Application protocol

Isolation, expansion, and phenotyping of bone marrow-derived mesenchymal stem cells (MSCs)

Mesenchymal stem cells or mesenchymal stromal cells (MSCs), are somatic or adult stem cells that can be isolated from various sources, including bone marrow, adipose tissue or umbilical cord. Isolated MSCs are fibroblast-like cells that demonstrate site-specific differentiation. MSCs may regenerate damaged or diseased tissues in vivo and potentially play a role in immunomodulation, which provides the basis for a variety of clinical applications. In this application protocol, we describe the enumeration, isolation, expansion and quality assessment of bone marrow-derived MSCs.

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.

For mononuclear cell isolation

  • PE buffer: Phosphate buffered saline (PBS), pH 7.2, and 2mM EDTA. Keep buffer cold (2–8 °C).
    ▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
  • 15 mL of Ficoll-Paque™ (ρ = 1.077 g/mL)
  • MACS® SmartStrainers (100 µm) (# 130-098-463)

For bone-marrow MSC quality control

  • MSC Enumeration Kit, human (# 130-106-646)

For MSC isolation

  • CD271 MicroBead Kit, human (# 130-099-023)
  • PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
    ▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
  • MACS Columns and MACS Separators: CD271+ cells can be enriched using MS or LS Columns. Positive selection can also be performed using the autoMACS Pro or the autoMACS Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×1072×108MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LS1×1082×109MidiMACS™, QuadroMACS™,
VarioMACS, SuperMACS II
autoMACS2×1084×109autoMACS Pro, autoMACS
▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
  • (Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD271 (LNGFR)-PE (# 130-091‑885), CD271 (LNGFR)-APC (# 130-091-884), or CD45-FITC (# 130‑080-202). Learn more about our antibodies and dyes.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometric exclusion of dead cells
  • (Optional) Dead Cell Removal Kit (# 130-090-101) for the depletion of dead cells
  • (Optional) Pre-Separation Filters, 30 μm (# 130-041-407) to remove cell clumps

For MSC expansion

StemMACS™ MSC Expansion Media Kit XF (# 130-104-182)

For MSC phenotypic and functional analysis

  • StemMACS AdipoDiff Media, human (# 130-091-677)
  • StemMACS ChondroDiff Media, human (# 130-091-679)
  • StemMACS OsteoDiff Media, human (# 130-091-678)
  • MSC Phenotyping Kit, human (# 130-095-198)
  • MSC Suppression Inspector, human (# 130-096-207)

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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.