RU2800991C2 - Method of biofabrication of a transplant in the form of cell spheroids for regenerative technologies for restoring a subject's cartilage based on the subject's own costal cartilage perichondrium and multipotent mesenchymal stromal bone marrow cells of the same subject - Google Patents

Abstract

FIELD: regenerative medicine.

SUBSTANCE: invention discloses a method of obtaining a transplant in the form of cell spheroids for the restoration of the subject's cartilage, which includes the isolation of cells from the cambial layer of the perichondrium of the own costal cartilage and multipotent mesenchymal stromal cells of the subject's own bone marrow, separate cultivation of the isolated cells in 2D- systems at the bottom of culture flasks, transferring cells to plastic microplates at the rate of 8,000 cells per 1 well, with the cells of two cell cultures being mixed in a 1:1 cell ratio, and their subsequent 3D cultivation in wells to form cell spheroids.

EFFECT: invention makes it possible to obtain a transplant with a pronounced regenerative potential, the effect of stimulating the regeneration of the surrounding cartilage and antimicrobial activity, for the effective restoration of cartilage, in particular articular cartilage, besides it is possible to use the obtained spheroids for bioprinting.

7 cl, 1 ex

Description

The invention relates to medicine, namely tissue engineering, transplantology, regenerative medicine, traumatology and orthopedics, plastic surgery and dentistry.

State of the art

Hyaline cartilage tissue is the most common type of cartilage skeletal tissue in the human body, which forms the skeleton in the fetus (forms a temporary embryonic skeleton, which is gradually replaced by bone), airways, etc., in the postnatal period of human development, hyaline cartilage tissue is represented in costal cartilages, cartilages of the nose, larynx (partially), trachea and large bronchi, covers all articular surfaces of bones, cartilaginous growth plates in tubular bones. The ability for reparative regeneration of hyaline cartilage is associated with the presence of the perichondrium in those organs where it covers the cartilage. The perichondrium is a dense, vascular-rich connective tissue membrane present on the growing bone (at the stage of endochondral osteogenesis in fetal development), costal cartilage, cartilage of the larynx and respiratory tract, etc. The perichondrium is a source of cambial cells, serves for the growth and regeneration of cartilage tissue. Complete regeneration of hyaline cartilage after traumatic injuries is possible in cases of damage to cartilage covered by the perichondrium and with the obligatory preservation of the latter after the destruction of the cartilage. Prechondroblasts of the perichondrium are activated upon injury, divide and differentiate into chondroblasts, which are capable of producing tissue-specific intercellular cartilage substance, filling the free space of a post-traumatic defect with cartilage regenerate. However, tissue-specific cartilage regeneration under the perichondrium is not always possible due to the advanced development of another regenerate from the surrounding young fibrous connective tissue, which quickly replaces the space of the cartilage defect, eventually turning from loose fibrous connective tissue into an atypical regenerate - a connective tissue scar. The condition for organotypic cartilage regeneration under the perichondrium is the preservation of its integrity, this connective tissue plate must be closed, and all its injuries sutured. This atypical regenerate has different strength characteristics, is weakly connected to the fibrous framework of the intact surrounding cartilage, is often damaged and disrupts the function of the cartilage tissue of the joints.

Articular cartilage is devoid of perichondrium. Therefore, the point of view still prevails that the body of an adult is not capable of producing new hyaline cartilage tissue after traumatic defects or osteoarthritis on the articular surfaces of bones, including after damage to cartilage tissue separately or together with bone tissue forming the articular surface. Articular cartilage has a reduced regenerative potential in the postnatal period of development, as there are not many skeletal stem cells in this tissue that can be activated, they cannot effectively counteract cumulative damage, such as increased stress on the joints, as well as injuries that lead to cartilage destruction and osteoarthritis. Various cells have been found in articular cartilage claiming to be stem/progenitor cells of cartilage origin (cartilage-derived stem/progenitor cells (CSPCs), articular cartilage-derived progenitor cells (ACPCs) found in mature cartilage, as well as progenitor cells in the groove Ranvier cartilage and other intra-articular tissues that have the potential to participate in cartilage regeneration Adult articular cartilage has limited repair capacity, chondrocytes are lacunae-bound and unable to migrate to damaged areas, cell division within cartilage is very slow, cartilage defects often progress to osteoarthritis.

The potential for hyaline cartilage repair is limited by the absence of vessels and nerves in the cartilage, the inability to supply inflammatory effector cells and progenitor cells with the blood stream, and the relatively small number and low mitogenic characteristics of chondrocytes located in a dense extracellular matrix. Cartilage cells in the articular cartilage is not more than 5%. The rest of the space is occupied by an extracellular matrix rich in collagen and proteoglycans.

Historically, the regenerative capacity of articular cartilage and hyaline cartilage in general after the end of the intrauterine period of development has been questioned, among other things, due to the slow rate of renewal of the cartilage matrix, especially the fiber backbone - cartilage collagen. The half-life of articular cartilage matrix proteins (collagens) is very long (more than 20 years), which indicates a low intensity of cartilage matrix renewal. However, it has been established that the physiological regeneration of cartilage in humans continues after the onset of puberty and the cessation of growth throughout a person's life. In different parts of the body, the physiological regeneration of cartilage proceeds at different rates. For example, in the ankle joint, this tissue was renewed much faster than in the knee joint, and more slowly in the hip joint (Ming-Feng Hsueh, Patrik Onnerfjord, Michael P. Bolognesi, Mark E. Easley, Virginia B. Kraus. Analysis of "old" proteins unmasks dynamic gradient of cartilage turnover in human limbs Science Advances, 2019; 5 (10): eaax3203 DOI: 10.1126/sciadv.aax3203).

The regenerative capacity of intact articular cartilage in humans has been demonstrated by clinical studies, with joint distraction in adult patients observing an obvious increase in joint space width and cartilage thickness on radiographs (Interna F., Van Roermund P. M., Marijnissen A. C. A., Cotofana S., Eckstein F., Castelein R. M., Bijlsma J. W. J., Mastbergen S. C, Lafeber F. P. J. G., Tissue structure modification in knee osteoarthritis by use of joint distraction: An open 1-year pilot study. Ann. Rheum. Dis. 70, 1441-1446 ( 2011)).

Since 1994, two-stage procedures known as autologous chondrocyte transplantation have been used in clinical practice and have been shown in various randomized trials to provide sustained clinical improvement in patients with articular cartilage defects. Three generations of this technique are known. In the third generation, chondrocytes were cultured in 3D without the use of any scaffold materials (3D-ACI, three-dimensional autologous chondrocyte implantation), offering complete autologous three-dimensional (3D) transplantation, which itself can be performed in a more gentle way - arthroscopically. To do this, the cells were cultured in high density, where chondrocytes form the so-called spheroids - rounded cell agglomerates (chondrocytes), which consist only of the patient's own chondrocytes and the extracellular matrix formed by them, which is synthesized by the chondrocytes themselves in 3D culture, is an important and integral part of the chondrocytes .

The disadvantages of all these generations of autologous chondrocyte implantation technology is the need for two consecutive operations on the joint. The first operation involves taking pieces of a healthy part of cartilage from a non-loaded surface to isolate cells and increase their number in the laboratory for subsequent autologous transplantation. It is known that persistent damage to the structural organization of the articular cartilage (a defect at the sampling site does not recover spontaneously) leads to a change in the distribution of forces and a loss of mechanical strength, such sampling can have certain negative consequences. The second operation involves transplantation of cellular material to replace cartilage defects. The patient is doomed to two postoperative periods, requires assistive devices for movement, long-term rehabilitation. Own cartilage chondrocytes are used, the proliferative potential of which is limited and these cells partially lose their tissue specificity during cultivation. It is known that expansion (multiplication) of chondrocytes is limited after population doubling in culture, as these cells tend to acquire a fibroblast-like appearance and lose their cartilaginous phenotype before becoming senescent.

New variants of this technique are being developed, most recently a variant using nasal cartilage biopsy has been proposed to bypass the need for prior autologous cartilage sampling at the knee joint. The disadvantages of the technology include strict state registration rules, additional trauma to the nose, a small amount of biopsy for expansion, which requires more mitoses, high costs and complex logistics associated with ex vivo expansion for subsequent implantation in humans, which limit the wide availability of this type of autologous cell implantation (chondrocytes).

A group of inventions is known (patent RU 2731314 "Method for the production of cellular spheroids for cartilage restoration") that makes it possible to obtain an autologous transplant in the form of cellular spheroids from the perichondrium of the rib of a subject with a pronounced regenerative potential for the restoration of articular cartilage. The method includes isolating cells from the perichondrium of the subject's own costal cartilage, culturing the isolated cells, transferring the cells to agarose plates at the rate of 20,000 cells per 1 well of the agarose plate, and then culturing them to form cell spheroids. EFFECT: method makes it possible to exclude additional traumatization of the joint, to take a sufficient amount of cellular material for expansion and is not associated with trauma to the nasal cartilage. Perchondrium cambial cells have some similar properties to stem cells, such as clonal expansion, extensive proliferation, and the potential to differentiate into chondrogenic and adipogenic clones.

Numerous attempts have been made to use mesenchymal stromal cells to repair articular cartilage, and clinical studies have been conducted. Despite the ability to generate cartilage tissue, MSCs tend to differentiate into hypertrophic chondrocytes and initiate subsequent unwanted endochondral ossification. Effective and recognized by the medical community, there are no medical regenerative technologies for replacing cartilage with these cells.

According to modern concepts, cultured MSCs after autologous transplantation do not function in the body as tissue precursors either in a normal stationary state or under conditions of illness or injury; they are not stem cells. According to the concept of Medical Signaling Cells, after transplantation (In vivo), MSCs are sources of trophic factors that they secrete, modulating the immune system and inducing their own stem cells, tissue-specific progenitor cells (responsible for the regeneration of damaged tissue) to restore damaged tissues, the lifespan of transplanted cells, even with autologous transplantation, is several days (Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jun;6(6):1445-1451. doi: 10.1002/sctm. 17-0051. Epub 2017 Apr 28. PMID: 28452204; PMCID: PMC5689741).

It has been established that MSCs have chondro-inductive effects in combination with transplantation of autologous chondrons for the treatment of focal cartilage defects (de Windt TS, Vonk LA, Slaper-Cortenbach ICM, Nizak R, van Rijen MHP, Saris DBF. Allogeneic MSCs and Recycled Autologous Chondrons Mixed in a One-Stage Cartilage Cell Transplantion: A First-in-Man Trial in 35 Patients Stem Cells 2017 Aug;35(8):1984-1993 doi: 10.1002/stem.2657 Epub 2017 Jun 23 PMID: 28600828). In this known method, the operation was performed through mini-arthrotomy of the knee joint. Cartilage defects were trimmed by removing the calcified cartilage layer and creating smooth edges of the intact surrounding articular cartilage. The surgical wound was temporarily covered with a sterile dressing. The resulting purified cartilage tissue was processed using a rapid enzymatic isolation protocol to obtain 100,000-400,000 chondrons (chondrocytes with their pericellular matrix) in the laboratory for subsequent retransplantation. Allogeneic MSCs were isolated from the bone marrow of two healthy donors (aged 2 and 5 years) and propagated in an adhesive culture. Until the moment of application, these cells were in a cryopreserved state. After thawing, MSCs were washed with a solution of 0.9% sodium chloride with 10% human serum albumin with a concentration of dimethyl sulfoxide remaining after cryopreservation of MSCs <0.001% in the final product. Before use, autologous chondrons and allogeneic MSCs were combined in a ratio of 10:90 (standard yield) or 20:80 (high yield), depending on the amount of chondrons isolated from the patient.

The cells were suspended in fibrin glue (Beriplast, CSL Behring, USA) using 1.5-2 million cells per milliliter of glue. Approximately 90 minutes later, the cell material was delivered to the operating room, the dressing was removed from the knee wound, and the cells were implanted into the joint using a fibrin glue carrier. Approximately 0.9 ml of cell product (cells + fibrin glue) was implanted per square centimeter of the defect, the product should adhere tightly to the bottom and edges of the cartilage defect. The authors point out that in previous in vitro and in vivo studies they were able to show the advantage of using a combination of chondrons and MSCs compared to chondrules or MSCs alone, which makes it unlikely to achieve the same results with the limited number of isolated autologous chondrules available. The above method is based on the preliminary sampling of a relatively small amount of cartilage around the articular cartilage defect to isolate the chondrules, so the possibilities of cartilage restoration in this case are limited by the small size of the cartilage defects to be closed. Freshly thawed allogeneic cells have poor viability when transplanted into a living organism, they need time before transplantation for intracellular regeneration of cryodamages under favorable conditions in vitro (this stage is not provided for by this technology), frozen cells also have all the characteristic and known disadvantages of allogeneic material. The use of donor material left over from bone marrow transplantation in children can make it difficult to widely implement the method in practical healthcare.

It is important to note that 3D aggregates - spheroids from MSCs are considered more physiological for cell cultures due to the enhancement of intercellular interactions and interactions with the matrix accumulated in the spheroid. Cells themselves (MSCs) cultured in spheroids, compared to 2D adhesive cultures, have enhanced anti-inflammatory and tissue reparative/regenerative effects with improved cell survival after transplantation (Cesarz Z, Tamama K. Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int. 2016 ; 2016:9176357. doi: 10.1155/2016/9176357. Epub 2015 Nov 16)

It has recently been established that, under laboratory conditions, adipose tissue MSCs can initiate cartilage formation by neonatal chondrocytes when mixed and co-cultivated in biomimetic hydrogels with certain properties. The technology is not intended for clinical use (Wang T. et al. Modulating stem cellchondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels// J. Mater. Chem. B, 2016, 4, p.7641-7650, DOI: 10.1039 /c6tb01583b).

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

The objective of the invention is to create a method for biofabrication of a transplant in the form of cell spheroids for regenerative technologies for restoring the subject's cartilage based on the perichondrium cells of the subject's own costal cartilage and multipotent mesenchymal stromal cells of the bone marrow of the same subject.

The technical result of this invention is to obtain an improved autologous graft based on cellular spheroids, which not only have a pronounced regenerative potential, but also the ability to activate reparative regeneration locally, both local tissues and cells of the perichondrium of the spheroid-transplant, as well as improve the integration of the regenerate growing from cellular spheroids with partially regenerating surrounding cartilage tissue of the recipient area for effective restoration of cartilage, in particular, articular cartilage, as well as to reduce the risk of infectious complications due to the synthesis of antimicrobial peptides (indoleamine-2,3-dioxygenase (IDO), cathelicidin LL-37, hepcidin) MSCs , which are part of the transplant-spheroid. Only one arthroscopic operation is required to fill the cartilage defect, the operation itself is faster, since the waiting stage is eliminated during which the collected cellular material is prepared for transplantation into the defect. The resulting spheroid has a higher regenerative potential compared to spheroids from perichondrium cells only.

Additionally, the method according to the invention makes it possible to obtain spheroids from the costal cartilage perichondrium and bone marrow MSCs faster and larger than spheroids based only on perichondrium cells, since 2 cell cultures are used simultaneously, the material is prepared in a personalized manner, and the cryopreservation step is excluded. It should be noted that the sampling of both perichondria and bone marrow sampling in the volumes necessary for the implementation of this method are performed on an outpatient basis under local anesthesia. Bone marrow is harvested from the iliac crests using bone marrow aspiration needles. Higher adhesive properties and the ability to mutually fuse such spheroids allow the use of smaller spheroids, which improves the viability of cells in the central part of the spheroid during 3D cultivation and the survival of spheroid cells during engraftment. MSCs create a favorable local environment for cartilage regeneration from the cambial cells of the perichondrium; as part of a spheroid, they have an enhanced anti-inflammatory and tissue reparative/regenerative effect, including preventing scarring, preventing apoptosis, and stimulating cell division effects.

The achievement of the specified technical result is ensured by the implementation of a method for obtaining a transplant based on cellular spheroids for regenerative technologies for restoring the subject's hyaline cartilage based on the perichondrium cells of the subject's own costal cartilage and MSCs of the subject's bone marrow, which includes the following steps:

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

b) isolating adherent stromal cells from the subject's own bone marrow;

c) separate cultivation of the cells isolated in steps a) and b) in adhesive 2D cultures separately without chondrogenic differentiation medium with the addition of 10% thrombolysate (PLTGold® Human Platelet Lysate, Merck);

d) transferring the cells obtained in step c) to NanoCulture® 3D culture plates (Corning® 1536-well microplate for spheroids with round bottom, ultra-low attachment, with lid, sterile (Corning® 1536-well Black/Clear Round Bottom Ultra-low Attachment Spheroid Microplate, with Lid, Sterile) and subsequent 3D cultivation with the formation of cell spheroids, and at this stage, mixed cultivation of perichondrium cells and MSCs in a ratio of 1: 1. In particular embodiments of the invention, the subject is a human. In embodiments, the cartilage is articular cartilage, cartilage of the nose, larynx, trachea and bronchi, auricle, or cartilage of the intervertebral discs.In particular embodiments, the cell culture in step c) is carried out for 20-30 days. In private embodiments of the invention, the transfer of cells in stage c) is carried out at the rate of 8000 cells of two grown cell cultures per 1 well of the microplate in a ratio of 1:1.

In particular cases, the fusion of cells into cell spheroids is carried out in the wells of a plastic microplate, where they stay for 5 days (3D cultivation is acceptable for no longer than 8 days). In private embodiments of the invention, the fusion of cells into cell spheroids is carried out after 5-8 days.

The present invention also relates to a transplant for cartilage repair of a subject, comprising cell spheroids based on cells of the cambial layer of the perichondrium of the subject's own costal cartilage and the subject's own bone marrow, and obtained by the method of the invention. In particular embodiments, the cartilage is articular cartilage, cartilage of the nose, larynx, trachea and bronchi, auricle, or cartilage of intervertebral discs.

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

The technical result of the present invention is also achieved through the use of cells of the cambial layer of the perichondrium of the own costal cartilage and MSCs of the subject's bone marrow in a ratio of 1:1 to obtain a transplant in the form of cell spheroids with a diameter of 420-460 μm. The importance of the presence of thrombolysate in the nutrient medium should be emphasized, since thrombolysate effectively supports the proliferative ability of cultured cartilage differon cells, moreover, thrombolysate maintains tissue specificity of cells in culture (De Angelis E. et al. Platelet lysate reduces the chondrocyte dedifferentiation during in vitro expansion: Implications for cartilage tissue engineering// Res Vet Sci 2020 Dec 133:98-105 doi: 10.1016/j.rvsc.2020.08.017 Epub 2020 Sep 6.).

Definitions and terms

Various terms relating 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 the description of this invention refer to well-known methods, including modifications of these methods and replacing them with equivalent methods known to those skilled in the art.

In the description of the present invention, the terms "comprises" and "comprising" are interpreted to mean "includes, among other things." These terms are not intended to be construed as "consisting only of".

In the description of this invention, the term perichondrium (perichondrus) is the connective tissue sheath of cartilage (with the exception of the cartilage of the articular surfaces of bones); consists of two layers: the outer (fibrous), denser, the layer passes without sharp boundaries into the surrounding connective tissue, the inner (chondrogenic) layer contains cells that can turn into chondroblasts that ensure the growth of cartilage.

In the description of this invention, the term biofabrication is the process of artificially designing and growing living, functional tissues or organs outside the human body for subsequent transplantation to a patient in order to replace or stimulate the regeneration of damaged organs or tissues, most often using bioprinting technology.

In the description of this invention, the term bone marrow stroma is a connective tissue, represented by all types of cells that are localized between the outer surface of the vessels of the bone marrow and the surface of the bone, which serves as an environment for hematopoietic (hematopoietic) cells.

In the description of this invention, the term spheroids is densely packed spherical cell aggregates formed by three-dimensional cultivation, an important property of which is the ability for mutual adhesion and subsequent tissue fusion, as well as for adhesion to the elements of the recipient's extracellular matrix; tissue spheroids have a tissue-like structure, they are often called three-dimensional microtissues created under artificial conditions that maximize intercellular interactions.

In the description of this invention, the term adhesive cultures are monolayer cultures of cells capable of attaching to cultural plastic or glass through their receptors, under certain conditions, these cells will attach to the substrate and will normally divide and spread in this way. Such cells have a dependence on the substrate - attachment to the substrate is a necessary prerequisite for cell proliferation.

In the description of this invention, the term scarring is the process of maturation of granulation tissue, ending in the formation of an atypical regenerate - a scar, morphologically characterized by a decrease in the number of vessels and cellular elements, the formation of fibrocytes and characteristically laid collagen fibers.

In the description of this invention, the term organ bioprinting is the robotic layer-by-layer creation of organ structures from tissue spheroids or cells according to a digital model.

Chondrification (also known as chondrogenesis) is the process of cartilage formation from compacted mesenchymal tissue. During cartilage formation, undifferentiated MSCs are highly proliferating and form dense aggregations of chondrogenic cells at the center of chondrification. These condrogenous cells then differentiate into chondroblasts, which then synthesize the cartilage extracellular matrix.

In the description of this invention, the term multipotent mesenchymal stromal cells (MSCs) is a heterogeneous population of stromal cells from the bone marrow and some other sources capable of differentiating into cells of mesenchymal origin: adipocytes, chondrocytes and osteocytes.

In the description of this invention, the term transplantation regeneration - transplant regeneration, stimulation of the regeneration process or the very possibility of recovery by transplantation of tissues that are further destroyed and resorbed, creates a model (framework) that allows local tissues to regenerate. The transplantation method is used to stimulate the regeneration process or to ensure the very possibility of recovery.

Subculturing is the process of transferring some or all of the cells from a previous culture to a fresh culture medium, often a new culture vessel.

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

In the description of this invention, PLT Gold®, a commercial product of the German pharmaceutical, chemical and biological company Merck, is an animal protein-free cell culture supplement, which, according to the manufacturer, is an alternative to fetal bovine serum, which has a number of significant advantages, is an unfractionated product, a lysate derived from human platelets that does not require the addition of heparin.

In the description of this invention, chondron - a group of cartilage cells of a single origin in combination with the adjacent intercellular substance, are the true morphological unit of articular cartilage. The chondrules (plural "chondron") have long been thought to function as the primary metabolic units of hyaline cartilage.

Detailed disclosure of the invention

The technical result is achieved by the fact that the cambial layer of the perichondrium is used as a source of chondroprogenitor cells for the production of spheroids, which is a cellular source of complete regeneration for cartilage, the proposed spheroids have higher indicators of regenerative potential due to the introduction of MSCs into the composition of the spheroid (in a ratio of 1:1 or 1 :2 to the cells of the cambial layer of the perichondrium). MSCs have well-known chondroinductive, anti-inflammatory effects that stimulate transplantation regeneration, which facilitates engraftment of the regenerate, tissue mutual fusion of spheroids, and adhesion of spheroids to elements of the extracellular matrix. An important antimicrobial effect of such spheroids is due to the production of MSCs, which are part of spheroids, antimicrobial peptides, which reduces the risk of postoperative infectious complications.

Isolation of cells from the cambial layer of the perichondrium occurs by its mechanical scraping under visual control using an operating microscope, followed by enzymatic dissociation of the extracted layer of the perichondrium in 0.1% collagenase II solution (Sigma-Aldrich, USA) in DMEM nutrient medium (Gibco, USA) with the addition of 2 mM L-glutamine (LLC PanEco, Russia) and penicillin-streptomycin, 100-fold solution, working concentration 10 ml/l (100-fold solution) (LLC PanEco, Russia), tissue fragments were incubated in for 24 hours at 37°C.

To obtain a primary culture from the cambial layer, cells isolated from the perichondrium are seeded in plastic flasks for cell cultivation; for work with attached cultures, a culture nutrient medium of the following composition is used: DMEM (or DMEM / F12) 450 ml (LLC "PanEco", Russia), L-glutamine 292 mg, thrombolysate (PLTGold®) 50 ml, penicillin 100 units/ml, streptomycin 100 µg/ml, at 100% humidity, temperature 37°C, gas mixture with 5% CO ₂ .

To obtain a primary cell culture from the bone marrow, a bone marrow aspirate was taken from the iliac bones, mononuclear cells were isolated on a density gradient during centrifugation (ficoll solution), it was found by flow cytometry that fibroblast-like cells obtained as a result of culturing the mononuclear fraction of bone marrow aspirate, had the following phenotype: CD13+, CD34-, CD44+, CD45-, CD49b+, CD54+, CD90+, CD105+, CD106+-, CD117+-, HLA- ABC+-, HLA-DR-. Cells were transferred to surface-treated plastic culture flasks using the following culture medium: DMEM (or DMEM/F12) 450 ml, L-glutamine 292 mg, thrombolysate (PLTGold®) -50 ml, penicillin 100 U/ml, streptomycin 100 μg/ml, at 100% humidity, temperature 37°С, gas mixture with 5% CO ₂ in air gas mixture.

Subculturing monolayer adhesive cell culture of the cambial layer of the perichondrium is carried out until the second or third passage with a change of medium every 2-3 days. After the third passage, the cells are removed from plastic vials with a 0.05% trypsin-EDTA solution with Hank's salts. The resulting cell suspension is washed from trypsin/EDTA, including using DMEM medium with 10% bovine serum, followed by centrifugation (10 minutes at 200g) and transferred in the amount of 4000 perichondrium cells and 4000 MSCs to each well of the microplate. Cellular aggregates of round shape - spheroids - are formed from cultured cells inside the wells from untreated cultural plastic. Thus, the formation of microtissues - cell spheroids occurs in a scaffold-free 3D cell culture using polystyrene microplate wells with an untreated surface.

For transplantation of spheroids into a living organism, they are removed from the wells of microplates into a sterile tray and transferred to a test tube (the temperature of the solution - the carrier of spheroids is maintained at 37°C all the time), where they settle to the bottom without additional centrifugation. The spheroids are washed from the culture medium. After that, a suspension of spheroids in the F-12 medium, without glutamine, is placed in a syringe and transplanted to the subject into the defect zone through an applicator with a diameter of at least 1 mm2, arthroscopically, at an ambient temperature of 37°C, fibrin glue may be added. The spheroids stick together and stick to the recipient surface in 20-30 minutes while maintaining the temperature in the transplantation area at least 37°C. The limb is immobilized for a day. These spheroids can be used for cartilage bioprinting, both in vivo bioprinting and tissue engineering structures in the laboratory.

Implementation of the invention

Example 1

The perichondrium was taken from the costal cartilage of sheep. Surgical access to the costal cartilage was performed. To separate the perichondrium from the underlying cartilage, a sterile nutrient medium F12 was injected under it with a syringe needle. The severed perichondrium was immersed in a sterile Petri dish with warm nutrient medium F12 (37°C). With constant wetting, the perichondrium was pulled over a sterile wooden block, upwards with the cambial layer. Under the control of an operating microscope, this layer was scraped off with a scalpel blade. Using a pipette, pieces of perichondrium were transferred to a container for enzymatic dissociation. We used 0.1% collagenase II solution (Sigma-Aldrich, USA) in DMEM nutrient medium (Gibco, USA) supplemented with 2 mM L-glutamine (PanEco, Russia) and penicillin-streptomycin, 100-fold solution, working concentration 10 ml/l (100-fold solution) (LLC PanEco, Russia), tissue fragments were incubated in a medium with collagenase in an incubator for 24 hours at a temperature of 37°C.

To obtain a primary culture from the cambial layer, cells isolated from the perichondrium are seeded in special plastic cell culture flasks designed for working with adhesive (attached) cell cultures. For cell expansion, a culture nutrient medium of the following composition is used: DMEM (or DMEM /F12) 450 ml (LLC PanEco, Russia), L-glutamine 292 mg, thrombolysate (PLTGold®) 25 ml (can be increased to 50 ml), penicillin 100 U/ml, streptomycin 100 µg/ml, at 100% humidity, temperature 370°C, gas mixture with 5% CO ₂ .

To obtain a primary culture of cells from the bone marrow, the bone marrow in the form of an aspirate was taken from the ilium, mononuclear cells were isolated on a density gradient by centrifugation (ficoll solution) by a well-known method. Using flow cytometry, it was found that fibroblast-like cells obtained by culturing the mononuclear fraction of bone marrow aspirate had the following phenotype: CD13+, CD34-, CD44+, CD45-, CD49b+, CD54+, CD90+, CD105+, CD106+-, CD117+-, HLA- ABC+-, HLA-DR-, typical for MSCs. The cells were transferred into plastic culture flasks with a treated surface, the following nutrient medium is used for 2D cultivation: DMEM (or DMEM/F12) 450 ml, L-glutamine 292 mg, thrombolysate (PLTGold®) - 25 ml, penicillin 100 units/ml, streptomycin 100 µg/ml, at 100% humidity, temperature 37°C, gas mixture with 5% CO ₂ in air gas mixture inside the vial.

Subculturing monolayer adhesive cell culture of the cambial layer of the perichondrium is carried out until the second or third passage with a change of medium every 2-3 days. After the third passage, the cells are removed from plastic vials with a 0.05% trypsin-EDTA solution with Hank's salts. The resulting cell suspension is washed from trypsin/EDTA, including using DMEM medium with 5% bovine serum, followed by centrifugation (10 minutes at 200g) and transferred in the amount of 4000 perichondrium cells and 4000 MSCs to each well of the microplate (8000 cells per 1 well). ). Cellular aggregates of round shape - spheroids - are formed from cultured cells inside the wells from untreated cultural plastic. Thus, the formation of microtissues - cell spheroids occurs in a 3D cell culture using polystyrene microplate wells with an untreated (non-adhesive) surface. The resulting tissue-engineering construct belongs to the scaffold-free type; inside the spheroid, cells form their own extracellular matrix, which improves cell viability.

For transplantation of spheroids into a living organism, they are removed from the wells of microplates into a sterile tray and transferred to a test tube (the temperature of the solution - the carrier of spheroids is maintained at 37°C all the time). Inside the tube, the spheroids settle to the bottom without additional centrifugation. The spheroids are washed from the culture medium. After that, a suspension of spheroids in the F-12 medium, without glutamine, is placed in a syringe and transplanted to the subject into the defect zone through an applicator with a diameter of at least 1 mm2, arthroscopically, at an ambient temperature of 37 ° C, it is possible to add fibrin glue or gel to the graft, based on hyaluronic acid obtained by a known method (Stevens M.M., Marini R.P., Schaefer D., Aronson J., Langer R, Shastri V.P. In vivo engineering of organs: the bone bioreactor. Proc Natl Acad Sci U S A. 2005 Aug 9; 102 (32): 11450-5). The spheroids stick together and stick to the recipient surface in 20-30 minutes while maintaining the temperature in the transplantation area at least 37°C. The limb is immobilized. These spheroids can be used for in vivo bioprinting of cartilage or fabrication of tissue engineering constructs in the laboratory.

The spheroids obtained from sheep, rabbit, and human cells have a high regenerative potential, which is manifested both in vivo and in organ models of cartilage defects.

While 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 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 clear that various modifications are possible without departing from the essence of the present invention.

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

1. Ming-Feng Hsueh, Patrik Önnerfjord, Michael P. Bolognesi, Mark E. Easley, Virginia B. Kraus. Analysis of "old" proteins unmasks dynamic gradient of cartilage turnover in human limbs. Science Advances, 2019; 5 (10): eaax3203 DOI: 10.1126/sciadv.aax3203

2. Interna F., Van Roermund P. M., Marijnissen A. C. A., Cotofana S., Eckstein F., Castelein R. M., Bijlsma J. W. J., Mastbergen S. C, Lafeber F. P. J. G., Tissue structure modification in knee osteoarthritis by use of joint distraction: An open 1 -year pilot study. Ann. Rheum. Dis. 70, 1441-1446 (2011)

3. Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. Jun 2017; 6(6): 1445-1451. doi:10.1002/sctm. 17-0051. Epub 2017 Apr 28. PMID: 28452204; PMCID: PMC5689741

4. de Windt TS, Vonk LA, Slaper-Cortenbach ICM, Nizak R, van Rijen MHP, Saris DBF. Allogeneic MSCs and Recycled Autologous Chondrons Mixed in a One-Stage Cartilage Cell Transplantion: A First-in-Man Trial in 35 Patients. Stem Cells. 2017 Aug; 35(8):1984-1993. doi: 10.1002/stem.2657. Epub 2017 Jun 23. PMID: 28600828

5. De Angelis E. et al. Platelet lysate reduces the chondrocyte dedifferentiation during in vitro expansion: Implications for cartilage tissue engineering// Res Vet Sci. Dec 2020; 133:98-105. doi: 10.1016/j.rvsc.2020.08.017. Epub 2020 Sep 6.

6. Cesarz Z, Tamama K. Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int. 2016; 2016:9176357. doi: 10.1155/2016/9176357. Epub 2015 Nov 16

7 Wang T. et al. Modulating stem cell-chondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels// J. Mater. Chem. B, 2016, 4, p. 7641-7650, DOI: 10.1039/c6tb01583b

8. Stevens M.M., Marini R.P., Schaefer D., Aronson J., Langer R, Shastri V.P. In vivo engineering of organs: the bone bioreactor. Proc Natl Acad Sci USA. 2005 Aug 9; 102(32):11450-5.

Claims (7)

1. A method for biofabrication of a transplant in the form of cell spheroids for regenerative technologies for restoring the subject's cartilage based on the perichondrium cells of the subject's own costal cartilage and multipotent mesenchymal stromal cells of the bone marrow of the same subject, characterized in that adhesive cells are isolated from the cambial layer of the perichondrium of the subject's own costal cartilage, adhesive stromal cells are isolated from the subject's own bone marrow, the isolated cells are cultivated separately in adhesive 2D cultures without chondrogenic differentiation medium, while in order to obtain a primary culture, the isolated cells are seeded in plastic vials with a culture medium of the following composition: DMEM or DMEM /F12 450 ml , L-glutamine 292 mg, PLTGold® Human Platelet Lysate, Merck - 50 ml, penicillin 100 U/ml, streptomycin 100 µg/ml, at 100% humidity, temperature 37°C, gas mixture with 5% CO ₂ , transfer resulting cells to NanoCulture® 3D Culture Plates Corning® 1536-well Ultra-low Attachment Spheroid Microplate, with Lid, Sterile Corning® 1536-well Black/Clear Round Bottom Ultra-low Attachment Spheroid Microplate, with Lid, Sterile and subsequent 3D cultivation with the formation of cell spheroids, while producing a mixed cultivation of perichondrium cells and MSCs in a ratio of 1:1 in the amount of 8000 cells per well. 2. The method of claim 1 wherein the subject is a human. 3. The method of claim 1, wherein the cartilage is articular cartilage, cartilage of the nose, larynx, trachea, bronchi, auricle, or intervertebral discs. 4. The method according to claim 1, in which the cultivation of adhesive stromal cells from the subject's own bone marrow, as well as separate cultivation of the isolated adhesive cells from the cambial layer of the perichondrium of the subject's own costal cartilage, is carried out for 20-30 days. 5. The method according to p. 1, in which the transfer of cells at the stage of separate cultivation of the isolated cells is carried out at the rate of 8000 cells of the perichondrium and MSCs in a ratio of 1:1 per 1 well of the agarose plate. 6. The method according to p. 1, in which the fusion of cells into cell spheroids is carried out in the wells of a plastic microplate, where the cells stay up to 6 days. 7. The method according to claim 1, in which the size of the spheroids is 420-450 microns.