High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me₂SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN₂ at −80 °C displayed the highest OSC viability. Different cooling rates ranging from −1 to −40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me₂SO) with VS3 (3 M PG and 3 M Me₂SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.     

Cryopreservation of swine colostrum-derived cells

Mesenchymal stromal cells (MSCs) have been demonstrated to possess anti-inflammatory and antimicrobial properties and are of interest in biotechnologies that will require cryopreservation. Recently, MSC-like cells were isolated from colostrum and milk. We used an interrupted slow freezing procedure to examine cryoinjury incurred during slow cooling and rapid cooling of MSC-like cells from swine colostrum. Cells were loaded with either dimethyl sulfoxide (Me₂SO) or glycerol, cooled to a nucleation temperature, ice-nucleated, and further cooled at 1 °C/min. At several temperatures along the cooling path, cells were either thawed directly, or plunged into liquid nitrogen for storage and later thawed. The pattern of direct-thaw and plunge-thaw responses was used to guide optimization of cryopreservation protocol parameters. We found that both 5% Me₂SO (0.65 M, loaded for 15 min on ice) or 5% glycerol (0.55 M, loaded for 1 h at room temperature) yielded cells with high post-thaw membrane integrity when cells were cooled to at least −30 °C before being plunged into, and stored in, liquid nitrogen. Cells cultured post-thaw exhibited osteogenic differentiation similar to fresh unfrozen control. Fresh and cryopreserved MSC-like cells demonstrated antimicrobial activity against S. aureus. Also, the antimicrobial activity of cell-conditioned media was higher when both fresh and cryopreserved MSC-like cells were pre-exposed to S. aureus. Thus, we were able to demonstrate cryopreservation of colostrum-derived MSC-like cells using Me₂SO or glycerol, and show that both cryoprotectants yield highly viable cells with osteogenic potential, but that cells cryopreserved with glycerol retain higher antimicrobial activity post-thaw.     

Methodology for processing mastectomy and cryopreservation of breast cancer tissue in a resource- poor setting: A pilot study

BackgroundThere is scarcity of breast cancer tissues derived from women of African origin available for patient - derived xenograft and organoid models.ObjectiveWe aim to create a versatile protocol for processing mastectomy and cryopreservation of breast cancer tissue.MethodologyAn immediate collection of breast cancer tissue from mastectomy was bathed in 4 °C HBSS and immediately transferred to 4 °C RPMI1640 containing HEPES, 10% FBS, Streptomycin and Penicillin. Tissues were processed over ice yielding nine samples of cold ischemic time (20–45 min) stored at 3 min interval. Cut samples were transferred into cryovials containing 4 °C cryoprotectant agent (90% FBS +10% Me₂SO) before snap -freezing in liquid Nitrogen vapour and final short-term storage in −80 °C Freezer. The histomorphology, tissue and molecular viability were assessed.ResultsThe cold ischemic times had no detrimental effect to the nine samples despite being processed in a resource poor setting, hence providing a reproducible and reliable protocol.     

The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth.Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of −5 °C/min to −20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of −5 °C/min to −20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin.The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034).The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.     

Quality improvements to mackerel (Scomber japonicus) muscle tissue frozen using a rapid freezer with the weak oscillating magnetic fields

Although freezing is the most popular long-term food preservation method, the formation of ice crystals during the freezing process often degrades the quality of the product. Recently, several reports have argued that oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between −30 °C and −35 °C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p = 0.061), and the increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.     

Viscosities encountered during the cryopreservation of dimethyl sulphoxide systems

This study determined the viscous conditions experienced by cells in the unfrozen freeze concentrated channels between ice crystals in slow cooling protocols. This was examined for both the binary Me₂SO-water and the ternary Me₂SO-NaCl-water systems.Viscosity increases from 6.9 ± 0.1 mPa s at −14.4 ± 0.3 °C to 958 ± 27 mPa s at −64.3 ± 0.4 °C in the binary system, and up to 55387 ± 1068 mPa s at −75 ± 0.5 °C in the ternary (10% Me₂SO, 0.9% NaCl by weight) solution were seen. This increase in viscosity limits molecular diffusion, reducing adsorption onto the crystal plane. These viscosities are significantly lower than observed in glycerol based systems and so cells in freeze concentrated channels cooled to between −60 °C and −75 °C will reside in a thick fluid not a near-solid state as is often assumed.In addition, the viscosities experienced during cooling of various Me₂SO based vitrification solutions is determined to below −70 °C, as is the impact which additional solutes exert on viscosity. These data show that additional solutes in a cryopreservation system cause disproportionate increases in viscosity. This in turn impacts diffusion rates and mixing abilities of high concentrations of cryoprotectants, and have applications to understanding the fundamental cooling responses of cells to Me₂SO based cryopreservation solutions.     

Understanding the Influence of State/Phase Transitions on Ice Recrystallization in Atlantic Salmon (Salmo salar) During Frozen Storage

Temperature fluctuations during storage and distribution of frozen foods lead to ice recrystallization and microstructural modifications that can affect food quality. Low temperature transitions may occur in frozen foods due to temperature fluctuations, resulting in less viscous and partially melted food matrices. This study systematically investigated the influence of state/phase transitions and temperature fluctuations on ice recrystallization during the frozen storage of salmon fillets. Using a modulated differential scanning calorimeter, we identified the characteristics glass transition temperature (T _g ′) of −27 °C and the onset temperature for ice crystal melting (T _m ′) of −17 °C in salmon. The temperature of salmon fillets in sealed plastic trays was lowered to −35 °C in a freezer to achieve the glassy state. The temperature (T) of frozen salmon fillets in sealed plastic trays was modulated to achieve a rubbery state (T > T _m ′), a partially freeze-concentrated state (T _g ′ < T < T _m ′) and a glassy state (T < T _g ′). We performed temperature modulation experiments by exposing packaged salmon to room temperature twice a day for 2 to 26 min during 4 weeks of storage. We also analyzed ice crystal morphology using environmental scanning electron microscopy and X-ray computed tomography techniques to observe the pore distribution after sublimation of ice crystals. Melt–refreeze and isomass rounding mechanisms of ice recrystallization were noticed in the frozen salmon subjected to temperature modulations. Results show that ice crystal growth occurred even in the glassy state of frozen salmon during storage, with or without temperature fluctuations. Ice crystal size in frozen salmon was greater in the rubbery state (T > T _m ′) due to the increased mobility of unfrozen water compared to the glassy state. The morphological/geometric parameters of ice crystals in frozen salmon stored for 1 month differed significantly from those in 0-day storage. These findings are important to the frozen food industry because they can help optimize storage and distribution conditions and minimize quality loss of frozen salmon due to recrystallization.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

The effect of ice formation on the function of smooth muscle tissue stored at −21 or −60 °C

The mechanisms by which single cells are injured during freezing are relatively well understood, but it is likely that additional factors apply to tissues and organs, factors that may be responsible for the poor suecess of attempts to cryopreserve complex multicellular systems. One such factor may be the formation of extracellular ice.

This study was designed to discover whether ice formation as such is detrimental to the contractile recovery of pieces of mammalian smooth muscle after storage at subzero temperatures. Strips of taenia coli muscle were equilibrated with 2.56 M Me₂SO in a buffered solution, cooled at either 0.3 or 2 °C/min to ?21 °C and then held at this temperature in the frozen state. Other muscle strips were bathed in a solution the composition of which mimicked that of the unfrozen phase of the previous solution at ?21 °C; it contained 4.49 M Me₂SO and 1.75 times the normal concentration of salts, and muscles equilibrated with this solution were also cooled at either 0.3 or 2 °C/min to ?21 °C, and then held unfrozen for the same length of time.It was shown that exposure to ?21 °C and the increased concentration of solutes had little effect on the contractile recovery of the muscles, whereas ice formation was damaging. Furthermore, the rate of cooling had a marked effect upon functional recovery in the frozen muscles, and this could be correlated with the known effect of these cooling rates on the pattern of ice formation in the tissue. The effect was also seen in muscles frozen at ?60 °C. Improved buffering increased the functional recovery of all groups, but the effect of ice, and of cooling rate in the presence of ice, was confirmed. These findings may have significant implications for attempts to cryopreserve complex tissues and organs.     

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation,cellular viability,and metabolism

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (−196 °C). The organic solvent dimethyl sulfoxide (Me₂SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me₂SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me₂SO during cryopreservation. We found that the addition of trehalose to Me₂SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to −40 or −80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me₂SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me₂SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me₂SO and additive solution produces acceptable in vitro platelet quality.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Two-step accelerating freezing protocol yields a better motility,membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.     

Preservation of female genetic resources of common carp through oogonial stem cell manipulation

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (−1 °C/min) and short-term storage (−80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me₂SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at −80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.     

Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA¹) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity.First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with Me₂SO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for Me₂SO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for Me₂SO. A mixture of Me₂SO, ethylene glycol and glucose (ratio 4:2:1– termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using Me₂SO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures.In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Cryobiological parameters of multipotent stromal cells obtained from different sources

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × V_o) and bone marrow (0.28 × V_o) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol⁻¹ for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me₂SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol⁻¹ and 27.4 ± 0.9 kcal mol⁻¹ for Am and BmMSCs respectively. In the presence of Me₂SO, activation energies were found to be 11.6 ± 0.3 kcal mol⁻¹ and 19.5 ± 0.5 kcal mol⁻¹ respectively. Furthermore, Me₂SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to −30 °C, followed by 3 °C/min to −80 °C.     

Effects of cryoprotective agents and freezing on abdominal ganglia of aplysia.

Isolated Aplysia depilans abdominal ganglia were exposed to 10 and 20% dimethylsulphoxide (Me₂SO) or glycerol at room temperature. Results indicate that Me₂SO induced an irreversible depression of extracellularly recorded ganglionic spontaneous spike generation while glycerol proved to be non-toxic. Intracellular recordings of individual nerve cell spontaneous activity during exposure to the cryoprotective agents were obtained in a few preliminary experiments. Both Me₂SO and glycerol induced a decrement in the nerve cell membrane potential. The main difference between the action of the two cryoprotectants was in the rate and the amount of depolarization, both being higher in the case of Me₂SO exposure.The Aplysia abdominal ganglia were frozen to ?20 °C and to ?196 °C. In all but one ganglia frozen to ?20 °C, including the preparations frozen in the absence of any cryoprotective agent, functional recovery was obtained after thawing. However, only the application of 20% glycerol improved the recovery of the preparations to a significant extent. In ganglia protected with 20% glycerol a full recovery of the action potential amplitude and frequency was obtained. In ganglia protected with 20% glycerol intracellular recordings of individual nerve cells demonstrated spontaneous spike activities before freezing and after thawing.No functional recovery was observed in ganglia frozen to ?196 °C in the absence of a cryoprotective agent. While in most preparations frozen with a cryoprotectant spontaneously generated spikes were recorded after thawing. However, the action potential frequency and amplitude were significantly depressed. It is concluded that further investigation is required to improve the freezing technique so that Aplysia ganglia may be preserved at low temperatures. It is suggested that intracellular exploration of the effects of cryoprotectants and freezing on identified nerve cell membrane may prove to be useful in future investigations.