Immortalized Human Adipose-derived Stromal Cells - Bmi-1/hTERT
|T0540||1x106 cells / 1.0 ml|
With its ability to maintain multipotency, mesenchymal stem cells provide new venues for cell-based therapy in restoration of damaged or diseased tissue. Most clinical studies utilize bone marrow-derived stem cells, however adipose-derived stromal cells (ASCs) is emerging as an alternative source for their ease of harvesting. The Immortalized Adipose-derived Stromal Cells- Bmi-1/hTERT is derived from human ASCs that have maintained their primary phenotypes and their ability to respond to osteogenic and adipogenic inductions. After 3 weeks of induced osteogenic differentiation, the immortalized ASCs show nuclear translocation of RUNX2, upregulation of alkaline phosphatase and calcium deposition. Furthermore, the immortalized ASCs-Bmi-1/hTERT exhibit normal karyotype and has been shown to exceed 140 population doublings, making these cells suitable for studies in regenerative medicine.
|Species||Human (H. sapiens)|
|Seeding Density||6,000 - 8,000 cells/cm2|
|Population Doubling Time||90 - 100 Hours|
|Immortalization Method||Serial passaging and transduction with recombinant lentiviruses carrying mouse Bmi-1 (pLOX-CWBmi1) [Ref. seq.: NM_007552.4] and hTERT (pLOX-TERT-iresTK) [Ref.seq.: NM_198253.2] gene|
For Research Use Only
|Unit quantity||1x106 cells / 1.0 ml|
|Cell Type||Immortalized Cells|
CD44, CD73, CD90 and CD105
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 IV medium available at abm, Cat. No. TM004. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 10%, L-glutamine (G275) to a final concentration of 1% and Gentamicin (G272) to a final concentration of 50 μg/mL. Optional for better growth: 1-5 ng/mL recombinant bFGF (Z101455)
1. Real Time PCR, immunofluorescence, and telemorase activity was used to quantify Bmi-1 and/or hTERT gene expression in the immortalized cell line. 2. Culture tested negative for mycoplasma
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."
|Depositor||Creative Cell Kft|
- Important Considerations for Immortalized Cells
- Immortalized Cell Handling Instructions Upon Arrival
- Subculturing Protocol
- Freezing Protocol
- Hepatocyte Instructions
- Differentiation Protocol
- Thawing Protocol
|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.
|Can T0540 differentiate into fully functional adipocytes and can they be used as a model to study adipocyte-derived EMVs?|
Concerning adipocyte differentiation, these cells produce large droplets of fat. The differentiation should be stable however the protocol will have to be optimized by the end user and this is not part of the guarantee. Further information regarding the osteogenic differentiation of T0540: This can be achieved via two different protocols: 1) Using Thermo-Fischer/GibcoSTEMPro Osteogenesis Differentiation kit (cat. n.:A1007201) OR 2) A "self-made" osteogenesis medium: Ascorbic acid 110 mg beta-glycerophosphate 540 mg Dexamethasone 250 microliter of a 100 micromol stock solution, (made in abs. alcohol); final concentration 1 nM Add 250 ml completed DMEM medium (including 10% FBs, glutamax/glutamine, antibiotics). Please note that the plated cell number is critical and will need to be optimized by the end user. You may start off by plating 1.3 x 104 (13,000 cells) / cm2 on day zero, followed by a changing of the media to the osgeogenic media the next day (day 1). Feed cells twice a week for 2-3 weeks. Kindly note that these are general guidelines only to help the end user start the differentiation process. Differentiation is not part of ABM's normal guarantee as further optimization by the end user is a critical step.
|What is the differentiation rate at late passages (i.e. 100, 150)?|
Most of the cells differentiate into adipocytes, even after hundreds of passages. The extinction (Photometer: 492 nm) of the extracted oilred O staining was determined , and these immortalized cells can be used as a positive control for other MSC work. Adipocytes produce different size of droplets, but most of the cells are more or less positive.
|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.