Figure 2.

Large-volume-hydrogel-encapsulation vitreous cryopreservation of stem cells using plastic straw (PS). (A) Typical images showing freezing, vitrification, and partial vitrification (p-vitri) of cell culture medium and four cryoprotective agent (CPA) solutions during cooling (top) and devitrification/recrystallization (devitri) during warming (bottom). The red material at the distal end of the straw is sealing wax. (B) Typical phase and live/dead (stained with acridine orange/ethidium bromide or AO/EB as green/red) fluorescence images of pADSCs encapsulated in the microcapsules after treated with CPA #1 (i.e., CPA addition and removal) or cryopreserved with the four different CPAs. (C) Typical fluorescence images of pADSCs with and without encapsulation subjected to CPA #1 treatment and cryopreservation with the four different CPAs. (D) Quantitative cell viability of pADSCs that are fresh, treated with CPA #1 without cryopreservation, or cryopreserved with four different CPAs (n = 4–5. * p < 0.05, ** p < 0.01). Culture medium: Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) with 10% (v/v) fetal bovine serum (FBS). CPA #1: 1 mol L⁻¹ 1,2-Propanediol (PROH), 1 mol L⁻¹ ethylene glycol (EG), 10% dextran T50 and 1 mol L⁻¹ trehalose; CPA #2: 1 mol L⁻¹ PROH, 1 mol L⁻¹ EG, 10% dextran T50 and 0.5 mol L⁻¹ Trehalose; CPA #3: 1 mol L⁻¹ PROH, 1 mol L⁻¹ EG and 1 mol L⁻¹ Trehalose; CPA #4: 1 mol L⁻¹ PROH, 1 mol L⁻¹ EG and 10% dextran T50. CPAs #1-#4 were prepared using DMEM/F12 with 80% (v/v) FBS. Fresh: cells kept in culture medium; CPA Treatment: cells subjected to addition and removal of CPA #1; Cryop: cells subjected to cryopreservation. W/O Encap: cells without encapsulation; W/Encap: cells with encapsulation.