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PubMed2006

[Establishment of immortalized lymphoblastoid cell bank of a keloid pedigree].

OBJECTIVE: To investigate a method for establishing immortalized lymphoblastoid cell bank of keloid pedigree so as to provide a long-term source of specimens for keloid research. METHODS: With Epstein-Barr virus transformation, fresh and frozen blood samples collected from all members of the keloid pedigree were used respectively to establish the immortalized lymphoblastoid cell lines of B lymphocytes. RESULTS: Twenty-seven immortalized lymphoblastoid cell lines of the keloid pedigree were obtained successfully, and all cell lines survived cryopreservation in liquid nitrogen. CONCLUSIONS: The immortalized lymphoblastoid cell bank of the keloid pedigree can preserve the whole gene information and provide long-term DNA sources for keloid research. Preparation of the cell lines with fresh blood is more efficient than that with frozen blood.

Adolescent↗
PubMed1994

Biologic resurfacing of a major joint defect with cryopreserved allogeneic periosteum under the influence of continuous passive motion in a rabbit model.

Periosteal autografts have been widely used for biologic resurfacing of full-thickness articular cartilage defects in rabbits. This study examined the effect of donor and recipient age on biologic joint resurfacing using fresh and cryopreserved periosteal allografts in a rabbit model. The study also assessed the ability of cryopreserved periosteal allografts to undergo neochondrogenesis. Eighty New Zealand rabbits were divided into six groups receiving combinations of fresh or cryopreserved allografts. After all the grafts were secured with fibrin glue, the animals were subjected to two weeks of continuous passive motion followed by four weeks of cage activity or intermittent active motion. The rabbit knees were analyzed at six weeks by gross examination, histochemical staining, and collagen typing. No significant difference in chondrogenic potential between fresh and cryopreserved periosteal allografts was observed. Grafts obtained from adult donors were much less chondrogenic than those from adolescents, but adults recipients experienced reasonably good results when they received allografts from young donors. No evidence of rejection was found in any of the samples as late as six weeks after transplantation.

Aging↗
PubMed2005

Sperm motility, plasma membrane integrity, and binding capacity to homologous zona pellucida of cryopreserved epididymal spermatozoa in the domestic cat.

We examined motility, plasma membrane integrity, and binding capacity to homologous zona pellucidae (ZP) of frozen/thawed epididymal cat sperm as a model species for endangered felines. Epididymal spermatozoa from 20 domestic cats were frozen with freezing egg-yolk extender containing 3.0% glycerol in 0.25-ml straws. Post-thaw motility and plasma membrane integrity of the frozen/thawed spermatozoa were 31.8 +/- 2.4% and 32.2 +/- 4.2%, respectively. The frozen/thawed spermatozoa were co-cultured with frozen/thawed immature homologous oocytes with intact ZP for 3 h to examine their ability to bind to the ZP. Sixteen of the 20 frozen/thawed sperm samples demonstrated the ability to bind to ZP. These results indicated that the freezing system for epididymal sperm used in the present study gives appropriate information for banking the genetic resources of wild felid species.

Animals↗
PubMedDate not supplied

In vitro and xenogenous capacitation-like changes of fresh, cooled, and cryopreserved stallion sperm as assessed by a chlortetracycline stain.

Like the human female, the mare experiences reproductive tract pathology that may sometimes be circumvented by the use of assisted reproductive technologies (ARTs). One such technology, gamete intrafallopian transfer (GIFT), may be used in mares that exhibit ovulatory, oviductal, or uterine abnormalities that limit the use of common ARTs, such as embryo transfer. Homologous GIFT has been successfully performed in the horse; however, the logistics, costs, and associated risks of surgically transferring gametes to the oviducts of a recipient mare are considerably high. Use of a less costly species in a heterologous or xenogenous procedure would therefore be beneficial. This study represents the preliminary investigation into the use of sheep as recipients for xenogenous GIFT procedures using equine gametes. We investigated the capacitation response of fresh, cooled, or frozen stallion sperm after 1) in vivo incubation in the reproductive tract of estrous and anestrous ewes as well as 2) in vitro incubation in a modified Krebs/ Ringer extender at 37 degreesC with and without the addition of heparin at 10 IU/mL for up to 8 hours. A chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm. Findings indicated that oviductal fluid samples recovered from estrous ewes had significantly higher numbers of sperm exhibiting capacitation-like staining patterns when compared to samples recovered from anestrous ewes (P < .05). Fresh semen yielded higher capacitation-like staining patterns after in vivo incubation than did frozen-thawed or cooled samples. A transition from majority CTC unreacted sperm to majority CTC non-acrosome intact sperm was demonstrated for both in vivo and in vitro studies. In vitro incubation of stallion sperm with heparin did not result in an increased capacitation-like staining response over time when compared with nonheparinized samples. Results from this study suggest that xenogenous capacitation of stallion sperm may occur in the estrous ewe.

Anestrus↗
PubMed2006

Analysis of ectopic and orthotopic bone formation in cell-based tissue-engineered constructs in goats.

Despite decades of extensive research, the application of cell-based bone tissue engineering in clinically relevant models remains challenging. To improve effectiveness, a better understanding of how the technique should work is crucial. In the current study, we investigated the onset time, rate, location and direction of bone formation in ectopically and orthotopically implanted clinically sized tissue-engineered constructs to gain insight the mechanism behind it. Bone marrow stromal cells (BMSCs) were obtained from 10 goats, culture expanded and cryopreserved. Porous biphasic calcium phosphate (BCP) disks of 17mmx6mm were per-operatively seeded with BMSCs or left empty. Both conditions were implanted intramuscularly and in bilateral critical-sized iliac wing defects. Fluorochromes were administered at 3, 5 and 7 weeks and samples were retrieved after 9 weeks. Histology showed abundant and homogeneous bone formation throughout the intramuscular BMSC samples and little bone in the controls. Histomorphometry and measurements of the fluorochrome labels of the ectopical BMSC samples indicated that osteogenesis started at the periphery and subsequent osteoconduction filled the whole scaffold within 7 weeks. In the orthotopically implanted disks, there was good integration with the surrounding bone, but minimal bone in the center of the implants, in both conditions. Bone was only derived from the interface with the surrounding bone, there was no early bone at the surfaces in contact to soft tissue as was seen in the ectopical samples. Apparently cell survival was minimal and insufficient for relevant additional bone formation. However, the speed of integration with surrounding bone and subsequent bone apposition on the BMSC-seeded orthotopic scaffolds were found to be significantly enhanced, which may be relevant especially in challenging environments.

Animals↗
PubMed2000

Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane.

The spermatozoa of most mammals behave as 'perfect osmometers'. The volume response to osmolality obeys the Boyle-Van't Hoff relationship (i.e. volume changes are determined by the osmotically active fraction of the cell volume (solids and water)). Most evaluations of osmotic sperm cell behaviour have been based on the mean volume of the cell population. In the present study, both mean and modal volumes of samples of sperm were evaluated. Both mean and modal volumes responded to environmental osmolality via the Boyle-Van't Hoff relationship; however, the modal volume showed a more sensitive response than the mean volume. This was confirmed for both ejaculated and epididymal spermatozoa. After incubation under capacitating conditions, the difference in modal and mean volume response of ejaculated sperm was considerably diminished and, in epididymal sperm, completely abolished. The sperm osmotic behaviour was still consistent with the Boyle-Van't Hoff equation, but the apparent osmotically inactive modal cell volume decreased after exposure to capacitating conditions in both ejaculated and epididymal sperm samples. The changes in epididymal sperm were more intensive. Due to its enhanced sensitivity to environmental osmolality and incubation under capacitating conditions, the modal volume could be used as a parameter for evaluating sperm population response, such as for detecting environmentally or cryopreservation-induced membrane changes.

Animals↗
PubMed1987

The evaluation of endothelial damage following corneal storage: a comparison of staining methods and the value of scanning electron microscopy.

Staining techniques and scanning electron microscopy (SEM) were used to assess damage to the endothelial cells following corneal storage. The aim was to establish whether these assays are useful in the assessment of endothelial integrity following corneal storage. Four groups ensured a range of normal and damaged endothelial cells: 1) fresh; 2) stored for 7 days in a moist chamber; 3) stored by the cryopreservation method of Capella and Kaufman; 4) damaged by rapid freezing with a cryoprobe. Trypan blue (TB), nitroblue tetrazolium (NBT), acridine orange (AO), fluorescein diacetate (FDA) and ethidium bromide (EB), were used to stain the endothelial layer. SEM was carried out on duplicate samples. Staining with NBT resulted in low cell counts due to loss of cells. There was no significant difference between the extent of damage measured by TB, AO or FDA, but it was shown that staining with FDA and EB can distinguish between damaged and intact cells. Tissue stored for 7 days in a moist chamber had a reduced number of intact cells compared to fresh tissue, and tissue stored by the Capella and Kaufman technique gave a reduced number of intact cells compared to both these control and storage groups. SEM showed surface perforation was characteristic of injured cells, rather than complete disruption. FDA has a theoretical advantage over the other stains, and should provide a more accurate appraisal of defects of cell membrane integrity. For this reason, the use of FDA with EB to stain the endothelium, with SEM carried out on duplicate samples, were preferred as assays to use in the development of corneal storage.

Animals↗
PubMed1985

Cryopreservation of Babesia bigemina for in vitro cultivation.

Babesia bigemina-infected RBC and merozoites were cryopreserved and used to initiate in vitro cultures in normal bovine RBC; the cryoprotectant was a final 10% polyvinylpyrrolidone in Vega y Martinez solution. A cooling rate of 20 C/min until -80 C and then rapid transfer to liquid N2 storage was satisfactory. Samples for culture initiation were rapidly thawed at 37 C, washed in Vega y Martinez solution and resuspended in complete culture media containing 10% normal bovine RBC. The optimum culture conditions to reestablish cultures were a 24-well plate (16 mm ID), 5 mm in depth, and an atmosphere of 2% to 5% O2, 5% CO2, and 93% to 90% N2.

Animals↗
PubMed2006

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells.

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.

Animals↗
PubMed2002

Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts.

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

Antigens, CD34↗
PubMed2001

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation.

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).

Animals↗
PubMed1983

Delayed kinetics of recovery of haemopoiesis following autologous bone marrow transplantation. The role of excessively rapid marrow freezing rates after the release of fusion heat.

Thirty-five patients were treated by intensive chemotherapy and/or whole-body irradiation followed by reinjection of cryopreserved autologous bone marrow. In 8 patients the kinetics of recovery of haemopoiesis was delayed (recovery to 10(9) leucocytes/litre beyond day 27 and recovery to 50 X 10(9) platelets/litre beyond day 25). This delay was directly responsible for the death of 3 patients and contributed to a fatal outcome in 2 others (mortality rate 9-14%). Retrospective analysis of these 8 cases revealed that failure of autologous transplantation was associated with poor recovery of CFUc, which was in turn related to an excessively rapid freezing rate after the release of fusion heat. Recovery of CFUc to 50% or more was achieved in 100% of cases when the freezing rate was less than 5 degrees C/min, 45% for freezing rates between 5 and 10 degrees C/min and 22% when the freezing rate exceeded 10 degrees C/min (n = 71, P less than 0.001). There was an inverse linear or logarithmic relationship between CFUc recovery and freezing rate after the transition phase (r = -0.46, r = -0.43, P less than 0.001). The quantity of nitrogen introduced into the freezing chamber to annul the fusion heat must therefore be calibrated with accuracy so that the desired shortening of the transition phase will not be accompanied by an overly marked increase in the freezing rate, which would result in the destruction of stem cells. To ensure an adequate freezing rate, it is crucial to monitor the temperature continuously in each sample of bone marrow during the freezing process. This study also suggested that other factors may have interfered with the kinetics of recovery after autologous bone-marrow transplantation. These factors include myelofibrosis, the presence of an Australia antigen and administration of compounds that are toxic for the bone marrow after reinjection of cryopreserved marrow. However, the responsibility of these factors cannot be stated with certainty.

Adult↗
PubMed1992

Analysis of electromagnetic heating patterns inside a cryopreserved organ.

Computer analysis of the induced electromagnetic field and heating distribution inside cryopreserved organs subjected to electromagnetic illumination at frequencies of 84 MHz, 434 MHz and 2.45 GHz were carried out using a spherical model for the organ, with special reference to heating in a single-mode resonant cavity. The interaction between the frequency of the incident field and the size and dielectric properties of the sample was investigated. It is shown that uniform heating of organs is likely to be achieved at lower frequencies, as might be expected. However, the ratio between the power penetration depth for plane waves and the size of the organ is not a sufficient basis on which to judge quantitatively the uniformity of the power absorption. Hot or cold spots can occur within the organ even when this ratio is greater than unity; the wavelength in the material is also an important factor. The results from this study indicate that the use of a resonant cavity as a heating applicator has advantages over plane-wave illumination. A sharp upper limit can be set to the frequency suitable for rapid and uniform heating of a given organ.

Animals↗
PubMed2006

Use of X-ray tomography to map crystalline and amorphous phases in frozen biomaterials.

The outcome of both cryopreservation and cryosurgical freezing applications is influenced by the concentration and type of the cryoprotective agent (CPA) or the cryodestructive agent (i.e., the chemical adjuvants referred to here as CDA) added prior to freezing. It also depends on the amount and type of crystalline, amorphous and/or eutectic phases formed during freezing which can differentially affect viability. This work describes the use of X-ray computer tomography (CT) for non-invasive, indirect determination of the phase, solute concentration and temperature within biomaterials (CPA, CDA loaded solutions and tissues) by X-ray attenuation before and after freezing. Specifically, this work focuses on establishing the feasibility of CT (100-420 kV acceleration voltage) to accurately measure the concentration of glycerol or salt as model CPA and CDAs in unfrozen solutions and tissues at 20 degrees C, or the phase in frozen solutions and tissue systems at -78.5 and -196 degrees C. The solutions are composed of water with physiological concentrations of NaCl (0.88% wt/wt) and DMEM (Dulbecco's Modified Eagle's Medium) with added glycerol (0-8 M). The tissue system is chosen as 3 mm thick porcine liver slices as well as 2 cm diameter cores which were either imaged fresh (3-4 h cold ischemia) or after loading with DMEM based glycerol solutions (0-8 M) for times ranging from hours to 7 days at 4 degrees C. The X-ray attenuation is reported in Hounsfield units (HU), a clinical measurement which normalizes X-ray attenuation values by the difference between those of water and air. NaCl solutions from 0 to 23.3% wt/wt (i.e. water to eutectic concentration) were found to linearly correspond to HU in a range from 0 to 155. At -196 degrees C the variation was from -80 to 95 HU while at -78.5 degrees C all readings were roughly 10 HU lower. At 20 degrees C NaCl and DMEM solutions with 0-8 M glycerol loading show a linear variation from 0 to 145 HU. After freezing to -78.5 degrees C the variation of the NaCl and DMEM solutions is more than twice as large between -90 and +190 HU and was distinctly non-linear above 6 M. After freezing to -196 degrees C the variation of the NaCl and DMEM solutions increased even further to -80 to +225 HU and was distinctly non-linear above 4 M, which after modeling the phase change and crystallization process is shown to correlate with an amorphous phase. In all tissue systems the HU readings were similar to solutions but higher by roughly 30 HU, as well as showing some deviations at 0 M after storage, probably due to tissue swelling. The standard deviations in all measurements were roughly 5 HU or below in all samples. In addition, two practical examples for CT use were demonstrated including: (1) glycerol loading and freezing of tissue cores and, (2) a mock cryosurgical procedure. In the loading experiment CT was able to measure the permeation of the glycerol into the sample at 20 degrees C, as well as the evolution of distinct amorphous vs. crystalline phases after freezing to -196 degrees C. In the mock cryosurgery example, the iceball edge was clearly visualized, and attempts to determine the temperature within the iceball are discussed. An added benefit of this work is that the density of these frozen samples, an essential property in measurement and modeling of thermal processes, was obtained in comparison to ice.

Animals↗
PubMed1994

Helicobacter pylori in a Costa Rican dyspeptic patient population.

Gastric biopsies from 65 Costa Rican dyspeptic patients were investigated for the presence of Helicobacter pylori DNA by polymerase chain reaction (PCR) amplification of 16S rRNA sequences. Both frozen and paraffin-embedded samples were used, and the results were compared with bacterial culture and histological examination. Helicobacter pylori DNA was detected by PCR in 60 (92%) of the patients, and Helicobacter pylori strains confirmed by PCR could be isolated from 37 of them. Altogether, 59 patients were shown to be infected using the combination of culture and histology as the reference method. The sensitivity of PCR analysis of frozen material was 98% (58/59). The PCR analysis of paraffin-embedded samples seemed less reliable than that of frozen biopsy material.

Adult↗
PubMed1992

The effect of storage in liquid nitrogen on the isolation of oral yeasts in human saliva.

Stimulated whole saliva samples were collected from a group of 127 6-yr-old schoolchildren. Each sample was divided into three parts. The first two, one of which contained added glycerol, were immediately frozen in liquid nitrogen and stored at -196 degrees C for 2 months. The third part was transferred to the laboratory and plated on selective medium for yeasts. Colony counts of the frozen and non-frozen samples were then compared. Statistical analysis showed a highly significant correlation between the counts for frozen and unfrozen samples. A high (40.1%) prevalence of yeast carriers was found, and Candida albicans was the most frequently recovered yeast.

Candida↗
PubMed1990

Transferrin polymorphism detected in human urine using isoelectric focusing followed by immunoblotting.

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.

Alleles↗
PubMed2006

Pre-fractionation of archival frozen tumours for proteomics applications.

We report a protocol for pre-fractionation of proteins from frozen tumours. Using this protocol two separate protein fractions are extracted: Fraction 1 is enriched for cytosolic and Fraction 2 for nuclear/nuclear membrane proteins. The accuracy and reproducibility of the protocol were demonstrated by Western blot analyses and SELDI-TOF-MS profilings. The resulting protein profiles were similar within the respective groups of Fractions 1 and 2 samples, without differences between freshly prepared tumours or those obtained as frozen tissue from biobanks.

Chemical Fractionation↗