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Purpose
Purpose
The goal of this lab is to learn the procedure of preparing cells for cryopreservation in liquid nitrogen and then bringing them out of liquid nitrogen.
Materials Required
HEK 293 Cell Line in 10 cm plate
Media:MEM, 10% FBS + PSG
Freeze down media: 10ml MEM, 10% FBS+PSG, 10% DMSO
Trypsin/EDTA
change pic: 15 ml tubes
One Nunc cryo tube
Two 75 cm2 flask
Pipet aid
Assorted 1,5,10, and 25 ml pipets
Varistaltic system and pasteur pipets
Biosafety II hood
Water bath at 37 degree Celsius
Cryosafe
Clinical centrifuge
-80 degree freezer
Procedure
Media and trpsin/EDTA were warmed up for 15 minutes in water bath at 37 degree Celsius
Biosafety hood and vacuum system were turned on
After thoroughly rinsing media bottle and TE with 70% EtOH, they were placed in biosafety hood
Media off cells was sucked up with pasteur pipet and then 4ml TE was added to cells
While cells were incubating for 5-10 mins at 37 degree Celsius, 10 ml media was added in 15ml tube and flask
After cells were lifted, cells+TE was transferred to 15 ml tube containing 10 ml of media
Cells were centrifuged at 30 speed for 3 mins and then rinsed with 70% EtOH and returned to hood
Carefully supernatant was sucked with pasteur pipet and cell pellet was gently resuspended by tituration with 8 ml of media
Cells were mixed and 0.5ml were transferred to flask containing 10ml of fresh media
Cells were mixed in 15ml tube and 20 microlitre of cells were counted with hemacytometer
Calculations were carried out as follow:
Calculate total number of cells in tube and volume of freeze down media needed to resuspend cells at concentration between 1x10^6 to 1x10^7 cells per ml:
count and add cells in all 4 sections of 16 squares: 60 + 50 + 55 + 35 = 200
take average: 200/4 = 50
Multiply by 10^4: 50 x 10^4 = 500,000 cells/ml
500,000 cells/ml x 7.5ml in tube = 3,750,000 cells in tube
volume of freeze down media needed: 3,750,000 cells/1000000 cells/ml =3.75 ml
While cells in 15ml tube were centrifuging at 30 speed for 3 mins, nunc cryo tube was labelled
After centrifuging, supernatant was sucked off with pasteur pipet and cells were resuspended with enough calculated freeze down media
After resuspending cells in freeze down media, using 1ml pipet, 1ml of cells were transferred to nunc cryo tube
Tube was placed in freeze down box located in -80 degree Celsius freezer
After more than 24 hours, nunc cryo tube with cells was transferred into the liquid nitrogen. The process of lowering the cells in liquid nitrogen is shown below in first video:
VID_20211015_125458.mp4VID_20211019_102036.mp4After three days in liquid nitrogen, cells were brought up using the same procedure as shown above in second video
Hood was ready for working with the freeze down, media was warmed up and 10ml of media was ready in 15ml tube
Cap was tightened on nunc cryo tube and freeze down tube was warmed up in water bath by rocking it back and forth
Once ice is melted, tube was sprayed down with 70% EtOH and placed in nunc tube rack in hood
Using 1ml pipet, cells were carefully resuspended and transferred to 15ml tube containing 10ml of media
Cells were centrifuged to remove DMSO cryoprotectant at 30 speed for 3mins and then supernatant was sucked off with pasteur pipet
Cells were resuspended in 10ml of media and 10ml of cells were transferred to 75 cm^2 flasks
To remove any residual DMSO from media in flask, media from flask was removed next day and 10ml fresh media was added to cells
After cells were allowed to grow for two days, picture of cells was taken using infinity capture
Results
Cells were allowed to grow for atleast two days after removing DMSO and adding fresh media
Picture was taken of cells using infinity capture microphotography
Conclusion
This lab was successful in cryopreserving the HEK 293 cells.
This lab allowed to learn the procedure of cryopreserving the cells in liquid nitrogen and then how to take them out to carry the experiment forward.
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