WO1996034090A1

WO1996034090A1 - Apparatus and method for sterilizing, seeding, culturing, storing, shipping and testing tissue, synthetic or native vascular grafts

Info

Publication number: WO1996034090A1
Application number: PCT/US1996/005151
Authority: WO
Inventors: Alvin Peterson; Lee K. Landeen; Joan Zeltinger
Original assignee: Advanced Tissue Sciences, Inc.
Priority date: 1995-04-27
Filing date: 1996-04-09
Publication date: 1996-10-31
Also published as: NZ306304A; JP4059301B2; KR19990008070A; EP0822978A4; JPH11504216A; EP0822978A1; AU703117B2; CA2219202A1; AU5484396A

Abstract

An apparatus and method for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts is disclosed. Specifically, the present invention relates to an apparatus and method for seeding and culturing vascular grafts with human cells. The apparatus includes a fluid reservoir (10), a pump (12), an alternating pressure source (16), and at least one treatment chamber (14). By alternating pressure to a support structure (32) within the treatment chamber (14) upon which a vascular graft scaffold (26) is positioned, a varying radial stress is placed on the scaffold (26). This stress results in a tissue-engineered vascular graft with cells and their fibers oriented in a manner which is more likely to possess long term dimensional stability and the patency of native vessels with normal physiologic function.

Description

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.

Claims

We claim:

1. An apparatus, comprising:

a chamber having a first port and a second port for flow of fluid therethrough;

a support structure located within said chamber configured and dimensioned to support a tubular prosthesis; and

means for imparting a radial stress to the prosthesis.

2. The apparatus of claim 1 wherein said support structure is moveable between a first position and a second position.

3. The apparatus of claim 2 wherein said imparting means comprises means for moving said support structure between the first and second positions.

4. The apparatus of claim 3, wherein said support structure comprises an expandable tubular member having an outer diameter that is variable in response to pressure within the tubular member, said tubular member adapted to receive the tubular prosthesis thereover.

5. The apparatus of claim 4, wherein said moving means comprises an alternating pressure source communicating with the tubular member for moving said support structure from the first position to the second position.

6. The apparatus of claim 1, wherein said support structure is comprised of a porous material.

7. The apparatus of claim 6, wherein said imparting means comprises means for forcing fluid flow through said support structure.

8. The apparatus of claim 7, wherein said forcing means comprises a pump providing alternating pressure.

9. An apparatus, comprising:

a chamber having a first port and a second port for flow of fluid therethrough;

a variable support structure located within said chamber configured and dimensioned to support a tubular prosthesis;

said support structure being moveable between a first position and a second position, wherein movement of said support structure between said positions creates varying radial stresses in the prosthesis supported thereby.

10. The apparatus of claim 9, wherein the support structure comprises an expandable member that is adapted to receive the tubular prosthesis thereover.

11. The apparatus of claim 10, wherein the expandable member comprises an expandable tubular member having an outer diameter that is variable in response to pressure within the tubular member.

12. The apparatus of claim 11, wherein the expandable tubular member comprises an angioplasty balloon.

13. The apparatus of claim 11 further comprising an alternating pressure source communicating with the tubular member for moving said support structure from said first position to said second position.

14. The apparatus of claim 13, wherein said alternating pressure source comprises:

a pump for providing a first level of pressure and second level pressure;

a valve connected in between said pump and said support structure for alternatingly allowing the first level of pressure and the second level of pressure to be placed on said support structure, wherein the first level of pressure corresponds to the first position and the second level of pressure corresponds to the second position.

15. The apparatus of claim 14, wherein the valve is connected to a timer for variably opening and closing the valve.

16. The apparatus of claim 11, wherein the expandable tubular member is sized such that in at least the first position the tubular member and prosthesis are spaced apart to define a passage that permits fluid to circulate between said tubular member and said prosthesis.

17. The apparatus of claim 16, wherein said prosthesis is held in place on said support structure by a grommet .

18. An apparatus for seeding and culturing vascular grafts, comprising:

a housing defining a seeding and culturing chamber with first and second ports to permit flow of a medium therethrough;

a vascular graft support structure mounted in the chamber, said structure adapted for supporting a vascular graft such that media flow between said ports is permitted to circulate around the surfaces of the graft; and

a pressure source for alternatingly expanding said vascular graft support structure from a first position to a second position to apply a varying radial stress on the graft.

19. The apparatus of claim 18, wherein said vascular graft support structure comprises an elastic tube.

20. The apparatus of claim 19, wherein said pressure source comprises: a pump for providing a first level of pressure and a second level of pressure;

a valve connected in between said pump and said support structure for alternatingly allowing the first level of pressure and the second level of pressure to be placed on said support structure, the first level of pressure forcing said support structure into the first position and the second level of pressure forcing said support structure into the second position.

21. The apparatus of claim 18, further comprising a plurality of said housings.

22. The apparatus of claim 18, wherein the first and second ports of said housing may be sealed for enclosing, sterilizing, storing, and shipping the vascular graft.

23. An apparatus for seeding and culturing vascular grafts, comprising:

a housing defining a seeding and culturing chamber with first and second ports to permit flow of a medium therethrough; and

a porous vascular graft support structure mounted in the chamber, said support structure fluidly connected to the first port such that medium flows from the first port through said porous support structure and out the second port;

wherein the medium flow through said support structure applies a radial stress on a graft mounted on said support structure.

24. The apparatus of claim 23, further comprising a pump for providing a medium flow to said housing with varying pressure.

25. The apparatus of claim 24, wherein said pump is a bladder pump.

26. The apparatus of claim 22, wherein said support structure comprises a rigid porous tube.

27. The apparatus of claim 22, wherein said support structure comprises an elastic porous tube.

28. A method for seeding and culturing a tubular prosthesis, comprising:

exposing the prosthesis to a fluid media for seeding and culturing; and

imparting a radial stress to the prosthesis during said seeding and culturing to encourage a desired alignment of cells on the prosthesis.

29. The method of claim 28, wherein said step of imparting radial stress comprises:

placing said prosthesis on a support structure; and moving said support structure between a first position and a second position so that the radial stress is imparted to the prosthesis.

30. The method of claim 29, wherein said support structure comprises an elastic tube.

31. The method of claim 30, wherein said step of expanding said support structure comprises moving said tube from the first to the second position by a pump, said pump alternatingly providing a first level and a second level of pressure .

32. The method of claim 28, wherein said step of imparting radial stress comprises:

placing said prosthesis on a porous support

structure; and

forcing the fluid media through the porous support structure so that radial stress is imparted to the prosthesis from the fluid media.

33. The method of claim 32, wherein the porous support structure comprises a rigid tubular member.

34. A method for treating a tubular prosthesis

comprising:

providing a fluid media source;

pumping the fluid media from said source to create a fluid stream;

holding the prosthesis in a media chamber communicating with the fluid stream; and

variably moving said prosthesis from a first contracted position to a second expanded position in said fluid stream.

35. The method of claim 34, wherein the diameter of said prosthesis is varied by said step of variably moving said prosthesis.

36. The method of claim 35, wherein said step of holding comprises placing the prosthesis over an elastic tube in the chamber and said step of variably moving comprises moving said tube from a first expanded position to a second contracted position.

37. The method of claim 36, wherein said step of moving said tube from said first expanded position to said second contracted position is accomplished through the use of a pump, said pump alternatingly providing a first and a second level of pressure to said tube.

38. The method of claim 35, wherein said step of holding comprises placing the prosthesis over a porous tube structure in the chamber and said step of variably moving comprises forcing the fluid media through said tube structure with varying pressure.

39. The method of claim 38, wherein said step of forcing the fluid media through said tube structure is accomplished through the use of a pump, said pump alternatingly providing a first and a second level of pressure to said tube structure.

AMENDED CLAIMS

[received by the International Bureau on 09 September 1996 (09.09.96); original claims 1-39 replaced by amended claims 1-23 (4 pages)]

1. An apparatus, comprising:

a housing defining a seeding and culturing chamber having a first port and a second port for flow of fluid therethrough;

means for imparting a radial stress to the prosthesis .

2. The apparatus of claim l wherein said support structure is moveable between a first position and a second position.

4. The apparatus of claim 3 , wherein said support structure comprises an expandable tubular member having an outer diameter that is variable in response to pressure within the tubular member, said tubular member adapted to receive the tubular prosthesis thereover.

6. The apparatus of any one of claims 1-5, wherein said support structure is comprised of a rigid or elastic material which may be porous.

7. The apparatus of claim 6, wherein said imparting means comprises means for forcing fluid flow through said support structure. 8. The apparatus of claim 7, wherein said forcing means comprises a pump providing alternating pressure.

9. The apparatus of claim 4, wherein the expandable tubular member comprises an angioplasty balloon.

10. The apparatus of claim 5, wherein said alternating pressure source comprises:

a pump for providing a first level of pressure and second level pressure;

11. The apparatus of claim 10, wherein the valve is connected to a timer for variably opening and closing the valve .

12. The apparatus of claim 4, wherein the expandable tubular member is sized such that in at least the first position the tubular member and prosthesis are spaced apart to define a passage that permits fluid to circulate between said tubular member and said prosthesis.

13. The apparatus of any one of claims 1-12, wherein said prosthesis is held in place on said support structure by a grommet.

14. The apparatus of claim 1, further comprising a plurality of said housings.

15. The apparatus of claim 1, wherein said housing comprises a first and second ports which may be sealed for enclosing, sterilizing, storing, and shipping the vascular graft.

16. The apparatus of claim 8, wherein said pump is a bladder pump.

17. A method for seeding and culturing a prosthesis, comprising:

exposing the prosthesis to a fluid media for seeding and culturing; and

imparting a radial stress to the prosthesis during said seeding and culturing to encourage a desired alignment of cells on the prosthesis. 18. The method of claim 17, wherein said step of imparting radial stress comprises:

19. The method of claim 18, wherein said support structure comprises a rigid or elastic tube. 20. The method of claim 19, which comprises moving said tube from the first to the second position by a pump, said pump alternatingly providing a first level and a second level of pressure.

21. The method of claim 17, wherein said step of imparting radial stress comprises:

placing said prosthesis on a porous support structure; and

22. The method of claim 21, wherein the porous support structure comprises a rigid tubular member.

23. The method of claim 17, wherein said prosthesis is tubular and its diameter is varied by said step of variably moving said prosthesis.