Tina:General cell culture protocols: Difference between revisions

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==Feeder independent hES cells culture==
==Feeder independent hES cells culture==
===Preparing Matrigel Solution===
===Preparing Matrigel Aliquots===


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


2. Add cold DMEM/F12 (1:1) to the Matrigel bottle. Pipet gently to avoid bubbles formation.
2. Place 15 mL conical tubes and an unopened box of 1000 uL tips at -20°C, overnight.


3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)
3. Aliquot 145 uL of Matrigel stock solution to a 15 mL tube as quick as possible. Switch tips every 5 tubes to ensure sterility and that the tips are chilled.
4. Store aliquots at -70°C (for up to 6 months).


* It is critical to keep everything cold while handling Matrigel.
* It is critical to keep everything cold while handling Matrigel.
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===Preparing Matrigel Plates===
===Preparing Matrigel Plates===


1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use.
1. Thaw a 145 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.
2. Add 12.5 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 the 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.
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.
* Matrigel plates are good for 1 weeks at 4°C, although fresh plates are preferable.  




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===Thawing hESCs on Matrigel plate===
===Thawing hESCs on Matrigel plate===


1. Prepare Matrigel plate 1-2 h before thawing hESCs.  
1. Prepare Matrigel plate 1-2 h before thawing hESCs. If cooled Matrigel plates are used, place the plates at room temperature for at least an hour prior to plating cells.


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


3. Thaw hES vial at 37°C water bath (with small frozen portion left).
3. Add 5 mL pre-warmed DMEM/F-12 media (37°C) to a conical tube. Transfer cells to the conical tube and wash the vial with another 5 mL of DMEM/F-12 media.


4. Trans cells to the conical tube and wash the vial with an extra 1 ml of mTeSR1 media.
4. Centrifuge at 300g for 5 minutes.  


5. Centrifuge at 300g for 5 minutes.  
5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of pre-warmed mTeSR1 media to the plate.


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. Resuspend pellet in 1 ml of mTeSR1 media.  
 
7. After centrifuge is completed, discard the media without disturbing the pellet. 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. Add cell suspension to the Matrigel plate in a drop wise manner.
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===Passaging hESCs on Matrigel plate===
===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 mTeSR1 media to the plate.


2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.
2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.
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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 media and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.
8. Aspirate PBS 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.  
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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 media and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.
9. Aspirate PBS 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.  

Revision as of 01:12, 14 April 2012

Feeder independent hES cells culture

Preparing Matrigel Aliquots

1. Thaw Matrigel stock solution at 4°C, overnight.

2. Place 15 mL conical tubes and an unopened box of 1000 uL tips at -20°C, overnight.

3. Aliquot 145 uL of Matrigel stock solution to a 15 mL tube as quick as possible. Switch tips every 5 tubes to ensure sterility and that the tips are chilled.

4. Store aliquots at -70°C (for up to 6 months).

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


Preparing Matrigel Plates

1. Thaw a 145 ul aliquot of Matrigel at 4°C for 1-2 h before use.

2. Add 12.5 ml of cold DMEM/F12 (4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.

3. Add 1 ml of the 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 1 weeks at 4°C, although fresh plates are preferable.


Thawing hESCs on Matrigel plate

1. Prepare Matrigel plate 1-2 h before thawing hESCs. If cooled Matrigel plates are used, place the plates at room temperature for at least an hour prior to plating cells.

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

3. Add 5 mL pre-warmed DMEM/F-12 media (37°C) to a conical tube. Transfer cells to the conical tube and wash the vial with another 5 mL of DMEM/F-12 media.

4. Centrifuge at 300g for 5 minutes.

5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of pre-warmed mTeSR1 media to the plate.

7. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of mTeSR1 media.

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 mTeSR1 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 PBS 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 PBS 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