Tina:General cell culture protocols: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>TinaLo
No edit summary
>TinaLo
 
(7 intermediate revisions by the same user not shown)
Line 1: Line 1:
==Feeder independent hES cells culture==
==General ES cell culture medium==
===Preparing Matrigel Solution===
*'''mouse ESCs'''
1. [[mESCs culture medium]]


1. Thaw the stock Matrigel overnight (at 4°C).
2. [[mESCs freezing medium]]


2. Add cold DMEM/F12 (1:1) to the Matrigel bottle. Pipet gently to avoid bubbles formation.
*'''human ESCs'''
3. [[hESCs culture medium]]


3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)
4. [[hESCs freezing medium]]


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


2. Add 7 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 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 2 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.
3. [[Thawing hESCs on Matrigel plate]]


2. Add 5 ml pre-warmed mTeSR1 media (37°C) to a conical tube.
4. [[Passaging hESCs on Matrigel plate (HUES)]]


3. Thaw hES vial at 37°C water bath (with small frozen portion left).
5. [[Passaging hESCs on Matrigel plate mechanically (H1, H9)]]


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.
==Osteogenic differentiation media==
 
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
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