cell harvest by scraping versus trypsinization - (Jul/13/2006 )

If you are passaging cells then always use trypsin rather than scrapping. Alot more damage is done to the cells through scrapping. But beware that leaving Trypsin on for long periods will increase the probability that it will be internalised and then damage intracellular proteins.
If you are harvesting the cells for other purposes such as getting protein for Westerns or looking for surface receptor expression, then scrapping will be the preferred technique.
Someone mentioned macrophage cell lines adhering to tissue culture plastic like glue. This is certainly true, but the way round that is to grow the cells in SUSPENSION CULTURE. Cell lines such as J774.A1 and Raw 264.7 grow fantasically well in suspension and therefore NEVER have to be trypsinised to passage on.
Another tip for cells that are difficult to trypsinise is to wash the cells in VERSENE (0.05% EDTA) a couple of times before the PBS"A" wash. We always do all our trypsinisations at RT and have never had a problem

yes ! it definitely depends on the cell type. ex. Caco -2 cell line grows in the form of a layer and requires trypsinization as long as 15-18 min. and MCF cell line on the other hand requires just 3-5 min.
also method of subculturing depends on the cell type. loosely adherent cell lines like SP2/O can be easily detached from the surface by tapping the flask gently.
scraping/ trypsinization for macrophages. trypsinization is the only method for Caco-2.
as aimikins said it depends on your cell line type, type of experiment. ex, for flow cytometry analysis surface molecules of your cells you cannot use trypsin . instead you can use hypotonic solution for disadhering your cells .
every method have their own advantages and disadvantages.
cheers

Hi,

Cells like SF-9 and other insect cells are so sensitive that neither trypsin nor cell scraper can be used to detach them. Both of these methods damage the insect cells. These cells are detached by means of tapping the flask or by pipetting the medium over the cell monolayer.

Where as most of the mammalian cells can be detached by trypsinization, scraping is a good option for cells which are sensitive to trypsin. Having said that researchers prefer to detach cells by scraper when the purpose of the experiment is to do western or enzyme assays.

Dear Exploresci,

We grow our Sf9 and Sf21 in suspension, plate for infection with virus, leave for 24hrs and then harvest by scraping. As long as your RPM is lower than 20, the cells grow like weeds in suspension. I can never understand why people persist in growing some cells as adherent cultures when they could save time, resources and effort growing them in suspension.

You need to use Biological stirring platforms like those from TECHNE (Cambridge UK). They operate in the humidified CO2 Incubator and have adjustable stirring speeds. The RPM is critical in that you do not want to spin too slowly and get cell aggregates, and you do not want to spin too quickly and get cell damage. The major advantage is that when you use the cells you do not need to trypsinise them... this gives the advantage of reducing the numbers of manipulations in the cabinets and that can drastically reduce contamination... and secondly reduces the spend on PBS, Trypsin, Tissue culture media and TC Flasks. The platforms and stirrer bottles are an initial expense, but I have had my platforms/stirrers for 15-20 years and have saved huge amounts. The bottles are autoclavable and can be easily re-siliconised when the glass deteriorates over time.

Also, a good couple of washes in PBS without calcium or magnesium makes trypsin more effective.
Additionally, using fresh trypsin that has not been repeatedly warmed up to 37 and then refrigerated is stronger than "worn out trypsin". I never heat my trypsin. Ever. For that specific reason.