RU2731314C1 - Method for production of spheroids for cartilage repair - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to tissue engineering, regenerative medicine, traumatology and orthopaedics, and discloses a method for obtaining a graft in the form of cell spheroids and a graft for reconstructing a cartilage of a subject obtained by said method. Method involves isolating cells from the perichondrium of the subject costal cartilage, culturing the recovered cells, transferring the cells into agarose plates in amount of 20,000 cells per 1 well of the agarose plate and subsequent cultivation thereof to form cell spheroids.

EFFECT: invention enables to obtain a transplant in the form of cell spheroids from the perichondrium of a subject rib having a marked regeneration potential, for effective cartilage regeneration, particularly articular cartilage.

12 cl, 4 dwg, 2 ex

Description

Technology area

The invention relates to medicine, namely to tissue engineering, regenerative medicine, traumatology and orthopedics.

State of the art

The integrity of the articular cartilage is a prerequisite for the normal functioning of the joint. The ability of the cartilage (covering the articular surface of the bone) to fully self-heal is limited, since it is an avascular and aneural tissue depleted of its own cambial cells (lacking perichondrium). Because of these structural features, circulating progenitor cells present in blood and bone marrow do not penetrate the damaged area in sufficient quantities to facilitate the repair process. It is quite reasonable to believe that an effective way to restore the integrity of cartilage has not yet been developed.

Cartilaginous and osteochondral defects currently remain an open problem in tissue engineering of the musculoskeletal system. Therefore, in case of significant damage to the articular cartilage, it is necessary to remove the patient's joint entirely and replace it with an endoprosthesis.

The problem is extremely urgent, as damage and degradation of articular cartilage leads to osteoarthritis (OA), mainly in the elderly, and this is one of the leading causes of pain and disability in this large age group of the population.

There are numerous methods of filling cartilage defects with tissue-engineered constructs made using scaffold technology - co-cultivation of cells with carrier matrices, currently called matrix-associated chondrocyte implantation (MACI). Various versions of matrix carriers (scaffolds) have been proposed, for example, a synthetic matrix based on hyaluronic acid benzyl ester (Hyalograft C, HYAFF 11) [1], a synthetic matrix based on polyglactin and poly-n-dioxanone (Bioseed C) [2], pork collagen membrane (ChondroGiude) [3]. The essential features of some variants of these technologies are attempts to maintain chondrogenic differentiation of their own chondrocytes during cultivation due to special chondrogenic differentiation media that restrain the growth of cell mass, which complicates the growth of chondrocytes in adults in the required quantities. The body has to independently dispose of the matrix framework due to the inflammatory reaction to the foreign body, which complicates the regeneration of the cartilage.

The disadvantages of culturing any cells on matrices (biodegradable carriers) are well known. The cells are unevenly distributed within the matrix, this is due to the presence of gradients of partial pressures of oxygen and carbon dioxide, as well as different availability of nutrients and the rate of metabolite removal, depending on the depth of the cells in the tissue-engineered construct. The weak link of such technologies is the lack of natural mechanisms of matrix adhesion to recipient surfaces (known methods of attachment are unreliable). The technology requires the destruction of the healthy part of the articular surface in order to harvest cartilage for cell isolation in the laboratory. Therefore, additional arthroscopic surgery and its anesthetic accompaniment are necessary.

There are known beskaffold technologies for cartilage restoration. Three-dimensional cultivation of chondrocytes in the absence of a substrate suitable for cell adhesion leads to the formation of cell spheroids (chondrospheres) [4]. Cartilaginous spheroids have a unique ability to tissue fusion, are capable of fusion with each other to form larger spheroids, as well as spreading on the surface of the adhesive plastic and the recipient bed [5].

There is a known method for the production of three-dimensional living cartilage-like micro tissues in vitro and their use as a transplant material (US 7.887,843 B2 Method for in vitro production of three-dimensional vital cartilage tissue and use thereof as transplant material (co.don® AG, Teltow, DE This method includes obtaining cell spheroids from cultured chondrocytes of the articular cartilage in 96-well plates made of non-adhesive plastic.An additional technological stage of work with the cell material is required - the mutual fusion of spheroids in a 24-well plate to increase the size of spheroids, which indicates the limited potential of spheroids' self-growth obtained in this way.The resulting spheroids are used for partial replacement (filling in a density of 3 to 70 spheroids per cm ² ) defects in the articular surfaces of bones in order to form new cartilage in the damaged area. bend ate cells, especially in the center of the spheroids. The cartilage tissue of the joint contains the only population of differentiated cells - chondrocytes, the proliferative potential of which is limited.

Also known from the prior art is a method for the production of spheroids from cartilage cells in agarose microplates containing 81 wells with a diameter of 800 μm or 256 wells with a diameter of 300 μm [5]. Spheroids were formed from cultured chondroprogenitor cells of the nasal septum in chondrogenic differentiation medium (DMEM, human albumin 1.25 μg / ml, ascorbic acid 50 μg / ml, insulin 6.25 μg / ml, penicillin 100 U / ml, streptomycin 100 μg / ml , insulin-transferrin - selenite (single concentration)). The seeding density was 2 million cells per microplate. Spheroids obtained by this method are characterized by the presence of a fibrillar extracellular matrix - collagen type 2. Also, large diameter spheroids (prepared in an 81-well plate) showed a tendency to grow (increase in diameter relative to the original). A significant disadvantage of this method is the limited amount of starting material taken for cell isolation, which can be obtained from the cartilage of the nasal septum without creating a noticeable cosmetic defect, which leads to the need for long-term cell cultivation to accumulate the required cell biomass, while in the process of spheroid formation, gradual death occurs cells, spheroids decrease in size.

Thus, despite the existing methods of cartilage restoration, all of them are characterized by a number of disadvantages and limitations, therefore, there remains a need to develop and create new methods and approaches to solving this problem.

Disclosure of invention

The objective of the present invention is to develop a new method of obtaining a transplant based on cell spheroids for the effective restoration of cartilage, in particular, articular cartilage.

It is proposed to obtain a transplant in the form of cellular spheroids to restore the cartilage of a subject based on the cells of the perichondrium of the subject's own costal cartilage.

The technical result of this invention is to obtain a transplant based on cellular spheroids with a pronounced regenerative potential for effective restoration of cartilage, in particular, articular cartilage, while obtaining spheroids is not accompanied by cell death in agarose wells. In addition, the method according to the invention eliminates the need to use differentiating chondrogenic media that slow down the build-up of cell biomass. In addition, the present invention provides a reduction in trauma to the subject during the collection of material for cartilage restoration, while additional hospitalization, anesthesia is not required, and surgical trauma to the intact part of the articular cartilage is excluded. The perichondrium of the subject's rib cartilage is harvested on an outpatient basis under local anesthesia.

Additionally, the method of the invention makes it possible to obtain spheroids from the perichondrium of the costal cartilage faster and of greater volume compared to spheroids based on cartilage cells. The use of a graft based on larger spheroids from the perichondrium facilitates their transplantation, and the restoration of cartilage is faster and more efficient. Reparative regeneration of cartilage occurs from the cambial (progenitor) cells of the perichondrium.

Achievement of the specified technical result is ensured by the implementation of the method of obtaining a transplant based on cellular spheroids for the restoration of the cartilage of the subject based on the cells of the perichondrium of the subject's own costal cartilage, including the following steps:

a) isolation of cells from the perichondrium of the subject's own costal cartilage;

b) cultivation of cells isolated at stage a) in an adhesive culture without a chondrogenic differentiation medium;

c) transfer of cells obtained in step b) into agarose plates and their subsequent cultivation with the formation of cell spheroids.

In particular embodiments of the invention, the subject is a human.

In particular embodiments of the invention, the cartilage is articular cartilage, cartilage of the nose, larynx, trachea and bronchi, auricle, or intervertebral disc cartilage.

In particular embodiments of the invention, the cell culture in step b) is carried out for 20-30 days. In particular embodiments of the invention, the transfer of cells in step c) is carried out at the rate of 20,000 cells per well of an agarose plate.

In particular variants, the fusion of cells into cell spheroids is carried out in agarose wells (wells of an agarose plate), where they stay up to 21 days. In particular embodiments of the invention, the fusion of cells into cell spheroids occurs after 7-15 days. In particular embodiments of the invention, the size of the spheroids is 250-700 μm.

The present invention also relates to a graft for the restoration of cartilage of a subject, comprising cell spheroids based on cells of the perichondrium of the subject's own costal cartilage and obtained by the method of the invention.

In particular embodiments of the invention, the cartilage is articular cartilage, cartilage of the nose, larynx, trachea and bronchi, auricle or intervertebral disc cartilage.

In particular embodiments of the invention, the subject is a human.

In particular embodiments of the invention, the size of the spheroids is 250-700 μm.

The technical result of the present invention is also achieved through the use of perichondrium cells of the subject's own costal cartilage to obtain a transplant in the form of cellular spheroids.

Brief Description of Drawings

Figure 1. Dynamics of changes in the size of spheroids from the articular cartilage and perichondrium of the rib during their cultivation after 1, 7 and 14 days.

Figure 2. Dynamics of changes in the size of spheroids from the articular cartilage and perichondrium of the rib during their cultivation after 1, 7 and 14 days:

a) articular cartilage, concentration of 10,000 cells per spheroid;

b) articular cartilage, concentration of 20,000 cells per spheroid;

c) perichondrium, concentration of 10,000 cells per spheroid;

d) perichondrium, concentration of 20,000 cells per spheroid.

Figure 3. Histological structure of spheroids from articular cartilage and perichondrium of the rib during their cultivation after 1, 7 and 14 days.

Figure 4. The process of covering the defect zone of the articular surface of the subject with spheroids obtained by the method according to the invention.

Definitions and terms

Various terms related to the objects of the present invention are used above and also in the description and in the claims. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing the present invention refer to well known techniques, including modifying these techniques and replacing them with equivalent techniques known in the art.

In the description of this invention, the terms "includes" and "including" are interpreted to mean "includes, among other things". These terms are not intended to be construed as “consists only of”.

In the description of this invention, the term "tissue" refers to a system of cells and non-cellular structures that have a common structure, in some cases, a common origin, and specialized in performing certain functions.

Used in this application, the term "graft" refers to spheroids, and the term "transplant" is the transfer of these spheroids into the body of a subject, usually to repair a cartilage defect.

As used herein, the term "cartilage defect" refers to cartilage tissue that is absent, reduced in quantity, or otherwise damaged. A cartilage defect can also be the result of a disease, treatment for a disease or injury.

Used in this application, the term "chondrogenic environment" means the environment necessary for the differentiation of cells in the chondrogenic direction.

As used in this application, the term "spheroids" refers to tightly packed ball-shaped cell aggregates formed by three-dimensional culture. Their important property is the ability for mutual adhesion and subsequent tissue fusion, as well as for adhesion to the elements of the extracellular matrix.

Detailed disclosure of the invention

The technical result is achieved in that the perichondrium is used as a source of chondroprogenator cells for the production of spheroids, which is a cambial zone for cartilage, supplying progenitor cells for its renewal and reparative regeneration, and, therefore, containing a greater number of cambial cells. It should be noted that costal and articular cartilage belongs to hyaline cartilage tissue. When spheroids are formed from perichondrium cells, their diameter does not decrease in this case.

Isolation of chondroprogenic cells from the perichondrium occurs by mechanical grinding it into small pieces, followed by their enzymatic dissociation.

To obtain a primary culture, cells isolated from the perichondrium are seeded in plastic flasks with a culture medium of the following composition: DMEM (or DMEM / F12) 450 ml, L-glutamine 292 mg, fetal bovine serum 50 ml, penicillin 100 U / ml, streptomycin 100 μg / ml, at 100% humidity, a temperature of 37 ° C, 5% CO ₂ (this environment and conditions are used at all stages of subsequent cultivation).

Subcultivation of a monolayer culture of chondroprogenic cells is carried out until the third passage with a change of medium every 2-3 days.

After the third passage, cells are removed from plastic vials with trypsin / EDTA. The resulting cell suspension is washed from trypsin / EDTA, including using DMEM medium with 10% serum, followed by centrifugation (10 minutes at 200g) and transferred into 81-well agarose plates with a well diameter of 800 μm at a concentration of up to 1.6 million cells onto the plate, where spheroids begin to form from the cultured cells in the wells. Thus, the formation of cell spheroids occurs in a scaffold-free 3-Dimensional Cell Culture Utilizing Micro-Molded Non-Adhesive Hydrogels.

To obtain agarose plates, a 3D Petri Dish silicone form (Microtissues®, USA) is used in accordance with the manufacturer's protocol: the agarose solution is distributed into silicone molds, resulting in a microformed (micromodulated) non-adhesive agarose hydrogel - an agarose (micro) plate (a form formed from an agarose hydrogel, having wells of the same size, in a particular embodiment, 81 wells with a diameter of 800 μm).

Spheroids stay in the wells of an agarose plate for up to 21 days with a change of medium every 2-3 days.

For transplantation of spheroids, they are collected from plates and transferred to a test tube, where they settle to the bottom without additional centrifugation. Thereafter, the spheroids are placed in a syringe and transplanted to the subject into the defect area through a needle or applicator with a diameter of at least 1 mm ² . Transplantation can be performed arthroscopically.

Implementation of the invention

Example 1

The perichondrium was taken from the costal cartilage of sheep. To separate the perichondrium from the underlying cartilage, an isotonic solution was injected under it. Fragments of articular cartilage were taken from the femoral condyles as a control in the same animals.

Isolation of cells from the perichondrium of the costal and articular cartilage was carried out by preliminary mechanical grinding followed by enzymatic dissociation of tissue pieces.

To obtain a primary culture, the isolated cells were seeded in plastic flasks with a culture medium of the following composition: DMEM 450 ml, L-glutamine 292 mg, fetal bovine serum 50 ml, penicillin 100 U / ml, streptomycin 100 μg / ml, at 100% humidity, temperature 37 ° C, 5% CO ₂ (this environment and conditions are used at all stages of subsequent cultivation). Subculturing of a monolayer culture of chondroprogenic cells was carried out until the third passage with a change of medium every 2-3 days.

After the third passage, cells were removed from plastic (plastic vials) with trypsin / EDTA. The resulting cell suspension was washed from trypsin / EDTA, including by DMEM medium with 10% serum, followed by centrifugation (10 minutes at 200g), then transferred to 81-well agarose plates with a well diameter of 800 μm at a concentration of up to 1.6 million. cells on the plate, after which spheroids began to form.

Cultivation of spheroids was carried out for 14 days with a change of medium every 2-3 days.

As a result, the spheroids from the perichondrium doubled in size during cultivation, and the spheroids from the articular cartilage decreased (Fig. 1, Fig. 2). Typically, spheroids derived from cartilage cells decrease in size within agarose wells over time. This is due to the biological characteristics of cartilage cells, non-optimal cultivation conditions, the death of some cells and the loss of structural elements. Therefore, the result obtained using perichondrium cells was not absolutely expected for a specialist.

Thus, when growing (obtaining) spheroids according to the method of the invention in agarose wells, spheroids are obtained from the perichondrium of the costal cartilage, which increase their biomass in the agarose wells, in contrast to spheroids from articular cartilage.

Histologically, the spheroids from the articular cartilage at a cultivation period of 14 days consisted mainly of cells, while the spheroids from the perichondrium according to the present invention already on the 7th day of cultivation contained a fibrous extracellular matrix. (Fig. 3). Thus, as a result of the experiment, it was shown that an equal initial number of cells produced a different biomass of spheroids: there were more spheroids obtained by the method according to the invention from the perichondrium than spheroids from articular cartilage, respectively, a larger volume of the defect can be filled. In addition, the production of fibrous extracellular matrix during 3D culture is evidence of a higher functional activity and cell viability.

Example 2

Spheroids from the perichondrium of the costal cartilage (hyaline), obtained according to the present invention, were placed in the defects of the articular surface with a diameter of 4 mm, formed on fragments of the condyles of the femur bones of sheep, until they completely covered the defect zone (Fig. 4). Spheroids from hyaline cartilage tissue - articular cartilage were used as a control. After filling with spheroids, fragments of sheep femur condyles were cultured in a plastic bottle with the following medium: DMEM 450 ml, L-glutamine 292 mg, fetal bovine serum 50 ml, penicillin 100 U / ml, streptomycin 100 μg / ml, at 100% humidity , a temperature of 37 ° C, 5% CO ₂ , within 7 days with a change of medium every 3 days.

It was noted that on the first day of cultivation the spheroids from the perichondrium merged with each other and interacted with the structures of the defect zone, spreading out on the recipient bed. By the seventh day of cultivation, the defect was completely covered with connective tissue from the introduced spheroids. The size of the spheroids from the perichondrium is optimal for effective closure of the articular surface defect, and due to plasticity (the ability to merge with each other and spread out on the surface of the defect) tissue fusion of spheroids and engraftment of spheroids occurs quickly.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are provided for the purpose of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

List of literature, which is included in the present description of the invention by reference:

1. Marcacci M Zaffagnini S, Kon E, Visani A, Iacono F, Loreti I. Arthroscopic autologous chondrocyte transplantation: technical note. [Journal] // Knee Surg Sports Traumatol Arthrosc. - 2002 - 3: T. 10. - pp. 154-159.

2. Ossendorf C Kaps C, Kreuz PC, Burmester GR, Sittinger M, Erggelet C. Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results. [Journal] // Arthritis Res Ther. - 2007 - 2: T. 9.

3. Behrens P Ehlers EM, Köchermann KU, Rohwedel J, Russlies M, Plötz W. New therapy procedure for localized cartilage defects. Encouraging results with autologous chondrocyte implantation [Journal] // MMW Fortschr Med. ... - 1999 - 45: T. 141. - p. 49-51.

4. Anderer U Libera J. In vitro engineering of human autogenous cartilage. [Journal] // J Bone Miner Res. ... - 2002 - 8: T. 17. - pp. 1420-1429.

5. Stuart MP Matsui RAM, Santos MFS, Côrtes I, Azevedo MS, Silva KR, Beatrici A, Leite PEC, Falagan-Lotsch P, Granjeiro JM, Mironov V, Baptista LS. Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel. [Journal] // Stem Cells Int. ... - 2017

Claims (15)

1. A method of obtaining a transplant in the form of cellular spheroids for the restoration of the cartilage of a subject based on the cells of the perichondrium of the subject's own costal cartilage, including the following steps: a) isolation of cells from the perichondrium of the subject's own costal cartilage; b) culturing the cells isolated at stage a) without chondrogenic differentiation medium, and to obtain a primary culture, the isolated cells are seeded in plastic flasks with a culture medium of the following composition: DMEM 450 ml, L-glutamine 292 mg, fetal bovine serum 50 ml, penicillin 100 units / ml, streptomycin 100 μg / ml, at 100% humidity, a temperature of 37 ° C, 5% CO2; c) transferring the cells obtained in step b) into agarose plates at the rate of 20,000 cells per 1 well of an agarose plate and their subsequent cultivation with the formation of cell spheroids. 2. The method of claim 1, wherein the subject is a human. 3. The method of claim 1, wherein the cartilage is articular cartilage, nasal, laryngeal, trachea, bronchial, pinna or intervertebral disc cartilage. 4. The method according to claim 1, wherein the culturing of the cells in step b) is carried out for 20-30 days. 5. The method according to claim 1, in which the transfer of cells in step c) is carried out at the rate of 20,000 cells per 1 well of an agarose plate. 6. The method of claim 1, wherein the fusion of the cells into cell spheroids is carried out in the wells of an agarose plate, where the cells remain for up to 21 days. 7. The method of claim 6, wherein the fusion of cells into cell spheroids occurs after 7-15 days. 8. The method of claim 1, wherein the spheroids are 250-700 microns in size. 9. A graft for restoring the cartilage of a subject, comprising cell spheroids based on cells of the perichondrium of the subject's own costal cartilage, obtained by the method of claim 1. 10. The graft of claim 1, wherein the cartilage is articular cartilage, cartilage of the nose, larynx, trachea, bronchi, auricle, or intervertebral discs. 11. The graft of claim 1, wherein the subject is a human. 12. The graft of claim 1, wherein the spheroids are 250-700 microns in size.

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