₂ wear aprons, shoes, and gloves to avoid injuries from Nitrogen splashes or explosion of imperfectly seal vials.
• Work with one cell line at a time.

1.    Documentation  

• Note the position of vial in cane and canister in the LN₂ register PR-33 and cell line history File PF-03.
• Check the level of LN₂ once in two weeks, at the time of revival and at the time of freezing and keep a record in LN2 register PR-33

2.    References used to draw SOP

• WHO Polio Laboratory Manual.
• S2, Supplement to the WHO Polio laboratory manual- adapataion of newly received cells to local condition