TISSUE MECHANICAL FRAGMENTATION DEVICE INTENDED FOR THE PREPARATION OF A COMPOSITION OF ISOLATED CELLS, AND CORRESPONDING METHOD

Abstract

A device for mechanical fragmentation of tissues facilitates preparation of the composition of isolated cells from a tissue sample, the preparation including mechanical fragmentation of the tissue, the device including a motorized support and a container, the motorized support including a motor having a rotor secured to a coupling member, the container internally including a rotary shaft with radial arms sweeping the inside of the container as the shaft rotates, the motorized support and the container including a complementary securing unit that allow the container to be immobilized on the motorized support, the coupling member and the rotary shaft being configured in such a way as to allow axial rotation of the rotary shaft when the rotor of the motor is turning. The radial arms are of two types: rigid vane-type arms, and flexible filamentary-type arms, notably made of nylon or of metal. A corresponding method is also disclosed.

Claims

1. A device for the mechanical fragmentation of tissues intended for the preparation of a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanically fragmenting the tissues in the device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container (1) as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein the radial arms (10, 12) are of two types: vane-type arms (10) and filamentary-type arms (12).

2. The device according to claim 1, wherein the vane-type arms (10) are rigid and the filamentary-type arms (12) are flexible.

3. The device according to claim 1, wherein the rotary shaft (9) is vertical, the container (1) is cylindrical or straight frustoconical in shape, with a lower bottom wall (8), an upper wall (5) and a cylindrical or frustoconical rising lateral wall (4), the rotary shaft (9) being central and coaxial to the lateral wall (4), and wherein the vane-type arms (10) are arranged in the lower portion of the rotary shaft (9), whereas the filamentary-type arms (12) are arranged above the vane-type arms (10).

4. The device according to claim 1, wherein each vane-type arm (10) has at least one bevelled longitudinal edge, said at least one bevelled edge being on the lateral side of the arm first sweeping the inside of the container as the rotary shaft (9) rotates.

5. The device according to claim 1, wherein each filamentary-type arm (12) is consisted of a nylon or metal wire.

6. The device according to claim 5, wherein the nylon or metal wires have a diameter comprised between 0.5 mm and 3 mm.

7. The device according to claim 3, wherein the cylindrical or frustoconical rising lateral wall (4) of the container (1) internally includes serrations.

8. The device according to claim 3, wherein the container (1) is impermeable to liquids and wherein the upper wall (5) includes two openings, the first opening (17) being closed by a filter (15), the second opening (20) being closed by a self-sealing septum which can be pierced by a piercing tubing and which seals automatically upon removal of the piercing tubing in such a way to be totally impermeable.

9. The device according to claim 8, wherein, as an alternative, the self-sealing septum is a self-closing valve (16) or flap.

10. The device according to claim 2, wherein each filamentary-type arm (12) is consisted of a nylon or metal wire.

11. The device according to claim 10, wherein the nylon or metal wires have a diameter comprised between 0.5 mm and 3 mm.

12. The device according to claim 2, wherein the rotary shaft (9) is vertical, the container (1) is cylindrical or straight frustoconical in shape, with a lower bottom wall (8), an upper wall (5) and a cylindrical or frustoconical rising lateral wall (4), the rotary shaft (9) being central and coaxial to the lateral wall (4), and wherein the vane-type arms (10) are arranged in the lower portion of the rotary shaft (9), whereas the filamentary-type arms (12) are arranged above the vane-type arms (10).

13. The device according to claim 12, wherein each filamentary-type arm (12) is consisted of a nylon or metal wire.

14. The device according to claim 13, wherein the nylon or metal wires have a diameter comprised between 0.5 mm and 3 mm.

15. A method for preparing a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanical fragmentation of the tissues in a device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein a device according to claim 1 is implemented for the fragmentation of the tissues, and the sampled tissue is introduced into the container (1), the motor is powered on, the container (1) having previously been fastened to the motorized support (3) so that the coupling member can rotate the rotary shaft (9) then, after the motor has been stopped, at least one portion of the container (1) content is collected.

16. A method for preparing a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanical fragmentation of the tissues in a device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein a device according to claim 2 is implemented for the fragmentation of the tissues, and the sampled tissue is introduced into the container (1), the motor is powered on, the container (1) having previously been fastened to the motorized support (3) so that the coupling member can rotate the rotary shaft (9) then, after the motor has been stopped, at least one portion of the container (1) content is collected.

17. A method for preparing a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanical fragmentation of the tissues in a device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein a device according to claim 3 is implemented for the fragmentation of the tissues, and the sampled tissue is introduced into the container (1), the motor is powered on, the container (1) having previously been fastened to the motorized support (3) so that the coupling member can rotate the rotary shaft (9) then, after the motor has been stopped, at least one portion of the container (1) content is collected.

18. A method for preparing a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanical fragmentation of the tissues in a device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein a device according to claim 4 is implemented for the fragmentation of the tissues, and the sampled tissue is introduced into the container (1), the motor is powered on, the container (1) having previously been fastened to the motorized support (3) so that the coupling member can rotate the rotary shaft (9) then, after the motor has been stopped, at least one portion of the container (1) content is collected.

19. A method for preparing a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanical fragmentation of the tissues in a device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein a device according to claim 5 is implemented for the fragmentation of the tissues, and the sampled tissue is introduced into the container (1), the motor is powered on, the container (1) having previously been fastened to the motorized support (3) so that the coupling member can rotate the rotary shaft (9) then, after the motor has been stopped, at least one portion of the container (1) content is collected.

20. A method for preparing a composition of isolated cells, in particular adipocytes and/or cells contained in the adipose tissue, from a tissue sample, obtained in particular during a liposuction, said preparation including at least one step of mechanical fragmentation of the tissues in a device, said device comprising a motorized support (3) and a container (1), the motorized support (3) comprising a motor having a rotor integral with a coupling member, the container (1) internally including an axially rotatable rotary shaft (9) and including radial arms (10, 12) capable of sweeping the inside of the container as the rotary shaft (9) rotates, the motorized support (3) and the container (1) including complementary detachable fastening means (11) for immobilizing the container (1) on the motorized support (3), the coupling member and the rotary shaft (9) being configured in such a way as to allow axial rotation of the rotary shaft (9) as the motor rotor rotates, wherein a device according to claim 6 is implemented for the fragmentation of the tissues, and the sampled tissue is introduced into the container (1), the motor is powered on, the container (1) having previously been fastened to the motorized support (3) so that the coupling member can rotate the rotary shaft (9) then, after the motor has been stopped, at least one portion of the container (1) content is collected.

Description

DETAILED DESCRIPTION OF AN EXEMPLARY EMBODIMENT

[0110] The following description with respect to the appended drawings, given by way of non-limitative examples, will permit a good understanding of what the invention consists in and of how it can be implemented.

[0111] In the appended drawings:

[0112] FIG. 1 shows an axial vertical cross-section, passing through the rotary shaft, of the container of the invention,

[0113] FIG. 2 shows a top view of the container of FIG. 1,

[0114] FIG. 3 shows a radial cross-section of the container of FIG. 1, and

[0115] FIG. 4 schematizes the different steps of preparation of composition of isolated cells using the device of the invention.

DEVICE

[0116] Within the framework of the solution of mechanical processing of the sampled tissue implemented in the invention, the separation of the cells from the tissue is based on attrition and shearing forces undergone by the tissue in a mixer.

[0117] Preferentially, this mixer is intended for crushing the adipose tissue and for obtaining cells composing the adipose tissue, mainly the cells of the SVF (Stromal Vascular Fraction) but it may also be used to purify cells contained in other tissues or organs.

[0118] The mixer of the invention mainly includes two components: a support including an electric motor, called motorized support, and a container capable adaptable on the motorized support. The motorized support includes a motor making it possible, when the container is fastened to the motorized support, to rotate a rotary shaft internal to the container, said rotary shaft including radial arms. The container is detachably fastened to the motorized support and a coupling means, which is disengageable from the rotational driving, is installed between the motorized support and the rotary shaft of the container.

[0119] The speed of rotation of the rotary shaft is preferentially of about 4000 rpm (rotation per minute), which provides an optimum yield of cell purification in terms of number of cells and of viability. In variants, a speed of rotation comprised between 2000 and 8000 rpm is provided, and the mixer may include means for adjusting the speed of rotation of the motor rotor and hence of the rotary shaft.

[0120] The rotary shaft, which is central/coaxial to the container, herein substantially frustoconical in shape, is provided with radial vanes at the base thereof, for a convection of the adipose tissue. Higher, along the rotative shaft, nylon or metal wires are fastened perpendicularly/radially to the rotary shaft in order to ensure efficient cutting and splitting of the tissue. The diameter of the nylon or metal wires is comprised between 0.5 mm and 3 mm. The number of nylon or metal wires is comprised between 3 and 15.

[0121] In order to optimize the cutting of the tissue by the nylon or metal wires, serrations may be inserted on the inner face of the container. The number thereof may be comprised between 2 and 12 and the thickness thereof may be comprised between 1 mm and 6 mm. The presence of the serrations makes it possible to create a turbulent flow of tissues allowing an optimal mixture. These serrations are nevertheless optional in the sense that it is possible to obtain cells without them, but their presence significantly increases the cell preparation yield.

[0122] The container has, in its upper part, a lid that includes two devices making it possible to reduce the contamination of the tissue during the implementation of the invention. The first device is a vent through the lid, provided with a filter of 0.22 μm, allowing exchanges of air devoid of infectious or inert particles. The second device is a valve or a septum allowing the passage of a cannula without passage of external air concomitantly with the passage of the cannula. Preferably, this valve or this septum let a 3-mm cannula, typically a liposuction cannula, pass through. This usable cannula diameter may however be from 1 mm to 5 mm. This valve or this septum allows the adipose tissue coming from the liposuction to enter directly the container for being crushed and makes it possible to sample the homogenate after mixing in the mixer.

[0123] Hence, preferably, the container is closed by a septum that is a silicone valve, accepting the passage of a hollow cannula, of diameter 1 to 5 mm, and that closes automatically upon removal of said cannula. This silicone valve may be completed by an external/top plug, to avoid a prolonged direct contact of the valve with the external air. This non-pierced silicone valve is impermeable, in particular in the outward direction from the container, to liquids, particles and cells. The silicone valve and the plug are impermeable to gas+liquid+particles+cells in the two directions.

[0124] In FIGS. 1 to 3 is shown an example of container 1 of the invention of vertical use and driving of its rotary shaft by the bottom. This container is herein consisted of a frustoconical single-piece bowl with a bottom wall 8 extended upward by a frustoconical rising lateral wall 4. The inner face of the lateral wall 4 of the container 1 includes serrations 19. The rotary shaft 9 is arranged centrally and coaxial to the lateral wall 4 and is herein formed by the assembly of several parts. The rotary shaft 9 passes through the bottom wall 8 in a sealed manner and includes, at its lower end, a means 7 for its fast coupling to a complementary coupling member (not shown) of a support 3 (see FIG. 4) including an electric motor and on which the container may be detachably fastened.

[0125] The motorized support 3 (see FIG. 4) includes means 13, 14 for the adjustment of its operating parameters, in particular on/off, duration and/or speed of rotation. To allow this detachable fastening, fastening means 11 that are complementary of those of the motorized support are implemented and, in the present case, with a quarter-turn screwing. The seal of the passage of the rotary shaft 9 through the bottom wall 8 is obtained by implementation of an O-ring seal 2.

[0126] The frustoconical bowl with a narrow lower base and a wide top is closed at its top by an upper wall 5 forming a lid. This lid 5 is in this example clipped and is not intended to be removed. It may even be welded. In a variant embodiment, this lid is removable. The lid 5 includes two through-openings. The first opening 17 is closed by a filter of 0.22 μm and a grating 18. The second opening 20 is closed by a self-closing valve 16 of 3-mm diameter and that ensures a total sealing when closed. This flap 16 gives way when a cannula is introduced into the second opening 20. In the case of a self-sealing septum of the silicone valve type, the latter closes after removal of the cannula. The second opening 20 is externally covered by a removable cap.

[0127] The rotary shaft 9 includes radial arms 10, 12 of two types. Towards the bottom of the rotary shaft are arranged rigid vane-type arms 10 and, above these latter, flexible or soft filamentary-type arm 12 made of nylon or metal. The vane-type arms 10 may include ridges 6 extending in a vertical plane substantially perpendicular to the radial extension of the arm 10. The radial arms, in particular the filamentary arms 12, are staggered over the height of the rotary shaft 9 and angularly distributed all over the latter.

Method

[0128] The steps of obtaining the desired cell composition are schematized in FIG. 4.

[0129] Beforehand, at step A, a sampling of adipose tissue has been performed on a patient 21 by a suction syringe 22 or an automatic vacuum-sucking device. At step B, the adipose tissue is introduced into the container 1 by passage of the cannula of the syringe 22 through the second opening 20. During the injection of the tissue into the container, the air that is over-pressurized inside the container 1 exhausts through the first opening 17 by passing through the filter 15. At step C, the syringe 22 is removed from the container 1. At step D, the container 1 is fastened to the motorized support 3 and the motor of the latter is activated to rotate the rotary shaft 9 of the container 1, which destructs the tissue and releases the cells. The operating parameters are adjusted by the adjustment means 13, 14 of the motorized support. At step E, the cells and other elements of the tissue that has been broken up are collected into a syringe cannula 23 introduced through the second opening 20. At step F, during the sucking by means of the syringe 23, external air may enter the container through the first opening 17 by passing through the filter 15. At step G, a centrifugation is performed in order to select the desired cells for a reinjection at step H.

[0130] Based on a sampled adipose tissue, the mixer of the present invention allows obtaining a cellular composition called SVF (Stromal Vascular Fraction) close to that obtained by the protocol using collagenase.

[0131] Typically, from the quantitative point of view, based on 25 ml of adipose tissue, about 200 000 of viable cells are obtained. From the qualitative point of view, the use of the mixer of the invention makes it possible to obtain about 17% of cells having a phenotype CD34+, CD31−, CD45− with respect to the total number of purified cells gathering the cells considered as progenitor cells (Mesenchymal Stem Cells) as well as the pericytes. Such a ratio is very close of that obtained with the purification protocol based on the use of the collagenase (19%).