Maintenance and freezing of pluripotent stem cells

Maintain human embryonic stem cells (ES) or induced pluripotent stem cells (iPS) on a standard cell attachment matrix (e.g., Matrigel® or Laminin-521) in StemMACS™ iPS-Brew XF medium. The medium is designed to enable  culture under feeder-free conditions and allows rapid culture initiation after cryopreservation. Follow the protocol of the medium data sheet.

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StemMACS iPS-Brew XF, human

Use the StemMACS™ Passaging Solution XF for gentle detachment of pluripotent stem cell colonies and dissociation into cell clusters. The solution is designed to minimize manipulation of the culture, eliminating lengthy inactivation, dilution, or centrifugation steps. Thus, transfer into new cell culture conditions is reproducible, standardized, and fast, ensuring optimal viability and attachment. Follow the protocol of the solution data sheet.

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StemMACS™ Passaging Solution XF

The panel for analysis of pluripotent stem cells includes antibodies for both surface markers and intracellular markers. Surface markers are stained first. Subsequently, cells are fixed and permeabilized for intracellular staining.

To set up the instrument and compensate for spectral overlap, single stainings for each antibody and an unstained cell sample are required. The unstained sample does not contain any antibody, but is otherwise treated in the same way as the stained samples regarding e.g. fixation and permeabilization. The pipetting scheme in the tables below provides an overview. As SSEA-1 is not expressed in pluripotent cells, the MACS® Comp Bead Kit, anti-mouse Igκ is used for compensation of PE-Vio® 770 instead of a cell sample.

▲ Note: Fluorescence-minus-one (FMO) controls might help you to set the gates more accurately. FMO controls contain all antibodies except for one.

1. Determine cell number. Use 1×10⁶ iPS cells per sample.
2. Centrifuge cell suspension at 300×g for 5 minutes. Aspirate supernatant completely.
3. Resuspend sample 1 in 85.6 μL of PBE buffer. This will be the cell sample stained with the complete panel.
4. Resuspend sample 3 in 107.8 µL buffer. Resuspend samples 2, 4, 5, 6, and 7 in 100 µL buffer. These will be the unstained and single-stained samples, respectively.
5. Add 10 μL of each of the following antibodies detecting the surface markers to sample 1: i) Anti-SSEA-4- VioGreen™ and ii) Anti-SSEA-5-VioBlue®. Add 2.2 µL of CD15-PE-Vio770 and Anti-TRA-1-60-PE.
6. Add 2.2 µL of Anti-TRA-1-60-PE to sample 3. Add 10 µL of Anti-SSEA-4-VioGreen to sample 4 and 10 µl of Anti-SSEA-5-VioBlue to sample 5, respectively.
7. Do not add antibodies to samples 2, 6, and 7 at this point.
8. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
9. Wash cells by adding 1 mL of PBE buffer per sample and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
10. Resuspend all samples in 100 μL of PBE buffer. Add 100 μL of Inside Fix per sample.
11. Mix well and incubate for 20 minutes in the dark at room temperature.
12. Wash cells by adding 1 mL of PBE buffer and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
13. Resuspend sample 1 in 105.6 μL of Inside Perm.
14. Resuspend samples 2–5 in 100 μL of Inside Perm. Resuspend samples 6 and 7 in 107.8 μL of Inside Perm.
15. Add 2.2 μL Anti-Sox2-FITC to sample 1 and 6.
16. Add 2.2 µL of Anti-Oct3/4 Isoform A-APC to samples 1 and 7.
17. Mix well and incubate for 15 minutes in the dark at room temperature.
18. Wash cells by adding 1 mL of Inside Perm and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
19. Resuspend cell pellet in a suitable amount of PBE buffer for analysis by flow cytometry. A volume of 1 mL of PBE buffer per sample is recommended.
    ▲ Note: Fixed and permeabilized cells are smaller than viable cells. Thus, FSC/SSC settings of the flow cytometer might have to be adjusted.

Instrument setup

1. Set up the scatter and voltages for all channels with the unstained cell sample (sample 2) (A in figure below). Specify the trigger.
2. Use single-color stainings (samples 3–7) to define the compensation (B in figure below).
3. To set up the PE-Vio 770 channel, temporarily lower the trigger to detect all the Comp Beads and define the compensation (C in figure below). Afterwards change the trigger settings back to the value specified in step 1.

Isotype controls

Isotype controls are used to check for non-specific binding of the various fluorochrome-conjugated antibodies to cells. Cells are incubated with the isotype control antibodies following the instructions above for preparing sample 1 and analyzed accordingly. The figure below shows a representative example of isotype control staining.

This antibody panel enables the simultaneous detection of both surface and intracellular markers for monitoring pluripotency. The figure below shows a representative result for the analysis of human induced pluripotent stem cells (iPS). The pluripotency markers SSEA-4, SSEA-5, TRA-1-60, Sox-2, and Oct 3/4 were expressed at high levels, whereas expression of the differentiation marker SSEA-1 (CD15) was low.

In contrast, cells differentiated towards the neural lineage expressed the pluripotency markers at low levels, or even shut off the expression of pluripotency markers, and up-regulated the differentiation marker SSEA-1 (CD15). Sox2 was expressed at high levels, as would be expected from neural precursors.

As a differentiation control, a second sample was stained with antibodies against PSA-NCAM and Pax6, which are markers for neuronal and neural progenitors, respectively. The large majority of cells (80%) co-expressed both markers.

PSC cultures were also monitored visually by light microscopy. The images show the typical morphology of PSC colonies (A in figure below) or cells differentiated into neural precursors (B in figure below).

Use the StemMACS™ Cryo-Brew for cryopreservation of pluripotent stem cells to ensure high viability and rapid recovery after thawing. To freeze as single cells, use Trypsin/EDTA for cell detachment. To freeze as cell clusters, use StemMACS™ Passaging Solution XF for detachment. Follow the protocol of the StemMACS Cryo-Brew data sheet.

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StemMACS Cryo-Brew

The following is a list of additional resources that may be of interest:

• Multicolor flow cytometry analysis of human pluripotent stem cell culture