Tina:Cardiomyocyte differentiation in H9 ESCs (CDH9): Difference between revisions

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*'''Cells''': Brainbow H9 constructs
*'''Cells''': Brainbow H9 constructs
==Experiment #1 (CDH9_E1_T1)==
==Experiment #1 (CDH9_E1_T1)==
*Study Purpose: Try 3 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
*Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
*Date: 4/10/2013 ~
*Date: 4/10/2013 ~
*Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3)
*Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded at 4/10. (1:6 dilution from the second passage of H9 ESCs under a feeder free culture system) OP9 cells were used as a negative control.
*Protocol used: Christian Metallo Lab protocol.  
*Protocol used: Christian Metallo Lab protocol.  
*Three induction medium used: Gln+ induction medium,DMK induction medium
*Two induction medium used: Gln+ induction medium,DMK induction medium
*Two cell density used: 100%, 80% confluency.
*Two cell density used: 100%, 80% confluency.
* Experimental design:
'''12-well plate #1'''
'''12-well plate #1'''
{| {{table}} border=1
{| {{table}} border=1
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| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''4'''
|-
|-
| align="center" style="background:#f0f0f0;"|A||Gln, repeat 1||Gln, repeat 2||Gln, repeat 3||Gln, repeat 4
| align="center" style="background:#f0f0f0;"|A||Gln, repeat 1||Gln, repeat 2||DMK, repeat 1||DMK, repeat 2
|-
|-
| align="center" style="background:#f0f0f0;"|C||DMK, repeat 1||DMK, repeat 2||DMK, repeat 3||DMK, repeat 4
| align="center" style="background:#f0f0f0;"|B||Gln, repeat 3||Gln, repeat 4||DMK, repeat 3||DMK, repeat 4
|-
|-
| align="center" style="background:#f0f0f0;"|B||Negative ctrl, repeat 1||Negative ctrl, repeat 2||Negative ctrl, repeat 3||Negative ctrl, repeat 4
| align="center" style="background:#f0f0f0;"|C||Negative ctrl, repeat 1||Negative ctrl, repeat 2||Negative ctrl, repeat 3||Negative ctrl, repeat 4
|-
|-
|}
|}
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| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''4'''
|-
|-
| align="center" style="background:#f0f0f0;"|A||Gln, repeat 1||Gln, repeat 2||Gln, repeat 3||Gln, repeat 4
| align="center" style="background:#f0f0f0;"|A||Gln, repeat 1||Gln, repeat 2||DMK, repeat 1||DMK, repeat 2
|-
|-
| align="center" style="background:#f0f0f0;"|C||DMK, repeat 1||DMK, repeat 2||DMK, repeat 3||DMK, repeat 4
|}
*Induction: 4/16 (Day 0). Cells reached 100% and 80% confluency, respectively. Aspirate mTeSR1 medium, wash once with PBS, add Gln+ and DMK induction medium accordingly. Pre-mix CHIR99021 (and DMK, for DMK group only) with culture media, then added to each well.
*4/17 (Day 1). Change medium (pre-mix DMK). Observed lots of cell death. Might due to the extremely high cell density and the sudden withdrawal of serum in differentiation medium.
*4/18 (Day 2). Still, lots of dead cells. Add 5 ul DMK to DMK group.
*4/19 (Day 3). Shake wells before change media to remove dead cells. Most of them has been removed after that. Change to combined medium.
*4/20~4/22. Change medium accordingly. Cells were still dying. But many survived also.
*4/23 (Day 7). Change to Insulin+ medium. Start to change medium every 3 days from now on.
*4/26 (Day 10). Observed one beating colony in 80% confluency, Gln+ group (Plate#2, A1). No signs of any other beating cells in other wells yet.
 
 
 
==Experiment #2 (CDH9_E1_T2)==
*Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
*Date: 4/20/2013 ~
*Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded at 4/20. ('''1:3''' dilution from the second passage of H9 ESCs under a feeder free culture system).
*Protocol used: Christian Metallo Lab protocol.
*Two induction medium used: Gln+ induction medium,DMK induction medium
*Two cell density used: 100%, 70% confluency.
'''24-well plate #1'''
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| align="center" style="background:#f0f0f0;"|'''4'''
|-
| align="center" style="background:#f0f0f0;"|A||100%, Gln, repeat 1||100%, Gln, repeat 2||100%, DMK, repeat 1||100%, DMK, repeat 2
|-
| align="center" style="background:#f0f0f0;"|B||100%, Gln, repeat 3||100%, Gln, repeat 4||100%, DMK, repeat 3||100%, DMK, repeat 4
|-
| align="center" style="background:#f0f0f0;"|A||70%, Gln, repeat 1||70%, Gln, repeat 2||70%, DMK, repeat 1||70%, DMK, repeat 2
|-
|-
| align="center" style="background:#f0f0f0;"|B||Negative ctrl, repeat 1||Negative ctrl, repeat 2||Negative ctrl, repeat 3||Negative ctrl, repeat 4
| align="center" style="background:#f0f0f0;"|B||70%, Gln, repeat 3||70%, Gln, repeat 4||70%, DMK, repeat 3||70%, DMK, repeat 4
|-
|-
|}
|}
*Cells seeded at 4/10. (1:6 dilution from the second passage of H9 ESCs under a feeder free culture system) OP9 cells were used as a negative control.
*Induction: 4/23 (Day 0). Cells reached 100% and 70% confluency, respectively. Start experiment. Mix CHIR99021 and DMK with medium before adding to each well.
*4/24 (Day 1). Change medium (pre-mix DMK). Still, observed lots of cell round-up. May be died or detached. can't tell.
*4/25~ . Change medium accordingly. Take pictures.

Revision as of 23:35, 26 April 2013

  • Purpose: To determine how Metallo Lab's modified cardiomyocyte differentiation protocol works on H9 ESCs.
  • Cells: Brainbow H9 constructs

Experiment #1 (CDH9_E1_T1)

  • Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
  • Date: 4/10/2013 ~
  • Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded at 4/10. (1:6 dilution from the second passage of H9 ESCs under a feeder free culture system) OP9 cells were used as a negative control.
  • Protocol used: Christian Metallo Lab protocol.
  • Two induction medium used: Gln+ induction medium,DMK induction medium
  • Two cell density used: 100%, 80% confluency.

12-well plate #1

100% confluency 1 2 3 4
A Gln, repeat 1 Gln, repeat 2 DMK, repeat 1 DMK, repeat 2
B Gln, repeat 3 Gln, repeat 4 DMK, repeat 3 DMK, repeat 4
C Negative ctrl, repeat 1 Negative ctrl, repeat 2 Negative ctrl, repeat 3 Negative ctrl, repeat 4

12-well plate #2

80% confluency 1 2 3 4
A Gln, repeat 1 Gln, repeat 2 DMK, repeat 1 DMK, repeat 2
  • Induction: 4/16 (Day 0). Cells reached 100% and 80% confluency, respectively. Aspirate mTeSR1 medium, wash once with PBS, add Gln+ and DMK induction medium accordingly. Pre-mix CHIR99021 (and DMK, for DMK group only) with culture media, then added to each well.
  • 4/17 (Day 1). Change medium (pre-mix DMK). Observed lots of cell death. Might due to the extremely high cell density and the sudden withdrawal of serum in differentiation medium.
  • 4/18 (Day 2). Still, lots of dead cells. Add 5 ul DMK to DMK group.
  • 4/19 (Day 3). Shake wells before change media to remove dead cells. Most of them has been removed after that. Change to combined medium.
  • 4/20~4/22. Change medium accordingly. Cells were still dying. But many survived also.
  • 4/23 (Day 7). Change to Insulin+ medium. Start to change medium every 3 days from now on.
  • 4/26 (Day 10). Observed one beating colony in 80% confluency, Gln+ group (Plate#2, A1). No signs of any other beating cells in other wells yet.


Experiment #2 (CDH9_E1_T2)

  • Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation.
  • Date: 4/20/2013 ~
  • Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded at 4/20. (1:3 dilution from the second passage of H9 ESCs under a feeder free culture system).
  • Protocol used: Christian Metallo Lab protocol.
  • Two induction medium used: Gln+ induction medium,DMK induction medium
  • Two cell density used: 100%, 70% confluency.

24-well plate #1

1 2 3 4
A 100%, Gln, repeat 1 100%, Gln, repeat 2 100%, DMK, repeat 1 100%, DMK, repeat 2
B 100%, Gln, repeat 3 100%, Gln, repeat 4 100%, DMK, repeat 3 100%, DMK, repeat 4
A 70%, Gln, repeat 1 70%, Gln, repeat 2 70%, DMK, repeat 1 70%, DMK, repeat 2
B 70%, Gln, repeat 3 70%, Gln, repeat 4 70%, DMK, repeat 3 70%, DMK, repeat 4
  • Induction: 4/23 (Day 0). Cells reached 100% and 70% confluency, respectively. Start experiment. Mix CHIR99021 and DMK with medium before adding to each well.
  • 4/24 (Day 1). Change medium (pre-mix DMK). Still, observed lots of cell round-up. May be died or detached. can't tell.
  • 4/25~ . Change medium accordingly. Take pictures.