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Print Version |
| BioSafety Level |
II |
| Organism |
Homo sapiens |
| Source Organ |
Mammary |
| Growth Properties |
Adherent |
| Morphology |
Cobblestone or Spindle-shaped |
| Recommended Seeding Density |
Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions. |
| Markers |
K5, K14, vimentin, E-cadherin, p63, K8, K18, K19, CD29, CD49f
a-SMA, CD10, Thy-1 when grown in DCFI-1 |
| Applications |
For Research Use Only |
| Immortalization Method |
Serial passaging and transduction with recombinant lentiviruses carrying hTERT gene |
| Description |
The immortalized human mammary progenitor cells – hTERT (K5+/K19+) is a clonal cell population of progenitors coexpressing normal mammary and stem cell markers. It has the ability to self-renew and differentiate into luminal and/or myoepithelial cell lineages. Through propagation in different optimized media, the cells are able to give rise to new population of cells with different morphology and characteristics, such as mucin-1 positive cells, vimentin-negative cells, or expressing basal, luminal, and other stem cell markers. Aldehyde dehydrogenase 1A3 enzyme is noticeably higher in expression, a marker commonly used for isolating normal and tumor mammary stem cells. K5+/K19+ cells have Wnt, Notch, and hedeghog gene regulatory pathways. It is a valuable tool in research in the field of biology, the stem/progenitor origin, and the heterogeneity mechanisms in breast cancer. |
| Procedure Overview |
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| Propagation |
Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels with the following conditions. To make DFCI-1 the base medium is Prigrow VIII and Prigrow IV (1:1, vol/vol) medium available from ABM (TM018 and TM004). To make the completed growth medium, add the following components to the base medium at final concentration: 0.01M HEPES (Gibco; 15630), 1% fetal bovine serum (TM999), 12.5 ng/ml (EGF) epidermal growth factor (Z100135), 6.5 ng/ml triiodothyronine (Sigma; T5516), 0.545 ng/ml beta-estradiol (Sigma; E2257), 1 µg/ml insulin (Z101065), 1 µg/ml hydrocortisone (Sigma; I5500), 0.006X ethanolamine (Sigma; E9508), 14.1 µg/ml phosphoethanolamine (Sigma; P0503), 10 µg/ml transferrin (Sigma; T2252), 2 mM glutamine (Gibco; 25030-081), 2.6 ng/ml sodium selenite (Sigma; S1382), 1 ng/mg cholera toxin (Sigma; C8052), 35 µg/ml bovine pituitary extract (Hammond Cell Tech), 1% Penicillin/Streptomycin (G255) or 10 µg/ml Gentamicin (Gibco; 15750-060), and 10 µg/ml ascorbic acid (Sigma; A4034). Adjust to pH 7.4. Atmosphere: air: 93.5%, CO₂: 6.5%; Temperature: 37.0°C.
Optimized Media:
To express basal, luminal, and stem cell markers with some myoepithelial cell markers (a-SMA, CD10, Thy-1), grow cells in DFCI-1.
For cells to adopt spindle-shape morphology around tight epithelial cell colony and the presence of mucin-1 positive cells grow cells in mammary epithelial growth medium (MEGM; Lonza CC-3151) supplemented with B27 (10ml/500ml medium), 20 ng/ml (EGF) epidermal growth factor (Z100135), 20 ng/ml FGF2 (Z101455), 4 µg/ml heparin, and Penicillin/Streptomycin (G255). Atmosphere: air: 93.5%, CO₂: 6.5%; Temperature: 37.0°C.
For luminal differentiation and the absence of myoepithelial differentiation, and the appearance of mucin-1 positive and vimentin-negative cells, grow the cells in DCFI-2 media where the base is α-MEM and Prigrow IV (1:1, vol/vol) medium available from ABM (TM004). Supplement with, 0.01M HEPES (Gibco; 15630), 12.5 ng/ml (EGF) epidermal growth factor (Z100135), 6.5 ng/ml triiodothyronine (Sigma; T5516), 0.545 ng/ml beta-estradiol (Sigma; E2257), 1 µg/ml insulin (Z101065), 1 µg/ml hydrocortisone (Sigma; I5500), 0.006X ethanolamine (Sigma; E9508), 14.1 µg/ml phosphoethanolamine (Sigma; P0503), 10 µg/ml transferrin (Sigma; T2252), 2 mM glutamine (Gibco; 25030-081), 2.6 ng/ml sodium selenite (Sigma; S1382), 1 ng/mg cholera toxin (Sigma; C8052), 0.05% bovine serum albumin (BSA), 1% Penicillin/Streptomycin (G255) or 10 µg/ml Gentamicin (Gibco; 15750-060), and 10 µg/ml ascorbic acid (Sigma; A4034). Adjust to pH 7.4. Atmosphere: air: 93.5%, CO₂: 6.5%; Temperature: 37.0°C.
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| Preservation |
1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO.
2. Storage Temperature: Liquid nitrogen vapour phase. |
| Quality Control |
1) Western blot to assess cell lineage-related and stem cell markers; 2) Immunofluorescence staining analysis for lineage markers; 3) Gene expression profiling for gene regulatory pathways |
| Disclaimer |
1. The CoA for this product (provided upon request) verifies the cell-type specific gene expression via RT-PCR only. 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.
2. We strongly 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).
3. 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.
4. 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. |
