Immortalized Human Bone Marrow Mesenchymal Stromal Cells - hTERT (iMSC3)
|T0529||1x106 cells / 1.0 ml|
Human mesenchymal stromal cells have extensive potential in research: uses for these including tissue repair and tissue engineering, gene therapy, the study of mesenchymal tumor oncogenes, and the analysis of cell fate determination and differentiation. The iMSC#3 cells express markers found in both primary human mesenchymal stromal cells and the multipotent form: CD90, CD44, NT5E, THY1, ENG, alanyl aminopeptidase (ANPEP), integrin beta I (ITGB1), integrin alpha 5 (ITGA5), major histocompatibililty complex, class I, A and B (HLA-A and B), and activated leukocyte cell adhesion molecule (ALCAM). These cells have also shown the ability to differentiate into adipocytes and osteoblasts. Furthermore, the cells have been verified for normal copy number, and lacks chromosomal aberrations and tumorigenicity.
|Species||Human (H. sapiens)|
|Tissue/Organ/Organ System||Bone Marrow|
|Cell Morphology||Spindle shaped|
|Seeding Density||20,000 cells/cm2|
|Population Doubling Time||45 - 55 hours|
|Immortalization Method||Serial passaging and transduction with recombinant retroviruses carrying hTERT gene (pBabe-puro-hTERT)|
For Research Use Only
|Unit quantity||1x106 cells / 1.0 ml|
|Cell Type||Immortalized Cells|
CD44, NT5E, THY1, ENG, CD90
Use of PriCoatTMT25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells inECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.
The base medium for this cell line is Prigrow III medium available at abm, Cat. No. TM003. To make the complete growth medium, add the following components to the base medium: heat-inactivated fetal bovine serum to a final concentration of 10%, and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.
|Preservation Protocol||1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO.|
2. Storage Temperature: Liquid nitrogen vapour phase.
(1) Human telomerase reverse transcriptase expression was verified with quantitative PCR; (2) Surface markers were determined with flow cytometry; (3) Gene expression profile was analysed using the Illumina WG-6 v2 Expression BeadChip and Illumina HumanHT12 v4; (4) Adiopocyte differentiation was detected with Oil Red O staining, while osteoblast differentiation was detected by staining cells with Alizarin Red and ALPL and quantification; (5) DNA methylation profiling was performed using the Illumina HumanMethylation450 BeadChip. Copy number verification was performed with high-resolution mapping using Affymetrix Genome-Wide Human SNP Array 6.0.; (6) Karyotype analysis was performed by arresting cells during metaphase and subjecting chromosomes to G-banding and subsequent imaging; (7) Tumorgenicity was verified in vivo with subcutaneous injection into immunodeficient athymic mice; (8) miRNA expression profile was determined using high-throughput sequencing.(9) Puromycin selection used (2 μg/mL) however not needed for long term culture.
1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.
2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected].
3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.
4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).
5. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.
6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.
7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."
- Important Considerations for Immortalized Cells
- Immortalized Cell Handling Instructions Upon Arrival
- Subculturing Protocol
- Freezing Protocol
- Hepatocyte Instructions
- Thawing Protocol
- Immortalized Cell Lines Flyer #1
- Cell Line Catalog (Volume 2)
- Immortalized Cell Lines Flyer #2
- Immortalized Cells FAQ
|I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?|
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
|How often do I need to change the media?|
The media should be changed every 2-3 days.
|Why do these cells need bio safety level II?|
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
|Do you sell ECM coated T75 flasks?|
Yes we can provide a coating service. Please inquire with [email protected]
|Is it necessary that we have to use the recommended T25 ECM-coated flasks for growing the cells? Can we use normal ECM coated 60mm/90mm petri plates for growing the cells?|
We strongly recommend using G299 T25 flasks to ensure recovery of these cells before testing other plates.
|What can I coat a larger dish to subculture?|
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
|How long can I store frozen vials for?|
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
|When are cells plated for live cell shipments?|
1 day prior to shipping
|Should the cap of the flask be changed before starting the cell culturing step?|
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
|What is the recommended storage temperature?|
In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
|How is cell density crucial for drug selection?|
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
|My cells are not detaching, what method do you recommend to trypsinize the cells?|
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
|Why is it important to determine the optimal seeding density?|
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
- Skårn, M et al. "Generation and characterization of an immortalized human mesenchymal stromal cell line" Stem Cells Dev 23(19):2377-89 (2014). PubMed: 24857590 .
- Naderi, EH et al. "Bone marrow stroma-derived PGE2 protects BCP-ALL cells from DNA damage-induced p53 accumulation and cell death" Mol Cancer 14(1):14 (2015). PubMed: PMC4323193.
- Bakshi, R., Hokugo, A., Zhou, S., Zhang, Z., Wang, L., Rezzadeh, K., … Jarrahy, R. "Application of Hydroxycholesterols for Alveolar Cleft Osteoplasty in a Rodent Model" Plastic and Reconstructive Surgery 143(5):1385–1395 (2019). DOI: 10.1097/prs.0000000000005528.
- Elsharkawi, I., Parambath, D., Saber-Ayad, M., Khan, A. A., & El-Serafi, A. T. "Exploring the effect of epigenetic modifiers on developing insulin-secreting cells" Human cell 1-9: (2019).