Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Significance of the inactivation of transport in thermal death of Escherichia coli.

Cells of Escherichia coli ML308-225, harvested from the exponential phase, were heated in 50 mM potassium phosphate, and the loss in viability and inability to transport lactose, proline, and alpha-methylglucoside was compared. After cells were heated at 48 degrees C for 15 min, there was a 16% loss in viability and a similarly small reduction in the steady-state accumulation of lactose at 25 degrees C. The initial rates of lactose and proline transport were severely inhibited by heating at either 48 or 50 degrees C, but substantial recovery occurred within 5 to 7 min at 25 degrees C. Heating at 50 degrees C for 15 min caused an 86% loss in viability, but only a 53% decrease in the steady-state accumulation of lactose and only a 24% reduction in the initial rate of alpha-methylglucoside uptake. Twice as much alpha-methylglucoside was accumulated at 50 degrees C as at 25 degrees C. Although alpha-methylglucoside phosphate leaked from the cells at 50 degrees C, the concentration retained within the cells was about 500 times that externally, when only about 14% of the cells were viable. Overall, these results indicate that cells made nonviable by heating at 50 degrees C still have significant membrane integrity.     

Significance of the inactivation of transport in thermal death of Escherichia coli.

Cells of Escherichia coli ML308-225, harvested from the exponential phase, were heated in 50 mM potassium phosphate, and the loss in viability and inability to transport lactose, proline, and alpha-methylglucoside was compared. After cells were heated at 48 degrees C for 15 min, there was a 16% loss in viability and a similarly small reduction in the steady-state accumulation of lactose at 25 degrees C. The initial rates of lactose and proline transport were severely inhibited by heating at either 48 or 50 degrees C, but substantial recovery occurred within 5 to 7 min at 25 degrees C. Heating at 50 degrees C for 15 min caused an 86% loss in viability, but only a 53% decrease in the steady-state accumulation of lactose and only a 24% reduction in the initial rate of alpha-methylglucoside uptake. Twice as much alpha-methylglucoside was accumulated at 50 degrees C as at 25 degrees C. Although alpha-methylglucoside phosphate leaked from the cells at 50 degrees C, the concentration retained within the cells was about 500 times that externally, when only about 14% of the cells were viable. Overall, these results indicate that cells made nonviable by heating at 50 degrees C still have significant membrane integrity.     

Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB

BACKGROUND: Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40, saline or albumin. However, the process of freezing and thawing results in loss of HSC numbers and/or function. METHODS: This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol, volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40, 10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. RESULTS: Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001), CD34+ recovery, viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). Discussion: These results suggest that improved HSC recovery, viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO.     

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate. We have established addition and elution protocols that prevent osmotic damage and have used these to investigate the effect of multimolar concentrations of Me(2)SO on membrane integrity and functional recovery. We have investigated the effect of freezing and thawing over a range of cooling rates and cryoprotectant concentrations. CD34(+) cells tolerate up to 60 min exposure to 25% w/w (3.2M) Me(2)SO at +2 degrees C with no significant loss in clonogenic capacity. Exposure at +20 degrees C for a similar period of time induced significant damage. CD34(+) cells showed an optimal cooling range between 1 degrees C and 2.5 degrees C/min. At or above 1 degrees C/min, increasing the Me(2)SO concentration above 10% w/w provided little extra protection. At the lowest cooling rate tested (0.1 degrees C/min), increasing the Me(2)SO concentration had a statistically significant beneficial effect on functional recovery of progenitor cells. Our findings support the conclusion that optimal recovery of CD34(+) cells requires serial addition of Me(2)SO, slow cooling at rates between 1 degrees C and 2.5 degrees C/min and serial elution of the cryoprotectant after thawing. A concentration of 10% w/w Me(2)SO is optimal. At this concentration, equilibration temperature is unlikely to be of practical importance with regard to chemical toxicity.     

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products

Background aimsThe question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.MethodsCryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003–14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay.ResultsSignificant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = −0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration.ConclusionsOnce frozen, HPC products were stable for up to 14.6 years at <−150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.     

Study of a new device for washing and concentrating cryopreserved hematopoietic stem cells and mononuclear cells: a single center experience

Background aimsCryopreserved cellular products, as parts of hematopoietic progenitor cell (HPC) transplants, mononuclear cell reinjections for donor lymphocyte infusion or extracorporeal photopheresis, can be washed before being reinjected into the patient or infused directly, depending on local practices. The aim of washing is to reduce the incidence and severity of adverse reactions (ARs) due to the dimethyl sulfoxide (DMSO) used as a cryoprotective agent and other factors, such as dead cell debris. At the authors’ cell therapy laboratory (CTL) in Poitiers, France, as in 76% of Etablissement Français du Sang (EFS) CTLs, all cryopreserved products undergo thawing in a water bath followed by washing with the COBE 2991. As this device will soon cease to be available, an alternative process needs to be assessed.MethodsThe authors compared two closed systems: the authors’ semi-automatic system using the traditional centrifugation method (COBE 2991) and an automated device using spinning membrane filtration (Lovo). A total of 72 HPC bags available for research were used. The authors first performed a paired comparison, processing one or two HPC bags washed by each device. A second study was carried out to compare two different washing solutions generally used by EFS CTLs along with variable storage conditions. Finally, the authors studied the efficiency of the Lovo with three or four thawed bags. The main parameters studied were viable CD34+ cell recovery and viability, CD3+ cell recovery, stability up to 6 h after washing, DMSO elimination and center feasibility.ResultsThe Lovo device showed better CD34+ cell recovery compared with the COBE 2991 while maintaining CD34+ viability and stability over 6 h. Moreover, Lovo efficiency seemed to be independent of the number of thawed bags processed and washing solution used in the authors’ study. CD3+ cell recovery met the authors’ internal specifications (cell recovery >50%), with similar results seen when processing with either the COBE 2991 or Lovo. Additionally, on average, 97% of DMSO was removed after washing with Lovo, minimizing the risk of ARs. The storage conditions post-processing indicated preferred storage conditions of 7 ± 3°C. Finally, if processing time seemed shorter using COBE 2991 for one bag washed, the Lovo device required only one staff member regardless of the number of HPC bags processed.ConclusionsThe Lovo device seems to provide an opportunity to standardize HPC processing, ensuring patient safety, with, on average, 97% of DMSO removed, while improving recovery of cells of interest and maintaining viability over time in case of delayed transplant. The Lovo device consequently seems to be a serious alternative to the COBE 2991.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies—A Validation Study

Background

Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps—including transport duration, temperature and, importantly, tumor cell enrichment techniques—are confounding factors for gene expression analysis in DTCs.

Methods

LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays.

Results

Neither storage at –80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes.

Conclusion

Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.     

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 degrees C or at room temperature in KGM, saline, Dulbecco's modified Eagle's medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 degrees C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.     

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.     

The effects of low temperature on fertilized rabbit ova in vitro,and the normal development of ova kept at low temperature for several days

Fertilized rabbit ova at the 2-blastomere stage kept in rabbit serum were stored at low temperatures for various lengths of time. They were then cultured at 38 degrees C. for about 24 hours to determine their viability. A number of the viable ova were finally transplanted into recipient does. It was found that rapid cooling of ova to 5 degrees or to 0 degrees C. was more harmful to the subsequent viability of ova than slow cooling. Rapid cooling was not more lethal to the ova than slow cooling, but did prevent their future normal cleavage. There was no difference between those ova cooled rapidly or slowly to 10 degrees C. It was concluded that temperature shock has an adverse effect on ova, especially at the lower temperatures, though temperature shock can be remedied by acclimatization (slow cooling). Thus, the physiological significance of temperature shock would seem to be broadened. The optimal temperature for the storage of ova was investigated. It was found that 10 degrees C. was the best temperature; at this temperature viable ova were obtained after storage for 144 to 168 hours. At 0 degrees , 5 degrees , or 15 degrees C. the ova were viable for 96 to 120 hours, while at 22-24 degrees C., only for 24 to 48 hours. The percentage of dead ova was low at a favorable temperature, increasing only at the end of the storage period. At an unfavorable temperature, however, the rate of death increased steadily from beginning to end of storage. The percentage of abnormally cleaved ova (arrested cleavage and fragmentation) remained at a low level at first at a favorable temperature, but then increased just before or during death of the ova. A critical time for the viability, the abnormal cleavage, and the death of ova was characteristic of each temperature. About 24 to 28 per cent of the viable ova remaining after being stored at 0-15 degrees C. for 2 to 4 days and cultured at 38 degrees C. for 24 hours were capable of development into normal young. The compatibility of serum and ova, the absence of a correlation between the viability of the ova and the source of the fertilizing spermatozoa, and the fertilization of superovulated ova (i.e., the percentage of fertile does in follicular phase and in luteal phase, the percentage of unfertilized ova and of fertilized ova at different stages, the percentage of does that had produced a normal number of ova or had produced a large number of ova, etc.), are reported. The possibility of a more efficient utilization of the germ cells of valuable animals by means of the present techniques, and the possibility of a new approach to the experimental investigation of mammalian genetics and development, have been mentioned.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

Clinically relevant preservation conditions for mesenchymal stem/stromal cells derived from perinatal and adult tissue sources

The interplay between mesenchymal stem/stromal cells (MSCs) and preservation conditions is critical to maintain the viability and functionality of these cells before administration. We observed that Ringer lactate (RL) maintained high viability of bone marrow–derived MSCs for up to 72 h at room temperature (18°C–22°C), whereas adipose-derived and umbilical cord-derived MSCs showed the highest viability for 72 h at a cold temperature (4°C–8°C). These cells maintained their adherence ability with an improved recovery rate and metabolic profiles (glycolysis and mitochondrial respiration) similar to those of freshly harvested cells. Growth factor and cytokine analyses revealed that the preserved cells released substantial amounts of leukaemia inhibitory factors (LIFs), hepatocyte growth factor (HGF) and vascular endothelial growth factor-A (VEGF-A), as well as multiple cytokines (eg IL-4, IL-6, IL-8, MPC-1 and TNF-α). Our data provide the simplest clinically relevant preservation conditions that maintain the viability, stemness and functionality of MSCs from perinatal and adult tissue sources.     

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HPC) is widely used for evaluation of graft adequacy of peripheral blood stem cell grafts, and is also useful in planning the apheresis sessions necessary to obtain these grafts. The state-of-the-art method to enumerate CD34+ cells makes use of a multiparameter definition of HPC based on their light scatter characteristics and dim expression of CD45, and the use of counting beads to derive the concentration of CD34+ cells directly from the flow cytometric assessment. This method can be extended with a viability stain and additional markers for further immunological characterization of CD34+ cells, and has been successfully implemented in multicenter trials. Thus, the lower threshold of a safe HPC graft in terms of short- and long-term hematopoiesis may be more accurately defined.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

Aldehyde dehydrogenase as an alternative to enumeration of total and viable CD34(+) cells in autologous hematopoietic progenitor cell transplantation

We validated the correlation of aldehyde dehydrogenase ALDH(br) cells with total and viable CD34(+) cells in fresh and thawed hematopoietic progenitor cell (HPC) products, and looked for a correlation with time to white blood cell (WBC) and platelet engraftment after autologous transplantation, using simple linear regression analyzes. We found a significant correlation between pre-freeze ALDH(br) cell numbers and pre-freeze total CD34(+) (P < 0.001), viable CD34(+) (P < 0.001) and post-thaw viable CD34(+) (P < 0.001) cell numbers. We suggest that ALDH(br) may be substituted for CD34(+) cell numbers when evaluating HPC. As post-thaw viability testing apparently adds no significant information, we suggest that it may not be necessary. Finally, neither marker correlated with time to engraftment in our patients, supporting previous data suggesting the existence of a threshold dose for timely engraftment around 2.5 × 10(6) cells/kg.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.