Amniotic fluid stem cells (AFSC) are clonogenic stem cells with the capacity to differentiate into cells of the endoderm, mesoderm, and ectoderm lineages. Derived from the amniotic fluid, AFSCs avert much of the controversy of human embryonic stem cells (hESCs) while offering the desired stem cell characteristics of self-renewal and pluripotency.

The following protocol outlines steps for amniotic fluid stem cell expansion using PRIME-XV AFSC Expansion Medium. This medium is manufactured under cGMP conditions and comes completely formulated and ready for use. For more information on this medium, please refer to the product insert.

• PRIME-XV AFSC Expansion Medium (FUJIFILM Irvine Scientific, Catalog #91133)
• Amniotic fluid stem cells (frozen)
• 15 mL conical, centrifuge tubes
• Culture vessels (T-25 or T-75)
• Phosphate Buffered Saline (PBS) without calcium and magnesium (FUJIFILM Irvine Scientific, Catalog #9240)
• 1X Trypsin EDTA (FUJIFILM Irvine Scientific, Catalog #9341)

The following amniotic fluid stem cell expansion protocols can be found below:

• Thawing Amniotic Fluid Stem Cells
• Expansion and Subculture of Human Amniotic Fluid Stem Cells

These procedures are a general guideline for culturing human AFSCs derived from human amniocentesis. Procedures for optimal growth conditions should be determined for each application and cell type as appropriate. Safe laboratory procedures should be followed and protective clothing should be worn when handing this medium. The acute and chronic effects of over-exposure to this medium are unknown.

Human amniotic fluid stem cell expansion in PRIME-XV AFSC Expansion Medium after six passages showed positive staining of the pluripotency markers OCT-4A (top) and Nanog (bottom). Nuclei were counterstained with DAPI (blue).

Thawing Amniotic Fluid Stem Cells

1. Thaw PRIME-XV AFSC Expansion Medium at 2-8°C overnight or at room temperature. Pre-warm the amount of medium needed for one procedure at 37°C (repeated warming of medium may reduce product performance). Store the remaining medium at 2-8°C. Thawed medium can be kept in the refrigerator for up to one week.
2. Rapidly thaw a frozen vial of AFSCs in a 37°C water bath by swirling the vial until all its content are liquid. The vial should still feel cold to touch. The process typically takes less than 2 minutes.
3. Pipette the content of the entire vial into a 15 mL conical tube containing 5 to 10 mL pre-warmed PRIME-XV AFSC Expansion Medium.
4. Centrifuge the cells for 5 minutes at 300xg at room temperature.
5. Resuspend the pellet in pre-warmed PRIME-XV AFSC Expansion Medium (follow the instruction of subculture to determine the required volume of culture medium), count the cells and determine total viable cell number. Transfer the cell suspension to an appropriate culture flask for continuous culture.

Expansion and Subculture of Human Amniotic Fluid Stem Cells

1. Aliquot the required quantity of PRIME-XV AFSC Expansion Medium and pre-warm the medium to 37°C.
2. Remove entire spent medium from culture flask and gently rinse the flask with equal volume of PBS without calcium and magnesium.
3. Add pre-warmed 1X Trypsin EDTA to the culture flask, 1mL for T-25 flask and 3mL for T-75 flask, and tilt the flask in all directions to disperse the Trypsin EDTA evenly over the cells.
4. Incubate the flask at 37°C, 5% CO₂ incubator for 5-10 minutes to detach the cells from culture surface. Tap the side of the flask to aid the detachment of the cells.
5. Add pre-warmed PRIME-XV AFSC Expansion Medium to the flask, 2mL for T-25 flask and 6mL for T-75 flask to stop trypsinizing. Disperse the cells by gently pipetting the media over the entire culture surface of the flask, and transfer the entire contents to a 15mL conical tube.
6. Centrifuge cells down at 300xg for 5 minutes. Aspirate off supernatant.
7. Resuspend the cell pellet in a small amount of pre-warmed PRIME-XV AFSC Expansion Medium and count the cells with a cell counter.
8. Inoculate 1.25x10⁵ cells into T-25 flask containing 5mL pre-warmed PRIME-XV AFSC Expansion Medium, or 3.75x10⁵ cells into T-75 flask containing 15mL medium. Note : It is recommended to seed cells at approximately 5,000 cells/cm² of culture vessel.
9. Feed the cells by replacing the spent medium with equal amount of fresh pre-warm PRIME-XV AFSC Expansion Medium every 2 days.
10. Subculture the cells when the cell confluence reaches 70%. It usually takes 4 days if the seeding density at Step 8 is used.