Tina:Osteogenic differentiation on mES brainbow cells (OSG)
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Seeding density test
- Purpose: To determine the seeding density of mESCs for osteogenic differentiation
- Test #1:
- Date: 7/5/2012 ~ 7/7/2012
- Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
- 7/5: P10 mES brainbow cells (on feeder) were trypsinized. Cells were then divided into two groups. Group one were seeded 1:2, 1:4, 1:6, 1:8 and 1:12 on gelatin coated glass-well immediately. Group two were let sit for 15 min and then seeded 1:2, 1:4, and 1:6 on gelatin coated glass-well.
- 7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.
- According to results, seeding at 1:6 with 15 min sitting time is the best seeding density for osteogenic induction (one day after seeding).
Osteogenic differentiation media composition test
- Purpose: To test the effects of osteogenic differentiation media on mESCs
- Test #1:
- Date: 7/1/2012 ~ 7/9/2012
- Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media (with 1% serum):
Component | Volumn | Final concentration |
MEM | 193ml | |
Serum | 2ml | 1% |
Dexamethasone | 200ul of 0.1mM stock | 0.1uM |
Ascorbic acid | 1ml of 10mg/ml stock | 50ug/ml |
b-GP | 2ml of 1M stock | 10mM |
PenStrep | 2ml | 1% |
Total | 200 mL |
- 7/1: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 10 min, and then seeded 1:5 (two wells) and 1:10 (4 wells) onto feeder free system (now become P10).
- 7/2 and 7/3: Cell density was still low. Induction should wait for one more day.
- 7/4: Cell density became too high. Several colonies were formed. Therefore, the best time point to induce osteogenic differentiation is ‘’1-2 days after seeding’’’ (need further confirmation).
- Add induction media to each well on 7/4 to observe the cell morphology changes at the beginning of osteogenic differentiation.
- 7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.)
- 7/6: Possible cause of cell death: (1)chemical concentration is too high (toxic). (2)serum concentration is too low.
Further tests should be arranged (with lower chemical concentration and/or higher serum concentration).
- Test #2:
- Date: 7/13/2012 ~ ongoing work
- Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media:
Basal media were changed from MEM into DMEM. Different concentration of serum were tested.
Component (volumn)/group | 5% serum | 15% serum | 2me |
DMEM | 193ml | 193ml | 193ml |
Serum | 5%(10ml) | 15%(30ml) | 15%(30ml) |
0.1 uM Dexamethasone | 200ul of 0.1mM stock | 200ul of 0.1mM stock | 200ul of 0.1mM stock |
50ug/ml Ascorbic acid | 1ml of 10mg/ml stock | 1ml of 10mg/ml stock | 1ml of 10mg/ml stock |
10mM b-GP | 2ml of 1M stock | 2ml of 1M stock | 2ml of 1M stock |
1% PenStrep | 2ml | 2ml | 2ml |
1% Glutamax | - | - | 2ml |
1% NEAA | - | - | 2ml |
0.1% 2me | - | - | 200ul |
Total | 200 mL | 200 mL | 200 mL |
- 7/13: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 15 min, and then seeded 1:6 (three wells) onto feeder free system (now become P10).
- 7/14(Day 0): PBS wash twice and then add induction media to each well.
- 7/15-7/16: Take pictures. Cells look ok. Scheduled to change media three days after induction.
- 7/17 (Day 3): Take pictures. Observed some cell death. Change media.
- 7/18: Take pictures. Number of cell death were under control. Important finding: Cell death occurred less in 5% serum group. This might suggests that cell death may be due to lack of nutrition. Managed to change induction media daily hereafter.
- 7/19-7/23: Change media and take pictures daily.