Hello everyone,
I’m start working on HEK293 cel culture and They have very slow growth rate. today is the the third day of cell culture even though the media DMEM contain 10% FBS penstrept and glutamine. how can I reach 90% confluence with healthy cell?
It looks like seeding cell density is too low. There is an RG discussion that may be helpful:
https://www.researchgate.net/post/What-is-the-optimum-HEK-293T-cell-seeding-cells-cm2-for-large-scale-culture-and-lentiviral-production
Hello Hind Alotaibi
Sometimes, incorrect thawing procedure could lead to loss of cell viability and slow growth of cells. Usually, thawing is done by placing the freezing vial in water bath at 37 degree C. On thawing, the cell suspension is added drop by drop to
pre-warmed culture medium and centrifuged at low speed to remove DMSO present in the freezing medium. The cell pellet is tapped and added to T-25 flask in 4ml culture medium. Usually after a few hours the cells attach to the substratum. After attachment, the culture medium is replaced with fresh medium to remove the traces of DMSO.
For HEK293 cells you should use DMEM (with high glucose) supplemented with 10% fetal bovine serum (FBS).
At times, freezing of cells if not done in a proper manner with gradual cooling and use of proper freezing medium, there is a high likelihood that on thawing you may obtain a low viability count and the cells may not be healthy.
From the pic, the cells seems to be too less. I would suggest for getting enough healthy cells you need to trypsinize these cells after a day or two and re plate them in a 6-well plate or 35mm culture dish since the cell density is low. Once you have enough cells you can move to a bigger flask like T-75 flask.
I hope this will help.
Good Luck.
Malcolm Nobre hello thank you so much for your answer and I want to ask about the speed the centrifuge what do you suggest and the for how long I used to use 1000 rpm for 5 min.
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