Mesenchymal Stem Cells - Stemcell · PDF fileMesenchymal Stem Cells ... including cord blood,...
-
Upload
nguyenngoc -
Category
Documents
-
view
219 -
download
1
Transcript of Mesenchymal Stem Cells - Stemcell · PDF fileMesenchymal Stem Cells ... including cord blood,...
Mesenchymal Stem CellsMesenCult™: Your High-Performance System
for MSC Isolation, Culture & Differentiation
2
Product Overviewfor Mesenchymal Stem Cells
IsolateObtain untouched MSCs from human bone marrow or mouse compact bone or select CD271+ MSCs from human bone marrow.• RosetteSep™ Human MSC Enrichment Cocktail: page 4• EasySep™ Human CD271 Positive Selection Kit: page 5• EasySep™ Mouse MPC Enrichment Kit for Compact Bone: page 13
Dissociate and Cryopreserve Dissociate and cryopreserve MSCs with animal component-free media.• MesenCult™-ACF Dissociation Kit: page 6• MesenCult™-ACF Freezing Medium: page 7• CryoStor®CS10, CS5 and CS2: page 7
Support Products• Hypoxia Chamber: page 15• ALDEFLUOR™ for Stem and Progenitor Cell Identification: page 15• Support Reagents, Antibodies and Primary Cells: page 15
STEMCELL Technologies’ products are manufactured under a Quality Management System (QMS) certified to ISO 13485. Unless otherwise indicated, products are provided for research use only, not for human or animal therapeutic or diagnostic use.
Your regulatory authority will provide guidance on the requirements for ancillary materials for cell therapy applications. Depending on the requirements, STEMCELL may be able to assist you in meeting your regulatory and quality requirements.
STEMCELL Technologies stands behind the quality of our products. We welcome onsite audits of our manufacturing facilities to ensure that your quality requirements are met. If you have any questions or would like to discuss the potential use of a product for your application please contact us.
CulturePerform CFU-F assays and expand human and mouse MSCs.• MesenCult™-ACF Medium for Human MSCs: page 6• MesenCult™-XF Medium for Human MSCs: page 8• MesenCult™ Proliferation Kit (Human): page 10• MesenCult™ Proliferation Kit with MesenPure™ (Mouse): page 12
New
DifferentiateDifferentiate MSCs to adipocytes, osteoblasts and chondrocytes• MesenCult™ Adipogenic Differentiation Medium (Human): page 11• Adipogenic Stimulatory Supplement (Mouse): page 14• Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF): page 11• Osteogenic Stimulatory Kit (Human): page 11• Osteogenic Stimulatory Kit (Mouse): page 14• MesenCult™-ACF Chondrogenic Differentiation Medium: page 11New
3
Mesenchymal Stem Cell Products
For Every Facet of Discovery
Mesenchymal stem cells (MSCs) are fibroblast-like cells isolated from bone marrow, adipose, and other tissues - including cord blood, peripheral blood, fetal liver, skeletal muscle, placenta, amniotic fluid and synovium.1-7 These are all vascularized tissues, and accumulating evidence suggests that MSCs are pericytes8 which closely encircle endothelial cells in capillaries and microvessels of multiple organs.8-15
MSCs are defined by properties exhibited following in vitro culture. Namely the ability to self-renew, differentiate into bone, adipose and cartilage tissue,16 the expression of CD105, CD73 and CD90, and the lack of expression of CD45, CD34, CD11b and HLA-DR. While originally coined mesenchymal stem cells,17 MSCs are also referred to by other terms, such as multipotent mesenchymal stromal cells,16,18 mesenchymal progenitor cells19 or medicinal signaling cells.20 No single term has been uniformly adopted, and as a result, the acronym MSC is commonly used to encompass all terminologies applied to these cells.
MSCs have potential utility in a range of cellular therapies, with applications relating to tissue engineering and regenerative medicine, and as vehicles for gene therapy.21-24 To realize the therapeutic potential of MSCs, studies in animal models are of fundamental importance. Because MSCs are rare, occurring at an estimated frequency of 1 in 100,000 cells in adult human bone marrow, they must be expanded in vitro to obtain sufficient numbers for research and therapeutic applications.25 The ability to expand human MSCs in xeno-free or animal component-free medium may alleviate concerns about immune rejection of transplanted cells or disease transmission, and is therefore an important consideration when the MSCs are to be used therapeutically.25
Advantages of MesenCult™
HIGH-PERFORMANCE CULTURES. Species-optimized media formulations provide superior performance compared to traditional and competitor media.
COMPREHENSIVE. A comprehensive and integrated range of products is available for the isolation, enrichment, expansion and differentiation of human and mouse MSCs.
STANDARDIZED. Standardized media and culture conditions minimize variability and lead to increased reproducibility between experiments.
EASY TO USE. Products are supplied with detailed technical information to guide researchers through the enrichment and culture of MSCs.
MesenCult™Your High-Performance System for MSC Isolation, Culture & Differentiation
Choose from a comprehensive range of specialty products for human and mouse MSCs designed to service scientists along the basic to translational research continuum.
The MesenCult™ product line was developed as a comprehensive and integrated suite of products to help MSC researchers standardize their cell culture system and overcome issues associated with cell culture media variability. Providing products for both mouse and human MSCs enables STEMCELL Techologies to serve scientists along the basic to translational research continuum.
4
Human MSCs can be rapidly and easily enriched directly from unprocessed bone marrow using the RosetteSep™ Human Mesenchymal Stem Cell Enrichment Cocktail and a simple density gradient centrifugation (Figure 1, Table 1).
The RosetteSep™ Enrichment Cocktail contains monoclonal antibodies to deplete cells expressing CD3, CD14, CD19, CD38, CD66b and Glycophorin A. The antibodies link unwanted cells to red blood cells present in the bone marrow sample, causing them to pellet together when centrifuged over the density gradient medium. Desired cells are left untouched and highly enriched at the density gradient medium : plasma interface.
Figure 1. Overview of RosetteSep™ Human Mesenchymal Stem Cell Enrichment procedure.
Table 1. CFU-F frequency and fold enrichment obtained for RosetteSep™-enriched human bone marrow samples (n = 9; Mean ± SD).
* Data normalized to density gradient centrifugation-separated bone marrow.§ p < 0.0001 when compared to density gradient centrifugation alone. The CFU-F assay was performed with the MesenCult™ Proliferation Kit (Human; Catalog #05411).
Add RosetteSep™ cocktail to bone marrow
Unwanted cells are linked to red blood cells (rosetted)
Incubate 20 minutesat room temperature
Layer over density gradient medium (e.g. Lymphoprep™)
Density gradient medium
PlasmaEnriched MSCs
Density gradient medium
Red blood cells and unwanted cells (rosetted)
Enriched MSCs are untouched
Label
Spin
Collect
Enrich Human MSCsWith RosetteSep™ Human MSC Enrichment Kit
PRODUCT QUANTITY CATALOG #
RosetteSep™ Human Mesenchymal Stem Cell Enrichment Cocktail
For labeling 40 mL whole bone marrow
15128
For labeling 200 mL whole bone marrow
15168
DENSITY GRADIENT CENTRIGUFATION
ONLYROSETTESEP™
Frequency of CFU-F
1 : 84,000 ± 37,000 1 : 12,500 ± 6,500§
Recovery of CFU-F
100% 64 ± 24%
Fold Enrichment*
1 8 ± 2
5
Mesenchymal Stem Cell Products
Isolate Human MSCsWith EasySep™ CD271 Positive Selection Kit
CD271 has been identified as a marker of MSCs and CD271+ cells isolated from human bone marrow display high CFU-F activity.26 CD271 is also known as the p75 neurotrophin receptor (p75NTR) or the low-affinity nerve growth factor receptor (LNGFR). The EasySep™ Human CD271 Positive Selection Kit is designed to select CD271+ cells from fresh bone marrow mononuclear cells with high purity and recovery (Figure 2, Table 2).
EasySep™ is a powerful cell separation tool that is fast, easy and gentle on cells. With the EasySep™ Human CD271 Positive Selection Kit, CD271+ cells are targeted for selection by linking CD271+ cells to magnetic particles. The tube containing the magnetically labeled cells is placed into an EasySep™ magnet, and after a brief incubation the unlabeled cells are poured off. The highly purified CD271+ cells remain in the tube. EasySep™ magnetic particles do not interfere with downstream applications such as flow cytometry and do not need to be removed to preserve cell functionality.
Figure 2. Overview of EasySep™ Human CD271 Positive Selection procedure.
Add EasySep™reagents
Incubate 15 minutes
Add EasySep™magnetic particles
Incubate 15 minutes
Place tube in magnet for 5 minutes
Pour off supernatant. Positively selected cells remain in tube.
Bone marrowcell suspension
Collect
Table 2. CFU-F fold enrichment obtained for EasySep™-enriched human bone marrow samples (n = 4; Mean ± SEM).
PRODUCT QUANTITY CATALOG #
SELECTION COCKTAIL
EasySep™ Human CD271 Positive Selection Kit
For 1 x 109 cells 18659
REQUIRED EQUIPMENT
EasySep™ Magnet 1 magnet 18000
EASYSEP™ HUMAN CD271+ SELECTION
CFU-F Fold Enrichment 23 ± 6
6
New: Culture Human MSCs With Animal Component-Free MesenCult™-ACF Medium
MesenCult™-ACF is a defined, serum- and animal component-free (ACF) culture medium to support efficient derivation and expansion of MSCs from primary human bone marrow (BM) and adipose tissue. When used in combination with MesenCult™-ACF Attachment Substrate, MesenCult™-ACF Dissociation Kit and MesenCult™-ACF Freezing Medium, MesenCult™-ACF Medium is part of a complete, animal component-free culture system which supports efficient attachment, clonogenic growth and long-term expansion of primary human MSCs.
MSCs cultured with MesenCult™-ACF exhibit robust suppression of T cell proliferation and significant reduction in hematopoietic cell contamination at early passages.
Advantages of MesenCult™-ACF
• Defined, animal component-free culture system
• Optimized for isolation of MSCs directly from primary human tissue
• No adaptation required when transitioning MSCs from serum-containing medium
• Significant reduction in hematopoietic cell contamination at early passages
• Supports efficient clonogenic growth and long-term expansion of human MSCs
Figure 3. Human BM-derived MSCs cultured in MesenCult™-ACF display multi-lineage differentiation potential.
Human BM-derived MSCs cultured in MesenCult™-ACF Medium for 3 passages (A) differentiate into adipocytes (B; Oil Red O staining), osteogenic cells (C; Von Kossa/ALP staining) and chondrocytes (D; Alcian Blue + Nuclear Fast Red staining).
BA
C D
Magnification: 40X
Magnification: 40X
Magnification: 40X
Magnification: 40X
PRODUCT QUANTITY CATALOG #
MesenCult™-ACF Culture Kit* 1 kit 05449
MesenCult™-ACF Medium§ 500 mL 05440
MesenCult™-ACF Dissociation Kit 1 kit 05426
*MesenCult™-ACF Culture Kit includes MesenCult™-ACF Medium (Catalog #05440) and MesenCult™-ACF Attachment Substrate (Catalog #05444).§MesenCult™-ACF Medium must be supplemented with L-Glutamine (e.g. Catalog #07100), and must be used in conjunction with MesenCult™-ACF Attachment Substrate and MesenCult™-ACF Dissociation Kit.
Figure 5. Human BM-derived MSCs cultured in MesenCult™-ACF display characteristic expression of MSC surface markers.
Human BM-derived MSCs cultured in MesenCult™-ACF were stained at Passage 4 with antibodies to mesenchymal markers (CD90, CD73 and CD105) and hematopoietic markers (CD45, CD11b and HLA-DR).
0
0 102 103 104 105
0
0 102 103 104 105
0
0 102 103 104 105
CD90 PE CD73 PE CD105 PE
0
0 102 103 104 105
0
0 102 103 104 105
0
0 102 103 104 105
CD45 PE CD11b PE HLA-DR APC
New
New
Figure 4. Human BM-derived MSCs cultured in MesenCult™-ACF expand faster than cells cultured in serum (FBS)-based or competitor media. (n = 3 except for Competitor 3 where n = 2; Mean ± SEM).
Competitor 2 (xeno-free medium)
P1 P2 P3 P4 P5 P6 P7 P8
Traditional formulation (FBS-based)
MesenCult™-ACF Medium
Competitor 1 (serum-free medium)
1010
109
108
107
106
105
104
103
102
Competitor 3 (chemically-defined medium)
Tot
al C
ell N
umbe
r (L
og)
Passage Number
7
Mesenchymal Stem Cell Products
Cryopreserve MSCs With Animal Component-Free Freezing Media
MesenCult™-ACF Freezing Medium is a defined, serum-free and animal component-free medium for the cryopreservation of MSCs. This complete and ready-to-use medium is recommended for cryopreservation of human MSCs previously cultured in MesenCult™-ACF (Catalog #05440), MesenCult™-XF (Catalog #05420) or MesenCult™ (MesenCult™ Proliferation Kit; Catalog #05411) media.
Advantages of MesenCult™-ACF Freezing Medium
• Defined, serum-free and animal component-free
• Reproducibly high recovery rates
• Preserves human MSC multipotency and expansion capacities
• Convenient, ready-to-use format
Figure 6. Human MSCs cryopreserved in MesenCult™-ACF Freezing Medium show reproducibly high post-thaw cell viability.
Human MSCs cultured in serum-free medium and cryopreserved in MesenCult™-ACF Freezing Medium show reproducibly high viability of cells after thawing. Viability is higher than observed with a competitor ACF freezing medium (n = 6 independent experiments, P = 0.001).
Figure 7. Human MSCs cryopreserved in MesenCult™-ACF Freezing Medium maintain multi-lineage differentiation potential.
Human MSCs cultured in serum-free medium and cryopreserved with MesenCult™-ACF Freezing Medium (A; 3 days after thawing) differentiate to the adipogenic (B; Oil Red O staining) and osteogenic (C; Alizarin Red staining) lineages.
BA C
PRODUCT QUANTITY CATALOG #
MesenCult™-ACF Freezing Medium
50 mL 05490
CryoStor®CS10 100 mL 07930
5 x 16 mL 07930
CryoStor®CS5 100 mL 07932
CryoStor®CS2 100 mL 07933
MesenCult™-ACF Freezing Medium
CryoStor®CS10, CS5 and CS2:
CryoStor® cryopreservation media products are formulated with 2, 5 or 10% USP-grade DMSO and are cGMP manufactured. These versatile media are designed to maximize post-thaw viability and recovery of a variety of cell types, including MSCs.
8
Reduce variability by culturing your human MSCs in a standardized xeno-free (XF), serum-free medium. MesenCult™-XF Medium is optimized for both the expansion of MSCs and their enumeration using the CFU-F assay.
MesenCult™-XF supports the serum-free isolation of MSCs from primary tissue. Passage 0 cultures obtained from primary human bone marrow (BM) show significantly reduced hematopoietic cell contamination compared to serum-based cultures (Figure 8). Furthermore, MSCs expanded in MesenCult™-XF for two passages showed no detectable hematopoietic cell contamination, indicated by the lack of CD34 and CD45 expression (Figure 9). MSCs cultured in MesenCult™-XF also robustly express the mesenchymal markers CD90, CD105 and CD73 (Figure 9).
MesenCult™-XF supports the long-term culture (>8 passages) of MSCs, with cells expanding faster in MesenCult™-XF than MSCs cultured in serum-based medium (Figure 10). MSCs cultured in MesenCult™-XF maintain the ability to differentiate into adipogenic, osteogenic and chondrogenic lineages (Figure 11). The chondrogenic differentiation potential of MSCs cultured in MesenCult™-XF is enhanced at early passages (e.g. P2) and better maintained after multiple passages (e.g. P4, P8) compared to cells cultured in serum-based medium (Figure 12). MSCs cultured in MesenCult™-XF also more robustly suppress T cell proliferation and reduce cell cycle division (Figure 13).
* MesenCult™-SF Culture Kit includes MesenCult™-XF Medium (Catalog #05420) and MesenCult™-SF Attachment Substrate (Catalog #05424).§ MesenCult™-XF Medium must be supplemented with L-Glutamine (e.g. Catalog #07100), and must be used in conjunction with the MesenCult™-SF Attachment Substrate and the MesenCult™-ACF Dissociation Kit.
Advantages of MesenCult™-XF
• Defined, xeno-free formulation
• Optimized for use with MSCs obtained directly from primary tissue
• Supports faster cell expansion compared to traditional serum-based medium
• Cultured MSCs exhibit robust suppression of T cell proliferation
• Minimal lot-to-lot variability
Figure 8. Primary human bone marrow-derived MSCs show less hematopoietic cell contamination when cultured in MesenCult™-XF (A) compared to a traditional serum-based medium (B).
Magnification: 5X
A
Magnification: 5X
B
Passage 0 Passage 0
REFERENCESMesenCult™-XF is the best defined culture system for umbilical cord MSCs (Hartmann et al. 2010).www.stemcell.com/references
Culture Human MSCsWith Xeno-Free MesenCult™-XF Medium
PRODUCT QUANTITY CATALOG #
MesenCult™-SF Culture Kit* 1 kit 05429
MesenCult™-XF Medium§ 500 mL 05420
MesenCult™-SF Attachment Substrate
1 kit 05424
9
Mesenchymal Stem Cell Products
Magnification: 20X
E
Magnification: 40X
A
Magnification: 10X
B
Figure 11. Human bone marrow-derived MSCs cultured in MesenCult™-XF display multi-lineage differentiation potential.
A. Oil red O staining of adipocytes generated from passage 1 MSCs.B. Alizarin Red detection of Ca++ deposits indicates the formation of bone structures in cells generated from passage 4 MSCs.C. Collagen II staining of chondrocytes generated from passage 2 MSCs.
Magnification: 10X
C
0 1 2 3 4 5 6 7 8 9 10
MesenCultTM-XF Medium
Traditional formulation
100
102
104
106
108
1010
1012
Figure 10. Human BM-derived MSCs cultured in MesenCult™-XF expand faster than cells cultured in a traditional serum-based medium (n = 3; Mean ± SEM).
Passage Number
Tota
l Cel
ls
Figure 13. MSCs cultured in MesenCult™-XF more robustly suppress T cell proliferation than MSCs cultured in a traditional serum-based medium.
Passage 2 MSCs generated in MesenCult™-XF or a traditional serum-based medium were treated with mitomycin C prior to co-culture with T cells. T cells were purified from peripheral blood using EasySep™ cell separation and fluorescently labeled with CFSE dye. 2 x 105 CFSE-labeled T cells were cultured with 1 x 105 MSCs in serum-free medium supplemented with 100 U/mL IL-2. T cells were stimulated with tetrameric antibody complexes against CD3ε, CD28 and CD2. On days 3 and 7, cells were harvested, stained with anti-CD45 antibody and propidium iodide and the T cell division history measured as CFSE dye dilution analyzed by flow cytometry.
CFSE
Day 3
Day 7
Unstimulated T cells Activated T cells FBS MSCs MesenCultTM-XF MSCs
1% (37%)
3% (95%) 82% (95%)
18% (37%) 1% (37%)
53% (95%)
Magnification: 4X
A
Magnification: 20X
B
Figure 12. MSCs cultured in MesenCult™-XF (A, B, C) better maintain chondrogenic differentiation potential than MSCs cultured in a traditional serum-based medium (D, E, F).
Magnification: 20X
C
Magnification: 4X
D
Magnification: 20X
F
Passage 2 Passage 4 Passage 8
Figure 9. Phenotype of MesenCult™-XF culture-expanded human MSCs.
Cou
nts
CD90 (Thy-1)+
0
10
20
30
40
50
100 101 102 103 104
CD105 (SH2)+
Cou
nts
100 101 102 103 1040
10
20
30
40
50
60
70
CD73 (SH3/SH4)+
Cou
nts
100 101 102 103 1040
10
20
30
40
50
60
CD34-100 101 102 103 104
Cou
nts
0
10
20
30
40
50
60
CD45-100 101 102 103104
Cou
nts
0
10
20
30
40
50
60
10
Standardize your human MSC cultures with the serum-based MesenCult™ Proliferation Kit (Human). The optimized medium formulation enables efficient expansion of MSCs in vitro (representative expansion data are shown in Table 3) and components are prescreened to minimize lot-to-lot variability. The culture-expanded cell population maintains multi-lineage potential and cells express CD105 (SH2), CD73 (SH3/SH4) and CD90 (Thy-1) and lack expression of CD45 and CD34 (Figure 14).
The MesenCult™ Proliferation Kit (Human) is comprised of MesenCult™ MSC Basal Medium (Human) and Mesenchymal Stem Cell Stimulatory Supplement (Human), and can be used for both the expansion of human MSCs and their enumeration using the CFU-F assay.
Table 3. Expansion of MSCs obtained from four consecutive human bone marrow (BM) samples that were cultured with the MesenCult™ Proliferation Kit (Human).
* The MesenCult™ Proliferation Kit (Human) comprises MesenCult™ MSC Basal Medium (Human; Catalog #05401) and Mesenchymal Stem Cell Stimulatory Supplement (Human; Catalog #05402).
Figure 14. Phenotype of culture-expanded human MSCs using the MesenCult™ Proliferation Kit (Human).
CD34-
Cou
nts
100 101 102 103 104
0
10
20
30
40
50
CD90 (Thy-1)+
Cou
nts
1000
10
20
30
40
101 102 103 104
50
Culture Human MSCsUsing MesenCult™ Proliferation Kit (Human)
BM SAMPLE FOLD EXPANSION DAYS IN CULTURE
A 3732X 40
B 6966X 58
C 1770X 41
D 230X 28
PRODUCT QUANTITY CATALOG #
MSC CULTURE MEDIUM
MesenCult™ Proliferation Kit (Human)*
500 mL 05411
PROLIFERATION KIT COMPONENTS
MesenCult™ MSC Basal Medium (Human)
450 mL 05401
Mesenchymal Stem Cell Stimulatory Supplement (Human)
50 mL 05402
CD105 (SH2)+
Cou
nts
1000
10
20
30
40
50
101 102 103 104
CD73 (SH3/SH4)+
Cou
nts
100 101 102 103 1040
10
20
30
40
50
CD45-
Cou
nts
100 101 102 103 1040
10
20
30
40
50
11
Mesenchymal Stem Cell Products
Optimize the differentiation of human MSCs with standardized media and supplements. New animal component-free MesenCult™-ACF Chondrogenic Differentiation Medium delivers efficient and robust chondrogenic differentiation of human MSCs with as few as 3 x 105 cells per 3D pellet and in as little as 14 days (Figure 15).
MesenCult™ Adipogenic Differentiation Medium (Human) provides robust adipogenic differentiation of human BM- and adipose-derived MSCs previously cultured in serum-containing or serum-free media. The easy-to-use protocol does not require cycles of induction and maintenance.
The complete Osteogenic Stimulatory Kit (Human) enables the differentiation of human MSCs into osteogenic progenitors, and the detection and enumeration of human colony-forming unit - osteoblast (CFU-O). The kit also supports CFU-O assays from rat bone marrow cells. For human MSCs previously cultured in MesenCult™-XF, use the Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF) for in vitro differentiation into osteogenic progenitors.
Culture in MesenCult™ Adipogenic Differentiation Medium (Human)
Adiopocytes derived from human MSCs differentiated in vitro with MesenCult™ Adipogenic Differentiation Medium (Human)
AdipogenicDifferentiation
Culture-expanded MSCs
Culture with the Osteogenic Stimulatory Kit.
Figure 16. Adipogenic and osteogenic differentiation of human MSCs.
OsteogenicDifferentiation
Differentiate Human MSCsTo Adipocytes, Osteoblasts and Chondrocytes
PRODUCT QUANTITY CATALOG #
ADIPOGENIC DIFFERENTIATION
MesenCult™ Adipogenic Differentiation Medium
250 mL 05412
OSTEOGENIC DIFFERENTIATION
Osteogenic Stimulatory Kit (Human)
1 kit 05404
Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF)
1 kit 05434
CHONDROGENIC DIFFERENTIATION
MesenCult™-ACF Chondrogenic Differentiation Medium
100 mL 05455
Bone nodule derived from human MSCs differentiated in vitro with the Osteogenic Stimulatory Kit (Human).
A. Phase micrograph of human bone nodule
B. Tetracycline labeled human bone nodule
C. SEM of human bone nodule showing mineralized collagen ECM
D. TEM of human bone nodule
SEM: scanning electron micrographTEM: transmission electron
micrographECM: extracellular matrix
A
CD
B
Photos kindly provided by Dolores Baksh, Peter W. Zandstra and John E. Davies, Institute of Biomaterials & Biomedical Engineering, University of Toronto
New
Figure 15. MesenCult™-ACF Chondrogenic Differentiation Medium induces robust chondrogenic differentiation of human MSCs with as few as 3 x 105 cells and as early as day 14.
Human BM-derived MSCs were cultured in MesenCult™-ACF Medium then differentiated to the chondrogenic lineage using MesenCult™-ACF Chondrogenic Differentiation Medium. Robust chondrogenic differentiation was observed (A) starting with as few as 3 x 105 MSCs, or (B) when differentiating for just 14 days starting with 5 x 105 MSCs.
A B
200 μm
12
Enrich and Culture Mouse MSCsUsing MesenCult™ Proliferation Kit with MesenPure™ (Mouse)
Establishing mouse MSC cultures is inherently difficult due to both the rarity of these cells in bone marrow (BM) and compact bone (CB) and because these cultures often contain a high proportion of hematopoietic cells.27-29
The MesenCult™ Proliferation Kit with MesenPure™ (Mouse) is a standardized medium for the expansion of mouse MSCs as well as their enumeration using the CFU-F assay. This optimized medium enriches for mouse MSCs in culture without serial passaging and frequent medium changes, steps routinely employed to deplete unwanted hematopoietic cells from mouse MSC cultures using other media. Primary MSCs treated with MesenPure™ appear homogeneous and mostly devoid of hematopoietic cells as early as passage 0 (Figure 17). Cultures also contain increased numbers of MSCs that display more robust differentiation (Figure 18 and 19).
Control With MesenPure™
P0
P3
100 µm
Figure 17. Mouse MSC cultures treated with MesenPure™ appear homogeneous and mostly devoid of hematopoietic cells as early as passage 0 (P0).
Primary BM-derived MSCs were cultured under hypoxic conditions in complete MesenCult™ medium without (Control) or with MesenPure™. In control conditions, many hematopoietic cells (viewed as highly refractile cells under phase contrast optics) are present at P0 and still present at P3 (left panels). In MesenPure™-treated cultures, low numbers of hematopoietic cells were observed as early as P0, with subsequent passages reducing the number further (right panels).
Figure 18. Flow cytometry analysis of P0 MSC culture treated with MesenPure™ demonstrates significant enrichment of CD45-/CD29+/Sca1+ cells.
Primary BM-derived MSCs were cultured for 14 days under hypoxic conditions in complete MesenCult™ medium without (Control) or with MesenPure™ prior to flow cytometry analysis being performed. ~9% of the control culture and ~70% of the MesenPure™-exposed culture was CD45-. Analysis of the CD45- population revealed ~75% of the control cells and ~96% of the MesenPure™-exposed cells co-expressed CD29 and Sca1.
SS
C
SS
C
Sca
1-F
ITC
Sca
1-F
ITC
Control With MesenPure™
CD45-
9.04%CD45+
90.53%CD45-
70.48%CD45+
28.88%
6.70% 74.88%
5.57%
1.22% 96.11%
1.29%
P0 Cultures
CD45- Selected Cells from P0 Cultures
12.84% 1.38%
CD29-PECY7 CD29-PECY7
CD45-APC CD45-APC
Mouse MSCs expand faster when cultured at 5% O2 compared to 20% O2. Use the hypoxia chamber (Catalog #27310) to generate a hypoxic environment (See page 15).
Advantages of MesenPure™
SAVES TIME. Fast enrichment of mouse MSC cultures without serial passaging.
HIGH-PERFORMANCE CULTURES. Homogeneous mouse MSC cultures display robust self-renewal and differentiation potential.
VERSATILE. Optimized for use with mouse bone marrow- and compact bone-derived MSCs and MEFs.
EASY TO USE. Simply add MesenPure™ to complete MesenCult™ proliferation medium just prior to use.
13
Mesenchymal Stem Cell Products
EASYSEP™ MOUSE MPC ENRICHMENT FOR COMPACT BONE
CFU-F Fold Enrichment 110 ± 74
Recovery of CFU-F 58 ± 19%
B
A
Figure 19. MSCs exposed to MesenPure™ require fewer cells to demonstrate robust differentiation.
(A) BM-derived MSCs initially cultured in complete MesenCult™ medium without (Control) or with MesenPure™ were plated at three different densities and then differentiated using MesenCult™ Osteogenic Stimulatory Kit (Mouse; Catalog #05504). Differentiation cultures were fixed and stained for Alkaline Phosphatase (ALP) activity (red areas) and mineralization (black areas). In control cultures, modest differentiation into the osteogenic lineage was only observed when cultures were seeded at the highest density. In MesenPure™-treated cultures, robust osteogenic differentiation was observed at all seeding densities. (B) 4.0 x 104 cells/cm2 of P2 CB-derived MSCs were used in osteogenic and adipogenic differentiation. 5.0 x 105 cells of P1 CB-derived MSCs were used for chondrogenic differentiation. MesenPure™-treated cultures displayed strong osteogenic, adipogenic and chondrogenic differentiation.
Number of cells/cm2 (x103)
40 60 120 40 60 120
BM-MSCs
CB-MSCs
Osteogenic Adipogenic
Number of cells/cm2 (x103)
Control With MesenPure™
Chondrogenic
*The MesenCult™ Proliferation Kit with MesenPure™ (Mouse) is comprised of MesenCult™ MSC Basal Medium (Mouse; Catalog #05501), Mesenchymal Stem Cell Stimulatory Supplements (Mouse; Catalog #05502) and MesenPure™ (0.5 mL; Catalog #05500)
PRODUCT QUANTITY CATALOG #
MesenCult™ Proliferation Kit with MesenPure™ (Mouse)*
1 kit 05512
MesenCult™ MSC Basal Medium (Mouse)
400 mL 05501
Mesenchymal Stem Cell Stimulatory Supplements (Mouse)
100 mL 05502
VIDEOIsolation of MSCs from Mouse Compact Bonewww.stemcell.com/CompactBoneVideo
Mouse MSC Enrichment from Compact Bone Using EasySep™
The EasySep™ Mouse MPC Enrichment Kit for Compact Bone is designed to enrich MSCs from mouse compact bone cell suspensions by depleting undesired cells (Table 4). It can be used as an alternative method for mouse MSC enrichment from compact bone if culture-based enrichment using the MesenCult™ Proliferation Kit with MesenPure™ (Mouse) is not desired.
EasySep™ is a powerful cell separation tool that is fast, easy and gentle on cells. With the EasySep™ Mouse MPC Enrichment Kit for Compact Bone, cells expressing CD45 and TER119 are targeted for depletion by linking them to EasySep™ magnetic particles via tetrameric antibody complexes. The tube containing the magnetically labeled cells is then placed into an EasySep™ magnet and after a brief incubation the unlabeled MSCs are poured off and collected. The unwanted cells remain in the tube.
Table 4. CFU-F fold enrichment and recovery obtained for EasySep™-enriched mouse compact bone samples compared to control samples in complete MesenCult™ medium without MesenPure™ (n = 6; Mean ± SEM).
* Note: The EasySep™ Mouse MPC Enrichment Kit for Compact Bone is optimized for the enrichment of mouse MSCs from compact bone, not bone marrow. A technical video outlining the procedure for the isolation of cells from mouse compact bone is available at www.stemcell.com/CompactBoneVideo
PRODUCT QUANTITY CATALOG #
ENRICHMENT COCKTAIL
EasySep™ Mouse MPC Enrichment Kit for Compact Bone*
For 4 x 108 cells
19771
REQUIRED EQUIPMENT
EasySep™ Magnet 1 magnet 18000
100 µm
Learn more about mouse MSC culture by watching the on-demand webinar “Tips & Techniques for Highly Enriched Mouse MSC Cultures As Early As Passage 0” available at www.stemcell.com/MesenPureWebinar.
14
Optimize the differentiation of mouse MSCs to Osteoblasts and adipocytes with standardized media and supplements.
Mouse MSCs or mouse embryonic fibroblasts (MEFs) achieve unparalleled levels of osteogenic differentiation with the MesenCult™ Osteogenic Stimulatory Kit (Mouse) compared to competitor formulations. Superior expression of key osteogenic transcripts involved in bone differentiation and maturation is observed for MSCs differentiated with the MesenCult™ Osteogenic Stimulatory Kit (Mouse) compared to cells differentiated with a leading commercially available medium for mouse cells (qPCR analysis, Figure 20a). Cultures also exhibit robust bone matrix deposition (Alizarin Red-staining, Figure 20c).
The Adipogenic Stimulatory Supplement (Mouse) efficiently differentiates mouse mesenchymal stem cells to adipocytes when combined with MesenCult™ MSC Basal Medium (Mouse) (Figure 21).
* The Adipogenic Stimulatory Supplement (Mouse) must be used in conjunc-tion with MesenCult™ MSC Basal Medium (Mouse; Catalog #05501).
Figure 21. Differentiation of mouse compact bone-enriched MSCs to adipocytes using the Adipogenic Stimulatory Supplement (Mouse) combined with MesenCult™ MSC Basal Medium (Mouse).
A. Unstained adipocytes generated from passage 3 cells previously cultured with the MesenCult™ Proliferation Kit (Mouse).
B. Oil red O staining of adipocytes generated from passage 3 cells previously cultured with the MesenCult™ Proliferation Kit (Mouse).
Figure 20. Differentiation of mouse compact bone-derived MSCs with the MesenCult™ Osteogenic Stimulatory Kit (Mouse).
A. Expression of key osteogenic transcripts in mouse compact bone-derived MSCs as measured by qPCR.
B. Undifferentiated mouse compact bone-derived MSCs do not produce bone matrix and appeared negative for Alizarin Red-staining.
C. Differentiated mouse compact bone-derived MSCs display robust bone matrix deposition as detected by Alizarin Red-staining.
100000
10000
1000
100
10
1
Competitor A
MesenCult™ Osteogenic Stimulatory Kit (Mouse)
Differentiate Mouse MSCsTo Adipocytes and Osteoblasts
Bsp
Fol
d-I
ndu
ctio
n (l
og s
cale
)
Osx Alpl Runx2
A
PRODUCT QUANTITY CATALOG #
OSTEOGENIC DIFFERENTIATION
Osteogenic Stimulatory Kit (Mouse)
1 kit 05504
ADIPOGENIC DIFFERENTIATION
Adipogenic Stimulatory Supplement (Mouse)*
100 mL 05503
MesenCult™ MSC Basal Medium (Mouse)
400 mL 05501
B C
A B
Mouse MSCs cultured using the MesenCult™ Proliferation Kit with MesenPure™ (see pages 12-13) can be differentiated to chondrocytes with MesenCult™-ACF Chondrogenic Differentiation Medium and culturing for 21 days under normoxic (20% O2) conditions.
15
Mesenchymal Stem Cell Products
Stem and Progenitor Cell IdentificationThe ALDEFLUOR™ reagent system detects viable stem and progenitor cells based on aldehyde dehydrogenase (ALDH) enzyme activity. Over 1000 publications have used it to detect viable stem and progenitor cells of various lineages, including human mesenchymal stem/progenitor cells.30
PRODUCT USE QUANTITY CATALOG #
Lymphoprep™
Density gradient separation of MNCs in bone marrow
250 mL 07801
500 mL 07851
PBS + 2% FBSEasySep™ and RosetteSep™ isolations
500 mL 07905
Ammonium Chloride Solution
Lysing red blood cells present in bone marrow
100 mL 07800
500 mL 07850
L-Glutamine Cell culture 100 mL 07100
100 mm Tissue Culture Treated Dishes
Cell culture10/pack 27125
240/case 27127
3% Acetic Acid with Methylene Blue
Nucleated cell counts
100 mL 07060
Trypan Blue Viable cell counts 100 mL 07050
PBS (without Ca++ or Mg++)
Washing cells 500 mL 37350
Trypsin-EDTADissociation; Detachment
500 mL 07901
Human Platelet Lysate
Cell culture supplement
50 mL 06960
100 mL 06961
500 mL 06962
Human Platelet Lysate, Fibrinogen Depleted
Cell culture supplement
50 mL 06963
100 mL 06964
500 mL 06965
Support Reagents for Cell Enrichment and Culture
PRODUCT QUANTITY CATALOG #
Hypoxia Chamber 1 chamber 27310
Single Flow Meter 1 meter 27311
References1. Campagnoli C, et al., Blood 98: 2396-2402, 20012. In't Anker PS, et al., Blood 102: 1548-1549, 20033. Zuk PA, et al., Mol Biol Cell 13: 4279-4295, 20024. Erices A, et al., Br J Haematol 109: 235-242, 20005. De Bari C, et al., Arthritis Rheum 44: 1928-1942, 20016. Kuznetsov SA, et al., J Cell Biol 153: 1133-1140, 20017. Tondreau T, et al., Stem Cells 23: 1105-1112, 20058. Caplan AI, Cell Stem Cell 3: 229-230, 20089. Andreeva ER, et al., Tissue Cell 30: 127-135, 199810. Doherty MJ, et al., J Bone Miner Res 13: 828-838, 199811. Bianco P, et al., Stem Cells 19: 180-192, 200112. Zannettino AC, et al., J cell Physiol 214: 413-421, 200813. Shi S, et al., Bone Miner Res 18: 696-704, 200314. Sacchetti B, et al., Cell. 131: 324-336, 200715. Crisan M, et al., Cel lStem Cell 3: 301-313, 200816. Dominici M, et al., Cytotherapy 8: 315-317, 200617. Caplan AI., J Orthop Res. 9: 641-650, 199118. Horwitz EM et al., Cytotherapy 7: 393-395, 200519. Deana S. et al., Stem Cells Int. 2010: 51902820. Caplan AI., Tissue Eng Part A. 16: 2415-2417, 201021. Horwitz EM, et al., Cytotherapy 10: 771-774, 200822. Jones BJ, et al., Exp Hematol 36: 733-741, 200823. Uccelli A, et al., Nat Rev Immunol 8: 726-736, 200824. Caplan AI, J Cell Physiol 213: 341-347, 200725. Thirumala S, et al., Expert Opin Biol Ther 13: 673-691, 201326. Quirici N, et al., Exp Hematol 30: 783-791, 200227. Phinney DG, et al., J Cell Biochem 72: 570-585, 1999 28. Anjos-Afonso F, et al., J Cell Sci 117: 5655-5664, 200429. Peister A. et al., Blood 103: 1662-1668, 200430. Gentry T, et al., Cytotherapy 9: 259-274, 2007
PRODUCT QUANTITY CATALOG #
ALDEFLUOR™ 40 tests/kit 01700
STEMCELL Technologies provides high-quality primary and secondary antibodies, as well as a range of primary cell products, for MSC research. For a complete listing visit www.stemcell.com/Antibodies and www.stemcell.com/PrimaryCells.
Support ProductsFor Mesenchymal Stem Cells
Hypoxia Chamber
A hypoxic environment, easily generated using a hypoxia chamber, mimics physiological conditions. Many cell types, such as mouse MSCs, undergo enhanced proliferation when cultured with lowered atmospheric oxygen tension.
Hypoxic Tissue Culture Environment
Copyright © 2015 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies and Design, STEMCELL shield, Scientists Helping Scientists, MesenCult, MesenPure, EasySep, and RosetteSep are trademarks of STEMCELL Technologies Inc. ALDEFLUOR is a trademark of Aldagen, Inc. All other trademarks are the property of their respective holders.
New
New
Scientists Helping Scientists™ | WWW.STEMCELL.COM
TOLL-FREE PHONE 1 800 667 0322 • PHONE 1 604 877 0713
[email protected] • [email protected]
FOR GLOBAL CONTACT DETAILS VISIT OUR WEBSITE
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY
AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.
DOCUMENT #28367 VERSION 3.4.4 JANUARY 2016