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PubMed1995

In vitro chemosensitivity assessed with the MTT assay in childhood acute non-lymphoblastic leukemia.

Cellular drug resistance is supposed to play a major role in chemotherapy failures which frequently occur in childhood acute non-lymphoblastic leukemia (ANLL). Therefore, we determined in vitro chemosensitivity to daunorubicin, doxorubicin, mitoxantrone, 6-thioguanine, etoposide, and cytosine arabinoside (Ara-C) in childhood ANLL using the colorimetric MTT assay. The 4-day MTT assay was successfully performed in 62/73 samples obtained from 53 children with ANLL. We obtained comparable results from bone marrow or peripheral blood samples, and from fresh or cryopreserved samples. In vitro chemosensitivity was not related to clinical features such as sex, age, white blood cell count, or FAB-types. The group of poor responders to chemotherapy was median 3-fold more resistant to Ara-C than the group of good responders, but identification of a threshold for Ara-C sensitivity predictive for individual responses was limited due to the great overlap of in vitro chemosensitivities between both groups. Children with relapsed ANLL were in vitro median 3-fold more resistant to Ara-C than the initial ANLL group. No significant differences for the other drugs were observed with respect to clinical response or disease status. These results suggest that in vitro resistance to Ara-C plays an important role in chemotherapy failures in childhood ANLL, but larger studies are necessary to establish the predictive value of Ara-C sensitivity assessed with the MTT assay.

Antineoplastic Agents↗
PubMedDate not supplied

Stimulation of cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline, and 2'-deoxyadenosine.

We have investigated the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on the motion characteristics and longevity of domestic cat spermatozoa. Freshly collected or cryopreserved domestic cat epididymal sperm were incubated with 0.01-20 mM caffeine, pentoxifylline, or 2'-deoxyadenosine for 15 minutes at 23 degrees C. The percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were determined for each group using computer-assisted semen analysis. Freshly collected domestic cat sperm exhibited a strong forward progressive movement, and treatment with caffeine, pentoxifylline, or 2'-deoxyadenosine did not consistently alter sperm motion. Following cryopreservation, spermatozoa exhibited decreased (P < 0.05) MOT, VCL, VSL, and ALH. Caffeine and pentoxifylline increased (P < 0.05) the MOT, VSL, VCL, and ALH of cryopreserved sperm at 0.01-20 mM, in a dose-dependent manner. 2'-Deoxyadenosine also increased (P < 0.05) both VSL and VCL at 1.0 mM, and MOT, VSL, VCL, and ALH at 10 mM. All treatments shifted the percentage of nonhyperactive sperm to either a transitional or hyperactivated state. The motility indices of cryopreserved samples were examined during a 6-hour incubation to assess the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on sperm longevity. Compared to untreated control samples, the longevity of stimulated cryopreserved sperm was not reduced. These results indicate that motility stimulants may prove useful for enhancing the fertility of cryopreserved cat sperm by increasing their motility and producing hyperactivated motion.

Animals↗
PubMed1976

Electrophoretic distributions of human peripheral blood mononuclear white cells from normal subjects and from patients with acute lymphocytic leukemia.

The electrophoretic mobilities of mononuclear white cells from the peripheral blood of normal subjects and leukemic patients were measured. Analysis of both fresh and cryopreserved samples of each type showed that cryopreservation had no distinguishable effect on the electrophoretic distribution of either normal or leukemic cells. Mode mobilities (mobilities at which maximum intensities are observed) of cells from nine leukemic patients were 7-28% below the average mode mobility of normal cells. The standard deviation of the electrophoretic mobilities of normal cells was 2%. Normal cells had an asymmetric, often bimodal, electrophoretic distribution; leukemic cells consistently had a single symmetric distribution.

Electrophoresis↗
PubMed1995

DNA preparation from cryopreserved bone marrow cell samples.

1. Quickly thaw cryopreserved MNCs (1 ml) in 9 ml PBS supplemented with DNase I immediately before use (final concentration: 0.1 mg/ml). 2. Incubate at 37 degrees C for 10 min. 3. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 4. Incubate with saturating amounts of selected MoAbs for 20 min at 4 degrees C. 5. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 6. Immunomagnetic bead separation of viable cells as described by Lea et al. (1). 7. DNA extraction. 8. Measure quality and yield of DNA by spectrophotometry. In conclusion, the DNA extraction method presented here, based on DNase I pretreatment of the cryopreserved cells followed by an immunological selection for viable cells, provides cell suspensions with close to 100% viability; thus, high-quality DNA can be extracted even from cryopreserved cell samples of low initial viability. Furthermore, using this method DNA analyses can be performed on selected cellular subsets if desired. We thus recommend this method for DNA analyses in hematological research when the only materials available are cryopreserved bone marrow samples of low viability.

Bone Marrow↗
PubMed1990

Cryopreservation of equine mononuclear cells for immunological studies.

A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time.

Animals↗
PubMed1999

Microprocessor-controlled freezing device for cryopreservation of cell samples.

A freezing device for cryopreservation of blood mononuclear cells has been developed. The device is microcontroller operated, allowing cell freezing by a fully automatic, unattended process. To ensure optimum preservation, the temperature in the cell suspension uniformly decreases from room temperature to -100 degrees C and then the samples are transferred to long-term storage. The performance of the device has been tested using both physiological solution and a sample of cell suspension. The control of temperature variation of cell suspension in the entire temperature range has been realised with an accuracy better than +/- 0.1%. The viability of cells recovered from the frozen samples was 95%. The nitrogen consumption for one cycle of cryopreservation was 1.51. In addition to the fully automatic mode, the manual and semi-automatic modes are available for research purposes. The device has been designed using low cost and widely used electronic components and materials, it is compact and simple to operate.

Algorithms↗
PubMed1998

Pseudo-rearrangement of the MLL gene at chromosome 11q23: a cautionary note on genotype analysis of leukaemia patients.

AIMS: The MLL gene on chromosome 11q23 is frequently disrupted by chromosomal translocations in association with haematological malignancies. Recently, a specific site within the 8.3 kb MLL break-point cluster region that is cleaved during the early stages of apoptosis has been identified. Because MLL gene rearrangements are used to identify patients with high risk leukaemia, it was the aim of this study to determine whether this DNA cleavage event could be triggered in diagnostic bone marrow samples solely through ex vivo incubation at room temperature. METHODS: Pretreatment bone marrow samples were collected from six paediatric leukaemia patients. Genomic DNA for Southern blot analysis of MLL gene rearrangements was isolated immediately after samples were obtained and compared to genomic DNA isolated after incubation of specimens for 24-60 hours at room temperature, simulating delays in processing that might occur when samples are delivered to reference laboratories. In addition, cryopreserved samples from 70 paediatric leukaemia patients were screened for evidence of site specific MLL cleavage. RESULTS: After ex vivo incubation of bone marrow samples, site specific MLL cleavage resulting in a pseudo-rearrangement of the MLL gene was detected in two of six patients. In addition, a third patient with a similar MLL pseudo-rearrangement in cryopreserved cells was identified. CONCLUSIONS: Pseudo-rearrangement of the MLL gene at chromosome 11q23 was caused by ex vivo incubation of bone marrow samples. This novel phenomenon, which could lead to misclassification of leukaemia patients, might also be of importance for genotype analysis by Southern blotting at other loci.

Blotting, Southern↗
PubMed1998

Comparison of various methods of processing human cryopreserved-thawed semen samples.

We compared the efficacy of various methods of processing cryopreserved-thawed samples for the recovery of functionally adequate spermatozoa as assessed by the response to the sperm stress test (SST), an index of temperature activated sperm membrane lipid peroxidation, and immediate and delayed changes in sperm viability and motion parameters. Donor semen samples (n = 28) were cryopreserved-thawed and divided into six equal parts, one part was used as control and the remaining parts were used to compare five methods of sperm processing as follows: direct Percoll gradient processing, washing by one-step or stepwise addition of the washing medium followed by Percoll processing, and washing by one-step or stepwise addition of the washing medium. Additional samples (n = 10) were evaluated for the immediate and delayed (6 h at 37 degrees C) impact of one-step and stepwise washing (without Percoll separation). Compared with wash-only methods, samples processed using Percoll had a significantly higher SST score (P = 0.001), motility, rapid spermatozoa (>50 microm/s), curvilinear velocity and motility index (P < 0.001). Comparing various Percoll methods, direct Percoll processing resulted in the highest number of motile spermatozoa recovered (P < 0.00001) and a higher SST score based on curvilinear velocity (P = 0.001). Stepwise washing gave a significantly higher number of motile spermatozoa (P < 0.001) but with a significantly lower SST score based on the concentration of motile spermatozoa (P = 0.001), motility (P = 0.001) and motility index (P = 0.01). Sperm viability and motion parameters after 6 h of incubation showed no difference between one-step and stepwise washing. In conclusion, compared with wash-only methods, Percoll processed samples resulted in the recovery of spermatozoa with superior quality as assessed by SST and motion analysis. One-step washing of the samples gave an overall comparable recovery compared to the samples prepared stepwise. Having significantly higher SST scores, similar viability and the maintenance of motility, one-step washing may be a better method of processing thawed samples than the stepwise washing.

Cell Survival↗
PubMedDate not supplied

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions.

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.

Cell Survival↗
PubMedDate not supplied

Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation.

Earlier studies on human sperm cryodamage have shown that plasma membrane stress is the primary process and that phospholipid peroxidation in cryopreserved samples is not inhibited by addition of antioxidants. One consistent effect of cryopreservation is loss of enzymatic activity of the peroxidation defense enzyme, superoxide dismutase (SOD). To clarify this aspect of the freeze-thaw process and to develop a more complete resolution of the reactions leading to cryodamage, we sought to identify which of the two most probable mechanisms, loss of enzyme protein from the cells of denaturation of the protein, operates. If the first operates, cellular enzymatic activity and enzyme protein as identified by immunocytochemistry should give a linear correlation. If the second operates, there should be no correlation. In this study, five individual samples were analyzed before and after cryopreservation for immunoreactive Cu/Zn SOD and cell intactness by flow cytometry, for SOD enzymatic activity by a highly sensitive fluorimetric method, and for motility characteristics by Hamilton-Thorn motility analyzer. Fresh samples were obtained by the "swim-up" method and had > 95% intact cells with > 78% motile cells. After freeze-thaw, about half the cells were intact. SOD enzymatic activity was determined on Triton X-100 cell extracts, a method that removes all enzymatic activity from the cell structure, and compared with immunoreactive SOD in the cells as determined by indirect immunofluorescence mean intensities. Residual immunofluorescence was observed in the cells after Triton X-100 treatment; if this was taken into account, a close linear correlation between SOD enzyme activity and SOD immunoreactivity was obtained (r = 0.90; P = 0.00014). There was no correlation between SOD enzyme activity ratios for cryopreserved and fresh cells and fraction of intact cells after freeze-thaw. We conclude that loss of SOD protein from the subset of cells undergoing acute membrane damage is the most probable primary mechanism of SOD enzymatic activity loss from the sample and that resistance to cryodamage and SOD activity in any given cell are quite independent of one another.

Adult↗
PubMedDate not supplied

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method.

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3. 3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46. 5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.

Animals↗
PubMed2003

Comparison between two cord blood collection strategies.

BACKGROUND AND OBJECTIVES: Collection strategy is the first step for collecting good quality cord blood (CB) units. There are two principal different techniques to collect CB from the umbilical vein: in the delivery room while the placenta is still in the uterus by midwives and obstetricians or in an adjacent room after placental delivery by CB-bank trained personnel. In this study, the benefits and disadvantages between two different CB collection strategies were evaluated in order to improve CB bank methodology. DESIGN AND METHODS: Valencia CB bank maintains the two different collection strategies aforementioned. Before processing CB units, volume was calculated and samples were drawn for cell counts. After processing and before cryopreservation, samples for cell counts, CD34 analysis, viability, clonogenic assays and microbiology were drawn directly from the bags. We compared the efficiency of the two collection techniques. RESULTS: Obstetric date and umbilical CB was obtained from 848 vaginal (484 collected in uterus and 364 collected ex uterus). The proportion of excluded CB units before processing was 33% for ex uterus and 25% for in uterus. The difference was statistically significant. A larger volume and a higher number of total nucleated cells, CD34+ cells and CFUs were harvested in the in uterus collection group. INTERPRETATION AND CONCLUSIONS: Based on our findings, we conclude that the mode of collection influences the hematopoietic content of CB donations. Collection before placental delivery is the best approach to CB collection and allows optimizing CB bank methodology.

Antigens, CD34↗
PubMed2003

Comparison between two strategies for umbilical cord blood collection.

The use of cord blood (CB) for transplantation has increased greatly in recent years. The collection strategy is the first step in collecting good-quality CB units. There are two main techniques for collecting CB from the umbilical vein: in the delivery room while the placenta is still in the uterus by midwives and obstetricians or in an adjacent room after placental delivery by CB bank trained personnel. In this study, the benefits and disadvantages between the two different CB collection strategies were evaluated, in order to improve CB bank methodology. Valencia CB bank maintains the two different collection strategies. CB was obtained from 569 vaginal and 70 caesarean deliveries and obstetrical and clinical charts were reviewed. Before processing CB units, volume was calculated and samples were drawn for cell counts. After processing and before cryopreservation samples were drawn for cell counts, CD34+cell analysis, viability, clonogenic assays and microbiology were drawn directly from the bags. We compared the efficiency of the two collection techniques. Obstetric data and umbilical CB were obtained from 569 vaginal (264 collected in utero and 305 collected ex utero) and 70 caesarean deliveries. The proportion of excluded CB units before processing was 33% for vaginal ex utero, 25% for vaginal in utero and 46% for caesarean deliveries. Differences were statistically significant. For vaginal deliveries a larger volume and a higher number of nucleated cells, percentage of CD34+ cells and colony-forming units (CFUs) were harvested in the in utero collection group. There was no statistical difference between CB collected after placental expulsion from vaginal and caesarean deliveries. Comparison between all vaginal and caesarean deliveries did not show any difference. We conclude that the mode of collection influences the haematopoietic content of CB donations. Collection before placental delivery is the best approach to CB collection and allows optimisation of CB bank methodology. Caesarean deliveries seem to contain similar progenitor content to vaginal deliveries.

Adult↗
PubMed1998

A cryopreservation method for Pasteurella multocida from wetland samples.

A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

Animals↗
PubMed2002

Cryopreserving human peripheral blood progenitor cells with 5-percent rather than 10-percent DMSO results in less apoptosis and necrosis in CD34+ cells.

BACKGROUND: The grade of toxicity experienced by patients when cryopreserved peripheral blood progenitor cells (PBPCs) are reinfused is related to the amount of DMSO present in the PBPC concentrate. This study was initiated to investigate whether cell viability, apoptosis, and necrosis would be altered in CD34+ cells if PBPCs were cryopreserved with 5-percent as opposed to the conventional 10-percent DMSO. STUDY DESIGN AND METHODS: Samples of PBPCs from consecutive patients were mixed in parallel with 5- and 10-percent DMSO, frozen at a controlled rate, and stored in liquid nitrogen for periods of 3 to 22 months. Two different flow cytometric methods were used to measure both the absolute count of total and viable CD34+ cells as well as the fraction of apoptotic and necrotic cells in the post-thaw samples frozen with 5- and 10-percent DMSO. RESULTS: Both the number of total and viable CD34+ cells were higher (n = 18) or equal (n = 1) in all the samples cryopreserved with 5-percent as opposed to 10-percent DMSO. The percentage of viable CD34+ cells in the PBPC sample was significantly higher, and the fraction of apoptotic and necrotic CD34+ cells was significantly lower in the samples frozen with 5-percent as compared to 10-percent DMSO. CONCLUSION: Cryopreserving PBPC with 5-percent rather than 10-percent DMSO results in improved CD34+ cell viability and possibly a higher potential for in vivo engraftment and ex vivo manipulations of HPCs.

Antigens, CD34↗
PubMed2003

No differences in colony formation of peripheral blood stem cells frozen with 5% or 10% dimethyl sulfoxide.

High-dose chemotherapy with autologous stem cell rescue usually requires cryopreservation of the cells. For several years, 10% dimethyl sulfoxide (DMSO) has been used as the standard cryoprotectant. Because DMSO infusion can lead to toxic clinical complications in a dose-related manner, we wanted to evaluate if reduction to 5% DMSO would be possible. We have compared colony formation in the myeloid, erythropoietic, and megakaryocyte lineages in peripheral blood progenitor cell (PBPC) samples cryopreserved in parallel with 5% and 10% DMSO. Twenty-seven PBPC samples from patients with malignant diseases were investigated after 3 months of cryopreservation in liquid N(2), and samples from 14 of these patients were investigated after 1 year. A significantly higher colony formation was demonstrated for colony-forming units-erythrocyte (CFU-E) and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) both at 3 months and at 1 year in the 5% samples. For CFU-granulocyte-macrophage (GM) and CFU-megakaryocyte (Mk) no significant difference was demonstrated neither at 3 months nor at 1 year in samples frozen with 5% and 10% DMSO. Also, there was a statistically significant correlation between the CFU-total and CFU-Mk-total, indicating that the CFU-total might be used as an evaluation of megakaryocyte progenitors. Viability testing with the Trypan Blue exclusion test showed that cells cryopreserved in 5% DMSO had significantly higher viability than the cells cryopreserved in 10% DMSO. We conclude that 5% DMSO is at least as good for cryopreservation of small-volume PBPC samples as the conventional 10% DMSO, and our results suggest that the possibility of using 5% DMSO for cryopreservation of autologous PBPC grafts should be further investigated in clinical studies.

Bone Neoplasms↗
PubMed1990

The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes.

The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.8 M of Me2SO during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me2SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-14C]isoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of alpha-amino[1-14C]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180 min, subsequent addition of labeled alpha-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at significantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined.

Amino Acids↗
PubMed1997

Effects in the Comet assay of storage conditions on human blood.

The Comet assay is a rapid and sensitive method for analyzing single cells for DNA damage. Using human lymphocytes, the assay is particularly useful for human monitoring studies, as well as for in vitro genotoxicity testing of chemicals. In such studies, it is not always possible to collect and process matched samples on the same day as the blood is taken. It would be useful if some samples could be stored and examined at a different time, without loss of viability or other factors affecting responses. It is thus important to understand the effects of storage conditions on blood to be used in such studies and how exposure or treatment might modify such responses. In a joint study in two laboratories, blood was taken from various donors and stored under different conditions. It was examined on day 1 (day on which sample was taken) and days 2, 3, 4, 5, or 8 at room temperature, 4 degrees C, or -20 degrees C. Cells were treated after storage (from day 2 onward) with bleomycin (BLM) and ethylnitrosourea (ENU). The data were analyzed either by eye (classifying cells with different categories of damage) or by using a computerized image analysis system (Kinetic Imaging Ltd., Liverpool UK. Software Package Comet 3.0) where the tail moment, which is considered to be a sensitive measurement, has been analyzed. There was no loss of cell viability at 4 degrees C or room temperature up to 8 days when measured by trypan blue dye exclusion. Findings suggest that on days 1-4 for the untreated samples at room temperature or 4 degrees C there were no biologically meaningful changes in both the different categories of cell damage and tail moment data. In treated cultures up to day 4, either at room temperature or at 4 degrees C, responses were only minimally affected and changes were considered not to be of biological significance. However, there was slightly less variability between samples at 4 degrees C than at room temperature in one laboratory. The reverse was true in the other. This would suggest that samples can probably be stored up to day 4 at 4 degrees C or room temperature without any untoward effects. Provided samples can be processed within this 4-day time frame, it would not seem necessary to cryopreserve samples at -196 degrees C.

Bleomycin↗