WO2011098367A2

WO2011098367A2 - Method for in-vitro production of tissue engineered auto- and allografts suitable for guarded cryopreservation

Info

Publication number: WO2011098367A2
Application number: PCT/EP2011/051336
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: WO2011098367A3

Abstract

The invention pertains to a method for the generation of a living cell based tissue engineered organ and/or tissue analogous structure having mechanical and/or functional properties comprising the steps of providing a organ and/or tissue, wherein the organ and/or tissue has the capacity of functioning or forming the native organ/tissue corresponding to the replacement; and wherein the tissue engineered organ and/or tissue analogous structure is made of/contains material incorporating cryoactive substances and/or particles. Furthermore the invention pertains to a scaffold for use in such a method, to a tissue engineered replacement structure made using this method, in particular in the form of a vessel and/or a heart valve.

Description

Method for in-vitro production of tissue engineered auto- and allografts suitable for guarded cryopreservation TECHNICAL FIELD

The present invention relates to methods and devices for the tissue engineering of 3D-auto- and allografts which are particularly adapted to subsequent guarded cryopreservation thereof. More particularly, the present invention is directed to compositions and methods to manufacture functionalized tissue-engineered grafts, scaffolds for use in such methods, as well as to tissue-engineered grafts obtained using such a method.

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.

Tissue engineering is a multidisciplinary science that utilizes basic principles from the life sciences and engineering sciences to create cellular constructs for transplantation. The first attempts to culture cells on a matrix for use as artificial skin, which requires formation of a thin three dimensional structure, were described for example in US 4,060,081, 4,485,097 and 4,458,678. They used collagen type structures as scaffolds which were seeded with cells, then placed over the denuded area.

US 4,520,821 describes the use of synthetic polymeric meshes as scaffolds to form linings to repair defects in the urinary tract. Epithelial cells were implanted onto the synthetic matrices, which formed a new tubular lining as the matrix degraded. The scaffold served a two fold purpose - to retain liquid while the cells replicated, and to hold and guide the cells as they replicated.

In EP 299 010 a method of culturing dissociated cells on biocompatible, biodegradable matrices for subsequent implantation into the body was described. This method was designed to overcome a major problem with previous attempts to culture cells to form three dimensional structures having a diameter of greater than that of skin. It was recognized that there was a need to have two elements in any matrix used to form organs: adequate structure and surface area to implant a large volume of cells into the body to replace lost function and a matrix formed in a way that allowed adequate diffusion of gases and nutrients throughout the matrix as the cells attached and grew to maintain viability in the absence of vascularization. Once implanted and vascularized, the porosity required for diffusion of the nutrients and gases was no longer critical.

To overcome some of the limitations inherent in the design of the porous structures which support cell growth throughout the matrix solely by diffusion, WO 93/08850 disclosed implantation of relatively rigid, non-compressible porous matrices which are allowed to become vascularized, then seeded with cells.

EP 1077072 on the other hand discloses a scaffold based tissue engineering method in particular for heart valves where during the growth process the seeded scaffold is subjected to a particular pressure/flow pattern in order to stimulate growth and/or induce the formation of structures under conditions as similar to the final use conditions as possible. Many tissues have now been engineered using these methods, including connective tissue such as bone and cartilage, as well as soft tissue such as hepatocytes, intestine, endothelium, and specific structures, such as ureters.

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 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. SUMMARY OF THE INVENTION

Unlike their natural counterparts, engineered tissues have flexibility in aspects of their design and production such as the scaffold material, cell density and even shape and size, etc. Consequently, the survivability of engineered tissue to cryopreservation can be addressed in the early stage of the construct design. To examine the effect of these "adjustable" design parameters on their cryopreservation and to integrate the preservation issue into early product design is of great practical interest to industry.

In order for the use of engineered tissue to become practical in the clinical setting, the cryopreservation process must require minimal post-thaw processing by the end-user (clinician). Therefore, cryoprotective agents (CPAs) such as dimethylsulphoxide (DMSO) should be used sparingly or even substituted by non-toxic CPAs, since DMSO removal from tissue can require complex and timeconsuming dilution procedures, especially when higher concentrations are employed.

The present invention relates to methods, scaffolds and cell-matrix replacement tissue structures which allow the guarded cryopreservation of tissue engineered 3D-auto- and allografts in general, and heart valves or vascular conduits in particular.

According to a first aspect of the invention, a method for the generation of a cell-matrix replacement tissue structure having mechanical strength and flexibility or pliability is proposed. This method is comprising the steps of

providing a scaffold formed of a biocompatible, biodegradable material,

seeding the scaffold with dissociated preferably human cells,

wherein the (human) cells have the capacity of forming the native tissue corresponding to the replacement; and

cultivating the cells under conditions allowing the development of the tissue replacement. In order to address the above problems with cryopreservation, according to the invention the scaffold is made of a material incorporating cryoactive substances and/or particles. Besides tissue engineered 3D-auto- and allografts having primarily mechanical strength and flexibility and/or pliability also tissue engineered organs can be used. More generally speaking, the invention relates to a method for the generation of a living cell based tissue engineered organ and/or tissue analogous structure having mechanical and/or functional properties comprising the steps of providing a organ and/or tissue, wherein the organ and/or tissue has the capacity of functioning or forming the native organ/tissue corresponding to the replacement; and wherein the tissue engineered organ and/or tissue analogous structure is made of/contains material incorporating cryoactive substances and/or particles.

From a general point of view methods as disclosed in EP 1 077 072 but also as more recently disclosed in Schmidt D, Mol A, Breymann C, et al.; "Living autologous heart valves engineered from human prenatally harvested progenitors"; Circulation; 2006;114:1125-131 as well as in Hoerstrup SP, Cummings Mrcs I, Lachat M, et al. "Functional growth in tissue-engineered living, vascular grafts: follow-up at 100 weeks in a large animal model"; Circulation. 2006;1 14:1159-166 are possible and the disclosure of these documents shall be included into the specification as concerns the tissue generation protocols.

Preferentially the cells used to the seeding of the scaffold are selected from the group of prenatal or perinatal (postnatal) autologous cells but also allogeneic cells, from different sources of the body, such as umbilical cord blood derived endothelial or mesenchymal progenitor cells, umbilical cord derived endothelial or mesenchymal cells, vain derived endothelial cells or fibroblasts, adipose tissue derived mesenchymal progenitor cells, etc. So the gist of the invention is thus to use a scaffold in such a tissue generation method which inherently carries cryoactive substances and/or cryoactive particles. Under the expression cryoactive substances and/or particles, systems are to be understood which allow to overcome limitations in temperature transfer inside of tissue engineered grafts during cryopreservation or components to improve cell viability during and/or after cryopreservation inside of the tissue. So the basic idea is to use a scaffold which carries such systems to be released/become active only once necessary, so during or after the cryopreservation step. Locating these systems in the scaffold has the advantage that they are located within the tissue structure so exactly where needed and do not have to be provided from the outside for the cryopreservation process (however additional provision of such systems from the outside, for example in the liquid for the cryopreservation process, is not excluded). In this way not only are these systems already located and buried within the tissue structure so exactly where they are needed to have and develop an optimum effect, they can also be used, due to the high efficiency possible due to the perfect location, in minimal concentrations. So these systems are actually incorporated within the tissue replacement structure, either in the intercellular space or even in the intracellular space. The cryoactive substances and/or cryoactive particles can either be cell-permeating cryoprotectants, such as ethylene glycol, or non-permeating cryoactive agents e.g. in the form of a macro molecule, to facilitate vitrification of the solution, or systems such as sucrose as a low molecular weight compound which causes cells to shrink.

Preferably in the scaffold such cryoactive substances are present in a concentration of 0.0001 - 10 weight% (w/w), preferably 0.01- 1 weight% or 0.1-0.5 weight%. In other words the scaffold preferably comprises or is built of a matrix material, normally of polymeric (synthetic or biologic) matrix material, including the cryoactive substances (embedded and/or attached). It is also possible that the actual matrix material is a cryoactive substance in the sense that the degradation products thereof or at least part thereof have a cryoprotective effect.

According to a first preferred embodiment, such components are selected from the group of temperature-conductor supporting molecular-, nano-, micro,- macro- particles and/or polymers, more preferably comprising carbon, teflon, metals, or ceramics.

According to yet another preferred embodiment, the material of the scaffold comprises a polymeric matrix to which cryoactive proteins and/or particles are linked, preferably covalently linked. This (covalent) linking is possible either by providing a monomer or oligomer precursor material onto which the cryoactive proteins and/or particles are covalently linked in a first step, and in a second step this starting material is polymerized and then cast into a mould for the formation of the actual three-dimensional scaffold structure, or cast into the mould and polymerize in the mould for the formation of the actual three-dimensional scaffold structure. In the alternative is possible to use the polymeric matrix material as the starting material, and to covalently attach the cryoactive proteins and/or particles thereto. The covalent link can be of the grafting type, but it can also be a terminal attachment. The expression covalent link shall also include linking by means of a strong hydrogen bonds, while however a true covalent linking is preferred. Linking can take place directly, so in that the cryoactive proteins and/or particles are directly linked to the matrix material, or it can take place via a linker structure.

Biodegradable matrices have proven effective as scaffolds to augment regeneration in vivo. Newly developed biomaterials intended for clinical use should be easy to handle and suitable for practical purposes and aspects in regenerative medicine. Consequently, e.g. Lutolf et al. developed a synthesis scheme based on conjugate addition reactions between conjugated unsaturations on end-functionalized poly ethylene glycol (PEG) macromers and thiol-bearing peptides that allows formation of bioactive networks by the mixing of aqueous buffered solutions under almost physiologic conditions. Reference is made in this respect to Jo YS, Rizzi SC, Ehrbar M, Weber FE, Hubbell JA, Lutolf MP; "Biomimetic PEG hydrogels crosslinked with minimal plasmin-sensitive tri-amino acid peptides"; J Biomed Mater Res A. 2009, as well as to Lutolf MP; "Biomaterials: Spotlight on hydrogels"; Nat Mater. 2009;8:451-453, the disclosure of which is included into this specification.

It is for example possible to attach cryoactive systems via S-S-bonds to the matrix material, so e.g. cryoactive proteins with thiol-groups to matrix materials which are vinylsulfone-functionalised to establish a covalent link.

Covalent coupling of cryoactive proteins/particles to biodegradable matrices is, according to a preferred embodiment, possible in different ways:

Tissue engineered heart valves using synthetic matrices containing cryoactive proteins/particles:

The synthesis of hydrogels can e.g. be accomplished through Michael-type addition reaction of thiol- (-SH) containing peptides, e.g. cysteines onto vinylsulfone-functionalized branched PEG (4arm PEG) and contains two functional steps. A typical adhesive and MMP-sensitive gel of 50 μΐ volume containing 10% (w/w) PEG can be formed by dissolving 5 mg PEG in 20 μΐ triethanolamine buffer (0.3 M, pH 8.0) and reacting this solution with 10 μΐ of 1 mM RGD (Ac-GCGYGRGDSPG-NH₂) and containing equal molar amounts of a cryoactive peptide/protein or several peptides/proteins in combinations containing two reactive thiol groups for binding and an MMP substrate (Ac-GPQGIWGQ- NH₂) in a first step. This solution is then mixed with 10 μΐ of a precursor solution (in the same buffer) containing a peptide containing optionally an MMP substrate (Ac-GCRD- GPQGIWGQ-DRCG-NH₂) flanked by charged amino acids (Arg-Asp) and two Cys residues to render it more soluble and allow formation of a network, respectively. MMP cleavage sites can be introduced if needed to adjust protein release and/or geldegradation using MMP-inhibitors to neutralize cellular released MMPs. To facilitate cell invasion into these normally nonadhesive hydrogels, gel formulations are augmented to covalently attach the integrin-binding RGD peptide Ac-GCGYGRGDSPG-NH₂ to the gel network as described and, to integrate cryoactive components, recombinant proteins can be physically entrapped into gels by mixing it with the PEG precursor before gelation, according to the previously described method. Therefore, single or multiple recombinant cell viability promoting proteins such as fibroblast growth factor, hepatocyte growth factor, vascular endothelial growth factor, epidermal growth factor, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, growth differentiation factor, insulin- like growth factor, myo statin, nerve growth factor and other neurotrophines, platelet-derived growth factor, thrombopoietin, transforming growth factor alpha, transforming growth factor beta, etc. or other cell-growth promoting agents such as hormones/steroids, anti-oxidants, anti-apoptotic agents, energy or oxygen providing agents, can physically entrapped into gels by mixing it with the PEG precursor before gelation. To form heart valves, as described by Schmidt et al. using synthetic hydrogels containing cryoactive peptides/proteins, hydrogels precursor solutions can be mixed to form heart valves in a ring-shaped device (diameter 20 mm) separated by a 1.0 mm spacer. Crosslinking should be allowed to proceed for 30 min at 37°C in a humidified atmosphere, and the gels are incubated overnight in 0.1M phosphate buffered saline (PBS, pH 7.4). In a next step, previously ex vivo expanded mesenchymal progenitor cells or fibroblasts can be seeded onto the scaffolds (3.5xl0⁶ cells/cm²). Constructs are positioned in a strain- perfusion bioreactor and perfused (4 mL/min) with medium. After 21 days, leaflets are endothelialized with endothelial progenitor cells (1.5xl0⁶ cells/cm²) on both leaflet sides and cultivated for an additional 7 days under exposure of the same mechanical conditions. Thereafter, tissue engineered heart valves containing cryoactive components can be explanted from the bioreactor and analyzed, cryopreserved, or directly implanted for therapeutic applications, respectively.

Tissue engineered heart valves using the biomatrix fibrin containing cryoactive proteins/particles:

Recombinant expressed transglutamin (TG)-domain containing proteins/particles for example spontaneously cross-link to fibrinogen by the transglutaminating (TG) activity of factor XIII during fibrin polymerization. In vivo, covalently tethered TG-protein/particle is consequently protected from diffusion, contrary to wild-type, uncoupled proteins admixed into fibrin, which diffuses out rapidly. In TG-protein/particle fibrin gels, gradual degradation of the bulk matrix fibrin by local fibrinolytic activities such as plasmin or MMPs results in concomitant, local liberation of low levels of TG-protein/particle into tissue. Protein/particle release can be inhibited in presence of plasmin or metalloproteinase inhibitors.

Using a ring-shaped device (diameter 20 mm) fibrin gel matrices can be prepared by mixing the following components at the final concentrations of 10 mg/mL fibrinogen (Fluka AG), 2U/mL factor XIII (Baxter AG, Vienna), and 2.5 mmol/L CaCl₂ TG- protein/particle within the fibrinogen solution before initiation of fibrin gelation by addition of thrombin (see e.g. Ehrbar M, Zeisberger SM, Raeber GP, Hubbell JA, Schnell C, Zisch AH. "The role of actively released fibrin-conjugated VEGF for VEGF receptor 2 gene activation and the enhancement of angiogenesis." Biomaterials. 2008;29: 1720-1729 as well as Zisch AH, Zeisberger SM, Ehrbar M, et al. "Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: Study of angiogenic signaling by ephrin-B2". Biomaterials. 2004;25:3245-3257, the disclosure of these documents is expressly included as concerns the specifics of the method). In a next step, previously ex vivo expanded mesenchymal progenitor cells or fibroblasts can be seeded onto the scaffolds (3.5x10⁶ cells/cm²). Constructs are positioned in a strain- perfusion bioreactor and perfused (4 mL/min) with medium containing MMP inhibitors. After 21 days, leaflets are endothelialized with endothelial progenitor cells (1.5xl 0⁶ cells/cm²) on both leaflet sides and cultivated for an additional 7 days under exposure of the same mechanical conditions in presence of MMP inhibitors. Thereafter, tissue engineered heart valves containing cryoactive components can be explanted from the bioreactor and analyzed, cryopreserved, or directly implanted for therapeutic applications, respectively.

Covalent coupling of proteins/particles to synthetic and biological scaffolds:

Combinations of the above two coupling methods as described above are possible. Synthetic, proteolytically degradable hydrogels containing coupled cryoactive proteins/particles can be used as an initial cell-substrate. Consequently, the cells will proteolytically digest this initial matrix and substitute it by the formation of a biological extracellular matrix. Particle, proteins or peptides bearing two cysteine residues for crosslinking to synthetic hydrogels could also contain an additional transglutamin (TG)- domain to allocate the recombinant cryoactive protein directly to the newly formed biomatrix via cross-link to fibrinogen by the transglutaminating (TG) activity of factor XIII during ECM formation.

According to yet another preferred embodiment, the material of the scaffold comprises a polymeric matrix into which vesicles containing cryoactive protein/particles are embedded. It is also possible that covalent linking is used as described above in combination with embedding. Phospholipid vesicles (liposomes) for guarded cryopreservation of tissue engineered constructs containing cryoactive proteins/particles is possible based on the following considerations: Phospholipid vesicles (liposomes) are a conventional organic-based nontoxic and biodegradable vesicle and have been approved by the FDA for different clinical uses in the past several years. For example, Doxil is a type of stealth liposome encapsulating the anticancer drug Doxorubicin. Controllable particle release from liposomes is another important issue for therapeutic applications. Heat triggering is one of the methods with the largest potential; for example, a temperature-sensitive liposome encapsulated doxorubicin (ThermoDox) used in treating female patients with locally recurrent breast cancer by hyperthermic induced doxorubicin release is currently developed. Among the several heating methods, alternative magnetic field (AMF) stimulation of iron oxide nanoparticles is a safe since it heats up only the local area filled with iron oxide nanoparticles; it can also be relatively efficient in releasing hydrophobic or hydrophilic cryoactive components compared to other methods (e.g. laser, high intensity focused ultrasound, radio frequency and microwaves).

Phospholipid vesicles (liposomes) for guarded cryopreservation of TE constructs containing cryoactive proteins/particles can for example be used according to the following different ways:

Preparation of liposomes containing cryoactive proteins/particles:

As described by Zeisberger et al. for the preparation of 40 ml of liposomes containing cryoactive particles the following method can e.g. be used: Soy phosphatidylcholine (4.0 g, Epikuron 200, Lukas Meyer, Hamburg, Germany), cholesterol (0.6 g, Fluka, Buchs, Switzerland) and D,L-a-tocopherol (0.02 g, Merck, Darmstadt, Germany) corresponding to 1 : 0.3 : 0.01 mol parts are prepared by freeze-thawing and filter extrusion. The dry lipid mixture is solubilized in a physiologic phosphate buffer (20 mM, pH 7.4) supplemented with mannitol (230 mM). For internalisation of cryoprotective agents 1-10 mol parts referred to SPC are added to the lipid mixture. The resulting multilamellar vesicles are freeze-thawed in 3 cycles of liquid nitrogen and water at 40°C, followed by repetitive (5- l Ox) filter extrusion through 400 nm membranes (Nuclepore, Sterico, Dietikon, Switzerland) using a LipexTM extruder (Lipex Biomembranes, Inc., Vancouver, Canada). Non-encapsulated particles are removed by dialysis (Spectrapore tube, 12-14Ό00 mol. wt. cut-off). Liposome size and homogeneity is routinely measured with a Nicomp laser light scattering particle sizer (Nicomp 370, Sta. Barbara, CA). Routinely prepared small unilamellar liposomes have a mean diameter of 135 ± 55 nm.

Preparation of sulfo-SMCC linker containing liposome with terminal cysteine residues, or transglutamin (TG)-domain for crosslinking to synthetic, or biological matrices, respectively:

Liposomes are composed of soy phosphatidylcholine SPC:CHOL:PE-PEG-NH₂ at a molar ratio of 1 :0.2:0.07. For internalisation of cryoprotective agents 1-10 mol parts referred to SPC are added to the lipid mixture. As cryoactive components the below-mentioned systems can be used.

Small unilamellar liposomes are prepared as previously described by sequential filter extrusion of multilamellar liposomal preparations in phosphate buffer (PB, 67 mM, pH 7.4) through NucleporeTM membranes with a LipexTM extruder. Size and stability of the liposomes can be analysed with a particle sizer.

Liposomes containing 0.07 mol parts amino -poly(ethylene glycol)-phosphatidyl- ethanolamine (PE-PEG-NH₂) referred to SPC in Phosphate buffer (PB, 67 mM, pH 7.4) are incubated with crystalline sulfo-SMCC (bearing terminal two cysteine residues, or a transglutamin (TG)-domain) at a molar ratio of PEG-amino to maleimide groups of 1 : 5 for 30 min at 30°C. Excess sulfo-SMCC is removed on a Biogel P6 column (BioRad, Glattbrugg, Switzerland) in HBSE buffer (10 mM HEPES, 150 mM NaCl, 9.1 mM EDTA, pH 7.5). Prepared sulfo-SMCC liposomes, containing cryoactive components, can be either linked via a terminally introduced cysteine residues to synthetic PEG-hydrogels or via terminally introduced TG-domain to biological matrices such as fibrin, as described above. Preparation of thermosensitive liposomes entrapping cryoactive proteins/particles:

As described by Tai et al. (Tai LA, Tsai PJ, Wang YC, Wang YJ, Lo LW, Yang CS. Thermosensitive liposomes entrapping iron oxide nanoparticles for controllable drug release. Nanotechnology. 2009;20: 135101) iron oxide nanoparticles can serve as a heating source upon alternative magnetic field (AMF) exposure. Iron oxide nanoparticles can be mixed with thermosensitive liposomes for hyperthermia-induced particle release, such as hydrophobic or hydrophilic cryoactive components. The leakage 'temperature window' can ranging between 35-37°C, but working slightly lower than the leaky 'temperature window', for example, at 33.5°C, keeps the particles entrapped inside the liposomes. Further, the leaky 'temperature window' can be adjusted by varying the amount of iron oxide nanoparticles.

Thermosensitive liposomes encapsulated with iron oxide nanoparticles are prepared by the thin- film hydration method coupled with sequential extrusion. An aliquot of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, MW = 734.05), DPPC:Chol = 5: 1, weight ratio (total lipid 10 mg), in chloroform is placed into a round-bottomed flask and heated over the higher Tm temperature of the composed lipid (50°C) under a water bath and the chloroform is removed to form the lipid film by a rotary evaporator, followed by evaporation under vacuum for 12 firs. Dry films are hydrated by adding 1 ml of suspension of iron oxide nanoparticles (Resovist 7, or 14 mg Fe ml-1) and carboxylfluorescein (100 mM) to be loaded in liposomes under 50°C water bath for 30 min. Dispersions are homogenized with a miniextruder at 50°C through 400 and 200 nm polycarbonate filters (Avanti Polar Lipids, Alabaster, AL) for 30 times.

Non-entrapped iron oxide nanoparticles are removed by repeated washing by filtration through a 0.1 μιη Amicon low-binding Durapore PVDF membrane (Millipore Corporation, Bedford, MA) using centrifugation at 2000 rpm. The iron concentration is measured by ferric ion assay: the iron in the sample is fully dissolved and oxidized to ferric ions by 1 M HCl and 0.3% H₂0₂ and a sodium thiocyanate solution is added to form the complex of ferric thiocyanate with a UV/vis absorption at 473 nm. A particle size analyzer (90 plus particle size analyzer, Brookhaven Instruments Corp., Long Island, USA) is used to determine the hydrodynamic diameters of iron oxide nanoparticles and liposomes.

More generally speaking, according to yet another preferred embodiment, particles are vesicles, preferably micro-vesicles, more preferably phospholipid vesicles, wherein the vesicles are encapsulating cryoprotective agents and/or cell viability promoting components, wherein the cryoprotective agents are preferably selected from the group of dimethylsulphoxide, glucose and derivates selected from the group of trehalose, sucrose, raffinose, stachyose, glycerol, dextran, sericin, albumin, modified gelatins, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, hydroxyethyl starch, and combinations as well as derivatives thereof.

According to a further preferred embodiment, the cell viability promoting components are selected from cytokines, preferably fibroblast growth factors, hepatocyte growth factors, vascular endothelial growth factors, epidermal growth factors, erythropoietin, granulocyte- colony stimulating factors, granulocyte-macrophage colony stimulating factors, growth differentiation factors, insulin-like growth factors, myostatin, nerve growth factors, neurotrophines, platelet-derived growth factors, thrombopoietin, transforming growth factor alpha, transforming growth factor beta, or cell-growth promoting agents selected from the group of hormones/steroids, anti-oxidants, anti-apoptotic agents, energy or oxygen providing agents, and combinations as well as derivatives thereof.

Preferentially, the cryoprotective agents and/or cell viability promoting components are bound/encapsulated and/or essentially inactive until they get released during or after a thawing process subsequent to cryopreservation of the replacement structure, wherein release is induced in particular by temperature change, radiation, ultrasound, and/or enzymes. In other words the cryoprotective agents/cell viability promoting components are essentially stored in a finely distributed way within the tissue structure but not interacting therewith until released specifically either actively from the outside by imparting a corresponding trigger (temperature change, irradiation etc), or by the change of the surrounding conditions so to speak automatically.

According to a further preferred embodiment, the material of the scaffold comprises a polymeric matrix selected from the following group of synthetic polymeric materials: po lylactic acid, starch-based polymers, aromatic aliphatic co-polyesters, polyhydroxyalkanoates, polylactide, trimethylene carbonate, polyethylene glycol, polylactide-co-glycolide acid, DegraPol, pluronic, polyglycolic acid and combinations as well as derivatives thereof or from the following group of biological polymeric materials: fibrin, alginate, collagen, matrigel, cellulose and derivatives as well as combinations thereof, or from a combination of biological and synthetic materials.

Furthermore the present invention relates to a three-dimensional scaffold for use in a method as described above, wherein the scaffold is based on a polymeric matrix selected from the following group of synthetic polymeric materials: polylactic acid, starch-based polymers, aromatic aliphatic co-polyesters, polyhydroxyalkanoates, polylactide, trimethylene carbonate, polyethylene glycol, polylactide-co-glycolide acid, DegraPol, pluronic, polyglycolic acid and combinations as well as derivatives thereof or from the following group of biological polymeric materials: fibrin, alginate, collagen, matrigel, cellulose and derivatives as well as combinations thereof, or from a combination of biological and synthetic materials, and wherein the matrix incorporates cryoactive substances and/or particles.

According to a preferred embodiment of such a scaffold, the cryoactive substances and/or particles are attached to the matrix and/or embedded in vesicles such that they only get released during a thawing process subsequent to cryopreservation of the replacement structure induced in particular by temperature, radiation, ultrasound, or enzymes. The attachment or linking can generally be a covalent attachment, but also other attachments are possible such as hydrogen bond attachment, ionic bond attachment, van- der-Waals interaction attachment and combinations thereof..

Furthermore the present invention relates to a method for making a scaffold as described above, wherein the polymeric matrix material or a polymeric matrix precursor material is mixed in liquid state with cryoactive substances and/or particles, optionally followed by a step of linking of the cryoactive substances and/or particles to the polymeric matrix material or polymeric matrix precursor material, and casting the liquid into a mould for the generation of three-dimensional scaffold. The method may also include a step of cross- linking/curing of the scaffold material.

Last but not least the present invention relates to a method for the cryopreservation of a tissue engineered replacement structure made using a method as given above, using preferably a scaffold as given above, wherein the replacement structure, preferably a vessel, tube and/or heart valve, is inserted into a liquid tight cavity of a cryopreservation device together with a liquid medium, preferably a cell growth promoting liquid medium such that the tissue engineered replacement structure is completely immersed in the liquid medium, wherein the cryopreservation device and its liquid tight cavity is preferably closed in a liquid tight manner, and wherein the tissue engineered replacement structure is cooled down in a vitrification process to temperatures below 100°C and stored at that temperature for at least two days, preferably for at least 10 days, and wherein just before, or during or just after a subsequent thawing process the cryoactive substances are released into the tissue engineered replacement structure.

The present invention also relates to a tissue engineered replacement structure, preferably a three-dimensional auto- or allograft structure made using a method as described above wherein it is preferably a vessel, tube and/or heart valve, wherein even more preferably it is an autologous tissue engineered replacement structure.

Furthermore the present invention according to a further aspect relates to the use of a tissue structure as given above for implantation in a living human body.

Further embodiments of the invention are laid down in the dependent claims.

DESCRIPTION OF PREFERRED EMBODIMENTS

As exemplified in the general description above, the problem of cryopreservation and storage of tissue engineered auto-and allografts is addressed by compositions, scaffolds and methods to manufacture functionalized tissue-engineered grafts, but also by profiled cryo-devices as shall be outlined below, or combinations thereof.

As outlined above, the invention provides a method to cryo-preserve tissue engineered auto- and allografts, characterized by providing a functionalised matrix/scaffold in order to improve temperature and/or mass transfer during cryopreservation inside of tissue engineered grafts, and/or to improve cell viability of the cryopreservation inside of tissue engineered grafts. These concepts can be combined advantageously by providing and using a specific cryo device which allows to improve homogeneous temperature transfer during cryopreservation inside of tissue engineered grafts.

The functionalized scaffolds suitable for guarded cryopreservation are composed out of synthetic components, 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, polyglycolic acid (PGA) and combinations thereof. Further, scaffolds may be composed out of biological components such as fibrin, alginate, collagen, matrigel, cellulose and combinations thereof. Matrices may be composed out of synthetic in combination with biological components (hybrid-matrices). The scaffolds incorporate components to overcome limitations in temperature transfer inside of tissue engineered grafts during cryopreservation. Such components can consist of temperature-conductor supporting molecular-, nano-, micro,- macro- particles and/or polymers, being composed of such as carbon, teflon, metals, or ceramics, etc.

The scaffolds, may comprise encapsulating particles to overcome limitations in mass transfer inside of tissue engineered grafts during cryopreservation. Such particles may be micro-vesicles of any composition, such as phospholipid vesicles (liposomes) or any other encapsulating particles containing CPAs and/or cell viability promoting components.

The scaffolds may comprise encapsulating particles to overcome limitations in mass transfer inside of tissue engineered grafts. Such particles can be loaded with CPAs. CPAs may be such as dimethylsulphoxide (DMSO), glucose and derivates (e.g. trehalose, sucrose, raffmose, stachyose, etc.), glycerol, dextran, sericin, albumin, modified gelatins, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol (PEG), hydroxyethyl starch (HES), and combinations thereof.

The scaffolds may comprise encapsulating particles to improve cell viability inside of tissue engineered grafts. Such particles can be loaded with cell viability promoting components. Such cell viability promoting components may be cytokines (such as fibroblast growth factor, hepatocyte growth factor, vascular endothelial growth factor, epidermal growth factor, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, growth differentiation factor, insulin- like growth factor, myostatin, nerve growth factor and other neurotrophines, platelet- derived growth factor, thrombopoietin, transforming growth factor alpha, transforming growth factor beta, etc.) or other cell-growth promoting agents such as hormones/steroids, anti-oxidants, anti-apoptotic agents, energy or oxygen providing agents, etc. which get released during the thawing process induced by e.g. temperature, radiation, ultrasound, or enzymes. Such cell viability promoting components can also be coupled directly or via a linker to the scaffold.

As mentioned above, the above proposed method for the making of the tissue engineered graft can be advantageously combined with the use of a specific device for the cryopreservation of tissue-engineered grafts. Such a cryo-device provides an adapter function with the aim to bring temperature-conducting material in close proximity with the tissue engineered graft, and which prevents any folding or the like of the tissue engineered structure before, during cryopreservation and during subsequent thawing. At the 'graft- device' interface the device contains an ultra-thin coated surface with 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. The general idea of the device is to provide a channel like, contiguous structure which is adapted to the three-dimensional shape of the tissue engineered structure. The tissue engineered structure is put into this profiled interior space such that the wall structures of the tissue engineered structure are either in contact with the profiled wall structures of the cryo device or at least the profiled wall structures are very close (in the millimetre or sub-millimetre range) proximity of the wall structures, preferably all over the wall structure of the replacement structure. Like this, as in this cavity also a liquid is present, any folding of the tissue structure is prevented and close contact/distance between the profiled cooling walls is ensured for homogeneous cooling/warming of the tissue engineered structure.

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 teflon, 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.

Tissue engineered heart valves using synthetic biodegradable matrices containing FGF and VEGF for guarded cryopreservation

A typical cell adhesive and MMP-sensitive vinylsulfone-functionalized branched PEG (4arm PEG) gel of 50 μΐ volume containing 10% (w/w) PEG is formed by dissolving 5 mg PEG in 20 μΐ triethanolamine buffer (0.3 M, pH 8.0) and reacting this solution with 10 μΐ of 1 mM RGD (Ac-GCGYGRGDSPG-NH₂) peptide and containing 10 μg each of recombinantly expressed human fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) containing an MMP substrate, charged amino acids (Arg-Asp) and one Cys (Ac-GPQGIWGQ-DRCG-NH₂) in a first step. This solution is then mixed with 10 μΐ of a precursor solution (in the same buffer) containing a peptide containing an MMP substrate as well (Ac-GCRD-GPQGIWGQ-DRCG-NH₂) flanked by charged amino acids (Arg-Asp) and two Cys residues, respectively. To form heart valves, hydrogels precursor solutions are premixed to form heart valves in a ring-shaped device (diameter 20 mm) separated by a 1.0 mm spacer. Crosslinking should be allowed to proceed for 30 min at 37°C in a humidified atmosphere, and the gels are incubated overnight in 0.1M phosphate buffered saline (PBS, pH 7.4). In a next step, previously ex vivo expanded fibroblasts are seeded onto the scaffolds (3.5xl0⁶ cells/cm²), in the presence of cell culture medium supplemented with MMP-inhibitors. Constructs are positioned in a strain-perfusion bioreactor and perfused (4 mL/min) with medium also supplemented with MMP-inhibitor. After 21 days, leaflets are endothelialized with endothelial progenitor cells (1.5xl 0⁶ cells/cm²) on both leaflet sides and cultivated for an additional 7 days under exposure of the same mechanical conditions. Thereafter, tissue engineered heart valves containing covalently incorporated FGF and VEGF are explanted from the bioreactor and cryopreserved in a cryocontainer as follows.

For vitrification a method described by Mukaida et al. (T. Mukaida, S . Wada, K. Takahashi, P.B. Pedro, T.Z. An and M. Kasai, Human reproduction (13) N 10, pp 2874- 2879) is adapted. As cell-permeating cryoprotectant, ethylene glycol (EG) is used. It is mixed with two non-permeating agents, Ficoll 70 (average molecular weight 70 000, Pharmacia, Uppsala, Sweden) as a macromolecule to facilitate vitrification of the solution, and sucrose as a low molecular weight compound which causes cells to shrink. EG is diluted to 40% (v/v) in freezing solution (Serum free basal medium containing 30%> w/v Ficoll plus 0.5 M sucrose) to a final concentration of 18% Ficoll and 0.3 M sucrose. The final vitrification solution does not contain MMP-inhibitors for enabling cell-demanded (secretion of MMPs) FGF- and VEGF release and degradation of the synthetic matrix. The tissue engineered heart valves are directly suspended in vitrification solution at RT and placed in a closed valve position inside of the cryocontainer. Two minutes after exposure of the tissue engineered heart valves to the vitrification solution, the cryocontainer is plunged into the vapour phase of nitrogen.

To thaw the tissue engineered heart valves, the cryo-container is taken out of the vapour phase of nitrogen and immediately plugged to a heating device warming the sample up to 37°C. The tissue engineered heart valves are expelled into culture medium containing 0.5 M sucrose. After 5 minutes, the tissue engineered heart valves are ready for further investigation or transplantation.

Claims

1. Method for the generation of a living cell based tissue engineered organ and/or tissue analogous structure having mechanical and/or functional properties comprising the steps of

providing a organ and/or tissue,

wherein the organ and/or tissue has the capacity of functioning or forming the native organ/tissue corresponding to the replacement; and

wherein the tissue engineered organ and/or tissue analogous structure is made based on cryoactive substances and/or contains material incorporating cryoactive substances and/or particles.

Method according to claim 1 for the generation of a cell-matrix replacement tissue or tissue analogous structure preferably having mechanical strength and flexibility and/or pliability, or of a replacement organ, or a combination thereof, comprising the steps of

providing a scaffold formed of a biocompatible, preferably biodegradable material,

seeding the scaffold with dissociated, preferably human, cells,

wherein the, preferably human, cells have the capacity of forming the native tissue or organ corresponding to the replacement; and

cultivating the cells under conditions allowing the development of the tissue or organ replacement,

wherein the scaffold is made of a material incorporating cryoactive substances and/or particles.

3. Method according to claim 1 or 2, wherein the cryoactive substances and/or particles are components to overcome limitations in temperature transfer inside of tissue engineered grafts during cryopreservation or components to improve cell viability during and/or after cryopreservation inside of the tissue, wherein preferably such components are selected from the group of temperature-conductor supporting molecular-, nano-, micro,- macro- particles and/or polymers, more preferably comprising carbon, teflon, metals, or ceramics.

Method according to any of the preceding claims, wherein the material comprises a matrix, preferably a polymeric matrix or a xeno- or allogenic decellularized matrix, to which cryoactive proteins and/or particles are linked, wherein the link is preferably selected from a covalent bonds, hydrogen bond, ionic bond, van-der- Waals bond, or combinations thereof.

Method according to any of the preceding claims, wherein the cell based tissue engineered organ and/or tissue analogous structure contains cryoactive proteins/particles..

Method according to any of the preceding claims, wherein the particles are vesicles, preferably micro-vesicles, more preferably phospholipid vesicles, wherein the vesicles are encapsulating cryoprotective agents and/or cell viability promoting components, wherein the cryoprotective agents are preferably selected from the group of dimethylsulphoxide, glucose and derivates selected from the group of trehalose, sucrose, raffinose, stachyose, glycerol, dextran, sericin, albumin, modified gelatins, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, hydroxyethyl starch, and combinations as well as derivatives thereof.

Method according to claim 6, wherein the cell viability promoting components are selected from cytokines, preferably fibroblast growth factors, hepatocyte growth factors, vascular endothelial growth factors, epidermal growth factors, erythropoietin, granulocyte-colony stimulating factors, granulocyte-macrophage colony stimulating factors, growth differentiation factors, insulin-like growth factors, myostatin, nerve growth factors, neurotrophines, platelet-derived growth factors, thrombopoietin, transforming growth factor alpha, transforming growth factor beta, or cell-growth promoting agents selected from the group of hormones/steroids, anti-oxidants, anti-apoptotic agents, energy or oxygen providing agents, and combinations as well as derivatives thereof.

Method according to any of the preceding claims, wherein the cryoprotective agents and/or cell viability promoting components are bound and essentially inactive until they get released during or after a thawing process subsequent to cryopreservation of the replacement structure, wherein release is induced in particular by temperature change, radiation, ultrasound, and/or enzymes.

Method according to any of the preceding claims, wherein the material comprises a polymeric matrix selected from the following group of synthetic polymeric materials: polylactic acid, starch-based polymers, aromatic aliphatic co-polyesters, polyhydroxyalkanoates, polylactide, trimethylene carbonate, polyethylene glycol, polylactide-co-glycolide acid, DegraPol, pluronic, polyglycolic acid and combinations as well as derivatives thereof or from the following group of biological polymeric materials: fibrin, alginate, collagen, matrigel, cellulose and derivatives as well as combinations thereof, or from a combination of biological and synthetic materials.

Three-dimensional scaffold for use in a method according to any of the preceding claims, wherein the scaffold is based on a polymeric matrix selected from the following group of synthetic polymeric materials: polylactic acid, starch-based polymers, aromatic aliphatic co -polyesters, polyhydroxyalkanoates, polylactide, trimethylene carbonate, polyethylene glycol, polylactide-co-glycolide acid, DegraPol, pluronic, polyglycolic acid and combinations as well as derivatives thereof or from the following group of biological polymeric materials: fibrin, alginate, collagen, matrigel, cellulose and derivatives as well as combinations thereof, or from a combination of biological and synthetic materials, and wherein the matrix incorporates cryoactive substances and/or particles.

11. Scaffold according to claim 10, wherein the cryoactive substances and/or particles are covalently attached to the matrix and/or embedded in vesicles such that they only get released during a thawing process subsequent to cryopreservation of the replacement structure induced in particular by temperature, radiation, ultrasound, or enzymes.

12. Method for making a scaffold according to any of the preceding claims 10-11, wherein the polymeric matrix material or a polymeric matrix precursor material is mixed in liquid state with cryoactive substances and/or particles,

optionally followed by a step of covalent linking of the cryoactive substances and/or particles to the polymeric matrix material or polymeric matrix precursor material,

and casting the liquid into a mould for the generation of three-dimensional scaffold.

13. Method for the cryopreservation of a tissue engineered replacement structure made using a method according to any of claims 1-9, wherein the replacement structure, preferably a vessel, tube and/or heart valve, is inserted into a liquid tight cavity of a cryopreservation device together with a liquid medium, preferably a cell growth promoting liquid medium such that the tissue engineered replacement structure is completely immersed in the liquid medium, wherein the cryopreservation device and its liquid tight cavity is preferably closed in a liquid tight manner, and wherein the tissue engineered replacement structure is cooled down in a vitrification process to temperatures below 100°C and stored at that temperature for at least two days, preferably for at least 10 days, and wherein just before, or during or just after a subsequent thawing process the cryoactive substances are released into the tissue engineered replacement structure.

14. Tissue engineered replacement structure made using a method according to any of claims 1-9, wherein it is preferably a vessel, tube and/or heart valve, wherein more preferably it is an autologous tissue engineered replacement structure.

15. Use of a tissue structure according to claim 14 for implantation in a living human body.