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. 2015 Mar 15;4(1):38–49.

Figure 3.

Figure 3

Recovery rate of hPSCs after thawing from various cryopreservation methods. A. Photos from left to right: Colonies of hPSCs (KhES-1) passaged without cryopreservation. Approx. 500 KhES-1 cells in AggreWell 400 after thaw at day 1. KhES-1 cells transferred to rhVTN-N-coated dish and cultured with TeSR-E8 at day 2. Cell clumps prior to the fist passage at day 4. Scale bar = 100 μm. B. KhES-1 cells cultured for four days via cell clump formation with AggreWell 400 plates were stained with DAPI, anti-Vimentin-, anti-β-tubulin-antibody at day 4 in 6-well dish (left panels). 6 × 104 KhES-1 cells seeded on rhVNT-N coated dish just after thawing without clump formation were cultured with TeSR-E8 for four days and stained with DAPI, anti-Vimentin-, anti-β-tubulin-antibody at day 4 in 24-well dish (right panels). Scale bar = 1 mm. Percentage of positively stained area with respective antibody using Image J software is shown in the right upper corner of the photo. C. Growth of KhES-1 cells after thawing, dissociated with seven dissociation reagents and frozen with four different cryopreservation mediums. Dissociation mediums: Acc:Accutage, Try:x1 TrypLE Select, pro:Pronase, EP:EDTA/PBS(-), 0.75T: x0.75 TrypLE Select diluted by 0.5 mM EDTA/PBS(-) [TrypLE Select: EDTA/PBS(-) = 3:1], 0.5T: x0.5 TrypLE Select diluted by EDTA/PBS(-) [TrypLE Select: EDTA/PBS(-) = 1:1], 0.25T: x0.25 TrypLE Select diluted by EDTA/PBS(-) [TrypLE Select: EDTA/PBS(-) = 1:3]. Cryopreservation mediums [cryo: CryoStem, CP1: CP-1, C+A: CP-1 with HSA, TC: TC protector]. Y axis: cell numbers and X axis: days after thaw. Freeze (Day-7<) mean 7 days or more prior to thaw. Means of results from five experiments are plotted and shown in line graphs. D. Numbers of colonies of KhES-1 cells scored in three wells of 6-well dish four days after thawing, dissociated with x1, x0.75, x0.5, or x0.25 TrypLE Select and frozen with CryoStem. Results from five independent experiments are plotted. Figures in graph are mean and error bars are standard deviation. E. Numbers of colonies of three hPSC lines (ESC:KhES-1, ESC:H9, iPSC:PFX#9) scored in 6-well dish four days after thawing via cell clump with AggrewWell 400. Cells were dissociated with x0.75 T (x0.75 TrypLE Select) and cryopreserved with CryoStem.