Tina:Cell seeding
Cell seeding density test (ASIC_E1)
- Purpose: To determine the cell seeding protocol of CellASIC ONIX system
Test #1 (ASIC_E1_T1)[edit]
- Cells: osteogenic differentiated hESCs, 3 days after osteogenic induction(from H9_Bb2 P9)
- Cell number: ~2.5*10e5/ml
- Well number: A
Plate operation:[edit]
precoating
1. Prepare Matrigel (coating solution).
2. Aspirate solution from wells 1 and 8. Leave bottom holes fill with PBS.
3. Aspirate PBS from well 6 and 7, including bottom hole.
4. Add 10ul coating solution to well 6 (add to the bottom hole).
5. Incubate at room temperature for 1 hr.
6. Wash step: aspirate coating solution from well 6 and 7. Add 10ul culture medium to the bottom hole of well 6.
Cell loading
1. Prepare cell suspension (Osteogenic differentiated hESCs from one 6-well plate, dilute 50X).
2. Aspirate solution from well 6 and 7, including bottom hole.
3. Pipette 10ul of cell suspension to well 6 (bottom hole). Monitor under microscope. Take pictures.
Preculture
1. Aspirate solution from well 7, including bottom hole.
2. Add 350ul osteogenic induction medium to well 1, and 50ul medium to well 7.
3. Observe under microscope. Place plate in incubator.
Results:[edit]
- Cell number was not enough.
- Cells were clustered (close to inlet 6). Cell loading protocol need to be improved.
- 350ul culture medium could last for 3-4 days.
- Some of the cells died after seeding, others expanded a bit and form colonies. No more expansion after 3 days.
- Image after cell loading
File:Tina Lo 021813 ASIC E1 T1 pic 1.jpg File:Tina Lo 022113 ASIC E1 T1 pic 2.jpg File:Tina Lo 022213 ASIC E1 T1 pic 3.jpg
Test #2 (ASIC_E1_T2)[edit]
- Cells: osteogenic differentiated hESCs, 4 days after osteogenic induction(from H9_Bb2 P9)
- Cell number: ~7.5*10e6/ml
- Well number: B
Plate operation:[edit]
precoating
same as ASIC_E1_T1
Cell loading
1. Prepare cell suspension (Osteogenic differentiated hESCs from five 6-well plate, dilute 100X, cell counts:~7.5*10e6/ml).
2. Aspirate solution from well 6 and 7, including bottom hole.
3. Pipette 10ul of cell suspension to well 6 (bottom hole). Monitor under microscope.
Results:[edit]
- Cells jammed in flow inlet. Cell number was too high.
Test #3 (ASIC_E1_T3)[edit]
- Cells: osteogenic differentiated hESCs, 6 days after osteogenic induction(from H9_Bb2 P9)
- Cell number: ~2.5*10e6/ml
- Well number: C
Plate operation:[edit]
precoating
did not do precoating
Cell loading
1. Prepare cell suspension (Osteogenic differentiated hESCs from five 6-well plate, dilute 1000X, cell counts:~2.5*10e6/ml).
2. Aspirate solution from well 6 and 7, including bottom hole.
3. Pipette 10ul of cell suspension to well 6 (bottom hole). Monitor under microscope.
(added steps) 4. Aspirate exceeded cell suspension from well 6 to induce capillary flow backward. from outlet 7 to inlet 6. in order to increase cell number at the left hand side.
5. Aspirate solution from well 6 and 7, including bottom hole.
6. Add culture medium to well 1 to induce capillary flow from inlet 1 to outlet 7. Use the increased pressure to even the distribution of cells inside the culture area.
Preculture
1. Aspirate solution from well 7, including bottom hole.
2. Add osteogenic induction medium to well 1 to 350ul, and add 50ul medium to well 7.
3. Observe under microscope. Place plate in incubator.
Results:[edit]
- Cell number was good.
- Cell density/distribution was good. The new protocol worked.
- Looks like these osteogenic differentiated cells did not proliferate much. Should be able to culture with the microfluidic system for a longer time.
- Image after cell loading
File:Tina Lo 022013 ASIC E1 T3 pic 1.jpg File:Tina Lo 022113 ASIC E1 T3 pic 2.jpg File:Tina Lo 022213 ASIC E1 T3 pic 3.jpg
Test #4 (ASIC_E1_T4)[edit]
- Cells: undifferentiated hESCs(from H9_Bb2 P7)
- Cell number: ~cell from 35mm dishes/700ul, loaded 30ul
- Well number: D
Plate operation:[edit]
precoating
same as ASIC_E1_T1, coated with Matrigel
Cell loading
same as ASIC_E1_T3
Preculture
same as ASIC_E1_T3, with mTeSR1
Results:[edit]
- Cell density/distribution was good. Cell loading number works for undifferentiated ESCs. could be a little more though..Can try to load more cells next time.
- Cells did not proliferate well for the first 3 days. Cells experienced some death at the beginning.
- Cells started to proliferate rapidly after Day 5.
- Once cells start to proliferate, it can be cultured for ~4 more days before it becomes to confluent. Sometimes it depends on the distribution of cells at the beginning. The actual culture days depends.
Test #5 (ASIC_E1_T5)[edit]
- Cells: undifferentiated hESCs
- Cell number:
- Well number: A
Plate operation:[edit]
precoating (modified)
1. Prepare Matrigel (coating solution, thaw at 4C, on ice, overnight).
2. Aspirate solution from wells 1 and 8. Leave bottom holes fill with PBS.
3. Aspirate PBS from well 6 and 7, including bottom hole.
4. Add 20ul coating solution to well 6 (add to the bottom hole).
5. Incubate at room temperature for 3 hrs.
6. Wash step: aspirate coating solution from well 6 and 7. Add 10ul culture medium to the bottom hole of well 6. Incubate at room temperature for another 3 hrs.