Tina:General cell culture protocols: Difference between revisions

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


1. Thaw Matrigel stock solution at 4°C, overnight.
2. [[mESCs freezing medium]]


2. Place 15 mL conical tubes and an unopened box of 1000 uL tips at -20°C, overnight.
*'''human ESCs'''
3. [[hESCs culture medium]]


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. [[hESCs freezing medium]]
4. Store aliquots at -70°C (for up to 6 months).


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




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


===Preparing Matrigel Plates===
*'''MEF'''
2. [[MEF culture medium]]


1. Thaw a 145 ul aliquot of Matrigel at 4°C for 1-2 h before use.
3. [[Preparation of inactivated MEFs]]


2. Add 12.5 ml of cold DMEM/F12 (4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.
*'''mouse ESCs'''
4. [[Thawing mESCs on feeder]]


3. Add 1 ml of the Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly.
5. [[Passaging mESCs on feeder]]


4. Leave the plate at RT for 1-2 h or put it at 4°C overnight.
*'''human ESCs'''
6. [[Thawing hESCs on feeder]]


* Matrigel plates are good for 1 weeks at 4°C, although fresh plates are preferable.  
7. [[Passaging hESCs on feeder]]


*'''Cryopreservation'''
8. [[Cryopreservation of ESCs]]


==Feeder independent ESCs culture protocol(Matrigel)==
1. [[Preparation of Matrigel plates]]


===Thawing hESCs on Matrigel plate===
2. [[Preparation of MEF conditioned medium]]


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.
3. [[Thawing hESCs on Matrigel plate]]


2. Thaw hES vial at 37°C water bath (with small frozen portion left).
4. [[Passaging hESCs on Matrigel plate (HUES)]]


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


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.
==Osteogenic differentiation 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
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

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