Serial culture and count your tissue stem cell-containing preparation using your usual culture conditions.
- Use any cell culture dish type you wish. Adherent or non-adherent tissue stem cells can be counted. Initiate culture with about 100,000 viable total cells per 25-cm2 attachment area; or for suspension cultures about 100,000 viable total cells per 2 mLs of culture medium.
- For adherent cells, 3-5 hours after the initial cell plating to start the analysis, replace the culture medium and count a parallel duplicate dish for the time = 0-day viable cell count.
- Serially passage the culture on a regular culture schedule (e.g., passage after every 3 days or every 5 days). It is okay to deviate from strict regular passage intervals, as long as the time of the actual serial passage is recorded. Record passage times as days and fractions of days as needed (e.g., 78 hours = 3.25 days).
- Maintain a constant split basis at each passage. Different constant split bases are recommended for different types of tissue stem cell preparations:
- CD34+-selected hematopoietic cells – 1:5
- Whole unfractionated cord blood – 1:20
- Adipose or dental bone-derived mesenchymal cells – 1:20
Important: Changing the split basis during the serial culture will prevent stem cell counting!
- Continue serial passaging until one of the following terminal proliferation endpoints is achieved:
- 1) No increase in total cell number above the input cell number for the passage is seen for two consecutive passages.
- 2) There are two consecutive passages for which no cells are detectable for counting.
Important: Termination of the serial culture before achieving a terminal proliferation endpoint will prevent stem cell counting!
At the end of each passage period, count and record the total number of live cells and dead cells in the culture dish, and the serial passage day number. Any live-dead basis may be used, including trypan blue dye-exclusion. Ideally, perform replicate sample counts at each passage and report the mean count values.
Enter your split basis and cell count data in the table below and submit. The picture below represents an example for a 3-day passage interval. Replace with actual interval times used.
The long-standing challenge of counting tissue stem cells specifically and accurately is met by Asymmetrex’s Kinetic Stem Cell Counting technology. In March 2020, Asymmetrex Director James Sherley presented the latest advance in the technology at the Perinatal Stem Cell Society’s Annual Congress in Salt Lake City, Utah. In the following video of his presentation, he describes both the theory and the practice of counting perinatal and postnatal tissue stem cells.