General

Maitanence - Freezing - Thawing

Maitanence

Material and reagent

1. 1xPBS
2. medium complete
3. trypsin/EDTA (T/E)
4. plate

Appropriate volumes for the cell culture vessel

• further information: Usefull numberes in cell culture

Protocol

1. Cells should be growing well or known to be in log phase (semi-confluent, aprox. 70 - 80 %)
2. Remove medium, wash once with pre-warmed PBS, decant it and add T/E
3. Incubate (3-5 min; 37°C), check the progress in microscope
4. Once the cells are rounded up and detached from the plastic surface, add pre-warmed media and vigorously wash any remaining cells from the bottom of the culture vessel
5. Transfer propriate volume of the cell suspension to a suitable culture vessel and add propriate volume of media
6. Incubate as usually (e.g. 37°C, 5% CO2)

ATCC Resources

Guide to Subculturing Cell Line Monolayers

Cell counting

Cell harvesting and freezing

Material and reagent

1. 1xPBS
2. medium complete
3. trypsin/EDTA (T/E)
4. cryoprotective medium (DMSO 10%; FBS 90%)
5. falcon (usualy 15mL-falcon)
6. cryovial(s)
7. Mr. Frosty device (pre-warmed to r.t.)

Note: Composition of cryoprotective medium can differ (see Usefull numberes in cell culture: cryoprotective medium)

Appropriate volumes for the cell culture vessel

Protocol

1. Cells should be growing well or known to be in log phase (semi-confluent, aprox. 70 - 80 %)
2. Remove medium, wash once with pre-warmed PBS, decant it and add T/E
3. Incubate 3-5 min at 37°C, check the progress of the enzyme treatment in microscope
4. Once the cells are rounded up and detached from the plastic surface, add pre-warmed media and vigorously wash any remaining cells from the bottom of the culture vessel
5. Transfer to falcon tube and centrifuge (1000 rpm; 3 min)
6. Decant supernatant, resuspend cell pellet by tapping and slowly add cryoprotective medium to give a final cell concentration of 1 to 2x106 cells/ml (usually 1 ml of cell suspension from 25cm2 per 1 cryovial)
7. Transfer 1 ml of cell suspension to appropriately labeled cryovial(s), place cryovial(s) into Mr. Frosty device (pre-warmed to RT) and place it to -80 C freezer for cca 24hrs
8. Then move the cryovials to appropriate storage place (-80 C freezer box for short term storage or to liquid nitrogen for long-term storage)

ATCC Resources

Cell thawing and recovery

Material and reagent

1. 1xPBS
2. medium complete
3. falcon (15ml-falcon)
4. plate

Appropriate volumes for the cell culture vessel

Protocol

1. Remove the cryovial from liquid nitrogen storage (in laminar hood, turn the cap a quarter turn to release any residual nitrogen that may be trapped, then re-tighten the cap), or from -80 C freezer and immediately transfer to 37°C water bath
2. While holding the tip of the cryovial, gently agitate the cryovial, being careful not to allow water to penetrate the cap or seal
3. When completely thawed, transfer contents of cryovial to falcone
4. Slowly add 5-10 mL pre-warmed media and centrifuge (1000 rpm; 3 min)
5. Decant supernatant containing the cryoprotective agent and resuspend the pellet in a volume of complete media appropriate for cell culture vessel
6. Transfer the cell suspension to a suitable culture vessel and incubate as usually (37°C, 5% CO2)

ATCC Resources

• Cryogenic Storage of Animal Cells
• Webinar: Best Practices in Cryopreservation

Usefull numberes in cell culture

Seeding

Seeding from literature

Plate	Surface area	Seeding density	Confluency	Trypsin	Growth medium
	(mm2)	Seeding density	Confluency	(mL)	(mL)
Dish
35 mm	962	0.3 x 106	1.2 x 106	0.5	1
60 mm	2 827	0.8 x 106	1.2 x 106	0.5	5
100 mm	7 854	2.2 x 106	1.2 x 106	1	10
150 mm	17 671	5.0 x 106	1.2 x 106	1	15
Well
96-well		XX x 106	1.2 x 106	1	1
24-well	200	0.05 x 106	1.2 x 106	1	1
12-well	401	0.1 x 106	1.2 x 106	1	1
6-well	962	0.3 x 106	1.2 x 106	1	1
Flask
T-25	2 500	0.7 x 106	2.8 x 106	3	1
T-75	7 500	2.1 x 106	8.4 x 106	5	1
T-160	16 000	4.6 x 106	18.4 x 106	10	1

Protocol

1. Remove medium, wash once with pre-warmed PBS, decant it and add T/E
2. Incubate 3-5 min at 37°C, check the progress of the enzyme treatment in microscope
3. Once the cells are rounded up and detached from the plastic surface, add pre-warmed media and vigorously wash any remaining cells from the bottom of the culture vessel
4. transfer propriate volume of cells onto a new plate
5. add upp prewarmed media
   
   optional step - for sensitive cells:
6. After trypsinisation; transfer to falcon tube and centrifuge (1000 rpm; 3 min)
7. Decant supernatant, resuspend cell pellet in pre/warmed media
8. transfer whole volume onto a new dish

Resources

• ATCC Culture Guides
• ATCC: Technical Documents
• ATCC Animal Cell Culture Guide
• ATCC: Video tutorial: Thawing, Culturing, and Cryopreserving Human Organoids

Acknowledgement

• Marketa Vančurová; Zdeněk Hodný group