APPARATUS AND METHOD FOR STERILIZING, SEEDING, CULTURING, STORING, SHIPPING AND TESTING TISSUE, SYNTHETIC OR
NATIVE VASCULAR GRAFTS
BACKGROUND OF THE INVENTION
Technical Field
The present invention relates to the sterilization, seeding, culturing, storing, shipping, and testing of
vascular grafts. Specifically, the present invention relates to an apparatus and method for sterilizing vascular grafts and then seeding and culturing the grafts with human cells, resulting in grafts populated with viable human cells.
Discussion of the Related Art
Vascular grafts are used by vascular and thoracic surgeons to repair or replace segments of arterial and venous blood vessels that are weakened, damaged, or obstructed due to trauma or disease such as aneurysm, atherosclerosis, and diabetes mellitus. Historically, vascular grafts have been either homografts, such as the patient's own saphenous vein or internal mammary artery, prosthetic grafts made of
synthetic materials such as polyester (e.g., Dacron),
expanded polytetraflouroethylene (ePTFE), and other composite materials, or fresh or fixed biological tissue grafts.
However, synthetic grafts generally have inadequate patency rates for many uses, while the harvesting of
homografts requires extensive surgery which is time- consuming, costly, and traumatic to the patient. Fixed tissue grafts do not allow for infiltration and colonization by the host cells, which is essential to remodeling and tissue maintenance. Consequently, fixed tissue grafts degrade with time and will eventually malfunction.
Due to the inadequacies of these currently available synthetic and biological grafts, as well as the cost and
limited supply of homografts, tissue engineered grafts are being developed which have been sterilized and then seeded and cultured, in vitro, with cells. These tissue engineered grafts may be superior to other grafts for use in replacement therapy in that they more closely display the long term dimensional stability and patency of native arteries and vessels with normal physiologic functionality.
Historically, the seeding and culturing of vascular grafts, and tissue in general, has taken place in a static environment such as a Petri or culture dish. However, there are disadvantages to seeding and culturing tissue in such an environment. For example, the lack of circulation of
nutrients in these static systems results in a slow and ineffective seeding and culturing process. Moreover, cells which are seeded and cultured in a dynamic environment are more likely to tolerate the physiological conditions which exist in the human body once implanted. Thus, there exists a need for a dynamic environment in which to seed and culture tissue-engineered vascular grafts and other prosthetic devices.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a dynamic environment for seeding, culturing, and testing vascular grafts of any desired length or diameter.
It is a further object of the invention to provide a precise mechanical device with a minimum of moving parts to provide such an environment .
It is yet a further object of the invention to provide a closed system free from contamination for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts .
In accordance with the present invention, there is provided an apparatus and method for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts. Specifically, the present invention is an apparatus and method for seeding and culturing vascular grafts with human
cells, resulting in a tissue-engineered vascular graft populated with viable human cells.
The apparatus according to the invention comprises a fluid reservoir, a pump, at least one graft treatment
chamber, a tube for supporting the graft in the treatment chamber, and an alternating pressure source for applying a radial stress to the prosthesis housed in the treatment chamber. Applying radial stress to the vascular graft scaffold located on the tube within the treatment chamber during seeding and culturing results in a vascular graft with cells and their fibers oriented so as to more likely tolerate the physiological conditions found in the human body. In this manner, the invention advantageously utilizes a
mechanically non-complex apparatus to create a dynamic environment in which to seed and culture tissue-engineered vascular grafts or other implantable devices.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention will become more readily apparent from the following detailed description, which should be read in conjunction with the accompanying drawings in which:
FIG. 1 ii a schematic diagram illustrating an apparatus according to the present invention for sterilizing, seeding, culturing, storing, shipping, and testing a prosthesis;
FIG. 2 is a block diagram illustrating a preferred embodiment of an alternating pressure source;
FIG. 3 is a schematic diagram illustrating an
alternative exemplary embodiment of the present invention for sterilizing, seeding, culturing, storing, shipping, and testing a prosthesis, in which a plurality of prostheses may be treated simultaneously; and
FIG. 4 is a schematic diagram illustrating yet another alternative exemplary embodiment of an apparatus according to the present invention for sterilizing, seeding, culturing, storing, shipping, and testing a prosthesis.
DETAILED DESCRIPTION OF THE INVENTION
The following embodiments of the present invention will be described in the context of an apparatus and method for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts, although those skilled in the art will recognize that the disclosed methods and structures are readily adaptable for broader application. Note that
whenever the same reference numeral is repeated with respect to different figures, it refers to the corresponding
structure in each such figure.
FIG. 1 discloses a system for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts. According to a preferred embodiment of the invention, this system primarily comprises a fluid reservoir 10, a pump 12, a treatment chamber 14, and an alternating pressure source 16.
Fluid reservoir 10 is used to store fluid for the system. Two illustrative suitable reservoirs are the Gibco- BRL 1L media bag and any rigid container capable of
sterilization. Reservoir 10 may include a one way filter so as to provide a direct source of gas to the fluid within the system. Examples of fluid which may be used in the system include, but are not limited to, sterilizing fluid, tanning fluid, fluid containing cells, or fluid containing a culture medium. It is to be understood that during testing, seeding, and culturing in a preferred embodiment, the fluid may be advantageously kept at human body temperature, and may be composed of a fluid which approximates the viscosity of human blood. One illustrative example of a solution which
approximates the viscosity of blood is saline with glycerol.
The fluid contained in reservoir 10 is retrieved through fluid line 18 by pump 12. Fluid line 18, as well as all other fluid lines in the system, may be made of any type of medical grade, durable tubing suitable for transporting the fluid in use. Pump 12 may be preferably any fluid pump which can achieve variable flow rates . One such pump is the
Masterflex L/S Digital Drive peristaltic pump manufactured by Cole-Palmer, although one skilled in the art could select
from a variety of commercially available pumps. Pump 12 propels the fluid from reservoir 10 to treatment chamber 14 through fluid line 20.
Treatment chamber 14 preferably may be composed of any biocompatible, rigid material capable of being sterilized such as Teflon, polycarbonate, PVC, or stainless steel.
Treatment chamber 14 may be comprised of two sections which are secured and made leak proof through any standard means such as inner and outer threads or the use of bonding agents . In order to view vascular grafts within treatment chamber 14, a viewing port may be placed at any point on the chamber, or alternatively, the chamber may be made of an optically clear material such as polycarbonate or PVC. Inlet port 28 and outlet port 30 of treatment chamber 14 allow for the
perfusion and/or circulation of fluid into and through the chamber. Inlet port 28 and outlet port 30 are also used to attach treatment chamber 14 to fluid lines 20 and 22
respectively. Fluid line 22 connects chamber 14 back to fluid reservoir 10 so as to create a closed system.
Treatment chamber 14 houses an expandable tube 32 upon which may be placed a vascular graft scaffolding 26. As is discussed in detail in both the patents incorporated by reference below, scaffolding 26 may illustratively consist of any knitted, braided, woven, felted, or synthesized materials that are bioresorbable and/or biocompatible, as well as any native graft scaffolding material. Tube 32 may be comprised of any suitable elastomeric material, such as PET or silicone angioplasty balloons, which is capable of expanding and contracting. Treatment Chamber 14 and tube 32 may be made any length or diameter so as to hold a vascular graft
scaffolding 26 of any length or diameter. This is
advantageous, as the system may be used to sterilize, seed, culture, store, ship, and test vascular grafts of any size, such as coronary, carotid, iliac, and peripheral leg grafts. A porous clip or grommet 33 may be placed on tube 32 at both ends of scaffolding 26 to hold the scaffolding firmly in place on the tube during treatment. However, one skilled in
the art will understand that any structure which allows for retention of the scaffolding 26 on tube 32 may be used.
Grommets 33 are beneficial, as the tubing can be made smaller than the grafts so as to allow for the perfusion and/or circulation of fluids in between the graft and the tube, without the possibility of slippage of the graft on the tube.
Tube 32 may be expanded and contracted by alternating pressure source 16, a preferred embodiment of which is shown in detail in FIG. 2. Specifically, FIG. 2 shows a pump 34 which may be any standard pump capable of providing both positive pressure and negative (or vacuum) pressure, such as a piston or diaphragm pump. Valve 36 accepts the positive pressure and negative pressure from pump 34 through lines 40 and 42 respectively. Due to signals- from timer 38, valve 36 causes alternating pressure to be applied to tube 32 from line 24. Valve 36 may be any type of inline valve capable of directing and regulating multiple pressure lines. One such valve is the MAC 45S, model 45A-AA1-DAAA-1BA.
By expanding and contracting tube 32 with alternating pressure source 16, tube 32 places a varying radial stress on vascular graft scaffolding 26. This radial stress is
advantageous as it can be detected by living cells attached to the scaffolding, thus causing the cells to align
themselves parallel to the axis of stress and to secrete extracellular matrix molecules which are also aligned
parallel to the axis of stress. In this manner, vascular grafts are formed with cells and their fibers configured so as to more likely tolerate the physiological conditions found in the human body.
The system according to the present invention may contain a plurality of chambers 14 for treating a plurality of vascular grafts. FIG. 3 discloses a system according to the present invention which contains two treatment chambers 14. Although FIG. 3 illustrates the connection of only two treatment chambers to the system, it will be apparent to one skilled in the art that any number of chambers may be
connected to the system in similar fashion. Specifically,
line 20 may be split to connect to each inlet 28, line 24 may be split to connect to each tube 32, and line 22 may be split to connect to each outlet 30 of each chamber 14 in the system. In this manner, a plurality of vascular grafts may be simultaneously seeded, cultured, or tested.
Alternatively, each treatment chamber 14 may be
connected to a separate reservoir 10 and pump 12 so that multiple treatment chambers in a system would only share a single alternating pressure source 16. It is to be
understood that a pump 12 with multiple pump lines may also be used so that each treatment chamber 14 in the system would use the same alternating pressure source and same pump 12 (each using a different pump line), but would be connected to a different media reservoir 10.
FIG. 4 discloses an alternative embodiment of the invention for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts. According to this embodiment of the invention, the system primarily comprises a fluid reservoir 10, a bladder pump 50, a treatment chamber 46, and an alternating pressure source 54.
Fluid reservoir 10 and the fluids which it may contain are described in detail in conjunction with FIG. 1. The fluid contained in reservoir 10 is retrieved through fluid line 60 by bladder pump 50. Fluid line 60, as well as all other fluid lines in the system, may be made of any type of medical grade, durable tubing suitable for transporting the fluid in use. Bladder pump 50 is comprised of a pneumatic pressure chamber 51 and a bladder 53, which may be comprised of an suitable elastomeric material. An illustrative
suitable bladder is the Cutter/Miles double valved hand activated blood pump. Bladder pump 50 forces fluid from reservoir 10 to treatment chamber 46 through fluid line 58 by being alternately compressed and expanded by alternating pressure source 54 in conjunction with valve 52 and timer 55. Alternating pressure source 54 preferably may be any standard pump capable of providing both positive pressure and negative (or vacuum) pressure, such as a piston or diaphragm pump.
Valve 52 accepts the positive pressure and negative pressure from pump 54 through lines 64 and 66, respectively. Due to signals from timer 55, valve 52 causes alternating positive and negative pressure to be applied to bladder 53 from line 62. Valve 52 may be any type of inline valve capable of directing and regulating multiple lines. One such valve is the MAC 45S, model 45A-AA1-DAAA-1BA.
When negative pressure is applied to bladder 53, fluid will be drawn from fluid reservoir 10 until bladder 53 is filled with fluid and is in a fully expanded state. During expansion of bladder 53, check valve 74 will ensure that no fluid is drawn from fluid line 58. Once the signal from timer 55 causes a positive pressure to be applied to bladder 53, the fluid contained in the bladder is forced out of the bladder and through fluid line 58 to treatment chamber 46. When fluid is forced out of bladder 53, check valve 72 will ensure that no fluid is forced back into fluid line 60. In this manner, a pulsitile, cyclic fluid flow to treatment chamber 46 is created.
Treatment chamber 46 preferably may be composed of any biocompatible, rigid material capable of being sterilized such as Teflon, polycarbonate, PVC, or stainless steel.
Treatment chamber 46 may be comprised of two sections which are secured and made leak proof through any standard means such as inner and outer threads or the use of bonding agents. In order to view vascular grafts within treatment chamber 46, a viewing port may be placed at any point on the chamber, or alternatively, the chamber may be made of an optically clear material such as polycarbonate or PVC. Inlet port 68 and outlet port 70 of treatment chamber 46 allow for the
perfusion and/or circulation of fluid into and through the chamber. Inlet port 68 and outlet port 70 are also used to attach treatment chamber 46 to fluid lines 58 and 56
respectively. Fluid line 56 connects chamber 46 back to fluid reservoir 10 so as to create a closed system. It is to be understood that although only one treatment chamber 46 is shown in FIG. 4, fluid lines 56, 58, and 60 may be branched
so as to connect more than one treatment chamber in parallel to the system.
Treatment chamber 46 houses a porous tube 48 upon which may be placed a vascular graft scaffolding 26. Scaffolding 26 is discussed in detail in conjunction with FIG. 1 above. Porous tube 48 may be comprised of any suitable rigid
material, such as Teflon, PVC, polycarbonate, or stainless steel, which may be made fluid permeable. One illustrative example of a suitable porous tubing is the porous plastic tubing manufactured by Porex Technologies. Alternatively, porous tube 48 may be comprised of any suitable elastomeric material, such as PET or silicone angioplasty balloons, which is capable of expanding and contracting, and which may be made fluid permeable. Treatment Chamber 46 and tube 48 may both be made any length or diameter so as to hold a vascular graft scaffolding 26 of any length or diameter. This is advantageous, as the system may be used to sterilize, seed, culture, store, ship, and test vascular grafts of any size. Porous clips or grommets 33 may be placed on tube 48 at both ends of scaffolding 26 to hold the scaffolding in place on the tube during treatment.
If tube 48 is comprised of a rigid porous material, then the varying fluid pressure caused by the action of bladder pump 50 will force fluid through the porous material. The fluid force through the porous material will place a varying radial stress on the vascular graft scaffolding.
Alternatively, if tube 48 is comprised of a porous
elastomeric material, tube 48 may be expanded and contracted by the varying fluid pressure provided by bladder pump 50. By expanding and contracting porous tube 48 with bladder pump 50, tube 48 places a varying radial stress on vascular graft scaffolding 26. Moreover, as is the case with a rigid tube 48, the fluid flow through the elastomeric porous material will also place a varying radial stress on scaffolding 26. In this manner, a cyclical radial loading of the scaffolding and cells supported thereon is created, resulting in vascular grafts which are formed with cells and their fibers
configured so as to more likely tolerate the physiological conditions found in the human body.
It is to be understood that the inlet port and outlet port of treatment chamber 14 (in FIGS. 1 and 3) and treatment chamber 46 (in FIG. 4) may be sealed in a known manner (e.g., luer locks or threaded plugs) so as to create a sealed treatment chamber free from contamination. The sealed chambers may be used to sterilize, store, and ship vascular grafts or other protheses. In particular, prior to placing a sealed chamber into the systems of FIGS, 1, 3, and 4, a vascular graft scaffolding 26 which is secured within the sealed chambers 14 or 46 may be sterilized by some chemical means such as ethylene oxide or peracetic acid, radiation means such as an electron beam or gamma rays, or by steam sterilization. Sealed treatment chambers 14 or 46,
containing the sterilized vascular graft scaffolding, may then be placed back into the systems of FIGS. 1, 3 and 4 for seeding and culturing and unsealed without contaminating the system or the vascular graft .
Seeding and culturing of the vascular graft in treatment chambers 14 and 46 is generally accomplished by known
techniques, with the added benefits and advantages gained from the radial stress placed upon the vascular graft during use. Examples of suitable seeding and culturing methods for the growth of three-dimensional cell cultures are disclosed in U.S. Patent No. 5,266,480, which is incorporated herein by reference. The techniques described in U.S. Patent No.
5,266,480 for establishing a three-dimensional matrix, inoculating the matrix with the desired cells, and
maintaining the culture may also be readily adapted by a person of ordinary skill in the art for use with the present invention.
Once the vascular graft has reached the desired level of cell density, a preservative may then be pumped into
treatment chambers 14 or 46. Once the treatment chambers are filled with the preservative, the inlet ports and outlet ports of the chambers may be closed, again creating a sealed
chamber which may then be used to store and/or ship the cultured and preserved vascular graft. Preferably, the preservative is a cryo-preservative so that the graft may be frozen in chambers 14 or 46. In this manner, sealed
treatment chambers 14 or 46 may be used to sterilize,
culture, store, and ship vascular grafts or other protheses.
Various embodiments of the invention have been
described. The descriptions are intended to be illustrative, not limitative. Thus, it will be apparent to those skilled in the art that modifications may be made to the invention as described without departing from the scope of the claims set out below.