A rotating tissue culture insert positioned within a rotatable roller bottle partially filled with liquid for culturing cells

Abstract

A rotating tissue culture insert (20, 40) with a scaffold (11) positioned within a rotatable roller bottle (100) partially filled with liquid for culturing cells, the scaffold (11) comprising a shaft (3) and at least two projections extending radially from the shaft (3), the projections (4) defining wheels of the rotating tissue culture insert (20, 40) with tracks and contacting a surface (25) of the roller bottle (100) when the roller bottle (100) is in a lying position.

Description

[Technical Field]

[0001] The present invention relates to a rotating tissue culture insert for positioning within a rotatable roller bottle partially filled with liquid for the cultivation of cells. [Background technology]

[0002] Roller bottles have been commonly used as a method and machine for culturing cells for over a decade. They are typically used for cell growth. In some rare applications, the roller bottles contain nutrients and / or liquid for culturing cells with cells and / or a scaffold for the deposition of the cells. Roller bottles are typically used for culturing cells in a horizontal position, meaning that the roller bottle rotates around a horizontal axis of rotation parallel to the longitudinal axis of the bottle, which is typically cylindrical.

[0003] After the tissue has grown on the surface of the scaffold, the cells are removed in a subsequent step by decellularization, and the resulting tissue, free of viable cells, can be used for various medical applications such as transplants and dressings without immunocompatibility issues. In some applications, there is a demand for modeled tissues for organ replacement, while in other applications there is a demand for tissues with specific collagen orientations. The rotating tissue culture insert of the present invention prepares tissue for all of the aforementioned uses.

[0004] Roller bottles and devices for moving roller bottles are well known in the art. The state of the art in this field has so far focused on adapting the roller bottle itself, as disclosed in EP 0 394 713. This publication discloses that porous polystyrene foam is the scaffold. Further methods of tissue engineering are disclosed by Australian Patent No. 2013277275 and Australian Patent Publication No. 2017202721. A further bioreactor for producing (stented) TEHVs (tissue engineered heart valves) is disclosed by US Pat. No. 8,192,981. Summary of the Invention [Problem to be solved by the invention]

[0005] Object of the invention A first object of the present invention is to provide the possibility to preferentially orient the ECM (extracellular matrix) in the longitudinal direction. A second object of the present invention is to provide the possibility to preferentially orient ECM in the circumferential direction.

[0006] A third object of the present invention is to enhance perfusion and oxygenation of the culture medium. A fourth object of the present invention is to simplify the use of roller bottles for culturing cell-based tissue constructs compared to the current standard in the art, where tissue includes any cell-based matrix construct that can be produced by the methods described below. [Means for solving the problem]

[0007] The rolling tissue culture insert (abbreviated RTCI) of the present invention is adapted for positioning within a rollable roller bottle partially filled with a liquid for culturing cells. The roller bottle is not part of the insert of the present invention. The rolling insert is configured to roll within the roller bottle. The roller bottle is normally in a lying position, i.e., the roller bottle is oriented horizontally.

[0008] Tissue culture inserts provide a surface, typically a scaffold, on which cells can accumulate and / or grow. The insert preferably provides a scaffold for culturing cells not only on the surface but also within the scaffold material. The scaffold is preferably formed from a biodegradable material that degrades during tissue formation and growth. The RTCI may further include a scaffold holder for holding the scaffold.

[0009] Preferably, the scaffold holder is a mesh-like cylindrical support used to hold the scaffold. More preferably, the scaffold holder is a Nitinol stent. The Nitinol stent may be covered with a non-adhesive coating and / or layer to facilitate scaffold removal. The non-adhesive layer is preferably made of parafilm.

[0010] Used as an integral part of the manufacturing process, nitinol stents may function as anchoring systems in the final product, such as implantable stent systems for clinical use. Nitinol material is just one example of a suitable stent.

[0011] According to the present invention, a rotating tissue culture insert comprises a shaft and at least two protrusions extending radially from the shaft. Other components, such as a scaffold holder, can also be part of the rotating tissue culture insert. The protrusions form two wheels of the RTCI, the wheels having tracks that contact the surface of the roller bottle when the roller bottle is in a substantially lying or horizontal position.

[0012] The present invention provides the ability to create tissue-engineered products with desired preferential collagen orientation by combining RTCI with a roller bottle system, and further allows for the rotation of the scaffold with the roller bottle to enhance perfusion and oxygenation and / or static stretching to impart preferential ECM orientation.

[0013] According to a preferred embodiment, RTCI can be used to provide preferential longitudinal extracellular matrix (ECM) orientation, and in another preferred embodiment, RTCI can apply static circumferential stretch over the tissue culture period to induce preferential circumferential ECM orientation.

[0014] A preferred application is the use of the inserts in cardiovascular tissue engineering production, such as in the production of tissue engineered heart valves (abbreviated TEHV). Further embodiments are the subject matter of the dependent claims.

[0015] To use the maximum surface area of ​​the insert, it is advantageous for at least two projections to be located at the distal ends of the shaft, the distal ends being at the ends of the body, the maximum radial height of the projections being greater than the thickness of the scaffold.

[0016] When an additional substrate, also called a scaffold holder, is used to hold a scaffold, which is arranged on the outer surface of the shaft, the radial dimension of the protrusion is greater than the sum of the thicknesses of the scaffold and the substrate. An optional further part of the insert may be a sleeve, such as a multi-part sleeve, arranged coaxially with the shaft. When the sleeve is part of the shaft, the protrusion is greater than the sum of the thicknesses of the scaffold, the substrate and the sleeve.

[0017] It is advantageous if the substrate and / or sleeve include a plurality of openings. Mesh, also understood as an opening in the context of the present invention, is preferred as a material for the substrate holder. The substrate is also called a scaffold holder and is preferably a removable part of the rotating tissue culture insert. The scaffold holder can be individually configured according to the shape, size, and function of the implant to be formed by combining the tissue and the substrate. In this case, the substrate, or in other words, the scaffold holder, can be configured together with the tissue, which can be removed from the rotating tissue culture insert after the tissue has formed.

[0018] Alternatively or additionally, the sleeve is preferably a multi-piece sleeve, has a longitudinal axis parallel to the shaft and includes a plurality of openings, and the scaffold holder is preferably disposed on the sleeve. In the absence of a sleeve, the scaffold holder can be disposed on the shaft, more preferably being part of a rotating tissue culture insert.

[0019] Alternatively, the formed tissue can be detached from the substrate and used for transplantation or other medical uses without the scaffold holder. For optimal rolling characteristics, the shaft is hollow. For optimal contact between the tissue and the liquid inside the roller bottle, the hollow shaft has multiple openings.

[0020] Since the scaffold and / or substrate have different thicknesses depending on the tissue to be formed, the projections can be detached from the shaft and are preferably connected to the shaft by a screw. In this way, projections of different sizes can be provided on the shaft. In this way, rotating tissue culture inserts of different sizes can be constructed using the same shaft. The projections preferably have the form of a circumferential ring.

[0021] The scaffold may further comprise a nonwoven layer to allow fluid to easily penetrate the surface of the scaffold. Preferably, the nonwoven layer is made of a biodegradable material, which comprises or consists of polyglycolic acid.

[0022] In a further preferred embodiment, said nonwoven layer may be provided with a coating, more preferably a biodegradable coating, most preferably a polyhydroxybutyrate film. Alternatively, or advantageously, to the idea of ​​the protrusions forming two wheels, a rotating tissue culture insert can serve as a stretcher to radially stretch the scaffold.

[0023] This system allows for easy adjustment of the size of the rotating tissue culture insert depending on the approach. This means that smaller or larger stent-like scaffold holders, or support structures, can be used to achieve the desired results without modifying the roller bottle system. Furthermore, disposable roller bottle flasks are easy to use, readily available, and can accommodate scaffolds of various sizes.

[0024] Preferred embodiments of the invention described above and / or embodiments in advantageous combination with the idea of ​​a lug in the form of a wheel are described by the further dependent claims. Advantageously, the shaft includes a longitudinal axis and the rotating tissue culture insert further includes one or more parts that are radially movable relative to the longitudinal axis.

[0025] In an advantageous configuration, the rotating tissue culture insert may include an adjustment mechanism for adjusting the radial distance of the one or more components relative to the longitudinal axis of the shaft. To better distribute the forces applied to the scaffold, the one or more components are arc-shaped plates. The arcuate plate may be provided with openings to allow better contact between the scaffold and the liquid.

[0026] In a preferred embodiment, the shaft is formed by at least two elements that are part of an adjustment mechanism and are linearly movable relative to one another, preferably including a conical portion in each element, and the adjustment mechanism is adjustable by means of a screw or in other words a screw mechanism.

[0027] In a further embodiment of the present invention, or as an independent inventive idea, the substrate may be essentially cylindrical and may include bendable discrete portions, preferably radially bendable, more preferably at a distal end of the substrate. This can be achieved, for example, by a mesh structure that includes different mesh sizes.

[0028] A further idea of ​​the present invention is to provide a method for manufacturing an implant, the method comprising the steps of: A: Providing a roller bottle with a liquid for culturing cells and a rolling tissue culture insert with a scaffold, the roller bottle in a lying position with the fill level of the roller bottle substantially parallel to the longitudinal axis of the roller bottle, said RTCI may be an inventive RTCI as described above.

[0029] The rotating tissue culture insert further comprises a substrate, also called a scaffold holder, for attaching a scaffold to the rotating tissue culture insert. B: A step of rotating the roller bottle, wherein the scaffold is spaced away from the wall of the roller bottle and the tissue culture insert rotates, preferably counterclockwise relative to the direction of rotation of the roller bottle, and tissue is formed on the surface of the scaffold during rotation of the roller bottle. C: Removing the construct comprising at least the tissue and the substrate from the rotating tissue culture insert. According to the present invention, the composition is provided for the formation of an implant. This means that the structure is already an implant or is converted into an implant.

[0030] The configuration may include an essentially cylindrical portion, wherein the radial diameter of a rotating tissue culture insert is adjustable, wherein the radial diameter of the rotating tissue culture insert adjusts to the variable diameter of the cylindrical portion. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 is a diagram disclosing a first embodiment. [Figure 2] FIG. [Figure 3] FIG. [Figure 4] A diagram of the scaffolding. [Figure 5] FIG. 10 discloses a rotating tissue culture insert. [Figure 6] FIG. 1 is a view showing the inner surface of a roller bottle. [Figure 7]FIG. 10 discloses a second embodiment of the present invention. [Figure 8] FIG. 10 discloses a second embodiment of the present invention, showing a complete RTCI. [Figure 9] FIG. 10 discloses a second embodiment of the present invention. [Figure 10] FIG. 10 discloses a second embodiment of the present invention. [Figure 11] FIG. 10 discloses a second embodiment of the present invention. [Figure 12] FIG. 10 is a diagram illustrating a fourth embodiment of the present invention. [Figure 13] FIG. 10 is a diagram illustrating a fourth embodiment of the present invention. [Figure 14a] FIG. 1 discloses histological images showing the progression of extracellular matrix (ECM) deposition and organization over the course of tissue culture. [Figure 14b] FIG. 1 discloses histological images showing the progression of extracellular matrix (ECM) deposition and organization over the course of tissue culture. [Figure 14c] FIG. 1 discloses histological images showing the progression of extracellular matrix (ECM) deposition and organization over the course of tissue culture. [Figure 15] FIG. 1 shows the results of uniaxial tensile tests performed on TEM samples. [Figure 16a] FIG. 1 illustrates the changes in mechanical properties that can be associated with different ECM orientations. [Figure 16b] FIG. 1 illustrates the changes in mechanical properties that can be associated with different ECM orientations. [Figure 16c] FIG. 1 illustrates the changes in mechanical properties that can be associated with different ECM orientations. [Figure 17a] FIG. 1 illustrates the changes in mechanical properties that can be associated with different ECM orientations. [Figure 17b] FIG. 1 illustrates the changes in mechanical properties that can be associated with different ECM orientations. [Figure 17c] FIG. 1 illustrates the changes in mechanical properties that can be associated with different ECM orientations. [Figure 18]FIG. 1 shows a substrate with a changed structure. [Figure 19] FIG. 1 shows a substrate with a changed structure. [Figure 20] FIG. 1 shows a substrate with a changed structure. DETAILED DESCRIPTION OF THE INVENTION

[0032] Some advantageous embodiments of the rotating tissue culture insert and the inventive embodiment of the manufacturing method of the present invention are described in more detail below in conjunction with the drawings. Particular parts of different embodiments can be understood as separate features that can also be realized in other embodiments of the present invention. The combination of features described in the embodiments should not be understood as limiting the present invention.

[0033] Figure 1 discloses a first embodiment, preferably having a substrate 2 for supporting a scaffold 11 in the form of a stent. The substrate 2 in this embodiment preferably has the form of a cylindrical mesh body. The substrate 2 can be further combined with parts into a rotating tissue culture insert 20, as shown in Figure 5. The scaffold 11 can be attached to the substrate 2 in the particular form shown in FIG. 1 by stitching or the like using medical thread, although other fastening methods are possible.

[0034] The rotating tissue culture insert 20 further comprises a hollow shaft 3 as shown in Figure 2. The substrate 2 is located on the outer surface of the hollow shaft 3.

[0035] The rotating tissue culture insert comprises at least two rolling protrusions 4 arranged parallel to each other on a hollow shaft 3. These rolling protrusions 4 are preferably arranged circumferentially around the hollow shaft 3, thus forming a ring shape. The rolling protrusions 4 are rigidly connected to the hollow shaft 3.

[0036] Each rolling projection 4 has at least one circumferential track along which it can contact and roll while contacting a surface such as the inner wall of a roller bottle. This track may be interrupted by grooves. The rolling projections 4 are provided as wheels firmly connected to the shaft 3. The substrate 2 and the footing 11 can be arranged between the projections, and the radial height of the projection is greater than the sum of the thicknesses of the substrate 2 and the footing 11, in other words, the sum of their radial heights.

[0037] At least two projections 4 are attached to each end of the hollow shaft 3. These projections are located at distal positions. In preferred embodiments, the projections are attached by a transition or interference fit, threaded fastening, and / or a combination of clearance fit and welding.

[0038] The further protrusions preferably have the same configuration as the protrusion 4 and can be mounted equidistant from the distally located protrusion 4 . As shown in Figure 2, the hollow shaft 3 has openings 5 ​​on its shell surface. The openings are preferably arranged in rows parallel to the longitudinal axis of the hollow shaft. The openings 5 ​​in one row are equidistant from adjacent openings. The shell surface of the hollow shaft 3 has a plurality of rows of openings distributed circumferentially.

[0039] Some, and preferably all, parts of the rotating tissue culture insert 20 are made of metal, preferably medical grade stainless steel.

[0040] Scaffold 11 is shown in Figure 4. The scaffold can be made of a polymer material, preferably a nonwoven fabric, more preferably a polymer material containing polyglycolic acid (PGA). The PGA nonwoven fabric is preferably coated with poly-4-hydroxybutyric acid, which will be described later as a biodegradable film for controlling cell matrix adhesion. Tip 12 is provided with a thread 13. Scaffold 11 is preferably hollow cylindrical, but may also be prismatic.

[0041] 5 discloses a rotating tissue culture insert 20 with a scaffold 11, simply referred to as RTCI. This insert can be used in a rotating cell culture system (RCCS), in which a rotating bioreactor is used for cell growth. The rotating bioreactor can be formed by a roller bottle, as shown, for example, in EP 0394713. The present invention is not limited to the use of the RTCI in the roller bottles disclosed herein.

[0042] Roller bottles are known in the art. These containers are reused in laboratories and elsewhere for culturing cells. They are generally cylindrical in shape and adapted to rotate about their axis. The interior surface of such roller bottles is intended to provide an active surface for growing cells. Liquid growth medium is introduced into the roller bottle. The rolling motion of the bottle keeps the interior surface wet with the liquid medium, encouraging cell growth. Rotating rollers are used in a suitable device to rotate the roller bottle. Typically, roller bottle apparatuses are installed in an incubator or culture room and are adapted to control the temperature of cell growth within the roller bottles, and a rotary drive system, such as a rotating roller, is employed as part of the apparatus to rotate the roller bottles.

[0043] Rotating the roller bottle with the RTCI causes the RTCI to rotate inside the roller bottle counterclockwise or clockwise in the direction of the roller bottle rotation.

[0044] 6 discloses the interior surface 25 of such a roller bottle 100. The interior surface is provided with a ribbon 27 that runs parallel to the rotational axis and longitudinal axis of the roller bottle 100. The ribbon 27 is provided to stop the rotation of the RTCI once the roller bottle comes to a stop. Here, a shaft with a protrusion 4 made of a transparent plastic material is shown to illustrate the placement and function of the RTCI inside the roller bottle 100.

[0045] The roller bottle preferably has a neck so that the bottle can hold the liquid for culturing cells in a horizontal position. The RTCI is configured with dimensions such that the RTCI preferably rotates at least 2.5 times, preferably 3 to 4 times, around its axis while the bottle rotates once. The RTCI must be sized to pass through the neck of the roller bottle.

[0046] 7-11 disclose a second embodiment of the present invention in which a multi-piece sleeve 32 is part of a further embodiment of a rotating tissue culture insert 40 and includes arcuate plates 6 as part of the jacket. Each plate preferably has at least one row of openings 5. A scaffold holder, or substrate, can be placed on the sleeve 32.

[0047] In the embodiment of Figures 7 to 11, there are four arcuate plates 6, but there may be another number of plates, thus forming a sleeve. The sleeve cooperates with a shaft 33 to adjust the diameter of the sleeve 32 and the gap between the plates 6. The shaft 33 is formed by an arrangement of at least two parts 7, 8 that are linearly movable relative to one another. In the present invention, linear movement can be achieved by threading the two parts 7, 8 together. For this purpose, one part 7 is provided with a threaded bolt 34 and the other part with a threaded sleeve 35. The aforementioned threaded connection of the parts can also be achieved by a similar arrangement, such as one central threaded pin and two distal threaded sleeves, or one central threaded sleeve and two distal parts with threaded bolts.

[0048] The two parts 7, 8 of the arrangement are provided with conical sections 36, 37, the axial distance between which can preferably be varied by screwing or unscrewing said sections. As the arcuate plates 6 approach the conical portions 36, 37, the arcuate plates 6 widen or are spaced further apart from each other and therefore have a greater radial distance from each other than when the conical portions 36, 37 are spaced further apart from each other.

[0049] Since there is sufficient space between the shaft 33 and the sleeve 32 formed by the arc-shaped plate 6, the shaft 33 does not need to have a hollow shape or opening 28 of the rotating tissue culture insert 40 of Figures 7 to 11 compared to the embodiment of the rotating tissue culture insert 20 of Figures 1 to 6, but in this embodiment a hollow shape of the shaft 33 is also preferred.

[0050] The shaft 33 has a thread at its distal end for attaching the rolling projection 4, as in Figures 1 to 6. In this way, the projections can be removed, cleaned separately, and reused. It is preferable that all parts of the RTCI are removable, especially since the connecting parts are difficult to clean in an acceptable manner. These parts, including the scaffold 11, are shown in Figure 11 and are attached to the RTCI of Figure 13.

[0051] 9, each arcuate plate 6 has a ramp 38 at each end, where the thickness of the plate is reduced. The ramp 38 provides a sliding surface for contact with the conical portions 36, 37.

[0052] Figure 8 discloses the complete RTCI 40. The scaffolding holds the plate 6 from falling. The conical portion provides an expansion force to the plate.

[0053] The aforementioned RTCI mechanism for radially expanding the scaffold 11 is a further feature of the present invention that can be realized independently of the provision of protrusions 4. However, combining both features provides a combination of two options for controlled growth of tissue on the surface of the scaffold 11 within one insert.

[0054] A further object of the present invention is to change the structure of the substrate 2 compared to the first and second embodiments described above, as shown in FIGS. In this case, the substrate 2 is a cylindrical mesh body as described in the first embodiment, but has meshes of different sizes provided on the substrate 2. The mesh body 42 has meshes of approximately uniform size and is provided with a circumferential mesh portion 43 that defines a central axis A. The mesh body 42 further has protrusions 44, 45 and a tapered end portion 46, and the protrusions 44, 45 extend from the mesh portion 43 toward the end portion in parallel to the central axis A. One protrusion 44 has one large mesh 48 having a mesh size at least three times the average mesh size of the mesh of mesh portion 43. The second protrusion 45 is provided by a mesh structure 47 having the same average mesh size as mesh portion 43.

[0055] In this manner, the second protrusions 45 are stiffer than the first protrusions 44. When tissue 51 is formed on or around the substrate 42, the portions 49 of tissue 51 formed on or around the first protrusions 44 are more flexible toward the central axis A than the portions 50 of tissue 51 formed on or around the second protrusions 45. As shown in FIG. 20 , the valve flaps are achieved by modifying the structure 42.

[0056] Further examples of use, further advantages of the present invention, and some further examples are further described below. Cardiovascular tissues, such as blood vessels and heart valves, are characterized by a highly organized extracellular matrix, also known as the ECM. Notably, ECM orientation is crucial in determining the mechanical properties of tissues and their ability to sustain hemodynamic loads. Typically, to obtain robust tissue-engineered (TE) constructs suitable for cardiovascular tissue engineering applications, such as TE heart valves, TEHVs, or vascular prostheses, complex bioreactor systems are used to mechanically condition cells by applying physiological flow and / or pressure conditions that result in preferential collagen orientation.

[0057] The present invention provides a new and simpler method for generating TE samples in which preferential ECM orientation can be controlled. To achieve this goal, a new tissue culture method was developed that is compatible with commercially available roller bottle systems, such as the Pfeiffer™ CellRoll™ system, to ensure perfusion and oxygenation of the medium, thereby favoring uniform ECM deposition over the tissue culture period. This new culture method is implemented in combination with three different options designed to control ECM orientation and achieve mechanically robust TEHVs.

[0058] The first option is the provision of an RTC insert, also known as an RTCI, whose protrusions function as two wheels, as shown in the first, second, and third embodiments. This system allows for preferential longitudinal alignment of the ECM in response to static stretching, determined by the method of suturing the scaffold or fixing it to the scaffold holder. The resulting TEM can be used to fabricate TEHVs.

[0059] A second option is to provide a radial stretcher with a mechanism, such as a screw, and a sliding part to manually increase the diameter, and an expandable stent-like scaffold holder to which the scaffold is sutured or otherwise secured. By continuously or stepwise increasing the diameter of the stretcher over the course of the tissue culture period, preferential circumferential ECM orientation can be achieved. The resulting TEM can be used to fabricate TEHVs. The sleeve can be part of the stretcher that cooperates with the shaft.

[0060] A third option is to provide a stent-like scaffold holder that uses a nitinol stent specifically configured for minimally invasive transcatheter aortic valve replacement (TAVR for short). This option allows for the growth of tissue engineered matrices (TEMs) directly on the TAVR stent used for implantation, greatly simplifying the fabrication of TEHVs by reducing the handling of the TEMs and fixation such as suturing. The resulting RTCI is preferably seeded with cells and inserted into pleated roller bottles for tissue culture in commercially available roller bottle systems.

[0061] As previously mentioned, some or all of the components of the rotating tissue culture insert of the above-described embodiments can be made from medical-grade materials such as stainless steel. Alternatively, suitable materials include 3D printed polymer materials and / or glass fiber reinforced polymer materials (such as nylon blends).

[0062] Figures 12 and 13 show a fourth embodiment of the invention, in which the hollow shaft 3 has been modified to include more openings than in Figures 1 to 6. The free space defined by the openings in this embodiment is at least 30% of the outer cylindrical surface of the shaft 3, whereas the free space defined by the openings 5 ​​in the first embodiment is less than 10% of the outer cylindrical surface of the shaft.

[0063] Two prototypes of the RTCI for option 1 according to Figures 1-6 and 12-13 and one prototype of the RTCI stretcher according to Figures 7-11 have been manufactured and tested in multiple cultures.

[0064] All experiments used polyglycolic acid (PGA)-based scaffolds coated with 1% P4HB. For testing, rectangular scaffolds (size: 9.5 x 4 cm) were sutured to a 28 mm stent-like scaffold holder and inserted into the RTCI described in Option 1 (n=5 at each time point of 2, 4, and 6 weeks of tissue culture) (see Figures 1-6) or into the stretcher described in Option 2 (n=4 constructs cultured for 6 weeks) (see Figures 7-11). Following standard protocols, neonatal human dermal fibroblasts (hDFB, passage 7-9) were seeded at 1x106 / cm2 using fibrin as a cell carrier.

[0065] The results using Option 1 are shown in Figures 14a-14c. To better assess the performance of the rotating tissue culture method using RTCI described in Option 1, TEMs were made using different tissue culture time points. The following samples have been produced and tested by macroscopic evaluation, immunohistology, mass spectrometry, and uniaxial mechanical testing:

[0066] Figure 14a shows n=6 test tubes cultured for 2 weeks, Figure 14b shows n=6 test tubes cultured for 4 weeks, and Figure 14c shows n=6 test tubes cultured for 6 weeks. Figures 14a-c show representative histological images of longitudinal TEM sections demonstrating the progression of extracellular matrix (ECM) deposition and organization over the course of tissue culture. Importantly, the ECM is longitudinally oriented, as indicated by the blue arrows.

[0067] Additionally, results using Option 2 were compared with those using Option 1. To compare the performance of the RTCIs shown in Figures 1-6 and the stretcher RTCIs shown in Figures 7-11, TEMs were fabricated using a 6-week tissue culture period. N=4 TEM samples were fabricated using the stretcher RTCI and examined by macroscopic evaluation, immunohistochemistry, and uniaxial mechanical testing. The results were compared to those shown in Figures 14a-14c. The results were compared to TEMs cultured using the RTCIs described in Option 1. Figure 15 shows the results after 6 weeks of culture, where the resulting TEMs were decellularized and removed from the RTCI and scaffold holder for further testing.

[0068] FIG. 15 shows the results of uniaxial tensile tests performed on TEM samples manufactured using the stretcher RTCI of FIGS. 7-11, labeled 101, or the RTCI described in option 1 of FIGS. 1-6, labeled 102. Uniaxial mechanical testing of samples in both the circumferential and longitudinal directions provided information on tissue anisotropy: stress at failure and stiffness were greater in the longitudinal direction for TEM tubes cultured using option 1. On the other hand, the stress and stiffness at failure are slightly greater in the circumferential direction for TEM test tubes cultured using the stretcher described in Option 2. These results, shown in Figures 16a-c and 17a-c, demonstrate the changes in mechanical properties that can be associated with different ECM orientations.

[0069] Figures 16a-c disclose histological evaluation of samples cultured for 6 weeks using RTCI Option 1, also referred to as Method 1, and stretcher RTCI Option 2, also referred to as Method 2. Figures 16a-c are histological images of longitudinally sectioned samples.

[0070] Figures 17a-c show histological images of circumferentially cut samples. This analysis shows that in Option 1, the ECM is primarily aligned longitudinally. In contrast, in Option 2 or Method 2 stretchers, the collagen is primarily aligned circumferentially, especially in the central portion of the TEM.

[0071] Option 3, in conjunction with Figures 18-20, discloses the potential and relevance of the present invention to cardiovascular tissue engineering. Figures 18-20 show the results after 6 weeks of culture using RTCI according to option 1. The resulting TEMs were used to fabricate tissue-engineered heart valves with TEHVs (n=6) mounted on nitinol stents compatible with transcatheter aortic valve replacement (TAVR). The valve was then inserted into a custom-made silicone holder with an inner diameter of 25 mm and tested in vitro for at least 1 hour using a pulse duplicator system under both pulmonary pressure (peak systolic pressure: 20 mmHg) and aortic-like pressure (peak systolic pressure: 120 mmHg), with favorable results. Similarly, after 6 weeks of culture using RTCI (Option 2), the resulting TEMs were used to produce TEHVs (n = 2). In vitro testing using a pulse duplicator system demonstrated good performance for 1 hour under pulmonary artery pressure conditions.

[0072] Next, detailed methods for preparing tissue using RTCI are described below: Scaffolding preparation hTEMs were fabricated using a nonwoven polyglycolic acid (PGA) mesh (1 mm thick, specific gravity 70 mg / cm3, Confluent) coated with 1% poly-4-hydroxybutyrate (P4HB, MW 1x106, TEPHA Inc.) in tetrahydrofuran (Sigma-Aldrich™) as the starting matrix, as previously described. The PGA / P4HB mesh was cut into scaffolds (width: 100 mm, height: 45 mm) and sutured to a cylindrical scaffold holder (height 30 mm, inner diameter 28 mm) based on a nitinol stent. The scaffold is then assembled onto the RTC insert according to Figures 1-6, and the entire construct is sterilized (incubated in 70% ethanol for 30 minutes, followed by incubation in PBS supplemented with 10% penicillin-streptomycin (Sigma) and 1% antibiotic-antimycotic solution (Sigma) for 30 minutes). Finally, the scaffolds were cultured overnight in cell culture medium (Advanced DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% GlutaMax (Gibco), and 1% penicillin-streptomycin (Sigma)).

[0073] Cell growth and seeding Human dermal fibroblasts (hDFB, CellSystems Biotechnologie GmbH, passage 7-10) were seeded into roller bottles (850 cm 2 , TufRol EZ, Nunc)™ and grown to confluence in cell culture medium using the CELLROLL system. At harvest, cells were seeded onto the scaffolds at a density of 1 x 10 cells / cm using fibrin as a cell carrier, as previously established. After seeding, the constructs were incubated overnight in cell culture medium to promote cell attachment.

[0074] hTEM culture The day after seeding, the constructs were transferred to pleated roller bottles (1450 cm², TufRol, Nunc) and cultured in 150 ml of tissue culture medium (cell culture medium supplemented with l-ascorbic acid 2-phosphate (0.26 mg / ml, Sigma-Aldrich) from day 1, and cell culture medium supplemented with 5 ng / ml TGF-p1 (Peprotech) from day 7), which was changed twice a week. Tissue culture was performed in a standard incubator setting (37°C, 5% CO2, 95% relative humidity) at a rotation speed of 1.5 rpm. After 2, 4, or 6 weeks of tissue culture, the hTEM tubes were washed with PBS and decellularized using an optimized protocol.

[0075] Decellularization hTEMs were incubated twice overnight in detergent solution (0.25% Triton X-100, sodium deoxycholate, and 0.02% ethylenediaminetetraacetic acid in DPBS) in roller bottles. Furthermore, the mixture was incubated four times with decreasing concentrations of benzonase (Novagen) (100 U / ml, 80 U / ml, 40 U / ml, 20 U / ml in 50 mM TRIS-HCl buffer at pH 8.0) to decompose the remaining DNA. After washing, the hTEM tubes were stored in PBS supplemented with 1% penicillin I streptomycin (Sigma) at 4°C until further use.

[0076] (immuno)histochemistry Qualitative, quantitative, and (semi)quantitative immunohistological evaluation of cross sections of the various fabricated human cell-derived TEMs (hTEMs) was used to obtain relevant information regarding the structure and deposition of ECM and the presence of scaffold residues.

[0077] For each tube, a longitudinal section of the hTEM was cut, fixed in 4% formalin, dehydrated, embedded in paraffin, and cut longitudinally (slice thickness 3 μm). The sections were stained with hematoxylin and eosin (H&E) to assess cell presence and tissue morphology. Elastica van Gieson (ELVG) was used to examine elastic and collagen fibers, and collagen 1 (COL1, Abeam, ab34710) and collagen 3 (COL3, Abeam, ab7778) were used to assess specific collagen deposition. The stained samples were imaged under a bright-field microscope (MiraxMidi Microscope, Carl Zeiss GmbH).

[0078] hTEM dimension evaluation The thickness of the hTEM, which is the sum of the outer and inner layers of the scaffold, and the thickness of the ECM were measured using Pannoramic Bluer software (3DHISTECH, Ltd.). For each hTEM sample, thickness was measured at 10 different locations per section and the average value was calculated. At each time point (2, 4, and 6 weeks) in tissue culture, n = 4 different hTEM samples were measured and the average values ​​were reported.

[0079] Semi-quantitative evaluation For each sample, a semiquantitative assessment of histology was performed by rating the maturity and presence of ECM (outer, inner, and central regions of the hTEM samples), the presence of polymeric residues, the efficiency of decellularization, and the presence of collagen 1, 3, and elastin from 0 (absent) to 5 (abundant). Analysis was performed by two independent observers for each hTEM sample. For each tissue culture time point (i.e., 2 weeks, 4 weeks, 6 weeks), n=4 different hTEM samples were analyzed.

[0080] biochemical analysis Biochemical assays were used to quantify collagen (via hydroxyproline, HYP) and glycosaminoglycan (GAG) content. Briefly, 5-8 mg of lyophilized hTEM samples (n = 4 per time point, technically triplicates) were digested with papain (Worthington) in digestion buffer, and a stepwise protocol was performed to quantify the GAG ​​and HYP content and measure light absorbance using a plate reader (Tecan infinite M1000 pro). Quantification was performed using GAG and HYP standards. Double-stranded DNA (dsDNA) analysis was performed using the Qubit dsDNA HS Assay Kit (Thermofisher). Data were normalized to the total dry tissue weight.

[0081] Uniaxial Tensile Test Uniaxial mechanical testing was performed to obtain information about how mechanical properties change over time in tissue culture. Longitudinal hTEM samples (n=3 for each tissue culture time point, technically triplicate) were cut to 9x5mm lengths, clamped in custom-made clamps, and then tested using a uniaxial materials testing machine (Zwick Z010 TN, ZwickRoell GmbH, Ulm, Germany) that recorded force-strain data. During testing, samples were stored in a chamber filled with room temperature PBS. After an initial preload of 0.025N (0% strain), samples were preconditioned to 5% strain five times, with a 30-second recovery period between each preload cycle. The sample was then ramped to failure at a constant strain rate of 100% strain per minute. Stress calculations were performed using an estimate of the sample cross-sectional area, calculated by multiplying the sample width (5 mm) by the tissue thickness. Tissue thickness was obtained based on histological images of adjacent samples. The thickness values ​​were then corrected with a volumetric shrinkage correction factor of 1.15 to account for the loss of tissue volume due to histological sample preparation. The stiffness (E, Young's modulus), stress at failure, and strain at failure of the samples were calculated in the linear region of the force-strain curve.

[0082] Heart valve manufacturing and in vitro testing A systematic in vitro evaluation (n = 4) of TEHV performance was performed using a pulse duplicator system HDT-500 (BDC Labs, USA) equipped with a transonic sensor TS410 (Transonic Systems, USA) for flow measurement and a BDC-PT pressure sensor (BDC Labs, USA). All TEHVs were first tested for 1 hour under simulated pulmonary pressure conditions (peak pressure: 20 mmHg, mean pressure: 10 mmHg, minimum pressure: 0 mmHg). After gradually increasing the applied pressure, the TEHVs were tested for 1 hour under simulated aortic pressure conditions (peak pressure: 120 mmHg, mean pressure: 100 mmHg, minimum pressure: 80 mmHg). Testing was performed in PBS supplemented with 0.05% xanthan gum (Sigma) according to ISO 5840 testing requirements. Data were collected for 3 seconds and functionality was assessed by averaging 20 simulated heartbeats using Statys software (BDC Labs, USA) to determine the regurgitant fraction (RF) expressed as a percentage of the closed volume (CV, regurgitant flow during valve closure), leak volume (LV) and forward flow (FFV), effective valve area (EOA, estimated using the Gorlin formula described in ISO 5840), and maximum and mean transvalvular pulmonary / aortic gradients (peak pressure difference, PPDmax and PPDmean, respectively, calculated as the difference between ventricular pressure and pulmonary / aortic pressure during systole). [Explanation of symbols]

[0083] List of symbols 2 Scaffolding holder 3-axis body 4 protrusions 5 aperture 6 plates 7 Shaft parts 8 Shaft parts 11 Scaffolding 12 Tips 13 Screws 20 rotating tissue culture inserts 25 Surface 27 Ribbons 32 sleeve 33 Axial body 34 Threaded bolt 35 Threaded sleeve 36 Cone-shaped part 37 Cone-shaped part 38 slope 40 RTCI 42 Mesh body 43 Mesh section 44 protrusions 45 protrusion 46 Tapered end 47 Mesh Structure 48 large mesh 49 pieces 50 portions 51 organizations 100 Roller Bottles 101 code 102 code

Claims

1. A rotating tissue culture insert (20, 40) positioned within a rotatable roller bottle (100) partially filled with a liquid for culturing cells, the roller bottle having a scaffold (11) provided for culturing cells, The rotating tissue culture insert (20, 40) further comprises a shaft (3, 33) and at least two projections extending radially from the shaft (3, 33); The protrusions (4) define wheels of the rotating tissue culture insert (20, 40) having a track and contact the surface (25) of the roller bottle (100) when the roller bottle (100) is in a lying position.

2. 2. The rotating tissue culture insert (20, 40) of claim 1, wherein at least two protrusions are located at the distal end of the shaft (3, 33).

3. 3. A rotating tissue culture insert (20, 40) as claimed in claim 1 or 2, wherein the radial height of the protrusions (4) is greater than the thickness of the scaffold (11) or greater than the sum of the thicknesses of the scaffold (11), the scaffold holder (2) and any sleeve (32) that is coaxial with the shaft (33) and is positioned on the shaft (33).

4. 4. A rotating tissue culture insert (20, 40) according to claim 3, wherein the sleeve (32) has a longitudinal axis parallel to the shaft (3, 33) which comprises a plurality of openings (5), the scaffold holder (2) is preferably arranged on the sleeve (32) and / or the shaft (3, 33) and is part of the rotating tissue culture insert (20, 40), and / or the shaft (3, 33) and / or the sleeve (32) is a hollow body preferably comprising a plurality of openings (5).

5. 5. A rotating tissue culture insert (20, 40) according to any one of claims 1 to 4, wherein the projection (4) is detachable from the shaft (3) and is preferably connected to the shaft (3, 33) by a screw (13).

6. 6. A rotating tissue culture insert (20, 40) according to any one of claims 1 to 5, wherein the projection (4) has the form of a circumferential ring.

7. 7. A rotating tissue culture insert (20, 40) according to any one of claims 1 to 6, wherein the scaffold (11) comprises a nonwoven layer, preferably made from a biodegradable material, the nonwoven layer preferably comprising a coating, more preferably a biodegradable coating, most preferably a coating comprising a polyhydroxybutyrate material.

8. A rotating tissue culture insert (20, 40) according to any one of claims 1 to 7, wherein the rotating tissue culture insert (20, 40) comprises a stretcher for radially expanding the scaffold (11), and the sleeve (32) is preferably part of the stretcher.

9. A rotating tissue culture insert (20, 40) as described in any one of claims 1 to 8, wherein the shaft (3, 33) includes a longitudinal axis, and the rotating tissue culture insert (20, 40) further includes one or more parts (7, 8) that are radially movable relative to the longitudinal axis and form a sleeve (32).

10. 10. The rotating tissue culture insert (20, 40) of claim 9, comprising an adjustment mechanism for adjusting the radial distance of the one or more parts (7, 8) relative to the longitudinal axis of the shaft (3, 33), the one or more parts (7, 8) being preferably an arc-shaped plate (6), more preferably an arc-shaped plate (6) having an opening (5).

11. 11. A rotating tissue culture insert (20, 40) according to any one of claims 1 to 10, wherein the shaft (3, 33) is part of an adjustment mechanism and is formed by at least two elements which are linearly movable relative to each other, the two elements preferably including a conical portion in each element.

12. 12. The rotating tissue culture insert (20, 40) of any one of claims 1 to 11, wherein the adjustment mechanism is threadably adjustable.

13. 5. A rotating tissue culture insert (20, 40) as described in claim 3 or 4, wherein the scaffold holder (2) is essentially cylindrical and has separate bendable sections, preferably bendable in the radial direction, and more preferably has separate bendable sections at the tip position of the scaffold holder (2).

14. A method for manufacturing an implant, comprising: A: providing a roller bottle (100) with a liquid for culturing cells and a rolling tissue culture insert (20, 40) with a scaffold (11), preferably made of a biodegradable material, according to any of claims 1 to 13, wherein the roller bottle (100) is in a lying position with the fill level of the roller bottle (100) approximately parallel to the longitudinal axis of the roller bottle (100), the rolling tissue culture insert (20, 40) further comprising a scaffold holder (2) for attaching the scaffold (11) to the rolling tissue culture insert (20, 40); B: rotating the roller bottle (100), with the scaffold (11) moving away from the wall of the roller bottle (100) and the rotating tissue culture insert (20, 40) rotating within the roller bottle (100), and forming tissue (51) on the surface of the scaffold (11) during the rotation of the roller bottle (100); C. removing a structure including at least the tissue (51) and the scaffold holder (2) from the shaft (3, 33) of the rotating tissue culture insert (20, 40); A method of manufacturing, characterized in that the structure is provided for forming an integral part of an implant.

15. 15. The method of claim 14, wherein the configuration comprises a cylindrical portion with an adjustable radial diameter of the rotating tissue culture insert (20, 40), and the radial diameter of the rotating tissue culture insert (20, 40) is adjusted to the variable diameter of the cylindrical portion.

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