Subculture of Cells

1.     Scope

·       This document contains procedure used for passaging of RD and L₂₀B Cell culture.

2.     Definition

• RD-     Human Rabdomyosarcoma cell line sensitive to Polio virus and many other Enteroviruses.
• L₂₀B -    Cell line derived from mouse L-cells transfected with polio receptors that will support the growth of Polioviruses and very few other enteroviruses.
• MEM- Eagles Minimum Essential Medium.
• PBS-    Phosphate Buffer Saline

3.    Material

• Flask having healthy monolayer of cells
• PBS without Ca⁺⁺ & Mg⁺⁺
• Trypsin-EDTA
• Sterile pipettes-1ml, 5ml, 10ml.
• 10%GM
• Neubauer chamber
• Trypan blue
• Incubator
• Tissue culture tubes
• Eppendrof tube
• Microscope
• Pipette-aid.
• Micro pipette
• Tips
• Sterile Flasks
• Discarding pan with 1% hypochlorite solution

4.     Procedure

• Take 0.3ml trypan blue in an eppendroff tube.
• Take 75/150cm² flask, which had complete monolayer of healthy cells.
• Wash the cell with 5/10ml PBS with sterile 5ml/10ml pipette.

Add 5/10ml Trypsin EDTA in such a manner that it should cover the monolayer completely.

• Leave it until streaks appears and then removes it with sterile 5ml/10ml pipette.
• Tap gently the flask so that the cell sheet will detach.
• To detach the cells completely add 5ml/10ml 10% growth medium with sterile 5ml/10ml pipette and mix it to break clumps of cells.
• Take 0.1ml cell with sterile 1ml pipette and add in 0.3ml Trypan blue to make 1:4 dilutions of cells. Count cells in Neubaur chamber under microscope.
• Prepare the flasks and tubes each as per cell-count given below (Dilute cells with growth medium as required for preparation of tubes or flasks).

Cell-line	25cm2	75cm2	150cm2	Tissue culture tube
L20B	1.5×106cells/flask	5.2×106cells/flask	10×106cells/flask	2.7×105/ml
RD	0.7×106cells/flask	2.3×106cells/flask	--	1.4×105/ml

• For new flasks to be prepared label with name of cell culture, passage of cell culture, date of preparation and volume of seeding cells.
• Take 30ml of 10%GM if 75cm2 flask to be prepared or 50ml of 10%GM if 150cm2 flask to be prepared with sterile pipette, seed the cells as per required count with sterile pipette, mix and incubate at 36Ċ in incubator. When complete monolayer is formed, it can be used.
• For preparation of tubes draw a line for alignment and label tube with name of cell culture, passage and date of preparation.
• Dilute the cells as per count and add 1.0ml of cell dilution each tube.
• Keep the tubes in the rack and incubated it in slanting position (5°Ċ).

1.      Safety conditions

·       Never handle different cell culture at the same time.

·       Be careful to prevent microbial contamination to cell culture and contamination of working environment with cell culture material.

2.    Documentation

·       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

1.    References used to draw SOP

·       WHO Polio Laboratory Manual.