RFP-Expressing Human Bone Marrow-Derived Mesenchymal Stem Cells

Frozen Vials in a Dry Ice Package.

Frozen vial

General Information#RFP-HBMMSCs (cAP-0050RFP) are selected from puromycin resistant HBMMSCs after transfected with RFP expressing lentiviral particles. RFP-HBMMSCs are supplied in cryovials at passage 3- 4. Each lot of cAP-0050GFP is tested to ensure the cells can be passaged at least for three times (i.e., approximately 9 to 10 population doublings) after thaw. RFP-HBMMSCs should be maintained in complete growth media (Mesenchymal Stem Cell Growth Medium (MSCGM), cAP-35) following the detailed protocol described below).
Characterization of the Cells#
1.: Positive for CD29, CD44, CD73, CD90, CD105, and CD166 (greater than 95% of the cell population expresses these markers by flow cytometry).
2.: Negative for CD14, CD31, CD34, and CD45 (less than 2% of cell population expresses these markers by flow cytometry).
3.: HBMMSCs are negative for HIV-1, -2, HBV, HCV, and Bacteria, Yeast and Mycoplasma.
Product Usage# The cells are offered for Research Use Only.
Handling of Arriving Cells# When you receive the dry ice package with cells in frozen vials, transfer the frozen vials of cells into a -80C freezer for short period storage or a liquid nitrogen tank for long- term storage.
Protocols for Thawing the cells and subculture#
HBMMSCs are contact inhibited. It is essential that the cells be subcultured BEFORE reaching confluence as post-confluent cells exhibit changes in morphology, slower proliferation, and reduced differentiation capacity after passaging.
A):Pre-coating of T25 flasks- Add 2ml each Quick Coating Solution (cAP-01) into a T25 flask to cover the whole surface of the flask, 5 mins later, dispose the excessive coating solution by aspiration and the flask is ready to be used (although solution containing other extracellular matrix, i.e. gelatin, collagen, and fibronectin, can be used, make sure to optimize the conditions in advance).
B):Thaw the frozen cell vial in a 37C water bath first, and then transfer the cells into the pre-coated T25 flask with 10ml of cAP-35 medium, cells usually become confluent with 5-7 days.
C):To passage the cells, rinse the cells in a T25 flask with 5ml HBSS (RT) twice; then add 2ml Trypsin/EDTA (RT) (cAP-23) into one T25 flask; gently dispose the excessive Trypsin/EDTA solution within 20 seconds by aspiration.
D):Leave the T25 flask with the cells at RT or 37C for 1 min (most cells usually will detach from the surface within 1-2 mins; or monitor the cells under a microscope until most of cells become rounded up, and then gently tap the flask against the bench surface, and the cells will move on the surface of the flask when monitoring under microscope.
E):Add 5ml Trypsin Neutralization Buffer and spin down the cells with 800g centrifugation for 5 mins.
G):Re-suspend the cell pellet with 10 or 15ml Full medium and transfer 5 ml each into 2 or 3 pre-coated T25 flasks (for 1/2 to 1/3 subculture ratio).
H):Change medium every 2 or 3days and the cells usually become confluent within 7 days (when split at a 1/3 ratio).