Revival of Cell

1.     Scope

·       This document contains the procedure used to grow cells.

2.     Definition

·       LN₂         - Liquid Nitrogen

·       D.D.W - Double Distilled Water

·       GM      - Growth Media

3.    Material

·       Pipette (10ml)

·       Flask (25cm²)

·       Growth medium, D.D.W

·       LN ₂  container with cell line

·       Forceps

·       Cryogloves

·       Goggles

·       Incubator

·       Glass beaker

4.     Procedure

·       Take D.D.W in a glass beaker and put it at 37⁰C in waterbath.

·       Take 10ml of 10% GM in the labeled flask with sterile pipette.

·       Identify the canister and position of vial to be revive from cane in the LN₂ container from the cell line history record.

·       Wear cryogloves, eye protection goggles and shoes while handling LN₂

·       Take beaker from the water bath .Take out the cane and remove vial with forcep.

·       Hold the vial with forceps and dip only frozen part in warm D.D.W.

·       Put cane in to canister and place back into the LN₂ container and close the lid tightly.

·       Take the vial in the beaker into the biosafety cabinet in the Cell culture lab.

·       Transfer label from the revived vial (showing type of cell line, passage no. and date of freezing cells) to 25cm² flasks and write the date of revival on 25cm² flask.

·       When contents are completely thawed, wipe outside of the vial with alcohol and take out 1 ml of 10% GM from the flask and with sterile 2ml pipette and add drop wise into the vial.

·       Mix and take the suspension with the same pipette and add drop wise in 25cm² tissue culture flasks and another person should shake the flask very slowly.

·       Spread the suspension evenly in the flask and keep in the incubator at 36ºĊ.

·       After 5hrs. Check the flask under microscope to see whether cells are stuck or not

·       If cells are adherent remove the medium and replace with pre-warmed 10%GM using sterile pipette.

·       Thawing of cells to be done first in the morning before starting any work.

1.      Safety conditions

• When content of the vial completely thawed wipe outside of the vial with alcohol to reduce bacterial contamination.
• When using LN₂ containers closed toed shoes, visors and heavy duty gloves must be weared to avoid injuries from nitrogen splashed of explosion of imperfectly sealed vials.

2.    Documentation

• Cell line history file PF-03
• L20B Cell line maintainance work sheet PR-37
• RD Cell line maintainance work sheet PR-38
• Cell line Flask Preparation register PR-34
• Cell line daily work register PR-35

3.    References used to draw SOP

• WHO Polio Laboratory Manual