A Simple and Highly Effective Method for Slow-Freezing Human Pluripotent Stem Cells Using Dimethyl Sulfoxide, Hydroxyethyl Starch and Ethylene Glycol

<div><p>Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional −80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.</p></div

Selection of cryopreservation medium for slow-freezing.

<p>(A) Recovery frequencies (rate, %) of iPSC (201B7) colonies treated with Pronase/EDTA dissociation followed by cryopreservation with 5 different media (Formulas A–E). Recovery frequencies (rate, %) were determined by the percentage of ALP+ colonies 5 days after thawing compared with those at day 5 after passaging with Pronase/EDTA without cryopreservation. Recovery frequencies (rate, %) are shown as bars with S.D. Formula A: [6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline]; B: [6% HES, 5% DMSO, and 50% D-MEM/F12 in saline]; C: [6% HES, 5% DMSO, and 4% BSA in saline]; D: [6% HES and 5% DMSO in saline]; E: [6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline]. Results of 3 independent experiments are shown. Differences between E and the others are significant. *; <i>P</i><0.05. (B) The effects of EG addition on cryopreservation efficacy of freezing media. Various concentrations (1, 2, 3, 4, 5, 7.5, 10, 12.5, or 15% v/v) of EG were added to cryopreservation Formula D (6% HES, 5% DMSO in saline). Recovery frequencies (rate, %) were determined by scoring the post-thaw number of ALP+ colonies and those without cryopreservation. Results of 3 independent experiments are shown. *; <i>P</i><0.05 (C) ALP staining of colonies of iPSC 201B7 maintained for 5 days after passage (left photo: post-plating, non-frozen control) and those 5 days after thaw (right photo: post-thawing, dissociation with Pronase/EDTA and cryopreservation with CP-5E). Magnified photos are attached. Scale bars indicate 500 µm. (D) Cell colonies of hiPSC cell lines (201B7, 253G1) or hESC cell lines (KhES-1, H1) were dissociated with Pronase/EDTA, followed by cryopreservation with CP-5E (Formula E: 6% HES, 5% DMSO, and 5% EG in saline). Recovery frequencies (%) were determined by scoring the number of ALP+ colonies after thawing for comparison with nonfrozen cells. Results of 3 independent experiments are shown.</p

Schematic overview of the protocol for hPSCs cryopreservation and thaw.

<p>Schema shows the protocol for the slow-freezing procedure with the combined use of Pronase/EDTA and cryopreservation medium CP-5E (left) and rapid thawing (right).</p

hPSCs maintained differentiation potential after cryopreservation with CP-5E.

<p>(A) hPSCs were cryopreserved with CP-5E. Differentiation of hiPSC (201B7) (blue bar) and hESC (KhES-1) (red bar) was initiated via EB formation after thawing. qRT-PCR was used to assess pluripotency–related genes (<i>OCT4</i>, <i>SOX2</i>, <i>NANOG</i>, and <i>REX1</i>) and 3 germ layer differentiation marker genes (ectodermal [<i>PAX6</i>, <i>SIX3</i>, and <i>MAP2</i>], mesodermal [<i>T</i>, <i>PDGFRα</i>, and <i>GATA2</i>] and endodermal [<i>CXCR4</i>, <i>SOX17</i>, and <i>GATA4</i>]) before and after thawing. Gene expression before and after differentiation were compared by theΔΔCt method. (B) Differentiation of hiPSC (201B7) and hESC (KhES-1) 5 passages after thawing was initiated via EB formation. Molecules related to 3 germ layer differentiation: β-tubulin (ectoderm), α-SMA (mesoderm), or AFP (endoderm) were detected with specific antibodies and visualized with secondary antibodies labeled with Alexa Fluor 488 (green) or Alexa Fluor 546 (red). Nuclei were stained with DAPI. Scale bars: 200 µm. (C) Assessment of post-thaw teratoma formation by hiPSCs. One million hiPSC (201B7) cells cultured for 5 passages after thawing were transplanted under the epidermal space of the left testes of NOG mice; saline was injected in the right testes of the mice as controls. Ten weeks after transplantation, all mice developed teratomas (n = 3). A: photo of a teratoma (left) and control testis (right). Scale Bar: 1 cm. (B–E) Histological analysis of teratoma. Sections were stained with hematoxylin and eosin. B: neural rosette (ectoderm), C: cartilage (mesoderm) and pigmented melanocytes (arrow heads), D: gut-like epithelium (endoderm), E: immature hepatocyte-like cells (endoderm). Scale Bars: 100 µm.</p

hPSCs retained self-renewal potential and pluripotency after cryopreservation with CP-5E.

<p>(A) Cell growth of hiPSC (201B7) before (blue line) and after (red line) thaw. Up to 3 passages (18–20 days) are shown. The experiments were performed in triplicate. (B)hiPSCs (201B7) or hESCs (KhES-1) were cryopreserved with CP-5E. Expression of pluripotency-related transcription factor genes (<i>OCT4, KLF4, SOX2, NANOG, and REX1</i>) before and 3 passages after thaw were determined by qRT-PCR. (C)Immunostaining of pluripotency-related molecules (OCT4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hiPSCs (201B7) or hESCs (KhES-1) after thawing. These molecules were detected by specific antibodies and visualized with secondary Alexa Fluor 488 (green)-labeled antibody. Nuclei were stained with DAPI. Scale bars, 200 µm. (D) Flow cytometric analysis of pluripotency-related surface markers (SSEA-3, SSEA-4, and TRA-1-60) in hiPSC (201B7) or hESC (KhES-1) after thawing.</p