text:
Madame Defarge from A Tale of Two Cities had it right—knitting is a badass skill to have during the best of times—or the worst of times…like when the eventual zombie apocalypse hits. This groovy tote bag reminds one and all to pick up the needles sooner rather than later.
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Ubuntu with Google Tools
Ubuntu with Google Tools
The DVD with this issue includes a special version of Ubuntu Linux with several of the most popular Google Tools configured to open directly from Ubuntu's Applications window.
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Read full article as PDF:003-003_dvd_KORR1.pdf (1.58 MB)
NoScript Flaw
Mozilla's script blocker add-on could be putting malware sites on the whitelist.
RIP SSLv3
The Internet community officially banishes the notoriously unsafe Secure Sockets Layer protocol.
Mate 1.1 Arrives
Popular desktop environment continues the Gnome 2 legacy – with new support for the Gnome 3 toolkit.
US Government Requires HTTPS
The Obama White House has issued a memorandum telling all US government agencies they must use HTTPS for all websites and web communication.
Mozilla Signs Firefox Add-Ons
New program will dial up security for the Firefox browser.
Fedora 22 Arrives
Red Hat's community distro embraces the cloud.
Code.org and College Board Team Reach Out for Talented High School Coders
New partnership will bring more and better CS training to US schools
A Peek Inside TeslaCrypt Ransomware
Criminals offer online help over Tor network
Mumblehard Still Spams
Sophisticated malware is still present on Joomla and WordPress sites around the world.
Launchpad Gets Git
Future versions of Ubuntu's code service will support the popular Git version control system used with Linux and other open source projects.

subset: commoncrawl


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Alex and Chloe GOUJON TRIPLE - ANNEAU DE DIAMANT (3MM) RING - I love the contrast of the diamond and oxidized sterling silver.#3576
Zeto Double Layer Gauze Coat. 100% Organic cotton. Soft touch fine cotton jersey. The double layered gauze to keep you warm and cozy. Pull over cowl neck with 3/4 sleeve, finished with elastics.#3572
Hangbag, metallic, light and decorative heart shaped hanger for bags and all hangable objects - by Ototo [Editor's Note - genius! bag hook + keychain!]#3568
Cynthia Rybakoff's heavyweight sterling silver handmade chain can be worn as a necklace or belt. With hand stitched royal purple double-faced satin ribbon ties, it can be made to order in other colors.#3567
Yoga Army - hand-made silk one shoulder dress (also comes in navy, blue and black)#3559
A luxury city jacket is all I need this season. I love this A-line coat by Lily and Jae. The cut is chic. I love that it's more fitted through the bust with a slight flare through the waist. The 100 percent cotton fabric reminds me of velvet.#3557
japanese label mastermind ss '09: military meets dandy. this collection is too cool for school, tailoring and fabrics built to perfection.#3554
Absolutely stunning photo shoot by Steven Meisel for Italian Vogue. The level of detail is remarkable.#3553
Pull these cable-knit knee-highs on over long johns, pour yourself a steaming mug of joe and gaze out over the snow covered valley below. By Eloise.#3549
I'm kinda wishing Modern Amusement made a women's version of this peacoat. I really like the trim on the lapels, and it even comes with a neat red feather pin.#3547
I want to keep the frightful weather at bay with this stunning cinched waist puffer by Rudsak. Definitely not your brother's North Face!#3544
Unruly Scarves of Amsterdam are designed by typographer/designer Niels Shoe Meulman, have 6 different hip styles in various colors and are 100% silk.#3542
This look by Best Behavior is perfect for the office or school and even a date. I love the sweater, it's perfect for those cold rainy California days.#3540
new bags from VAEL Project are looking real slick. im feeling the premium leather and copper accents.#3535
Worship Worthy just released their Holiday '08 collection online. It features printed and sequins bodysuits and leggings as well as some great tees.#3534
I'm loving this silk and pearl necklace from Candace Ang! It's very Lanvin-esque. As seen on MTV.COM#3533
Adding some edge to tradition, Peckham Rye's silk scarves for 20 LTD. are limited to ten each, one with marijuana leaves, the other with skulls and crossbones.#3532
Dark Tales features fantastical styling in a homage to the beauty and dark intrigue of fairy tales.#3531


text:
Featuring: Trinidad James
Song: Bet I
Fresh off earning reader approval for February's Feel It, ATLien R&B sensation Jacquees returns to our pages with another fresh single off his forthcoming EP. On this leisurely joint, the unsigned crooner revels in... More
Posted one year ago
Featuring: Rich Homie Quan | Lloyd
Song: Feel It
Last August, Jacquees earned positive reader reviews for Won't Turn It Down, a promo cut designed to be blasted at high volume. Now, the Chris Breezy protégé is back on our pages with a new single and, if you enjoyed... More
Posted one year ago
Featuring: Chris Brown
Song: Won't Turn It Down
You can call the cops, you can offer to pay, you can even threaten me with violence, but when it comes to the latest offering from Jacquees, I Won't Turn It Down. Can you blame me? Drumma Boy's beat alone—featuring... More
Posted one year ago
Song: Dream Girl
Months after singer Jacquees came Clean with his DJBooth debut, the musician makes his soulful mark on our pages yet again with his third feature Dream Girl. With summer at its peak the sultry, bell-laced production on Dream... More
Posted 2 years ago
Song: Candy Rain (Quemix)
If Jacquees was hoping to take me back to my middle school dances, he's succeeded. For the singer's new video he grabs Soul For Real's classic '94 jam Candy Rain and gives it his own, updated flavor (a Quemix, if you... More
Posted about 3 years ago
Featuring: Jacob Latimore | Issa
Song: Clean
If you were to reach out and touch Booth newcomer Jacquees, you'd hear an audible squeak; the ATL crooner is just that damn Clean. On his latest promo single, a refreshing synth beat by CornaBoyz alumnus Tyrell Bing... More
Posted 3 years ago
Sample Text - Sample Link


text:
What's the secret to running a successful business? Knowing who to trust.
Australian Business Solutions Group
Watch the clip below to find out more about the range of services we offer to help you run and grow your business.
Free-trade agreements- What they mean and how to unleash opportunities for your business seminar-
View the video of our speakers including full slide deck from our exporting seminar held at The Brisbane Club 3.3.15
29 June 2015
Employee Share Schemes Bill Passes Through Parliament
The Tax and Superannuation Laws Amendment (Employee Share Schemes) Bill 2015 was passed through Parliament yesterday, and the... More
22 June 2015
Business Law In Review
Welcome to our latest edition of Business Law in Review designed to keep you up-to-date with corporate and commercial legal... More
17 June 2015
ABLA secures big win for employers in annual leave test case
Last Thursday, a Full Bench of the Fair Work Commission handed down a landmark decision in which it granted numerous claims... More
16 July 2015
to 16 July 2015
Free Seminar | How to take your business to China
On the back of the historic China Australia Free Trade Agreement, Australian Business and Consulting Solutions (ABCS) presents ... More
21 July 2015
to 21 July 2015
Manufacturing for global markets made easy
Have you ever thought about exporting your products but felt you were 'too small' or 'too expensive' to be competitive on a... More
28 July 2015
to 28 July 2015
Exporters' test drive - Taking off in new markets
Have you recently begun exporting and want to refine your sales pitch to ensure you are putting your best foot
forward? More
©2014 Australian Business Solutions Group


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Badger Mountain Hikers Enjoy New Watering Fountain!
I've been hiking Badger Mountain again with my dogs. I'm trying to do it every OTHER day.
I've decided it's a pretty good leg work out to go straight up the mountain and then run down it. I did that 3 times over the weekend and my legs were sooo sore! Still are!
Anyway, I want to say thank you to whomever is responsible for putting the AWESOME water fountain at the base of the hill


text:
Disclosed herein are methods for harvesting adipose tissue so as to preserve an increased population of viable microvascular endothelial cells. Adipose tissue containing microvascular endothelial cells is harvested using a collection apparatus incorporating an elongate cannula having apertures with tissue cutting edges. A sub-ambient pressure is applied to a lumen in the cannula to draw the adipose tissue through the aperture where it is then severed using the cutting edge to disrupt the connective adipose matrix. This harvesting provides a cleaner, more homogeneous sample of adipose tissue, thereby increasing the population of viable microvascular endothelial cells obtained through further processing. Rapid and easy methods for the further homogenization of the harvested adipose tissue are also disclosed.
1. A method of collecting fat tissue from a patient, said method comprising the steps of:
providing an elongated tubular cannula having a distal tip end and an opposite proximal end, a lumen extending in said cannula, and an aperture opening outwardly from said lumen on said cannula adjacent to said distal tip end and defining a tissue cutting edge surface;
inserting said cannula into a patient so that said aperture contacts fat tissue;
cutting said fat tissue with said tissue cutting edge surface;
creating a suction force in said lumen to draw said fat tissue from the patient through said aperture into said lumen;
selectively controlling the amount of suction to said lumen; and
collecting said fat tissue from said lumen into a collection chamber, wherein said collection chamber includes an entrance port through which said fat tissue passes from said lumen in the step of collecting, said method further comprising the step of discharging said fat tissue from said collection chamber through said entrance port and through a homogenizing member having apertures with cutting edge surfaces and sized to lower the viscosity of the fat tissue by disrupting a connective matrix without exposing cellular components within said fat tissue to excessive shearing forces.
2. The method of claim 1, wherein said collection chamber comprises a syringe having a plunger, and wherein said step of selectively controlling the amount of suction to said lumen comprises manipulating said plunger.
3. The method of claim 2, further comprising the step of providing the proximal end of said cannula with means for retaining the plunger in a withdrawn position.
4. The method of claim 1, wherein said apertures have a diameter of about 0.2 to 3.0 mm.
5. The method of claim 1 further comprising the step of passing said fat tissue from said collection chamber through said homogenizing member into a second syringe.
6. The method of claim 1 further comprising the step of coating the homogenizing member with parylene.
7. The method of claim 1 further comprising the step of providing a plurality of apertures opening outwardly from said lumen on said cannula adjacent to said distal tip end, said apertures being elongated in a direction along the length of said cannula, wherein each aperture further comprises opposite distal and proximal ends, and opposite side edges, and wherein said distal and proximal ends, and opposite side edges of each aperture comprise a tissue cutting edge surface.
8. The method of claim 1, wherein said aperture is elongated in a direction along the length of the cannula, and the length of said aperture is about three times its width.
9. A method for homogenizing fat tissue obtained from a patient prior to digestion of said fat tissue, said method comprising the steps of:
obtaining fat tissue from a patient, said fat tissue comprising microvascular endothelial cells;
disposing said fat tissue in a collection container comprising a first port;
positioning a planar homogenizing member comprising a plurality of apertures adjacent to said first port, wherein said plurality of apertures comprise at least one tissue cutting edge surface;
discharging said fat tissue from said collection container first port in a first direction generally normal to said planar homogenizing member through said plurality of apertures, so that said tissue cutting edge surface of said plurality of apertures cuts said fat tissue so as to free said endothelial cells;
receiving said fat tissue discharged through said homogenizing member in a receiving container through a second port; and
discharging said fat tissue received in said receiving container through said second port in a second direction opposite the first direction and generally normal to said planar homogenizing member through said plurality of apertures into said collection container.
10. The method of claim 9, further comprising the step of discharging said fat tissue from said collection container in said first direction through said plurality of apertures by use of a syringe.
11. The method of claim 10, further comprising the step of discharging said fat tissue from said receiving container in said second direction through said plurality of apertures by use of a syringe.
12. The method of claim 10, wherein said collection container is formed by said syringe, and said step of obtaining comprises withdrawing fat tissue from the patient using said syringe.
13. The method of claim 9, wherein each of said apertures has a diameter of between about 0.2 mm and 3.0 mm.
14. The method of claim 9, further comprising the step of coating the homogenizing member with parylene.
15. The method of claim 9, further comprising repeating the step of discharging said fat tissue from said collection container to said receiving container.
16. The method of claim 15, further comprising repeating the step of discharging said fat tissue from said receiving container to said collection container.
17. The method of claim 9, further comprising the step of discharging said fat tissue from said receiving container in said second direction through said plurality of apertures by use of a syringe.
18. The method of claim 9, further comprising the steps of:
coupling said collection and receiving containers using a hub assembly attached to both said first and second ports; and
wherein said step of positioning includes mounting said homogenizing member within said hub assembly.
19. The method of claim 18, wherein said collection chamber is formed by a syringe having a plunger, the step of discharging said fat tissue from said collection container in said first direction being accomplished by depressing the collection chamber syringe plunger, and said receiving chamber is formed by a syringe having a plunger, the step of discharging said fat tissue from said receiving container in said second direction being accomplished by depressing the receiving chamber syringe plunger.
This is a continuation of U.S. patent application Ser. No. 08/270,073, filed Jul. 1, 1994, now abandoned.
The present invention is directed to a method for the harvesting of microvascular endothelial cells. More particularly, the present invention relates to a method for the collection of adipose tissue and the initial refinement of microvascular endothelial cells for deposition on the surface of synthetic prosthetics.
Arteriosclerotic vascular disease is a leading cause of death throughout the world. While sophisticated medical techniques such as arterial endarterectomy and percutaneous balloon dilatation are being applied more and more often to treat pathologic stenotic occurrences, quite often the most effective therapy is the surgical removal of the occluded section of the vessel. In such cases, the restoration of blood flow to ischemic tissue depends on the implantation of a vascular graft.
Although autologous vascular tissue is the most suitable material for use in such grafts, prior surgical intervention and advanced vascular disease often limit the availability of such tissue. Accordingly, it has become common in recent years to implant vascular grafts fabricated of synthetic materials. While commercially available synthetic grafts are extremely durable and may be used to successfully restore blood flow to occluded tissue, associated thrombogenic complications reduce their effectiveness. In particular, smaller diameter vascular grafts tend to become dysfunctional as they are blocked by the normal clotting mechanisms. Specifically, the synthetic surface of the graft promotes the deposition of fibrin leading to associated cellular adhesion and occlusion of the vessel. Consequently, the long term prognosis for non-coated synthetic grafts is relatively poor.
To circumvent the problems associated with non-coated synthetic vascular grafts, procedures are being developed for lining prosthetics with human endothelial cells to produce a non-thrombogenic cell surface such as exists in native human vessels. The endothelial lining of natural blood vessels is a highly complex, multifunctional cell surface which interacts with both the blood and the underlying vessel wall components to maintain physiological homeostasis. Tests with animals have shown that the deposition of a functional large vessel endothelial cell lining on the interior surface of synthetic vascular grafts decreases the formation of thrombogenic occlusions and minimizes the disruption of blood flow through the vessel. However, harvesting a sufficient number of large vessel cells from a donor is difficult at best.
Recent advances in molecular biology and tissue culture have allowed the isolation and subsequent propagation of large vessel endothelial cells. In practice, the use of cultured large vessel endothelial cells is expensive, complicated and subject to inherent limitations. One problem is that cell culture techniques are highly technical requiring trained personnel and the use of specialized equipment under laboratory conditions. Yet, even under the best of conditions, the yield of cultured large vessel endothelial cells may be low. Moreover, typical seeding procedures using cultured cells require the use of specialized media under complex conditions to assure the complete and even deposition of endothelial cells on the synthetic surface of the graft.
In addition, cultured cells are generally not derived from the patient receiving the graft and, accordingly, may precipitate a wide range of immunological complications. If the immune response of the patient is not attenuated, the transplanted endothelial cells will likely be attacked and stripped from the surface of the graft by the body's defenses. Conversely, if the patient's immune system is artificially suppressed it may lead to life-threatening, opportunistic infections.
In view of these and other complications associated with the use of large vessel endothelial cell treatments of prosthetic devices, alternative methods of reducing the inherent thrombogenicity of synthetic materials have been developed. In particular, it was quickly recognized that human microvascular endothelial cells could be effectively used to render synthetic grafts non-thrombogenic.
Microvascular endothelial cells are derived from capillaries, arterioles and venules and are present in an abundant supply in most body tissues. While endothelial cells may be isolated from tissues such as brain, lung, retina, adrenal glands, liver and muscle tissue, the use of fat tissue as a source for these cells is preferred due to its abundance, availability and because its removal should not adversely affect the patient being treated. Quite often, microvascular endothelial cells are present in concentrations of 106 cells per gram of fat or higher, providing an ample source of materials for high density deposition procedures. Moreover, as the microvascular cells used to treat the synthetic graft are usually autologous, that is, taken from the recipient of the vascular prosthesis, immunological complications may be obviated.
Typically, microvascular endothelial cells are isolated from autologous adipose tissues such as perinephric fat, subcutaneous fat, omentum, or fat associated with the peritoneal cavity. Harvesting usually takes place under sterile conditions with the required amount of fat removed in one procedure. The collected tissue may then be washed before being transferred to a buffered digestive solution generally containing proteolytic enzymes such as collagenase, papain, trypsin, and mixtures thereof.
The adipose tissue is digested at 37� C. for a selected period to disrupt the connective matrix and disperse the cellular components including microvascular endothelial cells. Following digestion, the cellular components may be separated by low speed centrifugation to provide a cell-rich pellet. The pellet may be washed and used in the deposition procedure or purified further using a continuous gradient. In either case, purified cells are diluted in buffer and subsequently incubated with the synthetic prosthesis to provide endothelialized surfaces.
Commonly, collection of the desired adipose tissue involves the use of a suction pump connected to a collection apparatus having a needle or cannula. For example, U.S. Pat. Nos. 5,035,708 and 4,834,703, incorporated herein by reference, disclose the collection of adipose tissue using a suction pump to provide the necessary vacuum. However, such collection devices and associated methods tend to employ strong, uncontrollable suction that is extremely rough on the microvascular cellular components of the collected tissue. The resulting disruption of the relatively fragile cellular membranes can substantially lower the viability of the harvested cells. This, in turn, dramatically reduces the efficiency of the deposition process. While such collection procedures may provide sufficient adipose tissue, samples collected using such techniques generally require several additional labor-intensive preparatory steps to assure an adequate concentration of relatively pure microvascular endothelial cells for eventual deposition.
Further, source tissue collected using suction pumps is often relatively dirty, contaminated with unwanted body fluids and non-adipose cellular debris. Rather than obtaining translucent, white samples as seen in relatively pure adipose tissue, samples collected using pump-generated vacuums often appear bloody, with concentrations of connective or membrane tissue dispersed within the fat.
The incorporated contaminants interfere with each step of microvascular endothelial cell isolation including the initial homogenization and preparation of the collected sample for digestion. Moreover, such contaminants directly inhibit the enzymatic activity of the proteolytic enzymes leading to incomplete digestion of the sample and a corresponding reduction in the yield of non-adipose cellular components subsequently obtained by centrifugation. Finally, those cells which are collected and pelleted contain increased level of non-endothelial components. The use of such contaminated pellets further lowers the efficiency of the cell deposition procedure and interferes with the homogeneous layering of endothelial cells on the prosthetic surface. Consequently, the patient may have to endure more extensive liposuction than would otherwise be required in order to provide a sufficient number of microvascular endothelial cells.
As the efficiency of the endothelialization process is lowered at each step along the way by contaminants, the importance of starting this procedure with a relatively clean sample is evident. That is, a small increase in the amount of contaminating materials initially collected can dramatically reduce the yield of viable microvascular endothelial cells available for deposition on the surface of the synthetic graft. In addition to increasing the amount of adipose tissue which must be initially collected, the inevitable reduction in cell viability due to contaminating materials must be compensated for by longer deposition times or additional purification steps, both of which reduce the operating efficiency of the entire procedure. This can be particularly detrimental if the cells are to be collected immediately prior to the implantation of the prosthetic device.
Accordingly, a need exists to improve the yield of viable endothelial cells recovered from adipose tissue collected from a patient preparatory to implantation of a synthetic prosthesis. That is, microvascular endothelial cells which are present in a fat specimen should be more efficiently separated from the fat cells, blood cells, connective tissue, and other materials that are present in the specimen, so that a larger number of such endothelial cells are available to be deposited onto the synthetic graft.
In addition to the actual problems associated with the collection of material, the use of a suction pump complicates the operating environment and interferes with the surgeon's ability to freely maneuver the adipose tissue collection apparatus. More particularly, the collection apparatus is usually attached to the vacuum source via thick, unwieldy hoses that severely compromise the maneuverability of the collection tip. Such pumps often do not allow the precise, real time control of the strength of the vacuum at the collection tip, making it difficult to maintain constant, even harvesting of the desired source tissue. This lack of convenience and precise control inevitably results in the aspiration of undesirable tissue, thereby increasing the contaminant level of the samples or resulting in the collection of less preferable adipose tissue containing lower levels of microvascular endothelial cells. Further, vacuum sources, especially those approved for use in medical procedures, are generally complicated instruments that are relatively expensive to maintain.
In view of the deficiencies of the related technology as outlined above, it is an object of the present invention to provide an efficient, cost effective method for the collection of adipose tissue containing microvascular endothelial cells.
It is another object of the present invention to provide a reliable convenient method for the collection of substantially pure adipose tissue containing high levels of microvascular endothelial cells with a minimum of blood cells, connective tissue and other contaminants.
It is still a further object of the present invention to provide a reliable convenient method for the rapid homogenization of adipose tissue to facilitate the subsequent separation of microvascular endothelial cells.
These and other objectives are achieved by the present invention which, in a broad aspect, is directed to efficient, reliable and cost effective methods for the harvesting of adipose tissue containing identifiable cellular components such as microvascular endothelial cells. More particularly, the present invention is directed to methods of harvesting adipose tissue so as to preserve an increased population of viable endothelial cells using a collection apparatus generally comprising a variable volume container, typically a syringe assembly, attached to an elongated cannula. The elongated cannula, in fluid conducting communication with the variable volume container or syringe, preferably includes apertures appropriately sized and configured to minimize stress placed on cellular components while disrupting the connective matrix of the adipose tissue. That is, by collecting adipose tissue using specifically configured cannular apertures, the yield of endothelial cells may be substantially increased. Further, the collection apparatus is inexpensive, lightweight, easy to manipulate and allows accurate control of the applied suction.
The tissue harvesting methods of the present invention generally begin by inserting at least a portion of the cannula of the collection apparatus into the patient and directing the cannula tip to the area where the adipose tissue is to be collected. Preferably the harvesting procedure is carried out under aseptic conditions. Optionally, a saline solution or other biocompatible liquid may be injected into the collection area of the patient prior to harvesting to loosen the adipose tissue matrix. Following the insertion and positioning of the cannula tip, sub-ambient pressure is generated in the central bore of the syringe by drawing back a displaceable piston affixed to a plunger. If desired the piston may be retained in this withdrawn configuration by a locking mechanism attached to the plunger and designed to interact with the body of the syringe. The locking mechanism frees the hands of the operator and, when combined with the light weight of the collection apparatus provides enhanced maneuverability. In any case, the sub-ambient pressure in the central bore suctions the adipose tissue from the selected collection area, into the disruptive apertures of the cannula, through the cannula body and into the syringe assembly. As the central bore of the syringe fills with collected tissue, the sub-ambient pressure slowly equilibrates. Once the central bore of the syringe is substantially filled with relatively homogeneous adipose tissue the cannula tip is removed from the patient.
Another aspect of the present invention allows for the collected adipose tissue to be readily homogenized and washed with aqueous solutions to remove contaminating matter. Following removal of the cannula tip from the patient, the cannula may be detached from the syringe assembly. A filter, contained in a filter hub, may then be attached to the syringe assembly where the cannula was previously affixed. A second syringe assembly, preferably the same size as the first is then attached to the opposite side of the filter hub. When so joined, the piston of the first syringe assembly is substantially rearward in the syringe and the piston of the second syringe assembly is in a substantially forward position. By using the plungers to displacing the two pistons, the collected fat may be rapidly homogenized as it is forced through the filter which transects the flow path of the sample tissue. Optionally, rinse solutions may be added during the homogenization to separate contaminants from the endothelial cell rich adipose tissue homogenate. After homogenization and rinsing, collected adipose tissue, now substantially free of intact connective tissue and other contaminants, may be transferred to appropriate containers for digestion and further purification.
Additional objects and advantages of the present invention will be apparent from a reading of the following detailed description and exemplary preferred embodiment of the invention taken in conjunction with the appended drawing figures in which like reference numerals denote the same feature or features which are analogous in structure.
FIG. 1 is a perspective view of an adipose tissue collection apparatus showing attachment of the cannula to a syringe assembly with the plunger in an inserted position;
FIG. 2 is a partial sectional view of an adipose tissue collection apparatus showing the plunger held in a withdrawn position by an exemplary locking mechanism;
FIG. 3 is a partial sectional view of two syringe assemblies interconnected by a filter hub illustrating a configuration of the present invention used for homogenization of harvested adipose tissue;
FIG. 4 is a cross-sectional view taken across line 4--4 of FIG. 3 showing a homogenization filter according to the present invention;
FIG. 5 is a partial perspective view of a tip of a cannula used to provide high yields of microvascular endothelial cells in accordance with the teachings of the present invention;
FIG. 6 is a cross-sectional view taken along line 6--6 of FIG. 5 showing the positioning of the collection apertures adjacent to the tip of the cannula;
FIG. 7 is a partial perspective view of a tip portion of an embodiment of a cannula used to harvest adipose tissue;
FIG. 8 is a cross-sectional view taken along line 8--8 of FIG. 7 illustrating the elliptical shape and positioning of the collection apertures adjacent to the tip of the cannula;
FIG. 9 is a partial perspective view of a tip portion of another alternative embodiment of a cannula used to harvest adipose tissue.
Although this invention may be embodied in many different forms, there are shown in the drawings and will be described in detail specific embodimentsthereof with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and isnot intended to limit the invention to the specific embodiments illustrated. In particular, it must be emphasized that the present invention provides for the association of a wide variety of syringe bodiesand cannulas beyond those shown in the figures.
Moreover, the present invention may be used to harvest any identifiable cells associated with the adipose tissue matrix and is not limited to the harvesting and isolation of microvascular endothelial cells. As used herein the term "identifiable cellular components" refers to those cells which may be recognized by commonly used immunogenic, chemical or physicalseparation methods or tests.
Similarly, while the endothelialization of vascular prosthetics is an important object of the present invention, those skilled in the art will appreciate that the cellular products collected may be used to treat otherimplantable devices. Implants which can be treated to produce an endothelial cell lining or covering include, but are not limited to, intravascular devices such as artificial hearts, valvular prosthetics, andnatural or artificial valve leaflets. The collection apparatus and methods of this invention for harvesting endothelial cell rich adipose tissue may be used in the treatment of surfaces comprised of known synthetic materials such as polyester, polytetrafluoroethylene, or fixed and unfixednaturally occurring materials such as veins, arteries, heart valves and other tissues from animal sources, including humans.
Turning now to the figures, FIGS. 1 and 2 show an adipose tissue collectionapparatus 10 essentially comprising a variable volume container in the formof syringe assembly 12. Syringe assembly 12 is in fluid tight communicationwith an elongated cannula 14 having a lumen 26 and at least one collection aperture 16 appropriately configured for the relatively homogeneous collection of adipose tissue containing microvascular endothelial cells. Syringe assembly 12 generally comprises a hollow tubular body 18 defining a central bore 20 having a displaceable piston 22 sealingly disposed therein. Preferably piston 22 is affixed to an elongate plunger 28 which extends through an opening in the rear of hollow tubular body 18. By manually displacing plunger 28, piston 22 may be reversibly moved along the length of central bore 20. Sealing rings 31 ensure that piston 22 maintains good contact with the interior of tubular body 18 as it moves longitudinally. An entrance port 24 at the forward end of hollow tubular body 18 provides for fluid conducting communication between cannula lumen 26 and central bore 20.
Preferably syringe assembly 12 is a Toomey-type syringe (Sherwood Medical Co., St. Louis, Mo.) having a tapered tip 30 defining entrance port 24. While a Toomey-type syringe is preferred due to the ease of switching cannulas or other attachments, syringe assemblies having other types of connection mechanisms such as threaded connectors, catheter tips or luer locks are compatible with the present invention. Typically, hollow tubularbody 18 of syringe assembly 12 is formed of an inexpensive, non-reactive material such as polypropylene or other rugged polymer composition. Of course, those skilled in the art will appreciate that the size and fluid capacity of syringe assembly 12 may vary based on the amount of adipose tissue to be collected. However, for obvious reasons it is preferable thatsyringe assembly 12 be of sufficient volume to harvest the desired amount of adipose tissue in one collection procedure. As the volume of adipose tissue needed to provide the necessary amount of endothelial cells for an average cell deposition procedure is on the order of 20 mls to 50 mls, a preferred syringe volume is approximately 60 mls. Gradations 32 on the sides of most commercially available syringes provide an easy method for monitoring the amount of fat collected.
Optionally, syringe assembly 12 is provided with a locking mechanism 34, attached to plunger flange 48 which reversibly engages annular flange 38 at the rear of hollow tubular body 18 to retain plunger 28 and attached piston 22 in a withdrawn configuration. This configuration is illustrated in FIG. 2. When plunger 28 and piston 22 are in a substantially forward position, as shown in FIG. 1 the inner surface of hollow tubular body 18 will act on shoulders 36 of locking mechanism 34 to maintain it in a closed position. However, when plunger 28 is withdrawn past a certain predetermined point where shoulders 36 are no longer restrained, locking mechanism 34 will spring open due to the elastic memory of the mechanism material. When in an open position, shoulders 36 of locking mechanism 34 will be positioned to engage annular flange 38 as well as plunger flange 48 thereby preventing plunger 28 from reentering hollow tubular body 18. However, plunger 28 may be easily moved to a forward position by manually compressing locking mechanism 34 to reduce the diameter of shoulders 36 where they disengage annular flange 38 and slide easily into hollow tubular body 18. Although various locking mechanisms are compatible with the present invention, one particularly suitable device is sold under the trade name Grazer-Grams Lock (Gram Medical, Costa Mesa, Calif.). In addition to being obtainable for a variety of syringe sizes, these lockingmechanisms are also available in a single shoulder embodiment.
The last major constituent of collection apparatus 10 is cannula 14 having a lumen 26, a proximal end and a distal cannula tip 40. Many commercially available cannulas having different lengths and diameters are compatible with the present invention and may be used with various syringe assemblies. Cannulas which are particularly compatible and provide relatively high yields when used in accordance with the teachings herein are sold under the trade name Mercedes (Grams Medical, Costa Mesa, Calif.)and have inner diameters ranging from approximately 1 mm to 8 mm. Particularly preferable inner diameters range from 1.5 mm to 4 mm. While generally formed out of metal alloys such as stainless steel for ease of resterilization and reuse, the surfaces of these cannulas may be coated with biocompatible polymers to reduce stress on the collected cellular components.
While the general configuration of cannula 14 is relatively consistent for the different embodiments i.e. generally elongated with at least one lumen26, other characteristics of compatible cannulas may vary markedly. For example cannula connection 42, shown in FIGS. 1 and 2 at the proximal end of cannula 14, is adapted to seat on and releasably engage a syringe having a tapered tip 30. Those skilled in the art will appreciate that, aswith syringe assemblies, many types of cannula connections are compatible with the teachings of the present invention as long as they are adapted toengage selected syringe assembly 12. For example, cannulas having luer connectors, catheter connectors, threaded connectors and compression fittings may be used for the harvest of adipose tissue as long as they arecompatible with the connector of the selected syringe assembly. Moreover, cannulas permanently affixed to a syringe assembly to form a collection apparatus are also within the scope of the present invention and may be used with comparable results.
Another important feature of the cannulas of the present invention which may vary depending on the desires of the operating physician are the configuration and position of the collection apertures. For example, FIGS.5, 6, 7, 8 and 9 all show different configurations of collection apertures.In accordance with the present invention is desirable that the shape and configuration of the collection apertures impose stresses during harvest which disrupt the macro structures and connective components of the adipose tissue to provide a relatively homogeneous yield. Further the collection apertures should be large enough to resist blocking by any non-disrupted tissue thereby necessitating the removal of the cannula fromthe patient and interruption of the harvest procedure. Based on such considerations, collection apertures preferably range from 1 mm to 4 mm and more preferably from 1.5 mm to 3 mm.
The aperture configuration of FIG. 5 displays elongated tissue cutting edges on apertures 42 which result in substantially increased cell yields.In contrast, the rounded or less abrupt aperture edges such as those shown in FIGS. 7 and 9, do not appear to provide tissue cutting edges sufficiently disruptive to the connective matrix of the tissue resulting in less homogeneous sample composition and lower cell yield. While tissue cutting aperture configurations disrupt the macro connective structure of the harvested tissue, they do not unnecessarily place stress or shearing forces on the delicate cellular components dispersed within the adipose tissue matrix. While the rounded edges of collection apertures 44 and collection apertures 46 assist in reducing these undesirable shearing forces, they do not provide for the collection of substantially homogeneous tissue and therefore lower the overall cell yield.
Just as various configurations and sizes of collection apertures are compatible with the methods of the present invention, so to are different aperture placement schemes and cannula shapes. For example there is no requirement that the collection apertures be limited to locations near distal cannula tip 40. While such placement may promote sample purity as the collection area can be gauged more accurately, apertures placed further away from the tip may work equally well. Similarly, there is no requirement that cannula 14, and by extension lumen 26 be cylindrical in nature. For example, other, more elliptical, shapes may provide the same cell yield as the perfectly cylindrical shape illustrated in FIG. 6. Accordingly, as with the syringe assemblies, a wide range of cannula shapes, sizes and configurations are with in the scope of the invention and may be chosen based on the preferences of the individual operator.
In any event, once a collection apparatus is selected and assembled, the actual harvesting of the adipose tissue and identifiable cellular components may begin. Preferably the entire procedure is carried out underaseptic conditions. Collection apparatus 10 may be pre-assembled and sterilized ahead of time or may be assembled in the operating area just prior to use. Typically, syringe assembly 12, minus locking mechanism 34, is commercially available in a disposable, presterilized and prepackaged form. Conversely, cannula 14 is typically reusable and has been cleaned, packaged and resterilized on site. Accordingly, following the selection ofcompatible components which may be releasably engaged, syringe assembly 12 and cannula 14 are usually mated to form collection apparatus 10 just prior to insertion in the patient. At approximately the same time as syringe assembly 12 is attached to cannula 14 optional locking mechanism 34 may be affixed to plunger 28 via plunger flange 48. Preferably, lockingmechanism 34 is affixed prior to engaging cannula 14 to reduce the chances of inadvertent sample contamination.
As an optional preliminary step, saline or other biocompatible solutions may be injected into the adipose collection area prior to harvesting the desired material. The introduction of liquids into the area appears to disrupt the adipose matrix and reduce the cohesion of the connective tissue. To assist this disruption the fluid injected tissue may be massaged vigorously or subjected to other external forces. As those skilled in the art will appreciate, the actual volume of saline injected, area of injection and the time of injection before harvesting will depend on the circumstances of the operation such as age and health of the patient, amount of tissue to be harvested and the location of the adipose collection area. Typically several milliliters of solution will be injected approximately thirty minutes to an hour before harvesting is undertaken. A standard syringe and injection needle are used for the procedure. While it appears to improve the homogeneity of the sample recovered, adequate microvascular endothelial cell yields may be obtained without the addition of fluids or the application of external forces priorto harvesting.
When the adipose tissue is considered ready for harvest at least a portion of cannula 14 is inserted in the patient near the adipose tissue to be taken. Given the typical size of cannula 14 and its relatively blunt distal tip 40, a small incision is usually made in the skin of the patientfor the insertion. Following insertion, distal cannula tip 40, and more particularly aperture or apertures 16, is maneuvered to the area where theadipose tissue is to be harvested. As previously discussed, the adipose tissue is usually taken from perinephric fat, subcutaneous fat, omentum, or fat associated with the peritoneal cavity. Given the light weight and relatively small size of collection apparatus 10 the operating physician will have little trouble guiding cannula 14 as desired and precisely positioning it in the proper location.
It is important to note that, during the insertion and positioning of cannula 14 in the body of the patient, plunger 28 and displaceable piston 22 are fully inserted in hollow tubular body 18. That is, forward surface 50 of displaceable piston 22 is seated flush against the forward end of hollow tubular body 18 adjacent to entrance port 24. At the same time optional locking mechanism 34 is retained in a closed position by the inner surface of hollow tubular body 18. With displaceable piston 22 in a fully forward position, substantial amounts of fluid and other bodily material are prevented from entering cannula 14 and syringe assembly 12. Displaceable piston 22 is retained in this configuration until cannula 14 is properly positioned and the physician is ready to begin harvesting the adipose tissue surrounding apertures 16.
To initiate harvesting of the microvascular endothelial cell rich adipose tissue sub-ambient pressure is applied to cannula 14. Typically plunger 28and affixed piston 22 are slowly drawn back through hollow tubular body 18 by the operator. If desired, optional locking mechanism 34 may be engaged with annular flange 38 to maintain the withdrawn configuration and sub-ambient pressure. The increase in sealed volume defined by forward surface 50 of piston 22 and the inner surface of hollow tube body 18 creates sub-ambient pressure in syringe assembly 12. This, in turn, creates a suction in lumen 26 of cannula 14 which is in fluid tight communication with central bore 20 through entrance port 24. Unlike prior art suction pumps which maintained a uniformly high suction force at the collection tip, the present invention provides a gentle sub-ambient pressure which is easily and instantaneously adjustable. Judging by the "feel" of collection apparatus 10, or the appearance of the adipose tissuebeing harvested, the operator can attenuate the suction applied at apertures 16 by adjusting the amount plunger 28 is withdrawn from hollow tubular body 18. By pushing plunger 18 the suction will be reduced while withdrawing it further (maintaining the sealable deposition of piston 22 hollow tubular body 18) will rapidly increase the suction at the aperture.Alternatively, the operator may simply rely on locking mechanism 34 to maintain a steady suction at apertures 16.
The easily controllable sub-ambient pressure, combined with the favorable tissue cutting characteristics of apertures 16 provide cleaner more homogeneous adipose tissue for processing. Obstructing connective tissue is preferably disrupted while preserving the integrity of the cellular components. Moreover, as the operator is able to easily and efficiently adjust the position of cannula 14, regions of higher contamination may be avoided further increasing the purity of the tissue obtained. Should therebe a problem with obstruction of apertures 16 or cannula 14, the operator may simply push plunger 28 slightly forward to apply positive pressure to the cannula and apertures thereby clearing the obstruction. Finally, as the plunger is under direct control of the operator, the amount of adiposetissue collected may be controlled more closely.
Following the harvest of the desired amount of homogeneous adipose tissue and pressure equilibration of collection apparatus 10, cannula 14 is removed from the patient through the initial insertion site preferably maintaining aseptic conditions. As previously discussed the actual amount of adipose tissue collected will depend on a number of factors including number of microvascular endothelial cells needed and the capacity of collection apparatus 10. Typical volumes range from approximately 10 ml toapproximately 100 ml with average volumes ranging from about 40 ml to about60 ml. Of course those skilled in the art will appreciate that smaller or larger volumes may be collected for the purification of microvascular endothelial cells or other cellular components using the methods of the present invention. After cannula 14 is removed from the patient, it is usually disengaged from syringe assembly 12 containing the harvested tissue for cleaning and resterilization.
At this point, the harvested tissue may be processed further or stored for later use. For storage, the tissue is usually removed from syringe assembly 12 by ejecting it through harvesting port 24 into a separate container which may then be chilled. For processing, the collected tissue may be similarly transferred to microvascular cell isolation apparatus such as the one described in copending U.S. patent application Ser. No. 08/086,778, which is incorporated herein by reference. The desired identifiable cellular components will then be separated from the adipose tissue using digestion and the other procedures previously discussed.
Alternatively, in accordance with the teachings of the present invention the harvested adipose tissue may be processed further using syringe assembly 12 for rinsing and homogenization. For example, water or other aqueous solutions could be introduced into central bore 20 with the collected sample and shaken. Afterward the mixture is allowed to settle, preferably in a syringe stand (not shown), and separate. The adipose cellsand associated tissue including the overwhelming majority of microvascular endothelial cells will float while connective tissue, red blood cells and other contaminants sink or our solubilized in the aqueous solution. The rinsed adipose cells and associated tissue may then be decanted. Of coursethe process may be repeated as many times as necessary.
In another procedure the harvested tissue may be homogenized and rinsed at the same time. Referring now to FIG. 3, syringe assembly 12, containing the harvested tissue, is releasably attached to filter hub assembly 60. A second syringe assembly 212, preferably the same size as syringe assembly 12, is releasably attached to the opposite side of filter hub assembly 60.For the purposes of clarity reference numerals previously used for syringe assembly 12 will be used in the following discussion. Corresponding components of syringe assembly 212 will use the same reference numerals with the prefix 2.
In the embodiment shown, syringe assemblies 12 and 212 are Toomey-type syringes having tapered tips 30 and 230 positioned at their respective front ends. However, as previously discussed, many types of connectors arecompatible with the teachings of the present invention. Accordingly, any type of tip which is releasably engageable with filter hub assembly 60 maybe used.
Filter hub assembly 60 comprises a male hub 62 and a female hub 64 which may be mated using releasably engageable male threads 66 and female threads 68. When so mated, male hub 62 and female hub 64 cooperatively define passage 70. Passage 70 traverses filter hub assembly 60 with openings on opposite faces adapted to releasably engage tapered tip 30 andtapered tip 230 thereby placing syringe assembly 12 and syringe assembly 212 in sealed fluid conducting communication with each other. Filter member 74, shown more clearly in FIG. 4, is positioned axially with respect to passage 70 transecting it as the filter is held in place by compression forces imposed by mated male hub 62 and female hub 64. By transecting passage 70, filter 74 interrupts any flow of tissue or fluid therethrough. Elastic grommet 72, adjacent to filter member 74 ensures that hub assembly 60 is sealingly engaged.
Filter member 74 is a flat, radial disk-like structure having a central portion indicated by arrowed line 78. Multiple filter apertures 80, positioned in central portion 78 traverse the thickness of filter 74 thereby allowing material pass through. Exemplary embodiments use a filtermember 74 having an outer diameter of 24 mm with filter apertures 80 havinga diameter of approximately 1 mm. FIG. 4 also shows female hub 64 surrounding filter member 74. Of course those skilled in the art will appreciate that other aperture diameters may be employed depending on the amount of homogenization desired.
Typically, filter member 74 is formed of a tough, resterilizable metallic alloy such as stainless steel. However, as previously discussed, the use of metallic components to process adipose tissue may be detrimental to theyield of viable microvascular endothelial cells as metallic alloys have inherently high surface energy. Accordingly, it is preferable if filter 74is formed of a material having a low surface energy or, if metal is used, that it is coated with a material such as parylene. By low surface energy,it is meant that the materials have a lower electrochemical energy in comparison with metals. Examples of materials having low surface energy and good biocompatability which may be used to practice the present invention include, but are not limited to, polyethylene, parylene, polypropylene, nylon and other fluoropolymers.
While the size and configuration of selected apertures 16 yield a relatively homogeneous sample of adipose tissue, further homogenization todisrupt connective tissue in the adipose matrix may improve cell yield if done gently. As indicated above, collected adipose material is retained insyringe assembly 12 following harvesting. The material may be in its natural, harvested state or rinsed as previously described. Optionally, liquids may be added to the collected material. Filter hub assembly 60, having filter member 74 positioned across passage 70, is releasably engaged to tapered tip 30. Syringe assembly 212 is similarly attached to filter hub assembly 60 on the side opposite syringe assembly 12. When connected in this manner, syringe assembly 12 is in sealed fluid conducting communication with syringe assembly 212 through passage 70.
Plunger 28 is then pushed forward into hollow tubular body 18 to discharge the harvested adipose tissue and any added liquids from harvesting port 24defined by tapered tip 30. The ejected material then traverses passage 70 passing through filter apertures 80 of filter member 74 before being received by syringe assembly 212. As the adipose tissue is forced through the appropriate size filter apertures 80, the connective matrix is disrupted without exposing the associated identifiable cellular componentsto excessive shearing forces. This, in turn, lowers the viscosity of the collected material allowing contaminants to be more easily removed as wellas improving the subsequent digestion of the sample and increasing the ultimate yield of endothelial cells. As the filtered, harvested adipose tissue enters syringe assembly 212 through tapered tip 230 positive displacement forces plunger 228 toward the rear of hollow tube 218. Of course, this procedure may be repeated by reversing the sequence of eventsto move the tissue from syringe assembly 212 to syringe assembly 12.
The improved yield of microvascular endothelial cells provided by the methods of the present invention is illustrated in the following nonlimiting example.
Human adipose tissue was collected from the thigh of a female Caucasian. Four different commercially available cannulas having apertures of varioussizes and configurations were used to withdraw the fat from the thigh with the harvesting procedure taking place approximately thirty to sixty minutes prior to the experiment. Then samples collected by each type of cannula were then processed separately.
In each case the adipose tissue was briefly rinsed with Dulbecco's phosphate buffered saline. One of the samples of the harvested material was then homogenized by running the tissue between two syringes having a filter member with 1 mm filter apertures interposed between them. 10 gramsof the respective fat sample and 10 mls of a collagenase solution (4 mg/ml,Boehringer Mannheim) were then combined in 50 ml Erlenmeyer flasks and placed in a shaker to incubate at 37� C. for twenty minutes at 100 cycles per minute.
The resulting digestion slurry was then poured into 15 ml conical centrifuge tubes and spun at 1800 rpm for seven minutes.
The endothelial cells and red blood cells precipitated at the bottom of conical centrifuge tubes. Dark collagenase solution formed a middle layer and the nonsoluble fat and associated adipose tissue formed a plug on top of the centrifuge tube. Both the dark collagenase solution and the fat were discarded.
The endothelial cell pellets were resuspended using 10 ml of 0.1% bovine serum albumin in Dulbecco's phosphate buffered saline, pooled in a new sterile conical centrifuge tube and spun at 1800 rpm for four (4) minutes.The supernatant was discarded and the endothelial cell pellets were resuspended with 14% human serum in Plasma-Lyte� (Baxter Healthcare Corporation) an FDA-approved medium for sodding with human blood serum.
The final volume of this solution in each case was approximately 9 ml. 0.2 ml of each resulting endothelial cell suspensions were diluted to 20 ml with Isoton� solution (Baxter Scientific Products). The cell yield andcell sizes in each suspension were determined using a Coulter Multisizer II. The cell yield was defined as the number of cells (larger than 7.78 μm) recovered per unit gram of fat. The yield of cells was used as the index of suitability of the design although cell viability was not studiedin detail at this time. The adherence of the isolated cells on the well plate was examined only occasionally with reasonably good results.
The results of different syringe and cannula configurations are shown in Table 1 immediately below.
TABLE 1______________________________________Type of Syringe/ Cell YieldCannula (No. of Cells/g fat)______________________________________Catheter tip/ 1.12 � 1063.7 mm MercedesCatheter tip/ 1.60 � 1063.7 mm Mercedes, 4 � filterToomey-type/ 2.13 � 1063.0 mm MercedesLuer lock/ 1.58 � 1061.5 mm luer lockToomey-type/ 1.15 � 1063.00 mm Curret Special______________________________________
These data clearly show the improvement in identifiable cell yield through the use of the methods of the present invention. In particular, the results illustrate that the selection of an appropriate aperture size and configuration can increase the yield of microvascular endothelial cells from a given source of adipose tissue. For example, use of the Curret Special having a 3 mm diameter and rather rounded apertures only yielded approximately half of the viable cellular components obtained using a Mercedes having a 3 mm diameter and apertures with well-defined tissue cutting edges. Moreover, it is important to note that the diameter of the cannula alone is not the determinative criteria for increasing cell yields. This is illustrated by the fact that the use of either a 3.7 mm Mercedes or a 3 mm Curret Special only provided approximately 70% of the viable cells provided by a 1.5 mm Luer lock cannula having apertures with better tissue disruptive capabilities. Finally, the data show that homogenizing the harvested adipose tissue in accordance with the teachingsof the present invention can substantially increase microvascular endothelial cell yields. For example, when samples are collected using identical 3.7 mm Mercedes cannula, homogenizing the adipose tissue by passing it four times through a filter member having 1 mm filter aperturesincreased the yield of viable cells by over 40%. Such increases in cell yields can easily prove to be the difference between successful endothelialization and incomplete coverage of the synthetic graft which may lead to the formation of life-threatening clots.
Those skilled in the art will further appreciate that the present inventionmay be embodied in other specific forms without departing from the spirit or central attributes thereof. In that the foregoing description of the present invention discloses only exemplary embodiments thereof, it is to be understood that other variations are recognized as being within the scope of the present invention. Accordingly, the present invention is not limited to the particular embodiments which have been described in detail herein. Rather, reference should be made to the appended claims to define the scope and content of the present invention.
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Lingoport Resource Manager Released
Today, Lingoport announced that it has released Lingoport Resource Manager (LRM) to support development projects in their localization efforts. By optimizing the manual and often error-prone process of resource file management, LRM effectively bridges the gap between development and localization, ridding the two areas of unnecessary testing iterations and file confusion.
In a press release sent out today, Lingoport President noted:
"During our product
Internationalization Quality of Online Map Services
Leandro Reis, Senior Globalization Program Manager at Adobe, recently wrote up a tremendous synopsis of the world-readiness of online map services on his blog.
The heart of internationalization and localization is location (or locale, geography, place, etc). Maps represent this at the purest form. From user interfaces available in 57 languages on Google Maps to units of measurement, each little piece of information pertaining to a specific locale is taken into consideration. Reis' article highlights …
Localization Strategies for Global E-Business
Lingoport is proud to have collaborated on Localization Strategies for Global E-Business by Professor Nitish Singh, PhD, Associate Professor of Int. Business & St. Louis University Director Alternative Delivery Programs. The book is out now and is available on Amazon and the Cambridge University press websites.
A major focus of Singh's book is on internationalization, a point of expertise of Lingoport. Lingoport President & CEO Adam Asnes is featured a number of times from describing …
Website Localization: Factors to Consider
With nearly one-third of the world population using the internet, more and more opportunities are arising for people to communicate and for companies to reach new markets. Adapting an e-commerce website to a new locale has become an essential way for online businesses to survive and thrive in new markets. In an insightful post from the GPI Translation Blog, we learn first hand some of the strategies that go into …
Facebook Set to Introduce Translation Feature on Comments
With the introduction and prominence of Google's web translator, companies like Facebook have a more prominent need to provide users with translation features within their display. Often times users, myself included, will leave their Facebook tab to translate something their friends commented on or made a status about. Since introducing Facebook in multiple languages, users have soared to 750 million people. Obviously in such a connected world, these users need the ability to communicate …
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Russell Moore, president of the Southern Baptist Convention's Ethics and Religious Liberty Commission, clarified that his recent criticism of a Christian talk radio program as missing a redemptive message was not aimed at all Christian talk radio programs.
During a speech at the ERLC's Leadership Summit in Nashville on April 22, Moore said, "I listened on the way back up here from my hometown to some Christian talk radio this week, against my doctor's orders. And, honestly, if all that I knew of Christianity was what I heard on Christian talk radio, I'd hate it, too. There are some people who believe that fidelity to the Gospel simply means speaking, 'You kids get off my lawn.' That is not the message that has been given to us."
In a broadcast two days later, Christian talk radio host Janet Mefferd said she was offended at what Moore said and called on him to apologize. Also, Bott Radio Network President Richard Bott reportedly sent Moore a letter asking him for a public apology. more >>
Speaking at the third annual "Washington: A Man of Prayer" event Wednesday, Pastor Robert Jeffress said that while members of Congress can debate immigration, healthcare and taxation policies, there are some issues that are beyond debate.
"Issues such as the value of life inside and outside the womb, the sanctity of marriage between a man and a woman, the right to religious freedom for every human being and the compassionate treatment of the poor. … For the Judge of the universe has already rendered His opinion," Jeffress said in Statuary Hall at the United States Capitol in Washington.
Jeffress, senior pastor of the 11,000-member First Baptist Church in Dallas, Texas, was joined by Fox News host and former Republican governor of Arkansas Mike Huckabee and Sen. Ted Cruz (R-Texas) at the event, which was held to commemorate "the events of April 30, 1789, when … President [George] Washington, accompanied by Congress, proceeded to St. Paul's Chapel where, as one of his first official acts, the president offered a prayer of dedication to God on America's behalf," according to the event website. more >>
Bart Barper, who pastors First Baptist Church in Farmersville, Texas, spoke about religious freedom and sexuality at last month's Ethics & Religious Liberty Commission's annual leadership summit that was held in Nashville, Tennesse.
Barper caught up afterwards with The Christian Post where he explained why he stuck up for the developers who wanted to build an Islamic Center near Ground Zero, and why "God bless America" makes him feel uncomfortable and why Hobby Lobby matters.
The following is an edited transcript of the interview. more >>
NASHVILLE — Trillia Newbell, who serves as a consultant for Women's Initiatives at the Ethics & Religious Liberty Commission, was one of two women at its leadership summit last month to address the dozens of Southern Baptist pastors and church leaders.
Newbell, who recently authored United: Captured by God's Heart for Diversity, caught up with The Christian Post afterwards and shared why she believes the church should talk more about female pornography addiction and how churches should handle sex abuse allegations.
CP: What do you think the church needs to talk about more with regards to women and sex? more >>
Mark Harris, pastor of a North Carolina megachurch who is seeking the Republican nomination for a U.S. Senate seat, has come under scrutiny for accepting church offerings during his campaign.
Harris is currently on leave from First Baptist Church in Charlotte to focus on his campaign win the state's May 6 primary election. However, he has made several recent church appearances as an invited preacher where he has accepted money that could be a violation of campaign finance laws.
"I would hope the pastor would make it very clear that we're taking up a love offering for Mark Harris the pastor, not for the campaign," Harris said, according to North Carolina-based WRAL-TV. "Some of these guys are very aware that I'm on a personal leave of absence without pay, and I have no income. So, should the church take up a love offering, that would be no different from them taking up a love offering when I was preaching revival there or preaching any other meeting." more >>
NASHVILLE—Tony Merida, the founding pastor of Imago Dei Church in Raleigh, North Carolina, spoke on the topic of human sexuality during the Ethics & Religious Liberty Commission's annual leadership summit that was held in Nashville last week.
Following his discussion on "the Gospel and human trafficking," Merida, an associate professor of preaching at Southeastern Baptist Theological Seminary, spoke with The Christian Post about the relationship between pornography and sex trafficking, explained why awareness is insufficient, and laid out how Christians can do more to fight "the darkest of sins."
The following is an edited transcript of the interview. more >>


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P.E. Central Lesson Plan: Plus One Volleyball
Prerequisites:Students need to have some skill in executing the forearm pass(bumping) and the volley. Students should also be able to under hand serve.
Purpose of Activity:For students to move towards the ball while maintaining a body position that permits them to pass the ball to their team mates.
Suggested Grade Level:9-12
Materials Needed:One volleyball net for every 8-12 students One volleyball for each net
Lesson Plan:Description of Idea
Divide the class into teams of 4 to 6. Arrange teams in formation on each side of the net, two groups at each net. A player serves the ball over the net. The receiving team must now play the ball twice before they may return the ball. The serving team must now play the ball three times before they are allowed to return the ball over the net. Play continues as each team forces the other side to "plus one" the previous achievement. Only the serving team scores points and players may not play the ball twice in succession. Players must take turns serving the ball.
Teaching Suggestions:
Emphasize moving the feet and getting the body at a low level.
Observe whether student are moving towards the ball while maintaining a body position that permits them to pass the ball to their team mates.
If students cannot pass the ball more than twice, see the adaptions below.
Adaptations for Students with Disabilities:
Have students move closer to the net when serving.
Allow students to catch the ball instead of using the volley/bump.
Allow the ball to bounce in between passes.
Lower the net.
Use a beachball.
Author:Evert TenBruggencate who teaches at Immanuel Christian School, Aylmer, Ontario Posted on PEC: 10/1/2001.
This lesson plan was provided courtesy of P.E. Central (www.pecentral.org).
Products for This Lesson:


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7 Pretty Summer Dresses
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ASOS Summer Midi Dress in Smudge Spot Print
A bit looser than polka dots, this oversize print has a cool tie-dye feel. The high-low hemline and cutout back keeps it modern.
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Featured June 2012


text: Iron factory's Evillord stands 4.8"/12CM tall and can transform into 4 different modes. Iron factory also announced a second run of the extremely popular Iron Giant's Maiden. Preoders up at TFSource here: Iron Factory - IF-EX11 - Evillord As well as all iron factory figures: http://tfsource.com/iron-factory/ Additionally badcube's evil bugs are now available for both single and sets for preorder at TFSource here: Badcubes – Evil Bugs Set & Singles


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I bet you are thinking that I lost my mind! You read that title correctly, today I am sharing Plastic Spoon Christmas Trees with you today! If you are new here, WELCOME! If you have been here for a while you might remember that I created a Chrysanthemum Clock using plastic spoons this summer! I'm… Continue Reading
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Comments about Music Accessories Rack Drawer / Sliding Shelf:
i have mine mounted in a raxxess black oak studio rack. it mounted easily with little effort and works great. would definitely buy another
can be used as a shelf or a drawer depending on how you mount it. versatile. i use mine as a drawer with a single rack space above it left open so i can fit pedals and other larger items in it.
this drawer seems to be very sturdy with good roller action.it snaps into place when it is closed so it stays snug. no worries about it rolling open on its own.
i think it is a bit pricey for a drawer but the quality is good.
Comments about Music Accessories Rack Drawer / Sliding Shelf:
Overall, very happy with the product. This is a drawer that has four sides (not a shelf) which required me to use a laptop stand to hold my multi-track recorder so I could access the ports on the side.
Fairly flexible mounting mechanism. I modified the mount to work with my rack.
High quality build - very sturdy once mounted for the price. Metal drawer construction.
Compared to the wood shelves this seems built very well for the price.
Comments about Music Accessories Rack Drawer / Sliding Shelf:
As mentioned in another review, this is a nice piece of hardware for the price point. The drawer action is smooth, seems strong, and there is a nice detent when fully closed that at least when brand new is keeping the thing from just flopping out on it's own unless you give it a tug. Materials were excellent with no out-of-place or badly tapped holes. Unfortunately, because of the way SKB cases are designed and the wing nuts that this drawer uses to adjust the front-to-back rail-to-rail length, and which causes the assembled drawer to extend almost as wide as your center-to-center distance between holes on the rack rails, I could only place this drawer in the top or bottom slots on my 10-space SKB and I suspect the same would have been true of my 4 and 6-space racks. I'm referring to the standard full size SKB, not the roto-mold series. Look into your SKB case at the plastic assembly that extends into the interior of the case behind the rack rail holes in all the middle spaces and you'll understand why any hardware item that needs that space will not be installable in an SKB case.Assembly would have been extremely easy were it not for the same wing nut that keeps the thing from just going anywhere in my rack, so hopefuly just a slight redesign will make this an even better value. In the meantime, you could substitute a regular nut but it won't work quite as well and will require a little parts shopping.Until then, either put up with the issue, use it in a different brand rack or be prepared to do a little simple creative modification.
Comments about Music Accessories Rack Drawer / Sliding Shelf:
I ordered two of these, one to store small gadgets like picks, couplers, and batteries and one to serve as a shelf for my Fostex MR16D. I was hesitant because I couldn't find on-line reviews, but I am glad I took the gamble.The hardware is well put together with nice, solid materials, unlike some flimsy bargain drawers I've encountered that cost nearly as much. The bearing mechanism is smooth and there is no rasping or sticking as the drawer opens and closes. Changing between drawer and shelf mode is easy, providing you remember that the release pushes the opposite direction when it's installed in the opposite position. A press-in release instead of an up/down might have been an ergonomic improvement. I was pleased to discover that my MR16D actually fits snugly in the 1U drawer with enough room in the back to connect cables, a bonus because I won't have to worry about the recorder sliding off! One gotcha is that if you, like me, have a 16" deep rack with no rear rails, installing the rear supports is a bit of a trick. I had to do a full assembly, mark the wood screw positions, disassemble, install the rear supports and reassemble. Have a marker and drill handy in addition to your Phillips screw driver and all should go smoothly.I thought about giving this item a four for quality because there's always room for improvement in mechanical systems, but in the end I gave it straight fives because compared to other rack hardware, I can't imagine a much better piece for the price I paid.
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Originally written on Fox Sports | Last updated 8/30/12
They've missed the playoffs the last two seasons and have one playoff victory the last four years. Their quarterback was a mess last year, accounting for a whopping 25 turnovers, the most in the NFL. And, perhaps only in soft and sunny San Diego, posting a double snowman - an 8-8 record - was good enough of a showing for embattled coach Norv Turner and polarizing general manager A.J. Smith to keep their jobs with the San Diego Chargers. Team president Dean Spanos didn't feel compelled to give Turner and Smith the heave-ho, much to the chagrin of a growing segment of the ticket-buying public. Something would certainly have to give at the top if the Chargers miss the playoffs a third straight season. Turner has never been on the hot seat like he is now, and it seems like he's been on it since right after the Chargers hired him before the 2007 season. In an effort to both fill their many holes and perhaps to ward off the hot seat for Turner and Smith, the Chargers loaded up on fre...
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text: The In Wild frontier Part 4 - Danger In the Hills wiki last edited by guttridgeb on 05/07/13 05:52AM View full history Wild Frontier, part 4 of 6, "Danger in the Hills," starring WolverineAnd Ye Shall Remember This Day, part 4 of 4, "Salvation," starring NovaServants of the Dead, part 7 of 8, "The Bride," starring Cable and Ghost RiderCable and Ghost Rider are not convinced that all is lost for the girl. They must then prove it to the God of the Dead."Class Clown," starring Speedball


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Fr. 11a $20 1861 Demand Note PMG Choice Fine 15 Net.. ...
2014 January 8 - 10 & 13 FUN US Currency Signature Auction - Orlando #3526
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Unique "for the" Twenty Dollar Demand NoteFr. 11a $20 1861 Demand Note PMG Choice Fine 15 Net.
From our sale of the Dr. Edward & Joanne Dauer Demand Note Collection in September 2008. We described it there as follows: "This fabled Fr. 11a is unique not only for its Friedberg number, but also for type. When Demand Notes were first printed, the words 'Register of the Treasury' and 'Treasurer of the United States' were printed at the bottom of the note below the signature lines. The Treasury clerks, who were authorized to sign for these officials, added the words 'for the' after their signatures. After a short while, it was realized that the words 'for the' should be added to the plate in front of 'Register of the Treasury' and 'Treasurer of the United States' to save a considerable amount of time and effort on the part of the signers. Five and Ten Dollar 'for the' notes are extremely rare in the marketplace. There are only ten 'for the' $5 notes known--nine of which are Fr. 1a. The $10 notes are equally rare with ten pieces known--most of which are Fr. 6a. In $20 Demand notes, the currently offered piece is absolutely unique with no other 'for the' notes known--not only for this Friedberg number, but for the type. The note has a minor, exceedingly well-done restoration of one of the corner tips, and has been graded Choice Fine 15 Net with the comment 'Professional Restoration.' To our eye, the note has the appearance of a solid problem-free Very Fine. The signatures, although a bit light, remain nice and clear, and the colors of the note, including the green overprint on the face as well as the red serial number, have all their original brightness. The elaborately engraved green back also gives the appearance of a solid VF if not slightly better. This would be a handsome and desirable Demand Note of any denomination or variety at this grade level. The words 'unique' and 'irreplaceable' are considerably overused in today's market; however, for this note, 'unique' is exactly correct and 'irreplaceable' is right on the money. The successful bidder who brings this note home tonight can bask in the certain knowledge that no other collector will ever own a 'for the' $20 Demand note until tonight's winner is willing to part with his treasure." The note remains unique both for Friedberg number and type. Any collector wanting a $20 "for the" Demand note needs this very piece.
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Nearing retirement, Finch stymies Japan
Updated: July 24, 2010, 2:15 AM ETAssociated Press
OKLAHOMA CITY -- Jennie Finch's farewell tour at the World Cup of Softball first featured a display that she can still knock the ball out of the park. Then she showed that she can still pitch with the world's best, too.
Finch took a no-hit bid into the fifth inning in one of her final starts for the U.S. national team, Kaitlin Cochran homered in her second straight game and the Americans beat top rival Japan 5-0 Friday night at the World Cup of Softball.
"I love having the ball in ...
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38 year old woman | Potomac, MD, USA | Seeking men 37-48 within 20 miles
Are you positive, fun and bilingual (English&Spanish)?
After working for 13 years to build a successful career in Spain, I found that English fluency is essential to keep advancing in the European business world. Therefore, last September I decided to take a break and become a full time student for a while. I chose the DC area because my best friend from Spain lives here, and I was already familiar with it. This is an exciting adventure for me and I'm truly enjoying it. I wish I could already speak English and not only write it , but I will keep attending school 20 hours/week and working hard till I reach that goal. In the meantime, I would really prefer to meet potential "matches" that are fluent in both English and Spanish, so we can get to know each other. Also, before you contact me, please make sure you are: single or divorced (not separated), positive/optimistic, affectionate, romantic, caring, professional and truly educated/worldly--you feel comfortable in any social situation (black tie event or picnic), and you enjoy interacting with people from different backgrounds/cultures. I believe I possess these qualities, so if you find some common ground, feel free to drop me a line ~
Book club , Coffee and conversation , Business networking , Cooking , Dining out , Museums and art , Music and concerts , Exploring new areas , Performing arts , Shopping/Antiques , Travel/Sightseeing , Volunteering , Watching sports , Wine tasting
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'6" (167cms)
|5'8" (172cms) to 6'7" (200cms)
|Body type:
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|No preference
|Light brown
|No preference
|No Way
|No Way
|Social Drinker
|Never, Social Drinker
|I'll tell you later
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