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

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 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

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

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

<div><p>Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.</p></div

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

Culture of hPSCs with SPM on rhVNT-N coated dishes.

<p>(A) Phase contrast microscopic observation of iPSC cell line PFX#9 at passage 15. (B) Expression of SSEA-3 and TRA-1-60 by flow cytometric analysis of PFX#9 in indicated culture conditions at passage 15. (C) Time course of cell proliferation in patterned dish from days 1 to 4 (left to right). (D) Cell proliferation area that was occupied inrhVTN-N-coated spot area (spot Φ = 1 mm, 0.79 mm²/spot, X axis). Plot also shows the number of spots and their areas (out of 199 rhVNT-N-coated spots, Y axis) on days 1 to 4 (left to right). (E) Microscopic observations of clump cultures, single cell non-patterned or single cell patterned cultures with the higher magnified area in red rectangles at passage 20. A representative undifferentiated clump colony is shown in the upper left photo. Scale bars are appended. (F) Time course (0–100 h) of the area occupied by PFX#9 cells (in mm²) in 5 randomly selected spots (0.79 mm²/spot) measured by captured image analysis software (ImageJ 1.450, National Institutes of Health, Bethesda, MD, USA) every hour. Average of cell occupation area at every hour is plotted as a dot. The dot graph shows representative results from 3 independent trials. (G) Cell density (cells/mm²) of PFX#9 in single cell non-patterned or in single cell patterned culture was calculated by dividing harvested cell number by 962 mm² (35-mm non-patterned culture dish) or dividing harvested cell number by 156 mm² (total 199 spots of 1 mm diameter in 35-mm patterned culture dish). The results were obtained by scoring harvested cell numbers from 18 passages of indicated cultures and are shown as a bar (mean) with error bar (standard deviation). The significance of difference between 2 groups, p = 1.45 x 10⁻⁹. Representative results of 3 independent trials are shown. (H) Growth curve of PFX#9 in non-patterned culture, patterned culture or clump culture are shown in logarithmic graphs. PFX#9 cells in patterned culture or non-patterned culture were passaged every 4 days and in clump culture on feeder-free every 6 days and on feeder (SNL) every 5 days respectively.</p

Differentiation potential of hPSCs.

<p>(A) Gene expression profiles of PFX#9 cells in the indicated culture condition (clump culture, single cell non-patterned culture or single cell patterned culture) before (undifferentiated state) and after induction of differentiation via embryoid body (EB) formation. Average of gene expression values for self-renewal (undifferentiated state), ectoderm-, mesoderm- or endoderm-related genes is shown in comparison with reference standards of TaqMan hPSC Scorecard Panel (Life Technology). (B) EB formation at day 14 from PFX#9 cells (top left). EB attached to culture dish and continued to differentiate (top right). Cells were then stained with antibodies against β-tubulin (ectoderm), α-SMA (mesoderm), AFP (endoderm) and DAPI. (C) Tissue section of teratoma in NOG mouse generated by inoculating PFX#9 cells maintained in cell clumps is shown after staining with HE. Three germ layers of tissue consisting of neural rosette (ectoderm), muscle/cartilage (mesoderm) and gut-like epithelium (endoderm) are observed. Scores in Tables are visualized in bar graph below.</p

Cryopreservation and rapid thaw of KhES-1 cells grown in single cell non-patterned culture.

<p>(A) 1x10⁶ KhES-1 cells in single cell suspension from single cell non-patterned culture were cryopreserved with freezing medium, STEMCELL BANKER. The cells were thawed and cultured for 3 passages as single cells before examining the gene expression profile with TaqMan hPSC Scorecard. (B) Phase contrast image of KhES-1 cells cultured with SPM as single cells on rhVNT-N-coated dishes 3 passages after thaw. (C)Expression of SSEA-3 and TRA-1-60 on KhES-1 cells before and 5 passages after thaw was evaluated by flow cytometry. (D) Growth curve of KhES-1 in single cell non-patterned flat culture after thaw (blue line) was examined in comparison with cells in single cell non-patterned culture without cryopreservation (red line). The cryopreserved KhES-1 showed the same proliferation rate as the control in the second passage after thaw. Scores in Tables are visualized in bar graph below.</p

Karyotyping.

<p>Karyotype of PFX#9 maintained with SPM on rhVNT-N-coated dish (single cell patterning culture) was analyzed by mFISH at passage 5 (left). PFX#9 from single cell non-patterned culture underwent G-Band analysis at passage 15 (right).</p