Sand-mediated ice seeding enables serum-free low-cryoprotectant cryopreservation of human induced pluripotent stem cells

Abstract

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and/or introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above -10 °C, which enables cryopreservation of hiPSCs with no serum, much-reduced cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by their pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.

Keywords: Cryopreservation; Ice seeding; Sand; Stem cell; iPSC.

Graphical abstract

Fig. 1

Preparation and characterization of sand-PDMS film. (A) A schematic illustration of the procedure for preparing the sand-PDMS film: The sand was first rinsed overnight with water, autoclaved, dried, and then sifted onto a thin and uncured PDMS layer over a fully cured PDMS film through a mesh strainer with 200 μm openings. The PDMS film embedded with sand was baked at 75 °C for 30 min to form the sand-PDMS film. The sand-PDMS film was cut into small pieces and each piece was stuck/attached onto the inner wall of a cryovial via its smooth face. (B) Morphology and size distribution of sands before and after sifting them through the mesh strainer. Also shown is a high-magnification view of the sifted sands where the sharp morphology of the sands is more appreciable. The size distribution was quantified based on the area of sand particles on the films. (C) Scanning electron microscopy (SEM) image, showing the presence and morphology of sands partially embedded in the PDMS film. (D) Energy dispersive X-ray spectroscopy (EDXS) quantification of the elemental composition of the sand-PDMS and pure PDMS films. The surface of the sand-PDMS film contains an increased amount of silicon (Si) and oxygen (O) than the pure PDMS surface. (E) Quantification of Si counts for the sand-PDMS and pure PDMS films using EDXS. Scale bars: 200 μm **, p < 0.01 (n = 3 independent runs).

Fig. 2

Sand enables ice seeding at high subzero temperature. (A) Representative thermal histories in water containing no film (Control), pure PDMS film (PDMS), and sand-PDMS film (Sand-PDMS) during cooling. A sudden increase in temperature indicates ice seeding (which releases latent heat) in the sample. (B) Quantitative data of the ice-seeding temperature in water under the aforementioned three conditions. **, p < 0.01 (n = 10 (for sand-PDMS film and pure PDMS film) or 20 (for Control), independent runs). (C) Cryomicroscopy images at different times showing ice nucleation and growth around the sand particle during cooling the cell cryopreservation solution at subzero temperatures. Scale bar: 100 μm.

Fig. 3

The immediate and long-term viability of hiPSCs after cryopreservation under various conditions. (A) Immediate (after 2 h incubation at 37 °C) viability of hiPSCs assessed by live/dead (green/red) staining after cryopreservation with different methods: conventional method (10% DMSO+10% serum with no ice seeding), sand-mediated ice seeding alone, no cryoprotectant and no ice seeding, 5% DMSO with no ice seeding, 2% DMSO with ice seeding, and 5% DMSO with ice seeding. Scale bar: 500 μm. (B) Quantitative data of the hiPSC immediate viability and attachment efficiency (i.e., long-term viability) after the various cryopreservation conditions. For quantifying the attachment efficiency, the cryopreserved cells were thawed and cultured for 15 h, and the number of attached cells was counted by hemacytometer. The attachment efficiency is calculated as the percentage of cells counted after cryopreservation out of the number of cells initially cryopreserved. **, p < 0.01 (n = 3 independent runs) for the comparison of both immediate viability and attachment efficiency.

Fig. 4

Cryopreservation with sand-mediated ice seeding and 5% DMSO does not significantly affect the hiPSC pluripotency and cell cycle. (A) Images of cryopreserved (Cryo) hiPSCs showing typical colony morphology and high expression of pluripotency protein markers OCT-4 and SSEA-4, similar to fresh (Control) hiPSCs with no cryopreservation. Scale bar: 100 μm. (B–C) Representative peaks (B) and quantitative data (C, n = 3 independent runs) form flow cytometry analyses, showing the cryopreserved hiPSCs highly express pluripotency protein markers SSEA-4 and OCT-4 similar to the fresh control hiPSCs with no statistically significant difference. The light blue peaks are isotype controls. (D–E) Representative peaks (D) and quantitative data (E, n = 3 independent runs) from flow cytometry analyses, showing no statistically significant difference in the cell cycle distribution between the cryopreserved and fresh control hiPSCs.

Fig. 5

Cryopreservation with sand-mediated ice seeding and 5% DMSO does not significantly affect the differentiation capacity of the hiPSCs. (A) The cryopreserved hiPSCs can efficiently differentiate into cells with typical neural cell morphology (neurites extending out of the cell body) and high expression of the neural specific marker TUJ-1. Cell nuclei are made visible by DAPI staining. (B) The cryopreserved hiPSCs can efficiently differentiate into cells that highly express the cardiac specific markers cTnT. Cell nuclei are made visible by DAPI staining. (C) The teratomas grown from the cryopreserved hiPSCs contain tissues from all the three germ layers including ectoderm (neural epithelium with hypernucelated neuroectodermal structures), mesoderm (the nidus of cartilage with surrounding condensed mesenchymal cells), and endoderm (gut epithelium with subnuclear vacuoles and tube-like structure). Scale bars: 100 μm.