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Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing.

ABSTRACT: Cryopreservation is widely used to maintain backups of cells as it enables the semipermanent storage of cells. During the freezing process, ice crystals that are generated inside and outside the cells can lethally damage the cells. All conventional cryopreservation methods use at least one cryoprotective agent (CPA) to render water inside and outside the cells vitreous or nanocrystallized (near-vitrification) without forming damaging ice crystals. However, CPAs should ideally be avoided due to their cytotoxicity and potential side effects on the cellular state. Herein, we demonstrate the CPA-free cryopreservation of mammalian cells by ultrarapid cooling using inkjet cell printing, which we named superflash freezing (SFF). The SFF cooling rate, which was estimated by a heat-transfer stimulation, is sufficient to nearly vitrify the cells. The experimental results of Raman spectroscopy measurements, and observations with an ultrahigh-speed video camera support the near-vitrification of the droplets under these conditions. Initially, the practical utility of SFF was demonstrated on mouse fibroblast 3T3 cells, and the results were comparable to conventional CPA-assisted methods. Then, the general viability of this method was confirmed on mouse myoblast C2C12 cells and rat primary mesenchymal stem cells. In their entirety, the thus-obtained results unequivocally demonstrate that CPA-free cell cryopreservation is possible by SFF. Such a CPA-free cryopreservation method should be ideally suited for most cells and circumvent the problems typically associated with the addition of CPAs.

SUBMITTER: Akiyama Y

PROVIDER: S-EPMC6475437 | biostudies-other | 2019 Apr

REPOSITORIES: biostudies-other

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Directional freezing for the cryopreservation of adherent mammalian cells on a substrate.

Project description:Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.

| S-EPMC5813933 | biostudies-literature

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

Project description:Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

| S-EPMC5946737 | biostudies-literature

Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media - Vitrification versus Slow Freezing Methods.

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| S-EPMC4758583 | biostudies-literature

Development of cryopreservation media for the slow-freezing of cultured primordial germ cells in chicken.

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| S-EPMC10085767 | biostudies-literature

Evaluation of methylcellulose and dimethyl sulfoxide as the cryoprotectants in a serum-free freezing media for cryopreservation of adipose-derived adult stem cells.

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| S-EPMC3139530 | biostudies-other

Short-term storage of human spermatozoa in electrolyte-free medium without freezing maintains sperm chromatin integrity better than cryopreservation.

Project description:Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.

| S-EPMC3159537 | biostudies-literature

Cryopreservation of Pig Semen Using a Quercetin-Supplemented Freezing Extender.

Project description:Reactive oxygen species (ROS) produced during freeze−thaw procedures cause oxidative damage to the sperm, reducing fertility. We aimed to improve the post-thaw quality of pig sperm by quercetin (QRN) supplementation to reduce the cryodamage associated with the freeze−thaw procedure. Four equal aliquots of pooled boar semen were diluted with a freezing extender supplemented with different concentrations of QRN (0, 25, 50, and 100 µM) and then were subjected to cryopreservation in liquid nitrogen. Semen analysis was performed following 7 days of cryopreservation. Results demonstrated that the semen samples supplemented with 50 µM QRN significantly improved the post-thaw sperm quality than those subjected to other supplementations (p < 0.05). Semen samples supplemented with 50 µM QRN showed significantly improved plasma membrane functional integrity (47.5 ± 1.4 vs. 43.1 ± 4.1, 45.3 ± 1.7, and 44.1 ± 1.4) and acrosome integrity (73.6 ± 3.4 vs. 66.3 ± 2.4, 66.7 ± 3.6, and 68.3 ± 32.9) as compared to the control, 25 µM, and 100 µM QRN groups, respectively. The mitochondrial activity of the 50 µM QRN group was greater than control and 25 µM QRN groups (43.0 ± 1.0 vs. 39.1 ± 0.9 and 41.9 ± 1.0) but showed no difference with the 100 µM QRN group. Moreover, the 50 µM QRN group showed a higher sperm number displaced to 1 cm and 3 cm points in the artificial mucus than other groups. Therefore, supplementing the freezing extender with QRN can serve as an effective tool to reduce the magnitude of oxidative damage associated with sperm freezing.

| S-EPMC9410179 | biostudies-literature

Cryopreservation of orchid seeds through rapid and step freezing methods.

Project description:Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.

| S-EPMC6051192 | biostudies-literature

Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide.

Project description:BackgroundMesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed.ResultsThe study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a 'straight freeze' protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods.ConclusionA 5% DMSO / 5% HES solution cryopreservation solution using a 'straight freeze' approach can be recommended for rat MSC.

| S-EPMC3465236 | biostudies-literature

Cryopreservation of Ovarian Tissue: Detailed Description of Methods for Transport, Freezing and Thawing.

Project description:Purpose: In many cases cancer therapy leads to an irreversible reduction or even loss of ovarian reserve. Cryopreservation of ovarian tissue with subsequent thawing and re-transplantation of tissue after the cancer is in remission constitutes a promising method to preserve fertility in women. To date, more than 25 cases of live births after re-transplantation of cryopreserved ovarian tissue have been published worldwide. In Germany the first live birth after re-transplantation of cryopreserved tissue was in 2011. Material and Methods: After surgical removal of ovarian tissue in the Gynaecological Clinic of Dresden University, the tissue was sent to the Gynaecological Clinic of Bonn University in a special transport container at 5?°C and was frozen the next day using 1.5?M dimethyl sulfoxide cryosolution. In 2010 this ovarian tissue was thawed using a sucrose solution in the Gynaecological Clinic of Erlangen University Clinical Centre and was laparoscopically re-transplanted into the patient. Results: The patient became pregnant, the pregnancy was uneventful, and she gave birth to a healthy boy. Conclusion: Freezing of ovarian tissue with subsequent re-transplantation as described here is a viable method to preserve fertility in cancer patients.

| S-EPMC4168540 | biostudies-literature

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