Dataset Information

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification.

ABSTRACT: Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow-rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post-thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post-thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from Parkinson's disease and controls. In a dual-center study, we compared the results by immunocytochemistry (ICC), fluorescence-activated cell sorting analysis, and RNA-sequencing (RNA-seq). Adherent vitrification was achieved in the so-called TWIST substrate, a device combining cultivation, vitrification, storage, and post-thawing cultivation. Adherent vitrification resulted in preserved confluency and significantly higher cell numbers, and viability at day 1 after thawing, while results were not significantly different at day 4 after thawing. RNA-seq and ICC of hiPSCs revealed no change in gene expression and pluripotency markers, indicating that physical damage of slow-rate freezing disrupts cellular membranes. Scanning electron microscopy showed preserved colony integrity by adherent vitrification. Experiments using smNPCs demonstrated that adherent vitrification is also applicable to neural derivatives of hiPSCs. Our data suggest that, compared to the state-of-the-art slow-rate freezing in suspension, adherent vitrification is an improved cryopreservation technique for hiPSCs and derivatives. Stem Cells Translational Medicine 2019;8:247&259.

SUBMITTER: Kaindl J

PROVIDER: S-EPMC6392398 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification.

Kaindl Johanna J Meiser Ina I Majer Julia J Sommer Annika A Krach Florian F Katsen-Globa Alisa A Winkler Jürgen J Zimmermann Heiko H Neubauer Julia C JC Winner Beate B

Stem cells translational medicine 20181119 3

Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow-rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post-thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post-thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from ...[more]

PMID: 30456912

Similar Datasets

Project description:BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.

Project description:Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (Danio rerio). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6-1.5 × 105 °C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86-100%). No significant differences (p = 0.6584) were shown in a volume capacity (5.7-6.0 μL) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 × 107 sperm/vial, pooling of samples from 3-6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization.

Dataset Information

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification.

Publications

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets