Nov 24, 2023

Media changes and Passaging in 2- or 5-layer CellStacks

This protocol is a draft, published without a DOI.
  • 1Cancer Research UK Cambridge Institute, University of Cambridge
Allan JW Lui
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Protocol Citation: Allan JW Lui 2023. Media changes and Passaging in 2- or 5-layer CellStacks. protocols.io https://dx.doi.org/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 01, 2022
Last Modified: November 24, 2023
Protocol  Integer ID: 73417
Keywords: layer cellstacks protocol, protocol, layer
Abstract
Protocol for handling CellStacks
Materials
Corning CellStacks (2-layer: Corning 3269, 5-layer:Corning 3313)
Complete cell culture media
Phosphate-buffered saline
Trypsin solution (usually 0.25%, with EDTA and phenol red; Gibco 25200)
Centrifuge
50ml centrifuge tubes
Before start
Ensure shelves of incubator are truly level with a spirit level or app
Media Changes
Per flask, Prepare and pre-warm:
Reagent2-layer5-layer
Complete media for plating220ml550ml


Take flask out of incubator, and place in upright position. Wipe down cap areas with 70% ethanol if desired.

Holding the flask with the long side facing downwards, uncap the bottom cap, and pour out media in flask onto a walls of a large 10L tub to prevent splashing, shake flask to remove the final few drops.
IMPORTANT: Handle flasks for cell culture in a suitable biosafety cabinet. Image taken for demonstration of flask handling only.

Place CellStack on its flat position.

For 2-layer CellStacks, pipette in new warm media 50-60ml at a time until 220ml has been added
For 5-layer CellStacks, pour in 550ml of new warm media (often an entire bottle)
To evenly distribute media between layers: Flip flask towards you such that it rests on its longer side.
For 2-layer CellStacks, place a finger on the bottom ledge to ensure flask is level.

IMPORTANT: Handle flasks for cell culture in a suitable biosafety cabinet. Image taken for demonstration of flask handling only.
Then tilt into the upright position:

Return flask to incubator, lying flasks flat while minimising sudden or large movements to avoid media spilling between layers
Passaging
8m
Per flask, Prepare and pre-warm:
Reagent2-layer5-layer
PBS250ml500ml
Trypsin40ml100ml
Complete media for trypsin inactivation40ml x2100ml x2
Complete media for plating220ml550ml



Pour out media in flask, wash out remaining media twice by:
  1. Adding PBS
  2. Distributing evenly between layers to cover the entire culture surface area
  3. Pouring out PBS
Add Trypsin
Incubate flask for around 00:03:00 in incubator
Tap flask to dislodge cells
3m
Add 1 aliquot of media for trypsin inactivation and distribute evenly between layers.
Collect cell suspension into duran bottles (250ml for 2-layer, 500ml for 5-layer)
Wash out remaining cells with the other aliquot of inactivation media similarly.

Reduce clumping by pipetting up and down multiple times with a 10ml serological pipette against the bottom of the bottle
Count cells using a Vi-Cell and calculate volume of cells for seeding
If necessary, centrifuge cells for replating at 200 x g, 00:05:00 and remove supernatant.

If plating into CellStacks:
  1. Resuspend cells in media for plating
  2. Transfer suspension into flask by pouring or pipetting

5m
Reseed cells at desired density.

If plating into CellStacks:
  1. Calculate the required volume of cell suspension for plating
  2. Remove the same volume of media from the pre-warmed aliquot of media for plating and replace with the cell suspension
  3. Transfer suspension into flask by pouring or pipetting


Pelleting cells
5m
Transfer desired number of cells (usually 5e7) into 50ml centrifuge tubes
Centrifuge cells at 200 x g, 00:05:00 and remove supernatant.
Resuspend with 5 mL PBS , centrifuge and remove supernatant again.

5m
Flick tube to loosen pellet and freeze on dry ice.