WO2010129422A1 - Appareil tendeur multiaxial et procédé de construction de tissus - Google Patents

Appareil tendeur multiaxial et procédé de construction de tissus Download PDF

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Publication number
WO2010129422A1
WO2010129422A1 PCT/US2010/033213 US2010033213W WO2010129422A1 WO 2010129422 A1 WO2010129422 A1 WO 2010129422A1 US 2010033213 W US2010033213 W US 2010033213W WO 2010129422 A1 WO2010129422 A1 WO 2010129422A1
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Prior art keywords
scaffold material
scaffold
tension
cells
cell
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PCT/US2010/033213
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English (en)
Inventor
John E. Mayer, Jr.
Danielle Gottlieb
George C. Engelmayr, Jr.
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Children's Medical Center Corporation
Massachusetts Institute Of Technology
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Publication of WO2010129422A1 publication Critical patent/WO2010129422A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • the present invention is in the field of tissue engineering; more specifically to devices and methods for tissue engineering.
  • PV pulmonary valve
  • vascular graft materials used in cardiovascular surgery are limited by thrombogenicity, calcification, shrinkage, mechanical fatigue due to cyclic loading, and lack of viability with limited potential for remodeling (Kannan, et al., J Biomed. Mater. Res B Appl Biomater., 74(l):570-81 (2005)).
  • the gold standard in pediatric valve replacement is the cryopreserved valved homograft.
  • homografts perform well in the short-term and potentially retain some degree of cellular viability (Mitchell, et al., Ann. Thorac.
  • THVs tissue-engineered heart valves
  • Functional ovine TEHV were initially fabricated by seeding autologous, carotid artery-derived vascular smooth muscle cells (SMCs) and endothelial cells (ECs) onto nonwoven synthetic polymer scaffolds (Hoerstrup, et al., Circulation, 102:11144-9 (2000)). Further, despite differences in the phenotype of the seeded cells versus native valvular interstitial (VIC) and endothelial (VEC) cells (Butcher, et al., Arterioscler. Thromb. Vase.
  • VIP native valvular interstitial
  • VEC endothelial
  • TEHV TEHV exhibited a tri-layered structure reminiscent of the native PV leaflet, with prominent vimentin expression and diminished alpha smooth muscle actin (a-SMA) expression localized to the immediate subendothelium (Rabkm, et al., J Heart Valve Dis., 11(3):308-14 (2002)).
  • a-SMA alpha smooth muscle actin
  • BMSCs bone marrow-derived mesenchymal stem cells
  • Heart valves require immediate valve function in vivo.
  • the leaflets of replacement heart valves must open with negligible pressure gradient (i.e., without narrowing (stenosis)), and upon leaflet coaptation during valve closure there must be minimal to no leakage around or through the leaflets creating backward flow (regurgitation).
  • the tube portion of the graft must withstand pulmonary pressure without leaking.
  • Current TEHVs suffer from non-uniform tissue growth which can lead to "bare" scaffold areas through which blood can leak and cause the fg ⁇ V to fail.
  • Current TEHVs also suffer from imperfect mechanical properties which can lead to failure by stenosis or leaking.
  • a multi-axial tension device, a tension device/scaffold material assembly and uses for forming tissues using these devices are described herein.
  • the multi-axial tension device contains two end pieces, at least one longitudinal rod(s), one or more hoops and two mounting portions.
  • the device contains two longitudinal rods.
  • the end pieces contain connectors for releasably connecting the mounting portions to the end pieces.
  • a biocompatible bioerodible, b ⁇ oabsorbable, or highly biocompatible non-degradable scaffold material may be attached to the mounting portions of the tension device to form a tension device/scaffold material assembly.
  • the tension device applies tension to a cell-seeded scaffold in both the longitudinal direction, such that each end of the scaffold material is pulled away from the opposite end along the length of the scaffold material, and radially outward from the center of the scaffold material.
  • the tension devices may be used to form a variety of different tissues, such as a vascular conduit, a valved vascular conduit, a tendon, a ligament, and skin.
  • the cell-seeded scaffold is used to generate a valved conduit for use as a pulmonary valve replacement.
  • Figure IA is a schematic of the preferred embodiment for the tension device, without the mounting portions.
  • Figure IB is a plan view of an end piece used in the device.
  • Figure 2 is a schematic of the preferred embodiment for the tension device, including the mounting portions.
  • Figure 3 is a schematic of the preferred embodiment for the tension device, depicting the mounting portions separated from a tubular heart valve scaffold to be subsequently (in Figure 4) attached to the mounting portions.
  • Figure 4 is a schematic of the preferred embodiment for the tension device, including the mounting portions and a tubular heart valve scaffold attached to the mounting portions.
  • Figure 5 is a schematic of an optional embodiment for the tension device, depicting the optional impeller mechanism.
  • Figure 6 is a schematic showing the time course of surgical application of tissue engineered heart valves in sheep and their functional evaluation. Events in the time course are symbolized as follows: O - bone marrow aspiration; A - control valve evaluation; ⁇ - implantation and echocardiography; ⁇ - MRI; X - explant and echocardiography.
  • Figure 7 is a line graph showing flow velocities through implanted tissue engineered heart valves at 3 post-operative weeks. Data are expressed as velocity (ml/s) as a function of time (ms).
  • Figure 8 A is a bar graph showing measurements of conduit diameters (cm) measured by MRI (left bar) and explant (right bar) as a function of time in weeks post implantation.
  • Figure 8B is a line graph showing changes in cusp length and width over time (indicated by arrows). Data are expressed as the dimension (cm) as a function of time (days and weeks).
  • a tension device (10) that applies tension mult ⁇ -axially when a scaffold material is attached thereto during cell seeding and proliferation is described herein.
  • the tension device applies tension to the cell- seeded scaffold in the longitudinal direction, such that each end of the scaffold is pulled away from the opposite end along the length of the scaffold, and radially outward from the center of the scaffold.
  • Figure IA is a schematic of the preferred embodiment for the tension device.
  • the tension device (10) contains two end pieces (20a and 20b), two longitudinal rods (30a and 30b), one or more hoops (40a and 40b) and two mounting portions ⁇ see Figure 2; 50a and 50b).
  • the parts of the tension device are fabricated using biocompatible and non- erodable materials or biocompatible and controllably degradable materials that are autoclavable, gas-sterilizable, and/or gamma sterilizable.
  • the size of the tension device as a whole is selected to accommodate the tissue that is to be generated using the device.
  • a first end piece (20a) is located at the distal end (12) of the tension device (10), while the second end piece (20b) is located at the proximal end (14) of the tension device (10).
  • Each end piece (20) contains one circular end plate (22a in one end piece and 22b in the other) and a connector (19).
  • Each end plate is preferably a single piece, with a circumferential groove machined about the center.
  • each end plate is fabricated from a contiguous piece of Teflon.
  • the circular end plates are each centrally fitted with a circular rubber o-ring mounted about a centrally located, circumferential groove machined or cast into the radially projecting surface of the end plates (24).
  • the end plates have substantially the same diameter (e.g.
  • the o-ring (24) has a smaller diameter in its stress-free (i.e., non-distended) configuration compared with the end plate diameter. Each o-ring is stretched to situate it within the grooves in the end plates. Following positioning of the o-rings on the end plates, then the diameter of the stretched o-ring is slightly greater than the diameter of the end plate (e.g. by about 1-2 mm diameter).
  • Each end plate (22) contains a center hole (26) and at least two additional holes (27a and 27b) in the periphery (28) of the end plate.
  • the periphery (28) of the end plate contains the same number of holes (27) as the number of longitudinal rods in the device, and at least one additional hole (29), preferably an even number of additional holes, more preferably two additional holes (29a and 29b).
  • the periphery of the end plates contains two holes (27a and 27b) into which the longitudinal rods (30a and 30b) are inserted, and two additional holes (29a and 29b) that allow media to flow through the endplates when the cells are growing on the scaffold material.
  • the holes (27) that are designed to accommodate the longitudinal rods are evenly spaced in the periphery of the end plate.
  • the holes that are designed to accommodate the longitudinal rods may be threaded to secure a threaded end of the longitudinal rod.
  • the holes that are designed to accommodate the longitudinal rods are preferably smooth to allow the smooth end of the longitudinal rod to slide within the hole.
  • the holes (27a and 27b) in the end plates at the proximal end (14) of the device are threaded to secure correspondingly threaded ends of the longitudinal rods (30a and 30b), while the holes (27a and 27b) in the end plates at the distal end (12) are smooth.
  • the holes for insertion of the longitudinal rods in the end plates at both the proximal end and the distal end of the device are smooth.
  • the ends of the longitudinal rods and associated holes may be fitted to complementary magnets, providing a semi-rigid attachment.
  • the attachment could be made via a biodegradable medium, providing progressive relaxation and ultimate detachment following a prescribed in vitro cultivation period.
  • each end piece contains a connector (19) attached in a fixed manner to the portion (23 a or 23b) of the end piece that faces in center of the device.
  • the connector (19) is attached to and removable from the portion (23) of the end piece that faces in center of the device.
  • each connector attaches to the center hole (26) of the end plates.
  • the center hole (26) is threaded to accommodate a correspondingly threaded end ( 18) of the connector (19).
  • the end (18) of the connector that inserts into the center hole is in the form of a hollow tube and/or contains one or more holes to accommodate media access to the device.
  • the connectors detachably connect the end pieces to the mounting portions, hi one embodiment, the end (17) of the connector that is proximal to the center of the device includes a means for releasable attachment of a mounting portion (50).
  • the means for releasable attachment should allow for easy and quick separation of the connector from the mounting portion, it must also contain a sufficiently strong attachment to keep the mounting portion in place, allow for the attachment of the scaffold material to the tension device during all steps leading up to release of the mounting portions (50a and 50b), and accommodate any longitudinal tension developed by cellular-mediated contraction of the scaffold material subsequent to cell seeding, proliferation, and remodeling.
  • Suitable means for releasable attachment include a quick disconnect with push button (e.g.
  • the attachment is made via a biodegradable medium to allow for progressive relaxation (and associated decreases in longitudinal tension) and subsequent detachment over the course of in vitro cultivation.
  • Removal of the mounting portions with an attached cell-seeded scaffold allows for the cell-seeded scaffold to be removed from the tension device and subsequently placed into another environment.
  • scaffolds containing developing tissue may be removed from the tension device after cell seeding and initial tissue growth, and then placed inside a bioreactor device where additional mechanical forces may be applied to the tissue to facilitate growth of the tissue.
  • the mounting portions may be fixedly attached to the end pieces so that they are not removable from the end pieces.
  • the device does not contain a separate connector.
  • B. Longitudinal rods At least one longitudinal rod (30) attaches to each end piece at each end (12 and 14) of the device. Although more than two longitudinal rods may be included in the device, preferably the device contains two longitudinal rods (30a and 30b). The number and placement of the longitudinal rods is selected to allow for maximum flow of fluid around and through the scaffold material and also provides minimum interference during the removal of the cell seeded-scaffold material from the tension device. Further, the longitudinal rods allow for the potential attachment of hoops, as detailed below.
  • the longitudinal rods are preferably evenly spaced around the periphery of the end plates.
  • At least one longitudinal rod(s) (30) connects the first end piece (20a) with the second end piece (20b).
  • the device contains two longitudinal rods, which connect the first end piece (20a) with the second end piece (20b) such that the proximal end (32a or 32b) of each longitudinal rod (30a or 30b) is inserted in a hole (27a or 27b) in the first end piece (20a) and the distal end (34a or 34b) of each longitudinal rod (30a or 30b) is inserted in a hole (27a or 27b) in the second end piece (20b).
  • the first and the second end pieces are aligned so that all of the holes designed to contain a longitudinal rod in the periphery of one end piece line-up with all of the holes designed to contain a longitudinal rod in the periphery of the second end piece.
  • one end ⁇ e.g. 34, as shown in Figure IA) of the longitudinal rods is threaded and is secured into threaded holes in one end piece.
  • the other end ⁇ e.g. 32, as shown in Figure IA) of the longitudinal rods may be smooth and fit into smooth holes on the opposite end piece. This arrangement allows the end piece attached to the smooth end of the longitudinal rods to slide along the rod.
  • the distance between the first and second end pieces is adjustable. This is useful to accommodate scaffold materials of differing lengths and may also be used to adjust the amount of tension applied to a scaffold material after it is attached to the device.
  • both ends (32 and 34) of each longitudinal rod are smooth and attach to smooth holes in the end pieces.
  • both end pieces may be moved along the longitudinal rods relative to each other.
  • both ends of the longitudinal rods are threaded and are secured in threaded holes in both end pieces.
  • the distance between the end pieces may be fixed, or if the distal and proximal connections are made respectively via left- and right- handed threads, then the distance between end plates can be controlled by rotating each of the longitudinal rods.
  • the end plates and rod can be fabricated from a single, contiguous block of material.
  • the material of construction in this embodiment can be a temperature-responsive shape memory material or a material that swells under physiological temperature, pH, or other parameters such as to expand upon contact with a fluid medium under various physiological conditions ⁇ e.g., 37 0 C), thereby imposing tension on the interposed cell-seeded scaffold material.
  • a suitable fixation means such as a shaft collar (36). Shaft collars can be locked into place along the longitudinal rods, thus determining how far along the longitudinal rods the end piece may move.
  • the shaft collars may necessitate modification of the connector in order to create an effectively rigid shaft collar-connector attachment, such as by inserting the shaft collar into an indentation (38) that is sized to fit the shaft collar and is located on a side of the connector.
  • the longitudinal rods are preferably fabricated from a pliable material to accommodate bending that may occur as a result of movement of one or more end pieces along the length of the rods.
  • the longitudinal rods are fabricated using polyetheretherketone (PEEK) or a similarly, more, or less pliable material (e.g., stainless steel, Teflon (polytetrafluoroethylene (PTFE)).
  • the longitudinal rods are attached to one or more hoops (40), which are generally perpendicular to the longitudinal rods.
  • the hoops can be inclined at any angle with respect to the longitudinal axis, thereby allowing the possibility of imposing both radial and longitudinal tension components, as well as controllable radial tension by way of bending the hoop (i.e., an effectively non-rigid hoop).
  • the diameters of the hoops are greater than or equal to the diameters of the mounting portions and attached scaffold material and provide an attachment point to apply radial tension to the scaffold material.
  • the diameters of the hoops can be less than the diameters of the mounting portions and the attached scaffold material and thereby provide an internal, preferably biodegradable, rib-like structure upon which the scaffold can contract (while simultaneously under longitudinal tension imposed by attachment to the end plates).
  • the device may be used for tissue engineering the tubular elements comprising the gastrointestinal tract, such as the esophagus and small intestine.
  • the hoops are attached to the longitudinal rods by inserting each hoop through one hole in each longitudinal rod. This arrangement locks the hoops in a fixed location along the length of the longitudinal rods. Alternatively the hoops may be placed around or inside the longitudinal rods, without attaching the hoops to the longitudinal rods.
  • the hoops may be in any suitable shape.
  • the hoops are circular. Circular hoops are useful to provide an effectively equal amount of radial tension at a particular longitudinal location around the circumference of a tubular scaffold (in conjunction with a running, purse- string suture line between the scaffold and hoop).
  • the hoops may be rectangular or square-shaped. Rectangular or square-shaped hoops may be used to provide a gradient of tension to a tubular scaffold.
  • the hoops should have a sufficiently large circumference such as to facilitate drawing the sutures tight by a desired amount so that the sutures can apply radial strain to the scaffold material.
  • the amount of radial tension is a functions of the tension in the purse string suture and the relatively elasticity/rigidity of the hoop.
  • the radius of circular hoops is larger than the radius of the holes in the end pieces that accommodate the longitudinal rods, causing the longitudinal rods to bend outward at the longitudinal locations of hoop attachment or insertion.
  • Each hoop is capable of applying radial tension to the scaffold at a fixed position along the scaffold's length depending on the placement of the hoop along the length of the longitudinal rod.
  • the use of multiple hoops may be suitable when a particular pattern of pure radial (i.e., hoops exactly perpendicular to the longitudinal axis) or radial and longitudinal components of applied tension (i.e. when one or more hoops are inclined at an angle other than 90 degrees to the longitudinal axis) is desired along the length of a scaffold.
  • the hoops are preferably fabricated from a rigid material, which is more rigid than the material that forms the longitudinal rods, to provide constant radial strain to the scaffold material.
  • the hoops are fabricated from stainless steel.
  • hoops may be fabricated from other materials, including non-degradable polymers such as PEEK and Teflon, or biodegradable polymers such as poly(glycolic acid), ⁇ oly-(L-lactic acid), or blends or copolymers thereof.
  • the hoops can exhibit circumferential variations in curvature, such as to allow or disallow local hoop bending along the radial axis.
  • the hoop can be made out of a bimetallic (or other shaped changing) material that undergoes expansion with increased temperature, thereby imposing radial tensions upon device submersion in 37° C fluid media.
  • the tension device does not contain any hoops.
  • the device may provide radial tension to the scaffold by attaching the scaffold using suitable attachment means directly to the one or more longitudinal rod(s).
  • the mounting portions (50a and 50b) and their placement within the tension device (10) are illustrated in Figure 2.
  • One mounting portion (50a) is detachably connected to the end (17) of each connector that is proximal to the center of the device.
  • the device contains two mounting portions (50a and 50b), with a first one mounting portion at the proximal end (14) and the second at the distal end (12) of the device.
  • the mounting portions have two ends, a connection end (52) that connects to the connectors (19) and a mounting end (54) that extends towards the center of the tension device and is designed for placement of the scaffold material.
  • the mounting end optionally contains any suitable means for attaching a tubular, linear or planar scaffold material.
  • the mounting portions include a plug and barb piece (50) (e.g. available from McMaster-Carr) that connects to the connector (19) via the connection end (52) and a mounting ring (58) at the mounting end (54).
  • the mounting end (54) contains a groove (59) around its circumference. The groove allows for a scaffold material to be secured to the mounting portion at the location of the groove.
  • a tubular scaffold material may be attached to the mounting end (54) by extending the ends of the scaffold material over the mounting end (54) and securing the scaffold material to the mounting end (54) by any suitable means, such as by applying pressure to the section of the scaffold over the groove in the mounting ring, such as via an elastic o-ring, or a friction fit.
  • Removal of the mounting portions with an attached cell-seeded scaffold allows for the cell-seeded scaffold to be removed from the tension device and subsequently placed into another mechanical environment.
  • scaffolds containing developing tissue may be removed from the tension device after cell seeding and initial tissue growth, and then placed inside a bioreactor device where additional mechanical forces may be applied to the tissue.
  • the mounting portions may be fixedly attached to the end pieces so that they are not removable from the end pieces.
  • the device does not contain a separate connector (not shown in figures).
  • the tension devices contain an impeller system on. one of the end pieces ⁇ see e.g. , Figure 5).
  • the impeller system may be used to draw fluid into the tension device. This may be useful while seeding a mounted scaffold to provide continuous mixing of the fluid to facilitate homogenous cell attachment to the scaffold.
  • the impeller system may also be useful to provide shear stress to a cell-seeded scaffold, mimicking physiological shear stresses.
  • the impeller system may include any suitable means for drawing fluid into the tension device.
  • the impeller system includes a magnetically-coupled impeller/stirrer with an impeller on the inside of an end piece that turns in response to a stirrer on the outside (i.e., external to the roller bottle or container) of the tension device.
  • FIG. 5 illustrates an exemplary tension device in a roller bottle (80) with an impeller mechanism (90).
  • the impeller mechanism is installed on the downstream end plate of the tension device and magnetically couples with a magnetic stirrer, which is external to the roller bottle.
  • This mechanism can provide non-contact means for introducing either continuous or pulsatile fluid flow through the lumen of the cell-seeded scaffold.
  • the impeller mechanism (90) contains an impeller (92) with one or more vanes (93 a and b) with one or more magnets (94a and b) embedded in the impeller vanes.
  • the impeller vanes are inside an impeller housing (95), which also contains a cavity (96), a shaft (97), and one or more shaft mounting rods (98) and a collar (99).
  • a magnetic stirrer (100) is outside of and connected to the roller bottle (90).
  • the magnetic stirrer is attached to the roller bottle via a pendulum mechanism (110), which allows the magnetic stirrer to remain in a relatively stationary position while the roller bottle rolls. H.
  • the tension devices described herein may be used to apply multi- axial tension to a cell-seeded scaffold. It has been discovered that application of multi-axial tension in the form of combined longitudinal and radial tensions using the disclosed devices results in cell-seeded scaffolds with more homogenous tissue growth as compared to cell-seeded scaffolds prepared in the absence of tension or using uniaxial tension.
  • the examples below demonstrate that when TEHVs were prepared using unconstrained scaffolds, the seeded cells grew heterogeneous tissue, both leaving areas of the scaffold devoid of tissue growth, as well as yielding spatially- heterogeneous shrinkage and partial local collapse of the valved conduit scaffolds.
  • TEHVs produced using the disclosed tension devices undergo significantly less average change in size, both over their length and width, during in vitro culture. While TEHVs prepared using uniaxial tension in the longitudinal direction had a significantly reduced change in scaffold length, the scaffolds contracted at the midpoint of the tube. When sheep were implanted with TEHVs prepared using uniaxial tension in the longitudinal direction as pulmonary valve substitutes, they survived the operation, but died from arrhythmia and other problems associated with right ventricular pressure load from a relatively stenotic valved conduit In contrast, TEHVs prepared using the tension devices described herein resulted in TEHVs that did not become stenotic and resulted in long-term survival.
  • a scaffold material (60) is attached to the tension devices (10) described herein to form a tension device/scaffold material assembly (70) (see Figure 4).
  • First each end (62 and 64) of the scaffold material is placed over and attached to the mounting end (54) of one of the mounting portions (52).
  • the scaffold material is attached to the mounting end by any suitable attachment means, such as by suture, flexible o-rings (e.g. silicone rubber o- rings), biodegradable (e.g., fibrin) or non-degradable, but biocompatible glue (e.g., cyanoacrylate), Velcro, or miniature barbs.
  • the scaffold material is attached to the mounting end by suture.
  • the scaffold material (60) is attached to the one or more hoops (40).
  • the hoops apply radial tension to the scaffold material by attaching to the scaffold material and pulling the attached surface of the scaffold outward relative to the center of the scaffold material.
  • the scaffold material may be attached to the one or more hoops by any suitable means. In a preferred embodiment, sutures are used to attach the hoops to the scaffold material.
  • the scaffold material when the scaffold material is designed to form a heart valve, the scaffold material is aligned with the hoops so that the leaflets (66) are located inside of the hoops. If two hoops are present, then one hoop (40a) is generally located in an area that corresponds with the top of the material that forms the leaflet (i.e., the sinotubular ridge), and the second hoop (40b) is generally located in an area that corresponds with the bottom of the material that forms the leaflet (i.e., the basal attachment).
  • Scaffold materials are porous structures fabricated using biodegradable polymers.
  • the scaffolds are fabricated to provide an effective substrate for cell attachment and tissue formation in vitro.
  • the pores in the polymeric scaffolds allow for integration of cells throughout the scaffold and formation of tissue.
  • the scaffold materials used with the disclosed tension devices are preferably biodegradable, thereby allowing for the scaffolds to become replaced by forming neotissue as they degrade in vivo.
  • the scaffold materials offer growth potential that is not possible with currently used synthetic vascular grafts.
  • the scaffolds can be made of a biocompatible, non-degradable material.
  • the scaffold materials may be of any suitable shape according to the intended use of the tissue.
  • the scaffold materials are substantially linear or planar.
  • the scaffolds are substantially tubular in shape with a round or substantially round cross section.
  • the tubular scaffolds have a lumen extending throughout the length of the scaffold.
  • the scaffolds are substantially tubular in shape and contain valve leaflets extending into the lumen capable of opening in response to changes in pressure.
  • the scaffold materials may be of any appropriate length and diameter that is suitable for the intended surgical use of the cell-seeded scaffold.
  • the porous polymeric scaffold materials may be fabricated using any appropriate method, such as melt processing, solvent processing, leaching, foaming, extrusion, injection molding, compression molding, blow molding, spray drying, extrusion coating, or microfabrication.
  • the scaffolds are produced by spinning of fibers with subsequent processing into woven or non- woven constructs, with preferred pore sizes ranging from 100-300 microns.
  • tubular scaffold materials are formed from a felt or sheet like material of the polymer that can be formed into a tubular conduit.
  • the device could be fabricated as a nonwoven, woven or knitted structure from extruded polymeric fibers.
  • the polymeric sheet may be formed using any textile construction, including, but not limited to, weaves, knits, braids or filament windings. Any suitable method, such as electrospinning, may be used to fabricate the nonwoven or woven polymeric textile. Textile sheets may be formed into appropriate shapes using molds. Overlapping areas of textile may be bonded together using any suitable method. In one embodiment, needle punching is used.
  • the cells are seeded onto the polymeric scaffold materials via an intermediary hydrogel or similar carrier.
  • the pore size ranges can be larger than in the case of seeding with no hydrogel.
  • the fabrication methods and materials are chosen to produce a scaffold with the desired mechanical properties.
  • the polymers and fabrication methods selected to fabricate the scaffold materials are suitable to produce grafts with biomechanical properties suitable for use as valved or non-valved vascular conduits. Biomechanical properties that are important for vascular graft function include burst strength and effective bending stiffness.
  • the burst strength of the polymeric vascular graft is between about 1000 mmHg and about 3000 mmHg, preferably between about 1500 mmHg and about 2000 mmHg.
  • the polymeric vascular grafts possess an effective bending stiffness between about 50 and 300, preferably between about 150 and about 250.
  • Any suitable biodegradable or bioabsorbable and biocompatible scaffold material may be used. Preferably the material is absorbed in vivo over a time period ranging from months to a year following implantation of the material.
  • the scaffold materials may be fabricated using any known biodegradable or bioabsorbable polymer, co-polymer, or mixture thereof. Many suitable biodegradable polymers are known in art.
  • the scaffold can be made of a biocompatible, non-degradable material.
  • biodegradable polymers include synthetic polymers that degrade by hydrolysis such as poly(hydroxy acids), such as polymers and copolymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co- caprolactone).
  • poly(hydroxy acids) such as polymers and copolymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co- caprolactone).
  • the foregoing materials may be used alone, as physical mixtures (blends), or as copolymers.
  • the scaffold material is a 50:50 blend of poly(lactic acid) fibers and poly(glycolic acid) fibers. These biodegradable polymers can be extruded into fibers and fabricated into nonwoven or woven felts to produce grafts with biomechanical properties suitable for use in a wide variety of surgical applications, including for use as vascular grafts and replacement heart valves.
  • the scaffold material is seeded with cells to form tissue on the scaffold that will replace the scaffold as it degrades in vivo.
  • This approach results in an implantable cell-seeded scaffold that is capable of immediate in vivo function, yet also provides the advantage of the potential for future growth of the resulting tissue.
  • the tension device/scaffold material assembly (70) is placed into a container (not shown in figures) for cell seeding.
  • the container is just large enough to house the tension device, allowing for the concentration of added cells to be as high as possible.
  • the container is preferably capable of gas exchange with the environment, typically through a vented cap or lid.
  • the container is a cylindrical bottle.
  • the container is a cylindrical glass bottle with a vented cap for gas exchange.
  • the tension device/scaffold material assembly is typically sterilized prior to cell seeding.
  • the tension device /scaffold material assembly may be sterilized separately from the container and subsequently placed inside the container, or more preferably the /scaffold material assembly is placed into the container prior to sterilization.
  • Sterilization may be by any suitable means, including, but not limited to, exposure to ethylene oxide gas or autoclaving. Following sterilization, all remaining steps are carried out under sterile conditions, such as in a biological hood.
  • the scaffold material is fabricated from relatively hydrophobic materials, it may be necessary to first expose the scaffold to a biologically compatible wetting agent prior to the addition of cells in an aqueous media.
  • suitable biocompatible wetting agents include, but are not limited to, lower alcohols (i.e. ethanol and isopropanol), Pluronic® F-68 (BASF Corp.), Pluronic® F-38, Pluronic® P-105, Pluronic®-1OR5, Tween 20, Tween 60, Tween 85, Brij 35, Brij 78, Myrj 52, PEG 600 and glycerin.
  • the wetting agent is a 70% ethanol solution.
  • the wetting agent is then removed and the tension device and scaffold are washed in a biologically relevant solution, such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the tension device/scaffold material assembly is then soaked in a serum solution for a period of time to precoat the scaffold fibers with serum-derived cell adhesive proteins, such as fibronectin and vitronectin.
  • the serum may be any suitable serum, including but not limited to, fetal bovine serum (FBS) and fetal calf serum (FCS).
  • FBS fetal bovine serum
  • FCS fetal calf serum
  • the serum may also be autologous serum derived from the individual that is the intended recipient of the cell-seeded scaffold.
  • the serum solution contains a mixture of antibiotics and/or anthnycotics to reduce the risk of microbial contamination of the scaffold.
  • cells are added to the scaffold material.
  • the cells may be autologous or allogenic to the subject to receive the cell-seeded scaffold, or derived from some other suitably non-immunogenic cell source.
  • the cells are autologous; that is, they are obtained from the individual in which the resulting tissue will be implanted.
  • the cell types used to seed the scaffold material may be selected based on the tissue to be formed and the intended use of the tissue. Suitable cell types include, but are not limited to, tenocytes, fibroblasts, ligament cells, endothelial cells, smooth muscle cells, epithelial cells, muscle cells, nerve cells, kidney cells, bladder cells, intestinal cells, chondrocytes and bone-forming cells, or precursors thereof.
  • a scaffold when a scaffold is being used to prepare skin tissue, it may be seeded with keratinocytes or keratinocyte precursor cells, and when a scaffold is being used to prepare tendons or ligaments, it may be seeded with fibroblasts or fibroblast precursor cells.
  • differentiated cells of an appropriate lineage has limitations such as the need for invasive procedures to isolate the cells, and a limited ability to expand the cells in culture to a number required for cell seeding.
  • the scaffolds may be seeded with stem cells or progenitor cells.
  • Stem cell are cells that are capable of self-renewing cell division to give rise to phenotypically and genotypically identical daughters for an indefinite time and ultimately can differentiate into at least one final cell type.
  • Stem cells are defined as cells that have extensive, perhaps indefinite, proliferation potential, differentiate into several cell lineages, and that can repopulate tissues.
  • stem cells are proliferated while maintained in an undifferentiated state and then seeded directly onto scaffolds.
  • stem cells may be proliferated while maintained in an undifferentiated state and subsequently differentiated into a desired cell type prior to seeding onto scaffolds.
  • Suitable stem cells for seeding of scaffolds include pluripotent, multipotent or totipotent stem cells.
  • Suitable stem cells may be obtained from adult, embryonic, or fetal tissue. Stem cells have been identified in most organ tissues including bone marrow, muscle, adipose, liver, heart, lung, and nervous system tissue.
  • the embryonal stem (ES) cell has unlimited self-renewal and multipotent differentiation potential. These cells are derived from the inner cell mass of the blastocyst, or can be derived from the primordial germ cells from a post-implantation embryo (embryonal germ cells or EG cells). ES and EG cells have been derived from mouse, and more recently also from non-human primates and humans. When introduced into blastocysts, ES cells can contribute to all tissues of the animal. When transplanted in postnatal animals, ES and EG cells generate teratomas, which demonstrate their multipotency. ES (and EG) cells can be identified by positive staining with the antibodies SSEAl and SSEA4.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • limb-bud mesoderm tissue that generates bone, cartilage, fat, skeletal muscle and endothelium.
  • Mesoderm also differentiates to visceral mesoderm, which can give rise to cardiac muscle, smooth muscle, or blood islands consisting of endothelium and hematopoietic progenitor cells. Primitive mesodermal or mesenchymal stem cells are therefore useful as a source for a number of cell and tissue types.
  • the tension device is used to generate a tubular tissue for use as a valved or an unvalved vascular conduit.
  • the scaffold material may be seeded with endothelial cells and smooth muscle cells. Additionally, or alternatively, the scaffold material may be seeded with MSCs. It has been shown that MSCs isolated from ovine bone marrow and seeded onto valved conduit scaffold materials are capable of differentiating into cell phenotypes reminiscent of native pulmonary valves (Sutherland, et al., Circulation, 111 :2783-91 (2005)).
  • suitable cells may be cultured under standard cell culture conditions to proliferate the cells to a suitable number for seeding of the scaffold.
  • the cells are cultured using standard methods and an appropriate media for the cell type being cultured. Appropriate media for a wide variety of cell types are known in the art.
  • the number of cells required to seed the scaffold will depend on many factors, including, but not limited to, the size of the scaffold to be seeded, the lineage and viability of the cells, and the degree of cell attachment to the scaffold.
  • the cells are placed into suspension in the media and added to the tension device /scaffold material assembly (70) inside the container.
  • the suspended cells are initially added to the center hole (26) in one end piece (20) of the tension device (10).
  • both the leaflets on the inside of the scaffold and the outside of the scaffold which forms the conduit wall are seeded.
  • the container holding the tension device /scaffold material assembly is then closed.
  • the container containing the tension device/scaffold material assembly is placed horizontally along its long axis.
  • the container may then be mildly agitated to achieve mixing of the media containing the cells. Mild agitation may be achieved using any suitable means, such as a rotary or orbital shaker on a low setting. If the container is a cylindrical bottle, then suitable agitation may be achieved by placing the container on a roller device.
  • the media is removed from the container, and new media is added.
  • the media that was removed from the container may optionally be centrifoged at an appropriate speed to pellet any cells that were removed.
  • the cells may be resuspended in fresh media and added back to the container.
  • the resuspended cells may be added in any combination to the center hole (26) and the additional holes (29) in the periphery of the end plate in the end piece that is proximal to the opening of the container, hi one embodiment, approximately equal volumes of the media containing resuspended cells are added to the center hole (26) and each of the additional holes (29) in the periphery of the end plate that is proximal to the opening of the container.
  • the container is then mildly agitated as before.
  • the media is replaced at regular intervals over a time period of a few days.
  • the rate of media change depends on a number of factors including, but not limited to the cell type, the number of cells and the media being used.
  • the media is changed at approximately every 8-12 hours.
  • any cells removed from the container may be pelleted, resuspended and added back to the container to optimize cell seeding.
  • the tension device with cell-seeded scaffold is removed from the container and placed into a larger container.
  • the larger container is filled with media and is mildly agitated as before.
  • the larger container allows for less frequent media changes than when the smaller container was used.
  • the cell-seeded scaffold remains in culture for sufficient amount of days to allow the cells to proliferate and form tissue on the scaffold material.
  • the culture time will vary depending on a number of factors, but must be long enough to allow the tissue to develop to a point where it will be functional for its intended in vivo application.
  • the cell- seeded scaffold is used to generate a valved conduit for use as a pulmonary valve replacement, In this embodiment, on average a total of 32 days is required to allow for sufficient deposition of extracellular matrix (ECM) on the scaffold for the valve to function properly under pulmonary pressure. If the scaffold is less porous, a shorter culture time period, such 1-2 weeks of culture time would allow for sufficient deposition of extracellular matrix (ECM) on the scaffold.
  • the scaffold is removed from the tension device for implantation.
  • the scaffold is removed by carefully removing the ends (62 and 64) of the cell-seeded scaffold material containing ECM from the mounting ends (54) and removing the attachments to the one or more hoops (40).
  • the cell-seeded scaffold material containing ECM may then be trimmed as necessary to fit into its intended implantation site.
  • the mounting portions (50a and 50b) are attached to the end pieces by connectors (19), creating a modular design that allows for the cell-seeded scaffold material to be removed from the tension device and subsequently placed into another mechanical environment.
  • scaffold materials containing developing tissue may be removed from the tension device after cell seeding and initial tissue growth, and then placed inside a bioreactor device where additional mechanical forces may be applied to the tissue.
  • a developing vascular tissue or heart valve may be removed from the tension device and placed into a pulsatile flow loop bioreactor by connecting the mounting portions to the bioreactor.
  • Pulsatile flow loop bioreactors have been designed to develop optimized mechanical conditioning protocols for TEHVs to enhance the in vitro stage of tissue formation (Hildebrand, et al., Ann. Biomed Eng., 32(8): 1039-49 (2004)); Hoerstrup, et al., Circulation, 102:11144-9 (2000)).
  • the tension device (10) is designed for the formation and growth of a variety of tissues, including any substantially tube-shaped tissue, such as vascular grafts including, but not limited to, blood vessels and heart valves inside a blood vessel.
  • the tension device may also be used to generate substantially linear or planar tissue, such as skin, tendons and ligaments.
  • the disclosed tension devices are used to prepare valved conduits for use as heart valve grafts.
  • Heart valve grafts may be used for pulmonary valve (PV) and/or right ventricular outflow tract replacement.
  • Other uses for valved conduits include, but are not limited to, replacement of the aortic, mitral, or tricuspid valves, venous valves, or the pyloric sphincter.
  • PV pulmonary valve
  • RV right ventricular outflow tract
  • the disclosed tension devices may also be used to generate other non- valved conduits for use as vascular grafts.
  • Vascular grafts may be used as venous, arterial or artero- venous conduits for any vascular or cardiovascular surgical application. Exemplary applications include, but are not limited to, congenital heart surgery, coronary artery bypass surgery, peripheral vascular surgery and angioaccess.
  • Vascular bypass grafting is most commonly performed for the treatment of vessel stenosis. However, vascular grafts are also used for the treatment of other conditions, such as arterial aneurysm or chronic renal failure (as access for hemodialysis). Vascular grafting can be performed by conventional surgery or endovascular techniques.
  • Coronary artery bypass grafting is one example of vascular bypass surgery.
  • a bypass graft is used to bypass the coronary artery distal to the site of stenosis or occlusion.
  • a vein graft When a vein graft is used, one end is anastomosed to the aorta and the other end is anastomosed to the coronary artery beyond the stenosis or occlusion.
  • an arterial graft is used, the proximal end is left undisturbed (thus preserving the artery's normal blood inflow), and the distal end is anastomosed to the coronary artery beyond the stenosis or occlusion.
  • an anastomosis i.e., the surgical union of tubular parts
  • suture materials include proline (extruded polypropyline) and ePTFE.
  • MSCs Isolation and culture ofmesencyhmal stem cells
  • MSCs were obtained from ovine bone marrow using the method of Pittenger, et al. (Pittenger, et al., Science, 284:143-147 (1999)).
  • PGA Polyglycolic acid
  • PLLA poly-L-lactic acid
  • Biodegradable heart valve scaffolds were assembled using 1-mrn PGA/PLLA nonwoven mesh and mounted on 50-mm sections of 3/4-inch PFA tube (Cole Palmer Instrument Co). Each scaffold was inverted inside a 140-mL glass hybridization tube and sterilized by exposure to ethylene oxide. Approximately 1 billion cells were resuspended in 25 mL culture medium and transferred to the hybridization tube. Tubes were sealed and rotated at 4 cycles per minute and 37° C. At 6 hours, the medium was aspirated and centrifuged at 1000 rpm for 5 minutes, and the cells were resuspended in fresh culture medium.
  • the cell-seeded scaffold was transferred to a 1750-cm 2 roller bottle and incubated in culture medium as described above, along with 20 ⁇ g/mL basic fibroblast growth factor and 0.4 mg/L L-ascorbic acid 2-phosphate (Sigma- Aldrich). Medium and CO 2 atmosphere were changed at 48-hour intervals, and culture was continued for 4 weeks.
  • a preliminary assessment of the tissue-engineered valves could be made by visual inspection of the surface of the graft through the roller bottle and by gently inverting the bottle so that fluid filled the outflow section of the conduit.
  • Biodegradable heart valve scaffolds were assembled using 1-mm PGA/PLLA nonwoven mesh as described above and were mounted onto the mounting portions of a tension device to apply strain in the longitudinal direction. For multi-axial strain, the scaffolds were further attached to the hoops of the tension device by suturing. The scaffolds attached to the tension devices were added to glass hybridization tubes and sterilized by exposure to ethylene oxide. Approximately 1 billion cells were resuspended in 25 mL culture medium and transferred to the hybridization tube. Tubes were sealed and rotated at 4 cycles per minute and 37° C. At 6 hours, the medium was aspirated and centrifuged at 1000 rpm for 5 minutes, and the cells were resuspended in fresh culture medium.
  • the cell- seeded scaffold was transferred to a 1750-cm 2 roller bottle and incubated in culture medium as described above, along with 20 ⁇ g/mL basic fibroblast growth factor and 0.4 mg/L L-ascorbic acid 2- ⁇ hos ⁇ hate (Sigma-Aldrich). Medium and CO 2 atmosphere were changed at 48-hour intervals, and culture was continued for 4 weeks. Results:
  • Cell-seeded scaffolds shaped for use as valved conduits were generated either using no strain, or under uniaxial or multi-axial strain.
  • the cell-seeded scaffolds were removed from the tension devices and cultured in vitro for an additional 32 days. The change in the dimensions of the valved conduits was measured.
  • the tissue that developed without any constraints showed the greatest change in length and width, with a change of about -75% in length and a change of about 15% in width.
  • the tissue developed in a device with tension in only one-direction, i.e. axial strain, showed a change of about -10% in length and -25% in width.
  • the tissue developed in a tension device applying tension mult ⁇ -axiaUy, as described herein, showed a change of about -17% in length and a change of about -5% in width.
  • Example 2 Surgical application of tissue engineered heart valves prepared using static tension.
  • Materials and Methods Animals Female sheep (Ovis aries, Dorset subspecies) were obtained at four weeks of age, weighing approximately 1 Okg. Bone marrow aspiration was performed twice on each animal; at five and six weeks of age, 10ml/kg of bone marrow was harvested from the iliac crest under general anesthesia. Use of experimental sheep was approved by the Institutional Animal Care and Use Committee of Children's Hospital Boston. Animals were cared for by a veterinarian in accordance with the "Guide for the Care and Use of Laboratory Animals.”
  • Bone marrow was centrifuged on a Ficoll gradient (Sigma) and the mononuclear fraction was plated on uncoated plates in medium containing DMEM with high glucose, 10% fetal bovine serum (FBS), 10% autologous serum, 1OmM HEPES buffer and Ix antibiotic/antimycotic.
  • Mesenchymal stem cells (MSCs) were isolated by their avid adherence to tissue culture plates as previously reported (Sutherland, et al., Circulation, 111(21):2783- 91 (2005); Perry, et al., Ann. Thorac. Surg.
  • Sheets of non- woven scaffold containing 50% fibers of polyglycolic acid and 50% fibers of poly-L-lactic acid were assembled into a valved conduit by manual and machine needle punching, based upon normal dimensions of the ovine right ventricular outflow tract, as described above in Example 1 and in Sutherland, et al., Circulation, l l l(21):2783-91 (2005).
  • Custom tension device and scaffold preparation for seeding In order to immobilize the valved conduit and maintain its dimensions during culture, and to provide a homogeneous surface for cell seeding, a custom device was fabricated for valved conduits in 3D culture.
  • the valved conduit scaffold was mounted onto gaskets and tied into position with silk suture material, then sewn circumferentially to the tension device at approximately the level of the cusp base and cusp free edge.
  • the valved conduit was then placed inside a 150mm glass hybridization bottle and sterilized with ethylene oxide gas. Once sterile, scaffolds were pre-wet with 70% ethanol, washed three times with phosphate buffered saline and immersed in a solution of 90% FBS and 10% antibiotic/antimycotic while cells were prepared for seeding.
  • Valve culture Once sterile, scaffolds were pre-wet with 70% ethanol, washed three times with phosphate buffered saline and immersed in a solution of 90% FBS and 10% antibiotic/antimyco
  • MSCs were seeded onto the 80cm 2 scaffold at a density of 0.5- 1.7x10 cells/cm by adding a dense cell suspension to the bottle housing the scaffold.
  • Valves were seeded in 60ml of medium containing DMEM high glucose, 20% FBS, 10% autologous sheep serum, 2x antibiotic/antimycotic, 1 OmM HEPES buffer, 82ug/ml ascorbic acid-2- ⁇ hos ⁇ hate and 2ng/ml bFGF.
  • the scaffold was rotated at Irpm, and medium was changed every 12 hours. After 72 hours, the valve was removed from the glass bottle and placed in a 850cm 2 roller bottle containing 500ml of medium as above without sheep serum. The medium was changed every three days for one month.
  • Twenty-two autologous valves were prepared according to these methods, nineteen of which were implanted in vivo. The remaining three valves served as in vitro controls and were analyzed in parallel in order to characterize tissues at the time of implantation.
  • a fourth interspace thoracotomy was performed, and under cardiopulmonary bypass with a beating heart, the native pulmonary valve cusps and a 1 -2cm main pulmonary artery segment were excised.
  • the engineered valved conduit was removed from the tension device and interposed between the cut pulmonary artery segments.
  • Epicardial echocardiography was performed in order to evaluate valve function at implantation. Maximum tra ⁇ svalvar gradient was obtained by continuous wave spectral Doppler methods.
  • trivial long axis color jet width ⁇ 1mm
  • mild color jet width between 1 and 2mm
  • severe — color jet width >50% of conduit width and presence of retrograde diastolic flow in the main pulmonary artery After chest closure, the animal was transferred from the operating table to a recovery sling, recovered until hemodynamically stable, allowed to wake from anesthesia, and extubated. All tubes and drains were removed prior to return to the cage on the evening of operation.
  • Valve explant procedure At each experimental endpoint, under general anesthesia, epicardial echocardiography was performed for evaluation of valve function, using the same methods as those performed at valve implantation. Pressures were directly measured from positions proximal and distal to the graft to detect pressure gradients. The animal was exsanguinated via an aortic catheter, and the animal's lungs and heart were removed en bloc after cardiac arrest.

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Abstract

La présente invention concerne des dispositifs tendeurs multiaxiaux, des ensembles de matériaux pour dispositif tendeur/support et des procédés d'utilisation des dispositifs pour former des tissus. Le dispositif tendeur multiaxial contient deux pièces d'extrémité, au moins une tige longitudinale, et deux parties de montage, et de préférence une ou plusieurs boucles. De préférence, les pièces d'extrémité contiennent des connecteurs destinés à les connecter de manière amovible aux parties de montage. Un matériau biocompatible, bioérodable ou bioabsorbable de support peut être attaché aux parties de montage afin de former un ensemble de matériaux pour dispositif tendeur/support. Lors de l'utilisation, le dispositif tendeur applique une tension à un support ensemencé avec des cellules dans la direction longitudinale et de manière radiale en partant du centre vers l'extérieur du matériau de support. Les dispositifs tendeurs peuvent être utilisés pour former une variété de tissus différents, tels qu'un conduit vasculaire, un conduit vasculaire à valve, un tendon, un ligament, et la peau. Dans un mode de réalisation préféré, le support ensemencé de cellules est utilisé pour générer un conduit à valve destiné à être utilisé pour remplacer une valve pulmonaire.
PCT/US2010/033213 2009-05-08 2010-04-30 Appareil tendeur multiaxial et procédé de construction de tissus WO2010129422A1 (fr)

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DE102012013232A1 (de) * 2012-07-04 2014-05-08 Gaudlitz Gmbh Haltevorrichtung für ein Gewebeteil mit tubulärer Struktur
WO2022203513A1 (fr) * 2021-03-25 2022-09-29 Meatable B.V. Appareil et procédé de culture tissulaire

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DE102012013232A1 (de) * 2012-07-04 2014-05-08 Gaudlitz Gmbh Haltevorrichtung für ein Gewebeteil mit tubulärer Struktur
WO2022203513A1 (fr) * 2021-03-25 2022-09-29 Meatable B.V. Appareil et procédé de culture tissulaire
NL2027836B1 (en) * 2021-03-25 2022-10-10 Meatable B V Apparatus and process for culturing tissue

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