STEM CELL CALCULATOR V - UNFRACTIONATED HUMAN UMBILICAL CORD BLOOD HEMATOPOIETIC STEM CELLS (HSCs)

APPLICATION: This calculator allows determination of the tissue stem cell-specific fraction (SCF) of human HSCs in unfractionated umbilical cord blood when cultured in the following commercial cell culture medium after lysis or removal of red blood cells:

CULTURE CONDITIONS: Stemcell Technologies, StemSpanTM SFEM II (Cat#09655); StemSpanTM CD34+ Expansion Supplement (Cat#02691); pen/strep.

CALCULATOR V

Culture and counting procedures

    1. In a 24-well plate, initiate evaluation cultures with ideally 50,000 to 100,000 total viable cells per 2.25 mLs of the culture medium specified above. Fewer cells can be used, but more cells should not be used. Recommend preparing 6 replicate wells.
    2. Use 3 of the cultures to determine the mean total cell count 4-5 hours after plating. This analysis sets time = 0 hours.
    3. Approximately 72 hours later, determine the mean total cell count in the remaining 3 cultures, time = “72 hours.” This period should not be less than 66 hours and not more than 78 hours (i.e., the culture period must be limited to the range in fractional days from 2.75 to 3.25).

    Note: Accurate tissue stem cell counting requires an increase in total cell number during the period of this analysis.

    Calculator procedure

        1. Enter, d, the number of days of serial culture from the first cryopreservation of the initial isolated primary tissue cell preparation (e.g., 0, 3.0, 6.0). Entry of fractional days is recommended for increased accuracy.

        Important Note: For d > 0, subsequent culturing must have occurred in the same culture medium as prescribed above. (Calculators can be provided for other cell culture media and conditions. Please inquire.)

        d

        2. Enter N0, the total number of cells (live and dead) in the evaluation cultures at time = 0.

        N0

        3. Enter N72, the total number of cells (live and dead) after approximately 72 hours of culture.

        N72

        4. Enter, in units of fractional days, T, the actual period of time between the N0 cell count and the N72 cell count.

        T

        Result:

        EXAMPLE USE:

        1. In parallel with plating of primary cord blood unit mononuclear cells for CFU assay, perform the HSC rapid-counting evaluation 72-hour cell culture.
        2. Relate the HSC rapid-count to your CFU assay results.
        3. Evaluate the HSC rapid-count for correlation with clinical outcomes. It is predicted to be a better potency measure than CFU.