WO2011098365A2

WO2011098365A2 - Cryopreservation device, methods for making the same and uses thereof

Info

Publication number: WO2011098365A2
Application number: PCT/EP2011/051332
Authority: WO
Inventors: Simon P. Hoerstrup; Steffen M. Zeisberger
Original assignee: Universität Zürich
Priority date: 2010-02-12
Filing date: 2011-01-31
Publication date: 2011-08-18
Also published as: WO2011098365A3

Abstract

The invention relates to a cryopreservation device (1), in particular for the cryopreservation of living cellular three-dimensional structures such as tissue engineered valves (16), tubes (21), vessels. The proposed cryopreservation device comprises a liquid tight cavity (19) with an outer form element (4) and an inner form element (10), wherein outer, optionally coated surface portions (13-15, 22) of said form elements (4, 10) are bordering a contiguous, three-dimensionally shaped portion of said liquid tight cavity (19) and wherein the distance between surface portions (13) of the inner form element (10) and surface portions (22) of the outer form element (4) is in the range between 0.1-10 mm, preferably between 0.5-5 mm.

Description

CRYOPRESERVATION DEVICE, METHODS FOR MAKING THE SAME AND

USES THEREOF TECHNICAL FIELD

The present invention relates to cryopreservation devices, in particular for the storage and/or conservation of tissue engineered three-dimensional structures (tissue engineered 3D-auto- and allografts) such as vessels, tubular structures, organs as well as heart valves, as well as to uses of such a device and methods for the cryopreservation of tissue engineered structures .

PRIOR ART

Tissue engineering is emerging to aim at solving the problem of organ and tissue deficiencies and to provide the next generation of medical implants.

Due to the long production cycle, e. g. 6-8 weeks e.g. for vascular grafts, preservation of the product is critical to ensure the off-the-shelf availability to clinicians.

Simple preservation techniques, such as refrigeration (4°C) or tissue culture, have drawbacks including sample overgrow, high cost, risk of contamination or genetic drift. Consequently, cryopreservation (<4°C) is a more reasonable option, an approach based on the principle that biological, chemical and physical processes are effectively preserved at cryogenic temperatures. The difficulty of developing high-viability cryopreservation procedures and/or devices becomes apparent when one considers the hostile environment to which cells and tissues are subjected during the freezing process. The temperature drops from +37°C to -196°C, loss of over 95% of cell water can be incurred, the electrolyte concentration inside and outside the cells can increase by several orders of magnitude relative to isotonic conditions, concentrated organic solvents in the freezing media permeate the cells, ice crystals intercalate the tissue and mechanically deform cells, and ice may form inside cells, disrupting intracellular structures.

Many examples exist in the literature which illustrate the complexity of tissue cryopreservation. During the cryopreservation of human arteries, different failure modes have been observed. The arteries have fractured; the endothelial cells have been severely damaged, the smooth muscle cells have lost their responsiveness, a substantial fraction of cells lost their viability after freezing, and only cells close to surface survived a cryopreservation process. Some authors however report cryopreservation of cardiac valve allografts stored in liquid nitrogen up to 13 years with only minor loss of cell viability due to freezing and thawing.

The success of cryopreservation on the other hand depends on the practicability of the freezing, the actual storage during cryopreservation and the subsequent thawing process. Therefore there is a need for specifically tailored devices for the introduction of tissue engineered three-dimensional structures into the cryopreservation process, for keeping them at the cryopreservation temperature and for safely bringing them up to temperatures above 4°C once the tissue engineered structure shall be available for implantation.

SUMMARY OF THE INVENTION

It is therefore the aim of the present invention to provide an improved cryopreservation device for three-dimensional tissue engineered structures. The device shall allow an as efficient and as homogeneous as possible fast cooling process allowing for very high cooling rates without problems of sticking of the tissue engineered structures with wall elements of the cryopreservation device and/or of wall portions of the tissue engineered structure at each other. The device shall in particular allow cryopreservation using true vitrification conditions, i.e. shall be suitable for immersion into low-temperature cooling agents such as liquid or gaseous nitrogen.

In accordance with the present invention a cryopreservation device is proposed, in particular for the cryopreservation of living cellular three-dimensional structures such as tissue engineered valves, tubes, vessels, wherein all these cellular three-dimensional structures are to be understood as non-flat structures, so structures which have a three- dimensional cellular wall structure/shape, and which typically enclose at least partially a cavity or indentation. The proposed cryopreservation device comprises a liquid tight cavity with an outer form element and an inner form element, wherein outer surface portions of said form elements are bordering a contiguous, three-dimensionally shaped portion of said liquid tight cavity and wherein the distance between surface portions of the inner form element and surface portions of the outer form element is in the range between 0.1-10 mm, preferably in the range of 0.5-5 mm. Preferentially this distance is in the range of 1-2.5 mm. The tissue engineered structure is to be put into this liquid tight cavity which is bordered in a very narrow contiguous, channel-like manner by the walls of the inner form element and the walls of the outer form element. In other words inner form element and outer form element comprise a complementary shape of opposing walls, wherein normally the distance between opposing walls is in the above-mentioned range and is preferably essentially the same over the whole contiguous channel like liquid cavity formed by this interspace. The distance between these two form elements is typically in the above range in all the regions where the tissue engineered structure is located in between.

This has the effect that on the one hand the tissue engineered structure, if not held firmly in place by a slight clamping by the walls, is at least kept in position and prevented from folding and/or substantial movement during the whole cryopreservation process. On the other hand it has the effect, due to the close contact or the small distance between the tissue engineered structure and the wall elements, that the heat energy can be transported very quickly and homogeneously away from the whole tissue engineered structure, which is particularly important if cryopreservation shall take place under vitrification conditions. The whole tissue engineered structure can thus be cooled down to a desired low storage temperature in a very highly controlled quick (high cooling rate) and homogeneous (very low temperature gradients in the tissue engineered product) cooling process, and it can also be reheated/thawed in a controlled quick or if necessary slow and homogeneous (very low temperature gradients in the tissue engineered product) reheating process. This allows for a particularly gently process which prevents damage to the cellular structures due to the conservation process.

According to a first preferred embodiment of the present invention, the outer form element is a circumferential tubular structure, preferably a cylindrical, most preferably a circular cylindrical wall structure.

If the cryopreservation device is particularly tailored for the cryopreservation of tissue engineered tubes and/or vessels, preferentially the inner form element takes the form of a cylindrical, preferably a circular cylindrical structure, the outer wall forming said surface portion of the inner form element.

If the cryopreservation device is particularly tailored for the cryopreservation of valves in particular of heart valves, preferentially the inner form element comprises a bottom profile element and a top profile element which at least in portions are distanced from each other such that a top surface of the bottom profile element and a bottom surface of the top profile element are distanced with the distance in a range between 0.1 - 10 mm, preferably between 0.5-5 mm, most preferably with the distance in the range of 1-2.5 mm. Again preferentially this distance range is maintained wherever the tissue engineered structure is to be located between these two elements. According to a preferred embodiment, the bottom profile element may be attached to a bottom plate and the top profile element may be attached to a top plate of the cryopreservation device for simple use. The elements are structured that the valve is stored essentially in the closed state, so in a state where the valve leaflets are in closed position.

Of course inner form element and outer form element may also be tailored to other tissue engineered structures such as branching vessel structures, three-dimensional tubular wall structures, hollow organs etc.

According to a further preferred embodiment of the proposed cryopreservation device for valve structures, the bottom profile element comprises a convex top surface portion, preferably with a convex, upwards pointing essentially conical central portion, and wherein the top profile element comprises a concave bottom surface portion, preferably with a form corresponding to the top surface of the bottom profile element.

Preferentially in order to avoid any detrimental sticky contact between the form elements and the tissue engineered structure, the surfaces of said form elements, at least in all the regions where contact with the tissue engineered structure is possible, are coated with an inert surface coating (inert with respect to the tissue engineered structure). Preferentially this is a polymer-based surface coating, most preferably a PTFE surface coating. Further possible materials for the coating layer are silicon, polyvinylchloride (PVC), synthetic hydrogels, such as polylactic acid (PLA), starch-based polymers, aromatic aliphatic co- polyesters, polyhydroxyalkanoates (PHA), polylactide, trimethylene carbonate, polyethylene glycol (PEG), polylactide-co-glycolide acid (PLGA), DegraPol, Pluronic, and/or polyglycolic acid (PGA) and combinations thereof. Further, biological bio-matrices such as fibrin, alginate, collagen, Matrigel, and/or cellulose and combinations thereof are possible.

Preferably generally speaking the thickness of such surface coating is in the range of 1 nm - 1 mm, preferably of 1 μιη (micrometer) - 0.5 mm, more preferably in the range of 0.05- 0.1 mm. And as thin as possible coating layer thickness is advantageous as the thermal conductivity of such a layer is usually lower than the thermal conductivity of the material from which the form elements are made.

Further preferentially the coating is applied over the entire surfaces facing the liquid tight cavity at least where contact with the tissue engineered structure is to be expected.

According to a further preferred embodiment, the outer form element and/or the inner form element (and/or bottom plate and/or top plate if present) has a thermal conductivity above 40 W / (m · K), preferably above 100 W / (m · K), most preferably above 200 W / (m · K). In order to further increase fast cooling through the whole device and to the tissue engineered structure, it can be advantageous if the outer form element and/or the inner form element is provided with channels and/or grooves and/or indentations or penetration and/or circulation of liquid and/or gaseous cooling medium from the outside.

A further preferred embodiment is characterised in that it comprises a bottom plate and a top plate between which the outer form element is located such as to enclose a cavity, wherein completely within this cavity the inner form element is located such that the liquid tight cavity is formed by the interspace between inner walls of the outer form element and outer walls of the inner form element as well as if applicable between walls of parts of the inner form element, wherein preferably sealing elements are provided between the outer form element and a bottom plate and a top plate, respectively, and wherein further preferably bottom plate, outer form element and top plate are pressed together by external clamping elements.

The inner form element and/or the outer form element and/or bottom plate and/or top plate may essentially consist of metal generally or more specifically steel, copper, aluminium, silver and/or alloys thereof. Also possible is the use of carbon, ceramic etc.

The present invention furthermore relates to the use of such a cryopreservation device, in particular for the cryopreservation of tissue engineered cellular structures, preferably for the cryopreservation of vessels and/or tubes such as trachea tubes or for the cryopreservation of valves such as heart valves preferably for the cryopreservation of autologous tissue engineered cellular structures of this kind.

Preferably the use is made in a vitrification cryopreservation preferably using immersion into cooling medium, preferably into liquid or gaseous nitrogen.

Furthermore the present invention relates to a method for the cryopreservation of tissue engineered cellular structures, preferably of vessels, tubes and/or heart valves, using a cryopreservation device as outlined above, wherein preferably the final tissue engineered cellular structure is inserted into the liquid tight cavity together with a liquid medium, preferably a cell growth promoting liquid medium such that the tissue engineered cellular structure is completely immersed in the liquid medium, wherein the cryopreservation device and its liquid tight cavity is closed in a liquid tight manner, optionally after having inserted the inner form element and/or additional parts thereof, preferably by putting a top plate, optionally with one part of the inner form element attached thereto, onto the device and closing it.

Further embodiments of the invention are laid down in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1 shows a cryopreservation device for a heart valve, wherein in a) a top view, in b) a central axial cut, in c) a cut perpendicular to the axis at half height and in d) the bottom view are given; and

Fig. 2 shows a cryopreservation device for a vessel or tube, wherein in a) a top view, in b) a central axial cut, in c) a cut perpendicular to the axis at half height and in d) the bottom view are given.

DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides a device for the cryopreservation of tissue-engineered grafts as exemplified in figures 1 and 2. The cryo-device provides an adapter function with the aim to bring temperature-conducting material in close proximity with the tissue engineered graft. At the 'graft-device' interface the device contains an ultra-thin surface coating with the intention to protect the tissue engineered-graft from direct contact damage to the temperature-conductor material. The cryo-device chamber is designed to bring a cellularized 3D tissue engineered graft in tight proximity to the cryo-device wall to guarantee efficient temperature-transfer.

Temperature-conductor supporting material may be composed out of materials such as metals, carbon, ceramic, etc.

The inside coating of the cryo-device may be composed out of materials such as PTFE, silicon, polyvinylchloride (PVC), synthetic hydrogels, such as polylactic acid (PLA), starch-based polymers, aromatic aliphatic co-polyesters, polyhydroxyalkanoates (PHA), polylactide, or trimethylene carbonate, polyethylene glycol (PEG), polylactide-co- glycolide acid (PLGA), DegraPol, Pluronic, and/or polyglycolic acid (PGA) and combinations thereof. Further, biological biomatrices such as fibrin, alginate, collagen, Matrigel, and/or cellulose and combinations thereof. Example 1 : Cryo-device for tissue engineered heart valve

As exemplified in Figure 1, the cryo-device 1 for a tissue engineered heart valve 16 is composed out of temperature-conductor materials such as metals, carbon, ceramic etc. The individual elements are a bottom plate 2 and a top plate 3 between which a cylindrical circumferential sidewall 4 is provided which forms the outer form element. To optimize temperature transfer, the cryo-device provides an adapter function to bring temperature- conducting cryo- vial- walls in close proximity (0.1-10 mm, normally 0.5 - 5 mm) to the tissue engineered heart valve 16. At the 'graft-device' interface the device contains an ultra-thin surface coating with the intention to protect the tissue engineered graft 16 from direct contact to the temperature-conductor material. The cryo-device for tissue engineered heart valves 16 is composed of 5 functional parts: a heart-valve positive profile or inner bottom profile element 11, a heart- valve negative profile or inner top profile element 12, an external cylinder 4, sealing rings 5 and four screws 6 keeping the cryo-device closely connected.

More specifically, there is provided a bottom plate 2 and a top plate 3 between which the cylindrical wall structure 4 is clamped. Bottom plate 2 as well as top plate 3 are provided with circumferential grooves 9 at the position where contact is made with the side wall 4 to take up sealing rings 5. Also the side wall 4 may be provided with a corresponding groove for the sealing ring 5. Bottom plate and top plate when pressed against the axial faces of the wall structure 4 enclose a liquid type large cavity. These elements are held together by axial screws 6 which are located in bores in bottom plate 2 comprising bottom heads 7 and which on the top surface penetrate through top plate 3 and are screwed together by locking elements 8.

In the interior of this large cavity there is provided an inner form element, in this case a bottom form element 11 and a top form element 12. Bottom form element 11 is attached to bottom plate 2 and top form element 12 is attached to top plate 3. Like this a three dimensional contiguous liquid tight cavity 19 is provided if the cryopreservation device is in the closed state as given in Figure 1.

If the screws 6 are loosened or removed and top plate 3 with attached inner form element 12 are removed, this liquid tight cavity 19 is open and a tissue engineered heart valve 16 can be put into this open cavity such that the circumferential vessel part 18 is located between wall 4 and the bottom element 11 and such that the axial valve leafs 17 come to lie on the top surface 14 of the element 11, which surface is three dimensionally shaped. The cavity is then subsequently filled with a sufficient amount of liquid suitable for the cryopreservation process and then top plate 3 with attached top form element 12 is put onto the cryopreservation device such that the bottom surface 15 of the top form element 12 comes into close proximity or even into contact with the portions 17 of the valve structure 16. Now the full valve structure 16 , i.e. all the wall structures thereof, is located in a cavity which encloses very closely all over the surface structure of the engineered construct 16 . The whole structure is immersed in liquid. Example 2: Cryo-device for tissue engineered vessel conduit

The cryo-device for a tissue engineered vessel conduit is composed out of temperature- conductor materials such as metals, carbon, ceramic etc. as given in Figure 2. To optimize temperature transfer, the cryo-device provides an adapter function to bring temperature- conducting cryo-vial- walls in close proximity (0.1 - 10 mm, normally 0.5 - 5 mm) to the tissue engineered vessel conduit 21. At the 'graft-device' interface the device contains an ultra-thin surface coating with the intention to protect the tissue engineered graft 21 from direct contact to the temperature-conductor material. The cryo-device for tissue engineered vessels is composed of 5 functional parts: a cap or bottom plate 2, a cylindric bottom profile or inner form element 10, an external cylinder or circumferential sidewall 4, sealing rings 5 and four screws 6 keeping the cryo-device closely connected.

In this specific case the inner form element 10 is provided as an essentially circular cylinder the outer wall 13 of which defines the inner proximity surface 13 for the tissue engineered structure 21, while the inner wall 22 of the outer form element 4 defines the outer proximity surface. In this case, but also in the embodiment according to Figure 1, it is, for an efficient and fast transport of heat out of the tissue engineered structure, possible to provide the inner structure 10 as a hollow body, the interior of which is accessible to cooling medium, or at least a body provided with channels, grooves or the like which can be penetrated by the cooling medium when the device is immersed into liquid nitrogen. Generally speaking, for heart valves, a device according to Figure 1 has a height H in the range of 20-30 mm and a diameter D in the range of 50-70 mm. In case of a device for a vessel structure the device has a height H and a diameter D of 50-100 mm. LIST OF REFERENCE SIGNS cryopreservation device top surface of 11

bottom plate 15 three dimensionally shaped top plate bottom surface of 12 circumferential sidewall, 16 tissue engineered heart valve outer form element 17 leaf of 16

sealing ring 18 circumferential vessel part of connecting bar 16

bottom head of 6 19 liquid cavity of 1

locking element 20 axis of 1

circumferential groove in 2/3 21 tissue engineered vessel/tube for sealing element wall

inner form element 22 inner surface profile of outer heart valve inner bottom form element

profile element 23 surface coating layer heart valve inner top profile H height of device

element D diameter of device circumferential outer

surface/wall of 10/11/12

three-dimensionally shaped

Claims

1. Cryopreservation device (1), in particular for the cryopreservation of living cellular three-dimensional structures such as tissue engineered valves (16), tubes (21), vessels, comprising a liquid tight cavity (19) with an outer form element (4) and an inner form element (10), wherein outer surface portions (13-15, 22) of said form elements (4, 10) are bordering a contiguous, three-dimensionally shaped portion of said liquid tight cavity (19) and wherein the distance between surface portions (13) of the inner form element (10) and surface portions (22) of the outer form element (4) is in the range between 0.1-10 mm.

2. Cryopreservation device (1) according to claim 1, wherein the outer form element (4) is a circumferential tubular structure (4), preferably a cylindrical, most preferably a circular cylindrical wall structure.

3. Cryopreservation device (1) according to any of the preceding claims, wherein it is for the cryopreservation of tissue engineered tubes and/or vessels (21), and wherein the inner form element (10) takes the form of a cylindrical, preferably a circular cylindrical structure, the outer wall (13) forming said surface portion (13) of the inner form element (10).

4. Cryopreservation device (1) according to any of the preceding claims, wherein it is for the cryopreservation of valves (16) in particular of heart valves, and wherein the inner form element (10) comprises a bottom profile element (11) and a top profile element (12) which at least in portions are distanced from each other such that a top surface (14) of the bottom profile element (11) and a bottom surface (15) of the top profile element (12) are distanced with the distance in a range between 0.1-10 mm, preferably between 0.5-5 mm, more preferably with the distance in the range of 1-2.5 mm.

5. Cryopreservation device (1) according to claim 4, wherein the bottom profile element (11) comprises a convex top surface (14) portion, preferably with a convex, upwards pointing essentially conical central portion, and wherein the top profile element (12) comprises a concave bottom surface (15) portion, preferably with a form corresponding to the top surface (14) of the bottom profile element (11).

Cryopreservation device (1) according to any of the preceding claims, wherein the surfaces (13-15, 22) of said form elements (4, 10, 11, 12) are coated with a preferably inert surface coating (23), preferably a polymer-based surface coating, most preferably a PTFE surface coating, wherein the thickness of the surface coating is preferably in the range of 1 nm - 1 mm, preferably of 1 micrometer - 0.5 mm, more preferably in the range of 0.05-0.1 mm, wherein preferably the coating is applied over the entire surfaces facing the liquid tight cavity (19).

Cryopreservation device (1) according to any of the preceding claims, wherein the outer form element (4) and/or the inner form element (10) has a thermal conductivity above 40 W / (m · K), preferably above 100 W / (m · K), most preferably above 200 W / (m · K).

Cryopreservation device (1) according to any of the preceding claims, wherein the outer form element (4) and/or the inner form element (10) is provided with channels and/or grooves and/or indentations or penetration and/or circulation of liquid and/or gaseous cooling medium.

Cryopreservation device (1) according to any of the preceding claims, wherein it comprises a bottom plate (2) and a top plate (3) between which the outer form element (4) is located such as to enclose a cavity, wherein completely within this cavity the inner form element (10-12) is located such that the liquid tight cavity (19) is formed by the interspace between inner walls (22) of the outer form element (4) and outer walls (13-15) of the inner form element (10) as well as if applicable between walls (14, 15) of parts (11, 12) of the inner form element (10), wherein preferably sealing elements (5) are provided between the outer form element (4) and a bottom plate (2) and a top plate (3), respectively, and wherein further preferably bottom plate (2), outer form element (4) and top plate (3) are pressed together by external clamping elements (6-8).

10. Cryopreservation device (1) according to any of the preceding claims, wherein the inner form element (4) and/or the outer form element (10) and/or bottom plate (2) and/or top plate (3) essentially consist of a metal such as steel, copper, aluminium, silver and/or alloys thereof or of ceramic or carbon or combinations of such materials.

11. Use of a cryopreservation device (1) according to any of the preceding claims for the cryopreservation of tissue engineered cellular structures, preferably for the cryopreservation of vessels and/or tubes such as trachea tubes or for the cryopreservation of valves such as heart valves (16) preferably for the cryopreservation of autologous tissue engineered cellular structures of this kind.

12. Use according to claim 11, wherein it is a vitrification cryopreservation preferably using immersion into cooling medium, preferably into liquid or gaseous nitrogen.

13. Method for the cryopreservation of tissue engineered cellular structures, preferably of vessels, tubes and/or heart valves, using a cryopreservation device according to any of claims 1-10, wherein the final tissue engineered cellular structure is inserted into the liquid tight cavity (19) together with a liquid medium, preferably a cell growth promoting liquid medium such that the tissue engineered cellular structure is completely immersed in the liquid medium, wherein the cryopreservation device and its liquid tight cavity (19) is closed in a liquid tight manner, optionally after having inserted the inner form element (10) and/or additional parts thereof, preferably by putting a top plate (3), optionally with one part (12) of the inner form element (10) attached thereto, onto the device and closing it.