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.     

Gradual Thawing Improves the Preservation of Cryopreserved Arteries

This study was designed to test a slow, controlled, automated process for the thawing of cryopreserved arteries, whereby specimen warming is synchronized with the warming of its environment. Segments of minipig iliac artery, 4-5 cm in length, were subjected to controlled, automated cryopreservation in a biological freezer at a cooling rate of 1 degrees C/min to -120 degrees C, followed by storage in liquid nitrogen at -196 degrees C for 30 days. Following storage, the arterial segments were subjected to rapid (warming rate of approximately 100 degrees C/min) or gradual (1 degrees C/min) thawing. Thawed specimens were processed for light microscopy and for scanning and transmission electron microscopy, Cell death was determined by the TUNEL method. Metalloproteinase (MMP) expression was estimated by immunohistochemical analysis. Most of the cryopreserved vessels subjected to rapid thawing showed spontaneous fractures, mainly microfractures, whereas these were absent in slowly thawed specimens. In rapidly thawed vessels, the proportion of damaged cells was double that observed in those thawed more gradually. Increased intensity and extent of MMP-2 expression was shown by rapidly thawed specimens. The slow-thawing protocol tested avoids the formation of spontaneous fractures and microfractures and the accumulation of fluid within the arterial wall tissue. This results in improved tissue preservation.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Cryopreserving human peripheral blood progenitor cells

High-dose chemotherapy followed by autologous peripheral blood progenitor cell (PBPC) transplantation is used in the treatment of chemosensitive malignancies. Cryopreservation of PBPC in 10% dimethyl sulfoxide (DMSO) has been the standard procedure in most institutions. Infusion of PBPC cryopreserved with DMSO can be associated with toxic reactions such as vomiting, cardiac dysfunction, anaphylaxia and acute renal failure. The grade of toxicity experienced by patients is related to the amount of DMSO present in the PBPC. Cryopreservation with lower DMSO concentrations would be expected to reduce the toxicity. In recent studies done with PBPC cells cryopreserved with 5%, 4% and 2% DMSO, using 10% DMSO as a reference control, CD34+ cells were investigated for preservation of viability, apoptosis, and necrosis. Also preservation of mature colony-forming (CFU) cells, specifically mature myeloid, erythroid progenitors, CFU-megakaryocytes and long-term culture-initiating cells (LTC-ICs) were investigated, using 5% and 10% DMSO as cryoprotectant. All samples were frozen in a rate-controlled programmed freezer and stored in the vapor phase of liquid nitrogen until used. Conclusion: 5% DMSO is the optimal concentration for cryopreserving human PBPC in vitro. Consequently, some hospitals have started using 5% DMSO as cryoprotectant for the autologous PBPC as a standard procedure.     

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.     

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10⁵ cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.     

Factors influencing myeloid stem cell (CFU-C) survival after cryopreservation of human marrow and chronic granulocytic leukemia cells

Human marrow stem cells obtained from 20 patients (9 with nonhematological malignancy, 11 with acute leukemia in remission) and peripheral blood stem cells from 27 patients with chronic granulocytic leukemia were cryopreserved in 10% dimethyl sulfoxide (Me₂SO). It was found that the optimal cooling rate for the human myeloid stem cells (CFU-C) ranged from 1 to 3 °C per minute. The myeloid stem cells (CFU-C) maintained their viability for up to one year of storage in liquid nitrogen, after an initial 20% reduction due to the freezing procedure. Myeldoid stem cells survived better when thawed and diluted at room temperature (RT) than at 4 °C. However, the viability of thawed stem cells decreased when stored at RT for more than 1 hr. The viability of stem cells cryopreserved in bags and ampoules was similar. No differences were noted in the surivial of normal human marrow stem cells and cells from patients with chronic granulocytic leukemia when cryopreserved under similar conditions.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.     

A technique for the separation and cryopreservation of myeloid stem cells from human bone marrow.

We have developed a technique for the cryopreservation of large volumes of human bone marrow, which reduces cell losses due to clumping and release of lysosomal enzymes from mature granulocytes. Mononuclear cells were separated from whole bone marrow by a large-scale Ficoll-Hypaque procedure. The agar colony assay for myeloid stem cells (CFU-C) was used to assess each step of the isolation and cryopreservation procedure. Conditions of varied cell and cryoprotectant concentrations and freezing and thawing rates were compared to obtain optimal recovery of mononuclear cells and CFU-C. This technique has been used to store bone marrow from 45 patients with hematologic and non-hematologic neoplasms. Up to 750 ml of marrow was obtained from each patient and separated by step-gradient centrifugation, and the cell fraction containing myeloid stem cells was cryopreserved. The mean recoveries following separation, cryopreservation, and thawing for 18 marrow storages from patients with hematological neoplasms were 8.8 ± 2.9% for mononuclear cells and 47.8 ± 20.8% for CFU-C. In comparison, values for 27 marrows from patients with non-hematological neoplasms were 14.5 ± 5.5% for cells and 57.7 ± 13.7% for CFU-C.     

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

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

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

Effect of thawing rate and post-thaw culture on the cryopreserved fetal rat islets: functional and morphological correlation

The ability of the fetal pancreatic islet cells to multiply rendered them a potential tissue for transplantation studies to cure diabetes. A bank of fetal islets could be created with proper storage in liquid nitrogen. The aim of this study is to evaluate the effect of thawing rate and post-thaw culture on the structural and functional integrity of isolated cryopreserved islets of rat fetuses. Fetal rat islets were isolated by the collagenase digestion, cultured for three days, and then cryopreserved using dimethylsulphoxide as cryoprotectant and the step-rate cooling to -40 degrees C before immersing them in liquid nitrogen. The islets were thawed by the slow or fast warming rates using hyperosmolar sucrose solution and then cultured for 1 or 2 days. Insulin and C-peptide contents of the slow thawed islets were higher than those of the control. In the fast thawed islets the contents were similar to those of the control. Insulin and C-peptide release in response to glucose for the slow thawed islets were lower than those of the control and in the fast thawed islets they were similar to that of the control. Histological examination showed irregular periphery and fragmented central part of the large slowly thawed islets, which showed also variable immunohistochemical reaction to anti-insulin serum, ranging from strongly positive reaction to markedly weak reaction. Fast thawed islets showed mostly regular periphery and their reaction to the anti-insulin serum was slightly weaker than that of the control islets. It was concluded that fast thawing and post-thaw culture is much better than slow thawing, as indicated by nearly normal insulin and C-peptide content and release and intact structural integrity.     

Cryopreservation of Suspension Cell Line Derived from the Protoplast Culture of Brassica campestris var. Pekinensis

Protected by DMSO, the suspension cell line derived from the protoplast culture of Brassica campestris var. pekinensis can be stored in liquid nitrogen (-196℃) for a long term. The addition of sorbitol and mannose can increase and decrease the protection, respectively. The medium also has an effect on cryopreservation. The relative survival rates of cells are little different in different days of cryopreservation. The highest rate of relative survival of cryopreserved cells reaches 75.4%. When the cryopreserved cells are thawed and resuspended, regrowth immediately occurs after just one day of lag period. Resuspended for six days, the cells increase 300–500%. It is much better for recovery of growth to resuspend in the dark than in the light. Like the non-cryopreserved control, the cryopreserved cells can be normally digested, producing a number of viable protoplasts which can be actively divided and form calli.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Long-term preservation of canine bone marrow: in vitro studies.

In vitro studies were performed on canine bone marrow frozen with DMSO and stored in liquid nitrogen for 2 to 6 months. The results are compared with previously reported parallel in vivo experiments that demonstrated no loss of stem cells. When studies were performed immediately after thawing, there was no substantial drop in the count of nucleated cells and, except for megakaryocytes, there was no alteration of the bone marrow morphology. After two washes, and removal of DMSO, the nucleated cell count dropped to 50% of its previous value. Optic and electron microscopy showed severe damage in mature myeloid elements. In some instances, the cells had a condensed nucleus similar to the red-purple inclusion body of LE cells (as observed in systemic lupus erythematosus), and electron microscopy showed heavy chromatin clumping. On the other hand, both optic and electron microscopy showed a good preservation of lymphocytes, plasmocytes, and erythroid precursors. Two-hour DNA synthesis slightly dropped after storage, and this drop appeared more consistent when related to a constant volume of bone marrow (50 microliters) rather than to a constant number of nucleated cells (10(6)). In five instances frozen and thawed bone marrow was grown in short-term cultures, and analysis of 98 metaphases showed no major aberrations of the chromosomes and only 2% of minor aberrations, such as breakages and fragments. These data, compared with the results of previous in vivo experiments that showed no loss of stem cells after 5 months storage, suggest that stem cells are less sensitive to freezing and thawing injury than myeloid elements and/or that it might be safer for the thawed bone marrow not to be manipulated before infusion.     

Bone marrow cryopreservation and clinical implications in autologous bone marrow transplantation

A simple, rapid and effective technique using the IBM (Cobe)-2991 cell processor for the concentration of buffy coat cells from large volume marrow has been well adopted (n = 16). Only about one-eighth of the original volume was obtained while retaining more than 90% of the total nucleated cells to be cryopreserved in polyolefine bags with TC-199 culture medium and final 10% dimethylsulfoxide (DMSO) (n = 9), processed by a computerized Nicool ST-20 (France) programmed freezer and stored in a vapor phase of liquid nitrogen at -196 degrees C. Stem cell assay by CFU-GM after thawing yielded a mean of 50.39 +/- 19.54% which has been satisfactory for clinical implementation. So far, three cases with hematological malignancies had been rescued by autologous cryopreserved marrow after supralethal doses of chemoradiotherapy. Two patients with acute nonlymphocytic leukemia transplanted in 1st remission as of Oct. 31 had been disease free for 178+ and 157+ days, respectively, after transplant which was taken at the corresponding age of 53 and 42 years. The other patient who was a victim of Hodgkin's disease, stage IV, and was transplanted in 3rd remission, expired on the 59th day because of the complication of idiopathic interstitial pneumonitis despite excellent granulocytopoietic reconstitution. The preliminary results are encouraging for further exploitation, especially for those who would otherwise be candidates for allogeneic bone marrow transplantation but are limited by age or lack of an HLA-identical sibling to serve as marrow donors.     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.     

Protocols for thawing and cryoprotectant dilution of heart valves

PURPOSE: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. METHODS: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). RESULTS: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. CONCLUSION: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.