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==HUES8 line expansion== *'''4/13''' **Thaw one vial of HUES8 cells. P10 to P20. **Cell location: locate in Rack #1, Box #9 (Box name: 1), Cell location: #18 of the N2(l) tank in CERC **Cell information labeled on the vial: P19, ~4*10E5 cells, processed on 11/16/06 by CFG *'''4/15''' **Change medium for the first time. *'''4/16 – 4/22''' **Change medium daily *'''4/23''' **Passage cells 1:1 in order to keep Matrigel plate fresh. From one 6-well plate to one 6-well plate. P20 to P21 **Dispase: lot number: 12A42800; expiration date: 01/13 **Matrigel plate: prepared freshly (4/23). **Steps: ***1.Add 1 mL Dispase and treat for 6 min ***2.Aspirate Dispase and wash the plate 3 times ***3.Add 1 mL DMEM/F-12 and scrape cells off with a P1000 pipette tip (mechanical method). ***4.Centrifuge at 250g, 5 min ***5.Resuspend in mTeSR with a 2 mL sterile Pasteur pipette (pipette up and down 5 times) and seed cells in one 35 mm Matrigel coated plate Thoughts: Scrape with pipette tip in vertical and horizontal lines is hard to cover the whole plate. Cell scraper is needed for passaging (make an order). *'''4/24''' **Change medium Thoughts: Still don’t see colony at this moment. Discussed with several people with previous ESC culture experience and then decided to adjust my culture strategy slightly… From now on, starting to change medium every 2 days in order to let the unknown factors (if any) secreted by these cells themselves to concentrate in culture medium. *'''4/26''' **Change medium Thoughts: Notice some color change of the working mTeSR culture medium store in the fridge. It is critical to aliquot and only warm medium for a volume of one-time usage to prevent constantly cool and warm. Thaw a new aliquot of mTeSR stored in -20C. *'''4/28''' **Change medium *'''4/30''' **Change medium *'''5/3''' **Change medium *'''5/4''' **Surprisingly, one colony has been found! **Passage cells 1:1 from one 6-well plate to one 6-well plate to keep Matrigel fresh. P21 to P22. **Matrigel plate: prepared freshly (5/4). **Steps: ***1.Circle the only one colony itself under microscope ***2.Add 1 mL Dispase and treat for 6 min ***3.Aspirate Dispase and wash the plate 3 times ***4.Add 1 mL DMEM/F-12 and scrape cells off with cell scraper. Scrape the circled area only to exclude other differentiated cells/less-pluripotent cells. ***(While observing under the microscope, cells were still attached…) ***5.Use the tip of a glass Pasteur pipette to scrape these cells off vigorously. ***6.Centrifuge at 250g, 5 min ***7.Resuspend in mTeSR with a 2 mL pipette (5 times) and seed cells in one 35 mm Matrigel coated plate ***(While observing under the microscope, cells were still aggregated as a whole…) ***8.Pipette cells up and down with a P1000 micropipettor (5 times, gently) to disrupt the cell cluster ***9.Incubate Thoughts: 1.Colonies may be more difficult to detach than cells. Longer incubation time with dispase (for instance, 7 min) may be needed next time. 2.Colonies may be more difficult to disrupt than cells. A P1000 micropipettor may do a better job than a 2 mL sterile Pasteur pipette (too gentle). *'''5/5''' **Medium change was not performed today to let cells rest and recover from passaging. *'''5/6''' **Change medium *'''5/7''' **Change medium *'''5/8''' **Passage cells 1:2 from one 6-well plate to two 6-well plate to avoid condensation caused spontaneous differentiation. P22 to P23. **Matrigel plate: prepared on 5/4 **Treat dispase for 7 min Thoughts: Took some time to do passaging, tried several different ways... Eventually, use cell scraper to detach these cells. It’s easy but can not separate differentiated cells from pluripotent colonies. However, it might be the best way to do cell passaging at this moment since it’s the safest way to prevent contamination. It’s better to get enough cell number first and to select the more pluripotent colonies later. *'''5/9''' **Observing lots of cell death, therefore, did not change medium today. Thoughts: The undesirable cell death might be due to the improper cell handling time during passaging yesterday (The unsuccessful microdisection method caused me an extra 20-min). 1.Along with yesterday’s conclusion, it is better to do cell passaging using only cell scraper for the first one or two passages. 2.Reduce the treated amount and treated time of dispase. 3.Process cells in mTeSR medium directly to shorten passage time. *'''5/10''' **change medium *'''5/12''' **change medium (added 2X volumn) *'''5/14''' **Passage cells 2:2 from two 6-well plate to two 6-well plate to avoid condensation caused spontaneous differentiation. P23 to P24. **Matrigel plate: prepared freshly (5/14) **Steps: ***1.Mark the less pluripotent colonies under microscope ***2.Add 500 uL Dispase and treat for 5 min ***(While observing under the microscope, the edges of colonies were not curled…) ***3.Incubate for another 3 min ***4.Aspirate Dispase and wash the plate 3 times ***5.Add 1 mL mTeSR and scrape cells off with both cell scraper and P1000 pipette tips. Avoid the marked area to exclude differentiated cells/less-pluripotent cells. ***6.Pipette cells up and down with a P1000 micropipettor (6 times, gently) to disrupt the cell cluster and then seed these cells directly onto Matrigel plates
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