Tina:General cell culture protocols

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Revision as of 19:27, 5 April 2012 by >TinaLo
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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 mTeSR1 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. 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