Cryopreservation and Pluripotency Evaluation

Frozen vials were thawed in a 37°C water bath for 2 min 30 s. Thawed cells were immediately diluted dropwise using TeSR-E8 medium, plated onto vitronectin-coated culture vessels without washing and placed into a 37°C 5% CO₂ incubator. The split ratio at thawing was 1:1 in the DE algorithm experiments and 1:6 otherwise. Twenty-four hours later, the post-thaw culture was washed with PBS containing Ca²⁺ and Mg²⁺ and stained for esterase activity using calcein AM (Invitrogen). Fluorescence intensity of the stained cells were measured using a Synergy HT microplate reader (BioTek) with an ex/em 485/528 nm filter set. Cells cryopreserved using DMSO and plated post-thaw were used as a control. The ratio of fluorescence measurement of each DMSO-free sample to that of the DMSO control (or post-thaw reattachment rate) was used as the metric of optimization. In addition, fresh cells with the same cell count as cryopreserved cells pre-freeze were used as a positive control, passaged at the same split ratio and stained with calcein AM 24 h post-passage.
Growth in post-thaw culture of cells cryopreserved in the optimized DMSO-free solution was assessed label-free by imaging the culture daily with a Cytation 1 cell imaging multi-mode reader (BioTek) with a 4× objective (NA 0.13, Olympus) in the bright-field and scan mode using default focusing method. Images were automatically analyzed by the Gen 5 software (BioTek) using boundary recognition to measure the confluence. On day 4 after thawing as a result of three stress cycles of freezing, thawing and culturing for one passage, the cell colonies were examined for protein expression by immunocytochemistry using antibodies to detect Nanog (R&D Systems) and Oct4 (Millipore), counterstained with Hoechst 33342 (Invitrogen) and quantified using FIJI (ImageJ). Cells dissociated from the post-thaw culture were examined with anti-Tra-1-60 antibody and its isotype control (Invitrogen) and measured using a BD Accuri C6 flow cytometer (BD Biosciences). Also on day 4, the post-thaw culture was differentiated into all three germ layers using a STEMdiff trilineage differentiation kit (STEMCELL Technologies) over 7 days. The endoderm was detected with anti-FOXA2 and anti-SOX17 antibodies (R&D Systems), the mesoderm with anti-BRACHYURY (R&D Systems) and anti-HAND1 (Invitrogen) antibodies, the ectoderm with anti-PAX6 (DSHB) and anti-NESTIN (STEMCELL Technologies) antibodies, and all counterstained with DAPI (Roche). Fluorescent secondary antibodies (Invitrogen) were utilized to detect primary antibody binding and imaged using the Cytation 1 cell imaging multi-mode reader with a 20× objective (NA 0.45, Olympus).