Alice:IPSC Reprogramming Lab Notes
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<calendar> name=Alice:LabNotes format=%name/%year-%month-%day date=2011/07/01 view=oneyear </calendar>
Cell Culture Preparation
293T Culture Medium: 500 mls [Final] DMEM 445 mls FBS 50 mls 10 % Pen/Strep 5 mls 1 X
Fibroblast (IMR90/CRL-2097/HFF/HHF) Growth Medium: Volume Stock [Final] DMEM 445 mls FBS 50 mls 10 % Nonessential AA 5 mls 10 mM 0.1 mM Pen/Strep 5 mls 100 X 1 X
MEF (CF-1) Culture Medium: Volume Stock [Final] DMEM 420 mls FBS 75 mls 15 % Pen/Strep 5 mls 100 X 1 X
hES/iPS Culture Medium: 250 ml 500 ml Stock [Final] DMEM/F12 193 ml 385 ml KOSR 50 ml 100 ml 20% Glutamine 2.5 ml 5 ml 1mM Nonessential AA 2.5 ml 5 ml 10 mM 0.1 mM β-Mercaptoethanol 0.5 ml 1 ml 55 mM 0.1 mM bFGF* 0.1 mg/ml 50ng/ml (add fresh to each plate every time) *Dissolve 100 ug in 1ml PBS. Make 50.0 ul aliquots and store at -20C or – 80C *Store prepared media at 4°C up to 14 days – bFGF added fresh day of use
2x Freezing Media (hES) [final] 10 ml iPS medium 20% 2 ml FBS – ES Cell Qualified 60% 6 ml [HyClone] DMSO 20% 2 ml Store 4°C up to 2 weeks.
TrypLE [Invitrogen] Trypsin (0.25%)/ 0.04% EDTA [Invitrogen]
Virus Production
Retroviruses: • pMXsGFP • pMXs-hOct4 • pMXs-hSox2 • pMXs-hKlf4 • pMXs-hc-Myc Packaging Plasmid: • pCL ampho
Procedure
Day 1 • Seed 9x106 293T per 15 cm plate (3 plates per retrovirus) Day 2 • Replace media on cells (20 ml per 15 cm) • Add 1500ul HBS to 15ml tubes (3 for each virus) • Transfection: o Add DNA to new separate 15ml tubes (Final volume of DNA-Calcium = 4500ul): • 90ug pMXs-GFP + 90ug pCLampho + ddh20 to 3942ul • 90ug pMXsIRES-hOct4 + 90ug pCLampho + ddh20 to 3942ul • 90ug pMXsIRES-hSox2 + 90ug pCLampho + ddh20 to 3942ul • 90ug pMXsIRES-hKLF4 + 90ug pCLampho + ddh20 to 3942ul • 90ug pMXsIRES-cMyc + 90ug pCLampho + ddh20 to 3942ul o Add 558ul of CaCl2 (2M) to DNA o Add 1500ul CaCl2/DNA to HBS slowly (drop-wise) while gently vortexing o Add 3000ul to cells drop-wise covering plate evenly o Return plates to incubator Day 3 • Check for GFP • Replace media on 293T cells (carefully) with 20ml fresh media Day 4 • Collect viral supernatant from wells and distribute 30 mls per 50ml tube (two tubes per virus) • Centrifuge at 1700rpm for 10 minutes to remove 293T cells and debris • Transfer supernatant carefully to new 50 ml tubes • Add 10 ml of Retro-X concentrator (Clontech) to 30 mls of retroviral supernatant and mix by gentle inversion • Incubate overnight at 4C Day 5 • Centrifuge supernatant at 3000rpm for 45 minutes at 4C • Label tubes while centrifuging • Discard supernatant to bleach waste bottle (keep pellets on ice as much as possible) • Very gently resuspend each pellet in 1 ml (PBS or medium) using p1000 (combine duplicate tubes of each retrovirus) • Aliquot (100μl-200μl) and return tubes to ice • Store all aliquots at -80C Test each batch for GFP infection (expect >80-90% from 25-50μl of virus per 6-well – 100,000 fibroblasts)
Reprogramming Human Fibroblasts
Retroviruses: 1. pMXs – hOct4 2. pMXs – hSox2 3. pMXs – hKLF4 4. pMXs - hcMyc Lentiviruses: 1. pLKO.1 GFP shRNA 2. pLKO.1 Target shRNA
Reprogramming protocol
Day -1 • Prepare and label gelatin coated 12-well or 6-well plates and leave 1 hour at RT • Collect hFibs using 0.25% Trypsin • Dilute cells to 1x106 per ml • Seed 0.5x105 (50μl) per 12-well or 1.0x105 (100μl) per 6-well Day 0 • Aspirate media from cells • Add 1 ml (12-well) or 2ml (6-well) hFib media containing: o Retroviruses • 12.5 - 25μl (12-well) or 25-50 μl (6-well) [batch and cell line dependent] each virus per well o 5μg/ml polybrene (0.5μl/ml of 10mg/ml stock) Day 1 • Aspirate media from cells • Add fresh fibroblast medium Day 5 • Replace fibroblast media • Gelatinize 10cm plates for 2 hours • Seed 4-5x106 feeders per 10cm plate Day 6 • Collect and count Fibroblasts • Re-seed 50,000 to 100,000 per 10cm plate of feeders Day 7 • Replace media with human ES/iPS media Days 7+ • Replace ES media on cells every day (occasionally skipping a day) Days 24-40 • AP stain or • Pick hiPS colonies
Picking hiPS colonies
Material: 10-15cm plate Needles (e.g. 27 gauge) Disposable knife PBS 24-well plate of feeders
1. Use needle to cut square around colony 2. Using p200 set to ~150, draw up ~50μl of media, place tip over colony and scrape/suck colony into tip 3. Transfer colony to inverted lid of 15cm plate, creating a bubble of media containing the colony 4. Continue collecting remaining colonies creating individual bubbles on the 15cm plate 5. Use knife to chop colonies rapidly into small pieces 6. Wash knife with PBS between colonies 7. Draw ~100μl into p200 and pipette each bubble up and down ~10 times before transferring to separate wells of 24-well feeder plate containing 1ml fresh hiPS medium
Culturing hiPS cell lines
Preparation of MEF-covered plates
Need: 0.1% gelatin Irradiated MEFs (less than p5) MEF culture media 1. Add 1.5 ml gelatin to 6-well plates (5 ml for 10cm), leave at RT or 37C for 2 hours to 24 hours 2. Aspirate gelatin from plate and seed 4-5 x 106 cells per 6-well or 10 cm plate in MEF media 3. Let cells attach overnight before plating hES/iPS Use following day (as fresh as possible)
Thawing hES/hiPS Cells
Material: Feeder coated plates hES/iPS Medium (bFGF added day of use)
1. Get hES cells from liquid Nitrogen or -150°C freezer 2. Thaw in 37°C water bath for a few minutes until last crystal is gone 3. Clean tubes with EtoH before opening 4. Pipette cells gently into 15ml tube using 5ml pipette 5. Add 4ml ES medium drop-wise to cells 6. Spin at 800rpm for 5min at 5-10°C 7. Aspirate media 8. Resuspend pellet in hES medium pipetting up and down gently a few times using a 5 ml pipette 9. Plate ES Cells – 1 vial for 1 well of a 6-well plate. 3mls per well. 10. Incubate at 37°C 24 hours 11. If necessary, collect super and re-seed on another well and replace media (in case not all colonies attach) 12. Replace media daily with fresh ES media
Grow 4-6 days and passage when colonies reach ~300-500 cells. Want colonies to be high in density (but not touching) and small (to reduce chance of differentiation).
Manual Passaging of hES/iPS Cells
Material: Feeder coated 6-well plates hES/iPS Medium (bFGF added day of use)
1. Prepare wells with feeders (1 day in advance) 2. Aspirate media off feeders and wash 1x with PBS and add 3 ml fresh media 3. Remove media from hiPS/ES and add 1 ml fresh media 4. Using p200 tip, scratch cells from plate in a cross hatch pattern 5. Using p1000, piptte up and down to dissociate 5-10 times 6. Plate cells directly into new wells: a. 1/3 split (of 1 well): Add 0.33ml cells b. 1/4 split (of 1 well): Add 0.25ml cells c. 1/5 split (of 1 well): Add 0.2ml cells 7. After 24 hours, replace media with fresh hES media (3mls total per 6-well)
Enzymatic Passaging of hES/iPS Cells
Material: hES/hiPS media + Y27632 (bFGF added day of use) 6-Well feeder plate
1. Aspirate media from cells 2. Wash 1x with PBS 3. TrypLE treat cells: a. Add 1ml TrypLE to each well (6-well plate) with hES cells b. Incubate 5-10 minutes at 37C c. Remove TrypLE before colonies detach completely d. Add 1ml media and detach by scraping e. Pipette using p1000 2-3 times to dissociate 4. Transfer 200μl (1:5) to 330μl (1:3) dissociated cells to fresh feeders (3 mls total media)
Note: - Manually passage iPS lines initially for several passages and freeze back multiple vials, then adapt to enzymatic passaging - For adapting cells to TrypLE use 1:2000 Rock inhibitor; once adapted use 1:5000 Rock inhibitor - Typically passages are 1:3 to 1:5 every 4-6 days - Replace media every day (can add 2 volumes of media for 48 hours e.g. over weekend)
Freezing hES/iPS Cells
Freeze cells at 1 well of 6-well plate/1ml of freezing solution. 1. Remove medium and add 0.5ml hES medium per 6-well 2. Collect colonies using manual method keeping colonies intact (no dissociation) 3. Gently transfer to freezing vial using 5 ml pipette 4. Add drop-wise equal volume of freezing media 5. Freeze -80°C overnight in freezing container (Styrofoam) and store at -150°C or liquid nitrogen long term