HeLa cell culture - (Nov/11/2013 )

Greetings everyone!

I recently cultured some HeLa cells as practice as I am relatively new in the lab.

Day 1 (Friday): Started with fast-thawing cells in a water bath (37 deg Cel).  Pipette cells in to a 15-cm culture dish of 28 mL DMEM culture medium. Left to incubate over the weekend. 

Day 4 (Monday): This was what I observed .  Aspirated the cell suspension.  Wash with PBS and then trypsinize (coupled with incubation 37 deg Cel) followed by neutralizing with DMEM.  Spin down 1.2k rpm for 4 min in swing bucket centrifuge.  Followed by transferring to a 10-cm culture dish containing 6 mL DMEM.  Allowed to incubate overnight.

Day 5 (Tuesday): This was what I observed . Subsequently, changed DMEM medium.  This time using 8 mL of DMEM.  Left to incubate overnight.

DMEM used was consistent.

Would like to know if I am on the right track?  I deviated quite a lot from the standard protocol especially when it comes to the amount of PBS, trypsin and DMEM used when transferring the cells from 15-cm dish to 10-cm dish.  I used 10 mL PBS to wash the cells in the 15-cm dish before aspirating it and adding 8 mL of trypsin.  Subsequently, I used 10 mL of DMEM for neutralization.  Attaining a total of 28 mL in the 15-cm dish.  I then pipetted half, 14 mL into a 15 mL Falcon tube for centrifugation to pellet the cells before discarding the supernatant and then later transferred cells into a 10-cm dish of 6 mL of DMEM.

Basically, no - the cells you see at day 4 are not healthy and are very few, this may be due to a few reasons, but the most likely is that you didn't seed many in the first place.  You would normally leave them until the cells cover about 70-80% of the area of the dish before re-seeding them (referred to as passaging).  If you re-seed when they are at low density and remain at low density, the cells will often not survive.

Having said that. HeLa are incredibly tough, so the fact that you killed them means that you need more practice.  The easiest way for you to get experience is to observe someone experienced in cell culture, and have them observe you as you perform it too.

The thing about trypsinising cells is that you need to use a small amount and neutralize it with an excess of medium so that you don't dilute the medium too much - this will result in either osmotic shock (if your trypsin isn't in PBS or similar), or in a lowered amount of nutrients for the cells (diluted in the trypsin).  

You might need to find a way to removed and cryopreservant. I use DMSO and depending on the cell line I spin down and change media or I inoculate into a T25 and change the media within 18 hours to remove the DMSO.

Thank you for your replies.  Well, I just checked my cells today.  This is what I got. 

- Day 6

compared to...

- Day 1

It seems to have grown?

Are the cells for Day 6 about 70-80% confluent?

Thank you.

Some obviously survived - good, it shows that you aren't doing it completely wrong.  However - have a think about this - if you took a population of mice and hit them all with a drug at a level that would kill 90% of the mice, and repeated that for 10 generations - would you expect the mice you got out the end to be the same as the mice you started with?  So would you now trust your cells to be HeLa?

bob1 on Wed Nov 13 08:24:20 2013 said:

Some obviously survived - good, it shows that you aren't doing it completely wrong.  However - have a think about this - if you took a population of mice and hit them all with a drug at a level that would kill 90% of the mice, and repeated that for 10 generations - would you expect the mice you got out the end to be the same as the mice you started with?  So would you now trust your cells to be HeLa?

That means to say that there could be a possibility of differentiation? That was a very good analogy btw.  I understand what you mean now.

Thank you.

Not necessarily differentiation, but more lab based selection/evolution.  It's pretty well recognized now, that lab based evolution of cell lines and bacterial strains is a big problem for the life sciences, so many journals now ask that your cell lines be evaluated to ensure they are what you say they are, before they will publish you.  

So in my analogy above, I was trying to get you to see that you have just selected for a population of cells that are resistant to excess trypsin and have the  ability to grow under low density conditions, which is not necessarily what HeLa are capable of (they are certainly capable of growing under low density, but the trypsin thing???).  So if you used these cells for an experiment, you might get a result, which when you repeated it with HeLa that have been treated in a more conventional manner, gave you a different result - which one of these is the true result??