Alice:IPSC Reprogramming Lab Notes: Difference between revisions

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==Culturing hiPS cell lines==
==Culturing hiPS cell lines==
Preparation of MEF-covered plates
===Preparation of MEF-covered plates===
   
   
  Need:
  Need:
Line 174: Line 174:
  2. Aspirate gelatin from plate and seed 4-5 x 106 cells per 6-well or 10 cm plate in MEF media
  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
  3. Let cells attach overnight before plating hES/iPS
 
  Use following day (as fresh as possible)
  Use following day (as fresh as possible)


 
===Thawing hES/hiPS Cells===
Thawing hES/hiPS Cells:
  Material:
 
  Need:  
  Feeder coated plates
  Feeder coated plates
  hES/iPS Medium (bFGF added day of use)
  hES/iPS Medium (bFGF added day of use)
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  Grow 4-6 days and passage when colonies reach ~300-500 cells.   
  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).  
  Want colonies to be high in density (but not touching) and small (to reduce chance of differentiation).


==Manual Passaging of hES/iPS Cells==
==Manual Passaging of hES/iPS Cells==
  Material:
  Material:

Revision as of 22:46, 3 October 2011

Material

293T Culture Medium:

500 ml [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 ml		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:

250ml 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)

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