Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Studies on cryopreservation of Cryptosporidium parvum

Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.     

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.     

Effect of different cryoprotectants and transfer temperatures on the survival rate of hemp (Cannabis sativa L.) cell suspension in deep freezing

Adequate cell dehydration is the precipitating element in the successful cryopreservation of plant cells and organs. This could be achieved by using different cooling rates, transfer temperatures and cryoprotectants. Experiments were performed to determine these critical points in the freeze preservation procedure of Cannabis sativa (L.) suspension cultures. The explants were frozen at a cooling rate of 2 degrees C/min, while the transfer temperatures were -10 degrees C, -20 degrees C, -30 degrees C, -40 degrees C and -50 degrees C. The applied cryoprotectants were the DMSO, glycerol, proline and PEG in different concentration. The highest viability (58%) was obtained by using 10% DMSO and at -10 degrees C transfer temperature. The optimum transfer temperature varied remarkably by different cryoprotectant concentrations indicating the importance of their interactions.     

Effect of cryoprotective agents on rat cutaneous nerves.

Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work.     

The effect of cooling rate and of dimethyl sulfoxide concentration on low temperature preservation of neonatal rat heart cells

The effect of five cooling rates, 1, 5, 10, 30, and 50 °C/min, and of four DMSO concentrations, 2.5, 5, 7.5, and 10%, on the survival of neonatal rat heart cells after freezing and thawing were studied. Growth area, contracting area and contraction frequency were used as viability parameters. Growth area and contracting area were measured in a number of fields in statistically adjusted locations of the culture dish on the second and on the fifth day of culturing.Without freezing, DMSO concentrations higher than 5% caused a considerable decrease of the growth area and of the contracting area. After freezing and thawing, biphasic survival curves were found with a narrow optimum at 2.5, 5, and 10% DMSO and a broad optimum at 7.5% DMSO. The survival based on the growth area and the survival based on the contracting area were about the same on the second day of culturing but differed on the fifth day. On the second day of culturing the highest survival was 73%, at a cooling rate of 5 °C/min and with 5% DMSO. On the fifth day of culturing the highest survival based on the growth area was 100%, at a cooling rate of 10 °C/min with 7.5% DMSO; the contracting area was the same as on the second day. The cooling rate of 5 °C/min was optimal at all DMSO concentrations tested. There was no correlation between the contracting area and the spontaneous contraction frequency after freezing and thawing when both were expressed as percentages of the control. The contraction frequency after freezing and thawing was independent of the cooling rate and was maximally 50% of the control value.     

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

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

In vivo enzyme control through a strong stationary magnetic field--the case of thymidine kinase in mouse bone marrow cells

The influence of a strong homogeneous and stationary magnetic field (SMF) on the activity of the enzyme thymidine kinase (TdR-K) in bone marrow cells, and as a consequence of this on the incorporation of 125I-labelled 5-iodo-2-deoxyuridine (125IUdR) into DNA of mice and into isolated bone marrow cells in vitro, was assayed after exposure of immobilized mice. No effect could be elicited in moving mice, in cells in suspension or in enzyme in solution. The response depended on the body temperature during exposure: at 27 degrees C and 29 degrees C there was an increase and at 37 degrees C and a depression of enzyme activity. The TdR-K activity at low temperature increased with the field strength ranging from 0.2 to 1.4T. Thirty minutes were required for full expression of the effect at 1.4T; 5-10 min were needed after exposure for a return to base-line levels. Mice were given total-body irradiation at a dose of 0.1 Gy 137Cs gamma rays and then exposed immediately to a magnetic field at 1.4T for 30 min at a body temperature of 27 degrees C; gamma irradiation no longer inhibited the enzyme. Exposure to the magnetic field further removed from the time of gamma irradiation, did not negate the inhibitory effect of gamma irradiation. The observed responses to given challenges in this complex system support the hypothesis that the magnetic field affects TdR-K activity by way of a mediating structure, such as a membrane.     

Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage

The effects of different extenders, and cryoprotectants on the motility of tropical bagrid catfish (Mystus nemurus) spermatozoa were evaluated after short-term storage. Three extenders, physiological saline, Ringer or saline at three levels of sperm to extender dilutions (1:20, 1:30, or 1:40) and four cryoprotectants (DMSO, ethanol, glycerol or methanol) at three concentrations (5, 10, or 15%) were examined in two separate experiments. In the first experiment, milt was suspended in the respective extender at the three milt to extender dilution ratios in two sets of tubes. Extended milt in the first set of tubes was stored at -4 degrees C, and motility assessed after 24h, while the second set was kept at 23 degrees C and sperm motility was assessed immediately and at 30-min intervals thereafter. Ringer retained sperm motility better than the other extenders at all dilution levels at temperatures of 23 and -4 degrees C respectively. At 23 degrees C, the sperm motility was almost completely lost after 150 min except for those in Ringer at 1:20 dilution level which still had a motility of 18% (compared to those kept at -4 degrees C for 24, which had motility from 39 to 71%, regardless of extender). In the second experiment, various cryoprotectants were added to solutions of milt (that was diluted in Ringer at 1:20 ratio and cryopreserved in liquid nitrogen for 15 days). Sperm cryopreserved in 10% methanol had the highest motility (58%) compared with those in the other cryoprotectants at all concentrations.     

Refrigerated storage and cryopreservation of black drum (Pogonias cromis) spermatozoa

Procedures were developed for the collection, refrigerated storage and cryopreservation of black drum spermatozoa. Sperm samples were collected by removing and slicing the testis, and suspending the spermatozoa in Hanks' balanced salt solution (HBSS) at 200 mOsm/kg. Threshold activation (10%) of black drum spermatozoa occurred at 370 mOsm/kg, and complete activation occurred at 580 mOsm/kg in HBSS. Sperm cells activated in artificial seawater had higher motility than those activated in HBSS at osmolalities from 350 to 500 mOsm/kg. Spermatozoa stored at 4 degrees C in HBSS or artificial seawater at osmolalities from 202 to 290 mOsm/kg retained motility longer than did those stored at other osmolalities Dilution rate had no effect on sperm storage time at 4 degrees C. Four chemicals were evaluated as cryoprotectants: dimethyl sulfoxide (DMSO), n,n-dimethyl acetamide (DMA), methanol, and glycerol. Glycerol and DMA at concentrations of 10% significantly reduced motility within 52 min. Spermatozoa were cryopreserved at 3 freezing rates (-27, -30, or -45 degrees C/min) in a nitrogen vapor shipping dewar or a computer-controlled freezer. Spermatozoa frozen using 10% DMSO had the highest post-thaw motility at a freezing rate of -27 or -30 degrees C/min. Spermatozoa frozen using 5% glycerol, 5% DMSO, or 10% DMSO had the highest post-thaw motility at a freezing rate of -45 degrees C/min.     

Isolation and low temperature preservation of adult rat heart cells

Adult rat heart cells were isolated by perfusion of the coronary system of the heart with a 0.05% collagenase solution.In one method (A), cells were finally isolated by shaking the heart fragments in a collagenase solution, after which the cells were washed and suspended in a Ca- and Mg-free buffered salt solution. The effect of different DMSO concentrations, 5, 10, 15, 20, 25, and 30% and the effect of the addition and dilution rate of DMSO on the number of trypan blue-excluding, intact, and contracting cells were studied. The highest DMSO concentration which was tolerated by the isolated adult heart cells was 15%. Variation of addition rate and the dilution rate of DMSO had no effect. After freezing at external cooling rates of 1, 5, 10, 30, and 50 °C/min to ?100 °C, and then rapidly to ?196 °C, in the presence of 5, 10, or 15% DMSO, reanimation of these cells was not achieved.In another method (B), heart fragments, after collagenase perfusion of the heart, were first treated with 5, 10, or 15% DMSO, after which the cells were isolated. If these cells were frozen at 1 °C/min with 10% DMSO, 15% of the cells, expressed as a percentage of the control, remained morphologically intact and 38% of the cells were contracting after thawing. Significantly higher survival percentages of 30 and 61%, respectively, were obtained if the heart fragments were left intact during freezing.     

Response of murine bone marrow granulocyte-macrophage colony-forming units to hyperthermia in situ

A detailed understanding of how bone marrow stem cell progenitors are affected by heat is prerequisite to predicting how whole-body or regional hyperthermia protocols may affect bone marrow function. This investigation reports the reproductive integrity of murine tibial bone marrow granulocyte-macrophage colony-forming units (CFU-GM) after in situ hyperthermia. Heat was applied by water bath immersion of the leg of male BALB/c mice anesthetized with 90 mg/kg pentobarbital given subcutaneously. Tibial and rectal temperatures were monitored in representative animals by microthermocouples (tip diameter approximately 100 microns). By approximately 3 min after immersion of the limb, marrow temperature was within 0.3 degree C of water bath temperature (O'Hara et al., Int. J. Hyperthermia 5, 589-601, 1989) and was within 0.1 degree C by 5 min after immersion. The CFU-GM were cultured in "lung-conditioned" McCoy's 5A medium supplemented with 15% fetal calf serum and 0.3% Bacto agar. In situ heating of tibial marrow to exposure temperatures of 42, 42.5, 43, 44, and 45 degrees C gave D0's (+/- 95% CI) of 91 +/- 44, 44 +/- 27, 27 +/- 2.2, 16 +/- 6, and 7 +/- 4 min, respectively. Heating to 41.5 degrees C for up to 180 min did not result in cytotoxicity. Development of thermotolerance after approximately 100 min of heating was apparent by the presence of a "resistant tail" of the 42 degrees C survival curve. A plot of D0 vs water bath temperature was bimodal with an inflection point at approximately 42.5 degrees C. The inactivation enthalpy for temperatures above 42.5 degrees C was 586 kJ/mol (140 kcal/mol) and for temperatures below 42.5 degrees C was estimated to be 1205 kJ/mol (288 kcal/mol). These results show that CFU-GM can be heated predictably in situ, can be inactivated with thermal exposures as low as 42 degrees C, and are capable of developing thermotolerance. These findings underscore the necessity to understand stem cell inactivation by hyperthermia in situ prior to widespread implementation of clinical hyperthermia protocols where bone marrow may be included in the treatment field.     

The mitogenic activity of endothelin-1 on megakaryocytopoiesis in vitro

Endothelin (ET)-1 induces proliferation of various cells including smooth muscle cells, fibroblasts, glomerular mesangial cells, endothelial cells and osteoblasts. ET-1 also stimulates synthesis of interleukin (IL)-6 in endothelial and bone marrow stromal cells of rat. It is well known that IL-6 modulates megakaryocytopoiesis. Some studies have indicated that megakaryocytes express both ET receptors and they are targets for ET. Therefore we planned to examine the effects of ET-1 on the growth of normal megakaryocytic cells in rat bone marrow primary cell culture. Bone marrow cells were cultured at 37 degrees C, in an incubator atmosphere of 5% CO2 in air and 95% relative humidity for nine days. ET-1 at 10(-7), 10(-8 ) and 10(-11) M, and control with saline were added at the beginning of the experiment protocol. At each day, plasma clots were stained using direct-coloring thiocholin method for acetylcholinesterase activity. Although 10(-7) M ET-1 did not change the proliferation of megakaryocytic cells, this could be due to the presence of over crowded fibroblasts in the same environment. 10(-8) M ET-1 stimulated megakaryocytic cell growth to 234% over the control on the fifth day. ET-1 at a concentration of 10(-11) M also rised the megakaryocytic cell number significantly reaching up to 86% at the sixth day. Our results indicate that ET-1 may modulate the growth of megakaryocytic cells by an autocrine and/or paracrine action.     

Effects of extender composition,cooling rate,and freezing on the motility of sea bass (Dicentrarchus labrax,L.) spermatozoa after thawing

A successful cryopreservation procedure for sperm must guarantee recovery of the morphological and functional characteristics of the cells following thawing so that preserved semen can to be used comparably with non-preserved semen. The aim of this work was to identify a species-specific freezing protocol for sea bass (Dicentrarchus labrax) spermatozoa by optimising all the stages in the cryopreservation procedure. In the first stage of the experiments, the cryoprotectants and the relative concentrations that had the least toxic effect on motility at room temperature were selected. The capacity of the selected cryoprotectant substances was then assessed in freezing tests as follows: dimethyl sulfoxide (Me(2)SO) 5% and 7%, ethylene glycol (EG) 7% and 10%, propylene glycol (PG) 7% and 10%. The cryoprotectant that gave the best results in this second stage of the experiments was EG 10%, and this was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15min and 6h), as well as the effects of adding an energy substrate (1.25mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent+Na-pyruvate+EG10%) and the adaptation procedure (6h at 0-2 degrees C) that had given the best results in the preceding stages of the experiments, four cooling rates were tested: 10, 12, 15, 24 degrees C/min. It was shown that the semen that was diluted immediately after collection in extender that contained the cryoprotectant (EG 10%), was equilibrated for 6h at 0-2 degrees C and then cooled at a rate of 15 degrees C/min, showed motility on thawing comparable to that of fresh semen (P=0.045).     

Recovery of a marine dinoflagellate following controlled and uncontrolled freezing

A free-living, marine dinoflagellate, Crypthecodinium cohnii, was successfully preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a 1 °C/min cooling rate. After storage for a minimum of 7 days at ?150 °C material frozen by this method demonstrated a 47.7% mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at ?55 °C for 1 hr. This material demonstrated a mean recovery of 30.8% with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after uncontrolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 68% recovery following controlled freezing.The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cause.     

Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.     

Three-step cooling: A preservation method for adult rat heart cells

Adult rat heart cells were exposed to two-step cooling to ?196 °C with different holding periods at different subzero temperatures between both steps. The highest survival based on the percentage of trypan blue-excluding cells was 25% with 10% DMSO and a holding period of 6 min, and 21% with 15% DMSO and a holding period of 30 min. The highest survival based on morphological intactness was about 10%; there was no difference in results after cooling with 10 and 15% DMSO, and after holding between 2 and 30 min. The optimal survival based on the percentage of contracting cells was 52%, with 15% DMSO and a holding period of 2 min.When the holding period was replaced by a programmed cooling stage, the results could be improved. With this threestep cooling method, the optimal values, based on the number of trypan blue-excluding, intact, and contracting cells, were 40, 32, and 60%, respectively. It appeared that in the presence of 10% DMSO, which provided better survival than 5 and 15%, no significantly different results were obtained when the starting temperatures of the second cooling step varied between ?10 and ?20 °C, when the end temperatures varied between ?30 and ?60 °C, or when the cooling rates of the second cooling step varied between 0.1 and 1 °C/min. Three-step cooling provided similar results as linear cooling from 0 to ?100 °C, followed by rapid cooling to ?196 °C.     

Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions.

In the presence of high concentrations of the nonspecific polymer polyethylene glycol (PEG), intermolecular cohesive-end ligation with the DNA ligase from Escherichia coli was stimulated by high salt concentrations: 200 mM NaCl or 300 mM KCl in 10% (w/v) PEG 6000 solutions, and 100-200 mM NaCl or 150-300 mM KCl in 15% PEG 6000 solutions. Intermolecular blunt-end ligation with this ligase was also stimulated at 100-150 mM NaCl or 150-250 mM KCl in 15% PEG 6000 solutions. The extent of such intermolecular ligation increased and the salt concentrations at which ligation was stimulated extended to lower concentrations when we raised the temperature from 10 to 37 degrees C.     

Identification and characterization of a super-stable Cu–Zn SOD from leaves of turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

We report a novel super stable superoxide dismutase (SOD) extracted from the leaves of Curcuma longa L.-a post-harvest waste. The scavenging activity of this SOD remains intact both in crude and purified forms before and after heating at boiling temperatures (80-100 degrees C) up to 20 min, autoclaving (6-20 bars up to 10 min) and microwaving (frequency of 2,450 megahertz (MHz) or million cycles per second for 1-3 min). This SOD has significant shelf life at room temperature (25-35 degrees C) and is stable for at least 18 months at 4 degrees C and with the retained activity of 82% at -10 degrees C and 88% at -20 degrees C without any infection or contamination. The heat stable enzyme is present both in cytoplasm and chloroplasts. The enzyme is also stable under wide range of pH, alcohol and SDS concentrations. The heat stability of this SOD protein is not due to any associated phenolic compound as no phenolic compound was bound to the novel thermo-stable SOD. The activity staining through native PAGE and purification of the enzyme protein have shown that this form of enzyme has a native molecular weight of 30.8 kDa and has two subunits of 15 kDa as shown by SDS PAGE. The characterized novel isoform is a Cu-Zn SOD as is indicated by its sensitivity to both H2O2 and KCN. Indian, US and PCT patents have been filed and products are being developed using this hyperthermophilic enzyme.     

A novel method to measure cryoprotectant permeation into intact articular cartilage

Successful cryopreservation of articular cartilage (AC) could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. However, successful cartilage cryopreservation is limited by the time required for cryoprotective agent (CPA) permeation into the matrix and high CPA toxicity. This study describes a novel, practical method to examine the time-dependent permeation of CPAs [dimethyl sulfoxide (DMSO) and propylene glycol (PG)] into intact porcine AC. Dowels of porcine AC (10 mm diameter) were immersed in solutions containing high concentrations of each CPA for different times (0, 15, 30, 60 min, 3, 6, and 24 h) at three temperatures (4, 22, and 37 degrees C), with and without cartilage attachment to bone. The cartilage was isolated and the amount of cryoprotective agent within the matrix was determined. The results demonstrated a sharp rise in the CPA concentration within 15-30 min exposure to DMSO and PG. The concentration plateaued between 3 and 6 h of exposure at a concentration approximately 88-99% of the external concentration (6.8 M). This observation was temperature-dependent with slower permeation at lower temperatures. This study demonstrated the effectiveness of a novel technique to measure CPA permeation into intact AC, and describes permeation kinetics of two common CPAs into intact porcine AC.