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Cryopreservation of human fat for soft tissue augmentation: viability requires use of cryoprotectant and controlled freezing and storage.

BACKGROUND: Autologous fat transfer for soft tissue augmentation has been increasing in recent years. Graft longevity may vary greatly from patient to patient, requiring repeat procedures, often using frozen adipose tissue. Storage usually involves placing syringes of fat directly into a -20 degrees C freezer. However, the viability of fat frozen in this way is controversial. OBJECTIVE: This study tested methods for the optimal storage of adipose tissue harvested by tumescent liposuction. MATERIALS AND METHODS: Aliquots of washed adipose tissue were frozen directly at -20 degrees C or mixed with cryoprotectants, frozen at 1 degree C/min, and subsequently stored in liquid nitrogen vapor phase. Aliquots were subsequently thawed, and adipocyte viability was determined by staining and culture methods. RESULTS: Viability of adipocytes frozen at -20 degrees C was very low when analyzed by staining, and no cultures could be established from any of the specimens. In contrast, viable adipocytes were recovered from samples that were controlled-rate frozen in the presence of cryoprotectants and stored in nitrogen vapor. CONCLUSION. Our results indicate that fat frozen at -20 degrees C is not viable and thus provides no advantage over inert fillers. The methods here described could readily be transferred to the clinical setting after further laboratory study.

Adipocytes↗

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos.

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.

Animals↗

The preservation of micro-organisms in biological specimens stored at-70 degrees C.

The preservation of micro-organisms that may be found on the skin was studied by storage in liquid media at--70 degrees C. In the first part of the study the performance of 12 varieties of suspending media was evaluated with pure cultures of 17 species of micro-organisms maintained in the laboratory. After storage for 1 year the best medium (Oxoid Nutrient Broth with 15% glycerol) showed a mean survival for all organisms studied of 83.8%, with no significant differences between organisms. Even the worst medium (distilled water) permitted greater than 40% survival at 1 year. No changes in the characteristics of these micro-organisms were detected after 6 months storage in glycerol broth. In the second part of the study nose swabs were suspended in one representative medium (Bacto Nutrient Broth containing 7% glycerol). The mean percentage survival of staphylococci in these suspensions after 1 year's storage at - 70 degrees C was 75.4%. These results indicate that coagulase-negative coagulase-negative staphylococci in samples of skin flora may be stored under these conditions for long periods, greatly reducing the work-load in epidemiological studies of infection.

Cryopreservation↗

Composition of ruminal bacteria harvested from steers as influenced by dietary energy level, feeding frequency, and isolation techniques.

The effects of isolation techniques and time of sampling on composition of ruminal bacteria were examined in four steers fed two energy levels (2.24 or 2.92 Mcal metabolizable energy/kg DM) at two feeding frequencies. Diets were alfalfa hay and corn silage or ground corn/corn silage and were fed twice or 12 times daily. Whole ruminal contents were collected at four time intervals over a 4-d period. Fluid- and particle-associated bacteria were isolated. Energy level, feeding frequency, and preisolation freezing had little effect on composition of bacteria. Sampling time did not affect composition of bacteria harvested from steers fed frequently but had linear and quadratic effects on concentrations of cell components of bacteria harvested from steers fed twice daily. Differences were observed in the composition of bacteria harvested from the fluid phase compared with particle-associated or mixed populations of ruminal bacteria. These differences translated into different estimates of bacterial N supplied to the small intestine depending on N:purine ratio used. Composition of bacteria may be affected by fraction of contents sample and by time of sampling for animals fed infrequently. Freezing of samples before isolation of mixed bacteria does not appear to affect composition or estimates of bacterial N flows to the small intestine.

Animals↗

[Isolation of free DNA from archived serum samples suitable for molecular genetic studies].

INTRODUCTION: Collected and archived serum samples could be important sources for genetic studies, once DNA suitable for molecular genetic studies could be obtained from them. METHODS: DNA was isolated from 54 archived sera samples, collected previously from the participants of a Hungarian allergy study, with commercially available isolation kit. The authors have determined the concentration of the isolated DNA (81.88 +/- 52.36 ng/ml) and the size of the isolated fragments was estimated using semiquantitative real-time PCR. Two primers were used producing two different fragment size, for the phospholipase 2A and the actin beta genes, and melting curve analyses was performed as quality control. RESULTS: The concentration of the phospholipase 2A product was 2.9798 +/- 5.4454 microg/microl and the actin beta gene was 0.0015 +/- 0.0011 microg/microl. The melting curve analysis served as a quality control for the determination of the size of PCR products. In the case of the phospholipase 2A all samples produced the 133 bp PCR fragments, except one, while in the case of actin beta gene only six sample showed the expected 178 bp product, all the others samples had smaller fragments. CONCLUSIONS: These results confirm the suitability of the DNA isolated from archived sera samples for further molecular biological studies (SNP analysis, mutation detection) and give an estimate for the product size of the isolated DNA. Sera samples have been collected years ago can be a good source of genetic information on different diseases.

Actins↗

Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa.

This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4 degrees C (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4 degrees C (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 x 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.

Animals↗

The role of alternative splicing of the adhesion molecule, CD44, in lymphoid malignancy.

AIM: To investigate the expression of CD44 isoforms containing variant exon 6 (v6) in a well characterised cohort of patients with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukaemia (CLL), and to correlate this with phenotype and disease course. METHODS: Cryostat sections of OCT embedded diagnostic nodal material from NHL patients and cryopreserved mononuclear preparations from CLL patients were used as sources of RNA. After reverse transcription, PCR was carried out with amplimers positioned at either side of the variant exon insertion site to amplify all possible CD44 isoforms. Those isoforms containing v6 were identified after Southern blotting and hybridisation with a radiolabelled oligonucleotide. RESULTS: Of 32 NHL samples analysed, 16 did not express CD44 isoforms containing v6, six expressed an isoform containing exon v6 alone, and 10 expressed v6 long isoforms which contained exon v6 in addition to other variant exons. These data did not correlate with lymphoma classification, disease staging, or the presence or absence of extranodal disease. However, those patients expressing v6 long CD44 isoforms had a worse overall survival than those that did not. The plateau of the survival curves was 50% compared with 82%. No v6 long isoforms were detected in the 21 CLL samples investigated. CONCLUSIONS: The expression of v6 long CD44 isoforms is associated with aggressive disease in NHL, independent of grade, stage, or presence of extranodal disease.

Alternative Splicing↗

Rosette formation with mouse erythrocytes by lymphocytes from normal donors and patients with various diseases.

Rosette formation of human lymphoid cells with mouse erythrocytes has recently been proposed as a marker for a subpopulation of B lymphocytes. In this work we studied the percentage of mouse rosette forming cells (MRFC) in normal and pathological conditions and compared them to the percentage of sheep rosette forming cells (SRFC) a marker for T lymphocytes. Peripheral blood lymphocytes (PBL) from normal donors contained 6.2 +/- 1.1% (mean +/- 1 S.D.) MRFC. High percentages of MRFC were found in CLL patients, and a slight increase was observed in patients with systemic lupus erythematosus. MRFC were absent in Bruton's type agammaglobulinaemia, but were normally present in patients with T cell defects. Cryopreservation of lymphocytes in 10% DMSO did not significantly affect the mean percentages of SRFC and MRFC, though a slight increase of the former and a small reduction of the latter was observed. Double binding experiments on peripheral blood lymphocytes showed a predominant association of MRFC with cells staining for surface IgM and/or IgD. In all samples tested, we also observed a small population of MRFC negative for sIgM or sIgD and a few sIgM or sIgD positive cells that did not rosette with mouse erythrocytes.

Agammaglobulinemia↗

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols.

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.

Antigens, CD34↗

Lymphocyte subset analysis on frozen whole blood.

An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at -80 degrees C, in liquid nitrogen (LN2), and at -20 degrees C. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3+4+ percentages for F/T (-80 degrees C storage for up to 1 year) and fresh WB was less than -0.2% (95% limits +/-3%, P = 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P = 0.74). Absolute CD3+4+ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/microl, P < 0.0001), but the results were highly correlated (r2 = 0.975, P < 0.0001). Results were more variable, although still highly correlated, for CD3+8+ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.

Blood Preservation↗

Simultaneous quantitative determination of cyclosporine A and its three main metabolites (AM1, AM4N and AM9) in human blood by liquid chromatography/mass spectrometry using a rapid sample processing method.

We have developed a sensitive and specific liquid chromatography/mass spectrometry (LC/MS) method for the simultaneous determination of cyclosporine A (CsA) and its three main metabolites (AM1, AM4N and AM9) in human blood. Following protein precipitation, supernatant was directly injected into the LC/MS system. Chromatographic separation was accomplished on a Symmetry C8 (4.6 x 75 mm, 3.5 microm) column with a linear gradient elution prior to detection by atmospheric pressure chemical ionization (APCI) MS using selected ion monitoring (SIM) in positive mode. This method can be applied to single mass equipment. The analytical range for each analyte was set at 1-2500 ng/mL using 100 microL of blood sample. The analytical method was fully validated according to FDA guidance. Intra-day mean accuracy and precision were 95.2-113.5% and 0.9-8.9%, respectively. Inter-day mean accuracy and precision were 95.8-107.0% and 1.5-10.7%, respectively. In blood all analytes were stable during three freeze/thaw cycles, for 24 h at room temperature and for 12 months at or below -15 degrees C. Stability was also confirmed in processed samples for 24 h at 10 degrees C and for 6 months at 4 degrees C in methanol. In addition, we confirmed the method could avoid matrix effects from transplant subjects' samples. This LC/MS technique provided an excellent method for simultaneous quantitative determination of CsA and its three metabolites for evaluation of their pharmacokinetic profiles.

Atmospheric Pressure↗

Follicle-stimulating hormone measured in unextracted urine: a reliable tool for easy assessment of ovarian capacity.

OBJECTIVE: To determine the presence of FSH in unextracted urine of perimenopausal women using a microparticle enzyme immunoassay kit on an AxSYM random access immunoassay analyzer. DESIGN: Controlled descriptive study. SETTING: A large teaching hospital and infertility clinic. PATIENT(S): Forty perimenopausal women aged 32-55 years admitted to our clinic for a gynecological operation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mean serum FSH level and urinary FSH in early-morning urine, in a random void urine sample, and in 24-hour urine on the same day. FSH in urine on the day of excretion and 1 and 4 weeks thereafter, stored under various conditions. FSH in urine before and after extraction. RESULT(S): The Pearson's correlation coefficient between mean serum FSH levels and urinary FSH in early morning urine was 0.904, in a random void 0.915, and in 24-hour urine 0.857. Determination of optimal storage conditions revealed that urine was best kept at 4 degrees C without any additive. The correlation between FSH in extracted and unextracted urine was 98.9%. CONCLUSION(S): In perimenopausal women, FSH can be reliably measured in unextracted urine. The correlation between urinary FSH and a random void urine sample and mean FSH from a serial serum sample is very high. Urine can be stored for 4 weeks at 4 degrees C without loss of FSH immunoreactivity.

Adult↗

On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device.

High-pressure freezing (HPF) is currently the only method which enables adequate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and preparation methods, the technique has not yet reached its full potential. While suspensions can be prepared easily for HPF, tissue preparation is restricted by the need to compromise between different requirements and difficulties. (i) In order to achieve optimal freezing quality, very thin samples are required. (ii) There is mechanical difficulty in cutting such thin samples without distorting the organization of the tissue. (iii) The cutting and the succeeding preparation steps of small samples require long handling times (minutes), which may result in physiological and hence structural alterations. Computerized heat transfer simulations are presented which confirm that the efficiency of heat extraction from cylindrical samples contained within thin-walled metal tubes is higher than from standard flat discoid samples sandwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the quick excision of such cylinders of soft tissues. The device utilizes sharp gold needles of an inner diameter of 200 microm and wall thickness of 50 microm. The frozen sample contained in the soft gold needle permits all the manipulations needed for conventional cryo-preparation techniques for electron microscopy (e.g. cryo-sectioning, freeze-fracturing, freeze-substitution).

Animals↗

Filtration of platelet-poor plasma specimens yields platelet-free plasma. Application for batch analysis of beta-thromboglobulin and platelet factor 4.

The authors investigated filtration of platelet-poor plasma through a 0.2-micron filter for the analysis of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) in frozen specimens. Platelets were detected by electron micrograph in the platelet-poor plasma preparation without filtration. No platelets or platelet fragments were seen after filtration. Freezing the unfiltered specimens resulted in a significant increase in the beta-TG and PF4 concentrations, presumably because of release of these proteins from the platelet alpha-granules during freezing and thawing. There was no change in the PF4 levels and only a slight decrease in beta-TG levels after freezing of the filtered specimens. The filtered samples could be frozen for later batch analysis, resulting in considerable cost savings to the laboratory.

Blood Platelets↗

Estimation of sperm viability in frozen-thawed semen from Swedish A.I. bulls.

From a total of 40 Swedish diary A.I. bulls, the number of viable spermatozoa in frozen-thawed semen was estimated using a commercial luminometer (SVT) to determine ATP contents and by fluorescence microscopic examination of samples stained with the combined fluorophore probes Calcein AM and Ethidium homodimer (CAM/EthD-1). The bulls were of two breeds (24 Swedish Red and White and 16 Holstein) and two age classes (1-2 and 6-10 years old, prior to and after progeny testing, respectively). The SVT-instrument was able to estimate a frequency of viable spermatozoa (47.0 +/- 14.4%; means +/- SD) close to that recorded by the fluorophore probes (spermatozoa with intact plasmalemma and progressively motile, 53.6 +/- 11.5%) and by subjective visual assessment of sperm motility (53.4 +/- 6.0%). The percentage of motile spermatozoa assessed under phase-contrast was positively (r = 0.33, P < 0.01) correlated with both the total ATP content, the percentage of viable spermatozoa recalculated from the SVT reading (r = 0.26, P < 0.05) as well as the frequency of spermatozoa having intact membranes as assessed by CAM/EthD-1 (r = 0.45, P < 0.001). The latter was also significantly correlated (r = 0.59, P < 0.001) with the total ATP contents. No significant variation in ATP contents was present between bulls, breeds or age. A significant difference, however, was seen between bulls for sperm membrane integrity. The results indicate that the SVT instrument seems to provide a quick and easy estimation of the number of viable spermatozoa in frozen-thawed bull semen. The assessment of membrane integrity with fluorophore probes allows the estimation of sperm subpopulations within the sample and seems therefore to be more useful and objective to determine sperm viability than visual assessment of sperm motility.

Animals↗

Freezing material without electricity.

When collecting in the field, biological samples can be kept cool at 0 C in ice slush, or frozen at -20 C in a crushed ice + salt (NaCl) cold mixture, in well-insulated containers, for several hours or days.

Animals↗

Longitudinal stability of coagulation, fibrinolysis, and inflammation factors in stored plasma samples.

We sought to assess the longitudinal stability of risk factors for atherosclerosis and thrombosis. including several coagulation. fibrinolysis, and inflammation factors, in frozen plasma samples stored at -70 degrees C for months or years. We reviewed data collected on 29 different control pools over periods ranging from 7 to 59 months for two functional assays (factor VII and fibrinogen) and seven antigen measurements (C-reactive protein. D-dimer, plasmin-alpha2-antiplasmin complex, plasminogen activator inhibitor-1, protein C, protein S, and tissue plasminogen activator), totaling more than 15,000 data points. Screening of the data using least squares regression revealed only sporadic associations between monthly means and time, with no consistent trends. Analysis by repeated measures and summary measure methods revealed no evidence of sample degradation over time for the factors studied. Our finding of longitudinal stability in the biochemical properties of frozen plasma strengthens the presumption of sample stability on which molecular epidemiologic studies are based.

Acute-Phase Proteins↗

Quality of NAT2 genotyping with restriction fragment length polymorphism using DNA isolated from frozen urine.

In large studies and under field conditions common to epidemiological research, factors outside of and inside the laboratory can introduce misclassification of genetic susceptibility markers. Few reports have been made on the accuracy of genotyping individuals using DNA extracted from frozen urine that was stored for approximately 20 years. This study was performed to determine the reproducibility and accuracy of N-acetyltransferase 2 (NAT2) genotyping by RFLP analysis using DNA from stored urine. To obtain long-term frozen urine and blood samples from the same person, the databases of two large prospective studies were linked by name and date of birth. Six polymorphisms within the coding region of NAT2 were determined in 65 urine and blood samples after which, genotypes and imputed phenotypes (rapid, slow) were derived. To test reproducibility, all of the six polymorphisms were determined twice in 47 urine-blood pairs. Reproducibility of imputed phenotypes was 91.5% in urine samples and 97.9% in blood samples. To test accuracy, results for all six polymorphisms were compared between urine and blood DNA. All of the kappa's were at least 0.85 except one. Identical results for all six polymorphisms were seen in 78.5% of urine-blood pairs. Taking blood samples as a reference standard, rapid acetylators were classified as rapid in 97% of subjects (95% confidence interval, 90-100%), and slow acetylators were classified as slow also in 97% of subjects (95% confidence interval, 91-100%), when using urine. This study shows that stored urine samples can be used for DNA genotyping in large cohort studies, when blood samples are not available.

Aged↗