Tina:General cell culture protocols: Difference between revisions

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==Feeder independent hES cells culture==
==General ES cell culture medium==
*'''mouse ESCs'''
1. [[mESCs culture medium]]


===Preparing Matrigel Plates===
2. [[mESCs freezing medium]]


1. Thaw a 145 ul aliquot of Matrigel at 4°C for 1-2 h before use.
*'''human ESCs'''
3. [[hESCs culture medium]]


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


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.
==Feeder dependent ESCs culture protocol==
*'''Coating'''
1. [[Preparation of gelatin coated plates]]


*'''MEF'''
2. [[MEF culture medium]]


3. [[Preparation of inactivated MEFs]]


===Thawing hESCs on Matrigel plate===
*'''mouse ESCs'''
4. [[Thawing mESCs on feeder]]


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.
5. [[Passaging mESCs on feeder]]


2. Thaw hES vial at 37°C water bath (with small frozen portion left).
*'''human ESCs'''
6. [[Thawing hESCs on feeder]]


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.
7. [[Passaging hESCs on feeder]]


4. Centrifuge at 300g for 5 minutes.  
*'''Cryopreservation'''
8. [[Cryopreservation of ESCs]]


5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of pre-warmed mTeSR1 media to the plate.
==Feeder independent ESCs culture protocol(Matrigel)==
1. [[Preparation of Matrigel plates]]


7. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of mTeSR1 media.
2. [[Preparation of MEF conditioned medium]]


8. Add cell suspension to the Matrigel plate in a drop wise manner.
3. [[Thawing hESCs on Matrigel plate]]
8. Incubate the plate at 37°C incubator. Medium should be changed daily.


4. [[Passaging hESCs on Matrigel plate (HUES)]]


 
5. [[Passaging hESCs on Matrigel plate mechanically (H1, H9)]]
===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





Latest revision as of 22:27, 7 May 2013

General ES cell culture medium[edit]

  • mouse ESCs

1. mESCs culture medium

2. mESCs freezing medium

  • human ESCs

3. hESCs culture medium

4. hESCs freezing medium


Feeder dependent ESCs culture protocol[edit]

  • Coating

1. Preparation of gelatin coated plates

  • MEF

2. MEF culture medium

3. Preparation of inactivated MEFs

  • mouse ESCs

4. Thawing mESCs on feeder

5. Passaging mESCs on feeder

  • human ESCs

6. Thawing hESCs on feeder

7. Passaging hESCs on feeder

  • Cryopreservation

8. Cryopreservation of ESCs

Feeder independent ESCs culture protocol(Matrigel)[edit]

1. Preparation of Matrigel plates

2. Preparation of MEF conditioned medium

3. Thawing hESCs on Matrigel plate

4. Passaging hESCs on Matrigel plate (HUES)

5. Passaging hESCs on Matrigel plate mechanically (H1, H9)


Osteogenic differentiation media[edit]

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