Application protocol

Isolation, expansion, and phenotyping of umbilical cord-derived mesenchymal stem cells

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 MSCs derived from umbilical cord.

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 dissociation of umbilical cord tissue

  • Umbilical Cord Dissociation Kit (# 130-105-737)
  • Phosphate-buffered saline, pH 7.2
  • DMEM
  • gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237)
  • MACS® SmartStrainers (100 µm) (# 130-098-463)
  • (Optional) 70% ethanol
  • (Optional) MACS Tissue Storage Solution (# 130-100-008)
  • (Optional) ART® 1000 REACH™ pipet tips for removal of dissociated material from the closed C Tubes

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.

Preparation of buffer for tissue dissociation

PE buffer for dissociation of umbilical cord tissue

Prepare a solution with 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).

Dissociate human umbilical cord to a single-cell suspensions by combining mechanical dissociation with enzymatic degradation of the extracellular adhesion proteins that maintain the tissue structural integrity. Use the gentleMACS™ Octo Dissociator with Heaters in combination with the Umbilical Cord Dissociation Kit, human. Follow the protocol of the kit data sheet.

Download data sheet

Umbilical Cord Dissociation Kit, human

Expand umbilical cord-derived MSCs using the StemMACS™ MSC Expansion Media Kit XF, an optimized and standardized complete medium for the reproducible and reliable generation and expansion of MSCs from umbilical cord and other tissue sources. Follow the protocol of the kit data sheet.

Download data sheet

StemMACS MSC Expansion Media Kit XF, human

Growing interest in using MSCs for clinical research necessitates a fundamental understanding of mechanisms and processes underlying their differentiation into specific cell types, as well MSC phenotype validation of cultivated cells. The MSC Phenotyping Kit, human is based on ISCT (International Society for Cellular Therapy) markers for human MSCs, and helps easily perform standardized phenotyping of culture-expanded MSCs by flow cytometry. Follow the protocol of the kit data sheet. See "Functional analysis of expanded MSCs" to assess the differentiation potential of isolated MSCs.

Download data sheet

MSC Phenotyping Kit, human

Mesenchymal stem cell culturing protocols


To plan a dedicated antibody panel for your MSC research project, use our antibody panel builder.

Analysis of differentiation potential

Differentiation potential is a meaningful tool to qualify and define a MSC population. Grade of differentiation into adipocytes, chondrocytes, and osteoblasts is influenced by MSC origin, so for example, MSCs from bone marrow show greater potential to differentiate into osteoblasts than MSCs from umbilical cord. Increased age of donor and passaging of cultured MSCs also decrease differentiation potential.

StemMACS™ AdipoDiff, OsteoDiff, and ChondroDiff Media, human are optimized differentiation media for human MSCs isolated from various tissue sources. Each medium supports differentiation capacity analysis or quality control of expanded MSCs, as well as in vitro studies on MSC differentiation processes, including gene expression and protein profiling. Follow the protocol of the relevant kit data sheet.

Download data sheet

StemMACS AdipoDiff Media, human

StemMACS OsteoDiff Media, human

StemMACS ChondroDiff Media, human

Analysis of immunomodulatory potential

In the last few years, scientists have directed their attention to the immunomodulatory potential of MSCs. MSCs derived from bone marrow have been observed to suppress proliferation of T cells.1,2 This function of MSCs can be analyzed using the MSC Suppression Inspector, human, which contains T cell stimulation reagents optimized for an MSC suppression assay. Follow the protocol of the kit data sheet.

Download data sheet

MSC Suppression Inspector, human

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

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