User Guide ♦> Supplement Specifications Brand: Duogenic StemCells corporation Form: Liquid Cells: BM-MSC, ADSC, UC-MSC Storage: -20°C (not up to -15°C) Shelf Life: 12 months ❖ Precaution and Disclaimer This product was manufactured for R&D use only. It is not intended for injection or diagnostic procedures. Do not use AllPhase-Supplement 20X beyond the expiration date indicated on the product. ❖ Storage/Stability AllPhase-Supplement 20X should be stored at -20°C in the dark. To ensure stability, AllPhase-Supplement 20X should be thawed at 4°C for 20-24 hours before use. Do not refreeze ❖ ..ion of use We recommend to use Iscove's Modified Dulbecco's Media (IMDM from Gibco or Simply) to prepare complete media in a sterile environment. Aseptically add 50 ml of AllPhase-Supplement 20X in 1 Liter of IMDM. AllPhase-Supplement 20X is an optimized serum-free and xeno-free supplement recommended for the growth and expansion of ex vivo human mesenchymal stem cells (hMSCs), including bone marrow mesenchymal stem cell (BM-MSC), adipose-derived stem cell (ADSC) and umbilical cord-derived mesenchymal stem cell (UC-MSC). Human MSCs cultured with AllPhase-Supplement 20X keep an excellent expansion rate after several passages and maintain their self-renewal and multilineage differentiation potential in coating-free condition. ❖ Preparation Complete AIIPhase-MSC medium Thaw AllPhase-Supplement 20X at 4°C for 20-24 hours before use. Avoid freeze/thaw cycles. Aseptically add 50 ml of AllPhase-Supplement 20X in 1 Liter of IMDM (Gibco or Simply). Store complete AllPhase-MSC medium at 2-8°C protected from light. We recommend the use of the complete medium within a period of 3 months. ❖ Culturing hMSC with AllPhase-Supplement 20X Recovery 1. Pre-warm 5 ml of AllPhase-MSC complete medium in a conical tube. 2. Thaw the vial at 37°C in a water bath for 1-1m30, a small amount of ice should remain. 3. Slowly transfer the cells into the AllPhase-MSC complete medium. 4. Centrifugate the cells at 200g for 3 min at room temperature. 5. Remove the supernatant and resuspend the pellet in 1ml of AllPhase-MSC complete medium. 6. Count the viable cells by using the Trypan Blue assay. 7. Seed the desired number of cells. The recommended seeding density is shown in Table 1. 9. Replace the medium with fresh complete AllPhase-MSC medium every 2-3 days. No.18, Ln.10, Taiping 21st St., Taiping Dist., Taichung City 41161, Taiwan (R.O.C.) (+886) 04-22766193 www.d uogenicsc. com

Table 1. Recommended seeding density limits Seeding density Table 2. Recommended amount of TrypLE or Trypsin+EDTA for a confluence of 80%. Culture ware No.18, Ln.10, Taiping 21st St., Taiping Dist., Taichung City 41161, Taiwan (R.O.C.) (+886) 04-22766193 www.d uogenicsc. com ❖ Coating No coating. We recommend the use of Corning® CellBIND® surface vessels as it provides a better attachment for WJ-MSC. However, the same expansion rate was observed by culturing WJ-MSC, ADSC, BM-MSC on tissue culture treated vessels. See table 3.1 and 3.2 the tested brands. ❖ Additional products • Iscove's Modified...

Table 3.1 Culture ware tested brand with similar hMSCs expansion rate. ❖ Quality Control AllPhase-Supplement 20X performance is tested for the maintenance and expansion of adipose-derived stem cells for 3 passages. Doubling time < 30 hours. Others tested parameters: pH (7.0-8.0), endotoxins (<1 EU/ml), mycoplasma and sterility. ❖ Quality Assurance AllPhase-Supplement 20X has been manufactured under ISO13485 and in compliance with GMP standards. The manufacturer's catalogue number The manufacturer's batch code Explanation of the warning symbols Indicates the temperature limits to which expose...