STEM CELL CALCULATOR VII - HUMAN ADULT MOBILIZED PERIPHERAL BLOOD CD34+ HEMATOPOIETIC STEM CELLS (HSCs)

APPLICATION: This calculator allows determination of the tissue stem cell-specific fraction (SCF) of HSCs in human adult CD34+-selected mobilized peripheral blood (MPB) cells 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 VII

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 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 USES:

          Application for quantifying the HSC-specific fraction or dosage of human CD34+-selected MPB cells

          1. After isolation of a preparations of human adult CD34+-selected MPB cells, remove a sample of cells and perform a 72-hour evaluation culture as specified for HSC rapid-counting.
          2. Enter the respective values of d (d = 0.0 days), N0, N72, and T.
          3. Relate the HSC-specific fraction or dosage to other experimental factors or conditions under investigation.

          Note: If the isolated cells are cultured with the conditions for rapid-counting evaluations, the same rapid-counting evaluation procedure will allow determination of the HSC-specific fraction at any time during the culture of the isolated cell preparation (i.e., for d > 0.0).

          Application for SCID Mouse Repopulating Cell Assay Analyses

          1. In parallel with SCID mouse injection of human adult CD34+-selected MPB cells, perform the HSC rapid-counting evaluation 72-hour cell culture. Or if you routinely perform a 72-hour expansion-culture before injecting cells, count the starting total cell number (“N0”) and the 72-hour total cell number (“N72”).

          2. Evaluate the HSC rapid-count for correlation with engraftment efficiency.  It is predicted to correlate with engraftment potency.