Maintenance and freezing 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.

Materials

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-mouse Igκ (#130-097-900) to set up the PE-Vio® 770 channel for detection of the differentiation marker SSEA-1 (CD15)

Detection antibodies

  • Anti-TRA-1-60-PE, human REA157 (# 130-100-347)
  • CD15-PE-Vio770, human VIMC6 (# 130-113-486)
  • 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 (# 130-117-709)

Isotype control antibodies

  • Mouse IgG1-VioBlue IS5-21F5 (# 130-113-205)
  • REA Control (I)-FITC REA293 (# 130-118-354)
  • REA Control (I)-APC REA293 (# 130-120-709)
  • REA Control (S)-PE REA293 (# 130-113-438)
  • REA Control (S)-VioGreen REA293 (# 130-113-444)

For cryopreserving pluripotent stem cell cultures

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

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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.