Tina:General cell culture protocols: Difference between revisions

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(Created page with '==Feeder independent hES cells culture== ===Preparing Matrigel Solution=== 1. Thaw a stock Matrigel (5 ml/bottle) overnight (at 4°C). 2. Add 5 ml of DMEM/F12 (at 4°C) to the …')
 
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===Preparing Matrigel Solution===
===Preparing Matrigel Solution===


1. Thaw a stock Matrigel (5 ml/bottle) overnight (at 4°C).
1. Thaw the stock Matrigel overnight (at 4°C).


2. Add 5 ml of DMEM/F12 (at 4°C) to the Matrigel bottle. Pipet gently to avoid bubbles formation.
2. Add cold DMEM/F12 (1:1) to the Matrigel bottle. Pipet gently to avoid bubbles formation.


3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)
3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)
* It is critical to keep everything cold while handling Matrigel.




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1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use.
1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use.


2. Add 7 ml of DMEM/F12 (at 4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.
2. Add 7 ml of cold DMEM/F12 (4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.


3. Add 1 ml of Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly.
3. Add 1 ml of Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly.
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===Thawing hESCs on Matrigel plate===
===Thawing hESCs on Matrigel plate===


1. Add 5 ml pre-warmed hES media (37°C) to a conical tube.
1. Prepare Matrigel plate 1-2 h before thawing hESCs.  


2. Thaw hES vial at 37°C water bath (with small frozen portion left).
2. Add 5 ml pre-warmed mTeSR1 media (37°C) to a conical tube.


3. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES media.
3. Thaw hES vial at 37°C water bath (with small frozen portion left).


4. Centrifuge at 300g for 5 minutes.  
4. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES media.


5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.
5. Centrifuge at 300g for 5 minutes.  


6. After centrifuge is completed, discard the media without disturbing the pellet after that. Re-suspend pellet in 1 ml of hES media.
6. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of mTeSR1 media to the plate.


7. Add cell suspension to the Matrigel plate in a drop wise manner.
7. After centrifuge is completed, discard the media without disturbing the pellet after that. Resuspend pellet in 1 ml of hES media.
(Try to maintain cell aggregates during this process!)
 
8. Add cell suspension to the Matrigel plate in a drop wise manner.
   
   
8. Incubate the plate at 37°C incubator.
8. Incubate the plate at 37°C incubator. Medium should be changed daily.






===Passaging hESCs on Matrigel plate (HUES)===
===Passaging hESCs on Matrigel plate===


1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.
1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.


2. Pre-warm hES media and dispase at 37°C.
2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.


3. Aspirate the hES media and wash the plate with 1X PBS.
3. Aspirate media from the hESC culture and wash the plate with 1X PBS.


4. Aspirate the PBS and add 500 ul Dispase? Accutase (for single cell)/ collagenase IV (for colonies) to the plate and incubate at 37°C for 1 min.
4. Aspirate PBS and add 500 ul Dispase (for single cell suspension)/ collagenase IV (for clumps) to the plate and incubate at 37°C for 7 minutes.


5. Observe the cells under microscope. Edges of the colonies should start to curl.
5. Observe the cells under microscope. Edges of the colonies should start to curl.


6. Add 2 ml of hES media and pipette up and down to detach the cells completely.
6. Add 2 ml of DMEM/F-12 basal media and pipette up and down to detach the cells completely.


7. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.
7. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.


8. Aspirate the media and re-suspend pellet in n ml (n = number of plates to seed) of hES media.
8. Aspirate media and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.


9. Add cell suspension to the Matrigel plates in a drop wise manner.  
9. Add cell suspension to the Matrigel plates in a drop wise manner.  


10. Incubate the plate at 37°C incubator.
10. Incubate the plate at 37°C incubator. Medium should be changed daily.




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1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.
1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.


2. Pre-warm hES media and dispase at 37°C.
2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.


3. Aspirate the hES media and wash the plate with 1X PBS.
3. Aspirate media from the hESC culture and wash the plate with 1X PBS.


4. Aspirate the PBS and add 500 ul collagenase IV to the plate and incubate at 37°C for 1 min.
4. Aspirate PBS and add 500 ul collagenase IV to the plate then incubate at 37°C for 1-20 min (to be determined).


5. Observe the cells under microscope. Edges of the colonies should start to curl.
5. Observe the cells under microscope. Edges of the colonies should start to curl.
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6. Unwrap a Pasteur pipette and scrape the colonies off.
6. Unwrap a Pasteur pipette and scrape the colonies off.


7. Add 2 ml of hES media to collect cells.
7. Add 2 ml of DMEM/F-12 basal media to collect cells.


8. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.
8. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.


9. Aspirate the media and re-suspend pellet in n ml (n = number of plates to seed) of hES media.
9. Aspirate media and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.


10. Add cell suspension to the Matrigel plates in a drop wise manner.  
10. Add cell suspension to the Matrigel plates in a drop wise manner.  


11. Incubate the plate at 37°C incubator.
11. Incubate the plate at 37°C incubator. Medium should be changed daily.
 
 
 
===Cryopreservating hESCs===
 
Freezing media: 90% FBS + 10% DMSO
 
 
 
===Osteogenic differentiation media===
MEM containing 0-20% heat-inactivated ES-FBS (to be determined), 10−7 M Dexamethasone, 50 μg/ml Ascorbic acid and 10 mM β-glycerolphosphate

Revision as of 01:02, 5 April 2012

Feeder independent hES cells culture

Preparing Matrigel Solution

1. Thaw the stock Matrigel overnight (at 4°C).

2. Add cold DMEM/F12 (1:1) to the Matrigel bottle. Pipet gently to avoid bubbles formation.

3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)

  • It is critical to keep everything cold while handling Matrigel.


Preparing Matrigel Plates

1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use.

2. Add 7 ml of cold DMEM/F12 (4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.

3. Add 1 ml of Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly.

4. Leave the plate at RT for 1-2 h or put it at 4°C overnight.

  • Matrigel plates are good for 2 weeks at 4°C, although fresh plates are preferable.


Thawing hESCs on Matrigel plate

1. Prepare Matrigel plate 1-2 h before thawing hESCs.

2. Add 5 ml pre-warmed mTeSR1 media (37°C) to a conical tube.

3. Thaw hES vial at 37°C water bath (with small frozen portion left).

4. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES media.

5. Centrifuge at 300g for 5 minutes.

6. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of mTeSR1 media to the plate.

7. After centrifuge is completed, discard the media without disturbing the pellet after that. Resuspend pellet in 1 ml of hES media. (Try to maintain cell aggregates during this process!)

8. Add cell suspension to the Matrigel plate in a drop wise manner.

8. Incubate the plate at 37°C incubator. Medium should be changed daily.


Passaging hESCs on Matrigel plate

1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.

2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.

3. Aspirate media from the hESC culture and wash the plate with 1X PBS.

4. Aspirate PBS and add 500 ul Dispase (for single cell suspension)/ collagenase IV (for clumps) to the plate and incubate at 37°C for 7 minutes.

5. Observe the cells under microscope. Edges of the colonies should start to curl.

6. Add 2 ml of DMEM/F-12 basal media and pipette up and down to detach the cells completely.

7. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.

8. Aspirate media and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.

9. Add cell suspension to the Matrigel plates in a drop wise manner.

10. Incubate the plate at 37°C incubator. Medium should be changed daily.


Passaging hESCs on Matrigel plate mechanically (H1, H9)

1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of hES media to the plate.

2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.

3. Aspirate media from the hESC culture and wash the plate with 1X PBS.

4. Aspirate PBS and add 500 ul collagenase IV to the plate then incubate at 37°C for 1-20 min (to be determined).

5. Observe the cells under microscope. Edges of the colonies should start to curl.

6. Unwrap a Pasteur pipette and scrape the colonies off.

7. Add 2 ml of DMEM/F-12 basal media to collect cells.

8. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.

9. Aspirate media and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.

10. Add cell suspension to the Matrigel plates in a drop wise manner.

11. Incubate the plate at 37°C incubator. Medium should be changed daily.


Cryopreservating hESCs

Freezing media: 90% FBS + 10% DMSO


Osteogenic differentiation media

MEM containing 0-20% heat-inactivated ES-FBS (to be determined), 10−7 M Dexamethasone, 50 μg/ml Ascorbic acid and 10 mM β-glycerolphosphate