Method For Cryopreservation Of Cells Research Articles

Freezing and thawing of cells on a microfluidic device: a simple and time-saving experimental procedure.

Developing cell cryopreservation methods on chips is not only crucial for biomedical science but also represents an innovative approach for preserving traditional cell samples. This study presents a simple method for direct cell freezing and thawing on chip, allowing for long-term storage of cells. During the freezing process, cells were injected into the microchannel along with a conventional cell cryopreservation solution, and the chip was packed using a self-sealing bag containing isopropyl alcohol and then stored in a -80°C refrigerator until needed. During the thawing process, microcolumn arrays with a spacing of 8µm were strategically incorporated into the microfluidic chip design to effectively inhibit cells from the channel. The breast cancer cell lines MDA-MB-231 and B47D demonstrated successful thawing and growth after cryopreservation for 1 month to 1 year. These findings offer a direct cell freezing and thawing method on a microfluidic chip for subsequent experiments.

A simple method for effective cryopreservation of antlerogenic periosteum of sika deer.

Antlerogenic periosteum (AP) is the unique tissue type that gives rise to antlers and their antecedents, the pedicles. Deer antlers are the only mammalian organ that can fully regenerate. Efficient investigation of the mechanism of antler formation and regeneration requires year-round availability of AP, but naturally AP can only be obtained less than two months in a year. In the present study we took the cryopreservation approach to store the sampled AP in ultra-low temperature to overcome the limited period of availability. First, we evaluated the suitability of vitrification and cell cryopreservation method for cryopreservation of AP, cell migration status of the AP tissue pieces confirmed that vitrification methods did not work as the only few AP cells migrated out, whereas migrated cell numbers in the cell-cryo group (conventional method for cryopreservation of cells) were comparable to those of the fresh AP group. To further evaluate the suitability of cell cryopreservation method for AP tissue, AP samples were allocated into three groups based on the different ratios of cryopreservation reagents (dimethyl sulfoxide [DMSO], dulbecco's modified eagle's medium [DMEM] and fetal bovine serum [FBS]): AP-Cell-1 (1:4:5), AP-Cell-2 (1:2:7) and AP-Cell-3 (1:0:9), the results showed that migrated cell number were again comparable to the fresh AP group. There was no significant difference between the cell-cryo groups (AP-Cell-1 and AP-Cell-3) and the fresh group: (1) in viability (p > 0.05) through trypan blue staining (91.2%, 90.8%, and 92.4%, respectively); (2) in the attachment day, and all on Day 5 after cell seeding; (3) in cell proliferation rate (p > 0.05) through Cell Counting kit 8 (CCK8) measurement; and (4) in number of the formed clones (Clonogenicity). In the in vivo trials, there was no visible difference in temporal differentiation sequence of the formed xenogeneic antlers between the fresh AP and cryopreserved AP (AP-Cell-1 and AP-Cell-3). Overall, we found that the AP tissue was well cryopreserved just using the conventional freezing and thawing methods for cells, and their viability and developmental potential comparable to the fresh AP both in vitro and in vivo. The long-term preservation of the AP tissue is of great significance for the study of the periosteum biology in general and the mechanism underlying xenogeneic generation and regeneration of deer antlers in specific.

Thin-Film Evaporation Heat Transfer of Liquid Nitrogen and Its Application in Cell Vitrification

Abstract Cell vitrification has been an important method of cell cryopreservation. The faster the cooling rate is, the higher the cell survival rate is. However, in conventional cell vitrification methods, film boiling forms a vapor-blanket on the surface, which hinders further improvement of the cooling rate. To eliminate the problem, this article attempted to replace film boiling with thin-film evaporation (TFE) of liquid nitrogen. The experimental system was developed to investigate the TFE heat transfer characteristics of liquid nitrogen. Then, prostate cancer cells were cryopreserved by TFE vitrification method, open pulled straw vitrification method, and equilibrium freezing method. The results showed that the vitrification method of TFE obtained a higher cooling rate and better cell survival rate than the two other cell cryopreservation methods. Thus, the feasibility of this method was preliminarily proved viable when applied to the cell vitrification process. In addition, both the cooling rate and the cell survival rate are affected by the concentration of the cryoprotectant in the cell suspension. The cooling rate decreases as the concentration of the cryoprotectant increases, but the cell survival rate increases first and decrease afterward with an increase in the cryoprotectant concentration, in which an optimum value exists. This study demonstrates the practicality of the new ultrafast cell vitrification method.

Growth and albumin secretion of mouse fetal liver cells cryopreserved within porous polymer scaffolds as a viable cell source for bioartificial livers

To clinically apply bioartificial livers (BALs), an effective liver cell cryopreservation method is required for a stable cell supply. In this study, we performed tissue-engineered construct (TEC) cryopreservation of fetal liver cells (FLCs) in which FLCs cultured within a porous polymer scaffold were cryopreserved. Growth and albumin secretion in TEC-cryopreserved FLCs after thawing were compared to freshly isolated FLCs (control experiments). The effect of preculture duration prior to cryopreservation (0-3 weeks) on these functions was also examined. In the three-dimensional cultures, the TEC-cryopreserved FLCs with preculturing showed constant growth, and this growth was comparable to controls. On the contrary, the TEC-cryopreserved FLCs without preculturing did not proliferate after thawing. Albumin secretion of TEC-cryopreserved FLCs with preculturing rapidly increased up to day 12 and high secretory activity comparable to controls was maintained thereafter in FLCs with 1- or 2-week preculturing, suggesting this as an appropriate preculture duration. Compared to conventionally cryopreserved FLCs, growth and albumin secretion in the TEC-cryopreserved FLCs were significantly higher, indicating their usefulness as a potent cell source for BALs.

Paper-Based Cell Cryopreservation.

The continuous development of simple and practical cell cryopreservation methods is of great importance to a variety of sectors, especially when considering the efficient short- and long-term storage of cells and their transportation. Although the overall success of such methods has been increased in recent years, there is still need for a unified platform that is highly suitable for efficient cryogenic storage of cells in addition to their easy-to-manage retrieval. Here, a paper-based cell cryopreservation method as an alternative to conventional cryopreservation methods is presented. The method is space-saving, cost-effective, simple and easy to manage, and requires no additional fine-tuning to conventional freezing and thawing procedures to yield comparable recovery of viable cells. It is shown that treating papers with fibronectin solution enhances the release of viable cells post thawing as compared to untreated paper platforms. Additionally, upon release, the remaining cells within the paper lead to the formation and growth of spheroid-like structures. Moreover, it is demonstrated that the developed method works with paper-based 3D cultures, where preformed 3D cultures can be efficiently cryopreserved.

Apatite nanoparticles mediate intracellular delivery of trehalose and increase survival of cryopreserved cells

Cryopreservation of tissue cells is an important method to maintain cell viability and cellular function. However, cell viability and function are less than ideal by conventional cell cryopreservation methods, which may result in apoptosis and necrosis of cells in cryopreservation. Trehalose plays a role in maintaining cell structure and protecting cells from stress. However, owing to the difficulty in transport of trehalose across the cell membrane, its antifreeze effect is limited. A large amount of trehalose (up to 237 ± 8.5 mM) can be delivered to smooth muscle cells incubated in a medium containing trehalose and apatite nanomaterials at 37 °C for 6 h. Our data showed that trehalose was efficiently delivered intracellularly with the aid of nanoparticles (NP), with a loading efficiency up to 137.3 ± 34.5%, thus allowing for cryopreservation of LMC with nontoxic sugar as the sole cryoprotectant. Colloidal bioelastic apatite NP were used as bioactive promoters for the cryopreservation of tissue cells with trehalose. The addition of apatite NP in the medium substantially increased aortic smooth muscle cell cryosurvival, up to 83.6% (30% improvement over control without NP), a level comparable to that associated with the traditional Me2SO cryoprotective regimen. Furthermore, the cytotoxicity of nanocapsules in the intracellular delivery of trehalose was negligible. This method provides a new option to enhance the activity of valvular cells for cryopreservation.

СУЧАСНІ УЯВЛЕННЯ ПРО МЕТОДИ КРІОКОНСЕРВУВАННЯ ЕРИТРОЦИТІВ ТВАРИН

Nowadays, the method of blood transfusion is often used in veterinary practice. Hypothermic storage allows to save blood cells for a limited time, while morphofunctional parameters are getting worse. Cryopreservation allows to save and receive high-quality cells for the use in veterinary practice. Therefore, the development of reserves of donor blood is possible with long-term preservation in the frozen state. The use of cryopreservation makes it possible to avoid a number of problems: finding a donor at the right time for transfusion, the cost of maintaining the donor, etc. These days high therapeutic efficacy of using cryopreserved red blood cells was confirmed in intensive therapy and hematological diseases. The survival of biological objects under cryopreservation conditions is due to the ability of cells to withstand a complex of negative factors, including: the formation of crystals, an increase in the concentration of salts and osmotic pressure, dehydration of macromolecules and phase transitions of membrane lipids. Fundamental studies of physicochemical processes in cell suspensions under cooling and freezing conditions revealed important patterns that determine the basic principles of damage and protection of biological objects. The safety of cells under the influence of factors of low-temperature exposure is ensured by the use of special agents  -  cryoprotectants. Cryoprotective agents belong to different classes of organic compounds and are able to protect cells according to endo-and exocellular principle. However, the high efficiency of cryopreservation of red blood cells is achieved mainly by using endocellular compounds which must be removed after thawing from cell suspensions in order to maintain osmotic stability of cells under physiological conditions. Alternative to this may be cell-free cell cryopreservation methods developed on the basis of exocellular cryoprotectants. It should be noted that the cryoprotective properties of different compounds selectively manifest themselves depending on the type of cells. Biological preservation of erythrocytes for the use in veterinary practice is based on technologies for achieving biological stability and, accordingly, preserving a viable state after prolonged storage. This article reviews the mechanisms of cryodamage of erythrocytes, the cryoprotectants used in cryopreservation, as well as the existing low temperature storage methods.

Evaporation heat transfer of liquid nitrogen on microstructured surface at high superheat level

Liquid nitrogen, as a coolant, is generally applied in cell vitrification cryopreservation. It takes heat from the carrier with cell samples through its violent evaporation on the carrier surface. As a result, the temperature of the carrier plunges dramatically. This article focuses on the unsteady evaporation heat transfer characteristics of liquid nitrogen on a microstructured surface etched into the frozen carrier surface at a high superheat level. The heat flux and evaporation heat transfer coefficient of liquid nitrogen were investigated using a lumped capacitance method. The experimental results showed that the cooling rate of the thin film evaporation on the microstructured surface is obviously higher than that of pool boiling, which is currently being used for cell cryopreservation. The heat flux and the evaporation heat transfer coefficient work together to present a parabolic trend with the superheat decreasing during this heat transfer process. Besides, the microstructure of the surface has an important effect on the evaporation heat transfer of liquid nitrogen. The larger the thin film evaporation zone is, the higher the heat transfer coefficient is. The current investigation results in a cell cryopreservation method through vitrification with relatively low concentrations of cryoprotectants.

Modeling and experimental studies of enhanced cooling by medical gauze for cell cryopreservation by vitrification

Vitrification is considered as an important alternative approach to traditional slow freezing method for cryopreservation of cells. A typical cell vitrification procedure involves a non-equilibrium cooling process commonly accomplished in liquid nitrogen, while in which film boiling is believed to greatly hinder heat transfer surrounding the sample, resulting in incomplete vitrification or a much higher critical concentration. In this study, we developed a simple while effective approach, wrapping traditional French-type straw with medical gauze, to greatly enhance convective heat transfer during cooling by suppress film boiling. We further established a coupled heat transfer model for cooling and warming of cell suspensions to investigate the inherent thermodynamic mechanism in this approach. The model describes both the macroscale thermal distributions in extracellular solution and the microscale ice crystallization inside the cells. The simulation indicated that straws wrapped with medical gauze would increase cell survival subject to vitrification cryopreservation by significantly increasing the cooling rate to inhibit intracellular ice formation (IIF). Our experiments on human umbilical vein endothelial cells (HUVECs) further confirmed the predictions in that the cell survival rate was significantly increased by wrapping straws with medical gauze.

Evaluation of two human dental pulp stem cell cryopreservation methods.

Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was to evaluate whether human dental pulp stem cells (hDPSCs) isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system. The hDPSCs were cryopreserved using the Kamath and Papaccio methods. Post-cryopreservation viability was measured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greater expression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservation time may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.

Preserving human cells for regenerative, reproductive, and transfusion medicine.

Cell cryopreservation maintains cellular life at sub-zero temperatures by slowing down biochemical processes. Various cell types are routinely cryopreserved in modern reproductive, regenerative, and transfusion medicine. Current cell cryopreservation methods involve freezing (slow/rapid) or vitrifying cells in the presence of a cryoprotective agent (CPA). Although these methods are clinically utilized, cryo-injury due to ice crystals, osmotic shock, and CPA toxicity cause loss of cell viability and function. Recent approaches using minimum volume vitrification provide alternatives to the conventional cryopreservation methods. Minimum volume vitrification provides ultra-high cooling and rewarming rates that enable preserving cells without ice crystal formation. Herein, we review recent advances in cell cryopreservation technology and provide examples of techniques that are utilized in oocyte, stem cell, and red blood cell cryopreservation.

137 Controlling freezing using infrared radiation

Controlling ice growth is a significant tool in many fields such as preventing frost damage to plants, improving cryo-preservation of cells, tissues and organs, the frozen food industry, and cryopreservation procedures. We suggest a control of ice growth in super-cooled solutions by a combination of infrared (IR) radiation and antifreeze materials, such as antifreeze proteins (AFPs), for the purpose of cryobiology and food science. AFPs, which are part of the ice-binding proteins family, inhibit the growth of an ice crystal. As a result, the freezing temperature is reduced below the melting point. The hypothesis is that ice crystals can be stabilized in super-cooled solution by the use of the difference in the IR absorption of ice and water. Additional super-cooling can be achieved by AFPs. We use a homemade temperature-controlled system that allows fine control of the temperature of small amounts of liquid enclosed in a microdevice (Celik, Drori et al., 2013). The system includes an IR radiation to selectively heat and melt ice, due to the ability of IR radiation to heat ice more than water when a particular wavelength is chosen (Ullman, 2001). To measure the temperature within the device, we use a near-IR (NIR) camera and a band filter. This method is based on the temperature dependence of the NIR absorption of water (Kakuta, Fukuhara et al., 2011). Initial results show the ability to melt ice within microfluidic devices. We also were able to measure temperature field within the water using the IR camera. Simulation guided experiments are developed to show the control of ice growth using IR radiation in coexistence of ice crystals in super-cooled water. The combination we propose to control ice within miniature samples with an IR radiation and AFPs, opens the possibility of a new method for cryopreservation of cells and tissues. Source of funding: The Israel Science Foundation (ISF). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il

Ultra-high cooling rate utilizing thin film evaporation

This research introduces a cell cryopreservation method, which utilizes thin film evaporation and provides an ultra-high cooling rate. The microstructured surface forming the thin film evaporation was fabricated from copper microparticles with an average diameter of 50 μm. Experimental results showed that a cooling rate of approximately 5[Formula: see text]10(4) °C/min was achieved in a temperature range from 10 °C to -187 °C. The current investigation will give birth to a cell cryopreservation method through vitrification with relatively low concentrations of cryoprotectants.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.

Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.

An improved cryopreservation method for a mouse embryonic stem cell line

Embryonic stem (ES) cell lines including the C57BL/6 genetic background are central to projects such as the Knock-Out Mouse Project, North American Conditional Mouse Mutagenesis Program, and European Conditional Mouse Mutagenesis Program, which seek to create thousands of mutant mouse strains using ES cells for the production of human disease models in biomedical research. Crucial to the success of these programs is the ability to efficiently cryopreserve these mutant cell lines for storage and transport. Although the ability to successfully cryopreserve mouse ES cells is often assumed to be adequate, the percent post-thaw recovery of viable cells varies greatly among genetic backgrounds and individual cell lines within a genetic background. Therefore, there is a need to improve the efficiency and reduce the variability of current mouse ES cell cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of a C57BL/6 mouse ES cell line by characterizing the membrane permeability characteristics and osmotic tolerance limits. These values were used to predict optimal cooling rates, warming rates, and type of cryoprotectant, which were then verified experimentally. The resulting protocol, generated through this hypothesis-driven approach, resulted in a 2-fold increase in percent post-thaw recovery of membrane-intact ES cells as compared to the standard freezing protocol, as measured by propidium iodide exclusion. Additionally, our fundamental cryobiological approach to improving cryopreservation protocols provides a model system by which additional cryopreservation protocols may be improved in future research for both mouse and human ES cell lines.

A simple and efficient cryopreservation method for primate embryonic stem cells

Human embryonic stem (ES) cells have the potential to differentiate into all cell types. As these cells may be able to provide an unlimited cell source for transplantation therapies, it is necessary to establish reliable methods for their handling and manipulation, including human ES cell cryopreservation. Here, we report the development of a simple and efficient cryopreservation method for primate ES cell lines using vitrification in conventional cryovials. Using standard slow-rate cooling methods, the cryopreservation efficiency for cynomolgus monkey ES cell lines was approximately 0.4%, while that for a human ES cell line was virtually 0%. Primate ES cell lines, however, were successfully cryopreserved by the present vitrification method using conventional cryovials yielding a survival rate of about 6.5% for monkey ES cells and 12.2% for human ES cells. Vitrified ES cells quickly recovered after thawing and exhibited a morphology indistinguishable from non-vitrified cells. In addition, they retained a normal karyotype and continued to express ES cell markers after thawing. Thus, our vitrification ES cell cryopreservation method expands the utility of primate ES cells for various research and clinical purposes.

Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment.

Conventional hematopoietic stem cell cryopreservation methods use a DMSO concentration of 10%. However, cells manipulated ex vivo may require more refined freezing protocols adapted to the specific cell suspension. In this retrospective study, we evaluated the results obtained with CD34+ cells purified from peripheral blood of 39 patients on the CEPRATE SC System and frozen in 7.5% DMSO with a view to transplantation. The post-freezing recovery of progenitor cells was 89.4 +/- 27.87% for CD34+ cells, 59.13 +/- 36.93% for CFU-GM, and 53.49 +/- 40.71 for BFU-E. Neither the purity of the suspension nor the nucleated cell density during freezing was predictive of cell recovery. No difference was observed between cells stored in vials and bags. Thirty-seven patients transplanted with the concentrated CD34+ fraction received 4.46 x 10(6) CD34+ cells/kg and 33.04 x 10(4) CFU-GM/kg. The median time to granulocyte (>0.5 x 10(9)/l) and platelet (>50 x 10(9)/l) engraftment was 11 and 13 days, respectively. Only cell density and the infused number of CD34+ cells and CFU-GM were significantly related to hematological recovery. Our data suggest that purified CD34+ cells can be successfully cryopreserved in 7.5% DMSO and may represent a first step in establishing freezing parameters for selected CD34+ cells.