Cryopreservation of pluripotent stem cell aggregates in defined protein‐free formulation

Abstract

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate-based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size-dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein-free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three-germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein-free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale.

Three-Dimensional Aggregates of Mesenchymal Stem Cells: Cellular Mechanisms, Biological Properties, and Applications

“…72 It has been suggested that cell proliferation is inversely related to aggregate size due to diffusion limitations, and large aggregates have reduced proliferation potential compared to small ones. 107,116 However, the MSC aggregates are heterogeneous in both molecular profiles and biomechanical microenvironments. Apart from biomechanical analysis at individual cell and aggregate levels, quantitative measurement and analysis of the spatial distribution of oxygen, nutrient, and regulatory macromolecules in the 3D aggregates are required to definitively determine the role of diffusion in the aggregates.…”

Extracellular Matrices Decellularized from Embryonic Stem Cells Maintained Their Structure and Signaling Specificity

“…The aggregates of ES-D3 cells were obtained by seeding the cells from ESC monolayer into UltraLow Attachment (ULA) six-well plates (Corning, Inc.) at a seeding density of 2 -4 · 10 4 cells/cm 2 in 3 mL of growth media. 34 The aggregates were subcultured every 2-4 days and serially passaged for 5-15 passages. For passaging, the aggregates were trypsinized with 0.05% trypsin-EDTA for 5 min at 37°C, neutralized with the media containing FBS, and resuspended in the fresh media.…”

“…34 The samples were permeabilized with 0.2-0.5% Triton X-100, blocked, and incubated with primary ECM antibodies, including rabbit polyclonal FN, laminin (LN), collagen IV (Col IV), and vitronectin (VN) (Abcam). For fluorescence staining, the samples were incubated with Alexa Fluor Ò 488 goat antiRabbit IgG (Molecular Probes), counterstained with 4¢,6-diamidino-2-phenylindole (DAPI), and visualized using a fluorescence microscope (Olympus IX70) or a Zeiss 510 confocal microscope (Leica TCS SP2 AOBS).…”

Stem Cells, Redox Signaling, and Stem Cell Aging

“…Research into the role of ROS in stem cells can lead to more efficient methods of cryopreservation, which allows for the long-term storage of cells so they can be used at later time points for therapeutic or research uses. The technique employs slowly freezing cells to a temperature of -196°C in the presence of dimethylsulfoxide, both elements that reduce the formation of damaging ice crystals (96). Although most mammalian cell types can be cryopreserved and recovered with standard protocols, efficient recovery of cryopreserved stem cells is much more challenging.…”

“…The increase in ROS after cryopreservation was shown to induce apoptosis in human embryonic stem cells and contribute to the poor recovery rate (123). In subsequent studies, incorporating methods that minimize ROS levels over the whole cryopreservation process led to increased viability and cell recovery (71,96).…”