Hi, I would like to grow HeLa cells in suspension since you can grow up more culture then as adherent cells. Therefore, I am wondering what I need to do with my HeLa cells which are growing adherent on plates at the moment? Do I need to use RPMI or DMEM media or some special media to get them by stirring into suspension culture? I really appreciate any advice from people who have done this already. Thanks in advance, Nicole
There are HeLa variants that exist for suspension culture but you can use your own adherent HeLa cells and turn them into a suspension line - it just requires time. I suggest using ExCell media from Sigma. Being grown in suspension may change your biology - as you will lose/modify anchorage/ integrin dependent processes in these cells.
1.Prep your cells by slowly lowering the serum in your culture over of the course of a week or two until cells are healthy and growing normally in about 2-3% serum - now dissociate cells using your standard protocol. Plate them for 48 hours in ExCell media from Sigma. Trypsinize and check viability.
2. Seed three to six magnetic spinner flasks with ~ 5 x 10(5) viable cells/ml. Culture normally overnight with gentle spin in ExCell media.
3.Observe the culture the next day - some cells may be attached to sides or spinner -just ignore these cells - only remove the liquid material - this contains all your potential cells for reculture. Count cells for viability - if you have at least 50% - that's ok - if there is a high number of dead cells - you can run a quick ficoll gradient to remove the dead cells and resuspend only the live cells. If the cells in suspension are clumped, they can be dispersed gently with versene and pipetting, collected by centrifugation, and then reseeded into the flask at the appropriate density. Pool suspensed cells from 2-3 spinner flasks into a single new spinner flask with fresh medium at 2.5 x 105 cells/ml.
4.After three days, collect the cells growing in suspension by centrifugation. Repeat. Check viability (it should be at least 85-90% by the second round).
5.It may take 2-3 weeks to get a culture going strong - depends on your cells. You know they are ready when they are growing at a consistent rate. For HeLa- it is about 23-24 hour doubling time in suspension. Now they can be moved into normal plastic dishes in the ExCell media - just be careful when passaging them not to disturb any cells that may get sticky - every now and then you may get cells that start to stick to the bottom.
Good luck.
There are HeLa variants that exist for suspension culture but you can use your own adherent HeLa cells and turn them into a suspension line - it just requires time. I suggest using ExCell media from Sigma. Being grown in suspension may change your biology - as you will lose/modify anchorage/ integrin dependent processes in these cells.
1.Prep your cells by slowly lowering the serum in your culture over of the course of a week or two until cells are healthy and growing normally in about 2-3% serum - now dissociate cells using your standard protocol. Plate them for 48 hours in ExCell media from Sigma. Trypsinize and check viability.
2. Seed three to six magnetic spinner flasks with ~ 5 x 10(5) viable cells/ml. Culture normally overnight with gentle spin in ExCell media.
3.Observe the culture the next day - some cells may be attached to sides or spinner -just ignore these cells - only remove the liquid material - this contains all your potential cells for reculture. Count cells for viability - if you have at least 50% - that's ok - if there is a high number of dead cells - you can run a quick ficoll gradient to remove the dead cells and resuspend only the live cells. If the cells in suspension are clumped, they can be dispersed gently with versene and pipetting, collected by centrifugation, and then reseeded into the flask at the appropriate density. Pool suspensed cells from 2-3 spinner flasks into a single new spinner flask with fresh medium at 2.5 x 105 cells/ml.
4.After three days, collect the cells growing in suspension by centrifugation. Repeat. Check viability (it should be at least 85-90% by the second round).
5.It may take 2-3 weeks to get a culture going strong - depends on your cells. You know they are ready when they are growing at a consistent rate. For HeLa- it is about 23-24 hour doubling time in suspension. Now they can be moved into normal plastic dishes in the ExCell media - just be careful when passaging them not to disturb any cells that may get sticky - every now and then you may get cells that start to stick to the bottom.
Good luck.
use trypsin to remove cells from the place. Consult appropriate papers. Do not know the details.
The simplest way is to use bacteriological petri dishes instead of those used in cell culture. Trypsinized HeLa cells will not adhere to their surface (at least those I used so far).
Ales prokop has suggested to use trypsin to remove cells from the place. We did as follows
1.Preparation of 10 x Trypsin stored buffer : 80 g NaCl, 3 g KCl 0.73 g Na2HPO3 x 2 H2O, 0.2 g KH2PO320 g, Glucose, 7 g EDTA , fill with endotoxin-free water to 1 Litre, Adjust pH 7.2 .
2.Preparation of working solution: Dilute stock solution 1:10 with sterile distilled water. Store at +4 °C. Durability approx. 1 week. The microorganisms are removed by sterile filtration. Good lock.
Give it to the little trypsin treatment in cold conditions so that cell will seperate from each other with gental shaking.
Trypsinize the cells as you would when you are sub-culturing, spin down and resusupend in RPMI and then simply put them in a vessel which is continuously in motion so they don't have a chance to attach.Anything suitable ie roller bottle or stirring or shaker.Good luck
All the above are good suggestions, and spinner cultures are ideal for this, but if you are trying to do it on the cheap, just tape a broken piece of pipette onto the top of your flask such that when you fill it with media with cells in, it does not reach the neck. Once they are growing, don't ever let them stick down again, because they will!
The key to growing cells as single cells in suspension is to use divalent cation-free media (i.e., no Ca+2 or Mg+2). Invitrogen/Gibco has Eagle's MEM (with Earle's salts, etc.) in both forms - for monolayer and for suspension culture. The shift from one format to the other usually taks a few generations to select for those best adapted to the new condition. Suspension cultures usually are grown in lower serum concentration (E.g., 5% suspension, 10-15% monolayer). Thie culture condition and serum concentration do affect several physiological features, particularly well seen in fibroblasts: e.g., the monolayer condition lets the cells spread out into a flat pancake-like morphology that changes the distances between chromosomes (2D vs 3D in suspension) and hence radiation sensitivity and chromosome exchange aberration frequencies. Thus, the altered culture conditions may not be physiologically equivalent even if the cells are the same. See: Trypsin-induced changes in cell shape and chromatin structure result in radiosensitization of monolayer Chinese hamster V79 cells. M. Kapiszewska, N.M.S. Reddy and C.S. Lange. International J. Radiat. Biol., 60(4), 635-646; Is there a cell-to-cell contact effect on the X-ray dose-survival response of mammalian cells? N.M.S. Reddy, K.A. Cieszka, S. Rozenblyum, and C.S. Lange. Scan. Microscopy, 8(3), 621-629; Nutrient dilution before and after X irradiation increases the radioresistance of log- and plateau-phase Chinese hamster V79 cells. N.M.S. Reddy, P.J. Mayer, D. Nori, and C.S. Lange. Radiat. Res., 147, 115-118.
Further to my note above, another option is to grow the cells as adherent cells (same media, etc.) but on microbeads. They attach to the beads, which are kept in suspension by a slowly rotating impeller. This has a near equivalent of the suspension culture densities and yields, but with the same physiology as with the adherent monolayer cultures. One trypsinizes to release the cells from the beads, lets them settle, and collects the cell-containing supernatant for centrifugation and replating (in either fomat). For an in vivo-like situation, though, one needs 3D cell:cell contacts and spheroids are another possible model.
Dear Nicole,
If you are trying to get a lot of Hela's on minimum space, you might use an adapted version of the FibraStage system of New Brunswick. They have kind of porous pellets in which the cells go in, adhere but the space is highly reduced. Of course they would like you to buy their bottles (VERY expensive: €1100 per bottle. one bottle had replaced 37 175 cm2 bottles in our incubator), but If I don't remember wrong, they sell the pellets sterile and coated (I think) so you would not have to adapt your cells or risk that their methabolism has changed or their growth rate.
Good luck
I think u go for mechanical dissociation. then add these cells to Mg and Ca free media with low serum content. but one thing Hela cells r robust in natur. They even grow serum deprived media u can try wid L-15+ 1% serum + 1% antibiotics as basic media composition. All the very best.
- Badges
- Science topic