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. This application protocol describes the enumeration, isolation, expansion, and quality assessment of bone marrow-derived MSCs.
Prepare a solution with phosphate buffered saline (PBS), pH 7.2, and 2 mM 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).
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 1:20 with autoMACS® Rinsing Solution. 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). BSA can be replaced by other proteins such as respective serum albumin, respective serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
Isolate mononuclear cells by density gradient centrifugation using Ficoll-Paque™. Use the protocol of the following special protocol.
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The BM-MSC Analysis Cocktail Kit, anti-human is especially designed to quantify human MSCs from fresh bone marrow aspirate (BMA) with a standardized flow cytometry protocol and gating strategy based on the expression of CD271 (LNGFR) and Anti-MSCA-1 (W8B2). The kit allows identification of CD45+ leukocytes and CD271 (LNGFR)+/Anti-MSCA-1 (W8B2)+ MSCs, which are also CD45dim. Follow the protocol of the kit data sheet.
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Bone marrow-derived human MSCs can be isolated by plastic adherence or by specific surface markers such as CD271. Marker-based MSC isolation provides more uniform cell populations that show higher clonality, compared to plastic adherence. Use the CD271 MicroBead Kit, human to isolate MSCs. Follow the protocol of the kit data sheet.
It is recommended to filter the magnetically labeled cell suspension to guarantee a single-cell suspension before separating cells on the column.
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Expand isolated human 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 bone marrow and other tissue sources. Follow the protocol of the kit data sheet.
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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 Cocktail Kit, anti-human, REAfinity™ 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.
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MSC Phenotyping Cocktail Kit, anti-human, REAfinity
Mesenchymal stem cell culturing protocols
To plan a dedicated antibody panel for a MSC research project, use the antibody panel builder.
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.
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StemMACS AdipoDiff Media, human
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.
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The following is a list of relevant resources that might be of interest:
Column | Max. number of labeled cells | Max. number of total cells | Separator |
---|---|---|---|
Positive selection | |||
MS | 1×107 | 2×108 | MiniMACS™, OctoMACS™, SuperMACS II |
LS | 1×108 | 2×109 | MidiMACS™, QuadroMACS™, SuperMACS II |
autoMACS | 2×108 | 4×109 | autoMACS Pro, autoMACS |
▲ Note: Column adapters are required to insert certain columns into SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
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