Sf9/21; how do they attach to the surface? - (May/23/2007 )

Hi.

I was wondering how insects cells attach to the surface. I grow sf9 cells in adherent cultures and I find it difficult to dislodge the cells by sloughing or tapping the flask. I read that subculturing by scraping the cells reduces cell's viability. In this regard, I was wondering if one can dislodge insect cell by trypsinazing them..

Many thanks in advance.

Anna

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According to Eckert, Insect cell morphology includes basement membranes and basolateral clefts and although insect systems (gas exchange, osmosis, etc) vary from those of mammals, many of their cellular mechanisms are similar.

Since Trypsin as a protease is specific to peptides in which a carboxyl group is provided by a arginine or lysine (Eckert Animal Physiology) and these peptides are found in basement membrane proteins, treating a cell matrix with Trypsin should work on insect cells as well as mammalian.

In our lab, we never use mechanical separation techniques because they may give false positives when measuring apoptosis (cells are lysed during rough mechanical treatment). I would give them a shot of trypsin and see what happens.

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We grow our sf9/21 cells in SUSPENSION...it works great. No need to scrape or trypsinise. Look at getting TECHNE stirrer bottles and stirring platforms.
Advantages for suspension culture:

i) Buy a stirrer bottle and re use 10,000 times...saves money on TC plastic.
ii) No trypsinisation...therefore saving on Trypsin, PBS, media etc.
iii) Subculturing takes alot less TIME...so money saved there.


There seems to be a lack of knowledge about how to grow cells in suspension out there. This is the second reply I have made today about suspension cultures. Your supervisors should know this information?

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Hi Cabinboy and Rhombus!

Thanks for your replies! I have one more question. Do I have to have cells at viability 95% or greater to start growing cells in suspension, as Invitrogen manual suggests ?
I am growing cells to initiate suspension culture. In the lab we have only 500ml- and 1000ml-spinner flasks. Minimum volume 50 ml (for 500ml spinner flask)So I have to grow up adherent cells for inoculation. In Invitrogen manual, they strongly recommend to use ONLY cells with 95% viability or above. It's my 8 passage but the cells are between 75- 85% viability. Do I really have to have cells at viability 95% or greater to start growing cells in suspension??
I sacrificed one vial to check viability of attached cells with trypan blue (took the medium out, added 1:10 diluted trypan blue sol'n).
The cells are ~ 90-92 % viable but after I dislodge them, the viability drops.
I tried
1) tapping the flask - only 20% of the cells detach..., tried to hit the flask against bench top hard... but cells hold on tightly and do not detach...
2) I tried to slough them by repeatedly pipetting medium over cell monolayer... takes a lot of time and viability is 80-85% .
3) I tried to use scrappers ... viability is 80-85%....

That is the reason why I asked if it's possible to add some trypsin to dislodge the cells. So, let me ask my question again : Do I have to have cells at viability 95% or greater to start growing cells in suspension??

Thank you again for your help!!

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Dear Anna122,

I have not ever heard about this 95% Viability. I initiate my frozen cells directly into the stirrer bottle. The viability from frozen IS ALWAYS below 95% and my cells are still fine. I do every 2 months throw my cells away and initiate a freh bottle. The sf9 /21 when on plastic have 2 distinct morphologies:
i)Fibroblastic
ii) Round and golden appearance.

The ratio should be 80% Round and Golden, 20% fibroblastic. Over time this ratio reverses and the level of infection goes down. This is why you need to get fresh cells out every 2 months.

P.S.
I always use New Zealand FCS, it is the best quality and will always give higher viability and infectivity of your insect cells.

Have fun growing your cells
.
Rhombus

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