High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells  Ji Yeon Lee, M.Sc., Jeoung Eun Lee, M.Sc., Dong Ku Kim, Ph.D., Tae Ki Yoon, M.D., Hyung Min Chung, Ph.D., Dong Ryul Lee, Ph.D.  Fertility and Sterility  Volume 93, Issue 3, Pages 976-985 (February 2010) DOI: 10.1016/j.fertnstert.2008.10.017 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunocolocalization of GFP and SSEA-4 in hESCs. (A) Map of EF1α-EGFP lentiviral vector. (B and C) FACS analysis of H9-EF1-GFP and CHA-hES3-EF1-GFP lines; 97.9% of H9-EF1-GFP and 89.1% of CHA-hES3-EF1-GFP were positive for both SSEA-4 and GFP. (D–H) Undifferentiated H9-EF1-GFP cells expressed both SSEA-4 and GFP. (I–M) Differentiated H9-EF1-GFP cells expressed GFP, but not SSEA-4. (N–R) Undifferentiated CHA-hES3-EF1-GFP cells expressed both SSEA-4 and GFP. (S–W) Differentiated CHA-hES3-EF1-GFP cells expressed GFP, but not SSEA-4. (D, I, N, and S) Morphology of hESC colonies observed under a differential interference contrast (DIC) microscope. (E, J, O, and T) DAPI-stained hESC colonies. (F, K, P, and U) GFP expression in hESC colonies. (G, L, Q, and V) hESC colonies stained with an SSEA-4 antibody. (H, M, R, and W) Merged images of DAPI, EGFP, and SSEA-4. Scale bar = 50 μm. DAPI = 4,6-diamidino-2-phenylindole; FACS = fluorescence-activated cell sorting; hESC = human embryonic stem cells. Fertility and Sterility 2010 93, 976-985DOI: (10.1016/j.fertnstert.2008.10.017) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Schematic overview of human embryonic stem cell cryopreservation methods. There are four groups in the experiment. In the first group, cells are frozen in the cryocontainer by one-step method. In the second group, cells are frozen in the program-controlled freezer by one-step method. In the third group, cells are frozen in the cryocontainer by stepwise method. In the fourth group, cells are frozen in the program-controlled freezer by stepwise method. Fertility and Sterility 2010 93, 976-985DOI: (10.1016/j.fertnstert.2008.10.017) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Analysis of survival rates for thawed human embryonic stem cells (hESCs) . (A) Thawed colonies cultured in 12-well plates were stained by alkaline phosphate substrate to identify surviving colonies. 1 and 6: Nonfrozen control subjects for H9-EF1-GFP and CHA-hES3-EF1-GFP hESCs, respectively. 2: Thawed H9-EF1-GFP hESC colonies cryopreserved using one-step method and a cryo-container. 3: Thawed H9-EF1-GFP hESC colonies cryopreserved using one-step method and a program-controlled freezer. 4: Thawed H9-EF1-GFP hESC colonies cryopreserved using stepwise method and a cryo-container. 5: Thawed H9-EF1-GFP hESC colonies cryopreserved using stepwise methods and a program-controlled freezer. 7: Thawed CHA-hES3-EF1-GFP colonies cryopreserved using one-step method and a cryo-container. 8: Thawed CHA-hES3-EF1-GFP colonies cryopreserved using one-step method and a program-controlled freezer. 9: Thawed CHA-hES3-EF1-GFP colonies cryopreserved using stepwise method and a cryo-container. 10: Thawed CHA-hES3-EF1-GFP colonies cryopreserved using stepwise method and a program-controlled freezer. Magnification ×40. (B) Relative percentage of survival hESC colonies for each cryopreservation method. ∗,∗∗,∗∗∗,∗∗∗∗,∗∗∗∗∗,∗∗∗∗∗∗P<.05. Fertility and Sterility 2010 93, 976-985DOI: (10.1016/j.fertnstert.2008.10.017) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Fluorescence-activated cell sorting (FACS) analysis of undifferentiated SSEA-4+/GFP+ human embryonic stem cells (hESCs) after thawing. Summary of FACS analysis results (n ≥ 4 replicates) showing histograms representing relative percentage of undifferentiated hESCs (∗,∗∗P<.05). Fertility and Sterility 2010 93, 976-985DOI: (10.1016/j.fertnstert.2008.10.017) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Characterization of human embryonic stem cells (hESCs) after thawing. (A and E) Alkaline phosphatase (AP) staining of H9-EF1-GFP and CHA-hES3-EF1-GFP. (B and F) SSEA-4 staining of H9-EF1-GFP and CHA-hES3-EF1-GFP. (C and G) TRA-1-60 staining of H9-EF1-GFP and CHA-hES3-EF1-GFP. (D and H) TRA-1-81 staining of H9-EF1-GFP and CHA-hES3-EF1-GFP. Magnification ×200. (I) Karyotype of H9-EF1-GFP (46 XX). (J) Karyotype of CHA-hES3-EF1-GFP (46 XY). (K) Reverse-transcription polymerase chain reaction analysis of three germ layer specific genes. H9-EF1-GFP and CHA-hES3-EF1-GFP hESCs were cultured without a feeder for 3 weeks to allow spontaneous differentiation. Oct-4 was used as a marker for undifferentiated hESCs. α-Fetoprotein (AFP) and amylase genes were used as endoderm markers. The keratin-15 gene was used as a marker for ectoderm. Brachyury was used as a marker for mesoderm. 18S rRNA was used as an internal standard. Lane 1: undifferentiated H9-EF1-GFP; lane 2: differentiated H9-EF1-GFP; lane 3: undifferentiated CHA-hES3-EF1-GFP; lane 4: differentiated CHA-hES3-EF1-GFP. Fertility and Sterility 2010 93, 976-985DOI: (10.1016/j.fertnstert.2008.10.017) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions