Application protocol

Maintenance and cryopreservation of pluripotent stem cells

Miltenyi Biotec solutions for pluripotent stem cell culture, passaging, and cryopreservation ensure gentle handling and recovery with high viability. In this application protocol, we introduce the use of these solutions, as well as a multicolor flow cytometry protocol that allows for the simultaneous quantification of intracellular and surface markers for easy phenotyping of human pluripotent stem cell cultures.

Methods

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

General reagent and instrument requirements

  • Buffer: Dulbecco’s phosphate-buffered saline (DPBS) without Ca2+ and Mg2+
  • A small molecule ROCK inhibitor, e.g., StemMACS™ Y27632 (# 130-103-922) or StemMACS Thiazovivin (# 130-104-461) to improve cell attachment and survival
  • Cell attachment substrate. Validated substrates are, e.g., Matrigel®, Geltrex®, Laminin-511, Lamin-521, iMatrix-511, vitronectin, or CTS™ CELLstart™ substrate
  • 0.05% Trypsin/EDTA (alternatively, Accutase® or TrypLE™) and Soybean Trypsin Inhibitor (0.5 mg/mL) for single-cell splitting
  • 15 mL conical tubes

For maintaining pluripotent stem cell cultures

  • Medium: StemMACS iPS-Brew XF, human (# 130-104-368)

For passaging pluripotent stem cell cultures

  • StemMACS Passaging Solution XF (# 130-104-688)

For phenotyping pluripotent stem cells

  • PBE buffer consisting of PBS, pH 7.2, 0.5% BSA, and 2 mM EDTA. Prepare PBE buffer by diluting MACS® BSA Stock Solution (#130-091-376) 1:20 with autoMACS® Rinsing Solution (#130-091-222). Keep the buffer cold (2−8°C). 
  • Inside Stain Kit (#130-090-477) for intracellular staining
  • MACS Comp Bead Kit, anti-REA (#130-104-693) to set up the PE-Vio® 770 channel for detection of the differentiation marker SSEA-1

Detection antibodies

  • Anti-TRA-1-60-PE, human REA157 (# 130-100-347)
  • Anti-SSEA-1-PE-Vio 770, human and mouse REA321 (# 130-104-938)
  • Anti-SSEA-4-VioGreen™, human REA101 (# 130-098-341)
  • Anti-SSEA-5-VioBlue®, human 8e11 (# 130-106-657)
  • Anti-Sox2-FITC, human and mouse REA320 (# 130-104-940)
  • Anti-Oct3/4 Isoform A-APC, human and mouse REA338 (# 130-105-555)

Isotype control antibodies

  • Mouse IgG1-VioBlue IS5-21F5 (# 130-094-670) 
  • REA Control (I)-FITC REA293 (# 130-104-611)
  • REA Control (I)-APC REA293 (# 130-104-615)
  • REA Control (S)-PE REA293 (# 130-104-612)
  • REA Control (S)-VioGreen REA293 (# 130-104-608)
  • REA Control (S)-PE-Vio 770 REA293 (# 130-104-616)

For cryopreserving pluripotent stem cell cultures

  • StemMACS Cryo-Brew (# 130-109-558)
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Protocol


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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

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Detachment of pluripotent stem cells using the StemMACS Passaging Solution XF. After a few minutes exposure to the passaging solution, the cells begin to round up at the edges, at which point the solution can be removed.

Protocol

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, e.g., fixed and permeabilized, in the same way as the stained samples. 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-REA 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.

Pipetting scheme for surface and intracellular staining. Cell surface staining refers to steps 3–8 of the protocol below. Intracellular staining refers to steps 14–19.
Cell surface staining
SamplePBE BufferAnti-TRA-1-60-PEAnti-SSEA-1-PE-Vio 770Anti-SSEA-4-VioGreenAnti-SSEA-5-VioBlue
1. Multicolor panel70 µl10 µl10 µl10 µl10 µl
2. Unstained sample100 µl
3. Single-color staining100 µl10 µl
4. Single-color staining100 µl10 µl
5.. Single-color staining100 µl10 µl
6. Single-color staining100 µl
7. Single-color staining100 µl
Intracellular staining
SampleInside PermAnti-Sox2-FITCAnti-Oct3/4 Isoform A-APC
1. Multicolor panel90 µl10 µl10 µl
2. Unstained sample100 µl
3. Single-color staining100 µl
4. Single-color staining100 µl
5. Single-color staining100 µl
6. Single-color staining100 µl10 µl
7. Single-color staining100 µl10 µl
  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 70 μL of PBE buffer. This will be the cell sample stained with the complete panel.
  4. Resuspend samples 2–7 in 100 μL of PBE buffer. This will be the unstained and single-stained samples.
  5. Add 10 μL of each of the following antibodies against surface markers to sample 1: i) Anti-TRA-1-60-PE, ii) Anti-SSEA-1-PE-Vio 770, iii) Anti-SSEA-4-VioGreen™, and iv) Anti-SSEA-5-VioBlue®.
  6. Add 10 μL of one of the antibodies detecting a surface marker to samples 3–5.
  7. Do not add antibody 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 90 μL of Inside Perm.
  14. Resuspend samples 2–7 in 100 μL of Inside Perm.
  15. Add 10 μL of each of the following antibodies against intracellular markers to sample 1: i) Anti-Sox2-FITC, and ii) Anti-Oct3/4 Isoform A-APC.
  16. Add 10 μL of one of the antibodies for intracellular markers to samples 6 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
  19. completely.
  20. 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.
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Setting up the flow cytometer for compensation of spectral overlap. (A) Adjustment of scatter and voltage using an unstained sample. PE vs. FITC is shown as an example. (B) Compensation using a PE-stained cell sample; PE vs. FITC is shown as an example. (C) Compensation of the PE-Vio 770 channel using MACS Comp Bead Kit Anti-REA according to the kit protocol. PE-Vio 770 vs. PE is shown as an example.

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.

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Isotype control staining. Cells were incubated with the various isotype control antibodies (red line) or left unstained (black line) and analyzed by flow cytometry on the MACSQuant® Analyzer 10.

Representative results

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 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. 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.

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Multicolor flow cytometry analysis of undifferentiated (A) and differentiated (B) human iPSCs. Cells were stained with the antibodies as indicated and analyzed by flow cytometry on the MACSQuant Analyzer 10. Unstained cells were used as a control for gating. Numbers in the heatmaps specify percentages of single-positive (bold numbers) and double-positive cells.

Pluripotent stem cell cultures monitored visually by light microscopy show the typical morphology of pluripotent stem cell colonies or cells differentiated into neural precursors.

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Microscopy of cultured pluripotent stem cells. Cultures of undifferentiated human induced pluripotent stem cells (A) and induced pluripotent stem cells differentiated towards the neural lineage (B). Shown are light microscopy images of the same cultures that were used for flow cytometry above.

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

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