text:
|Publication number
|Publication type
|Application number
|Publication date
|Dec 8, 1981
|Filing date
|Jun 18, 1979
|Priority date
|Jun 18, 1979
|Publication number
|Max W. Scheiwe, Gunter Rau
|Original Assignee
|Helmholtz-Institut Fur Biomedizinische Technik
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (7), Referenced by (42), Classifications (19)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to a process and apparatus for freezing living cells.
In recent years processes for the treatment of cancers and tumors have been developed in which, after chemotherapy, the patient is transfused with specific body cells. Since it is not possible, because of the danger of fatal rejection, to use cells of donors other than the patient himself, it has been necessary to remove the specific body cell from the patient, prior to treatment and then store such cells until they are needed. To this end it is necessary to store the withdrawn cells for prolonged periods, often several months. To preserve such cells cryogenic preservation at low temperatures, have been employed since it is yet no other means available to store living cells.
Relatively large quantities of such cells are needed for the post-therapy infusion and most importantly, such large quantities must have a relatively large ratio of living cells. Nevertheless, none of the known cryogenic freezing processes is capable of freezing large cell quantities in a single sample nor are such processes capable of maintaining the high levels of living cells required for optimum therapeutic purposes. While the importance of improved cancer therapy might justify a high cost level, it is almost impossible, no matter at what cost, with the current processes to obtain the required large quantity of cells as for example autologous lymphoid blood corpuscles or medullar cells.
There are several factors in the cryogenic preservation processes which are extremely important. Among these are the steps of:
(1) The taking of the cell sample from the blood, marrow or tissue and the concentration therefrom of the required cell type;
(2) The transfer of this concentration to a freezing container;
(3) The mixing of the concentrated sample with a freeze protectant;
(4) The controlled freezing of the specific sample;
(5) Prolonged storage below minus 130� C. (143� K.); and
(6) Thawing of the sample in precise time/temperature relationship; and revitalization of the cells, i.e., their gradual dilution and elutriation.
Of the foregoing factors the most vital step and the one which has up until now presented the greatest difficulty is the controlled freezing of the sample.
According to the known freezing processes the cell samples were placed in closed freezing chambers wherein the temperature was reduced at a constant rate. It was found, however, that the cooling curve of the sample did not conform to the linear curve of the temperature drop in the cooling chamber, nor did the cooling curve of the temperature take into account the cooling curve of the sample, but to the curve as seen in FIG. 1. The sample would initially follow the curve of the temperature of the chamber, until a point below the freezing level TF at which time it would rise to a plateau defined at its upper limit by the freezing temperature TF at its lower limit TP (phase transformation temperature) constituting a pleateau where it would remain for some time. After some minutes, the temperature of the sample would again drop below the plateau TP at an extremely steep descent until it almost reaches the curve of the chamber temperature and thereafter follows in parallel the curve of the chamber temperature. Therefore, freezing processes have been developed which take into account the described abnormal thermal behaviour of aqueous samples. But, these processes don't take into account the actual freezing curve of the freezing samples, i.e. they don't use the sample's temperature for the regulatory system. Because of practical problems, the sample's mass is mostly not equal to the mass, the freezing curve has been established for. In this case, the freezing chamber generates a freezing curve of the samples which may be similar to that shown in FIG. 1, resulting in increased damage to the cells. The slightest variations from the freezing curve of the chamber for the particular cell drastically reduces the number of living cells in the sample and produces undesirable ice crystals and other harmful effects. Furthermore, all of the freezing units currently available, freeze laboratory samples only, that is, small samples having a volume no greater than about 2 ml. Consequently, the known freezing systems do not meet the increased requirements of large quantities of cells for broad spectrum therapy.
To be therapeutically effective, large quantities, as for example in the case of medullar cells, lymphocyte cells, granulocyte cells, amounts of about 1�1010 (range 1�109 to 1�1011) are required and for thrombocytes (platelets) amounts of about 1�1011 (range 1�109 to 1�1011) are required. These increased requirements can not be met by the prior art, since
(a) the cells must be frozen in volumes of about 100 to 200 ml in each unit sample, as otherwise the loss of time and sterility in filling smaller samples is too great. Steritily is insured in the prior art only, when the techniques of transfusion medicine are used and the storage the small samples is very expensive. For example, the refrigerant costs for storing a vessel of volume of 320 liters are approximately 10,400 DM (about $6,000) annually. The present invention permits the collection and storage of large amounts and at reduced costs.
(b) The therapeutic dose must contain after thawing at least 80% living cells as otherwise a lesser amount of living cells is ineffective in carrying out the vital therapeutic function. In the prior art test cells are sufficient at 50% viability, while generally, the best of the prior art samples do not produce more than 70% viable cells. On the other hand, cells frozen by the present invention are viable concentrations normally between 80-90% and sometimes to about 95% or more. Of course, not only the viable concentrations of cells give therapeutic effectiveness, but also the absolute number of living cells, which is the total recovery. This is the viable concentration times the absolute number of cells ready for transfusion divided by the number of cells originally in the sample before freezing. Though a sample may for example contain more than 70% viable cells, a total recovery may be near 10%. When cells are frozen by the present invention, the total recovery exceeds 80% on an average.
(c) Commercial freezing equipment show temperature fluctuation in the freezing chamber in about a theoretical value of �3� C. This fluctuation in temperature reduces the viability of the cells along the edge zones of the container and reduces the viability even more.
(d) The known commercial freezing units have a temperature/time function which is not influenced by the behavior of the sample itself, but only by the behavior of the freezing chamber. Therefore, the freezing in such equipment will proceed in the desired manner only if the sample volume is not changed relative to the predetermined setting, and irrespective of the actual freezing of the sample itself. Thus, when different sample geometry is used, a new program control must be employed with such freezing units. On the other hand, the control system according to the present invention renders such a step unnecessary.
In order to avoid the foregoing difficulties, and to obtain the objects of the present invention, the present invention proposes to use not only the temperature of the freezing chamber itself but a measured temperature of the sample being frozen for the control of the various phases of the cooling process and to determine both the sample and chamber temperatures simultaneously.
According to the present invention, a process of freezing cell suspensions within a freezing chamber is provided comprising the steps of lowering the chamber temperature at a first defined cooling rate to a given temperature value at which time the sample becomes cooled to its freezing temperature; holding the temperature of the chamber at this value until the temperature of the sample leaves the phase transformation plateau and decreases to a second predetermined temperature; heating the chamber to an intermediate temperature which is below the second predetermined temperature of the sample; maintaining the temperature of the chamber at the intermediate temperature until at such time as 85% of the mass of the sample is present in a frozen state, and; thereafter lowering the chamber temperature at a second defined cooling rate until the temperature at which substantially all the mass of the sample is frozen. Only when these relative values are matched exactly, it is possible to freeze reproducably relatively large quantities of cell suspensions and to obtain after the thaw a high survival rate.
Further, according to the present invention there is provided apparatus for carrying out the foregoing process comprising a freezing chamber having room for the mounting of a plurality of containers each having a volume of cells to be frozen, the containers being spaced from one another to provide uniform flow of the refrigerant media. The chamber has an inlet for the refrigerant, a fan for the circulation thereof, and a heater. The chamber defines an enclosed path for circulation of the cooling and heating media. The apparatus further contains a test sample supplied with a thermo-couple, and a heat sensing probe located in the chamber. In this manner the temperature of the chamber and of the samples to be frozen is constantly monitored, and the inlet of the freezing media and the heating media can be continuously and simultaneously monitored.
This apparatus is provided with a programmed control system wherein the process, aforementioned, can be automatically carried out. The program control may be a micro-processor the input of which, relative such a constant as predetermined cell freezing temperature, etc., can be made, and wherein the operation is made responsive to the actual temperature of the freezing chamber and samples being frozen.
The present invention further provides novel containers and freezing trays for the storage and for freezing of relatively large samples. Still further, the present invention provides, through the use of the process and the apparatus large concentrations of living cells, having a high degree of viability.
FIG. 1 is a graph, wherein the abscissa t is time in minutes and the ordinate T is temperature in degrees centigrade, showing the cooling curves of the chamber and the samples following the prior art;
FIG. 2 is a perspective view of a sample bag and freezing tray;
FIG. 3 is a schematic drawing of a specific freezing chamber used in the apparatus of the present invention;
FIG. 4 is a schematic drawing of the apparatus of the present invention; and
FIG. 5 is a graph similar to that of FIG. 1 showing the cooling curves for the chamber and samples according to the present invention.
Before turning to a description of the present invention it is to be pointed out that a great deal of literature exists on the nature of the cells in question and in particular those characteristics concerning them during freezing and upon being frozen, namely the rates at which the cells cool at a given temperature and time conditions, the plateau temperature of the cells at the freezing state, and the point at which at least 85% of the cells in any given sample are frozen. It is for this reason that the present invention does not go into such detail except wherein it is necessary for an understanding of the present invention. In speaking of cells in the present disclosure, cells, i.e. corpuscles found in the liquid portions of the body which can be extracted from blood, glandular secretions, marrow, as well as tissue cells are to be considered.
In general, cells intended for use in therapeutic infusion media are initially collected by known medical techniques and are separated, as from the blood, liquid or tissue, with a known cell separator such as a Haemonetics Mod. 30 or Aminco cell separator under periods of two to six hours, preferably, however, within an average time of about three to four hours, or until a sufficient amount of cells is available. for freezing. Another way of cell separation may be performed by density gradient centrifugation obtained by centrifugation or gravitational force, with or without specific filtering apparatus, without the use of a cell separator as described above. In many cases, the latter procedure is used to obtain thrombocytes from normal blood units. The concentration of the cell component, upon hematocrit should be at least between 20 to 50% and preferably about 35% which may contain an anticoagulant. The hematocrit values are of great importance for the viability and recovery of the cells after the freeze-thaw cycle. This is because the cells are dehydrated during freezing and thereby dilute the extracellular medium by the outcoming water. This fact is especially important for erythrocytes preserved by HES (hydroxyethylstarch), as was shown in respective investigations. Another important fact is the aggregation tendency of white blood cells and thrombocytes, which is extremely increased when the hematocrit exceeds the desired values indicated above. The hematrocrit, the concentration of the cryoprotectant and the cooling rate are dependent on one another, and this fact is responsible for the drastically decrease in recovery obtained when the indicated values are not chosen. Tests have shown that many living cells, for example, lymphacites, are short-lived in vitro and that damage to these cells already starts before they are subjected to freezing. The amount of the damage is time dependent. In the prior art, loss of time due to the procedures for transferring the cells, filling predetermined small volume containers, was generally considerable and often lasted more than one hour over the optimun range. As damage to the cells before freezing has a multiple effect on the damage after thawing, the saving of time in the process of handling the cells leads in itself to an increased cell vitality. As will be seen hereinafter, much of the transfer operation is avoided, according to the present invention.
According to the present invention, the cells are collected, i.e. by separation, in large quantities as for example, bone marrow, lymphacites, granularcites may all be collected in the range of 1�109 to 1�1011 cells per single sample, while the thrombocites may be collected in sample amounts of 1�1010 to 1�1012 ; stem cells may be collected in the range of 1�106 to 1�108 cells per single sample. These corpuscles being all cells capable of suspension in a fluid, are then suspended in a volume of approximately 100 to 300 ml of isotonic water or the like, as well as plasma or similar body fluids which may contain an anticoagulant. The volume is actually determined by weighing. Due, however, to the manner in which the corpuscles are obtained, the amount of cell material in any given volume is subject to fluctuation and it is not known for cell separation what quantity of the required cell is obtained. Therefore, by extracting large volumes of blood marrow or the like by repeated cell separation, one can be assured that at least a sufficient amount of cells is obtained at one time after separation.
Once the required cell quantity is obtained and suspended in its aqueous carrier, it is further admixed with a freeze protectant and/or stabilizers. Generally, two parts of the cell suspension can be mixed with 0.5 to 1.5 parts of the freeze protectant, such as a 20 to 40 T solution of dimethylsulphoxide (DMSO) in a 60 to 80% amino acid glucose solution. For erythrocytes cells the freeze protectant may be a hydroxyethyl starch. To this there may also be added an anti-coagulant and stabilizer.
A representative suspension may then be for example 10% DMSO, 23% mino acid glucose solution, 35% cell component, 29% plasma and anti-coagulant 3% by weight, based on total weight. The plasma may be body fluid, blood or aqueous carriers. Another representative suspension may be for example 10% polyethyleneglycol, 23% aminoacid glucose solution, 35% cell component, 29% plasma and anticoagulant 3% by weight.
Wi th the use of the freeze protectant, the osmotic load of cells is minimized. Further delay in osmotic reaction can be obtained by placing the cell suspension in a conventional tempering bath.
The collection of such large samples and the suspension thereof in protective solutions, lends itself to the direct filling of large flexible bags, such as those seen in FIG. 2 and generally depicted by the numeral 10, which can be shaped into relatively thin plates having a thickness within the range of 3 mm for approximately 100 ml volume to 10 mm for 240 ml volume. It has been found that in a thickness of between 4 to 10 mm such volumes can be easily and swiftly frozen with almost no damage to the cells. Secondly, the use of flexible bags enables the sample to be more easily handled before freezing, during the freezing process, during thawing, and during the infusion into the patient thereafter; allowing the single bag to be disposed of as a throw-away item after use. High sterility is maintained at relatively low cost. Further, such bags may be hermetically sealed by electric welding or heat welding avoiding problems of leakage inherent in the currently used sheetmetal bottles, sheetmetal plates, sheetmetal containers or other sheetmetal containers which are simply plugged with a stopper. Suitable flexible bags can be formed of polyethylene polyamide, teflon, rapton or polypropylene components which are generally inert to the plasma body fluid and human cell.
For the freezing process itself, the flexible bags are placed in a metal outer tray generally depicted by the numeral 10, as seen in FIG. 2. These trays comprise a bottom and a cover hinged together along one side and having interleafing side edges. These metal trays are highly heat conductive and have the advantage in allowing manipulation of the bags without damage thereto. Furthermore, the metal trays allow the flexible bag to assume an exact shape optimum for the freezing. That is, the flexible bag may be placed with as large a surface area relative to the desired thickness indicated above. Thus, the containers themselves may have a thickness of anywhere between the 4 to 10 mm range. For convenience and greater accuracy in use, a series of containers in the ranges set forth above may be supplied with each freezing unit. The covered trays are preferably formed of copper sheet of approximately 2 mm thickness and may be provided with means for clamping the tray and cover together.
As seen in FIG. 3, the freezing apparatus may be made of an insulated parallelepiped housing 14, having a hollow interior in which is located a central insulated or insulating core 16 defining a continuous oval duct 18; one portion of which defines per se the freezing chamber 20, in which are located open shelves or wall flanges on which the collecting trays 12 may be mounted so as to be spaced one from each other to permit circulation around and about each tray. An inlet 22 for the introduction of the freezing media such as a source of pressurized liquid nitrogen (LN2) extends through the housing wall. A fan 24 and suitable baffles 26 for equalization of the flow of the media are arranged between the inlet and the freezing chamber. A heating unit 28 such as an electric coil, provided with relay switch 29 is located downstream of the freezing chamber. Extending into the freezing chamber is a thermal transducer 30 in the form of a probe capable of sensing with great accuracy the instantaneous temperature of the freezing chamber and converting the same into an electrical signal.
At least one of the freezing trays 32 is supplied with a pair of thermocouples 34 extending through the wall thereof so as to contact the center of the sample in the flexible sample bag held therein. The thermocouples are copper-constant thermocouples having a tip diameter of 0.5 mm and are fixed by a suitable insulating spacer means through the wall of the reference tray. The reference tray is filled with a flexible bag containing a suspension consisting precisely of the components of the cell suspensions to be frozen except for the absence therein of the cell component itself. In place of the cell component, the reference sample contains an isotonic salt solution. Thus, the reference sample may contain 10% DMSO, 23% amino acid glucose solution, 35% isotonic salt solution, 29% plasma, and 3% anti-coagulant or stabilizer. The adjustment consists of variance detector, PI-section, and pulse generator containing the variable pulse-duty factor for the electromagnetic valve. The reference input is generated digitally and then converted into an analog signal. The microprocessor used is a model Z 80 (Zilog, USA) one, in which the user programs are inserted as hard ware. The desired parts of the cooling curve are selected by code switches in a very easy way thereby avoiding errors of the operator.
The basic control for the refrigeration system according to the present invention is shown schematically in FIG. 4. The freezing chamber 18 is supplied through its inlet 22 with a source 38 of liquid nitrogen through a hydraulic or electrically controlled valve 36. The liquid nitrogen is maintained under suitable pressure in its container 38 (schematically illustrated as a Dewar vessel) by a pressure source 40 which may itself comprise nitrogen. The thermocouples 34 from the reference sample 32, and the thermal sensor 30, sensing the temperatre within the freezing chamber itself, are coupled to a measuring device 42, wherein the temperatures are determined and converted into electrical signal outputs. These electrical outputs are fed to a computer control device 44 comprising a memory storage wherein a selected program (cooling rates, freezing levels, etc.) as required for the particular sample are entered and stored. The control system includes a comparator for converting the electrical signal from the thermocouples and thermosensors with the stored program and an analyzer and adequate means for controlling the operation of the supply valve 36 for the liquid nitrogen refrigerant and the operation of the relay 29 for the heating unit 28. The control system also includes a recording device by which the actual cooling curve can be visually demonstrated. It will be obvious to those skilled in the art that they or persons skilled in the art of electric or electronic control systems will be able to fashion together the specific details to form a suitable computer, recorder, input and control means, etc., without any difficulty or problem involved. Micro-processor systems, currently available, may preferably be used.
Before the sequence or cycle of the freezing process begins the desired characteristics of program are entered in the program control by quickly settng the switches of a simple microprocesser system. This data can, if desired, be changed during the freezing process so as to enable the study of variations in the cooling curves as they may effect cell vitality. Additionally, fixed programs can be entered which for example allow the checking of the existing setting data if necessary.
Strict adherence to the cooling curves of the present process are obtainable throughout the sample and not only at its center but also near its edges by employing the platelike containers described hereinbefore and by providing the continuous oval duct and freezing chamber and flow of the air through the freezing chamber. The flow rate of the refrigerant media can be maintained at about 20 m/s. In order to reduce the lamina boundary layer and/or the turbulent boundary layer along the surface and edge of the containers, the edges of the containers may be provided with special detachable shapes and forms providing low profiles and air foils. Although the chamber temperature may fluctuate about a theoretical value on the average of plus minus 0.5� C., this extremely slight fluctuation is not measurable on the sample itself which lies inside the foil itself inside the copper tray container. The resolution of the thermocouple devices is �0.1� C. and such fluctuation within the sample itself is not measurable.
The freezing chambers design is such that the refrigerant circulates in a cyclical or oval path. The construction of the freezing chamber permits the parallel freezing of a plurality of samples which are all pressurized with the flow in the same manner. The homogeneous flow is obtained by additional guide-plates or baffles mounted upstream of the circulating fan. The supply line for the refrigerant positioned downstream of the fan is a further reason for the only slight nominal/actual difference in the fluctuation of the chamber temperature.
The actual temperature of the cooling media in the chamber is basically a function of the LN2 feed (at predetermined pressure), flow rate (fan speed) and the concurrent selective use of the heater. By employing an electric heater, the heater may be turned on and off quickly, and its heatup and cool-time calculated with a high degree of accuracy. Thus, the selective feed of refrigerant, and use of the heater, sometimes concurrently can be made to control the chamber and sample temperature, through the sensor probes in each and the computer responsive control valves and relays.
Within the limits of the bag volume ranges disclosed and of the container thickness disclosed, the present freezing process is not critically affected. This is so because the present invention relates chamber freezing rates with the actual temperature of the samples to be frozen by simultaneous sensing of both chamber and sample temperatures and modifying one relative to the other in response to each.
A second form of container tray may be made from aluminum. Shaped samples of a volume of 100 ml, aluminum container having a wall thickness of the aluminum of 1 mm (k value of the vessel wall=2950 W/m2 � C. The foil bag may have a wall thickness of 0.08 mm. This container may give the cell suspension to be more frozen a thickness of 3 mm.
The process of present invention lends itself to the automatic control by the computer control system previously aforementioned although it is not limited to this specific form. By pre-storing within the computer system those known factors such as the freezing temperature, the plateau temperature, the phase transformation temperature as well as 85% frozen mass temperature relating to the desired curve of freezing for the given sample, as well as the initial and secondary cooling rates for the chamber, the system can thereafter be automatically controlled, without the dependence upon time for any particular step to proceed through each of the successive steps set forth. By the use of control system the data desired can be changed during the freezing process, to enable the study of variations in the cooling curves that may effect cell vitality, and to take into account the possible unknown variations in the suspensions itself. Additionally, fixed programs can be entered to allow the checking of the existing data during the freezing process per se, rather than relying upon the success of automatic control. On the other hand, it will be clear that the use of conventional freezing units may also be made with more manual controls, to carry out the present invention.
The actual freezing of the samples follows the program set forth in FIG. 5 which constitutes the process of the present invention. Samples, generally within the temperature range 0� C. to +32� C., preferably at 0� C., inserted in the foil bag and the tray container well dried on the exterior and interior are mounted within the freezing unit as previously indicated, and initally the temperature of the freezing unit is lowered at a predetermined rate BI upon which the samples to be frozen follow suit in an almost parallel curve, until the freezing temperature TF is reached. Whereupon, the temperature of the freezing chamber is reduced sharply at an increased rate BIa until a level TU is obtained. During this short interval the temperature of the sample dips below the freezing point and then sharply rises back to the freezing level which is in fact the plateau at which phase transformation takes place. The phase transformation is defined at the upper level of the freezing temperature TF and at the lower level by the temperature TP which is of course slightly lower. The temperature of the chamber is thereafter maintained constant at the temperature level TU until the amount of phase transformation in the sample causes the temperature of the sample itself to dip below the lower plateau limit TP. At this time, the heating unit is activated and the level of the temperature of the chamber is caused to sharply rise at a rate BIa to an intermediate level TO ; i.e., intermediate the lower leveled TU and the temperature of the sample at this point. The temperature of the chamber is again maintained for a period of time at the level TO so that removal of the latent heat of phase transformation is almost completed. During this period the temperature of the sample decreases to a point TII equivalent to the condition wherein the mass of the same is no longer a radiant source of heat and where at least 85% of the mass of the sample is presumed in the frozen state. At this point the temperature chamber is further cooled at the pre-determined rate BII passing level TH, until TIIa when virtually all of the remaining mass is considered frozen. The temperature of the chamber is thereafter reduced sharply along curve BIIa until all of the mass is frozen and reaches its lower most limit, (143K). It is noted that the temperature level TO is less than the temperature level TH, preferably by at least one degree. Upon passing the point TH the heater is discontinued. The point TH is of course easily detected by the automatic control which determines the coolent consumption required to keep the chamber temperature at the desired temperature. TH is not a fixed temperature but depends rather on the actual cooling process; therefore, it is detected automatically.
The freezing point of each of the solutions or suspensions will of course be slightly different. These freezing points are well known and can be read from standard tables or can be measured osmometrically. (For example, for a 10% DMSO solution the freezing point is -4.0� C.). The automatic control detects the freezing point of the solution itself by detecting the plateau temperature of the freezing sample. This actual temperature is then used to determine TP precisely. This allows the use of simple thermocouples, which may have a slightly different outputvoltage than theoretically desired, which otherwise would result in misleading temperature measurements. TF is pre-stored in the control system as well, because BIa should start even when the sample supercools. This method gives accurate cooling curves, though supercooling may appear or not and may not be reproducable. In this phase i.e., at the freezing temperature, the sample present is of course in its non-frozen state. The cooling rate BI is also determinable with respect to the given suspension so that an optimum cooling rate favorable for the vitality of the particular cells is obtained. It will be found that, at cooling rate greater or lesser than the optimum, demonstrable damage (thermal shock) occurs, presumably due to transposition within the cellular membrane buildig blocks and the impairment of the meachanism of the active substance transport (K+ Na+--pump). The transition temperature BIa is of course reduced at a rapid rate to minimize the α TU and in order to obtain the desired length of the freezing plateau, which is calcuable for a constant temperature of the chamber (i.e. TU). Rate of -150� C./min or greater are permissable.
The magnitude of the lower freezing limit TU effects heat transfer during the phase change of the sample, i.e., its freezing state, during which latent heat is released. It should be maintained so that a mean migration rate of the ice front within the range of 1.00 to 2. mm/min. is obtained preferably the average of 1.5 mm per minute, corresponding to a temperature plateau lasting 1.6 minutes, has proven particularly advantageous for 4-10mm thick plates containing white blood cells (lymphocytes, granulocytes, stem cells) or bone marrow cells. As for freezing of erythrocytes with HES, the migration rate is highly increased by lowering Tu to 15 to 25 mm/min, preferably to 21 mm/min. The influence of the migration rate has been studied by variation of that parameter without affecting the other parts of the freezing curve (that are BI, BII, BIIa). The migration velocities afore mentioned are optimized values. Deviations from these values resulting in either smaller or larger velocities cause damage to the cell to be frozen. Although the action of this effect on the cells is the subject of research, it can be said that the ice front presumably compresses the cells too much at lower values while at higher values the cells may be mechanically damaged by the growth of sharp ice crystals.
The chamber is heated at rate BI b generally at approximately 50� per minute except in the case of freezing erythrocytes with HES, where BI is given a value of 400� C./min, by connection the heating unit while the infusion of the liquid nitrogen refrigerant is either decreased or shut off completely, until the temperature of the chamber reaches the intermediate temperature TO at which the balance between the heat and the refrigerant maintains this temperature constantly. Since due to the binary or ternary nature of the generally aquueous suspensions, the sample continues to form ice by remaining just below the freezing temperature, the phase transformation plateau is maintained at a constant temperature during this period, the lower limit thereof TP being easily calculableas TP =TF -0.5� C.
The simultaneous lowering of the temperature of the sample during the maintenance of the chamber temperature at TO results in the further concentration of the residual solution. The level of TO at a determined difference from that of the temperature of the sample itself should be as great as the requirement for latent heat removal necessitates and for the maintenance of the desired temperature drop in the sample. It is desirable to shorten the length of these the sample is at the freezing plateau and therefore it is beneficial to optimize heat transfer therein and achieve a corresponding heat transfer during the phase when the chamber is held at TO. This is effected by the accelerated temperature rise of the chamber after passing the phase transformation temperature TP. This optinal in heat transfer results from the required cooling rate BII and the heat transfer coefficient between outer wall of sample and the refrigerant in the interior of the chamber. This coefficient is 110 W/m2 � C., as is seen from measurements on container plates in the freezing chamber. The heat transfer coefficient depends mainly on the evaporation enthalpy of the refrigerant droplets charged in the chamber. Without this evaporation effect the heat transfer is 80% less, as follows from the calculation of the heat transfer coefficient from Nusselt's heat transfer law for smooth plates at the laminar boundary layer for air. The additional latent heat is released to the point where the respective eutectic reaction is obtained (a salt water solution -21.2 C.). The eutectic point of the respective solution gives the minimum possible temperature for the existence of any liquid. Below this temperature, the whole sample is solidified when in thermodynamic equilibrium. Until the sample has reached an intermediate point between the freezing plateau and the eutectic reaction, i.e., the point TIIa, this process plays an important thermal roll in obtaining the advantages of the present invention. Only at this point can the sample be regarded as a frozen body without any inherent heat source and the cooling only takes plate until this point is achieved by stabilizing the chamber at TO. Once the sample passes through this intermediate point TII the chamber can then be cooled at a rate, BII until the sample reaches the TIIa level. The heating unit is turned off when the sample reaches TH. When the sample temperature is reduced to the point TIIa at which most of the cell suspension is frozen, the cooling can be continued at the more rapid rate BIIa until the conclusion of the cycle. The point TIIa has been detected in experiments as to be that temperature, at which BIIa may be used without additional damage in order to shorten the time needed for freezing. In all cases, when TIIa is not known, BIIa should have the same value as BII.
From the freezing point TF until the reaching of the point TIIa wherein the cooling sample reaches about -35� C. considerable changes in the state of the cell and in the original homogenous suspension liquid takes place, which are of utmost importance for the vitality of the cells. After the solidification temperature TF is reached the new phase ice forms, thereby water is removed from the residual solution and the concentration of the liquid takes place. The cell reacts with release of water, which however does not occur at any desired speed but is limited by the membrane permeability of the cells themselves. When freezing more slowly than specified, the cells remain closed to the concentrated solution too long, resulting in destruction of the cells as the shrinkage is too intense and also the proteins are denatured. When cooling faster than specified, water remains in the cell after the point TII has been reached. It forms intracellular ice and thereby destroys the cell from within.
Studies have shown that the effects occur even with minor deviations from the specified cooling rate appropriate for the type of cell. It was surprising to see that in the process of the present invention one can cool several cell species up to 700� /min. The migration of rate of the ice front can be brought (by the action of the length of the temperature plateau) to the optimum value of the cells other than erythrocytes with HES as a cryoprotectant, to 1,5 mm /min. For erythrocytes with HES, the migration velocity is 21 mm/min. Even those types of cells which are normally frozen at higher cooling rates can likewise be frozen in a controlled manner utilizing the present system. Thrombocytes for example are frozen at 30� /min (glycerine glucose method), erythrocytes (HES-meflood) are frozen at 700� C./min and action on the temperature plateau is still possible in this case due to the high maximum cooling rates.
(1) Lymphocytes
A sufficient quantity of lymphocyte cells are collected, and suspended in an aqueous solution of 10% DMSO, 23% amino acid glucose solution 35% cell component, 29% plasma and 3% anti-coagulant stabilizer as previously described or in any known manner, and then filled in flexible bags at about 150 ml volume. These bags are then individually inserted in a copper tray container having an inner thickness of about 5 mm which are then placed in the freezing chamber. Simultaneously, a reference sample is prepared, identical to the suspension but without the cell component and inserted into the reference copper tray. If the obtained volume is for example 80 ml, the same flexible bag may be used as described above. Using a container having an inner thickness of 5 mm doesn't afford any new settings for the freezing process automatic control, neither does the use of a container of smaller inner thickness, i.e. 3 mm. The use of the smaller thickness gives a better filling ratio for a single bag. This example is given in order to demonstrate the universality of the regulatory system, which therefore has a very broad field of applications; as concerning to the different volumes described, all examples may be treated in that manner.
(2) Thrombocytes
A sufficient quantity of thrombocytes are collected, and suspended in an aqueous solution of 5% Glycerine, 4% Glukose, 30% plasma, 5% ACD and 56% aminoacid solution including the thrombocytes in a concentration of 800 to 1200�103 cells per microliter. The suspension is filled in flexible bags at about 100 ml in volume. The bags are individually inserted in a copper tray container having an inner thickness of about 3 mm which are then placed in the freezing chamber. Simultaneously, a reference sample is prepared, identical to the suspension but without the cell component and inserted into the reference copper tray. The freeze protocol is the same as described under (1), lymphocytes, except for the cooling rate, the freezing point and the plateau end, which may be taken from the table below.
(3) Granulocytes
A sufficient quantity of granulocytes cells are collected, and suspended in an aqueous solution of 5% glycerine, 4% glukose, 7% Dextran T10, 23% aminoacid glukose solution, 45% cell component, 23% pLasma and 3% anticoagulant as previously described or in any known manner, and then filled in flexible bags at about 150 ml volume. These bags are then individually inserted in a copper tray container having an inner thickness of about 5 mm which are then placed in the freezing chamber. A reference sample is prepared simultaneously, identical to the suspension but without the cell component. The freeze protocol is the same as described under (1), lymphocytes.
The chamber temperature is initially lowered at rate BI equal to 6� C./min. The sample temperature follows this cooling until the freezing point TF of the solution is reached. The freezing point can be read in tables or measured (osmometrically) and is e.g. for the 10% DMSO solution, etc. of the example -4.0� C. In this phase the sample is present in the non-frozen state. Upon reaching the freezing point TF, the chamber is rapidly cooled at -150�/min. to TU equal to -55� C.
The end of the phase change at constant temperature is recorded when the previously determined temperature TP equal to -4.5� C. is reached. The chamber is now heated at +50�/min by connecting the electric heating unit until it reaches the temperature T0 equal to -13� C., and thereafter maintaining this temperature constant until the sample has reached -12� C. (TII).
Upon the passing of the sample through TII, the chamber is cooled further at the rate BII equal to 2� C./min until the chamber reaches TIIa equal to about -35� C. At the intermediate point the TH the heating unit is turned off. This occurs automatically by determination of the required cooling power to remove the heat produced by the heater after TO has been reached. At -35� C. the cooling is continued at a decreasing rate equal to 10� C./min to -130� C. This concludes the controlled freezing process.
__________________________________________________________________________ Cooling Cooling Freezing Plateau StartCell type rate rate point end BII:Example BI BII TF TP TII TU TO__________________________________________________________________________(2) Granulocyte.sup.(a) -6� C./min -2-3� C./min -4� C. -4.5� C. -12� C. -55� C. -13� C.(3) Bone marrow.sup.(a) -2� C./min -1� C./min -4� C. -4.5� C. -12� C. -55� C. -13� C.(4) Thrombocyte.sup.(b) -30� C./min -30� C./min -2� C. -2.5� C. -20� C. -65� C. -25� C.(5) Erythrocyte -700� C./min -700� C./min -2� C. -2.5� C. -20� C. -130� C. -90� C.(6) Medullar.sup.(a) -2� C./min -1� C./M -4� C. -4.5� C. -12� C. -55� C. -13� C.(7) Thrombocyte.sup.(c) -6� C./min -2-3� C./min -1.5� C. -2.0� C. -10� C. -50� C. -11� C.(8) Lymphocyte.sup.(d) -4� C./min -4-6� C./min -2.5� C. -3.0� C. -13� C. -55� C. -14� C.(9) Granulocyte.sup.(e) -6� C./min -2-3� C. -2.5� C. -3.0� C. -12� C. -55� C. -13� C.__________________________________________________________________________ .sup.(a) DMSO process (10% in the freezing solution) .sup.(b) Glycerineglucose process .sup.(c) DMSO process (4% in the freezing suspension) .sup.(d) Polyethylene process (10% in the freezing suspension) .sup.(e) Glycerineglukose-dextran process (5% Glycerine, 4% Glukose, 7% Dextran T10 in the freezing suspension)
The foregoing table set forth additional examples utilizing the steps previously set forth with the given parameters.
The cell suspension frozen according to the examples were stored under cryogenic storage conditions for a lengthy period of time. Upon thawing, at conventional time/temperature parameters and gradual dilution and elutriation, the cell suspension ready for transfusion contains an amount of at least 20% compatible plasma or autologous plasma. Retransfusion of autologous lymphocytes stem cells, and thrombocytes after freezing by the described process were carried out in 24 cases, after these cancer patients had been treated by chemotherapy. No problems due to the cryopreservation of cells arose during or after transfusion.
The concentration of viable cells was determined by known in-vitro tests, including fluorescence, hyper blue exclusion specific culture tests, detection of the released cell content and counting or hematocit determinations to be as follows:
______________________________________ total recoveryExample % of living cells � stand. dev. % � stand dev.______________________________________(1) 87.6 � 9.3 83.4 � 7.3(2)(3)(4) 88.5 � 10.6 84.2 � 8.13(5) 98.7 � 2.3 75.4 � 11.2(6) 119.9 � 44.3 98.3 � 32.5______________________________________
While the present invention finds obvious advantage in utilizing the flat tray-like containers, the process may be satisfactorily used for the freezing of cylindrical samples. The cells for such purposes may as for test purposes, be suspended in tubes holding a volume of 2 ml or more. The freezing solution is the same as that described earlier and there may be however only 1�107 cells per sample. A reference sample in the form of a tube similarly frozen, filled and provided with a thermal couple is also employed. Although more extended cylindrical samples can be similarly cooled by the present process a sample layer thickness in the main heat releasing direction in excess of 10 mm results in a diminished cell quality value. For this reason therefore the use of large cylindrical metal bottles holding 600 ml or more, commonly used in the preservation or red blood cells, is not suitable for the more complicated freezing of other cell components.
It will be seen from the foregoing, that the process by monitoring the temperature of both the ambient chambers, and the temperature of the samples permits the use of a flexible control system, by which the advantages of the present process are obtained, without dependence upon predetermined time limit. The measured sample temperature is used for controlling the various phases of the chamber cooling. Another important aspect is the use of compensatory heating, with which the heat-up characteristic of the chamber is adjusted as needed. This results in the following advantages:
Selection of the duration of the plateau temperature to the optimum value given by sample geometry and refrigerant temperature;
Rapid determination of the cooling curve optimal with respect to recovery for a cell type/protectant combination;
Variation of the cooling rate above and below the freezing point separately and independently of the duration of the plateau temperature;
Possible changes of single sample mass as well as number of samples without change of program;
Collection of large samples and the elimination of division or filling up of the cell containers;
Optimum definition of the obtained cells fro the freezing process;
Increased assurance that the samples are sterile.
Various modifications, changes and embodiments have been shown and referred to in the foregoing disclosure. Others will be obvious to those skilled in this art. Accordingly, it is intended that the foregoing disclosure be taken as illustrated only and not as limiting the scope of the present invention.
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Under the direction of new offensive line coach Joe D'Alessandris, the San Diego Chargers turned a weakness into a strength last season.
The Chargers struggled to protect quarterback Philip Rivers in 2012. But with a switch to a short passing game, Rivers was sacked just 30 times last season. San Diego's offensive line also paved the way for a career-high 1,255 rushing yards for Ryan Mathews.
Rookie D.J. Fluker proved ready to compete at the next level. Free-agent additions King Dunlap and Chad Rinehart performed better than expected.
And even with six different starting offensive line combinations, San Diego still played consistent up front. But center Nick Hardwick turns 33 in September, and right guard Jeromey Clary is 30, so the Chargers need a long-term answer at interior offensive line.
San Diego also could use some more depth at tackle, with Dunlap suffering through concussion issues last season. So it would not be a surprise if San Diego looks to take an offensive lineman early in this year's draft.
Best OL drafted since 2004: Selected in the third round of the 2009 draft, Louis Vasquez was one of the strongest lineman in his draft class, starting 54 games in four seasons for San Diego. Vasquez signed a lucrative, multi-year deal with the Denver Broncos last year, did not give up a sack and earned his first Pro Bowl invitation.
Worst OL drafted since 2004: Selected in the fifth round of the 2005 draft, Wesley Britt was released during final roster cuts his rookie year, and signed with New England's practice squad. The University of Alabama product played 16 career games in three years with the Patriots, including two starts.
OL depth chart: Starters – LT King Dunlap ($1.75 million in total compensation in 2014), LG Chad Rinehart ($2.75 million), C Nick Hardwick ($4.4 million), RG Jeromey Clary ($4.55 million), RT D.J. Fluker ($923,295). Reserves – C/G Rich Ohrnberger ($795,000), C Nick McDonald ($662,750), T Willie Smith ($645,000), T Mike Harris ($570,000), T Nick Becton ($495,000), T Kenny Wiggins ($495,000) and G Johnnie Troutman ($495,000).
A review of offensive linemen San Diego might select in each round of this year's draft:
First round, No. 25 pick: Xavier Su'a-Filo, 6-foot-4, 307 pounds, UCLA
Eric's rationale: He's versatile, fits San Diego's scheme and plays with a mean streak.
Second round, No. 57 pick: Joel Bitonio, 6-4, 302, Nevada
Eric's rationale: An offensive tackle in college, but projects to guard in the NFL.
Third round, No. 89 pick: Gabe Jackson, 6-3, 336, Mississippi State
Eric's rationale: Big, physical and a road grader in the running game.
Fourth round, No. 125 pick: David Yankey, 6-6, 315, Stanford
Eric's rationale: A good athlete who could pick things up quickly in terms of scheme.
Fifth round, No. 165 pick: Brandon Thomas, 6-3, 317, Clemson
Eric's rationale: Projected to go higher, but suffered a torn ACL during a workout.
Sixth round, No. 201 pick: Garrett Scott, 6-5, 307, Marshall
Eric's rationale: A developmental project that could fill in depth at tackle.
Seventh round, No. 240 pick: Brian Clarke, 6-2, 300, Bloomsburg
Eric's rationale: Good athlete and strong run blocker.


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The Five Least Laborious Foods To Make For a Labor Day Party (Videos)
Throw a labor day party without the labor with these simple recipes.
Rachael Ray Magazine
Yesterday, Clean Plate Charlie shared five easy recipes for tasty libations without the labor. We realized that at some point, you're going to need food to go with those drinks.
Lucky for us, celebrity chefs have simplified cooking to the point where it seems that you could make an entire four-course meal in about seven minutes.
From appetizer to dessert, we're bringing you a collection of the easiest dishes to make for your Labor Day weekend party so you can spend more time eating and drinking, and less time laboring!
1. Peppered Deviled Eggs - Alton Brown shows us an insanely simple way to make peppered deviled eggs. Here's a hint - grind pepper in the coffee grinder!
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4. Grilled Sweet Potato Wedges - If you're going to make burgers, you need something to go with them. Try these grilled sweet potato wedges instead of fries for a healthier alternative.
5. Patriotic Fruit Cups - Rachael Ray is the queen of super duper easy. These berry cups may be a no-brainer, but they're fresh, light and the red, white and blue colors fit in perfectly with a Labor Day party theme.
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This video shows you how to make seared bay scallops in a simple sherry cream sauce. It's very fast and easy—it's ready in about the time it takes your pasta to be ready! You'll sear the bay scallops on high, and then add butter, minced garlic, lemon zest, red pepper flakes. Give the pan a quick deglaze of dry sherry (or white wine) and then finish things off with a healthy splash of cold heavy cream and chopped parsley. And top it off with freshly grated Parmesan cheese. It's incredibly easy and just as delicious. Get the recipe for Creamy Bay Scallops with Spaghetti.
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- September 30, 2016 Manning Music Series Concert: IBMA Bluegrass Festival FREE CONCERT ON THE LAWN William Peace University invites you to a FREE bluegrass concert on Friday, Sept. 30, 2016 at 4 p.m., featuring The Counterclockwise Stage Band (4 p.m.) and the South Carolina Broadcasters (5 p.m.) on our Main Stage, as part of the World of Bluegrass (International Bluegrass Music Association) festival in Raleigh. This free concert kicks off our 5-concert Manning Music Series and the celebration of the Series' 10th Anniversary. The concert will be performed on the Main Lawn. Free parking in any of our University lots. Registration is required. REGISTER HERE. Bring a picnic, your lawn chairs, or a blanket and enjoy this free bluegrass concert made possible through the Manning Music Series Endowment Fund! Refreshments available. (NOTE: Alcohol is prohibited.) #IBMA #WPU #MANNING10 William Peace University wishes to make every event accessible to all people in accordance with the Americans with Disabilities Act. If you need a reasonable accommodation to fully participate in this event please contact the Office of University Advancement at 919.508.2680. Please make your needs known as soon as possible to allow sufficient time for effective accommodations, preferably at least five business days prior to the event view Event
- October 15, 2016 Open House Our Office of Admissions would like to invite you and your family to take part in one of our Open House Events. This will give you the opportunity to meet students and faculty, tour our beautiful campus, and learn about the excellent education opportunities available at William Peace University. No matter your level of interest, WPU has something for you! After hanging out with us, be sure to tour #OurRaleigh and take advnatage of our amazing downtown location. Grab lunch or coffee over at Seaboard Station, or pop over to the North Carolina Museum of History or the Governor's Mansion. To RSVP for Open House, please click here. view Event
- October 28, 2016 WPU Golf Tournament 2016 Tee It High and Let It Fly! Join local and regional golfers at William Peace University's fall golf tournament on October 28, 2016. Proceeds benefit WPU's Pacer Club (Athletics Dept.). REGISTER HERE Hedingham Golf Club 12:00 p.m. Check-In 1:00 p.m. Shotgun Start $125 entry fee/$400 per team. Tee Sponsorships $100 per hole Tickets include: Cart and Greens fees Range Balls Dinner Two drink tickets Individual awards Team prizes Hole In One Contest Closest to the Pin Contest Become a Tee Sponsor Tee Sponsorship - $100 Click here and then enter "New Gift" designated to "The Loyalty Fund" restricted to "tee sponsorship" and in the memo line enter your business name. If you would like to send your logo to us after you pay securely online, send it to firstname.lastname@example.com. Please send a high res EPS or JPG. Questions? Contact Coach Charlie Dobbins at email@example.com or 919-508-2330 or Coach Chris Duty at firstname.lastname@example.com or 919-508-2331. view Event
William Peace University's Lynn Owens, Ph.D., Named A World Journalism Education Congress FinalistBy William Peace University on August 6, 2013 1:44 pm
William Peace University's Lynn Owens, Ph.D., Named A World Journalism Education Congress Finalist
Associate Professor Of Communication For Four-Year Baccalaureate University Co Authors Research Paper That Garners International Recognition At World Journalism Education Council Conference
RALEIGH, N.C. – William Peace University (http://www.peace.edu), a private four-year university located in downtown Raleigh, has announced that Lynn C. Owens, Ph.D., co-authored a research paper that recently received international recognition by the World Journal Education Congress. Dr. Owens, who serves as the university's associate professor of communication and communication department chair, co-authored the research paper with Deb H. Wenger, associate professor of the University of Mississippi. Their paper on "The Evolution of Journalism Skills Required by Top U.S. News Companies" won third place at the World Journalism Education Council Conference. Among more than 100 submissions, Owens and Wenger's paper was the only U.S. paper to place, and the two professors are now in the process of publishing the document.
Through their research, Owens and Wenger explored the growing skills needed for employment across the journalism profession and what is most critical in delivering appropriate journalism instruction. By utilizing content analysis from the same three-month period in 2008, 2009, 2010 and 2012, the research evaluated all employment opportunities posted by the top 10 newspaper and broadcast companies across the United States. The results indicated a significant increase in the desire for mobile and social media skills as well as an increased emphasis on multimedia capabilities. For educators, the research paper argues the need to create more methodical instructional approaches in an effort to enhance technology-related skills that directly impact the journalism industry overall.
"We cannot ignore the fact that every industry around us is continuing to grow, expand and improve itself at a rapid pace, which means that future employees will need to be better equipped with the necessary skills to serve as effective industry leaders and professionals," said Debra M. Townsley, Ph.D., president of William Peace University. "That is why we are so proud to have professors like Lynn Owens as a member of our faculty to guide and direct education as it continues to evolve. Lynn's research is outstanding, and we look forward to having her work published and shared with the general public, so that other institutions can begin to reevaluate whether they are teaching the skills that are most imperative to today's journalism students."
"As educators, it critically important that we understand the changes that face our industry and revise and modify our methods of instruction to ensure that students graduate and can fully contribute to the needs of future employers," said Owens. "Our research uncovers the changes in the field of journalism and the growing skills that are needed now and in the future. We are honored that the World Journalism Education Council recognized our findings, and we look forward to implementing these skills within our own institutions and those around us over the coming years."


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2013 Audi A5 Convertible 2.0T Prestige quattro 2dr Convertible AWD (2.0L 4cyl Turbo 8A)
Amazing car, luxury, comfort, and great looks-pleased for 3 yrs and NO issues at all
2013 Audi A5 Convertible 2.0T Premium Plus quattro 2dr Convertible AWD (2.0L 4cyl Turbo 8A)
My 2013 Audi A5 has been a delight to own. The car is incredibly functional with all wheel drive, comfortable interior, great looks and good fuel economy. The convertible works effortlessly, and it is incredibly quiet with the top up, as well as warm during the winter. The attention to detail is exceptional with high quality materials and options that will impress most drivers. The car is very competent on the road at high speeds, but does equally well as a relaxed touring car at slower speeds with the top down. I never thought I would ever enjoy owning a car like this, especially driving it year-round! I picked mine up used with very low miles, and for the price it was just too good to pass up. It still has the balance of the factory warranty, and the dealer has been great so far.A few highlights: The Bang and Olufsen stereo will not disappoint, even with the top down. The MMI Nav system has an incredible amount of features, but it does take time to master, so keep your owner's manual close. The heated seats coupled with the neck warmers and wind deflector will keep you cozy with the top down even on very cold days. The Quattro system is incredibly competent on slippery roads. This is just such a complete package in a convertible that's designed to be used as a daily driver.Audi's quality seems to have increased significantly in recent years, so hopefully the car will stand the test of time. So far I have had great luck with the dealer for servicing. Seems as though Audi is trying very hard to compete successfully against the other luxury brands.Update on 7/19/16: Been putting a lot of miles on the A5, and now at 21,000. It continues to be a great car to own. I did need to replace the tires though because of an alignment issue which caused the Pirellis to make a lot of noise. Other than that, it has been pretty much a trouble free ride with up to 30 mpg on the highway. It's an easy car to live with on a daily basis.
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Established in 1986, we specialize in high-quality footwear for women, men and children alike. Our wide variety of comfort, fashion and accessories will make even your most challenging gift recipient ecstatic!
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Mark Berry, Acting CEO of Phoenix Rising, presents the Board of Directors' open letter to Queen Mary University of London (QMUL) urging them to release the PACE trial data, and hopes that other non-UK organisations will join British charities in the same request...
Discuss the article on the Forums.
Anybody tried or are on Amantadine?
Discussion in 'Antivirals, Antibiotics and Immune Modulators' started by Dr.Patient, Jul 19, 2014.


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WebRTC Signaling Protocols and WebRTC Transport Protocols are two separate things. Here's what to make of them.Read More
What if WebRTC SDP Munging was Prohibited?
SDP munging is on the standards table with an aim to prohibit it. What's SDP munging, why is it bad and what happens if and when it is gone?Read More
Scalability, VP9, and what it means for WebRTC
There are various ways to achieve video scalability in WebRTC, and VP9 is going to add some intereting new opportunities in this regards. Learn what simulcast and SVC means and how they fit into the bigger picture in WebRTC.Read More
Standards are for Losers
When looking at a standard, make sure to check who is behind it and who is missing in action. It usually tells you the power play that is taking place. In the case of WebRTC, it all boils down to the change in hegemony over VoIP from vendors to browsers.Read More
The Future of WebRTC Live Broadcast
WebRTC live broadcast is getting more traction, and with it, there needs to be a change in the architecture of how we build a winning infrastructure.Read More
Get Over it: WebRTC isn't Peer-to-Peer
Is WebRTC P2P or not? That's something that is easy to resolve – it both is and isn't, and if you miss that, you are missing a lot of what WebRTC means.Read More
WebRTC Mobile to Web? Make Sure You Think at Web Speeds
Getting WebRTC mobile to work properly in a use case that requires browsers as well has its own set of challenges.Read More
4 Good Reasons for Using HTTP/2
There are some pretty good reasons to adopting HTTP/2 in your website – and it isn't only about speeds.Read More
Do we Care about ORTC on Edge?
Microsoft just announced support for ORTC in both Edge and Skype. Here are a few of my thoughts about this move and how it relates to WebRTC.Read More


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GarageDesigning and installing quality custom garage cabinets and wall mounted storage systems is what we do. All day, every day. Garage cabinets are mounted off the floor to prevent water damage and provide easy cleaning in your garage area. The fully integrated backing provides stability and helps keep cabinets free from dirt and dust. Self-closing, concealed hinges are standard with all of our garage products.
Garage OrganizationWhether you need more storage space or you desire a full-blown man cave, our garage organization experts will work with you to transform your garage into a functional and stylish space that reflects your unique lifestyle.
ClosetFrom modern to traditional, basic to elaborate, we will design spaces for you that meet your unique style, budget and storage needs. Whether you need a closet, pantry, home office, entertainment center or other space our custom made cabinetry is made of the highest quality products that are guaranteed for life. Organizing your life is just that simple. And we guarantee our product for life.
Closet OrganizationOur cabinets have a 3/4″ thermofused melamine finish, which resists scratches and stands up to heat, moisture, stains and direct more effectively than other products. All cabinet doors come standard with soft-close hinges.
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Office OrganizationWhatever the situation, you need a customized solution that is both functional and comfortable to make it your own special place.
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Unique Space OrganizationWhen it comes to organizing your unique spaces, we offer many options. To see what would work best for your home, request a complimentary in-home design estimate.
Great Spaces For All Your Places provides high quality custom cabinetry, garage flooring and organization systems for the home. Whether it is a garage, closet, pantry, home office, laundry room or other storage area, our professional design consultants will create a space that fits your unique style and budget.
Meet The Team
David is a US Army Veteran who served his country for 22 years and has recently returned to the Army Reserves. His active duty years included three deployments and 10 years as a US Army Recruiter. He is currently in training with the Las Vegas Metropolitan Police Department and will be an LVMP officer in late 2015.
Prior to co-founding Great Spaces, Melanie was an account manager for Henriksen Butler Design Group Nevada where she worked on Commercial Interior projects. Previously, she spent 25 years in Commercial Real Estate Brokerage and interior design for new RE Developments. Melanie serves as the primary point of contact for customers and manages the day to day operations of the company.
Patrick is a graduate of Oklahoma State University with a degree in Entreprenural Studies. While in school he was a restaurant manager in Stillwater, Oklahoma. With management and customer service experience and extensive installation training he directs and supervises every aspect of project completion and ensures customer satisfaction.


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Churchill: The Greatest Briton Unmasked: The Greatest Briton Unmasked"I have not always been wrong. History will bear me out, particularly as I shall write that history myself."—Winston ChurchillIn the popular imagination, Winston Churchill is the greatest Briton. He was the lone voice that warned of the threat from Nazi Germany and the steady and defiant hand that guided Britain through its darkest days to ultimate victory in World War II.In this new
Churchill the Greatest Briton Unmasked"I have not always been wrong. History will bear me out, particularly as I shall write that history myself."--Winston Churchill In the popular imagination, Winston Churchill is the greatest Briton. He was the lone voice that warned of the threat from Nazi Germany and the steady and defiant hand that guided Britain through its darkest days to ultimate victory in World War II. In this new
How to make your car look great and how to sell it fastA quick sale and a decent price ... are they what you want when selling your car? If so, following this guide should increase your chances of achieving that most satisfying of sales one where you make good, honest money. Even if you are not planning to sell, this book shows you how to make your car your treasured pride and glory again using simple techniques and expert skills developed over more
World War One: History in an HourLove history? Know your stuff with History in an Hour. The 'Great War', from July 1914 to November 1918, was without parallel. It brought to an end four dynasties, ignited revolution, and forged new nations. It introduced killing on an unprecedented scale, costing an estimated nine million lives. It was the war that destroyed any notion of romance or chivalry in battle; it pulled in combatants
How to Draw from Photographs: Learn how to make your drawings "picture perfect"
Learn how to make your drawings "picture perfect"Photographs are valuable reference tools that most artists use. Complex subjects are generally easier to draw from photos-plus other factors, such as weather, movement, and lighting, can make drawing from life nearly impossible. Children and animals, for example, rarely sit still when you want them to, making it much easier to draw them from photographs. However, many beginning artists aren't
The Cold War: History in an HourLove history? Know your stuff with History in an Hour. From the end of World War Two to the collapse of the Soviet Union in 1991, Russia and America eyed each other with suspicion and hostility as the world lived in the shadow of the Cold War. As post-war Europe was rebuilt, Churchill, Roosevelt and Stalin struggled to maintain peace among the former Eastern and Western Allies. Two ideologies, two
$15.99Perfect for the aspiring artist! Covers basic techniques and guides reader to finished projectsIncludes an array of inspirational artworkCompetitive pricing, authoritative advice and a popular subject make this title a must-have for aspiring artists. Perfect for beginners and leisure artists, this book guides the reader from the most basic shapes and objects through to fully developed and varied
Winston Spencer Churchill: Defender of the Realm, 1940-1965Spanning the years of 1940-1965, THE LAST LION picks up shortly after Winston Churchill became Prime Minister-when his tiny island nation stood alone against the overwhelming might of Nazi Germany. The Churchill conjured up by William Manchester and Paul Reid is a man of indomitable courage, lightning fast intellect, and an irresistible will to action. THE LAST LION brilliantly recounts how
Dogs & Cats / Drawing: Learn to Draw Step by StepWhether they prefer the company of dogs, cats, or both, aspiring artists will learn how to create faithful depictions of all their furry friends with this essential drawing book. Inside, gifted artist Nolon Stacey provides fundamental techniques and complete instructions for drawing several different breeds of cats, kittens, dogs, and puppies. He also demonstrates how to portray specific canine
The Storm of War: A New History of the Second World War
A New History of the Second World WarFrom "Britain's finest military historian" (The Economist) comes a magisterial new history of World War II and the flawed axis strategy that led to their defeat. The Second World War lasted for 2,174 days, cost $1.5 trillion, and claimed the lives of more than 50 million people. What were the factors that affected the war's outcome? Why did the Axis lose? And could they, with a different
Why do My Skin Tones Look Lifeless? Plus 25 Solutions to Other Portrait Painting Peeves: Tips and Techniques on Oil Painting Portraits, Mixing Skin Colours, Eyes, Hair and MoreA troubleshooting guide for portraiture.Portraiture is often seen as the last frontier of representational art as every detail has huge consequences upon whether a portrait looks like the person depicted. The beginner need not venture far before encountering a possible minefield of problems. Common issues might be why a portrait painting looks childish, eyes look like marbles, hair looks like a
A Practical, Common-Sense Guide to Residential ConstructionIn How to Build a House, author and professional engineer George Michael Rentz, PE is informative and entertaining while taking the mystery out of residential construction. With more than thirty-five years in the construction industry, Rentz provides an overview of the information necessary when you are considering buying or building a new home. From the basics of site selection and design to cost


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My grandmother is in town from Colorado for 10 days. Today she stepped out of the car wrong and twisted her ankle. She doesn't want to go to the doctor - but she's having a hard time getting around on her foot. It's not swollen - but she really seems to be in pain. I'm trying to find a place that rents wheelchairs - that's open on the weekend. Does anyone know where I could get a wheelchair for a few days without waiting until Monday?
Thank You!!


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LSR Performance Billet Toe Links - 2010-2011 Camaro
Billet rear toe link for Camaro SS and V6 models. Crafted from billet 6061 aluminum for strength and beauty. These replace the factory, stamped steel, toe links and provide decreased unsprung weight and increased stiffness to improve suspension response.They also include significantly firmer bushings to reduce suspension compliance, and prevent excessive toe deflection upon cornering, braking, and acceleration. Designed using Finite Element Analysis to optimize strength to weight. Easy to install and require no permanent modifications to the car.
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Quote Of The Day: Pink's Bittersweet Success
"It feels very confusing. It's bittersweet. It's funny, it's empowering, it's 'Screw you,' it's all of that. It's amazing to be number one but it's ironic that it took [Carey Hart and I] breaking-up to get there. It's a testament to how often people break-up, because everyone can relate to what I'm saying. – Pink,… More »


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Which mobile network offers the best range of phones?
Call charges, price plans and services are all very well and good but most people are still attracted to mobile phone networks because of a particular deal on a particular handset. Things have evened out a little since the iPhone hit six out of the seven UK operators this time around, but there's still plenty of exclusive handsets in the network stores and it's worth knowing who's more likely to have the goods when your contract is near an end and you're in a good position to ask for a hardware upgrade.
We looked at both the PAYG phones and pay monthly phones on offer at the time of writing on all the UK networks. We ignored colour variations and only considered the number of different models and the number of brands each network represented.
The table above shows our findings. What we then did was look at the quality of the handsets themselves and, after thorough judging, this was the way we lined them up. So, as far as the actual phones go, these are the best UK networks to be with.
1st - O2
Not only did O2 have the largest number of different models in stock but the company scored very highly in terms of variety of brand as well. All the top phones of the moment are available with perhaps the only omission the popular HTC Legend or even the Hero. On top of that, O2 is the only network still to feature the webOS Palm phones and is the first to stock the Dell Streak in the same way as it was the first to stock a certain Apple handset too.
You rather get the feeling that you can rely on this network to have the latest and greatest as well as an impressive selection of cheaper feature phones as well.
2nd - Vodafone
It seems to have taken a while for Vodafone to catch on that you need a little handset sex appeal to help get the flag of quality service noticed, but the arrival of the iPhone and a few others seems to have levelled the playing field once more.
All the top Android phones are represented with even an exclusive on the Nexus One, and the sheer number of feature phones make an excellent range for the the whole market to pick from. We could have done with an appearance from a BlackBerry Storm or a Palm phone but certainly a solid choice here that gives you some confidence for the future.
3rd - 3
Despite the relatively small number of models on offer, 3 has done an excellent job of picking the right ones to stock. There's only one BlackBerry phone on the books but it's the best one; not a world of Nokias but the E72, 6700 and X6 are all represented.
The Samsung section has probably been rather too well pruned, but that's made up for by the Skypephone and INQ exclusives that you'll only get from this network. They're not for everyone but both are uniquely useful handsets not to be sniffed at by the premium smartphones doing the rounds.
4th - T-Mobile
While not providing such a range of phones and brands as you might expect, T-Mobile has done an excellent job of axing the poxy ones or at least just keeping them in the PAYG section. There's a good selection of BlackBerrys, including the Storm 2, the HTC Desire and Wildfire, and a really well chosen small set of quality Samsung smartphones as well as the big players you'd want from Nokia and Sony Ericsson.
This lot might not be your first port of call if you're all about the hardware but you can remain nice and confident that the company won't be pushing rubbish on you when it's time to upgrade. After all, 18 months is a long time and, in case it's escaped your attention, 24 months is even longer.
5th - Orange
Orange might feel a little hard done by to have wound up in fifth with such a good turn out in terms of numbers. There's a good glut of feature phones in the store and, although most of the more significant smartphones are all present and correct, it would have been nice to see something that no one else has got. The Motorola DEXT being pushed cut price does not count.
6th - Virgin Mobile
Not stocking the iPhone would be a brave decision but it's probably about not being able to afford it rather then anything else. In truth, VM seems to be going for a different end of the market to that of Apple charges. Instead, it's largely about feature phones with the odd turn up like a BlackBerry or two and the HTC Desire as well.
Virgin Mobile isn't going to knock your socks off with handsets, but you can at least appreciate that they know a good one when they see it. The current run of V-Festival inspired limited editions phones is worth a commendable mention.
7th - Tesco Mobile
So, there's the iPhone and, um, yeah.
Vodafone UK is the smart choice for customers who want to make the most of smartphones, according to the latest pieces of research from YouGov.
YouGov's Technology & Telecoms Consulting team's latest survey, Smartphone, Mobile Internet, eXperience (SMIX), positionsVodafone UK as the top operator for mobile internet, coming top in 8 out of 10 areas and beating its rivals in the most important measure of quality – reliability during the day.
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Photo Special10 Best Celeb Quotes This Week
The secret to puckering up to Matt Damon in Candelabra? Michael Douglas tells all. Plus, more from Kim Kardashian, Amanda Seyfried and other stars
Credit: Claudette Barius/HBO
Updated: Friday May 24, 2013 | 06:00 AM EDT By: Karen J. Quan
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- Created 3/4/16 by Pete
You've heard me say it before and I'm sure you will it again, many times, from me, but great food does not always have to be complicated. Oftentimes all that great food requires is starting with a great product and using good technique. A perfect example of this is a very simple and straightforward roast chicken. With just a few, simple ingredients and good technique one can turn the ordinary into something sublime. Just ask most chefs and they will tell you that a perfectly roasted chicken is a thing of beauty and one of their favorite dishes. This recipe kind... read more


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