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General Stem Cell Protocols

互联网2013-11-13

1250

实验试剂

1. Complete Culture Media Formulation for ES (Embryonic Stem)
For 500 ml of ES complete media:
• 100 ml fetal bovine serum (F2442)
• 5 ml 100x non-essential amino acids, NEAA (M7145)
• 5 ml 100x Pen-Strep (P4333 or P0781)
• 5 ml 100x sodium pyruvate (S8636 or P4562)
• 5 ml beta-mercaptoethanol
Stock solution = 3.5 µl diluted to 5 ml neat beta-maerpactoethanol (M7522) with tissue culture
grade water (W3500) — make fresh, do not store diluted solution
• 380 ml DME, high glucose (D5796) [See M3942 for reduced serum MegaCell formulation]
• 1 ml 106 Units/ml LIF (L5158)
For positive and negative selections, add:
• 5 ml 100x G418 (G8169)
• 0.1 ml 5000x Ganciclovir (use G2536 at 10 mM in PBS, P5368)
• Filter-sterilize as needed, store media at 4 °C, pre-warm to 37 °C before use.

2. Cryopreservation Media Formulations
2x Freezing media preparation
• 20% fetal bovine serum (F2442)
• 20% DMSO (D2650)
• 60% ES cell media
• Store freezing media at –20 °C and thaw to 37 °C or room temperature before use.
Ready-to-Go Freezing media
• Serum-free cyropreservation medium (C6295)
Contains MEM, 8.7% DMSO, and methylcellulose
• DMSO-free cryopreservation medium (C6039)
Contains MEM, glycerol, calf and fetal bovine serum
• Standard cryopreservation medium (C6164)
Contains MEM, DMSO, calf and fetal bovine serum

3. Complete Culture Media for Mouse Embryonic Fibroblast (MEF) Feeder Cells
For 500 ml media:
• 445 ml DMEM (D5796)
• 50 ml fetal bovine serum (F2442)
• 5 ml 100x Pen-Strep (P4333 or P0781)
Gelatin-treated culture ware
All dishes/plates need to be treated with 0.2% gelatin for 5 minutes at room temperature. 0.2% gelatin is made from diluting 2% gelatin solution (G1393) with PBS (P5368) and autoclave to sterilize. The volume depends on culture vehicle, plates, slides, etc. It is necessary to cover the entire culture surface.

4. Freezing Medium
The best recovery rate was observed in freezing medium consisting of 90% FBS-10% dimethyl sulfoxide (DMSO). However, Oct-4 expression in the thawed cells was lower than in cells frozen in hES cell growth medium with 10% DMSO. Nevertheless, by the next passage, the expression and distribution of Oct-4 and other markers of undifferentiated cells were indistinguishable between these two freezing conditions. We routinely use the 90% FBS-10% DMSO medium.

5. Materials needed:
• Chilled freezing medium: 90% FBS, 10% DMSO
• Cryovials, labeled with the line, passage number, and date
• Cryovial rack (rack with ice reservoir by Corning) (CLS430525)
• Styrofoam rack from packaging for 15 ml centrifuge tubes
• –80 °C freezer
• Trypsinize the cells; centrifuge in PMEF medium.
• Resuspend the pellet in the cold freezing medium. We recommend freezing one confluent 35 mm plate per vial in 0.5 ml of freezing medium. Work quickly and keep the cells on ice after the addition of the freezing medium.
• Aliquot cell suspension into prechilled freezing vials and sandwich the vials between two Styrofoam racks: tape to prevent the two racks from separating and transfer to a –80 °C freezer overnight.
Transfer the cryovials to liquid nitrogen for long-term storage.

实验步骤

1. ES Cell Cryopreservation on 96-well Plates
    1) Aspirate media and wash once with PBS.
    2) Freeze down the 3 x gelatinized only plate at –20 °C for DNA prep.
    3) For the 2 x plates with feeder cells, add 25 µl trypsin STV and incubate at 37 °C for 5 minutes.
    4) Disaggregate cells with 100 µl ES media and spin cells at 1K for 5 minutes.
    5) Take off supernatant. Be careful not to suck cells from bottom.
    6) Resuspend cells in 75 µl ES media.
    7) Add 75 µl 2x freezing media.
    8) Cover plates with parafilm, place plates in plastic bags and then in Styrofoam box and put in –80 °C freezer.

2. Expansion and Irradiation of MEF Feeder Cells
    1) Plate one vial primary MEF cells in one T75 flask with 20 ml MEF media.
    2) Expand 1x T75 flask of cells into 1x T175 flasks.
    3) Split 1x T175 flask of cells into 3 T175 flasks.
    4) Split 3x T175 to 6x T175 flasks if more feeders are needed.
    5) Trypsinize and pool all cells, spin down and resuspend cells in 50 ml MEF media.
    6) Irradiate cells (gamma irradiation at 3000 rads).
    7) Spin down and resuspend cells in MEF media.
    8) Add an equal volume of 2x Freeze media.
    9) Mix well and freeze for overnight in 0.5 ml aliquots at –80 °C.
    10) Store in liquid nitrogen.
Each T175 flasks of cells can be aliquoted into 6 vials. Each vial can then be plated onto 1x T25 flask. You may need to adjust these numbers according to plating efficiency.

3. ES Cell Maintenance
To maintain a healthy and undifferentiated culture, cells need to grow at high density (above 106 to 107/ 8 cm2 or one well of a 6-well plate) and they should be passaged frequently. As a general rule, the media should be changed daily and cells should be passed every other day with a 5 fold split.
Cells are often ‘shocked’ when coming out of freeze and may show a lag time from initial culture set up to the first passage, perhaps 3 to 4 days.
4. Thawing Clones
    1) Prepare feeder cells (50 µl/well) on 96-well plates.
    2) Remove plates from –80 °C and let plates sit in warm bath.
    3) After clones are thawed spin plates at 1K for 5 minutes.
    4) Remove supernatant from well.
    5) Resuspend clone in 100 µl ES media and add clone to fresh well with feeder cells.
    6) After cells grow to confluence pass cells to 1 well of 24-well plate.

5. Freezing hES Cells
Many of the established hES cells have low recovery upon thawing, as low as 0.1 to 1%. This may be because of the method of passaging the cells. Mechanical picking or using collagenase dispersion usually results in large cell aggregates, which presumably do not get cryopreserved as efficiently as smaller clumps. Trypsinized cells in our lab have a recovery rate of about 10 to 20% or higher and do not require more complicated procedures such as vitrification.

6. Freezing Protocol
Select a high-quality confluent culture with good morphology for freezing. We also recommend taking a picture of a sample field and staining for molecular markers characteristic of undifferentiated hES cells for future reference.

7. Thawing hES Cells
Thawing hES cells is a relatively simple procedure. The main rule to follow is to do everything quickly.

    1) Preparation
        a. Prepare mitomycin C-treated PMEFs a day before thawing.
        b. Make a thawing medium. We use 70% hES cell growth medium supplemented with 2x hLIF and 8 ng/ml bFGF with 30% hES cell- or PMEF-conditioned medium.
        c. Change the medium on the PMEF plate to the thawing medium; equilibrate in the CO₂ incubator for one hour. For 35 mm plates, use 1.5 ml medium; for four-well plates, use 0.5 ml medium per well.
        d. Prepare a 50 ml conical tube with 10 to 15 ml of warm hES cell growth medium.

    2) Thawing
         a. Thaw the vial in a 37 °C water bath, constantly agitating while ensuring that the neck of the vial is above the water level. Check the content of the vial after about 40 seconds and at 10-second intervals until only a small piece of ice remains.
         b. Quickly spray the vial with 70% isopropanol, then using a 1 ml pipetteman, add warm hES cell medium to the contents of the vial dropwise with gentle agitation. Do it quickly but very gently.
Immediately transfer the contents into the prepared 50 ml tube with warm hES cell medium; centrifuge at 160 x g for 5 minutes.
         c. Remove the medium completely without touching the pellet.
         d. Add 0.5 ml of hES cell thawing medium, gently resuspend the cells using a 1 ml pipetteman (2 to 4 repetitions), and transfer to prepared PMEF plates with equilibrated hES cell-thawing medium.
Spread the cells evenly throughout the well by moving the plate several times in two directions at 90 degrees to each other; avoid swirling.
         e. Check the cells the next day; if there are many dead cells or the medium has changed color,
change two-thirds of the medium; otherwise, do not change it for another day.
         f. The colonies usually begin to appear in 3 to 4 days and can be ready for splitting in 5 to 10 days.

8. hES Cell Quality Control
Although the morphology of hES cells is often used for evaluating the quality of the culture and its readiness for passaging or freezing, this criterion alone cannot be used for an assessment of ES cell pluripotency. Staining for the expression of Oct-4 or alkaline phosphatase even in colonies of "perfect" morphology can result in one or both of these markers appearing in the cells only at the periphery of the colony. It is important, therefore, to regularly assess the cells by analyzing the expression of markers of pluripotent cells. We look at Oct-4, SSEA-3, SSEA-4, TRA 1-60, and TRA 1-81 by immunostaining, or we perform an enzyme assay for alkaline phosphatase. The procedures for such assays and available antibodies are described elsewhere.

注意事项

Tips:
1. Do a visual check of ES cultures. Observe for nesting (cells are in contact with each other).
2. Undifferentiated ES cells should appear as compact colonies with prominent nucleoli.
3. When cells are colonial or ‘nested’ individual cell borders should not be distinguishable but the colony should have a well defined boundary and may appear a little refractile under a phase microscope.
4. To separate ES cells from MEF’s, plate co-cultures on non-coated plates for 30 minutes at 37 °C.
5. Feeder cells will attach leaving a relatively pure ES cell population in suspension.
To pass ES cells:
1. Aspirate media off the plate and rinse once with PBS.
2. Trypsinize cells with 0.05% trypsin (T3924) at 37 °C for 3 to 5 minutes.
3. Add 5 ml of ES media and pipet vigorously to get single cell suspension.
4. Spin at 1000 rpm for 5 minutes.
5. Plate cells out onto irradiated feeder cells.

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