WO2020250121A1

WO2020250121A1 - Rotation supporting unit for biological samples / scaffold and mass transfer method using the same

Info

Publication number: WO2020250121A1
Application number: PCT/IB2020/055397
Authority: WO
Inventors: Diana Nadia Caterina MASSAI; Diego Gallo; Gianpaolo SERINO; Giovanni PUTAME; Dario CARBONARO; Stefano GABETTI; Cristina BIGNARDI; Clotilde CASTALDO; Franca DI MEGLIO; Veronica ROMANO; Immacolata BELVISO; Anna Maria SACCO; Stefania MONTAGNANI
Original assignee: Politecnico Di Torino; Università degli Studi di Napoli Federico II
Priority date: 2019-06-11
Filing date: 2020-06-09
Publication date: 2020-12-17
Also published as: EP3983522A1

Abstract

A method to promote the mass transport of a solution through a sample, for example a biological tissue to be decellularized, includes the step of rotating the sample through a support group inside the solution. A support assembly comprising angularly equally spaced pocket arms houses cassettes for histological inclusion, biological samples or scaffolds of different rigidity during rotation and has windows to facilitate the transport of the solution through the sample or the scaffold.

Description

ROTATION SUPPORTING UNIT FOR BIOUOGICAU SAMPUES / SCAFFOUD AND MASS TRANSFER METHOD USING THE SAME.

Description

Technical field

The present invention relates to the biological, pharmaceutical, medical and veterinary sectors, for example for research and application in the fields of regenerative medicine, biotechnology, pharmaceutical industry and in general of bioprocesses and concerns a rotation support assembly for embedding cassettes histological or biological samples or even scaffolds of different rigidity, said assembly to be inserted inside a container to be used preferably in biology and biomedical laboratories and a method exploiting said assembly for the convective transport of a solution or more generally for the transport mass through samples for the realization of processes such as decellularization, recellularization, functionalization or other treatments. State of the art

Shaking a solution inside a container is a method often used during laboratory procedures to mix reagents and/or to promote the transport of the solution through dipped samples or scaffolds for decellularization, cell seeding, cell culture and functionalization with molecules or medications. By scaffold is meant a porous three-dimensional support, made of biocompatible material, capable of promoting adhesion and cell proliferation until the formation of the new tissue.

By exploiting the convective motion, the stirring method guarantees a faster process and a better result than the static methods, for which longer treatments are necessary with, sometimes, ineffective results. To guarantee the convective motion of the solution, a laboratory stirrer is commonly used which, by applying a rotating magnetic field, causes the rotation of a magnetic bar immersed in the container and the consequent mixing of the solution.

This method is widely adopted because it is cheap and easy to put into practice, however, in the case of samples directly immersed in the solution, the agitation of the fluid inside the container could cause a random suspension of the samples and their damage due to high shear forces and/or trapping of the sample around the magnetic bar. To overcome this problem, the samples can be placed in inclusion cassettes, however the random suspension of the cassettes in the container can cause an irregular and non- homogeneous transport of the solution through the samples, leading to a process that is difficult to reproduce and or to inconsistent results.

In the case of the decellularization process, for example, a solution containing a detergent is used to act on the cell membranes, causing their breakage. To ensure complete decellularization of the biological sample, the solution must penetrate the entire depth of the sample, but in parallel to preserve the extracellular matrix it is necessary to limit the time of exposure of the sample to the detergent which also acts on the extracellular matrix. As an alternative to stirring, the perfusion method is used to force the passage of the solution through the sample to be treated. In detail, the sample is placed inside a sealed chamber connected to a perfusion circuit consisting of tubes, a pump and a reservoir containing the solution. Thanks to the pump, the solution is drawn from the reservoir and pumped into the sealed chamber and then through the sample, using the native vascular network in the case of organs. In order to preserve the integrity of the sample, in addition to the perfusion circuit, there must also be a control system to set, control and monitor the working conditions of the circuit, such as for example perfusion pressures. Despite the effectiveness demonstrated by perfusion, this method is more complicated, more expensive and requires more work spaces than agitation.

Therefore, in the state of the art, there is no known economic instrumentation and methodology, easy to use and capable of solving problems relating to agitation processes.

Scopes and summary of the invention

The purpose of the present invention is to provide a rotation support assembly for cassettes, samples, scaffolds for use in agitation-based processes. This support assembly allows to keep the samples immersed in a solution inside a container and, when said assembly is equipped with a magnetic bar and the container is placed on a magnetic stirrer, to expose the samples to convective transport making the transport of the solution more efficient and repeatable and protecting the samples from the fluid dynamic structures/turbulent motion that can be generated in the container and that could damage the samples.

These purposes are achieved by means of a rotation support assembly for cassettes, samples, scaffolds that keeps them immersed in a stable manner in a solution inside a container, so as to avoid floating in agitation processes. Once the position of the samples has been fixed and known during the rotation, it is possible to calculate and establish the pressure range applied to them, ensuring greater control and repeatability of the entire process.

Said assembly is preferably equipped with a magnetic bar and, according to an example, has a cross shape. Placed inside a container and above a magnetic stirrer, this assembly enables to expose the samples homogeneously as the solution passes through them, improving mass transport, protecting the samples and increasing the efficiency and reproducibility of the process. The structure is designed so that the samples can be separated from the seat, rotated 180° and reassembled in the same seat so that, on the whole process, both sides of the samples are exposed to the same fluid dynamic field, moreover each sample can be managed independently. The design, in particular the number of arms, can be adapted in relation to the number and size of the samples.

Brief description of the drawings

Further features and advantages of the invention will emerge from the description of a preferred but not exclusive embodiment of a rotation support assembly for histological inclusion cassettes or biological samples or scaffolds of different stiffness and a method for decellularization, recellularization, functionalization or other treatments, based on the convective transport of a solution through the samples, object of the present patent application, illustrated only by way of non-limitative example in the drawings where:

- Fig. 1 represents a perspective view from above of a rotation support assembly for cassettes or samples or scaffold;

- Fig. 2 is a side view of the support assembly of Fig. 1;

- Fig. 3 is a perspective view of the support assembly for cassettes or samples or scaffolds in which at least two pocket arms (of which only one is illustrated) are coupled to a base to house a cassette or directly a sample or a scaffold ;

- Fig. 4 is a side view of the support assembly of Fig. 3;

- Fig. 5 is a perspective view of the base of the support assembly;

- Fig. 6 is a side view of the base of Fig. 5;

- Fig. 7 is a perspective view of a pocket arm of the support assembly;

- Fig. 8 is a side view of the pocket arm of Fig. 7;

- Fig. 9 depicts the pressure isocurves (in Pascal) in a cross section of the support assembly; - Fig. 10 depicts the velocity isocurves (in m/s) in a cross section of the support assembly.

Detailed description of the invention

According to a preferred - but not limiting - embodiment, the present invention relates to the sector of research and application in the fields of regenerative medicine, biotechnology, pharmaceutical industry and in general of bioprocesses and concerns a rotation support assembly 1 for embedding cassettes histological or biological samples or scaffolds of different stiffness and a method using said assembly for decellularization, recellularization, functionalization or other treatments based on the convective transport of a solution through the samples, with the exclusion of tanning.

The assembly 1 of the present patent application is to be used in stirring processes and comprises:

- a revolving frame with at least two pocket arms 2 for housing cassettes or directly samples or scaffolds;

- a base surface 3 extending transversely to at least two opposing pocket arms 2 to stabilize the frame.

The cassettes are to be used if the sample to be processed is soft, while when said sample is rigid, such as for example a fragment of bone tissue, said cassettes may not be used.

Said base surface 3 of assembly 1 is preferably defined by:

- four branches 4 converging on

- a central body 5 extended along a rotation axis of the assembly and perpendicular to the support surface of the four branches 4.

In the upper part of each of the four branches 4 there is a longitudinal groove 6 and the central body 5 has in the upper part a recess 7 for each pocket arm 2. On the bottom of assembly 1, facing base surface 3, the cross groove (not shown) houses a magnetic bar (or other material capable of generating a torque induced by a rotating magnetic field) preferably in the shape of a cross and known in the art.

Pocket arms 2 are coupled to the respective branches 4 by means of the grooves 6 and are adapted to contain samples, scaffolds or cassettes (not shown) in pockets 8 which in turn carry a sample. Pockets 8 can also directly contain rigid samples, for example of bone tissue.

Each pocket 8 of pocket arm 2 is delimited by a abutment blocking in the radial direction for the cassettes or samples or scaffold and is characterized in the upper part by an opening or mouth 9 inside which the cassette or sample or scaffold is inserted slidingly or in a slid-like manner in the longitudinal direction. Alternatively, the pocket arm is closed both at the top and at the bottom and a ridge substantially parallel to the rotation axis has a door mobile between an open position that allows a cassette to be inserted radially and a closed position that defines the blocking abutment during rotation.

On the major faces of pocket arm 2, respective windows 10 expose the cassette and the sample included therein to a convective flow when the assembly is rotating. On the upper part of the thin sides of each pocket arm 2 two T-shaped elements 11 allow the relative pocket arm 2 to be connected bi-directionally to the central body 5 by means of recesses 7.

In general, said pocket arms 2 are:

- angularly equispaced and converging towards an axis of inertia of the frame coinciding with the rotation axis;

- detachable from the central body 5 and each arm 2 comprises a first and a second side spaced from each other by the larger windowed faces, both sides being configured to be releasably connected, for example via recesses 7 and T-elements 11, to the central body 5 thus allowing to mount each arm in a first position in which the first side is connected to the central body 5 and is proximal to the rotation axis and a second position in which the second side is connected to the central body 5 and it is proximal to the rotation axis. When each pocket arm 2 is disconnected from the central body 5 and undergoes a rotation around its vertical axis of 180° it must be inserted again in the same longitudinal groove 6 of the branch 4 so as to cause an area previously proximal to the central body 5 is arranged distally and vice versa. Therefore, after a cycle comprising two phases of equal duration and rotation speed and with the arms in the two configurations, the variability of the pressure conditions applied on each face of the box is reduced. In particular, as a consequence of the 180° revolution, the areas of the sample exposed at low speed of the solution will be exposed at high speed and vice versa, resulting in the same consequence as above in which the sample on both faces is subjected to the same convective flow.

Assembly 1 is adapted to be inserted in a container which is then placed on a rotating magnetic field stirrer.

Said assembly 1 is also made of plastic material such as for example polymethylmethacrylate (PMMA) or acrylonitrile butadiene styrene (ABS) and allows for example a quick and effective decellularization of samples with a 30-50% reduction in the treatment time compared to to a procedure based on samples included in cassettes and floating freely in solution.

Assembly 1 is preferably used according to the following steps:

- inclusion of samples inside the cassettes;

- insertion of the cassettes inside pocket arms 2;

- coupling of arms 2 to branches 4 of assembly 1 ;

- insertion of the magnetic bar on the bottom of assembly 1 ;

- positioning of assembly 1 inside a container; - filling the container with a solution;

- activation of the stirrer with a predetermined rotation speed of the magnetic field.

When the stirrer is on, the magnetic bar, which is placed inside the cross groove on base 3 of assembly 1, induces the continuous rotation of the support assembly and therefore of the samples dipped in the solution. During the agitation, group 1 self-centers. Rotation can be controlled e.g. via a control unit of the stirrer and be at constant or variable speed according to a predefined sequence of accelerations, decelerations and time intervals at constant speed.

This allows the samples to be exposed to the convective flow with calculable working parameters, for example through computational fluid dynamics. This improves the control of solution transport, leading to greater reliability and repeatability of the process result compared to a conventional method. By means of the T-shaped elements 11 , the pocket arms 2 can rotate 180° so that both windows 10 in which, with or without cassette, the sample is contained can be subject to the same convective flow even without inverting the rotation speed.

Furthermore, rotation and subsequent coupling in the same angular position allows radially internal areas to become radially external and vice versa. In this way, there is less difference between the local pressure conditions applied to the sample during the entire process including the 180° rotation of the pocket arms with respect to the relative angular position.

In addition, the sample included in the cassette is protected from the fluid dynamics / turbulent motion structures that can develop inside the container and which could damage the sample.

Differently from what is known in the state of the art, the following advantages are obtained with the present invention: - keep the samples immersed and expose them to the convective motion of the solution favoring mass transport through them;

- protect the samples from fluid dynamic structures / turbulent motion by including them in boxes;

- possibility to treat each sample easily and independently due to the modular structure and the separable pocket arms;

- possibility to change assembly 1 both in terms of size and number of samples;

- possibility to use assembly 1 quickly and easily;

- possibility of using assembly 1 together with embedding cassettes, magnetic stirrer, magnetic bar and containers commonly used in biological and biomedical laboratories;

- possibility of creating assembly 1 by 3D printing;

- greater reproducibility and efficiency of processes.

The agitation-based method for the convective transport of a solution through samples for the realization of processes such as, for example, decellularization for use in research, biological, medical, pharmaceutical or veterinary fields comprises the following steps:

- providing at least two pocket arms 2 for housing cassettes, samples or scaffolds and having larger faces with windows and arranged in an angularly equidistant way through a frame;

- housing in pocket 8 of each arm 2 a cassette or a sample to be decellularized or treated;

- immerse arms 2 in the treatment solution;

- rotate arms 2 around an axis of rotation, each arm 2 providing a stop to radially block the cassette or the sample in contrast to the centrifugal acceleration. Since pocket arms 2 comprise a first and a second radial flank spaced from each other by the major windows, in the method according to the invention the step of inverting the position of each pocket arm 2 to pass from a first position can also be included wherein the first flank is proximal to the rotation axis to a second position wherein the second flank is proximal to the rotation axis with reference to the same angular position on the central body 5.

When the support group 1 is moving, in Fig. 9 the computational fluid dynamics simulation shows that, for a rotation speed imposed on the magnetic stirrer equal to 150 revolutions per minute, the rotation of assembly 1 induces a pressure drop which promotes the transport of the solution through the sample by advection. Further in Fig. 10 the computational fluid dynamics simulation shows that, for a rotation speed imposed on the magnetic stirrer equal to 150 revolutions per minute, the solution speed increases with the radius and the samples are exposed to a convective flow that facilitates the transport of the solution through the samples.

The materials and dimensions of the invention as described above, illustrated in the accompanying drawings and later claimed, may be any according to requirements. Furthermore, all the details can be changes with other technically equivalent ones, without thereby departing from the protective scope of this patent application.

For example, it is possible to provide that, if it is necessary to intensify the production of treated samples, the rotating magnetic field stirrer with the relative magnet are replaced by a machine having a dedicated structure so as to bring the pocket arms into rotation inside its own container for the solution with a base surface.

Claims

1. Method for mass transport in a sample or scaffold for use in biological, medical, pharmaceutical or veterinary fields including the steps of:

- supplying at least two pocket arms (2) for housing cassettes or samples or scaffolds having windowed larger faces and arranged in an angularly equispaced manner via a frame;

- place a cassette or sample or scaffold to be treated in a pocket (8) of each arm (2);

- dip the arms (2) in a treatment solution;

- rotating the arms (2) around a rotation axis, each arm providing an abutment to block the cassette or the sample or the scaffold against the centrifugal acceleration.

2. Method according to claim 1, wherein each arm (2) comprises a first and a second radial side spaced apart from each other by the larger faces, and further comprising the step of rotating each arm (2) around its vertical axis by 180 ° to pass from a first position in which the first side is proximal to the axis of rotation to a second position in which the second side is proximal to the axis of rotation, with the angular position of the arm (2) being equal.

3. Method according to one of claims 1 or 2, comprising the steps of:

- associate to the pocket arms (2) a magnet or a ferromagnetic component via a frame;

- apply a rotating magnetic field interacting with the magnet to provide the step of rotating.

4. Rotation support assembly for cassettes or samples or scaffolds for mass transport for use in biological, medical, pharmaceutical or veterinary settings, including: - a rotating frame with at least two pocket arms (2) to house histological embedding cassettes or directly biological samples or scaffolds of different stiffness and having larger faces with windows to expose the sample or scaffold to the action of a solution in which the frame is dipped in use;

- a base surface (3) extending transversely to the arms 2 to stabilize the frame during rotation about an axis;

- the arms (2) being angularly equispaced and converging towards an axis of inertia of the frame coinciding with the axis of rotation, and each pocket (8) comprising:

- an abutment in the radial direction for the cassettes or samples inside the arms (2); and

- a slit inlet (9) to out the relative cassette or sample.

5. Support assembly according to claim 4, wherein the arms (2) can be disconnected from a central body (5) of the frame and each arm (2) comprises a first and a second radial side spaced apart from each other by the larger faces, both sides being configured to releasably connect to the central body (5) thus allowing each arm (2) to be mounted on the central body (5) in a first position in which the first side is connected to the central body (5) and is proximal to the axis of rotation and a second position in which the second side is connected to the central body (5) and is proximal to the axis of rotation, with the angular position of the arm (2) on the central body (5) being equal.

6. Support assembly according to one of claims 4 or 5, wherein the frame defines the base surface (3), comprises branches (4) for each arm (2) and each arm (2) is coupled in a relative groove (6) of the branches (4) when the relative first or second side are in a use position.

7. Support assembly according to any one of the preceding claims, defining a seat crossed by the axis of rotation and adapted to house a magnet so that the support assembly (1) can be dragged in rotation by a rotating magnetic field agitator.

8. Support assembly according to claim 7, in which the frame is made of plastic material.

9. Support assembly according to any one of claims 4 to 8, comprising the cassette or sample carried by the arms (2) for performing a treatment by means of a solution.