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Tina:Osteogenic differentiation on mES brainbow cells (OSG)
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=='''Seeding density test (OSG_E1)'''== *'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation <br> === Test #1 (OSG_E1_T1) === *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). =='''Medium composition test (OSG_E2)'''== *'''Purpose:''' To test the effects of osteogenic differentiation media on mESCs <br> === Test #1 (OSG_E2_T1)=== *Date: 7/1/2012 ~ 7/9/2012 *Cells: P10 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media (with 1% serum): {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volumn''' | align="center" style="background:#f0f0f0;"|'''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% |- | style="background:#f0f0f0;"|Total | style="background:#f0f0f0;"|200 mL | style="background:#f0f0f0;"| |} *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 ββ1-2 days after seedingβββ (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 (OSG_E2_T2) === *Date: 7/13/2012 ~ 7/27/2012 *Cells: P10 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media: Basal media were changed from MEM to DMEM. Different concentration of serum were tested. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Component (volumn)/group''' | align="center" style="background:#f0f0f0;"|'''5% serum''' | align="center" style="background:#f0f0f0;"|'''15% serum''' | align="center" style="background:#f0f0f0;"|'''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 |- | style="background:#f0f0f0;"|Total | style="background:#f0f0f0;"|200 mL | style="background:#f0f0f0;"|200 mL | style="background:#f0f0f0;"|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. *7/19-7/26: Change media and take pictures daily. *7/27: Perform Alizarin Red S staining. (Fixed with 3.7% paraformaldehyde for 20 min; washed with Ca- Mg- PBS; stained for 1 hour.) === Test #3 (OSG_E2_T3) === *Date: 7/30/2012 ~ 8/3/2012 *Cells: P12 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media: 0, 2.5, 5, 7.5, 10, 12.5, and 15% serum in osteogenic differentiation media were tested. *7/30: P11 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:6 onto gelatin coated 6-well plates (now become P12). *7/31(Day 0): PBS wash twice and then add induction media to each well. *Change media daily. *8/3 (Day 3): Cell proliferated too fast. Cell colony became very big. It's really difficult to observe osteogenic differentiation. Therefore, cells were discarded. *Cell seeding density needs to be adjusted. Try 1:12 or 1:18 next time. === Test #4 (OSG_E2_T4) === *Date: 8/3/2012 ~ ongoing work *Cells: P13 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media: 5, 7.5, 10, 12.5, and 15% serum in osteogenic differentiation media were tested. *8/3: P12 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:8 onto gelatin coated 6-well plates (now become P13). *8/4 (Day 0): PBS wash twice and then add induction media to each well. 0%, 1%, 5%, 10%, 15% OSG: each for five 6-wells. *8/5 (Day 1): PBS wash twice and change media. There are cell death occurred in 0% and 1% serum. So, made some adjustment: 0% were changed into 7.5% OSG; 1% were changed into 12.5% OSG. *Change media daily (PBS wash once). =='''Induction efficiency test (OSG_E3)'''== *'''Purpose:''' To test the osteogenic differentiation efficiency of mESCs-derived osteoblasts <br> === Test #1 (OSG_E3_T1)=== *Date: 7/30/2012 ~ 8/4/2012 *Cells: P12 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media: 5% serum in osteogenic differentiation media. *7/30: P11 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:6 onto gelatin coated 25Ts (now become P12). 7/31(Day 0): PBS wash twice and then add induction media to each well. Change media daily. 8/4 (Day 4): Like OSG_E2_T3, also observed lots of cell death. Cell proliferated too fast. Cell colony became very big. Therefore, cells were discarded. Cell seeding density needs to be adjusted. Try 1:12 or 1:18 next time. === Test #2 (OSG_E3_T2)=== *Date: 8/3/2012 ~ ongoing work *Cells: P13 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media: 5% serum in osteogenic differentiation media. *8/3: P12 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 15 min, and then seeded 1:18 onto gelatin coated 25T (now become P13). *8/4 (Day 0): PBS wash twice and then add induction media. *Change media daily (PBS wash once). *8/7 (Day 3): Collect sample from one 25T. Store at -80C. === Test #3 (OSG_E3_T3)=== *Date: 8/6/2012 ~ ongoing work *Cells: P14 InsCAGGsAlov3 (mES brainbow cells) *Osteogenic differentiation media: 5% serum in osteogenic differentiation media. *8/6: P13 mES brainbow cells (feeder free) were trpsinized (TrpLE), let sit for 30 min, and then seeded 1:18 onto gelatin coated 35mm dishes (now become P14). *8/7 (Day 0): PBS wash twice and then add induction media (5% serum). Perform Alizarin red S staining in one 35mm dish. (Fix for 30 min; Stain for 20 min.) *Change media daily (PBS wash once).
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