Structural integrity of collagen and elastin in SynerGraft® decellularized-cryopreserved human heart valves

SynerGraft® (SG) decellularized–cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.     

Retained structural integrity of collagen and elastin within cryopreserved human heart valve tissue as detected by two-photon laser scanning confocal microscopy

Cryopreservation is commonly used for the long-term storage of heart valve allografts. Despite the excellent hemodynamic performance and durability of cryopreserved allografts, reports have questioned whether cryopreservation affects the valvular structural proteins, collagen and elastin. This study uses two-photon laser scanning confocal microscopy (LSCM) to evaluate the effect of cryopreservation on collagen and elastin integrity within the leaflet and conduit of aortic and pulmonary human heart valves. To permit pairwise comparisons of fresh and cryopreserved tissue, test valves were bisected longitudinally with one segment imaged fresh and the other imaged after cryopreservation and brief storage in liquid nitrogen. Collagen was detected by second harmonic generation (SHG) stimulation and elastin by autofluorescence excitation. Qualitative analysis of all resultant images indicated the maintenance of collagen and elastin structure within leaflet and conduit post-cryopreservation. Analysis of the optimized percent laser transmission (OPLT) required for full dynamic range imaging of collagen and elastin showed that OPLT observations were highly variable among both fresh and cryopreserved samples. Changes in donor-specific average OPLT in response to cryopreservation exhibited no consistent directional trend. The donor-aggregated results predominantly showed no statistically significant change in collagen and elastin average OPLT due to cryopreservation. Since OPLT has an inverse relationship with structural signal intensity, these results indicate that there was largely no statistical difference in collagen and elastin signal strength between fresh and cryopreserved tissue. Overall, this study indicates that the conventional cryopreservation of human heart valve allografts does not detrimentally affect their collagen and elastin structural integrity.     

Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6?mol/l cryoprotectant (4.65, 4.65, and 3.31?mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at ?80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4?mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P?<?0.05). After 3 and 6?months in vivo valve function was determined by two-dimensional echo-Doppler and at 7?months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P?=?0.0403, P?=?0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P?<?0.05) and infiltrating CD3+ T-cells (P?<?0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at ?80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.     

Transplantation of cryopreserved human corneas in a xenograft model

An ideal model to test methods of corneal storage for transplantation would simulate the environment of the grafted human cornea and predict the success of clinical corneal transplants (human to human). In this study, we tested such a model, the corneal xenograft (human to cat). Nine pairs of human corneas were transplanted into both eyes of nine recipient cats. One cornea of each pair was cryopreserved at -196 degrees C in 2.5 M dimethyl sulfoxide while the other was stored in preservative medium at 4 degrees C (control) for 6 +/- 2 (mean +/- SD) days before transplantation. One week after transplantation, the cats were euthanized and the eyes were examined. Three of the grafts (all cryopreserved) were clinical failures and showed no survival of donor corneal endothelial cells on scanning electron microscopy. The remaining six pairs of grafts were examined with a specular microscope and showed endothelial cell losses of 48 +/- 16% in cryopreserved and 8 +/- 16% in control corneas (p < 0.05). This survival is similar to survival in an earlier corneal perfusion model. The nine cryopreserved grafts were thicker than the control grafts, had fewer surviving keratocytes in the central stroma, and had more apoptotic central keratocytes (TUNEL assay). This failure rate in cryopreserved corneas clearly shows that this technique of cryopreservation was not adequate for clinical use. The corneal xenograft model can be used to study cellular survival and apoptosis in vivo after preservation as well as to test new methods of corneal preservation before initiating clinical trials.     

Characterization of a simplified ice-free cryopreservation method for heart valves

The aim of the present study was to characterize the hemocompatibility of ice-free cryopreserved heart valves in anticipation of future human trials. Porcine pulmonary heart valves were infiltrated with either an 83 % cryoprotectant solution followed by rapid cooling and storage at ?80 °C or with 10 % DMSO and control rate freezing to ?80 °C and storage in vapor phase nitrogen as conventional frozen controls. Cryopreserved leaflets were compared with fresh, decellularized and glutaraldehyde-fixed control valve leaflets using a battery of coagulation protein assays after exposure to human blood. Von Willebrand Factor staining indicated that most of the endothelium was lost during valve processing prior to cryopreservation. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen leaflets. Hemocompatibility differences were observed for cryopreserved leaflets versus both decellularized and glutaraldehyde fixed controls. In conclusion, the hemocompatibility results support the use of ice-free cryopreservation as a simplified preservation method because no statistically significant differences in hemocompatibility were observed between the two cryopreservation methods and fresh untreated controls.     

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from −147 °C to −47 °C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (−147 °C) and cryopreserved with temperature changes from −147 °C up to −47 °C and back to −147 °C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay.With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation.Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.     

Effects of sample collection and storage conditions on DNA damage in buccal cells from agricultural workers

Buccal cells are becoming a widely used tissue source for monitoring human exposure to occupational and environmental genotoxicants. A variety of methods exist for collecting buccal cells from the oral cavity, including rinsing with saline, mouthwash, or scraping the oral cavity. Buccal cells are also routinely cryopreserved with dimethyl sulfoxide (DMSO), then examined later for DNA damage by the comet assay. The effects of these different sampling procedures on the integrity of buccal cells for measuring DNA damage are unknown. This study examined the influence of the collection and cryopreservation of buccal cells on cell survival and DNA integrity. In individuals who rinsed with Hank's balanced salt solution (HBSS), the viability of leukocytes (90%) was significantly (p<0.01) greater than that of epithelial cells (12%). Similar survival rates were found for leukocytes (88%) and epithelial cells (10%) after rinsing with Listerine(?) mouthwash. However, the viability of leukocytes after cryopreservation varied significantly (p<0.01) with DMSO concentration. Cell survival was greatest at 5% DMSO. Cryopreservation also influenced the integrity of DNA in the comet assay. Although tail length and tail moment were comparable in fresh or cryopreserved samples, the average head intensity for cryopreserved samples was ～6 units lower (95% CI: 0.8-12 units lower) than for fresh samples (t(25)=-2.36, p=0.026). These studies suggest that the collection and storage of buccal samples are critical factors for the assessment of DNA damage. Moreover, leukocytes appear to be a more reliable source of human tissue for assessing DNA damage and possibly other biochemical changes.     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft?Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, were port an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at ～4 °C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85%glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchangedmechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.     

Processing of ovine cardiac valve allografts: 3. Implantation following antimicrobial treatment and preservation.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 °C, or cryopreservation with 10%Me₂SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85%glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Keratocyte injury in human corneas cryopreserved under standard conditions

This study was conducted to characterize ultrastructural damage to human corneas cryopreserved by a standard protocol. The materials used were seven human corneas that were unsuitable for transplantation due to the presence of positive bacteriological cultures; they were cryopreserved according the standard procedure. After freezing and thawing, samples were obtained for scanning and transmission electron microscopy studies. Marked damage was observed in keratocytes with signs of apoptotic cellular injury. However our observations have shown that apoptosis contribute less significantly than necrosis to cellular death in keratocytes of human corneas and although the control of apoptosis is clearly desirable, in order to improve the success of cryopreserved corneas for transplant, we need to continue our investigation to reduce the effects of the necrotic process. This revised version was published online in July 2006 with corrections to the Cover Date.     

Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants

Current methods of platelet storage are unsatisfactory because of the short shelf life of platelets and the rapid loss of platelet viability. We have developed a cryopreservation method that results in less damage from freezing and higher recovered function of platelets. Platelets were cryopreserved using a combination of epinephrine (EPN) and dimethyl sulfoxide (Me(2)SO) as cryoprotectants. The response of platelets to agonists was studied by flow cytometry and aggregation tests. Cryopreserving platelets with Me(2)SO decreased platelet annexin V binding due to freezing. The combination of EPN with Me(2)SO enhanced Me(2)SO cryoprotection and decreased platelet microparticle generation, suggesting that cryopreserving platelets using this combination is associated with increased platelet integrity. Platelet cryopreservation with an Me(2)SO/EPN combination also increased platelet aggregability, which was demonstrated by decreasing the lag phase and increasing the aggregation density to 66.39% +/- 6.6 that of fresh platelet-rich plasmas. We conclude that adding EPN as a combined cryoprotectant improves the quality of Me(2)SO-frozen platelets. As a method of aggregation of cryopreserved platelets, this method is comparable to that of normal fresh platelets and may improve the conditions for platelet transfusion.     

The addition of albumin improves Schwann cells viability in nerve cryopreservation

The purpose of the current study was to establish a valid protocol for nerve cryopreservation, and to evaluate if the addition of albumin supposed any advantage in the procedure. We compared a traditional cryopreservation method that uses dimethyl sulfoxide (DMSO) as cryoprotectant, to an alternative method that uses DMSO and albumin. Six Wistar Lewis rats were used to obtain twelve 20 mm fragments of sciatic nerve. In the first group, six fragments were cryopreserved in 199 media with 10% DMSO, with a temperature decreasing rate of 1 °C per minute. In the second group, six fragments were cryopreserved adding 4% human albumin. The unfreezing process consisted of sequential washings with saline in the first group, and saline and 20% albumin in the second group at 37 °C until the crioprotectant was removed. Structural evaluation was performed through histological analysis and electronic microscopy. The viability was assessed with the calcein-AM (CAM) and 4′,6-diamino-2-fenilindol (DAPI) staining. Histological results showed a correct preservation of peripheral nerve architecture and no significant differences were found between the two groups. However, Schwann cells viability showed in the CAM-DAPI staining was significantly superior in the albumin group. The viability of Schwann cells was significantly increased when albumin was added to the nerve cryopreservation protocol. However, no significant structural differences were found between groups. Further studies need to be performed to assess the cryopreserved nerve functionality using this new method.     

The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium

The effect of cryopreservation on ciliary activity of human nasal respiratory epithelium was evaluated. Samples were cryopreserved in a solution containing nutrient medium, 10% fetal calf serum, and two different concentrations (10 or 20%) of dimethyl sulfoxide and stored in liquid nitrogen at -196 degrees C for 2 weeks. Ciliary beat frequencies (CBF) of the samples before and after cryopreservation were compared. Mean CBF values did not differ significantly with both concentrations of dimethyl sulfoxide. The mean intrasample coefficient of variation of the CBF decreased significantly after cryopreservation. After thawing, CBF remained unchanged for at least 4 hr. It is concluded that normal ciliated epithelial cells can be frozen and stored in liquid nitrogen at 196 degrees C while maintaining their CBF.     

Cryopreservation of periodontal ligament cells with magnetic field for tooth banking

The purpose of this study was to establish a long-term tooth cryopreservation method that can be used for tooth autotransplantation. Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide (Me₂SO) using a programmed freezer with a magnetic field. Cells were cryopreserved for 7 days at −150 °C. Immediately after thawing, the number of surviving cells was counted and the cells were cultured; cultured cells were examined after 48 h. Results indicated that a 0.01 mT of a magnetic field, a 15-min hold-time, and a plunging temperature of −30 °C led to the greatest survival rate of PDL cells. Based on these findings, whole teeth were cryopreserved under the same conditions for 1 year. The organ culture revealed that the PDL cells of cryopreserved tooth with a magnetic field could proliferate as much as a fresh tooth, although the cells did not appear in the cryopreserved tooth without a magnetic field. Histological examination and the transmission electron microscopic image of cryopreserved tooth with a magnetic field did not show any destruction of cryopreserved cells. In contrast, severe cell damage was seen in cells frozen without a magnetic field. These results indicated that a magnetic field programmed freezer is available for tooth cryopreservation.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Cell viability improves following inhibition of cryopreservation-induced apoptosis

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.