Additional details include multiple questions:
1) Can mouse iPSC be cultured in feeder free conditions. Kindly provide the details of the media and matrix that could be used for the feeder free culture of mouse iPSC.
2) Passaging of mouse iPSC can be done using Trysin?? Or other methods including mechanical dissociation/milder enzymes such as collagenase or dispase are required??
3) Can the somatic cells after protein transduction be directly maintained in feeder free condition with the media supplement of the conditioned iPSC media from mouse MEF's?? Or its is important to grow cells in presence of feeder layer ducring initial reprogramming phase???
Kindly reply
Regards,
Tarun
Hello
ANSWER Q1
Mouse iPS cells must be grown either on a feeder cell layer or on Matrigel. Appropriate feeder cells for mouse iPS cells are mouse embryonic fibroblasts (MEFs), or human foreskin fibroblasts (HFFs).
NOT :Mouse iPS cells from SBI are provided at passage 10 and can be
passaged 50 times before differentiation
A-The culture conditions for MEF and HFF cells are described in
1-Reagent MEF Medium
DMEM containing 10% FBS, 2 mM glutamine,
1x10-4 M nonessential amino acids and 50 U
and 50 µg/ml penicillin and streptomycin.
2-Reagent 2x Cold Freezing Media
20% DMSO and 80% FBS
3-Reagent Mitomycin C solution 1 mg/ ml
B-Gelatin treatment of plates for MEF feeder cells :
1) Add enough sterile/ autoclaved 0.1% gelatin to cover the
bottom of the wells.
Approximate amounts:
10cm plate – 5 ml
6 well – 1.5 ml/ well
24 well – 0.5 ml/ well
96 well – 200 µl/ well
2) Incubate the gelatin-coated dishes for at least 15 min at 37°C.
3) Aspirate excess gelatin solution before using
C-Thawing MEF cells
To insure the highest level of viability, be sure to warm medium to 37°C before using it on the cells. Cells should be plated at a minimum cell density of 1 x 104 cells/ cm2
1)Remove the vial from liquid nitrogen and thaw quickly in a 37°C water bath.
2) Remove the vial from the water bath as soon as the cells are half-thawed, and sterilize the tube by spraying with70% ethanol.
3) Transfer the cells with 10 ml of MEF medium to a 15 cm conical tube and pellet the cells by centrifugation at 200 g for 5 min.
4) Discard the supernatant and resuspend the cells with 10 ml fresh MEF medium and plate the cells at seed density density of 104 cells/ cm2
5) Incubate at 37°C with 5% CO2, until the cells reach 80-
90% confluency.
6) Change medium twice a week or when pH decreases
5) Incubate at 37°C with 5% CO2, until the cells reach 80-90% confluency.
6) Change medium twice a week or when pH decreases
ANSWER Q2
Passage of iPS cells
1. Refresh media on newly thawed cells EVERYDAY until ready to be split
(This may take 2-5 days)
2. Day before splitting cells, seed feeders as described above.
3. When cells are confluent, aspirate media, wash 1x with 1xPBS, add 2 ml
0.05%Trypsin-EDTA and incubate at 37C for 2-5 min.
4. Dislodge and resuspend trypsinized cells with 8 mls iPS cells media; spin cells
at 0.2 rcf for 5 min and aspirate media.
5. Resuspend cells in fresh ES media to achieve a split ratio of 1:5-1:10 onto
plates with new feeders. Using this split ratio, mouse iPS cells are
usually passaged every 2-3 days.
If a large number of cells is not needed for experiments, 6-well or 12-well
plates are usually used for maintaining iPS cells
Or follow the Protocol in this video http://www.youtube.com/watch?v=54lmv26tu0g
ANSWER Q3
iPSC Protein Protocol
The following protocol is based on human and mouse fibroblasts and will be applicable to most other celltypes.
1. Seed HDF cells at 5x104
cells per well in a 6-well plate. On the next day, media was
changed to the protein transduction media, which was prepared by mixing the recombinant
reprogramming proteins at the final concentration of 32µg/ml (8 µg/ml per factor) with
iPSC medium (protein transfection reagent G288 is highly recommended to use for a higher
transduction efficiency).
2. After overnight culture in the protein transduction media, media was changed to normal
iPSC medium, and cells were cultured for additional 36 hours before repeating the same
protein transduction cycle.
3. Repeat protein transduction for at least 4 times to sustain activity of reprogramming
proteins for 7-10 days in the cells for the reprogramming process.
4. Change medium every 48 hours.
5. Once iPSC colonies form, prepare Mytomycin C treated MEF (mouse embryonic fibroblasts)
feeder cells in 24-well plate (80% confluent) at a concentration of 10 μg/ml for 3 hours in an
incubator at 37°C followed by 2X PBS wash.
6. Pick colonies manually into 96-well plate and trypsinize in 96-well plate.
7. Transfer the trypsinized cells from each of the 96 wells into 24-well MEF coated plate.
8. Wait for another 1-2 weeks for iPSC colonies to develop (change medium every 48 hours)
9. When MEF cells become too old (about 2 weeks) or a lot of iPSC colonies have developed
in the 24-well plate, prepare MEF feeder layer as described above in 6-well plate to expand.
10. Trypsinize the cells (both MEF and iPSC cells) and spin down at 1000Xg.
11. Seed iPSCs into 6-well MEF coated plate for expansion.
12. Using the same procedures above, iPSCs can be further expanded to bigger culture vessels to meet your lab needs for iPSC analysis or down-stream applications.
Good luck
There are various ways to culture mouse iPSCs. One important thing to do: we should document exact culture conditions in each passage. Different matrices and media may render the cells with different properties (e.g. distinct pluripotent states). These cells can be grown and passaged as single cells and often have genomic alterations.. Routinely, we need to do karyotyping and gene copy number analyses to evaluate mouse cell culture conditions.
Hi Tarun:
You will get all your answers in this publication:
http://www.ijdb.ehu.es/web/paper.php?doi=10.1387/ijdb.130173to
For humans:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0087151
Hope that helps!
Bw,
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