RU2785112C2 - Automated method for obtaining human or animal tissues for transplantations - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to the field of obtaining human or animal tissues in order to use them for transplantations, in particular to a method for obtaining a tissue matrix for allo- or xenotransplantations. For implementation of the specified method, after sampling of biological tissue, tissue is prepared for administration in a sterile medium. Then, the specified tissue is treated in such a way that to obtain a tissue matrix. After that, the obtained tissue matrix is packed. The specified stages are carried out in an automated way in a sterile reactor. At the same time, the reactor for implementation of the specified method contains a sluice chamber for administration and preparation of biological tissue to its administration to the sterile medium, means for treatment of biological tissue for obtaining a tissue matrix, means for packing the obtained tissue matrix, an output sluice chamber for the packed tissue matrix, and robotic means for movement of biological tissue from the input sluice chamber to means for treatment of biological tissue, and robotic means for movement of the tissue matrix from treatment means to package means and further to the output sluice chamber.

EFFECT: present invention allows for provision of traceability of batches in the process of obtaining tissue matrices.

11 cl, 3 dwg, 1 ex

Description

The present invention relates to the field of obtaining human or animal tissues for use in transplantation (allotransplantation or xenotransplantation).

Allotransplantation involves the presence of a donor and a human recipient. Autotransplantation involves only one human being.

Xenotransplantation involves the presence of an animal as a donor and a human as a recipient.

The increasing number of human musculoskeletal disorders makes it necessary to treat by bone reconstruction or replacement, both in the field of dentistry (maxillofacial restoration with a dental implant) and oncology (due to the removal of bone tumors) or orthopedic surgery (due to a fracture or to connect the vertebrae ).

Currently, bone restoration is performed through autografting, usually by puncturing the bone at the level of the iliac crest of the patient in need of treatment. However, such a puncture, in addition to being painful, is not reversible. Indeed, the pelvic bone does not regenerate and therefore the amount of available bone tissue is limited. In addition, autotransplantation requires a double surgical intervention for the same individual, which also has a significant impact on postoperative recovery.

Another approach is to use bone substitutes that are widely available and inexpensive, such as ceramic, coral or glass prostheses. Unfortunately, due to the lack of market orderliness, numerous problems regarding durability, material compatibility, even toxicity, underlie many postoperative difficulties, and medical professionals increasingly distrust such substitutes.

Allotransplantation is also applied. In practice, bone tissue is taken from the individual, for example, the head of the femur, removed during the installation of a hip prosthesis. This tissue contains the bone matrix and donor cells. To eliminate the risk of rejection or inflammatory reaction during transplantation, it is necessary to process the tissue to clear it of any "genetic" traces of the donor, i.e. remove traces of cells, blood or fat. A method to purify or "cell-remove" bone tissue to obtain cell-free bone matrix is described in Dufrane et al. Biomaterials. 2002 Jul.; 23(14):2979-88 and in Dufran et al. Eur Cell Mater. 2001 Jan 10; 1:52-8; discussion 58.

The main steps of this method are:

- centrifugation of tissue taken from a donor in order to separate blood and fat;

- cutting the fabric, as a rule, into cubes of different sizes;

- chemical treatment to remove traces of cells and deactivate viruses and/or bacteria;

- lyophilization to obtain a stable demineralized bone matrix;

- package.

Although allografting has been the most reliable method of bone regeneration to date, patient access to this technique is severely limited by the method and cost of producing a bone matrix with cells removed.

Indeed, at present, all these steps are performed manually by hospital staff in a bright, sterile, so-called "classified" room, i.e. created in accordance with a number of standards and subject to strict control. Each of these steps should be carried out primarily in different classified sterile chambers or isolators within the production area, with each isolator containing adequate equipment for each step and maintained under sterile conditions. This results in a large dead space for the movement of personnel between each isolator. In addition, special precautions must be taken when moving batches of product from one isolator to another.

It is then necessary to ensure that the batches are traceable to the origin of the fabric. This does not exclude erroneous actions on the part of personnel, which can lead to the withdrawal of batches.

In addition, the financial aspect cannot be ignored. Indeed, the production of a batch takes several weeks, and this period may be affected by the presence of hospital staff, who must provide basic care. The need for working time and the special space mentioned above leads to significant costs for the medical institution. For these reasons, many medical institutions do not invest in the production of cell-removed bone matrices. Therefore, access to such a bone source for allograft purposes is currently very limited.

Summarizing the above, it should be noted that the existing problems are due to the fact that:

- production is manual, which includes:

- classification at different stages;

- subsequent need for a working space;

- availability of storage space;

- difficulties with regard to traceability;

- the actions of operators can be a source of errors and adversely affect the quality of the product;

- the duration of the production process generates too high costs.

Relative classification should be understood in this case as keeping the biological tissue under sterile conditions, not only at each stage, but also between stages, during the probable physical movement of the tissue between two sterile zones. These production problems are not limited to bone tissue, they also apply to other biological tissues used in allotransplantation, such as skin, tendons, bladder, etc., or adipose tissue. The same problems arise in the production of therapeutic cellular agents. In order to simplify the access of patients to the mentioned source of biological tissue for allotransplantation, the applicant has created a method that solves the above problems.

Solution according to the invention

Therefore, the present invention proposes a method for obtaining a tissue matrix for allotransplantation or xenotransplantation, in which, after the selection of biological tissue:

- this fabric is classified;

- it is processed;

- packaging the obtained tissue matrix;

characterized in that these stages are carried out by automated reception in the same "classified" reactor.

To solve a set of problems, the applicant boldly selected a problem from it, in this case, a problem that is a priori the most difficult to solve, and trying to exclude the classification as much as possible, achieved the solution of all other difficulties. Therefore, the present application relates to a problematic invention.

The term "classify" the tissue should be understood as its preparation for use in the method under sterile conditions that meet strict standards. Also, a “classified” reactor is a reactor that complies with the same standards.

Packaging refers to any type of packaging, for example, placing the tissue matrix in a vacuum-sealed plastic bag or placing the tissue matrix inside the syringe in a usable form, while the syringe itself is packaged sterile in plastic packaging.

For certain types of biological tissue, the method of the invention may include the step of cutting or shredding the tissue. In addition, the method may include a drying or freeze-drying step prior to packaging.

Fabric processing may include one or more of the following steps:

- tissue centrifugation,

- chemical processing,

- biological treatment.

Chemical treatment involves any effect on biological tissue by chemical agents or reagents, for example, shaking the tissue in a solution of an antibacterial agent or in a solution whose pH is set by an acid or base by adding ionic compounds. Biological tissue can be subjected to several sequential chemical treatments with different end goals, such as defatting or inactivating bacteria or viruses or prions, or demineralization to improve osteoconduction.

Biological treatment uses biological agents, such as growth factors or proteins, to induce cell differentiation in the respective tissue.

The invention also relates to a classified reactor for carrying out the process according to the invention, comprising:

- lock chamber for the introduction and classification of biological tissue,

- means of processing biological tissue, intended to obtain a tissue matrix,

- means of packaging the fabric matrix produced,

- lock chamber for the release of the packed tissue matrix,

- robotic means for moving biological tissue from the lock chamber for introduction and classification to means for processing biological tissue,

- robotic means for moving the tissue matrix from the processing means to the packaging means, then from the packaging means to the sluice outlet chamber.

The sluice chamber for the introduction and classification of biological tissue, as well as the sluice chamber for the release of the packed tissue matrix, are designed to maintain sterile conditions inside the reactor that correspond to the standards required for the method. Therefore, tissue classification is carried out only in the area of the inlet airlock. The outlet lock is also quite obviously provided to maintain sterile conditions during the release of the product. Thus classified reactor can be made compact, without allowing, of course, the access of the individual inside. Thus, all steps of the method according to the invention can be carried out in an optimal sequence.

The invention will be better understood from the following description of several applications of the invention with reference to the attached drawing, which shows:

fig. 1 is a schematic representation of the method according to the invention;

fig. 2 is a schematic representation of a reactor for carrying out the invention;

fig. 3 is a perspective view of the interior of a reactor according to the invention.

Referring to FIG. 1, it can be seen that the biological tissue 1 taken from the donor is introduced into the reactor 2 through the inlet lock chamber 3. The tissue passes through several stages: centrifugation 4, cutting 5, chemical processing 6, lyophilization 7 and packaging 8. Then the resulting packaged tissue matrix 10 is removed from the reactor 2 through the outlet lock chamber 9.

The tissue 1 can be, for example, the femoral head taken from a donor when applying a hip prosthesis. In order for such a raw femoral head to be used to prepare a bone matrix for use in bone reconstruction in another patient, it must undergo several stages of transformation under controlled sterile conditions. Therefore, the femoral head is first introduced into the so-called classified reactor 2, in that part of it where sterile conditions are provided that correspond to the required standards for such transformations.

The introduction of the femoral head 1 takes place through an inlet sluice 3, which is configured such that no contamination from outside can enter the reactor 2. This sluice 3 can be provided, for example, with a double door system, each door being opened in turn, and conventional laminar air flow directed outward from the lock chamber. For internal insertion, the femoral head is placed in a special container, which facilitates movement during the steps of the method. Thus, the lock chamber 3 allows the introduction and classification of biological tissue, in this case the femoral head 1 is placed in the reactor 2.

As can be seen from FIG. 2, an automaton 13 or a number of interacting automata 13 is installed inside the reactor 2, containing various functional units and designed to carry out the steps of the method. Preferably, the machine 13 is controlled by an information system 14, into which, if necessary, the operator enters commands through a control station 15, in this case a computer. The reactor can have different shapes and sizes. For example, it can be made of metal in the form of a container for transportation by land or sea, or contain partially glazed walls. The only limitation for such a reactor is the need to provide and maintain sterile conditions inside that meet the necessary standards when carrying out the manufacturing method. These requirements and regulations may vary by geographic area. Such a reactor can be supplemented with some elements to ensure sterility, for example, devices for ventilation, disinfection or air conditioning. In this case, the machine 13 ensures that the femoral head 1, optionally together with its container, is introduced into the centrifuge 16 where it is subjected to centrifugation to separate the bone matrix from unwanted residues 11 such as brain, blood or fat residues. The femoral head 1 thus cleaned is then fed to the cutting device 17, which is configured to obtain bone pieces of a predetermined size. After that, these bone pieces are introduced into the chamber with the bath 18 for chemical treatment, in which they are mixed for a certain predetermined time. The treatment bath can be, for example, a disinfectant solution for inactivating bacteria, or any other bath appropriate to the type of tissue to be treated.

The chemical treatment chamber 18 can be designed in such a way that at the end of the treatment, the spent chemical solution 12 can be discharged into a suitable container 19 in communication with the reactor 2. For example, it can be provided that the container is located outside and is decoupled from the reactor by means of valves to replace it. when filled without disturbing the sterile conditions in the reactor. Removal of residues after centrifugation is possible in the same way. It is also possible to connect clean solution containers that serve to feed the chemical baths. The contents of these containers are easily traceable by the information system 14, for example by means of a bar code.

The bone pieces may be subjected to several consecutive chemical treatments in the same processing chamber or in chambers separate from the interior of reactor 2. This treatment may be supplemented by washing steps. The pieces of the cell-free bone matrix thus treated are then sublimated in the sublimation unit 20, i. e. dried until their moisture content is, for example, less than 5%, then packed in a vacuum or in a protective gas atmosphere to obtain a packaging unit 21. This achieves optimal stability of the bone matrix 10, allowing it to be stored at room temperature for several months. It is possible to provide for the labeling of packages to ensure the traceability of the produced batches. The packed tissue matrix 10 is withdrawn from the reactor 2 through an outlet lock 9 which is similar to the inlet lock 3. It is also possible to design the reactor 2 so that it has only one lock for inlet and outlet.

The control or regulation station 15 contains an information system 14 that contains information about the method of production of the packaged fabric 10. The information system 14 controls the machine 14 in the reactor 2. Some parameters may need to be entered by the operator, such as the type of fabric, in the case when the machine can control the production of several types of fabric, the duration of chemical treatment or the required percentage of moisture in the packaged fabric matrix 10.

Therefore, the manufacturing method described above makes it possible to introduce "raw" biological tissue into the reactor and obtain a decontaminated biological tissue suitable for use in transplantation. This method includes only one classification step when the fabric 1 is introduced into the reactor 2. The continuity of the classification is ensured by the limited and controlled space of the reactor, from which the fabric does not exit until the fabrication is completed. Since no operator can interfere with the flow of the process, erroneous actions are excluded, traceability is guaranteed, the need for space and manipulation is reduced to a strict minimum. This results in optimal production costs.

Another advantage of the method according to the invention lies in the possibility of continuous production in "small" volumes. Usually, when production is done manually in a medical center (through a tissue bank), hospital staff have to wait for a sufficient amount of source material, several dozen femoral heads, for example, 48, before proceeding with production, and this is for the purpose of optimization. costs. The method according to the invention makes it possible to start production with only one femoral head and ensure continuous production or every time a femoral head is present.

The reactor 2 can preferably be configured to produce several batches of tissue matrix simultaneously, in parallel or in an unconventional manner. Those. the two raw biological tissues may be introduced and treated at the same time, or the second biological tissue may be introduced at a time when the first tissue is already being processed. Batch traceability of the tissue matrix is ensured for each biological tissue.

For a specialist it is obvious that tissue 1 can also be obtained from an animal donor. The processing of the femoral heads has been described here, however, any other bone tissue, skin, or any other tissue suitable from the point of view of a specialist can be processed. The method according to the invention is not limited to the manufacture of tissue matrices. For methods of cell therapy or biotherapy, the method according to the invention can also be used. These methods of therapy are a type of autotransplantation, which is often used in oncology, in which individual differentiated cells are produced on the basis of cell strains taken from an individual for re-implantation in the organ to be treated of this individual. The preparation for such therapies, produced until now by hand, has the same problems as the production of tissue matrices. Their manufacture also includes the stages of classification, processing and packaging.

Ideally, the reactor 2 is easily moved by any vehicle and can be easily installed anywhere in the world. The method according to the invention does not require the intervention of highly qualified hospital personnel and can be brought into line with current regulations in the place where they operate.

A particular embodiment of the reactor according to the invention is shown in detail in FIG. 3 showing the interior of the reactor according to the invention. For clarity, the reactor-bounding walls are not shown, but it is clear that they enclose, like a sealed container, the equipment inside the reactor. By "sealed" is meant that there are no openings in the reactor, except for lock chambers and openings specially provided for technical needs.

The reactor 302 includes a lock 303 for introducing and classifying biological tissues and a lock 309 for discharging a packaged tissue matrix, each lock being located in this case at the end of the reactor. Cups 327 containing biological tissues are shown here in an airlock 303. Between both airlocks are located next to each other 5 devices: a centrifuge 316, a cutting device 317, two stations 318a and 318b for chemical processing, each of which is located in a fume hood 322a and 322b , a lyophilizer 320, and a packaging station 312, in front of which are respectively five robotic articulation arms 313, 323, 343, 353, and 363, and a robotic articulation arm 333 installed between the cutter 317 and the chemical treatment station 318a. All the elements described above are located on the floor 325, under which a space 326 is provided.

Since the elements of the reactor 302 have been described, the articulation between them for carrying out the method according to the invention will be described in detail below.

The airlock 303 for introducing and classifying biological tissues is similar to the airlock 3 in FIG. 1 and 2. After opening the outer door of the lock chamber, the operator places the biological tissue to be treated in the cup 327 into the lock chamber 303. The air flow from inside the lock chamber is such that no contamination from the outside can enter the lock chamber as long as the outer door remains closed. The automated processing method begins by opening the inner door (to the reactor) of the lock chamber 303. The robotic arm 313 grasps the cup 327 in the lock chamber 303 and places it in the cavity of the centrifuge 316. The cup 327 can be made, for example, to fit the working cavities of the centrifuge. The cup may have a double wall, wherein the inner wall is made openwork so that the extracted substances of the biological tissue during centrifugation pass through the openwork wall and thus physically separated from the biological tissue. Multiple batches in multiple dishes can be centrifuged at the same time if necessary. When implementing the method, it is possible to monitor the position of each batch in any device, without a possible failure.

After centrifugation, the robotic arm 327 removes the cup 327 from the centrifuge 316. The arm 323 removes biological tissue freed at least partially of unwanted elements from it for cutting.

It is also possible to provide for the presence of an intermediate platform on which the cups containing the centrifuged biological tissue are stored until the lever 323 is moved.

The cutting can be done in the usual way with a band saw or cutter. However, these methods have several disadvantages. First of all, friction with the participation of biological tissue generates heat with a temperature above 47°C, which damages the tissue. In particular, when it comes to bone, its cavities become clogged due to the heat and pressure created on the cutting surface. It is then necessary - theoretically - to change the cutting belt to eliminate any contamination between batches, which is rarely done when the method is carried out by technicians in a hospital. Finally, methods that have little geometric flexibility (4-axis machines) cause a large loss of biological tissue.

To overcome these shortcomings, the Applicant has successfully introduced cutting with a high-pressure, non-abrasive water jet into an automated process. This technique has already been applied manually by surgeons in the operating room, as reported in the Journal of the Mechanical Behavior of Biomedical Materiaks, 62, 2016, 495-503, with partial bone cutting during surgery. The use of a high-pressure water jet makes it possible, on the one hand, to cut very precisely and accurately without increasing the temperature that could damage the fabric, and, on the other hand, not to contaminate the batches with each other. Another advantage associated with the use of a high-pressure water jet is that it can be applied to any tissue, be it bone or skin. The pressure of the water jet can be adapted to the tissue being cut. Cutting is clear and has microscopic precision. If we are talking about bone, then the alveoli of the bone on the cut surface remain undamaged.

In practice, the tissue-retaining arm 323 is movable along several axes simultaneously and positions and moves the tissue being cut by the water jet in any given direction. Because the articulated arm is movable in multiple axes, any cutting geometry is possible, which also allows for optimized cutting to limit waste and achieve higher tissue matrix yields at the end of the process.

The high-pressure water jet is produced by a device that is well known to the person skilled in the art, which contains a compressor and an outlet nozzle, for example, made of sapphire or diamond to reduce its wear, the diameter of which is variable to adjust the pressure and cutting accuracy.

Although high-pressure water jet cutting is technically more advanced, the invention is not limited to this method only, as any conventional cutting methods can be applied.

After cutting, the fabric pieces are collected one at a time by the arm 333, for example on a sieve, before they are taken away, and placed into the basket lot by lot, so that pieces from different lots are not mixed and full traceability is ensured.

The baskets are placed by lever 343 into the chemical treatment baths as described above, and then removed when the treatment is completed. Also, several intermediate chemical sequential different types of processing can be carried out on platforms 318a and 318b. Ovens 322a and 322b are conventional laboratory ovens that allow the quality of air in the reactor to be controlled over chemical solutions. Once the chemical treatment is completed, the robotic arm removes the baskets and places them near the robotic arm 353. The robotic arm 353 picks up the treated pieces of biological tissue one by one and places each in a pre-sealed tube, i. into a tube that has already been capped but not welded, then each tube is inserted into the 320 lyophilizer. After lyophilization is completed and before the lyophilizer is opened, the plate is placed on the tube seal caps, each containing a piece of tissue matrix ready for use as a graft.

The arm 363 then completes the packaging of the pipes and, for example, applies a label with all the necessary information for batch tracing, then the pipes with the packed fabric matrix enter the outlet lock chamber 309 for removal by the operator.

The space 326 under the floor 325 can be used for all cables and pipes, whether electrical, informational, for fluids, necessary for connecting devices, as well as for the disposal of waste generated during the process.

Robotic arms can, of course, perform other movements than those described above to ensure reliable application of the whole method. The arrangement of devices described here is linear, however, any other arrangement that allows the method according to the invention to be carried out is also possible. In particular, it is possible to use a smaller number of robotic arms, even just one that performs all operations with devices installed around it.

The robotic tools described above are represented here as one or more floor-mounted robotic arms. However, any other configuration or equivalent device may be provided, be it suspended means and/or rail means capable of gripping or specifically designed for precision equipment. The concept of "robotic arm" should be understood in a very broad sense as an automated, programmable and/or remotely controlled tool. The conveyor belt, if necessary, can also be used to move fabrics, matrix, waste and any other item to be moved.

The devices described here are conventional devices such as a centrifuge or a lyophilizer. Other types of equipment can be used according to the fabric to be processed, or custom equipment can be designed. Means for processing, cutting and packaging should be taken in a broad sense and contain any elements known to a person skilled in the art.

Claims (24)

1. A method for obtaining a packaged tissue matrix for allo- or xenotransplantation, in which, after the selection of biological tissue: - preparing said tissue (1) for administration in a sterile environment; - processing the specified tissue in such a way as to obtain a tissue matrix; and - packaging the resulting tissue matrix, wherein said steps are carried out in an automated manner in a sterile reactor (2), characterized in that the reactor for implementing said method contains: - a lock chamber for the introduction and preparation of biological tissue for its introduction into a sterile environment; - means of processing biological tissue to obtain a tissue matrix; - means of packaging the resulting tissue matrix; - outlet lock chamber for packed tissue matrix; and - robotic means for moving biological tissue from the inlet lock chamber to the biological tissue processing means and robotic means for moving the tissue matrix from the processing means to the packaging means and further to the outlet lock chamber. 2. The method according to claim 1, which includes the step (5) of cutting the tissue. 3. The method according to any one of paragraphs. 1, 2, which includes a lyophilization step (7) before a packaging step (8). 4. The method according to any one of paragraphs. 1-3, in which the processing of the tissue (1) includes at least one step from the group including: tissue centrifugation (4), chemical treatment (6), biological processing. 5. The method according to any one of paragraphs. 1-4, in which the tissue matrix (10) is a cell-free bone matrix. 6. The method according to any one of paragraphs. 1-4, in which the tissue matrix (10) is intended for cell therapy. 7. The method according to claim 2, in which the tissue is cut with a high-pressure water jet. 8. The reactor for obtaining a packed tissue matrix (10) for allo- or xenotransplantation, including: a lock chamber for the introduction and preparation of biological tissue for its introduction into a sterile environment; means for processing biological tissue to obtain a tissue matrix; packaging means for the resulting tissue matrix; an outlet lock chamber for the packed tissue matrix; and robotic means for moving the biological tissue from the inlet lock chamber to the biological tissue processing means and robotic means for moving the tissue matrix from the processing means to the packaging means and further to the outlet lock chamber. 9. The reactor according to claim 8, which further includes means for cutting biological tissue. 10. The reactor of claim 9, wherein the cutting means comprise a non-abrasive, high-pressure water jet. 11. The reactor according to any one of paragraphs. 8-10, wherein the biological tissue moving robots and/or the tissue matrix moving robots comprise robotic arms.

Images