text:
|Publication number
|Publication type
|Application number
|Publication date
|Sep 8, 1992
|Filing date
|Jun 4, 1990
|Priority date
|Jun 4, 1990
|Fee status
|Also published as
|Publication number
|Ross A. Tubo, Susan F. Schaeffer, Alexander Schermer, Richard Odessey
|Original Assignee
|Biosurface Technology, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (5), Non-Patent Citations (23), Referenced by (51), Classifications (10), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to cryopreservation and long-term storage of cultured epithelial tissue sheets useful as skin wound dressings in a manner which maintains cell viability and colony-forming efficiency.
It has been a priority in the medical community to develop a skin wound dressing which will encourage new cell growth while preventing fluid loss and infection following skin wounds from burns, ulceration, or surgical excision. Since traditional dressings fail to protect large-scale wounds adequately, several alternatives have been developed. Among these alternatives are split- and full-thickness grafts of cadaver skin, porcine skin, and human allografts and autografts. Most have proved unsatisfactory since all but autografts eventually are rejected by the body in the absence of immunosuppressive therapy. In addition, use of conventional autografting techniques is not practical for massive burn injury involving large body surface areas.
Green et al. have developed a method of culturing epithelial cell sheets several cells thick for repairing burns, ulcerations and other skin wounds. U.S. Pat. No. 4,016,036 discloses the method for serially culturing keratinocytes to produce stratified sheets of epithelium. U.S. Pat. No. 4,304,866 discloses the method of producing transplantable cell sheets by culturing keratinocytes and detaching the sheet from its anchorage substrate using an enzyme such as dispase. U.S. Pat. No. 4,456,687 discloses agents useful to promote growth of epithelial cells. The disclosures of these patents are incorporated herein by reference. In the culture system developed by Green et al., epithelial cells divide rapidly on the surface of tissue culture dishes or flasks, and ultimately form a confluent, modestly stratified sheet of tightly interconnected cells. These confluent cultures can be released as a cohesive cell sheets by treatment, for example, with the enzyme dispase (see U.S. Pat. No. 4,304,866). The cultured sheets can then be attached to petrolatum impregnated gauze or other non adherent backing, transported in culture medium to the operating room, and applied to the patient.
Large burn surface areas can be covered with autograft materials prepared by these methods, but the autografts require time to culture. While the cells for autografting are being cultured, it is possible to maintain the wound with allograft material which is effective as a temporary wound dressing. Allograft material also promotes healing of chronic skin ulcers and split-thickness graft donor sites. Cultured autograft material prepared by the method of Green, et al. are now available commercially from Biosurface Technology, Inc., of Cambridge, Mass. Allograft material is available for experimentation and clinical testing.
A severe, very practical limitation on the use of cultured epithelial grafts is their inherent limited shelf-life. The viability and colony-forming efficiency of the sheets falls rapidly after they are removed from the substrate on which they grow. This restricts the time and distance the sheets may be shipped from production facility to the operating room. The cell sheets are extraordinarily fragile. They are normally able to maintain their ability to resume growth and to form colonies when applied to wounds only for about six to eight hours or less after dispase release. These temporal limitations preclude maintenance of a large inventory. Development of a cryopreservation method for extending the storage interval of the cultured sheets would permit maintenance of large inventories for shipment throughout the world.
The art is replete with descriptions of various tissue preservation methods including cryopreservation, use of special cell media, and certain packaging techniques. Cryopreservation allows for long-term storage by freezing the material in the presence of a cryoprotective agent. This agent displaces the aqueous material in and around the cells and thereby prevents ice crystals from forming. Numerous disclosed protocols vary the nature or amount of cryoprotective agent, and/or the time, course, or the temperature of the freezing process in an attempt to retain cell viability after a freeze/thaw cycle. See, for example, U.S. Pat. No. 4,559,298, U.S. Pat. No. 4,688,387.
Storing tissue by means of cryopreservation is a complicated and expensive process, capable of yielding highly variable results. However, no other approach has been shown to extend storage viability of animal tissue beyond very short periods, i.e., eight hours. See, for example, Pittelkow et al., 86 J. Invest. Dermatol. 4: 410-17, 413-14 (1986). For years skin banks have used frozen, human skin on burns as temporary allograft coverage. However, this frozen skin is not truly viable. Although banked skin is metabolically active, it is uncertain whether the epidermal cells can reproduce themselves. Heimbach, D., et al., "Artificial Dermis for Major Burns: a Multi-Center Randomized Clinical Trial", Ann. Surg. 313-320 (September 1988).
Large scale production of cultured epithelial autografts, as described by Green et al, promotes permanent coverage of large surface area wounds with the patients own skin. Although large amounts of cultured epithelium can be produced for patients, the limited shelf-life of the epithelium is a major concern. To this end, Cancedda and DeLuca have developed a protocol whereby confluent sheets of cultured keratinocytes are frozen in culture medium containing 10% glycerol (see EP 0 296 475). However, experience with this procedure indicates that cell recovery is variable and generally very low. In addition, the narrow time ranges of incubation makes this method impractical in large scale production. Moreover, grafts frozen with any protocol and then thawed after storage in liquid nitrogen often crack. Although wound coverage may be possible, the actual condition of the confluent sheets at the cellular level remains in question.
Early work using high molecular weight cryoprotectants showed that polyvinylpyrrolidone (PVP) or dextrans (MW 30-100Kd) alone prevents destruction of erythrocytes during cryopreservation Pegg, D. E., "Banking of Cells, Tissues and Organs at Low Temperatures", in Current Trends in Cryobiology (A. Smith, ed. 1970). Dextran and hydroxyethyl starch combined with glycerol will partly maintain the motility of frozen and thawed ram spermatazoa Schmehl et al., "The Effects of Nonpenetrating Cryoprotectants Added to Test-Yolk-Glycerol Extender on the Post-Thaw Motility of Ram Spermatazoa" 23 Cryobiol. 6:512-17 (1986).
Using trypan blue dye exclusion as a measure of viability, hydroxyethyl starch (HES) alone has been demonstrated to improve the survival of cryopreserved cells of hematopoietic origin, but only if the cryoprotectant was added and later removed in a slow, time-consuming manner (Conscience and Fischer, "An Improved Preservation Technique for Cells of Hemapoetic Origin" 22 Cryobiol. 5:495-98 (1985). This parameter of viability provides information only about short-term membrane stability and offers no data about the proliferative potential or long-term survival of the tissue. These authors concluded that HES did not offer any advantage for cryopreservation of cells of epithelial origin. In another study, HES was successfully used in the cryopreservation of cells of hematopoietic origin as determined by cell proliferation assays, a parameter that is more reflective of long-term viability (Wang, et al, Cryobiol. 24:229-237 (1987).
Studies using high molecular weight, non-penetrating (glass-forming) agents as cryoprotectants tend to focus on cells in suspension such as red blood cells and lymphocytes as described above. However, the need to maintain the integrity of a cohesive sheet of cells during cryopreservation places severe limitations on the recovery of viable cells (i.e., those with the capability of tissue regeneration).
It is an object of this invention to provide a cryopreservation methodology, capable of preserving a living, cultured sheet of epithelial cells, where the method maintains the structural integrity of the sheet, and preserves the mitotic competence of cells in the sheet, to permit formation of an epithelial tissue useful in wound healing.
Novel methods have now been discovered for cryopreserving confluent sheets of living, cultured epithelial cells so that they maintain their utility as a skin wound dressing. This methodology maintains the colony-forming efficiency of the cells in the sheet, i.e., preserves a significant number of living cells in the epithelial sheet in a mitotically competent form, such that regeneration of a healthy epithelium occurs. The methods also permit harvesting of cultured epithelial sheets when they are mature and warehousing the sheets in a cyropreserved form for future use.
The methods involve the following steps: 1) immersing the sheet in a cryoprotectant solution containing at least a non-cell-penetrating, glass-forming agent and, preferably, a cell-penetrating, glass-forming agent; 2) freezing the sheet by cooling it to a temperature at or below at least about -65� C., more preferably at or below -120� C. (the glass transition temperature of water), and most preferably, at or below -180� C. particularly for longer periods of storage. The preferred method involves cooling in the range of approximately -180� C. to approximately -196� C., preferably by exposure to the vapors of liquid nitrogen. The process further involves 3) storing the sheet at a temperature ranging from and below at least -65� C. and down to approximately -180� C.; and 4) thawing to produce an intact sheet wherein the cells have a colony-forming efficiency of at least 35%, often 40%, and in many cases 50% or more.
The cryoprotectant solution contains (on a weight percent basis) about 5% to 20% of a non-cell-penetrating, glass-forming agent, preferably about 15%, and about 10% of a cell-penetrating glass-forming agent. The cell-penetrating glass-forming agent may be glycerol, propylene glycol, ethylene glycol, dimethyl sufoxide (DMSO), and mixtures or derivatives thereof. Preferably, the cell-penetrating glass-forming agent is glycerol. The non-cell-penetrating glass-forming agent may be dextran, polyvinyl pyrrolidone, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, and mixtures or derivatives thereof, and is most preferably dextran or hydroxyethyl starch.
In preferred embodiments, the freezing step entails slowly cooling the sheet at least from about 4� C. to approximately -80� C., and further cooling to a temperature in the range of approximately -180� C. to approximately -196� C., i.e., the temperature of liquid nitrogen vapors. The slow cooling is conducted at a rate of about 1� C./minute. Additionally, the thawing step preferably entails heating the sheet, e.g., in air or other gas, from the low storage temperature up to the range of -120� C. to about -80� C. in a time between about 1 minute and 5 minutes, i.e., at a heating rate of about 20� C./min. to about 100� C./min. The sheet then may be incubated in an aqueous bath or otherwise rapidly heated to increase the temperature further up to about 20� C. to about 37� C.
Prior to use as a skin wound dressing, the thawed sheet is rinsed free of cryoprotectant using, for example, an isotonic buffer solution at physiological pH, preferably lactated Ringer's solution, in which cells can be stored temporarily before use. The epithelial cells of sheets used in the method preferably are cultured keratinocytes. The confluent sheet is several cells thick, and comprises differentiating layers.
Other objects and features of the invention will be apparent from the drawing, description, and claims which follow.
FIG. 1 is a cross section of a stratified cultured keratinocyte sheet after being released from a culture flask with Dispase. The photomicrograph is representative of a stratified cultured keratinocyte sheet used in the practice of this invention;
FIG. 2 is a bar graph illustrating the effects of the use of non-cell-penetrating agents in cryoprotection. Cultured keratinocyte sheets were equilibrated in cryoprotective medium (CPM) containing either 10% glycerol for 6 minutes (gly c-6) and 30 minutes (gly c-30), or 10% glycerol with 15% dextran (70 kd) for 30 minutes, then cooled to -180 C, and stored 2-3 days at -180 C. After thawing the viability was measured by assessing the total cell recovery (R), colony forming efficiency (CFE), and survival of colony forming cells (CFC) after disaggregation of the cell sheet. CFC=cells recovered X CFE of the recovered cells (expressed as a percentage of non-frozen, non-stored control sheets);
FIG. 3 is a bar graph illustrating the effect of dextran and Hespan (hydroxyethyl starch) on viability of cryopreserved cultured epidermal grafts. Grafts were equilibrated in CPM containing either 15% Hespan, 15% dextran (70 kd), or 15% dextran (500 kd), in addition to 10% glycerol, for 30 minutes, then cooled to -180� C. and stored for 2 days. After thawing viability was measured by assessing R, CFE, and CFC. Results are expressed as a percentage of non-frozen, non-stored control grafts;
FIG. 4 is a bar graph illustrating the effect of cryoprotective medium prior to freezing on viability of cryopreserved cultured epidermal grafts. Grafts were equilibrated at room temperature in cryoprotective medium containing 15% dextran (70 kd) and 10% glycerol for 30, 60, and 120 minutes. After thawing, cells in grafts were assayed for R, CFE, and CFC. Results are expressed as a percentage of control grafts;
FIG. 5 is a bar graph illustrating the effect of storage temperature on viability of cryopreserved cultured epidermal grafts. Grafts were equilibrated at room temperature in CPM containing 15% dextran and 10% glycerol for 30 minutes, frozen to -85� C. and then stored at about -180� C. in liquid nitrogen vapor, at -80� C. in a mechanical freezer, or with solid carbon dioxide (dry ice, sublimation point -77� C.) at a temperature of about -65� C. After thawing, R, CFE, and CFC of the grafts were measured. Results are expressed as a percentage of control non-frozen, non-stored grafts; and
FIG. 6 is a bar graph illustrating the effect of post-thaw treatment of cryopreserved culture epidermal grafts. Grafts were equilibrated for 60 minutes in cryoprotective medium prior to freezing. After freezing and storage at -180� C. for 1-3 days, grafts were thawed rapidly, removed from CPM after 0, 30, or 60 minutes post thawing and rinsed for 1 or 2 hours in lactated Ringer's solution. Cell recovery, CFE, and CFC of the treated grafts were measured and expressed as a percentage of control non-frozen, non-stored grafts.
Cultured human epithelial cell sheets can function as permanent autograft material for repair of skin wounds. As temporary allograft material, the sheets can promote healing of chronic skin ulcers and split-thickness graft donor sites and may also provide a highly effective burn wound dressing. The sheets are produced using a culture system developed by Rheinwald and Green, wherein serially cultured epithelial cells divide rapidly on the surface of tissue culture dishes or flasks and ultimately form a confluent, modestly stratified polarized sheet of tightly interconnected cells. A photomicrograph of a cross section of such a cultured sheet is shown in FIG. 1. Stratified epithelial cultures can be released as cohesive cell sheets by treatment with an enzyme such as dispase, stapled to gauze impregnated with an nonadherent material, e.g., petrolatum or Vaseline�, transported in culture medium to the operating room, and applied to the patient.
A significant limitation in the use of cultured epidermal grafts is their extreme fragility and short shelf life. Experiments indicated that cell viability in the grafts decreased substantially when the grafts had been separated from their culture substratum for longer than 8 hours, as measured by the ability of disaggregated cells to resume growth and form colonies when replated under optimal culture conditions. For this reason, sales of cultured epithelial sheets have been limited geographically, i.e., to those hospitals near enough to a production center so that the grafts could be prepared, transported, and applied to the patient within about 8 hours or less from the time dispase was first added to the cultures to initiate detachment at the culture facility. Actual time in transit could only be a few hours as time was consumed in preparing the grafts. Operating room scheduling and time of arrival of the grafts had to be coordinated carefully. Storage of the sheets for any significant period has been impossible, and therefore an inventory of ready-to-use product could not be maintained.
A possible explanation for such a short period of viability of detached epithelial cell sheets was provided by the observation that epithelial cells are found to lose the potential for further division and commit to terminal differentiation when, as single cells disaggregated from cultures by trypsin and EDTA treatment, they are temporarily maintained under conditions that prevent them from reattaching to a surface.
Experiments assessing viability beyond eight hours revealed that the temperature at which the dispase-treated grafts were maintained was extremely critical to cell viability as measured by colony forming efficiency (CFE) and the total number of colony-forming cells recovered (CFC, or total cells recovered X CFE). Maintenance of the cultured epithelial sheets at or slightly below physiological temperature failed to maintain the CFE of fresh tissue. Similarly, maintenance at 4� C. had no apparent positive effects, despite careful control of media conditions, pH, and CO2 /O2 balance. CFE can be maintained for greater than about 20-30 hours if the cultured sheets are maintained within a critical temperature range of 10� C. to 25� C., preferably 13� C. to 23� C. However, no method is currently available for storing the cultured sheets for more than one day while maintaining their utility as a skin wound dressing, and maintenance of an inventory of a ready-to-use product for wound healing applications has not been possible
The history of cryopreservation methodology has shown that the optimization of a cryopreservation protocol for a particular cell does not necessarily give good results when used with another cell type or when used with the same cell type from a different species, or with other cells in a tissue. Methods for freezing a suspension of keratinocytes yield poor results when used for intact sheets. In fact, freezing intact tissues for use in implants has not, to our knowledge, been successful for any tissue type (but see Cancedda et al., EP 0 296 475).
The methods disclosed herein of cryopreserving a confluent sheet of living, cultured epithelial cells after separation from their culture substratum maintain the mitotic competence, or colony-forming efficiency of the cells in the sheet at acceptable levels, and maintain the integrity of the sheet. Briefly, the method comprises four steps. First, the cell sheet is equilibrated in a cryopreservative solution for a time sufficient to allow the cryopreservative to mix thoroughly with and/or displace the water within and between the cells. Second, the sheet is cooled preferably to approximately -180� C. to -196� C., at a rate slow enough for the cryoprotected cells to avoid ice crystal formation and subsequent damage. The frozen sheets may be stored for long periods at approximately 31 180� C. or for shorter periods at higher temperatures, e.g., as high as about -65C. Before use, the sheets are warmed at room temperature in air or other gas for about 1-3 minutes, and then thawed completely by rapid warming in, for example, a water bath. Fourth, the cryoprotectant is removed from the cultured epithelial cell sheet by rinsing in an isotonic buffer such as lactated Ringer's solution.
Details of the procedure are disclosed below.
Epidermal cells (keratinocytes) are seeded into T150 culture flasks (Costar) at densities that will reach confluence in 10-12 days. Cultures from frozen cell suspensions from several epidermal cell strains may be used for allografts; cells from biopsies of burn patients for autografts. Cultures are maintained in gas tight flasks at 37� C. in "FAD" medium (one part Hams F12 supplemented with adenine and 3 parts Dulbecco's modified Eagle's medium (DME) plus 10% fetal bovine serum (FBS), 0.4 μg/ml hydrocortisone, 1�10-10 M cholera toxin, and 2�10-9 M triiodothyronine) and grown in the presence of lethally irradiated 3T3 fibroblasts. See U.S. Pat. No. 4,016,036. Ten ng/ml epidermal growth factor is included from the first feeding.
The cell cultures are used to prepare grafts once they have reached confluence. The supernatant medium is aspirated and 40 ml of Dispase II (Boehringer Mannheim) at a final concentration of 2.5 mg/ml (approximately 1.2 U/ml) is added to the flasks and incubated at 37� C. When the edges of the sheet become detached (˜45 min.), the upper portion of each flask is removed by burning with a soldering iron.
The enzyme solution is replaced by 20 ml of DME medium. The sheet of epithelial cells then is rinsed again with 20 ml of DME. After aspirating all but 3-4 ml of the second rinse, 5�10 cm pieces of Petroleum jelly (Vaseline�)-impregnated gauze (Cheesebrough Ponds) are placed over each detached sheet of cells with the superficial cells facing the gauze dressing. The cohesive cell sheet is then attached to the dressings with 12-15 staples (Ligaclips, Ethicon/J&J). The sheet is cut widthwise, stapled, and the grafts are then transferred to 100 mm dishes with the epithelium facing up. The edges of the graft are pressed to the dish with a rubber policeman to prevent the graft from floating. Twelve ml of DME is gently added and the dish is transferred to the storage container. (FIG. 1 illustrates in cross-section a typical cultured sheet made in accordance with the foregoing process). Cell recovery (R) and assay of colony forming efficiency (CFE) are used to determine the total number of mitotically competent cells (CFC). In this manner, the optimal conditions for preserving viability during the cryoprotective process can be determined, e.g. composition of cryoprotective medium, equilibration time in cryoprotective medium prior to freezing and after thawing, freezing rate, storage temperature, thawing process, rinsing procedure, and subsequent storage.
Colony-forming efficiency (CFE) assays are performed on the grafts prepared as set forth in the examples to follow, after storage for 1-3 days after freezing. Non-frozen graft are assayed as controls. Ligaclips are removed with forceps and the released cell sheet is dissociated to a single cell suspension in a mixture of trypsin (0.05%) and EDTA (0.01%) in isotonic buffer. Enzymatic action is arrested by addition of calf serum and is followed by two serial 1:10 dilutions made by adding 0.5 ml cell suspension to 4.5 ml FAD. An aliquot of the initial cell suspension is counted in a hemocytometer. A final concentration of 1,000-2,000 cells/ml is prepared and 1 ml of cell suspension is plated into 100 mm dishes containing lethally irradiated 3T3 cells in 12 ml of FAD medium.
After 10-14 days, cultures are fixed with 10% formalin in phosphate buffered saline and stained with a mixture of 1% Rhodamine and 1% Nile Blue A. Colonies are counted under a dissecting microscope and scored as either growing or aborted. CFE is calculated as follows: ##EQU1##
Using protocols of the invention, % CFE's typically exceed 35%, usually exceed 40%, and often are greater than or equal to 50%.
Development of a cryoprotective methodology requires optimization of each individual component in the process through independent study followed by an integrated approach, combining optimal components, to identify the final process. Optimal freezing, storing, thawing, and rinsing procedures that are compatible with maintaining maximal viability must be identified. These components are identified by colony forming efficiency assay as described previously.
Standard cryoprotective medium is composed of a physiologically balanced salt solution (e.g., cell culture medium) supplemented with bovine serum and glycerol, a cell-penetrating glass-forming agent. Although used successfully for cryopreserving cells in suspension, its ability to preserve viability of cells from cell sheets is less clear. FIG. 2 demonstrates the beneficial effects of supplementing standard cryoprotective medium with an additional component, a non-cell-penetrating glass forming agent, such as dextran. Note the increased cell recovery and, therefore, greater viability (CFC) obtained when the non-cell penetrating agent is used in conjunction with the cell-penetrating agent, as compared with using the cell-penetrating agent alone.
FIG. 3 illustrates the general phenomenon of increasing viability through use of non-cell penetrating glass forming agents. These agents are high molecular weight forms of complex carbohydrates. The non-cell-penetrating glass-performing agent is preferably a high molecular weight dextran of approximately 50-500 kilodaltons (kd), preferably 50-70 kd, chondroitin sulphate, polyvinylpyrrolidone, polyethylene glycol or a hetastarch such as hyroxyethyl starch. The cell-penetrating glass-forming agent is preferably glycerol, but may include propylene glycol, ethylene glycol, dimethylsulfoxide, and other penetrating glass-formers known in the art.
The cryopreservation process first requires immersion of the cell sheet to be frozen in cryoprotective medium for a time sufficient to permit equilibration of the cells with cryoprotectant. FIG. 4 demonstrates the effect of long-term equilibration of cultured epidermal sheets in cryoprotectant prior to freezing on viability. The data show that the sheet may be equilibrated for up to two hours in cryoprotectant prior to freezing without affecting the viability of cells within cryopreserved sheets. The equilibration is conducted more typically for approximately 30-60 minutes, at about 17� C. to 30� C., typically room temperature, in a cryoprotective solution, in a shallow storage dish.
Following equilibration, the dish containing the sheet and the cryoprotectant solution is sealed so that it is gas and water-tight. The sheet in the sealed container is cooled to at least about -65� C. (e.g., with dry ice), preferably below -120� C., and to Promote longer term storage, to approximately -180� C. to about -196� C. The cooling rate preferably is slow (e.g., ≦1� C./min.) from about 0� C. to at least -80� C. This serves to discourage ice crystal formation. Preferably, cooling is conducted at the outset in a rate-controlled cooling device such as a commercial programmable cell freezer (Cryomed, Inc. No. 1010/2700) to a temperature of -40� C. to -100� C., Preferably about -80� C. to -85� C., and then transferred to a liquid nitrogen storage vessel and maintained in vapors of liquid nitrogen to reduce its temperature further.
The preferred freezing protocol cools the sheet in the sealed container until the tissue is approximately 4� C. Then, the sheet is cooled at about 1� C. Per minute. Once the temperature of the sheet reaches at least -65� C., and preferably at least -85� C., the container is transferred to a liquid nitrogen refrigerator and stored at approximately -180� C. (nitrogen vapors) or -196� C. (liquid nitrogen).
Storing the tissue at or below -180� C. maintains the colony-forming efficiency of the cells better than storing the sheet at higher temperatures, as shown in FIG. 5. The data clearly indicate that storage at -180� C. in liquid nitrogen vapor is superior to storage at -80� C. in a mechanical freezer or at about -77� C. in dry ice. Since there is a dramatic decrease in viability during the first 2 or 3 days of storage at -80� C. and -77� C., in contrast to stable viability at -180� C. over longer periods. Grafts preferably are shipped at -180� C.
To thaw the sheet, the sealed container is removed from the liquid nitrogen refrigerator and preferably kept at room temperature in air for about 1 minute and up to about 3 to 5 minutes. This produces a warming rate of between about 20� C./min. to about 100� C./min. The graft may then be heated to room temperature without regard to the rate of heating. Preferably the last stage is conducted by submerging the sealed container in a water bath until the graft is thawed. This prevents the frozen sheet from cracking. Thawing is accomplished in about 1.25 minutes in a water bath at 37� C. If the water bath is 25� C., thawing takes about 1.5 minutes. Alternatively, the waterbath may be eliminated, and the sheet thawed at room temperature. However, this takes about 27 minutes and often has the effect of reducing cell viability.
The thawed sheet is removed from the cryoprotectant within about 1 hour, preferably as soon as possible. Once the sheet is thawed, the container may be opened and the cryopreservative solution replaced with an isotonic buffer solution at physiological pH (about 6.8-7.4), preferably FAD medium or lactated Ringer's solution to dilute out the cryoprotectant. Table 1A shows that not all isotonic buffered solutions at physiological pH are acceptable for dilution of cryoprotectant. Phosphate buffered saline and standard saline reduce viability significantly, as judged by CFE. The thawed sheet is equilibrated in rinsing buffer preferably for 15 minutes and may remain for up to 4 hours with a slight decrease in CFE (Table 1B).
TABLE 1A______________________________________Removal of cryoprotective medium fromcryopreserved cultured epidermal grafts after thawing.Use of isotonic buffers at physiological pH asrinsing solutions. % of cellsBuffer forming colonies rCFE______________________________________FAD 9.0 35.0PBS 3.4 13.1Normal saline 3.0 11.5______________________________________
Grafts were removed from cryoprotective medium and placed in rinsing solution for 15 minutes at room temperature. Colony forming efficiency assays were performed and the recovery of CFE (rCFE) was calculated as a percent of non-frozen, non-stored control grafts. FAD, F12/DME keratinocyte growth medium; PBS, Phosphate Buffered Saline; Normal saline, 0.9% saline.
TABLE 1B______________________________________Effect of increased rinsing time on thawed grafts % of cellsTime forming colonies rCFE______________________________________1 hour 6.4 71.12 hours 5.2 57.84 hours 4.5 50.0______________________________________
Grafts were removed from cryoprotective medium and placed in FAD rinsing solution for times indicated above at room temperature. Colony forming efficiency assays were performed and the recovery of CFE (rCFE) was calculated as a percent of non-frozen, non-stored control grafts.
Some of the limits of the final process of cryopreservation were tested and are shown in FIG. 6. The data show that cultured epithelial sheets can be equilibrated in cryoprotectant prior to freezing for up to one hour, frozen, thawed, left in cryoprotectant up to one hour post-thaw and finally rinsed in lactated Ringer's solution for up to 2 hours, producing an intact sheet of cells which are viable and capable of resuming normal metabolic function.
A sheet prepared as disclosed above may be placed surgically over a clean skin wound such as a burn or ulcer, using the gauze backing as a dressing, with the germinative layer in contact with the surface of the wound. Allograft sheets eventually are sloughed off, but prior to rejection provide safe and effective protection to the wound, and often promote wound healing. Autograft sheets typically take permanently and differentiate to produce intact, normally stratified skin. The higher CFC and CFE values that are achieved with this cryopreservation methodology demonstrates that greater numbers of individual cells in the sheet are mitotically competent, and therefore implies that a greater number of epithelial cell colonies will form at the wound site.
The invention will be illustrated further by means of the following, non-limiting Examples.
The harvested cultured graft is placed in a shallow storage dish in a cryoprotectant solution comprising 15% dextran (70,000 mw) and 10% glycerol in medium. The container is sealed so that it is gas and water-tight, and contains enough liquid such that little or no air space remains and the grafts are equilibrated at room temperature for 30 min.
The sheet is cooled in the sealed container until the tissue is approximately 4� C. The sheet is then cooled at 1� C. per minute to approximately -85� C. Once the temperature of the sheet reaches -85� C., the container is transferred into a liquid nitrogen refrigerator and thereafter stored in liquid nitrogen vapor (about -180� C.).
To thaw the sheet, the sealed container is removed from the liquid nitrogen freezer and kept at room temperature in air for approximately 1 minute. It is then placed in a 37� C. water bath for approximately 75 sec., until thawed. Once thawed, the container is opened and the sheet is placed in lactated Ringer's solution at physiological pH for 5 min. The buffer then is changed, and the sheet is equilibrated in the medium for at least another 10 min.
The graft is equilibrated in a shallow storage dish in a cryoprotectant solution comprising 15% hydroxyethyl starch (Hespan�) and 10% glycerol at room temperature for 15 min. Following equilibration, the container is sealed so that it is gas and water-tight.
The sheet in the sealed container is cooled until the tissue is approximately 4� C. Following this step, the sheet is cooled at 1� C. per minute to approximately -85� C. Once the temperature of the sheet reaches -85� C., it is transferred into a liquid nitrogen refrigerator and stored in liquid nitrogen (approximately -196� C.).
To thaw the sheet, the sealed container is removed from the liquid nitrogen freezer and is kept at room temperature in air for approximately 1 minute. It is then placed into a water bath until thawed. Once thawed, the container is opened and the sheet is placed in lactated Ringer's solution or serum free DME/F12 keratinocyte growth medium at physiological pH for 5 min. After 5 min., the buffer is changed and the sheet equilibrated in the medium for up to 4 hours.
The invention may be embodied in other specific forms. Other embodiments are within the following claims.
|Cited Patent
|Filing date
|Publication date
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|Union Carbide Corp
|Process for cell preservation
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|Istituto Nazionale Per La Ricerca Sul Cancro
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|Istituto Nazionale Per La Ricerca Sul Cancro
|Method for preserving transplantable sheets of epithelium cultured in vitro
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|Coriell Section I.A. [3] Preservation, Storage and Shipment; in Methods in Enzymology vol. LVIII, pp. 29-31; Jakoby & Pastan, Eds. Academic Press; New York, San Francisco, London 1979.
|Coriell Section I.A. 3 Preservation, Storage and Shipment; in Methods in Enzymology vol. LVIII, pp. 29 31; Jakoby & Pastan, Eds. Academic Press; New York, San Francisco, London 1979.
|Cuono et al., 80(4) Plastic and Reconstructive Surgery 626 (1987), Composite Autologous Allogeneic Skin . . . .
|Cuono et al., 80(4) Plastic and Reconstructive Surgery 626 (1987), Composite Autologous-Allogeneic Skin . . . .
|De Luca et al., Symposium Cultured Epithelium (28 Mar. 1987), Human Epidermis Culture for the Treatment . . . .
|Eldad et al., 13(3) Burns 173 (1987), Cultured Epithelium as a Skin Substitute.
|Hefton, et al. Cryopreservation of Cultured Epidermal Grafts A.B.A. (Abstract) Mar., 1988.
|Heimbach et al., 208(3) Ann. Surg. 313 320 (1988), Artificial Dermis for Major Burns: A Multi center . . . .
|Heimbach et al., 208(3) Ann. Surg. 313-320 (1988), Artificial Dermis for Major Burns: A Multi-center . . . .
|Korber et al., 23(6) Cryobiology 576 (1986), Cryopreservation of Human Platelet . . . .
|May et al., 73 J. MAG. 233 (1984), Recent Developments in Skin Banking and the Clinical Uses . . . .
|May, 23(6) Cryobiology 569 570 (1986), Hypothermic Preservation of Skin: A Review of Current . . . .
|May, 23(6) Cryobiology 569-570 (1986), Hypothermic Preservation of Skin: A Review of Current . . . .
|McGann et al., 23(6) Cryobiology 574 575 (1986), Comparison of Dimethyl Sulfoxide and Dimethyl Sulfone . . . .
|McGann et al., 23(6) Cryobiology 574-575 (1986), Comparison of Dimethyl Sulfoxide and Dimethyl Sulfone . . . .
|Neronov, et al. Cryopreservation of Rabbit Corneas by Combination of Dextran 40, Human Albumin and DMSO . . . Cryo Letters 10(5) 289 298 1989.
|Neronov, et al. Cryopreservation of Rabbit Corneas by Combination of Dextran-40, Human Albumin and DMSO . . . Cryo Letters 10(5) 289-298 1989.
|Praus et al., 17 Cryobiology 130 (1980), Skin Cryopreservation.
|Taylor, (3(2) Cryobiology 192 (1966), Cryopreservation of Skin: Discussion and Comments.
|Citing Patent
|Filing date
|Publication date
|Dec 12, 1991
|Jul 12, 1994
|Robyn Fisher
|Cold and cryo-preservation methods for human tissue slices
|Feb 2, 1993
|Aug 9, 1994
|Lifecell Corporation
|Method for processing and preserving collagen-based tissues for transplantation
|Dec 30, 1991
|Aug 23, 1994
|Cobe Laboratories, Inc.
|Lyophilization of erythrocytes
|May 31, 1991
|Jun 20, 1995
|Cryopharm Corporation
|Method of reconstituting lyophilized cells
|Sep 15, 1993
|May 21, 1996
|Organogenesis Inc.
|Cryopreservation of cultured skin or cornea equivalents with agitation
|Jun 5, 1995
|Mar 25, 1997
|Cryolife, Inc.
|Method of preparing transplant tissue to reduce immunogenicity upon implantation
|Jun 5, 1995
|May 27, 1997
|Cryolife, Inc.
|Treated tissue for implantation and methods of preparation
|Mar 29, 1995
|Jul 15, 1997
|Cobe Laboratories, Inc.
|Lyophilization of cells
|Dec 1, 1993
|Sep 23, 1997
|Development Biotechnological Processes Snc Di Pelliccia Maria Teresa
|Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial sheets
|Feb 26, 1993
|Jun 30, 1998
|Colorado State University Research Foundation
|Treated tissue for implantation and methods of treatment and use
|Jun 7, 1995
|Sep 1, 1998
|Cobe Laboratories, Inc.
|Method of freezing cells and cell-like materials
|Jan 27, 1997
|Dec 1, 1998
|Cryolife, Inc.
|Treated tissue for implantation and methods of preparation
|Sep 12, 1996
|Jan 5, 1999
|Colorado State University Research Foundation
|Treated tissue for implantation and methods of treatment and use
|Jun 5, 1995
|Jan 26, 1999
|Colorado State University Research Foundation
|Treated tissue for implantation and methods of treatment and use
|Jan 30, 1995
|Apr 6, 1999
|Organogenesis Inc.
|Cryopreservation of harvested skin and cultured skin or cornea equivalents by slow freezing
|Jun 5, 1995
|May 4, 1999
|Cryolife, Inc.
|Treated tissue for implantation and methods of preparation
|Jun 15, 1994
|Sep 28, 1999
|Cobe Laboratories, Inc.
|Method of freezing cells and cell-like materials
|Jun 7, 1995
|Dec 28, 1999
|Cobe Laboratories, Inc.
|Method of freezing cells and cell-like materials
|Apr 28, 1998
|Feb 13, 2001
|The American National Red Cross
|Method for preparing organs for transplantation after cryopreservation
|Apr 27, 1998
|Apr 24, 2001
|Kimberly Ann Woodhouse
|Biodegradable polyurethanes
|Aug 21, 1998
|Sep 18, 2001
|Advanced Tissue Sciences, Inc.
|Cells or tissues with increased protein factors and methods of making and using same
|Jan 2, 2001
|Feb 19, 2002
|Organogenesis Inc.
|Ice seeding apparatus for cryopreservation systems
|Apr 28, 2000
|Oct 29, 2002
|Interthyr Corporation
|In situ growth, freezing and testing of cultured cells
|Jan 16, 1998
|Apr 1, 2003
|Celadon Science, Llc
|Methods for promoting healing of corneal resurfacing wounds
|Nov 9, 1995
|Apr 15, 2003
|Celadon Science, Inc.
|Sheets of cultured epithelial cells used for wound repair and their cryopreservation
|Jun 20, 2002
|Jun 24, 2003
|Celadon Science, Llc
|Wound repair dressings and methods for their preservation
|Dec 26, 2001
|Oct 28, 2003
|Ortec International, Inc.
|Processes for making cryopreserved composite living constructs and products resulting therefrom
|Jan 16, 1998
|Mar 30, 2004
|Celadon Science, Llc
|Methods for promoting healing of skin resurfacing wounds
|Jun 20, 2002
|Nov 8, 2005
|Advanced Biohealing, Inc.
|Wound repair dressings and methods for their preservation
|Sep 9, 2002
|Mar 11, 2008
|Diagnostic Hybrids, Inc.
|In situ growth, freezing and testing of cultured cells
|Jan 20, 2005
|Apr 15, 2008
|Lifecell Corporation
|Particulate acellular tissue matrix
|Jun 7, 2002
|Nov 29, 2011
|Lifecell Corporation
|Acellular dermal matrix and method of use thereof for grafting
|Jan 6, 2011
|Jan 14, 2014
|Hememics Biotechnologies, Inc.
|Desiccated biologics and methods of preparing the same
|May 16, 2006
|Apr 7, 2015
|Tigenix, S.A.U.
|Use of adipose tissue-derived stromal stem cells in treating fistula
|Jun 20, 2002
|Dec 19, 2002
|Walid Kuri-Harcuch
|Wound repair dressings and methods for their preservation
|Jun 7, 2002
|Feb 20, 2003
|Lifecell Corporation, A Delaware Corporation
|Method for processing and preserving collagen-based tissues for transplantation
|Jul 7, 2005
|Sep 21, 2006
|Lifecell Corporation, A Texas Corporation
|Method for processing and preserving collagen-based tissues for transplantation
|Sep 22, 2008
|Mar 26, 2009
|Hememics Biotechnologies, Inc.
|Desiccated Biologics And Methods Of Preparing The Same
|May 16, 2006
|Apr 22, 2010
|Cellerix, S.L.
|Use of adipose tissue-derived stromal stem cells in treating fistula
|Feb 3, 2010
|Aug 5, 2010
|Wood Fiona M
|Cell Suspension Preparation Technique and Device
|Jun 4, 2010
|Feb 3, 2011
|Hememics Biotechnologies, Inc.
|Desiccated Biologics And Methods Of Preparing The Same
|Jan 6, 2011
|Apr 28, 2011
|Hememics Biotechnologies, Inc.
|Desiccated Biologics And Methods Of Preparing The Same
|Mar 13, 2014
|Sep 18, 2014
|Stratatech Corporation
|Cryopreservation of viable human skin substitutes
|Jan 8, 2014
|Jan 22, 2015
|Steminent Biotherapeutics, Inc.
|Cryopreservation storage device for cell collection bag, and using method thereof
|Sep 5, 1997
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|Universal Preservation Technologies Inc
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|Nov 14, 1996
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|Encelle, Inc.
|Hydrogel matrix for cellular tissue storage
|Nov 14, 1996
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|Sep 14, 1994
|Mar 23, 1995
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|Cryopreservation of cultured tissue equivalents
|Dec 26, 2001
|Aug 1, 2002
|Ortec International, Inc.
|Processes for making cryopreserved composite living constructs and products resulting therefrom
|Dec 26, 2001
|Dec 11, 2003
|Alla Laufer
|Processes for making cryopreserved composite living constructs and products resulting therefrom
|Oct 13, 2015
|Apr 21, 2016
|Cellular Dynamics International, Inc.
|Generation of keratinocytes from pluripotent stem cells and mantenance of keratinocyte cultures
|U.S. Classification
|435/1.3, 424/574, 435/374, 62/62
|International Classification
|Cooperative Classification
|European Classification
|Jun 4, 1990
Owner name: BIOSURFACE TECHNOLOGY, INC., MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:TUBO, ROSS A.;SCHAEFFER, SUSAN F.;SCHERMER, ALEXANDER;AND OTHERS;REEL/FRAME:005339/0228
Effective date: 19900529
|Feb 20, 1996
Owner name: GENZYME CORPORATION, MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BIOSURFACE TECHNOLOGY, INC.;REEL/FRAME:007833/0032
Effective date: 19960207
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|Feb 28, 2000
|Fee payment
Year of fee payment: 8
|Mar 24, 2004
|Maintenance fee reminder mailed
|Sep 8, 2004
|Lapse for failure to pay maintenance fees
|Nov 2, 2004
|Expired due to failure to pay maintenance fee
Effective date: 20040908


text:
Category:Bolivian expatriates in Argentina
From Wikipedia, the free encyclopedia
This category has only the following subcategory.
- ► Ambassadors of Bolivia to Argentina (2 P)
Pages in category "Bolivian expatriates in Argentina"
The following 16 pages are in this category, out of 16 total. This list may not reflect recent changes (learn more).

subset: commoncrawl


text:
In the heat of corporate crisis
Companies often need to react quickly in the face of unexpected challenges—whether industrial accidents, financial misdeeds, cyberattacks, or any number of disasters. Individual responses, particularly at the Board / C-Suite level, are crucial through all stages of a crisis.
A crisis of confidence
Awareness of threats is not the same as preparation to deal with them. More than three-quarters (76%) of surveyed board members believe their companies would respond effectively if a crisis struck tomorrow. But less than half (49%) of their companies have taken steps to be truly crisis-ready.
Corporate Regulatory and Ethical Compliance
Deloitte's annual Regulatory and Ethical Compliance survey of Corporate Compliance Officers' looks at benchmarking the progression and challenges faced by the Compliance profession in the UK.
A directors' guide to integrated reporting
In a rapidly changing unpredictable world, where every company has to build its licence to operate on foundations of trust, and where technology and international communications makes everything visible, leadership and relationships are the only consistent building blocks of success.


text:
Innovative Bookseller Claims the Kim Scott Walwyn Prize
Miriam Robinson, who has risen from bookseller to Head of Marketing at Foyles in less than four years, was tonight named the winner of the Kim Scott Walwyn Prize 2013.
The award, which celebrates both the professional achievements and promise of women in publishing, was announced at a ceremony packed with publishers and industry professionals at the Free Word Centre in Farringdon.
Joan Smith, human rights activist, journalist and novelist, gave the keynote speech before Denise Johnstone-Burt, co-chair of the Kim Scott Walwyn Committee, awarded Miriam with her Prize. The Prize, worth £1000, is sponsored by the Society of Young Publishers and also includes a two-day course of Robinson's choice, courtesy of the Publishing Training Centre.
Before embarking on a career in publishing, Miriam completed a degree at the University of Virginia in English and French Literature, and then taught English to University students in China. Upon her return to the States, Miriam completed a Publishing Certificate Programme in New York, before travelling again - this time to Paris, where she secured an internship at the well-reputed Shakespeare & Company.
This placement eventually brought Miriam to London, and she joined the fiction department of Foyles on Charing Cross Road in 2007. In just four years she has worked her way up to her current role, as Foyles' Head of Marketing. Within this position Miriam has been able to deploy her creativity and passion for bookselling, as well as championing original initiatives and orchestrating hugely successful debut events.
Denise Johnstone-Burt, co-chair of Prize Advisory Committee and the judging panel said:
'Miriam Robinson is an outstanding winner from a superb shortlist. Her enthusiasm for book-selling has already had a resounding impact, not just on Foyles but on the trade as a whole, and her spirited innovation and commitment give confidence that there is indeed a great future for the high street book shop.'
Viv Bird, Chief Executive of Book Trust, who managed the Kim Scott Walwyn Prize added:
'Miriam's innovative, forward-thinking approach as well as her passion for the bricks and mortar bookshop make her a well-deserved winner of the Kim Scott Walwyn award. The launch of the Charing Cross Road Festival, in particular, shows that she has the creativity and drive to push the success of high street bookselling in a challenging environment. I am delighted that Miriam has won this prestigious award for women in publishing.'
Miriam joins an impressive list of Prize alumni including: last year's winner Rukhsana Yasmin, Editor at Saqi Books; Kay Peddle, Editor at the Bodley Head; Kathy Rooney, Managing Director of Bloomsbury Publishing; Clare Alexander, Literary Agent at Aitken Alexander Associates; Annette Thomas, CEO of Macmillan; Penelope Hoare, Deputy Managing Director at Chatto & Windus (although now semi-retired); and the inaugural winner Lynette Owen OBE, Copyright Director of Pearson Education.
Andrew Turner, former chair of the SYP and member of the judging panel, commented:
'I was blown away by Miriam's passion and dedication to bookselling. Her desire to do all she can to promote the bookshop in the current age of digital sales and Amazon is something the book industry should praise and look to emulate. She has managed to take the negative impact of things out of her control and create unique and successful events out of them. Her creativity and passion should be an inspiration to us all.'
The Prize was founded in honour of Kim Scott Walwyn, a Publishing Director at Oxford University Press, who died in 2002 at the age of 45. She was an outstanding editor and a generous and inspirational manager whose career was widely recognised and celebrated during her life. The Prize, which has been awarded seven times, is open to any woman who has worked in publishing in the UK for up to seven years. It is managed by Book Trust and sponsored by the SYP and PTC.
Miriam fought off strong competition from the 2013 diverse shortlist, who each received a one-day training course at the PTC. They were:
- Laura Austin -Founder of Bookmachine, Ebook, Marketing & Account Manager, YUDU Media
- Juliet Mushens -Literary Agent at the Agency Group
- Laura Palmer - Co-founder & Editorial Director at Head of Zeus
The full line-up of judges for the 2013 Prize is:
- (Co-chair) Catherine Clarke - literary agent and managing director, Felicity Bryan Agency
- (Co-chair) Denise Johnstone-Burt - publisher, Walker Books
- Nicola Crossley - SYP vice-chair 2010; Trade Non Fiction, Orion Books
- Andrew Turner - SYP vice-chair and treasurer 2013; Nelson Croom
- Rukhsana Yasmin - winner of the 2012 Prize and commissioning editor at Saqi/The Westbourne Press/Telegram


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Timofey Nastyukhin vs Kotetsu Boku
ONE Championship Defending Honor watch video >>
Two seasoned strikers from Japan and Mongolia finally meet for the first time in the cage. Watch what happened. watch video >>
Narantungalag Jadambaa beats Kotetsu Boku by Submission (Von Flue Choke) watch video >>
Jadambaa Narantungalag vs Kotetsu Boku
One Championship - Ascent to Power watch video >>
Kotetsu Boku vs Vincent Latoel
One Championship 38 - Tribe of Warriors watch video >>
Kotetsu "No Face" Boku vs Major "Majee" Overall
One Championship - Pride of Lions watch video >>
Two of the most feared strikers in the ONE FC Lightweight Division are set to clash on 2 May in Manila. Fans will never forget the wars Eduard Folayang has been in, nor the come-from-behind knockout watch video >>
Kotetsu "The Korean Tiger" Boku vs. Arnaud "The Game" LepontOne FC 14 - War of NationsStadium Negara, Kuala Lumpur, Malaysia, MalaysiaMarch 14, 2014 watch video >>
Kotetsu Boku vs. Vuyisile ColossaOne FC 10 - Boku vs. ColossaIstora Senayan, Jakarta, Jakarta, IndonesiaSeptember 13, 2013 watch video >>
Willy Ni vs. Vincent LatoelOne FC 10 - Boku vs. ColossaIstora Senayan, Jakarta, Jakarta, IndonesiaSeptember 13, 2013 watch video >>


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The technical support is free for all users - registered or not. Please describe the issue or feature in detail, if possible with screenshots.
If you have a sales related problem ( lost license key, purchase renewals, etc. ) login on avangate.com using your email address and 'forgot password'. There you have full control over your registration profile.


text:
"Real World" legend David "Puck" Rainey is officially locked up in a California state prison ... TMZ has learned -- after he was convicted of stalking...
Read Whole Story
Jade proved herself a master at real-time reinvention, crafting a character that viewers would eagerly consume, pacing her scenes and delivered her lines and photos ops with an expert sense of timing.
Get top stories and blog posts emailed to me each day. Newsletters may offer personalized content or advertisements. Learn more.


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Browse By:
All Agencies » Multinational » North Atlantic Treaty Organization
North Atlantic Treaty Organization (1 - 10 of 133 items)
Afghanistan Security: Long-standing Challenges May Affect Progress and Sustainment of Afghan National Security Forces
GAO-12-951T: Published: Jul 24, 2012. Publicly Released: Jul 24, 2012.
The Department of Defense (DOD) and the North Atlantic Treaty Organization (NATO) report progress developing capable Afghan National Security Forces (ANSF), but tools used to assess the performance of ANSF units have changed several times. In April 2012, DOD reported progress increasing the number and capability of ANSF, with 7 percent of army units and 9 percent of police units rated at the highe...
Afghanistan Security: Department of Defense Effort to Train Afghan Police Relies on Contractor Personnel to Fill Skill and Resource Gaps
GAO-12-293R: Published: Feb 23, 2012. Publicly Released: Feb 23, 2012.
U.S. government (USG), non-USG coalition, and DOD contractor personnel perform various roles in the ANP training program. These roles include: (1) serving as advisors and mentors to build ministerial capacity in areas such as financial and human resource management at the Afghan Ministry of Interior; (2) serving as mentors and trainers to develop Afghan commanders abilities to operate traini...
Nuclear Weapons: DOD and NNSA Need to Better Manage Scope of Future Refurbishments and Risks to Maintaining U.S. Commitments to NATO
GAO-11-387: Published: May 2, 2011. Publicly Released: May 2, 2011.
U.S. nuclear weapons are aging, with key components reaching the end of their service life. In September 2008, the Department of Defense (DOD) and the National Nuclear Security Administration (NNSA) began a study of military requirements and design options for extending the B61 bomb's service life. The B61 is used to support the U.S. strategic deterrent and the North Atlantic Treaty Organization (...
Afghanistan Security: Afghan Army Growing, but Additional Trainers Needed; Long-term Costs Not Determined
GAO-11-66: Published: Jan 27, 2011. Publicly Released: Jan 27, 2011.
Developing capable Afghan National Army (ANA) forces is a key element of the U.S. and North Atlantic Treaty Organization (NATO)-led coalition effort to counter the insurgency and create sustainable security in Afghanistan. Since 2002, the United States, with assistance from NATO and other coalition nations, has worked to develop the ANA. The Department of Defense (DOD) leads U.S. efforts to train...
NATO Partnerships: DOD Needs to Assess U.S. Assistance in Response to Changes to the Partnership for Peace Program
GAO-10-1015: Published: Sep 30, 2010. Publicly Released: Nov 10, 2010.
The North Atlantic Treaty Organization (NATO) established the Partnership for Peace (PfP) to increase cooperation with former Warsaw Pact members and provide many of these countries with a path to NATO membership. As NATO confronts new security challenges, including the war in Afghanistan, its relationships with partner countries have grown in scope and importance. Additionally, NATO is developing...
The Strategic Framework for U.S. Efforts in Afghanistan
GAO-10-655R: Published: Jun 15, 2010. Publicly Released: Jun 15, 2010.
The United States and its international partners from over 40 nations have been engaged in efforts to secure, stabilize, and rebuild Afghanistan since 2001. In an effort to establish clear and specific U.S. strategic goals, the President of the United States, in March 2009, outlined the U.S. Strategy for Afghanistan and Pakistan. This strategy emphasizes a strategic goal to disrupt, dismantle, and...
Afghanistan and Pakistan: Oversight of U.S. Interagency Efforts
GAO-09-1015T: Published: Sep 9, 2009. Publicly Released: Sep 9, 2009.
GAO has identified Afghanistan and Pakistan as two of the most urgent issues facing this Administration and this Congress. In March, the President announced a strategy for Afghanistan and Pakistan, with a broad strategic goal of disrupting, dismantling, and defeating Al-Qaeda in Afghanistan; destroying its allies and safe havens in Pakistan; and preventing their return to Pakistan or Afghanistan....
NATO Enlargement: Reports on Albania and Croatia Respond to Senate Requirements, but Analysis of Financial Burdens Is Incomplete
GAO-08-1165R: Published: Sep 22, 2008. Publicly Released: Sep 22, 2008.
On April 2, 2008, the North Atlantic Treaty Organization (NATO) invited Albania and Croatia to begin accession talks for NATO membership. NATO wants new members to be democracies, have harmonious relations with neighboring countries, modernize and restructure their defense capabilities, protect civil liberties and human and minority rights, and have open market economies. The admission of new memb...
Darfur Crisis: Progress in Aid and Peace Monitoring Threatened by Ongoing Violence and Operational Challenges
GAO-07-9: Published: Nov 9, 2006. Publicly Released: Dec 11, 2006.
In 2003, violent conflict in Darfur, Sudan broke out between rebel groups, government troops, and government-supported Arab militias, known as the Janjaweed. The conflict has displaced about 2 million Darfurians and has so affected over 1.9 million others that they require assistance. Since October 2003, the U.S. government has provided humanitarian assistance in Darfur and supported African Union...
Applying Agreed-Upon Procedures: House Interparliamentary Groups
GAO-05-236R: Published: Feb 3, 2005. Publicly Released: Feb 3, 2005.
GAO agreed to assist Congress in evaluating the extent to which the Schedules of Receipts, Disbursements, and Fund Balance for five Interparliamentary Groups appropriately reflect the actual cash receipts and disbursements and related fund balances for the years ended December 31, 2002, and 2001. These five groups were the Mexico-United States Interparliamentary Group, Canada-United States Interpa...


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The Tomy Sleepcurve cot mattress will provide a comfortable sleep for baby. The SleepCurve® mimics the way a mother naturally cradles her baby in the crook of her arm, providing a space for the bulge on the back of baby's head and relieving pressure on baby's soft skull bones. This is the optimum position as the spine and head are both supported neutrally, so baby's chin won't be pushed forward onto the chest and the airways can be open fully. Baby's head will be supported and pressure areas will be relieved. The unique shape of the SleepCurve® mattress allows free head and neck movement for your baby. It is clinically proven to aid the correction of Flat Head Syndrome (FHS).

Sometimes babies get some sort of reflux during their first 3 months and it can be very unsettling, which is why a gentle slope has been incorporated into the SleepCurve® mattress so baby's airways can keep clear.

It is very important not to let your baby overheat which is why specially designed channels have been incorporated into the SleepCurve® mattress to dissipate the warm air underneath your baby, helping them to keep cool. The curve also allows hot air to flow freely away from baby's head.

The mattress comes with a pre-fitted hypo-allergenic, breathable protective cover.

Catalogue Number: 211-0977
- 120x60cm
- Promote correct body posture and aids breathing by preventing the chin being pushed forward into the chest
- Relief from pressure on the skull preventing/ remedying flat head syndrome and helps prevent reflux. Supplied with a pre-fitted hypoallergenic washable protector
Sorry, there are currently no sellers for this product.
Large Kitchen Appliance Services
- Free Delivery
- Free Packaging removal
- Product Recycling £9.99
- Product Installation from £9.99
Installation & Product recycling services are available on selected Hotpoint & Indesit products. Applicable installation services will be made available once you have added your appliance to your basket.
For the Installation services listed below, there's no need to worry about arranging a separate installation as our qualified engineers can do this for you:
|Washing Machine
|Electric Cooker
|Gas Cooker
|Tumble Dryer (Condensing only)
|Fridge or Freezer
Unfortunately, we are unable to install built-in appliances or vented tumble dryers and there may be restrictions based on your postcode. Our engineers will install appliances to pre-prepared locations within one metre of existing utility points. Product recycling is on a like for like size basis. For a full list of terms & conditions please see our information page (link - http://www.tesco.com/direct/help/delivery-info.page?). Installation is not available on appliances from our Partners.
Clubcard Boost now on Make your vouchers go further
How does it work?
- Your vouchers are waiting for you at the Tesco direct checkout
- Add the vouchers you want to use and they will double automatically
- The value of your Boost vouchers will be taken from your order, saving you money
Bonus: If the value of your Boost vouchers is more than your order, you'll receive the difference in Clubcard points!Continue shopping


text:
Illinois Public Media News
In-depth reporting from WILL, NPR, the Associated Press, and other sources
Contact WILL News at email@example.com
August 20, 2013
(Courtesy of the Kochiyama family/UCLA Asian American Studies Center)
August 19, 2013
The Japanese-American Internee Who Met Malcolm X
(Sean Powers/WILL)
August 19, 2013
Champaign Writing Rules For Backyard Chickens
Page 547 of 1124 pages ‹ First < 545 546 547 548 549 > Last ›
WILL Newscast audio
NPR News
17-Year-Old Transgender Boy Wins Texas Girls' Wrestling Championship
Father Of Navy SEAL Killed In Yemen Raid Has Harsh Words For Trump
Minus The U.S., Asian Economic Powers Meet To Form Trade Deal
Headstones Vandalized At Jewish Cemetery In Philadelphia
'People's Court' Judge Joseph Wapner Dies At 97
Navy Secretary Nominee Withdraws From Consideration
In A Dramatic Finish, 'Moonlight' Takes Best Picture At The 2017 Oscars
The Early 2000s Are Calling: New Nokia 3310 Phones Revealed
After Kansas Shooting, More Than $1 Million Donated For Victims' Families
Bill Paxton, Prolific Actor And Star Of 'Titanic' And 'Apollo 13,' Dies At 61
More NPR News
WILL Highlights
A short film series examining the severe underrepresentation of African-Americans in the sciences.
Classic Mornings
Get more out of your classical music experience and check out Vic Di Geronimo's blog.
The 21st
21st century radio for the 21st state. Weekdays at 11 am on WILL-AM 580.


text:
Why Days in Diocese?
World Youth Day is a unique celebration of faith and friendship. It is a good idea to extend such a celebration – therefore we are inviting you to stop and catch your breath before the culmination of the World Youth Day in Cracow. We want to experience that Jesus Christ unties us – regardless of cultures, races, languages. That it is He and He alone who is able to make us a real community. This way we, with multiplied joy of faith, will be able to fruitfully listen to the inspiration of the Holy Spirit, not only during the World Youth Day, but also later on every path that the Lord is leading us.
Why the Archdiocese of Szczecin-Kamień?
Despite the fact that it is one of the biggest dioceses in Poland, our Archdiocese is young and it is dynamically developing. We want you to contribute to the history of this community so that your heart can beat in the rhythm of the Church on the Oder and the Baltic Sea. We want you to take from the spirituality of the Church and enrich it.
Why here?
Szczecin is a remarkable part of Poland. Every nook and cranny is filled with an interesting history. The tumult of the big city is merging here with the plenitude of trees, and good mood is brought by an onshore wind. Between the sound of waves and the silent beaches, between blue lakes and green forests, amongst charming houses, beautiful churches and kind people you will certainly spend your time well – those moments will stay in your heart for years.


text:
I don't know how to love
I don't know who to trust.
My heart is gone,
Dissolved into nonexistence.
But yet I still felt the deep,
Stabbing pain when you said
That this is the end?
How can it be
That we live these lives,
So dark and secretive,
So full of hate and distrust?
How can I feel so empty and lost?
We've no more blood to bleed,
Yet it covers our hands?
Why are we so sad,
When we caused this?
Who is left to clean up the mess?
Never have I felt so alone,
Never have I felt so gone.
© Copyright 2017 Trainman238. All rights reserved.
Status: Finished
Genre: Poetry
wow. that was great. i loved it =]
wow woow omg i love it
this song is great
i love it
This was amazing! Very good.
Poem / Poetry
Short Story / Fantasy
Poem / Young Adult
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The Dublin Gaels football team won Friday's home conference game against Acalanes by a score of 32-29.
The Dublin Gaels football squad scored 32 points and restricted the visiting Acalanes Dons to 29 in the Gaels league victory on Friday.
The Gaels now possess a 5-3 record. They put it on the line next when they travel to Miramonte for a Diablo Foothill battle on Friday, November 4. Dublin will face a Matadors squad coming off a 42-6 league loss to Campolindo (Moraga, CA). The Matadors record now stands at 7-1.
Coming up next, the Dons face the Las Lomas Knights (Walnut Creek, CA) in a Diablo Foothill battle, on Thursday, November 10. Acalanes will attempt to advance on its 4-5 season record.
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At Fault Lines, Noel Erinjeri writes about the ACLU's lawsuit against the Orleans Public Defender and its chief, Derwyn Bunton, following the decision to refuse to take on more cases than it can ethically and constitutionally handle.
In its introduction, the complaint on behalf of three detained defendants both explains the obvious and makes a critical error:
3. However, OPD has refused to accept Plaintiffs as clients due to budgetary shortages and excessive caseloads. OPD has instead placed Plaintiffs on a waiting list for appointed counsel. OPD's refusal to represent Plaintiffs means that they must languish indefinitely in jail without counsel until OPD secures adequate resources to provide them with an attorney.
That these three defendants "languish indefinitely in jail" is certainly true. That they do so because of OPD, however, is certainly wrong. The Orleans Public Defender didn't arrest them. They didn't set bail. They certainly didn't set unmakeable bail. And the OPD neither has the wherewithal to "secure adequate resources," nor has somehow neglected to do whatever it is the ACLU apparently thinks they should have done to "secure adequate resources."
4. As long as OPD refuses to represent Plaintiffs, their prosecutions cannot proceed. Meanwhile, Plaintiffs have no access to an attorney for critical pretrial functions that would ordinarily be performed by defense counsel, such as conducting a preliminary examination to challenge their arrests and bail conditions; investigating the allegations; filing motions to preserve potentially exculpatory evidence; or negotiating with the prosecution.
That these three criminal defendants are constitutionally entitled to effective assistance of counsel is beyond question. That this fault is attributable to OPD, however, is not. The Orleans Public Defender didn't tell the state, "no thanks, guys. Keep your money. We just don't feel like representing any more defendants." Had that been the case, the ACLU's complaint would have been directed at the right party doing the wrong thing. It's not.
5. The public defender's budgetary crisis and its denial of counsel to Plaintiffs both result from the State of Louisiana's chronic underfunding of its public defender system. While the legislature yearly appropriates funds for public defense, the public defender system overwhelmingly relies on locally generated fines and fees for revenue.
As shown, the ACLU, unsurprisingly, notes the obvious, that OPD suffers from "chronic underfunding." Most public defenders suffer from the same malady, and one can find almost no public defender office claiming that it's funding is so generous that it doesn't need more money, thank you very much.
But then, the ACLU blames the underfunding in Orleans on the state's method of funding public defense. That too is a mistake. How the state chooses to establish funding is nobody's business but the voters. That the state must, somehow, adequately fund public defense to fulfill a constitutional mandate is the point.
Tax 'em. Collect fees, to the extent that doesn't otherwise violate the Constitution, rent out rooms in the governor's mansion if they want. Whatever. Fund public defense.
9. Plaintiffs bring this class action under 42 U.S.C. § 1983 on behalf of themselves and those similarly situated who have been or will be denied counsel indefinitely because OPD has refused to represent them.
10. Plaintiffs seek a declaratory judgment that OPD's indefinite denial of counsel violates their Sixth and Fourteenth Amendment rights to counsel, as well as their Fourteenth Amendment right to due process and the equal protection of the laws.
So OPD lacks the money to hire sufficient public defenders to handle the caseload, and it's OPD violating the constitutional right to counsel? Does the ACLU not see the gaping hole in its reasoning? Noel tees up the obvious conundrum:
Just what the hell are they supposed to do? Turn people away and they'll rot in jail, and if and when they get counsel precious time has been lost. Take them all in and it's like trying to drink from a firehose…along with a constitutional mandate that not a single drop of water hit the floor. And the defendants are caught in the middle.
Is there a solution? There is, but no one wants to hear it. Provide the money to adequately staff and fund the public defender's office, and let them do their jobs.
So why does the ACLU sue the OPD when it must realize that the OPD is neither at fault nor in disagreement with the central contention that defendants need to be defended and that it takes money to hire sufficient lawyers to handle the caseload? Is it that "no one wants to hear it," or that everybody knows it but nobody outside of the handful of people paying attention gives a damn? Poor criminal defendants are hardly as much of a fun way to allocate tax revenues compared, say, with a football stadium.
So why didn't the ACLU sue the state? Why didn't the ACLU bring writs of habeas corpus on behalf of detained and unrepresented defendants who "languish" in jail because judges have set bail while the state refuses to fund a constitutionally adequate defense?
I suspect that this is the first step in an unfortunately necessary strategic plan by the ACLU, to establish the existence of a constitutional violation and to do so with a defendant, the OPD, who is both disinclined to dispute the existence of that violation, and can't raise the defenses that the state could, such as separation of powers when it comes to compelling the state to allocate funds to public defense.
Thrown in, for good measure, is the probability that no Louisiana judge wants to end his career by being the guy who ordered the state to spend its money on poor criminals, as it would be perceived, rather than good times.
Had the ACLU gone after the real culprit, the legislative and executive branches of government, they could expect a response that they have done their part, establishing a public defense office, and it's now up to the OPD to do its job of defending. Problem solved!
The adequacy of the solution would be hotly disputed, but it then gets into the vague question of what, in a hypothetical sense, is sufficient for effective assistance of counsel. For anyone familiar with Strickland v. Washington, it's clear that the demands are so low as to be satisfied by the fact that a lawyer can stand up (not to be ableist, but) and breathe. Thanks, Supreme Court.
Or to look at this differently, OPD's inadequate funding creates an obviously untenable situation, which is being paid for by indigent defendants languishing in jail for lack of counsel. While OPD can't do a damn thing to fix the problem, other than provide ethically incompetent representation, the challenge to the constitutionality of this massive failure has to start somewhere.
Whether this strategy will prevail remains a mystery, as the bottom line remains that the state just doesn't want to pay for enough attorneys to do the job, fulfill the mandate and provide effective assistance of counsel, as the Sixth and Fourteenth Amendments demand. But something has to give, as the wheels of justice can't keep grinding up the bodies of the indigent because the rest of government has better, funner, things to do with its money. And because there is no clear and certain means by which to compel government to fulfill this constitutional mandate.


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|Property type
|Minimum Stay
|2 nights
Stylish to the max, Black Fern Matakana is a private retreat surrounded by native bush and vineyard views. Get your friends and family together and bring in a gourmet chef to cater a sumptuous meal. Or DIY at the outdoor kitchen by the heated pool. With a spacious open plan living area and large fully equipped kitchen, four ensuite bedrooms plus a separate one bedroom cottage sleeping up to 10, plenty of space!
With spectacular 360o views of the native bush, the valley and vineyards below, the Lodge at Black Fern features a spacious floor plan with four ensuite bedrooms. The fully equipped kitchen is a foodies? delight! The heated swimming pool is surrounded by landscaped gardens with easy access to the outdoor entertainment centre with its gas BBQ grill, fully equipped kitchen, sitting area and gas fireplace.
The open plan living area with vaulted ceiling boasts spectacular views over the neighbouring Ascension vineyard and hills beyond, a 50" wide-screen high definition television with DVD player and Sky TV and a built-in gas fireplace. The kauri wood dining table can accommodate 12 people and the large kitchen area features a gas cooktop and eat-in bar.
The spacious master bedroom also has panoramic countryside views, a large HD TV, and the master bath has ?his and her? basins, an enclosed shower and a bathtub. All bedrooms have king beds and private access to individual decks.
The ?Barn? is a separate fully self-contained cottage with its own king size bedroom, bathroom, kitchen and living area. It can be rented in addition to the Lodge, (but not on its own) to bring the sleeping capacity up to 10 people. Free wireless high-speed internet is available throughout the property.
Nestled in native bush but with expansive views into the surrounding hills and valleys, this retreat is the perfect place to unwind among the natural splendour and renowned culture of the Matakana area. Black Fern is available year-round. Book your stay today!
Special conditions:
Please note we charge by bedrooms occupied, not people.
Minimum stay requirements:
2 nights over statutory holiday weekends.
4 nights over Christmas andamp; New Year (December 19, 2016 - January 4, 2017) andamp; Easter (April 14-18, 2017).
Single Night Supplement: $250 Special Event Supplement $300.
Security Deposit - $400 or 20% of the rental fee, whichever is
greater. All prices include GST. Pets may be allowed with prior approval.
Please see our website blackfernmatakana.com for details.
Extras: Cleaning (included); Linen (included); Refundable Security Deposit (Required, NZ$0.00 per stay); Special Event Supplement (Required, NZ$300.00 per stay);
Ideal for: Couples retreats, family holidays, small weddings/accomodations, executive conferences
Amazing property. Clean and comfortable with fabulous amenities. Would love to stay again.
Beautiful home! Recommend to All.
Beautiful location & views and the pool area was fantastic!
Most beautiful farm house! With countdown and food stores 5min away, covinient location, beautiful views. Kind land lord! Perfect stay :) we used the bbq outside, it was such a nice place with the heater and fairy lights. Definitely well worth the price
|Rate Period
|Weekend Night
Two Bedroom
25 Feb 2017 - 25 May 2018
2 night minimum stay
|Notes: Rate applies for up to 4 people. NZ$1.00/adult and NZ$1.00/child for extra occupants.


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Click on the "Convert & Download Video + MP3" button to start the video conversion. After the video conversion is finished, you can download the converted video in the formats MP4 or 3GP (for mobile phones) or you can download the converted audio as a MP3 music file. This online service is completely for free.
Visit This Channel http://www.youtube.com/user/RossiTania Rosina Mia Cumi era bella rusina mia - Club Dance Remix tarantella tarantula calabria Rosina Mia Cu...
DMCA: Are you the owner of this convertable material and do you want to disable the conversion of your media on clip.dj? Then you can request a conversion block for this video.268
Rosina Mia Cumi era bella Rusina mia Club Dance Remix.
Grazie Calabria by Angelo Mauro. videos by Stan Carrizosa is a musical fantasy by xElixinox Und "eine der besten Folk-Songs mit ...
Angelo Mauro - Rusina Mia - Album Grazie Calabria.
Rusina Mia.


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Tropical Fish Keeping - Aquarium fish care and resources (http://www.tropicalfishkeeping.com/forum.php)
- Cyprinids and Atherinids (http://www.tropicalfishkeeping.com/cyprinids-atherinids/)
- - denisoni barbs (http://www.tropicalfishkeeping.com/cyprinids-atherinids/denisoni-barbs-3422/)
denisoni barbs
we finally got hold of a couple of these! the only ones we could find were real expensive. we went to a different store and found they had some juveniles so got a couple for a bargain price. my bf had wanted these for such a long time. they r so active, ill try to get pics but they dont stay still for long!
Good luck with getting the pics.
They really are beautiful fish, and I look forward to seeing them.
You do know that these fish can reach 6 inches and require a large tank and fast current?
i done research on these fish and have found that they usually grow to 4 inches and i do have current in my tank, thanks, mike some of the comments u make and the way u make them always make me feel like ive done somethin wrong!!!!
thanks kate :)
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text: Share on Facebook Follow on Facebook Add to Google+ Connect on Linked in Subscribe by Email Print This Post See Image Gallery. Ignacio Robles, #9, rushes the ball down the side of the field towards Fallbrook Warrior Miguel Calderon heads the ball to a teammate on their way downfield for a scoring attempt. Fallbrook Warriors battle Longhorns in close game added by Newsroom on February 21, 2013 View all posts by Newsroom → Leave a Reply Cancel Reply Your email address will not be published.


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Edward told the stunned nation during a radio broadcast, "I have found it impossible to carry the heavy burden of responsibility and to discharge my duties as king as I would wish to do without the help and support of the woman I love."
TAUBE: Descending from the Spanish throne →
"The glue that holds the monarchy together is the queen, but there is a great deal more skepticism and even cynicism about the next generation," he said. "You have a dysfunctional family, so it really falls on William and Kate to actually be an upright couple that can engage with the public and be glamorous at the same time. It's fine with the queen on the throne, and Charles won't reign for long, so it really does fall to the next generation to rescue them."


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|Hydra talks about Blizzard's Mist of Pandaria Release Party in Irvine, CA. Fimlys talks about his Release Night. We each talk about our own first impressions and how we are doing so far. We have lots of stories. And Fim is addicted to skinning!
Copyright © 2017 Twisted Nether Blogcast - All Rights Reserved
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So What Does 15 Years Of KDE Look Like?
So, I thought I would take a quick look at what KDE community "looks" like after 15 years under development. So here I will briefly show off three visualisations with no particular comment. I will just leave them here for your amusement.
So let's start with the now-infamous green blobs:
For the uninitiated, a quick lesson: Each column in this visualisation represents the commit history of everyone who has committed to KDE SVN. Each row represents a week, with the most recent weeks being at the top. If the contributor committed during that week, they get a green blob, otherwise it is left empty. For each column the committer, the date of their first commit and the % of weeks in which they committed (of those they /could/) is given.
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Created attachment 568465 [details] [diff] [review]
Conditionalize inspect context menu items.
Test fails when the highlighter is disabled. This is preventing us from turning off the highlighter on aurora.
Where can the testcase be found to verify this fix?
Not sure what you need, but this modified browser/base/content/test/test_contextmenu.html
Without it, the test would have failed in aurora 9 when we disabled the highlighter.


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Noel Gallagher Jams With Paul Weller In London
Former Oasis star Noel Gallagher joined Paul Weller on-stage at the Modfather's intimate London show.
Gallagher -- who split with Oasis last year - guested last night (April 21) on guitar and vocals on versions of Oasis' 2005 song "Mucky Fingers" and Weller's own "Echoes Round the Sun," which featured Gallagher and Oasis guitarist Gem Archer when it was released in 2008. Archer also joined Weller for the night's last song, a version of the Jam's "Start." Weller also played the Jam's "Strange Town" alongside tracks from throughout his solo career.
Weller played the 600-capacity Relentless Garage in Highbury, north London, for U.K. cable TV/Internet service provider Virgin Media. Weller is staging a "takeover" of Virgin's Music on Demand TV service during April, featuring exclusive interview footage. Last night's gig will air on Music on Demand in June.
Weller's new album, "Wake Up the Nation" (Island) was released on Monday (April 19), and was No. 1 on the Official Charts Co.'s midweek albums chart update.


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Maintain a centralized database for customers with service history, purchase history, balances, preferences and more.
Personalize experiences. Know your high spenders, problem makers, customers that are returning after a long absence and more. Our spa loyalty software helps you make every interaction count.
Everything you need to secure the next visit and the visit after that! Our software automates and supports all types of spa loyalty programs.
Set up memberships once and automate the rest. Our software tracks balances, makes redemptions easy, tracks expiry, partial payments, supports recurring memberships and more.
Reward customer loyalty & grow repeat business with our built-in loyalty program. Paperless, hassle free and designed to drive real business results for you.
See which employees and service categories are best at retaining customers and track how well you retain first time visitors. We give you the tools you need to boost loyalty and drive retention.
Customers enter feedback digitally while they're still at your spa. You increase feedback collection rates, the customer gets privacy and you get authentic, tamper-free feedback.
Follow-up immediately on negative feedback. You'll be notified through alerts and our issue management system makes sure nothing slips through the cracks.
Share positive feedback on Facebook, Twitter, your website and even in your own digital menu, all with a click of a button.
Enrich Salons grew their feedback collection from 8% to 45% and saw amazing returns. Read the case study >
"I recently transitioned my business to Zenoti software after researching several cloud solutions. Zenoti is user friendly and has extensive back-end capabilities. Now that I've implemented Zenoti, I've found the staff to provide exceptional customer service. I highly recommend Zenoti for both the software and the support."
- Paulina Saliba (General Manager | Results Laser Clinic)


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Makes about 4 cups of shaved ice
There may be no more elegant granita, sweet and tart. It's a fine finish to any meal, especially alongside some fresh macaroons.
2 cups Champagne, Prosecco, or sparkling wine
1 cup unsweetened apple juice or white grape juice
1/2 cup sugar, preferably superfine
1. Whisk all ingredients together in a bowl until the sugar dissolves and the foaming just starts to subside (some foaming will help the granita stay light and airy as it freezes). Pour into a 9 x 13-inch baking pan. Freeze on the floor of the freezer for about 2 hours, stirring with a fork every 20 minutes. When the granita is frozen but soft, smooth out the crystals and cover the pan with plastic wrap. Tightly sealed, the granita can stay in the freezer for up to 2 weeks.
2. Serve by scraping up the ice crystals with a fork and mounding them in cups, bowls, or glasses.
Wine Granita: Substitute red or white wine for the champagne; increase the sugar to 3/4 cup.
Sweet Dessert Wine Granita: Omit the apple juice and use 3 cups vin santo sweet Marsala or port; decrease the sugar to 1/4 cup.
The Ultimate Frozen Dessert Book
A Complete Guide to Gelato, Sherbert, Granita, and Semmifreddo, Plus Frozen Cakes, Pies, Mousses, Chiffon Cakes, and More, with Hundreds of Ways to Customize Every Recipe to Your Own Taste
by Bruce Weinstein and Mark Scarbrough
Morrow Cookbooks
256 pages; Paperback; $16.95; $23.95 (CAN)
Recipe reprinted by permission.
This page created June 2005
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text: Pull off a flattering silhouette with this Andrea spot print blouse from Joules. Fashioned from a soft, flowy material, this relaxed fitting piece sports a longer back hem, elbow-length sleeves, a round neckline and a contrast colour back. Team with skinny jeans and canvas trainers for the ideal weekend combination.Check stock and price


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Traditional freelancers and home workers - women assembling clothing or packing envelopes, for instance - will be increasingly outnumbered by 'teleworkers', home-based operatives electronically linked to central computer systems.
Techno-optimists, such as Bill Gates of Microsoft (who lives in a computer-controlled mansion) praise teleworking - its autonomy and flexibility. But one obstacle to the brave new world of the 'virtual office' has not received the attention it should have - the cramped dimensions of the typical British home.
As Andrew James, who publishes Home Run magazine from an office at the top of his house in Chiswick, points out, the person working from home needs, above all, dedicated space. The space should be quiet, well ventilated, private, and safe. 'Sharing the breakfast table is not really satisfactory. You don't want to have to pack and unpack your materials every day,' he says.
But few people have such spare space. Teleworkers in particular are likely to be low- paid employees and live in relatively low-priced housing, where quality standards are falling rather than improving.
Linda Sheridan, at Liverpool University, who with Professor Valerie Karn at Manchester University recently published a study of new British homes for the Rowntree Foundation, believes there are 'tremendous problems involved with home working because of the inadequacy of British homes.' British houses are now smaller than houses in any other country in Europe. New housing association houses are 'of lower amenity value than low-rise housing built by councils in the 1960s and 1970s' - not a flattering comparison - and privately built houses follow a similar trend.
The outlook for a future in which millions of British employees work from their homes is therefore not particularly good. Although various laws provide guidelines on minimum standards for space and amenities, these are very basic (11 cubic metres per person in an office for instance).
As Ann Goodwin, at the Institute of Environmental Health Officers, says: 'What local authority has the resources to check on relatively low-risk workplaces? Dangerous activities, such as the handling of chemicals and the use of machines have to take precedence.' And which local authority has the resources to grapple with giant corporations (who as employers are legally responsible for the safety of employees under the Health and Safety at Work Act) to enforce standards over thousands of individual workplaces?
Some people are already thinking of ways around the space problem. A contributor to Home Run magazine recommended using a garden shed as an office, although this only suits people with gardens. US firm Rubbermaid is aiming a foldaway workstation specifically at space-starved teleworkers. Mike Gowen, UK chief executive, admitted demand was moderate at present. But he added: 'There is a huge potential. People don't have the space at home to make an office and need something that can be folded up and put away.'
So while optimists dream of the flexible worker in his own electronic castle, the reality may be more prosaic - someone sitting in a corner of the living room tapping away on the keyboard, trying desperately to ignore the distracting noise and activity all around.
(Photograph omitted)Reuse content


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Talk about spitting images. These little celeb apples certainly didn't fall far from the tree; they're carbon copies of their gorgeous mothers.
Everything in this slideshow
Soleil Moon Frye's daughter Jagger
Case in point is Soleil Moon Frye's daughter Jagger who is just a Punky Brewster 2.0. It's hard to tell her apart from her mom flashing back to 30 years ago when she starred in the hit TV show. The actress's other daughter Poet, 8, is also a look-a-like and with baby number 3 on the way, there's definitely going to be more happy chaos.
Photo courtesy of Instagram
Reese Witherspoon's daughter Ava
As Reese Witherspoon's only daughter, she is always going to draw comparisons with her Oscar winner mother. But Ava's likeness to her mom is spooky. From the same golden blond hair, fine features, and pointy chin, the 13-year-old (shown here at age 10) has been, and is becoming, a replica of her total beauty of a mother.
Photo courtesy of Getty Images
Cindy Crawford's daughter Kaia
Cindy Crawford may have had a change of heart about allowing her 11-year-old daughter Kaia to get a jump start on her modeling career, but there's no denying that the youngster has a bright future ahead of her in the beauty industry. Looking the spit of her mom in every way, the brunette beauty, shown here in 2011, can also be assured that she will age amazingly well just like her 47-year-old mom.
Photo courtesy of Getty Images
Maggie Gyllenhaal's daughter Ramona
Maggie Gyllenhaal and her 6-year-old daughter Ramona have similar brown bob hairstyles and piercing blue eyes showing the continuation of the stunning family gene pool is intact. And already with a quirky style like her movie star mom, Jake Gyllenhaal's little niece (pictured in January of last year) is one to watch in Kid Hollywood.
Photo courtesy of Getty Images
Jennifer Garner's daughter Violet
If there was one facial characteristic Jennifer Garner could have passed on to daughter Violet to send her off the cuteness scale, then those adorable dimples are definitely a must-have. The seven-year-old, seen shopping last summer, also hints of her hunky dad Ben Affleck but she's definitely a mama's girl.
Photo courtesy of Getty Images
Nicole Richie's daughter Harlow
Nicole Richie's less than simple life as mom to Harlow Madden must be even more sweet given that the adorable munchkin looks just like her. From the golden wavy hair and cherubic face, this darling gets a 10 out of 10 for being a marvelous mini-me.
Photo courtesy of Getty Images
Katie Holmes' daughter Suri
As Suri Cruise gets older, she's looking more and more like her mom Katie Holmes. Of course, traces of her dad Tom Cruise are clear to see, but this mother-daughter, duo is a beauty force to be reckoned with. The silky straight dark hair and wide eyes and button nose is all Katie.
Photo courtesy of Getty Images


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Browse other items in the Evo collection from Wayside Furniture in the Akron, Cleveland, Canton, Medina, Youngstown, Ohio area.
The Evo Queen Bedroom Group by Legends Furniture from Wayside Furniture. We proudly serve the Akron, Cleveland, Canton, Medina, Youngstown, Ohio area. Product availability may vary. Contact us for the most current availability on this product.
Crafted from premium select hardwoods and veneers, the Evo collection presents beautiful furnishings for your home. Featuring English dovetail drawers with ball bearing glides, felt-lined drawers, tall legs, a sleek and modern design and an upholstered headboard, the pieces highlight contemporary style. Accommodating and elegantly constructed, these furnishings will serve you well for years to come.
The Evo collection is a great option if you are looking for Contemporary furniture in the Akron, Cleveland, Canton, Medina, Youngstown, Ohio area.
These images are representative of this group as a whole and may differ from this item.
Nothing on this page may be copied or reproduced without explicit permission.
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