Tina:Osteogenic differentiation on mES brainbow cells (OSG): Difference between revisions

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==Lentiviral production==
=='''Seeding density test (OSG_E1)'''==
*'''Purpose:''' To generate and concentrate Cre lentivirus
*'''Purpose:''' To determine the seeding density of mESCs for osteogenic differentiation
*'''Components:'''
<br>
**Lentiviruses: (1)pLenti CAG Cre IRES Puro (2)pLenti CMV GFP Puro (virus packaging control)
=== Test #1 (OSG_E1_T1) ===
**Packaging plasmids: psPAX2 and pMD2.G
*Date: 7/5/2012 ~ 7/7/2012
*'''Batch #1:'''
*Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
**Date: 6/26/2012 ~ 6/29/2012
*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.
**Cells: 293T cells
*7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.
**Virus packaging components:  
*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
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|''Component''''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Cre lentivirus'''
| align="center" style="background:#f0f0f0;"|'''Volumn'''
| align="center" style="background:#f0f0f0;"|'''Control'''
| align="center" style="background:#f0f0f0;"|'''Final concentration'''
|-
| MEM ||193ml||
|-
| Serum ||2ml||1%
|-
|-
| pLenti CAG Cre IRES Puro ||90ug||-
| Dexamethasone ||200ul of 0.1mM stock||0.1uM
|-
|-
| pLenti CMV GFP Puro ||-||30ug
| Ascorbic acid ||1ml of 10mg/ml stock ||50ug/ml
|-
|-
| psPAX2 (packaging plasmid) ||60ug||20ug
| b-GP ||2ml of 1M stock||10mM
|-
|-
| pMD2.G (packaging plasmid) ||30ug||10ug
| PenStrep ||2ml||1%
|-
|-
| style="background:#f0f0f0;"|Transfected 293T cell number
| style="background:#f0f0f0;"|Total
| style="background:#f0f0f0;"|27*10^6 cells
| style="background:#f0f0f0;"|200 mL
| style="background:#f0f0f0;"|9*10^6 cells
| style="background:#f0f0f0;"|
|}
|}
**Virus production: Exp was carried out at Dr. Yang Xu’s Lab (with Dr. Blue Lake’s help). 293T cells were seeded on 6/26 and transfected on 6/27. Virus were collected and concentrated on 6/29. Aliquot (105 ul) virus are stored at -80゚C at CERC.
*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).




==LentiGFP infection==
*'''Purpose:''' To confirm the efficiency of virus packaging
*'''Test #1:'''
**Date: 7/2/2012 ~ 7/6/2012
**Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto one gelatin coated glass-well.
**'''Components:''' Lentiviruses: pLenti CMV GFP Puro
**Infection on 7/3:
(1)Take pictures of cells before viral infection


(2)Change media of each well (2 ml/well)
=== Test #2 (OSG_E2_T2) ===
 
*Date: 7/13/2012 ~ 7/27/2012
(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency
*Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
 
*Osteogenic differentiation media:  
(4)Add 50 ul LentiCre virus in culture medium, and then mix gently
Basal media were changed from MEM to DMEM. Different concentration of serum were tested.
 
{| {{table}} border=1
(5)Put cells back into incubator
| align="center" style="background:#f0f0f0;"|'''Component (volumn)/group'''
 
| align="center" style="background:#f0f0f0;"|'''5% serum'''
(6)Take pictures of cells 2-3 days after infection.
| align="center" style="background:#f0f0f0;"|'''15% serum'''
 
| align="center" style="background:#f0f0f0;"|'''2me'''
**Results:
|-
Observation: Successful infection of GFP vectors. Both mESCs and MEFs are expressing GFPs now. Efficiency is higher in MEFs.
| DMEM ||193ml||193ml||193ml
|-
Pictures:
| 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
 
|-
==LentiCre infection==
| 50ug/ml Ascorbic acid ||1ml of 10mg/ml stock||1ml of 10mg/ml stock ||1ml of 10mg/ml stock
*'''Purpose:''' To confirm the effect of Cre recomninase in mES brainbow cells
|-
*'''Test #1:'''
| 10mM b-GP ||2ml of 1M stock||2ml of 1M stock||2ml of 1M stock
**Date: 7/1/2012 ~ 7/5/2012
|-
**Cells: P10 mES brainbow cells. P9 mESCs were passaged 1:6 on feeder dependent system (total: three wells).  
| 1% PenStrep ||2ml||2ml||2ml
**'''Components:''' Lentiviruses: pLenti CAG Cre IRES Puro
|-
**Infection on 7/3:
| 1% Glutamax ||-||-||2ml
(1)Take pictures of cells before viral infection
|-
| 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.)


(2)Change media of each well (2 ml/well)


(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency


(4)Add 0/10/25/50 ul LentiCre virus in culture medium, and then mix gently
=== 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.


(5)Put cells back into incubator


(6)Take pictures of cells 2-3 days after infection.


**Results:
=== 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).




*'''Test #2:'''
=='''Induction efficiency test (OSG_E3)'''==
**Date: 7/2/2012 ~ 7/6/2012
*'''Purpose:''' To test the osteogenic differentiation efficiency of mESCs-derived osteoblasts
**Cells: P10 mES brainbow cells. P9 mESCs were passaged (immediately, no sitting time) 1:4 onto gelatin coated glass-well (total: four wells).  
<br>
**'''Components:''' Lentiviruses: pLenti CAG Cre IRES Puro
=== Test #1 (OSG_E3_T1)===
**Infection on 7/3:
*Date: 7/30/2012 ~ 8/4/2012
(1)Take pictures of cells before viral infection
*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.


(2)Change media of each well (2 ml/well)


(3)Add 1ul polyBrene (millipore, TR-1003-G) in culture medium (1ul/2ml medium) to increase transfection efficiency


(4)Add 0/10 ul LentiCre virus in culture medium, and then mix gently
=== 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.


(5)Put cells back into incubator


(6)Take pictures of cells 2-3 days after infection.


**Results:
=== 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).

Latest revision as of 01:19, 9 August 2012

Seeding density test (OSG_E1)[edit]

  • Purpose: To determine the seeding density of mESCs for osteogenic differentiation


Test #1 (OSG_E1_T1)[edit]

  • 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)[edit]

  • Purpose: To test the effects of osteogenic differentiation media on mESCs


Test #1 (OSG_E2_T1)[edit]

  • 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 (OSG_E2_T2)[edit]

  • 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.

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.
  • 7/19-7/26: 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)[edit]

  • 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)[edit]

  • 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)[edit]

  • Purpose: To test the osteogenic differentiation efficiency of mESCs-derived osteoblasts


Test #1 (OSG_E3_T1)[edit]

  • 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)[edit]

  • 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)[edit]

  • 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).