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Powered by BiozHuman Mesenchymal Stem Cells (hMSC)
Primary Human Mesenchymal Stem Cells. Available from adipose tissue, bone marrow and umbilical cord matrix.
Human Mesenchymal Stem Cells from Bone Marrow (hMSC-BM)
Unit
500,000 cells
Cat.No.
C-12974
Only %1 left
$922.00
Mesenchymal Stem Cells (MSC), also termed Mesenchymal Stromal Cells, are multipotent cells that can differentiate into a variety of cell types and have the capacity for self renewal. MSC have been shown to differentiate in vitro or in vivo into adipocytes, chondrocytes, osteoblasts, myocytes, neurons, hepatocytes, and pancreatic islet cells. Optimized standard and GMP grade media are available to support both the growth of MSC and their differentiation into several different lineages. Recent experiments suggest that differentiation capabilities into diverse cell types vary between MSC of different origin.
Our hMSC are harvested from normal human adipose tissue, bone marrow, and umbilical cord matrix (Wharton’s jelly) of individual donors.
The cells are tested for their ability to differentiate in vitro into adipocytes, chondrocytes, and osteoblasts. Our hMSC show a verified marker expression profile that complies with ISCT* recommendations, providing well characterized cells.
*ISCT (International Society for Cellular Therapy) Cytotherapy (2006) Vol. 8, No. 4, 315-317
- Request our GMP grade cell culture media for mesenchymal stem cells.
- Our hMSC are now also available from HLA-typed donors.
| Recommended plating density | 4000 cells per cm2 |
| Passage after thawing | P2 |
| Tested markers | Differentiation capacity to adipogenic, osteogenic and chondrognic cells tested, CD105 positive, CD73 positive, CD90 positive, CD45 negative, CD34 negative, CD14 negative, CD19 negative, HLA-DR negative |
| Guaranteed population doubling | > 10 |
NOTE: Umbilical cord matrix hMSC are currently not available.
Figure 1.Human Mesenchymal Stem Cell Culture isolated from bone marrow in MSC Growth Medium in phase contrast.
Figure 2.Human Mesenchymal Stem Cell Culture isolated from bone marrow in MSC Growth Medium XF in phase contrast.
Figure 3.Human Mesenchymal Stem Cell Culture isolated from adipose tissue in MSC Growth Medium in phase contrast.
Figure 4.hMSC-BM after in vitro differentiation into adipocytes using PromoCell Mesenchymal Stem Cell Adipogenic Differentiation Medium.
Figure 5.hMSC-BM spheroid after in vitro differentiation into cartilage using our Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Alcian Blue staining).
Figure 6.hMSC-BM after in vitro differentiation into osteoblasts using our Mesenchymal Stem Cell Osteogenic Differentiation Medium (Alizarin Red S staining).
Figure 7.hMSC-BM after in vitro differentiation into neuronal cells using our Mesenchymal Stem Cell Neurogenic Differentiation Medium.
At PromoCell we guarantee for our primary human cells ≥ 500;000 viable cells after thawing. For this; we dispense > 500;000 cells per cryovial before cryopreservation as there will always be a certain percentage of dead cells after freeze/thaw.
In order to know the number of cells that survived the procedure; we defrost a representative number of vials per lot during QC; determine the cell viability using an electronic counting device and then calculate the number of viable cells that can be recovered after thawing. Both numbers - the calculated number of viable cells and the viability - can be found on the lot-specific Certificate of Analysis (CoA) that can be downloaded from our website.
Example: When the CoA indicates 600;000 viable cells and a viability of 80%; this means that the vial actually contains 750;000 cells (viable + dead); 80% thereof (600;000) were viable after thawing in our QC. We do not indicate the total number of cells per vial but just the number of expected viable cells which can be recovered when the recommended thawing protocol is used. You don't have to calculate any viabilities by yourself.
When the recommended plating density for your cell type is 5;000 - 10;000 cells/cm²; then the 600;000 viable cells can be plated e.g. in a T75 (corresponding to 8;000 cells/cm²) or in a T75 + a T25 (corresponding to 6;000 cells/cm²).
Our customers have successfully used TC flasks and dishes from all the leading cell culture plastic suppliers to grow PromoCell's primary human cells. We do not have any knowledge whether the dishes from local TC plastic suppliers work in the same way. We recommend to first test whether these brands provide the same good performance as the plastic of the leading manufacturers.
The differentiation rate into the osteogenic lineage is 70-100%.
Our MSC Growth Medium XF (Ready-to-use) provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors. Therefore; culture vessels must be precoated with 10 μg/ml human or bovine fibronectin.
Protocol: Fibronectin coating
Dilute the Fibronection Solution to 10 μg/ml final concentration in Dulbecco´s PBS w/o Calcium and Magnesium. Overlay the culture surface of your tissue culture vessel with an amount of the diluted Fibronectin Solution sufficient to effectively coat the complete surface. Be sure that the entire surface is covered. Place flasks on a level surface at RT for 60 min. Aspirate the excess Fibronectin Solution and use immediately or let air-dry the open vessel under a laminar flow bench. Unused vessels may be stored at 4°C for up to 2 weeks.
Usually 90-100% of the hMSC show a neuronal morphology after differentiation with our Mesenchymal Stem Cell Neurogenic Differentiation Medium (C-28015). 60-80% of them are positive for nissl bodies after a nissl stain. However the differentiation capacity depends on the origin of the cells and the number of population doublings they have undergone.
Differentiation of hMSC into mature adipocytes takes approx. 2 weeks. You can keep the adipocytes for up to 3 weeks in the MSC Adipogenic Differentiation Medium. After 3 weeks we recommend to switch to PromoCell's Adipocyte Nutrition Medium (C-27438).
The population doubling times for PromoCell hMSC (hMSC-BM; hMSC-AT; hMSC-UC) are typically ≤ 30 hrs when using Mesenchymal Stem Cell Growth Medium 2 (C-28009). If you seed the hMSC at 4.000 cells/cm2; it will take between 4-7 days until they reach subconfluency.
The time needed to detach our primary cells depends on many different factors like the cell type; cell density; lot #; trypsin concentration; the efficiency of the washing step before adding the trypsin and the trypsinization temperature.
For most cell types we recommend trypsinization at room temperature and direct observation of detachment under the microscope. This way; you can find out your individual trypsinization time and keep the contact time between cells and trypsin to a minimum. Most cells detach after 2-8 min.
Please refer to the instructions in the Manual. For some cell types; trypsinization at 37°C or the use of Accutase or another Detachment Solution is recommended.
Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing they are in P2.
These cells are frozen at the end of 2nd culture. Thawing and seeding results in passage 2 (3rd culture). We recommend that they be used for differentiation experiments not later than passage 5. The differentiation potential of hMSC in vitro is reduced with ongoing population doublings; meaning the earlier differentiation is induced; the higher the differentiation rates.
Short protocol:
- Trypsinize the cells as usual
- Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 106 cells/ml
- Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision; which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight.
- Transfer the vials into liquid nitrogen for long-term storage
Our Normal Human Cells have been cultured and tested in our growth media and have adapted to these conditions. Using other media may yield unsatisfactory results due to suboptimal supplies of nutrients and growth factors. PromoCell can only guarantee good cell growth (as stated in the Certificate of Analysis) when the cells are grown in the recommended media.
The human MSC derived from bone marrow adipose tissue and umbilical cord matrix are from different origins but with comparable biological properties and function. Depending on the tissue of origin they may have a higher preference for differentiation into one particular cell type and a lower preference for another one but they all still retain the differentiation potential for the mesenchymal lineage.
- Adipogenic differentiation can be identified morphologically and without any staining by the formation of intracellular lipid vesicles.
- In contrast; when MSC differentiate into bone cells; there is no significant change in morphology. It is recommended to perform Alkaline Phosphatase staining to detect osteoblastic differentiation or Alizarin Red S staining to show osteoblast mineralization.
- Chondrogenic differentiation is generally performed as spheroids in 3-D cell culture and not in 2-D monolayer culture. Staining with Alcian Blue to visualize the differentiation process is indispensable.
- Neurogenic differentiation can be detected using neuron specific markers (e.g. beta-3 tubulin; NeuN; MAP2) and by their typical neuronal morphology.
We have tested the differentiation capacity of our hMSC into adipocytes chondrocytes and osteoblasts over time and still see good differentiation rates after 10 population doublings i.e. at passage 5. However the differentiation potential declines with ongoing population doublings. To obtain optimal differentiation rates experiments should be performed as early in culture as possible.
For chrondrogenic differentiation it is important that the cells do not adhere to the wells. During differentiation the cells form spheroids which float in the medium. Therefore there are no special requirements for the wells as long as they are U bottom shaped and suitable for suspension culture.
PromoCell Growth Media have special formulations and are much more complex than DMEM or RPMI + FBS. In comparison to immortalized cell lines primary cells have much higher nutrient and growth factor requirements. Classical media do not usually achieve good performances with our cells.
It is not necessary to use coated flasks for (most of) our Normal Human Cells but it can be done. As coating with extracellular matrix proteins can affect cellular metabolism; it is recommended to use the same coating material for a complete set of experiments.
Cells that need to be grown on coated dishes:
- Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need Fibronectin-coating when grown in PromoCell MSC Growth Medium XF (C-28019) and when differentiated in MSC Neurogenic (C-28015); Adipogenic (C-28016); or Osteogenic (C-28013) Differentiation Media.
- Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055; C-28056).
- For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020); the TC plates should be pre-coated with collagen type I.
The population doubling time or generation time (tg) is usually calculated during the logarithmic phase of growth. It specifies the time (t) in hours needed by the culture to double its cell number.
tg = t / n
n: number of population doublings
Yes but the experimental starvation conditions have to be determined individually for each cell type. Usually the cells are maintained in basal medium with reduced growth factor concentrations or lower FBS content. The cells need to be in a good condition and the starvation should be kept as short as possible as prolonged serum and/or growth factor deprivation induces apoptosis.
Cell type-specific markers are usually determined one passage after thawing.
Example: After thawing; HUVEC are in P1. Markers (CD31 expression; Dil-Ac-LDL uptake) are tested in P2.
Please note: Cells that are frozen directly after isolation (blood cells) are tested directly after thawing with no further culturing step.
The passage number varies depending on the cell type. Please refer to the Manual for your cells under "Specifications".
You will also find the information in the Certificate of Analysis (CoA).
The recommended plating density after thawing/subculture may vary depending on the cell type. Please refer to the data sheet for your cells under "Specifications".
The Certificates of Analysis can easily be downloaded from our PromoCell website:
https://www.promocell.com/certificate-of-analysis/
Simply type in the lot number indicated on the cryovial/TC-flask and click the SEARCH button.
In principle; both types of liquid nitrogen storage are acceptable; each having its advantages and disadvantages.
- Liquid phase storage provides a consistent temperature of -196°C; a longer holding time and a greater vial capacity but involves the risk of contamination issues.
- Storage in the gas phase is very safe with respect to contaminations but the holding time of the cells is shorter and the vial capacity is reduced.
PromoCell guarantee 15 population doublings (PD) for most Normal Human Cells (unless otherwise indicated on the Certificate of Analysis) when the recommended PromoCell media and the PromoCell DetachKit are used. For more information; please check the respective Manual; section "Specifications".
PromoCell does not determine the number of passages but instead we calculate the population doublings (PD) that can be performed with the cells. The term passage only describes the process of detachment and replating and does not take into account different split ratios. The optimal split ratio is calculated from the actual cell yield after trypsinisation and the recommended plating density. In most of our cell types; the split ratio is usually between 1:3 and 1:6. Using 1:4 splits (i.e. increasing the growth surface by factor 4 each time); 15 doublings are achieved after 6-8 passages.
For recommended plating densities; please view the respective Manual; section "Specifications".
Freshly isolated cells that are plated in a tissue culture vessel for the first time are named primary cells or primary culture (corresponding to P0). As soon as they have been subcultured; they should correctly be termed normal cells (> P1).
Generally; the medium should be changed every 2-3 days.
Please note: Following thawing; the first medium change should be performed after 16-24 hours to prevent cell damage due to residual freezing medium.
General protocol for recovery of anchorage-dependent primary cells:
Remove vial from liquid nitrogen; transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min; until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress.
Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min).
The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.
Your observation can be either ascribed to a change of the culture medium to a cross-contamination with another cell type or to differentiation or senescence. The attached trouble shooting guide should help you to identify the reason for your observation.
Microbiological contaminations can be introduced into cell cultures by unsterile working techniques contaminated water baths media plasticware etc. Please check the attached trouble shooting guide for more detailed information.
Normal cells have a finite life span and therefore eventually stop growing and become senescent. In addition a cease in proliferation or cell death can be induced by other factors. For more details check the trouble shooting guide below.
Slow growth after subculture can be caused by over-trypsinization or other suboptimal culture conditions. Please see attached trouble shooting guide for possible reasons.
Several different methods for the detection of mycoplasmas have been described like e.g. cultures on agar in liquid or semi-solid media staining with DAPI mycoplasma-specific antibodies biochemical methods and PCR-based assays. PCR-based detection is very sensitive detects all mycoplasma species that occur in cell cultures and is completed within 3-5 hours.
Yes; you can use 4.5% neutral buffered formalin. Paraformaldehyde should work as well.
Osteogenic differentiation of hMSCs with PromoCell MSC Osteogenic Differentiation Medium takes ∼12-14 days (please view the respective Application Note for a detailed protocol). Use fibronectin-coated plates and change the medium every third day.
The bone cells tend to detach from the plastic after approximately 2 weeks; when differentiation is complete. Therefore; the tests should be performed promptly and the cells should be maintained in MSC Osteogenic Differentiation Medium until then.
You can also use Oil Red O to stain lipid droplets. At PromoCell; we used to use Oil Red O as well; but switched to Sudan III some time ago for organizational reasons.
Light flocculation may be seen upon thawing the supplements containing ECGS/heparin or BPE. This does not affect the activity of our media.
Optionally; the precipitate can be removed by centrifugation under sterile conditions.
We recommend to thaw the supplements (SupplementMix or SupplementPack) at 15-25°C.
Yes; there are a few differences:
- We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding; as opposed to 16-24 hours after seeding for most other cell types/growth media.
- When MSC Growth Medium XF; MSC Neurogenic Differentiation Medium; MSC Adipogenic Differentiation Medium 2 or MSC Osteogenic Differentiation Medium are used; flasks have to be coated with 10 µg/cm² (human or bovine) fibronectin according to the instruction manual.
- We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used; contact time should not exceed 2 min.
The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense.
Leave the PBS on the cells after staining/washing and analyze the sample immediately; as the dye may bleed upon prolonged storage without embedding.
We guarantee > 500;000 viable cells per vial. To do this; we need to freeze more than 500;000 cells; as we do not yet know the viability after thawing at the time of freezing.
For technical/organizational reasons; the initial cell number may also vary from lot to lot; so 2 lots with the same viability may not necessarily contain the same number of cells.
Based on negative feedback we have received from customers using bead baths; we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells.
If you don’t have a “normal” water bath but only a bead bath in your lab; thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand; and not in a floater; as described in the thawing protocol.
Our cells are not manufactured according to GMP guidelines and are intended for in vitro use only.
Our EXCiPACT™ GMP certification only applies to the processes related to the media.
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