Embryonic Stem Cell Propagation and Differentiation
Protocol
Dr. Marek Michalak’s Lab
September-October, 2005
1) PROPAGATION:
MATERIALS:
Propagation/ES Media*
Medium
ES FBS
L-glutamine (200mM)
500mL DMEM Multicell (#319-005-ES)
35mL (7.5%) Multicell (#80150) (@ -80°C)
5mL (2mM) GIBCO (#11779) (5mL aliquots,-20°C, cover
MEM Non-Essential Amino Acids 5mL (0.1mM)
w/ foil)
GIBCO (#01845) (cover w/ foil, 4°C)
(10mM)
Β-mercaptoethanol
Plasmocin
Pen/Strep
NaPyruvate
500uL BioShop (#MER-002) (50uL/7mL PBS, 4°C)
50ul
5ml
5ml (1mM) GIBCO (#1679) (4°C)
*Media is only usable for 1 month.
*Do NOT need to add Na Pyruvate to this type of media but others may have to:
Na Pyruvate (100mM) GIBCO (#1679)  add 5mL to make 1mM
0.1% gelatin autoclaved in PBS
LIF
Freezing Media**
ES FBS
DMSO
90% Multicell (#80150) (@ -80°C)
10%
**Prepare a large amount (10:1, ES FBS:DMSO), aliquot in 5mL in 15mL Falcon Tubes,
freeze at -20°C and thaw when needed**
a) Thawing ES Cells:
i) Preparation:
(1) Gelatin coating plates:
(a) For each cell line thawing, prepare one 6cm and one 10cm plate.
(b) Add 4mL or 6mL of 0.1% gelatin in PBS to the 6cm and 10cm plates,
respectively.
(c) Put in a 37°C incubator for one hour.
(d) Aspirate gelatin.
(i) Since gelatin is in PBS, do not need to dry plates (if in water,
plates need to be dried for 3-4 minutes because water will cause
osmotic shock.)
(ii) Make sure to aspirate all gelatin as to not dilute LIF.
(e) Add 5mL or 10mL of ES media to the 6 cm and 10cm plate,
respectively, the LIF (1:1000) and put in the 37°C incubator until use.
(2) Centrifuge tubes:
(a) For each cell line, put 10mL of warm ES Media in a 15mL Falcon
Tube and LIF (1:1000).
(3) Thaw LIF
(a) From the -80°C freezer.
ii) Procedure:
(1) Remove cells from the liquid nitrogen with tweezers and put into a
container of dry.
(2) With tweezers, gently thaw in 34-37°C water bath one vial at a time.
(3) With p1000, pipette cells into the prepared 10mL of ES Media in the
15mL Falcon Tube. Pipette up and down two times in media and rinse out
vial with this media into the 15mL Falcon Tube.
(4) Repeat steps 2 and 3 for all vials.
(5) Centrifuge tubes at 1200rpm for 5 minutes.
(6) Check size of pellet. If small, use a 6cm plate and in large, use a 10cm
plate.
(7) Aspirate media off the pellet and take 1mL of media from plate and resuspend the pellet.
(8) Tilt plate gently add back to the media.
(9) Repeat steps 6-8 for all pelleted cells.
(10) Gently nudge plates to spread out cells and incubate at 37°C for 24 hours.
b) Changing Media:
i) Preparation:
(1) Warm Media:
(a) Warm up ES Media in 37°C water bath
(2) Thaw LIF:
(a) From the -80°C freezer.
ii) Procedure:
(1) One plate at a time, aspirate the media.
(2) Add 10mL of warm, fresh ES Media gently to the side of the plate.
(3) Add LIF (1:1000) to each plate by tilting the plate and pipetting up and
down.
(4) Incubate at 37°C for 24 hours.
c) Splitting ES Cells:
i) Preparation:
(1) Check the plates:
(a) Using the microscope, check to see that there are round colonies of ES
cells.
(b) Some fibroblasts will also be present (if propagated previously with
MEFs).
(c) Cells should be 60-80% confluent. Split accordingly.
(2) Gelatin coating plates**:
(a) For each cell line prepare the decided number of 10cm plates for
splitting.
(b) Add 6mL of 0.1% gelatin in PBS to the 10cm plates.
(c) Put in a 37°C incubator for one hour.
(d) Aspirate gelatin.
(i) Since gelatin is in PBS, do not need to dry plates (if in water,
plates need to be dried for 3-4 minutes because water will cause
osmotic shock.)
(ii) Make sure to aspirate all gelatin as to not dilute LIF.
(e) Add 9.5mL of ES media to the 10cm plate, add LIF (1:1000) and put
in the 37°C incubator until use.
**(a) – (c) can be done the night before if splitting on a weekend **
(3) Warm Media:
(a) Warm up ES Media in 37°C water bath.
(4) Thaw LIF:
(a) From the -80°C freezer.
(5) Warm up Trypsin:
(a) Warm up ES Media in 37°C water bath.
ii) Procedure:
(1) Aspirate the media from plates (at maximum, do 2 plates at a time.)
(2) Wash twice with 7-10mL of PBS by gently adding PBS to side of the
plate.
(3) Add trypsin to each plate so that each plate split into will receive 500ul of
trypsin with cells.
(a) If splitting 1:4 add 2mL of tryspin or if splitting 1:3 add 1.5mL of
trypsin.
(4) Incubate at 37°C for 5 minutes.
(5) Pipette up and down to re-suspend cells and using the microscope, check
that the cells are in single cell suspension.
(a) Appear round and are floating in the trypsin.
(6) Add LIF (1:1000) by tilting plate and pipetting up and down.
(7) Tilt the plate and gently add 500ul to of the cells suspended in tryspin.
(8) Incubate at 37°C for 24 hours.
d) Freezing ES Cells:
i) Preparation:
(1) Chill freezing box:
(a) The freezing box MUST be chilled for 24 hours at 4°C prior to
freezing ES cells.
(2) Thaw Freezing Media:
(a) Thaw the necessary amount of freezing media in 37°C water bath from
the -20°C freezer.
(3) Prepare Falcon Tubes:
(a) Put 10 mL of ES Media/plate in a 50 mL Falcon Tube and add LIF
(1:1000).
ii) Procedure:
(1) Aspirate the media.
(2) Gently wash the plates 2 times with PBS by adding to the side of the plate.
(3) Add 2mL of trypsin to each plate and incubate for 5 minutes at 37°C.
(4) Pool the plates, if necessary, pipette up and down then check for single
cell suspension.
(5) Add the trypsin/cell suspension to the Falcon Tubes and centrifuge for 5
minutes at 1200rpm.
(a) if the -/- cell line does not pellet, can spin at 1500rpm for 5 minutes
(cannot do for other cell lines.)
(6) Aspirate the media and re-suspend pellet in of freezing media
(a) density for freezing cells is 5 x 106 – 10 x 106 cells/mL of freezing
media.
(7) Split into three freezing vials and put immediately into freezing box.
(a) DMSO at room temperature will kill the cells
(8) Place for 24 hours at -80°C and then into liquid nitrogen.
2) Differentiation:
MATERIALS:
Differentiation/EB Media
Medium
ES FBS
MEM Non-Essential Amino Acids
L-glutamine (200mM)
500mL DMEM Multicell (#319-005-ES)
100mL (20%) Multicell (#80150) (@ -80°C)
5mL (0.1%) GIBCO (#11779) (cover w/ foil, 4°C)
5mL (2mM) GIBCO (#11779) (5mL aliquots,-20°C, cover
Β-mercaptoethanol
Plasmocin
Pen/Strep
NaPyruvate
500uL BioShop (#MER-002) (50uL/7mL PBS, 4°C)
50ul
5ml
5ml (1mM) GIBCO (#1679) (4°C)
w/ foil)
NO LIF
Petri Dishes
Trypan Blue
0.1% gelatin autoclaved in PBS
a) Hanging Drops: DAY 0
i) Preparation:
(1) Petri Dishes:
(a) Add 6mL of PBS to each Petri Dish used for hanging drops.
(2) Trypan Blue:
(a) Put 50ul of Trypan Blue into Eppendorf tube(s).
(3) Prepare Falcon Tubes:
(a) In two 50mL Falcon Tubes, put 10mL of EB Media.
(b) In two 50mL Falcon Tubes, put 5mL-10mL of EB Media/plate
(depending on cell density)
ii) Procedure:
(1) Aspirate ES Media.
(2) Gently wash plates two times with PBS by pipetting into the side of the
plate.
(3) Add 2mL of trypsin to each plate and incubate for 5 minutes at 37°C.
(4) Pipette up and down, combine plates if necessary, and check for single cell
suspension.
(5) Transfer the trypsin/cells to the Falcon Tubes with 10mL of EB Media.
(a) washing in EB Media to remove the LIF from the cells from the ES
Media.
(6) Centrifuge for 5 minutes at 1200 rpm.
(7) Aspirate the media and re-suspend in the prepared EB Media in the Falcon
Tubes at 5mL of media/plate.
(8) Do a cell count to plate cells at 2.5 x 104 cell:
(a) 1:1 of Trypan Blue to Suspended Cells (prepared 50ul of Trypan
Blue:50ul of suspended cells)
(b) With the Haemocytometer and microscope, count four squares of cells
(1 in each corner) and then average the number of cells to get a more
precise concentration.
(c) Calculate how much volume needed: (must not exceed 1mL)
# cells want (cells)
# cells/plate (cells/mL)
2.5 x 104 cell
# cells/plate (cells/mL)
(9) Put this volume in the 50mL Falcon Tube prepared with 10mL of EB
Media and pipette up and down to re-suspend
(10) Put this cell suspension into an empty Petri Dish.
(11) Take a multi-pipetter and set to 20ul. Make rows of drops (6 rows of 8
drops, 6 rows of 7 drops when starting) on the lid of the Petri Dishes.
(12) Carefully flip lid onto Petri Dish and incubate at 37°C for 48 hours.
b) Suspending Embryoid Bodies (EBs): DAY 2
i) Preparation:
(1) EB Media
(a) warm EB Media to 37°C.
(2) Petri Dishes:
(a) Put 10mL of EB media in Petri Dishes (1 Petri Dish/Lid of EBs)
ii) Procedure:
(1) Wash drops with EBs off the lid and pipetting down the plate.
(2) Gently pipette EBs into the center of the Petri Dish.
(a) will see EBs floating.
(3) Leave at 37°C for 72 hours.
c) Plating EBs: DAY 5
i) Preparation:
(1) Gelatin Coat Plates:
(a) put 6-8mL of gelatin in 10cm tissue culture plates for at least 1 hour.
(b) coat 1 plate/plate of suspended EBs.
ii) Procedure:
(1) Aspirate the gelatin and add 10mL of EB Media gently to the plate
(2) With a p1000, pick out floating EBs.
(a) do not pipette more then 1mL of media.
(3) Add EBs gently to the side of the plate and nudge the plates to gently
spread.
(4) Incubate for 24 hours at 37°C.
d) Changing EB Media: DAY 6
i) Preparation:
(1) Warm up EB Media
ii) Procedure:
(1) Count how many EBs do not stick and write it on top of the plate.
(2) With the microscope, check for the EBs sticking.
(3) Aspirate the media.
(4) Gently pipette 10mL of EB media to side of the plate and incubate at 37°C
for 24 hours.
** Change Media every two days after (DAY 8, DAY 10, DAY12…)**