JP2011041472A - Spheroid for cell transplantation treatment composed of cell mixture and method for preparing the same - Google Patents

Abstract

To provide a spheroid for cell transplantation treatment comprising a cell mixture, which can be effectively and substantially used clinically, and a method for producing the same.
A spheroid for cell transplantation treatment comprising a mixture of cells expressed as cartilage-like tissue.
[Selection] Figure 10

Description

  The present invention relates to a spheroid for cell transplantation treatment comprising a cell mixture for use in the fields of cartilage tissue, bone tissue biology, medicine, and the like, and a method for producing the same. In the present invention, each cell material that can constitute a cell mixture that is expressed as a cartilage-like tissue is prepared for separation, and each separated cell is subcultured and expanded, and then one or more types are prepared. Cell spheroids for cell transplantation treatment by preparing a cell suspension by shaking or culturing the cells in a culture solution alone or mixed, and then culturing them by shaking. Isolate. The separated spheroids for cell transplantation treatment can be moved to the damaged area and subjected to cell transplantation treatment.

  Japan, South Korea, and countries in Europe and the United States have already entered an aging society, and the average life expectancy is at the highest level in the world. People's hope has come to focus on the quality of life (QOL) of living better and healthier than just prolonging life. One of the attentions is motor dysfunction. Arthritis that causes motor dysfunction includes various diseases, but in the United States, over 70 million patients in the United States complained of some arthritis or chronic joint symptoms in 2002. This is expected to reach 1 in 3 adults and double by 2020. It is the second most common cause of problems in daily life after heart disease, and costs 86.2 billion dollars a year. The most common cause of knee osteoarthritis in an aging society is one of the main causes, and it is estimated that there are 24 million people in Japan alone in a survey conducted by the University of Tokyo's 22nd Century Medical Center. . There are many patients in Japan, with a prevalence of 30% at the age of 45 to 65 and 63 to 85% at the age of 65 and over, and it is believed that 900,000 new patients occur each year. Unlike organ diseases, musculoskeletal diseases such as osteoarthritis deprive human limbs and reduce their QOL significantly. These musculoskeletal diseases are expected to increase with the aging of the future, and human and social losses due to such disabilities are extremely large.

  Most of these musculoskeletal diseases are caused by inflammation or damage of cartilage tissue and bone tissue. Currently, in the case of severe diseases, artificial joints made of metal and ultra-high molecular weight polyethylene are used for the treatment. However, there is a limit to the application patient because it wears after 10 years or more after implantation and various undesirable biological reactions are caused by the abrasion powder. In order to solve these problems, studies have been made to improve the wear resistance, but a limit is predicted in the wear resistance. In recent years, a number of techniques for culturing cells in vitro and applying the cultured cells or tissues to patients have been reported as new solutions. In this case, the cells or tissues may be used alone, or one or two or more biocompatible materials or scaffolds for tissue regeneration formed of artificial biomaterials made of bioabsorbable materials, or a culture solution or transportation. The treatment of cartilage tissue and bone tissue using tissue engineering technology and regenerative medicine, which is used as a structure constructed at the same time as a solution such as a solution and regenerates the tissue in vivo by self-healing ability, has attracted attention. . As a method for this treatment, a method of transplanting cartilage cells or bone cells cultured in the affected area, and cartilage tissue and bone tissue produced therefrom is considered.

  In 1994, Brittberg et al. Collected articular cartilage tissue from the non-overweight part of the joint, cultured the isolated cartilage tissue cell, and transplanted it into the osteochondral full thickness defect part of the overweight part (autologous chondrocyte transplantation, Autologous Chondrocyte Implantation) (Brittberg et al., New England Journal of Medicine, 331 (14), 889 (1994)) has been approved by the FDA in 1997, has been commercialized, and over 20,000 cases worldwide Has been implemented in a number of cases. The mid- to long-term results of 219 cases after 2 to 10 years were good, and 89% showed functional improvement (Peterson L, 6th Annu. Meet., American Academic Orthopaedic Surgery (1998)). On the other hand, there were reports of death cases due to bacterial infection after transplantation in 2002, and 41 postoperative infection cases were found in the CDC survey. I was reminded that there are case examples and there are some issues that should be treated carefully. In addition, this method cannot be used for the treatment of osteoarthritis with a wide range of cartilage tissue and bone tissue degeneration and partial defects that frequently occur in the elderly, and has been demanded to be improved.

  In Japan, chondrocytes were produced by tissue engineering using chondrocytes isolated from non-overweight articular cartilage and bone marrow-derived mesenchymal stem cells. Yes. However, these clinical application examples are traumatic osteochondral injury and isolated osteochondritis, and the range of cartilage defect was originally limited to only small cases (Japanese Patent Application No. 2001-384446 (Japanese Patent Application Laid-Open No. 2001-384446)). No. 2003-180819), Japanese Patent Application No. 2002-216561 (Japanese Patent Laid-Open No. 2003-111831), Japanese Patent Application No. 2003-358118 (Japanese Patent Laid-Open No. 2004-136096) Currently, the treatment results of artificial joint replacement are stable. Therefore, it cannot be said that it has entered into the treatment of osteoarthritis with extensive cartilage and bone tissue degeneration and partial defects, and most of these technologies other than those produced from cultured cells. This technology requires scaffolding of proteins, sugars or artificial biomaterials. The effects on the living body such as in-vivo compatibility are being taken up as problems, and there is a problem that clinical application is not easy in practice. Development is anxious.

  On the other hand, Hunziker et al. Who are seriously working on the treatment of osteoarthritis focusing on the fact that osteoarthritis is caused by degeneration of cartilage and partial defects that do not reach the subchondral bone, We are conducting basic research using an articular cartilage partial defect model. At this time, we have found that the central role of cartilage repair and regeneration is not chondrocytes but synovial cells (Hunziker et al., The Journal of Bone and Joint Surgery, 78-A, 721 (1996)). However, since this technique is also not practical because it has a narrow range of treatment, the discussion here is not directly related to the treatment of osteoarthritis. The establishment of early treatment technology was eagerly desired.

  The existence of adult stem cells has been confirmed in each tissue, and the localization of mesenchymal stem cells is being elucidated from all tissues. In particular, synovial cells and bone marrow cells are differentiated into tissues expressed as cartilage-like tissues by using mesenchymal stem cells that retain the ability to differentiate into chondrocytes in vitro and in vivo. Many studies have been reported on successful reconstruction at. Adult stem cells can be differentiated and regenerated into all tissues constituting the living body, and have a much higher proliferation ability than cultured somatic cells derived from general tissues in vitro. Such mesenchymal stem cells possessing an extremely high proliferation rate are advantageous for developmental and differentiation into cartilage tissue. On the other hand, Sekiya et al. Have confirmed good regeneration and repair of cartilage tissue defect sites in vitro or in vivo using synovial cells (Sekiya et al., Stem cell, 25, 689 (2007). )). Chen et al. Have confirmed differentiation into cartilage tissue as a result of co-culture of synovial cells with nucleus pulposus cells (Chen et al., Spine, 34-12, 1272 (2009)). Anderer et al. Succeeded in cartilage regeneration without using cartilage differentiation-inducing medium supplemented with proteins such as growth factors other than those produced from cultured cells using normal culture medium for differentiation into cartilage tissue (Anderer et al., Journal of Bone and Mineral Research, 17-8, 1420 (2002)).

  In view of these research results, mesenchymal stem cells were selected as a material to be used for the preparation of spheroids for cell transplantation treatment of cell mixtures. In particular, the ability to proliferate mesenchymal stem cells under normal cell culture conditions and the ability to easily differentiate into cartilage tissue is intended for cell transplantation treatments of a predetermined target amount within a short period of time. Spheroids can be produced. On the other hand, cells and tissues that have been prepared and transplanted in vitro for repairing cartilage or bone defects, or cartilage tissue and bone tissue using tissue engineering technology / regenerative medicine have been developed from almost one type. That was the mainstream.

Brittberg et al., New England Journal of Medicine, 331 (14), 889 (1994) Peterson L, 6th Annu. Meet., American Academic Orthopedic Surgery (1998) Hunziker et al., The Journal of Bone and Joint Surgery, 78-A, 721 (1996) Sekiya et al., Stem cell, 25, 689 (2007) Chen et al., Spine, 34-12, 1272 (2009) (Anderer et al., Journal of Bone and Mineral Research, 17-8, 1420 (2002))

JP 2003-180819 A JP 2003-111831 A Japanese Patent Application Laid-Open No. 2004-136096

  This inventor made it the subject which should be solved to provide the spheroid for cell transplantation treatment which consists of a cell mixture which can be utilized efficiently and substantially to clinical, and its manufacturing method.

  The present inventors diligently studied to solve the above-mentioned problems, prepared to separate each cell material constituting a spheroid for cell transplantation that is expressed as a cartilage-like tissue, and subcultured each separated cell. After one or two or more types of cells were grown in a single or mixed manner in a culture solution, a cell suspension was prepared, and the cells were attached to each other by culturing with shaking. The present inventors have succeeded in producing a spheroid for cell transplantation treatment by preparing a cell mixture and separating the thus produced cell spheroid for cell transplantation treatment. The spheroids for cell transplantation treatment of the present invention can be used for cell transplantation treatments, and further can be used as a system for evaluating physiological effects such as pharmacological effects and toxicity of compounds, drugs or toxicants. In the present invention, each cell material constituting the spheroid for cell transplantation treatment is not only a single use of chondrocytes, but also shows any proliferating ability and any stem cell or cartilage having an ability to easily differentiate into cartilage tissue. It is also possible to prepare a large amount of spheroids for cell transplantation expressed as cartilage-like tissue by using progenitor cells, etc., and the extent of extensive osteochondral damage and damage that occurs frequently in elderly people is the surface layer of cartilage tissue The present invention provides a spheroid for cell transplantation treatment that is extremely useful in establishing a treatment technique for partial defects that do not reach the subchondral bone from the end.

That is, according to the present invention, the following inventions are provided.
[1] A spheroid for cell transplantation treatment comprising a mixture of cells expressed as cartilage-like tissue.
[2] The cell mixture is chondrocyte, cartilage progenitor cell, synovial cell, synovial stem cell, osteoblast, bone marrow derived cell, living stem cell or mesenchymal stem cell, adipose derived cell, adipose derived stem cell, embryonic stem cell (ES cell) or a spheroid for cell transplantation treatment according to [1], which is a mixture of one or more cells selected from induced pluripotent stem cells (iPS cells) obtained by reprogramming differentiated cells .
[3] The spheroid for cell transplantation according to [1] or [2], wherein the cell mixture is a mixture of cells derived from autologous, allogeneic or heterogeneous tissues.
[4] The spheroid for cell transplantation according to any one of [1] to [3], wherein the size of the cell mixture is 50 to 999 μm.
[5] The method according to any one of [1] to [4], which is used for transplanting a part or all of cartilage tissue to an affected part damaged or missing, or a part of bone tissue damaged or missing. Spheroids for cell transplantation treatment.
[6] The spheroid for cell transplantation according to [5], wherein the cartilage tissue is articular cartilage, meniscus, intervertebral disc, costal cartilage, nasal septum, or auricular cartilage.
[7] The cell according to any one of [1] to [6], which is used for the treatment of arthritis, arthropathy, cartilage injury, osteochondral injury, meniscal injury, intervertebral disc degeneration, or osteoarthritis. Spheroids for transplantation treatment.
[8] The spheroid for cell transplantation according to [1], which is used for evaluating the efficacy or toxicity of a test substance.

[9] A method for producing a spheroid for cell transplantation treatment, comprising the following steps (1) to (4).
(1) A step of preparing for separation of one or more cells constituting the cell mixture;
(2) a step of subculturing and proliferating the cells isolated in step (1);
(3) One or two or more types of cells grown in step (2) are singly or mixed in a culture solution to prepare a cell suspension, and the cells are maintained in a high-density floating state and cultured. A step of producing a cell mixture in which cells are adhered to each other; and (4) a step of separating the cell spheroids produced in step (3):
[10] The cells prepared for separation in step (1) are chondrocytes, cartilage precursor cells, synovial cells, synovial stem cells, osteoblasts, bone marrow derived cells, living stem cells or mesenchymal stem cells, adipose derived cells [9], one or more cells selected from adipose-derived stem cells, embryonic stem cells (ES cells), or induced pluripotent stem cells (iPS cells) obtained by reprogramming differentiated cells Of producing spheroids for cell transplantation treatment.
[11] For cell transplantation treatment according to [9] or [10], in step (1), the target tissue is chopped and chopped, pulverized, and then treated with a protease to isolate cells. A method for producing spheroids.
[12] The target tissue is provided with a concave bottom, and has a side wall that is long enough to scatter away from the receiving equipment where the target tissue is located when cut during shredding. The method for producing a spheroid for cell transplantation according to [11], wherein the spheroid is placed in the bottom of the container and chopped by using a curved blade for operation longer than the long axis of the side surface of the container.
[13] The cell transplantation treatment according to [11] or [12], wherein the target tissue is digested with stirring using an electromagnetic stirrer under conditions of 37 ° C. and 5% CO ₂ when treated with a protease. For producing spheroids.
[14] The cell transplant according to any one of [9] to [13], wherein in the step (2), when the cells are chondrocytes and cartilage precursor cells, the number of subcultures is 3 or less. A method for producing therapeutic spheroids.
[15] In the step (3), when the culture in which the cells are maintained in a high-density floating state is caused by the flow of the culture solution resulting from the continuous repetitive motion resulting from the artificial manipulation, each cell is placed at the bottom of the culture vessel. The method for producing a spheroid for cell transplantation treatment according to any one of [9] to [14], wherein the suspension is a suspension culture that can maintain a suspended state in a culture solution without remaining, during the period of shaking culture.
[16] The method for producing a spheroid for cell transplantation according to [15], wherein the shaking culture is performed for 1 to 7 days.

[17] The cell transplantation treatment according to [15] or [16], wherein the cell density of the cell suspension in the shaking culture is 1 × 10 ⁴ cells / ml to 3,000 × 10 ⁴ cells / ml. A method for producing spheroids.
[18] In the step (3), when the cell suspension is subjected to shaking culture, the cells are favorably adhered to each other, the cell spheroid formation efficiency is improved, and the expression of the phenotype to a cartilage-like tissue suitable for transplantation is enhanced. The method for producing a spheroid for cell transplantation treatment according to any one of [9] to [17], which comprises one or more microcarriers for maximizing the therapeutic effect due to.
[19] A microcarrier is a molecule for promoting mutual adhesion between cells in a cell suspension, and maintains good adhesion between each cell and the microcarrier. [18] 8 A method for producing a spheroid for cell transplantation treatment according to 1.
[20] The microcarrier is collagen, collagen derivative, hyaluronic acid, hyaluronic acid derivative, rubricin, mucin, chitosan, chitosan derivative, polyrotaxane, polyrotaxane derivative, chitin, chitin derivative, urethane, cellulose, agarose, Gelatin, fibronectin, fibrin, platelet-rich plasma, heparin, heparin derivatives, Fragmin (generic name: dalteparin sodium), protamine sulfate, Avidin, Streptavidin, Biotin, laminin, 2-Octyl Cyanoacryleate , Calcium alginate, polylactic acid, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polypropylene, polyglycolic acid, polycaprolactone, polylactic acid polyglycolic acid copolymer, [18] or [19] formed of one or more biocompatible materials or bioabsorbable materials selected from the group consisting of a polylactic acid polycaprolactone copolymer and a polyglycolic acid polycaprolactone copolymer ] The manufacturing method of the spheroid for cell transplantation treatment of description.
[21] In the step (3), primary cells spheroids in which cells are adhered to each other are produced by culturing the cells while maintaining the cells in a high-density floating state, and then further focusing on the primary cell spheroids. Secondary cells in which the primary cell spheroids and the additional cells are adhered to each other are prepared by mixing one or more types of cells in a culture solution to prepare a cell suspension and then culturing. The method for producing a spheroid for cell transplantation according to any one of [9] to [20], wherein a cell spheroid is produced.
[22] The method for producing a spheroid for cell transplantation according to any one of [9] to [21], wherein the culture solution used in the step (3) is a cartilage differentiation induction medium.
[23] Production of a spheroid for cell transplantation treatment according to any one of [9] to [22], wherein in the step (4), the cell spheroid is shaped to be usable for endoscopic or arthroscopic surgery. Method.
[24] The method for producing a spheroid for cell transplantation according to any one of [9] to [23], wherein the production process of the spheroid for cell transplantation is within 14 days.

  In the spheroid for cell transplantation treatment of the present invention, each cell constituting the cell mixture not only uses a chondrocyte alone, but also exhibits one or more kinds of high proliferative ability, and can easily be applied to cartilage tissue. By using all kinds of stem cells or cartilage progenitor cells having a sufficient differentiation potential, it becomes possible to prepare a large amount of spheroids for cell transplantation expressed as cartilage-like tissue. The spheroid for cell transplantation treatment of the present invention is extremely useful in establishing a therapeutic technique for a wide range of osteochondral damage and partial defects that do not reach from the cartilage surface layer to the subchondral bone, which frequently occur in the elderly. Furthermore, in the present invention, since no periosteum patch is used, the burden on the patient side can be reduced compared to normal autologous cartilage transplantation, and the surgical invasion can be reduced because the incision is minimized.

  On the other hand, the present inventors have so far conducted research on carrier production suitable for cartilage regeneration by tissue engineering techniques, research on construction of optimal extracellular environment, research on cartilage regeneration by allotransplantation of tissue engineering cartilage, In addition, they have obtained results such as research on cartilage repair and regeneration using a chondrocyte sheet and a regenerated cartilage plate that do not use a scaffold of artificial materials. From these results, in order to treat the injury, tissue engineering cartilage component as a minimal cartilage induction initiator necessary for tissue repair and regeneration under the condition that the presence of mesenchymal stem cells mobilized to the damaged site is essential. Has found that the existence of is important. On the other hand, considering the above findings of the present inventors, the partial defect that does not reach from the cartilage tissue surface layer to the subchondral bone has pluripotency and proliferative ability, and possesses the ability to easily differentiate into cartilage tissue. There are many cases where only incomplete treatment with insufficient mobilization of mesenchymal stem cells resulting in insufficient tissue repair and regeneration can be expected. The spheroids for cell transplantation prepared by using mesenchymal stem cells and chondrocytes derived from synovium, bone marrow, etc. as cell materials of the cell mixture according to the present invention satisfy the above-mentioned conditions, and therefore have good tissue repair / regeneration results. I can expect.

FIG. 1 shows an embodiment of the present invention, in which a cell mixture in which cell materials are bonded to each other by preparing a single cell material suspension in a culture solution and culturing with shaking is shown in one embodiment of the present invention. It is a schematic diagram shown. FIG. 2 shows an embodiment of the present invention. In one embodiment of the present invention, two types of cells, cell material A and cell material B, are prepared by mixing and culturing a mixed cell material suspension in a culture solution. It is a schematic diagram which shows the cell mixture made to mutually adhere. FIG. 3 shows another embodiment of the present invention. In another embodiment of the present invention, a plurality of stages of shaking culture are repeated two or more times, and the primary cell spheroid, which is the result of the first shaking culture, is further added to one or two types. A cell suspension of a seed or more is prepared alone or mixed in a culture solution to prepare a cell suspension, followed by second or more shaking culture, thereby producing a cell spheroid as a result of the first shaking culture. It is a schematic diagram which shows the cell mixture formed with the secondary cell spheroid which is a cell mixture which mutually adhered cell materials. FIG. 4 is a schematic view showing a spheroid for cell transplantation of a cell mixture of a transplantation method using an auxiliary sheet-like structure in order to satisfactorily fix the cell mixture to an affected area in one embodiment of the present invention. is there. FIG. 5 shows an example of a transplantation method using an auxiliary sheet-like structure (chondrocyte sheet) for satisfactorily fixing a cell mixture to an affected area in one embodiment of the present invention. It is a figure which shows one example of a schedule about 14 days about the total preparation process of the spheroid for cell transplantation treatments including a culture period. FIG. 6 shows, in one embodiment of the present invention, the ratio of chondrocytes and synovial cell-derived cells, (2) shaking culture of a cell suspension of 75% synovial cell-derived cells and 25% chondrocytes. FIG. 6 is a phase contrast microscopic image showing a cell mixture in which cell materials are bonded to each other. FIG. 7 shows the ratio of chondrocytes and synovial cells derived from one embodiment of the present invention (4) shaking culture of a cell suspension of 25% synovial cells and 75% chondrocytes. It is a figure of the phase-contrast microscope image which shows the cell mixture which made cell material mutually adhere. FIG. 8A shows, in one embodiment of the present invention, a cell suspension of one type of cell (ie, (5) 0% synovial membrane cell / 100% chondrocyte) in a single cell material suspension in a culture solution. Is a diagram showing a cell mixture in which shaking culture is performed while moving the sample in a planar circle and cell materials are adhered to each other. FIG. 8B shows a cell suspension of one type of cell (ie, (5) 0% synovial membrane cell / 100% chondrocyte) in a culture solution according to one embodiment of the present invention. Is a diagram showing a cell mixture in which shaking culture is performed while moving the sample to a plane-left-right type and cell materials are adhered to each other. FIG. 9 shows, in one embodiment of the present invention, the ratio of chondrocytes and synovial cells derived from (1) a cell suspension of 100% synovial cells and 0% chondrocytes, and (2) 75% synovial cells. Cell suspension of 25% chondrocytes, (3) cell suspension of 50% synovial cells, cell suspension of 50% chondrocytes, (4) cell suspension of 25% synovial cells, 75% chondrocytes (5) Phase contrast microscopic image showing a cell mixture in which cell culture is carried out by shaking culture of cell suspensions of (5) 0% synovial cell-derived cells and 100% chondrocytes, respectively. It is. FIG. 10 shows, in one embodiment of the present invention, the ratio of chondrocytes and synovial cell-derived cells (3) shaking culture of 50% synovial cell-derived / 50% chondrocyte cell suspension for 5 days. It is a figure which shows the image which it implemented and observed under the confocal laser microscope. FIG. 11 shows another embodiment of the present invention. In another embodiment of the present invention, a plurality of stages of shaking culture are repeated two or more times, and one or two or more primary cell spheroids, which are the result of the first shaking culture, are used. Cell spheroids as a result of the first shaking culture are prepared by preparing a cell suspension by separately or mixing seeds or more cells in a culture solution, and then culturing the cells by shaking for the second time or more. A phase-contrast microscope when cell materials are prepared simultaneously for the formation of pre-formed primary cell spheroids and secondary cell spheroids, intended to be formed with secondary cell spheroids, which are cell mixtures in which cell materials are bonded together. FIG. FIG. 12 shows macroscopic findings of cell spheroids 12 hours after the start of shaking culture. It was observable macroscopically from 12 hours after the start of the shaking culture, and was observable during the entire shaking culture period.

   The present invention relates to a cell transplantation treatment for the treatment of osteoarthritis, arthritis, arthropathy, cartilage injury, osteochondral injury, meniscus injury or intervertebral disc degeneration comprising a cell mixture that is expressed as a cartilage-like tissue. It relates to spheroids.

  In the present invention, the “cell mixture” means a spheroid for cell transplantation expressed as a cartilage-like tissue, and includes a chondrocyte, a cartilage precursor cell, a synovial cell, a synovial stem cell, a bone derived from a donor tissue Blast cells, bone marrow-derived cells, living tissue stem cells or mesenchymal stem cells derived from any tissue, adipose-derived cells, adipose-derived stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells with all differentiated cells embryonic stem cells One type that has therapeutic effects when transplanted into an affected area such as a cell that has the same pluripotency and proliferative ability as ES cells, such as (iPS cells), and has the ability to easily differentiate into cartilage tissue Alternatively, it may be a cell mixture in which two or more types of cells are mixed. This is because pluripotent cells are undifferentiated cells, and therefore, after transplantation, it is predicted to differentiate into cells corresponding to the transplanted living tissue. The cell mixture is preferably an autologous tissue-derived cell for ideal cell transplantation without rejection or ethical problems, but the cartilage tissue is normally free from blood vessels, nerves, and lymph vessels, and white blood cells It is derived from the same or different types of tissues because it is known as a tissue with low immunogenicity and immune immunity that is unlikely to cause immune rejection during transplantation because invasion of inflammatory cells such as is impossible Or two or more of these cells.

  In the present invention, the spheroid for cell transplantation treatment of a cell mixture preferably has a size in the range of 50 to 999 μm for treatment. The size of spheroids for cell transplantation treatment is preferably about 600 μm, but this is because spheroids composed of general somatic cells are gas under normal culture conditions up to a distance of about 300 μm from the center to the surface layer of the spheroids. In addition, the diffusion of nutrients can be performed more smoothly, the life span of the cell spheroids in vitro can be extended, and the growth can be promoted to further beneficially exert the original function of the implant. Spheroids with a relatively thin cell mixture demonstrate the original function of cell spheroids by allowing more smooth diffusion of gases and nutrients, extending the life of the implant, and maintaining phenotypic expression as cartilage-like tissue This is because it works even more beneficially. On the other hand, cartilage tissue is normally an extracellular matrix secreted mostly from chondrocytes, and only a few percent of chondrocytes are contained therein. In addition, chondrocytes, which are extremely low in metabolism, are expected to have a longer distance from the center of the spheroid to the surface of the spheroid, which allows better gas and nutrient diffusion, than the other cells when in the form of spheroids in the cell mixture. However, those smaller than 999 μm are preferable.

The present invention relates to a method for producing a spheroid for cell transplantation treatment comprising the following steps (1) to (4).
(1) A step of preparing for separation of one or more cells constituting the cell mixture;
(2) a step of subculturing and proliferating the cells isolated in step (1);
(3) One or two or more types of cells grown in step (2) are singly or mixed in a culture solution to prepare a cell suspension, and the cells are maintained in a high-density floating state and cultured. A step of producing a cell mixture in which cells are adhered to each other; and (4) a step of separating the cell spheroids produced in step (3):

  In step (1) of the production method according to the present invention, each cell material constituting the cell mixture is prepared for separation. First, collect each donated tissue that is the source of each cell material that makes up the cell mixture, and remove skin tissue, subcutaneous tissue, muscle tissue, subchondral bone, ligament, meniscus, and other connective tissues After that, the material is chopped by physical means such as homogenizer, pulverizer such as mortar, blender, scalpel, syringe, forceps, ultrasonic device, etc. At this time, as an aseptic device for receiving each corresponding donated tissue, a device such as a culture plate, a resin material device including a centrifuge container, a glass material device including a watch glass, etc. is used. However, when the corresponding provided tissue is an elastic tissue such as a cartilage tissue, the cut tissue may be scattered away from the receiving equipment where the cut tissue is located during the chopping operation. Such a phenomenon may reduce the amount of collected tissue after chopping of the corresponding donor tissue, or return the cut tissue that has been scattered away from the receiving equipment positioned to separate each cell material below chopping. Sometimes it is difficult to carry out shredding for the purpose of increasing the possibility of contamination with bacteria and fungi. This is due to concerns about contamination with bacteria and fungi during primary culture, including the process of isolating and preparing each cell material from the corresponding donor tissue as described above, and aseptic operation is required to prevent such contamination. Because it is done.

  The target tissue is provided with a concave bottom, and when cut during shredding, the target tissue has a side wall portion that is long enough to scatter away from the receiving equipment where the target tissue is located. It is preferably located in the bottom of a container or the like and chopped using curved scissors. Usually, commercially available 50 ml capacity centrifuge containers have side walls that are long enough to prevent the target tissue from splashing away from the centrifuge container when chopping. For example, in the case of elastic tissues such as cartilage tissue Even so, it does not scatter away from the space inside the centrifuge container. In addition, according to the present method, the curved blade for surgery is bent, so that close contact with the concave bottom of a commercially available 50 ml capacity centrifuge container used for cell culture can be easily obtained. In general, the target tissue is affected by gravity and is always located at the concave bottom of the centrifuge container. The cutting motion of the concave bottom position of the centrifuge container of the surgical curved blade that continuously repeats such a phenomenon makes the target tissue gather at the bottom, and the elastic curved cartilage of the powerful surgical curved blade The cutting motion can be sustained, and even if the provided tissue is an elastic tissue such as a cartilage tissue, it can be cut into smaller sizes in a short time. In order to easily achieve the above chopping, it is preferable to use a surgical bending blade longer than the long axis of the centrifuge container.

After finely chopping by chopping, it is treated with at least one proteolytic enzyme selected from neutral protease, trypsin, serine protease, elastase, collagenase, etc., and set to the same temperature as the body temperature of the individual Cells consisting of a corresponding tissue and proteolytic enzyme solution in an environment such as an incubator for cell culture that provides water set to the same temperature as the body temperature of water or an individual body such as a thermostatic water tank that supplies the heated water The suspension is digested without inducing fluid agitation. In the present invention, the temperature and time for treating the proteolytic enzyme vary depending on the type of proteolytic enzyme and the species of the individual, but an electromagnetic stirrer (37 ° C. and 5% CO _{2 in} a normal cell culture incubator) It is preferable to digest with stirring using a magnetic stirrer. The reason can be explained as follows. In this method, a normal cell culture incubator, a sterile glass bottle, and a sterile magnetic bar for an electromagnetic stirrer are used in the process of digestion with proteolytic enzymes, but these cell culture equipment are kept clean. In addition, the cell suspension consisting of the corresponding donated tissue and proteolytic enzyme solution chopped into a sterile glass bottle easily induces agitation due to flow, and slightly covers the glass bottle lid. By loosening and releasing, it can be directly connected to the gas tonality such as temperature and humidity in the space in the incubator through this gap, and the gas tonality in the incubator ideally adjusted for cell culture is applicable. It is stable and advantageous for each cell in the cell suspension composed of the donor tissue and the protease solution.

  In step (2) of the production method according to the present invention, the cells thus separated are subcultured and grown. At this stage, any medium for cell growth or cartilage differentiation induction medium known in the art may be used as the medium, but the components necessary for viable cell growth (inorganic salts, carbohydrates, hormones, essential Basic medium containing amino acids, non-essential amino acids, vitamins (eg, Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture) A basic medium such as F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM)) is used. Ascorbic acid necessary for collagen synthesis is inevitably added to such a medium, and other growth factors or differentiation inducing factors such as FGF (Fibroblast Growth Factor), HGF (Hepatocyte Growth Factor), and IGF (Insulin-like Growth Factor). ), TGF (Transforming Growth Factor), EGF (Erythrocyte Growth Factor), BMP (Bone Morphogenetic Protein), TNF (Tumor Necrosis Factor), vitamins, interleukins, heparin, heparin derivatives, heparan sulfate, collagen, fibronectin, fibrin , Platelet-rich plasma, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement (including insulin, transferrin, selenite, bovine serum albumin, linolenic acid), dexamethasone, sodium bibrate, proline, L-glutamine Etc. are added as necessary. In addition, if necessary, additives for acid / base adjustment such as HEPES, antibiotics such as antibacterial agents and antifungal agents, 10% FBS (fetal bovine serum) which is the percentage during normal use or more If high growth is preferred, 10% or more of FBS may be included, and in view of ease of clinical application, serum derived from the patient's own patient may be contained at 10% to 10% or more.

  In the present invention, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, bone marrow-derived cells, living tissue cells or mesenchymal stem cells derived from any tissue, fat-derived cells, fat-derived stem cells, embryos Has the same pluripotency and proliferative ability as ES cells, such as sex stem cells (ES cells), induced pluripotent stem cells that have initialized all differentiated cells, and embryonic stem cells (iPS cells). Each cell material that has a therapeutic effect by being transplanted to an injured affected area such as a cell having a sufficient differentiation potential is proliferated by repeated subculture for about 2 weeks. Chondrocytes exhibiting a high degree of differentiation are 2 to 20 times or more, pluripotency and proliferation ability similar to those of ES cells, and undifferentiated cells having the ability to easily differentiate into cartilage tissue are 20 to 100 times or more. Preferably, any cell growth medium known in the art that can be used is a medium prepared with 10% FBS, ascorbic acid inactivated by heat.

When the culture is continued to reach about 90% confluency, it is treated with trypsin / EDTA to peel off the proliferated cell material and subcultured in a new medium. In the present invention, the subculture of chondrocytes and cartilage progenitor cells is preferably limited to 3 times or less, but this is because the chondrocytes are dedifferentiated particularly after the fourth subculture by repeated subculture. (De-differentiation), the original properties are lost, and the type of collagen in the extracellular matrix secreted from chondrocytes changes from fibroblast-like, such as from II to I. is there. The subculture of the chondrocytes and cartilage precursor cells in the present invention is a culture in which cells are seeded at a high density. The cell density at the time of seeding of the chondrocytes and cartilage progenitor cells shown in the present invention varies depending on the cells to be cultured, but is preferably 5,000 cells / cm ² or more, preferably 10,000 cells / cm ² or more. More preferably, the density is 20,000 / cm ² or more, and the density at which cartilage tissue or bone tissue is most efficiently regenerated is about (2.0 ± 0.4) × 10 ⁴ / cm ² . In subculture, when the cell density of seeded cells is 1,000 / cm ² or less, the cultured cells often change like the fibroblasts, that is, the degree of phenotypic expression is in the case of chondrocytes. It is weak and cannot achieve the purpose of this technology. One characteristic of the spheroid for cell transplantation treatment of the present invention is that the cell mixture is expressed as a cartilage-like tissue. When expressed as a cartilage-like tissue, it expresses a genetic trait such as SOX9 or HAS, or expresses a differentiation trait such as collagen II involved in matrix formation.

  When chondrocytes are cultured under the above conditions for about 2 weeks (that is, the time required until the number of passages is 4 times or more), they can grow at least 2 to 20 times or more. On the other hand, undifferentiated cells having the same pluripotency and proliferative ability as ES cells and having the ability to easily differentiate into cartilage tissue were isolated for about 2 weeks (that is, there was no limit on the number of continuous passages). The time required for the cells to be subcultured and proliferated) When cultured, the cells can proliferate at least 20 to 100 times or more.

  In addition, any cell growth medium known in the art, 10% FBS inactivated by heat, medium adjusted with ascorbic acid, other growth or differentiation to further increase the growth rate If a factor is added as needed, it can proliferate more than said proliferation.

  In step (3) of the production method according to the present invention, one or two or more types of cells are singly or mixed in a culture solution to prepare a cell suspension, and the cells are maintained in a high-density floating state. By doing so, cell materials are bonded together to produce a cell mixture. In this stage, it is preferable to add an excessive amount of cell material so that the cell mixture sufficiently adheres the cell materials to each other to form a spheroid for cell transplantation treatment. Cells originally have the property of trying to adhere to each other by various adhesion factors that are created by the cells themselves and expressed on the cell surface, or by electrical attraction and chemical bonding, and the tendency varies depending on the type of each cell. In addition to this property, there is an additional shaking motion, which increases the chance of artificial contact between cells. Each cell does not stay at the bottom of the culture container due to the flow of the culture solution resulting from the continuous repetitive shaking motion resulting from such an artificial operation, but during the shaking culture period in a suspended state floating in the culture solution. Can be maintained. Therefore, many other cell materials will quickly adhere to each other centering on the mixed cell complex of small size that has become a cell spheroid in the initial stage of shaking, so that the spheroid for cell transplantation treatment will grow. become. In particular, cells expressed as chondrocytes or cartilage-like tissue return to a state more similar to the original cartilage tissue while maintaining its size when present at a high density. In other words, in an environment where normal cartilage differentiation suitable for cartilage tissue can be maintained, chondrocytes produce and secrete collagen type II, which is a unique extracellular matrix, resulting in secretion in the body. Thus, the number of cells is relatively small, and the extracellular matrix exhibits the same properties as abundant normal cartilage.

In said process (3), each culture medium as described in process (2) of the preparation method by this invention can be utilized. Shaking culture is performed in an environment with the same temperature as the body temperature of the individual by setting the shaking intensity to 60 to 80 rpm using a shaking culture shaker that can be moved to a solid 8-shape, planar circle, or planar left and right. It is preferable. In particular, the shaking culture period is from 1 day to 7 days, and each cell material that has been able to proliferate sufficiently during the subculture period is an existing autologous chondrocyte that requires a cell culture period of 4 weeks or more. The culture period can be shortened compared to transplantation, and eventually, spheroids for cell transplantation treatment can be produced in a short period of time. Such shortening of the period also leads to the establishment of a method that can be applied clinically to elderly patients and patients with a wide range of injuries, etc. From the viewpoint of shortening the hospitalization period of patients and contributing to reducing medical expenses. The culture period is preferably 1 to 3 days, and the total production process of the spheroid for cell transplantation treatment including the subculture period and the shaking culture period is preferably about 14 days. As a culture vessel, a non-adhesive culture dish designed to prevent cells from adhering to the surface of the culture container (that is, a suspension culture dish, for example, HydroCell ^TM which becomes hydrophilic at about 37 ° C. and does not adhere to any cells) Use a culture dish (CellSeed. Co.)) or spinner flask in the case of mass culture. Through the above operation, spheroids for cell transplantation treatment having a size of 50 to 999 μm can be produced. For shaking culture, the cell material suspension is applied at a cell density of 1 × 10 ⁴ cells / ml to 3,000 × 10 ⁴ cells / ml. The cell density of the cell material suspension within the above range will be comprehensively judged according to the type of cell material to be used, the nature of the culture equipment, the space, volume, size, etc. of the culture vessel during shaking culture. May be set.

  In the step (3), the microcarrier is a molecule for promoting mutual adhesion between cells in the cell material suspension, and has good adhesion to each cell and the microcarrier. Hold. By utilizing this microcarrier, it becomes possible to enhance the expression of a cartilage-like tissue suitable for transplantation by improving the mutual adhesion between cell materials, improving the efficiency of cell spheroid formation, during shaking culture. The microcarriers may be used alone or in combination of two or more as required. When two or more kinds are used in combination, the different properties of the two microcarriers may react to serve as new microcarriers that exhibit further beneficial properties. For example, among the following, Fragmin and Protamine Sulfate form microcarriers due to different electrical charges, and this is due to the good adhesion between cells and said microcarriers (Nakamura et al., J Biomed Mater Res A , May-12, Epub ahead of print (2009)).

  Examples of the microcarrier include collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, lubricin, mucin, chitosan, chitosan derivatives, polyrotaxane, polyrotaxane derivatives, chitin, chitin derivatives, urethane, cellulose, agarose , Gelatin, fibronectin, fibrin, platelet-rich plasma, heparin, heparin derivatives, Fragmin (generic name: dalteparin sodium), protamine sulfate, Avidin, Streptavidin, Biotin, laminin, 2-Octyl Cyanoacryleate, calcium alginate, polylactic acid, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polypropylene, polyglycolic acid, polycaprolactone, polylactic acid polyglycolic acid copolymer Those formed of one or two or more biocompatible materials or bioabsorbable materials selected from the group consisting of a polymer, a polylactic acid polycaprolactone copolymer and a polyglycolic acid polycaprolactone copolymer are preferred. In addition, the microcarrier is clinically used by related organizations from the viewpoint of making a spheroid for cell transplantation treatment of a cell mixture that can be effectively and substantially used clinically and easily used in a treatment method using the spheroid. In this case, it is preferable to obtain permission and authorization.

  In the above step (3), the shaking culture will be described below with reference to FIG. 1, FIG. 2, and FIG. Cell material can be obtained by preparing and culturing a cell material suspension of a single type or two or more types (cell material A and cell material B in FIG. 2) alone or mixed in a culture solution. The cells can be adhered to each other, and the cell mixture can be subjected to shaking culture only once in the production process, or multiple stages of shaking culture that are repeated two or more times. When performing multi-stage shaking culture, the primary cell spheroid, which is the product of the first shaking culture, is used as a center, and one or more cells are further singly or mixed in the culture solution. A secondary cell that is a cell mixture in which cell spheroids and cell materials, which are the result of the first shaking culture, are adhered to each other by preparing a cell suspension and subsequently culturing the cells for the second or more times. A new cell mixture can be prepared with spheroids. Thus, by preparing a cell suspension in which the cell material is further mixed in the culture solution with the primary cell spheroid as the center, and subsequently performing the second shaking culture, the result of the first shaking culture is as follows. Immediately after adding the primary cell spheroid and the cell material of the second shaking culture in the process of producing a new cell mixture with the secondary cell spheroid, which is a cell mixture in which the product cell spheroid and cell material are adhered to each other The result of observing this image under a phase contrast microscope is shown in FIG.

  By culturing for the second time or more, it becomes possible to artificially change the structure of the resulting cell spheroid. For example, if the cell material of secondary cell spheroid closer to the surface layer is chondrocyte and the cell material of primary cell spheroid located deeper is synovial cell, the synovial cell having high proliferation rate In addition to the formation of primary cell spheroids that represent most of the cell spheroids, in the case of relatively elderly patients, chondrocytes with a lower growth rate can be prepared with less cellular material within a limited culture period. Even if not, it is sufficient to form the secondary cell spheroids described above, and therefore comprehensively determined in terms of the proliferative ability of each cell material, anatomy, histology and embryology in vivo, A production method may be selected in consideration of the types and combinations of the cell materials for the multi-stage shaking culture.

  In step (4) of the production method according to the present invention, cell spheroids produced for cell transplantation treatment are separated. The separated cell spheroids can further move to the affected area. In the above step (3), when the shaking culture is performed for 1 to 7 days, various sizes of spheroids for cell transplantation treatment are present in the culture dish. In order to separate them simply and according to size, in one embodiment, using a sterilized micropipette tip (Tip), cell transplantation treatment with a micropipette while observing under a phase contrast microscope A method of inhaling spheroids for use and replacing them with a new culture dish (floating culture dish) can be used. At this time, the culture medium and the cell suspension used in the process of producing the spheroid for cell transplantation treatment are collected using a micropipette tip so that the culture dish is moved in a circular motion and collected by the centrifugal force. Inhalation of spheroids for transplantation treatment of appropriate size according to the diameter of the chip when inhaled at the same time as the transport solution etc., which are similar to body fluids such as suspension, microcarrier-containing solution, various buffers or physiological saline be able to. Similarly, a spheroid for transplantation treatment may be prepared in a form that can be used for endoscopic or arthroscopic surgery in the process of transplanting cells into an affected area using an injection needle and syringe.

  When the produced cell spheroids are moved to place the cell spheroids at the transplant site, which is the site of injury, these cells are formed by the cells that also make up the surface of these cell spheroids. It has the property of trying to adhere to each other by various adhesion factors expressed on the cell surface, electrical attraction and chemical bonds. Therefore, the cell spheroids whose surfaces are constituted by such cells can be easily combined with each other even in the vicinity of adjacent cell spheroids. In addition, the microcarrier may be used when better fixation of the cell mixture to the damaged affected part and quick binding between the transplanted affected part and the cell spheroid in a short period of time are desired. Microcarriers include collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, lubricin, mucin, chitosan, chitosan derivatives, polyrotaxane, polyrotaxane derivatives, chitin, chitin derivatives, urethane, cellulose, agarose, gelatin, fibronectin , Fibrin, platelet-rich plasma, heparin, heparin derivatives, Fragmin (generic name: dalteparin sodium), protamine sulfate, Avidin, Streptavidin, Biotin, laminin, 2-Octyl Cyanoacryleate, calcium alginate , Polylactic acid, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polypropylene, polyglycolic acid, polycaprolactone, polylactic acid polyglycolic acid copolymer, polylactic acid poly It is preferably formed of one or more biocompatible materials or bioabsorbable materials selected from the group consisting of a licaprolactone copolymer and a polyglycolic acid polycaprolactone copolymer. The material of the microcarrier can be appropriately selected in consideration of the properties of the microcarrier, the configuration of the spheroid for cell transplantation treatment, the environment of the transplantation site, and the like.

  In addition to the use of a single microcarrier used to fix the above cell mixture to the damaged affected area, the transplanted site including the damaged site is provided as described above, considering the flat shape of the damaged site and the surrounding site. Tissue-derived chondrocytes, cartilage precursor cells, synovial cells, synovial stem cells, osteoblasts, bone marrow-derived cells, living tissue cells or mesenchymal stem cells derived from any tissue, adipose-derived cells, adipose-derived stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells in which all differentiated cells are initialized, embryonic stem cells (iPS cells), etc. have the same pluripotency and proliferative ability as ES cells, and can be easily applied to cartilage tissue A culture in which cells having differentiating potential have a therapeutic effect by transplanting to the affected area, cultured for a certain period from one or more cells, and composed of the cells and extracellular matrix in a sheet form Cell Alone or the cultured cell sheet and collagen, collagen derivative, hyaluronic acid, hyaluronic acid derivative, rubricin, mucin, chitosan, chitosan derivative, polyrotaxane, polyrotaxane derivative, chitin, chitin derivative, urethane, cellulose, Agarose, gelatin, fibronectin, fibrin, platelet-rich plasma, heparin, heparin derivatives, fragmin (general name: Dalteparin sodium), protamine sulfate, Avidin, Streptavidin, Biotin, laminin, 2- Octyl Cyanoacryleate, calcium alginate, polylactic acid, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polypropylene, polyglycolic acid, polycaprolactone, polylactic acid polyglycolic acid A sheet formed of an artificial biomaterial of one or more biocompatible materials or bioabsorbable materials selected from the group consisting of a polymer, a polylactic acid polycaprolactone copolymer, and a polyglycolic acid polycaprolactone copolymer A structure or various shaped structures may be supplementarily coated or supplemented on the surface of cartilage or bone tissue. Here, FIG. 4 shows an example of a mode in which the cell mixture is used in an auxiliary manner by using a sheet-like structure in order to satisfactorily fix the cell mixture to the damaged area.

  In the present invention, cell transplantation is first a cellular component of a spheroid for cell transplantation treatment in a cell mixture, but a molecule secreted from the constituent cells in the cell spheroid and a production extracellular matrix, and a process for producing the spheroid for cell transplantation treatment Is used to transfer the culture solution and the cell suspension, the microcarrier-containing solution, various buffers or physiological solutions such as physiological saline used in the preparation to the wounded lesion for treatment at the same time. Cell transplantation treatment that can exert its original function as expressed in a tissue is possible.

  In another aspect of the present invention, the spheroid for cell transplantation treatment can be used with a system for evaluating the physiological effect and toxicity of a medicinal effect and toxicity of a compound, drug, toxicant, etc. separately from the purpose of treatment. Is possible. The use of the spheroid for cell transplantation treatment is not particularly limited, and it can be used for all tests / researches using cell spheroids other than the purpose of treatment and treatment of diseases using cell spheroids. Can do. For example, all adult stem cell spheroids obtained by the method of the present invention are treated with factors such as IGF, TGF, transferrin, insulin, FBS, GA-1000, ITS-supplements, dexamethasone, ascorbic acid, sodium pyruvate, proline and the like. Thus, differentiation into chondrocytes can be induced, and stem cell therapy by autologous cell transplantation can be achieved by returning the differentiated cells thus obtained to the patient. However, the use of the spheroid for cell transplantation treatment of the present invention is not limited to the above specific embodiment.

  In another aspect of the present invention, a plurality of stages of shaking culture that are repeated two or more times, and one or more kinds of primary cell spheroids, which are the product of the first shaking culture, are used. Cells are singly or mixed in a culture solution to prepare a cell suspension, followed by second or more shaking cultures. As a result of the first shaking culture, the resulting cell spheroids and cell materials It becomes possible to produce a new cell mixture with a secondary cell spheroid which is a cell mixture in which the two are adhered to each other. By applying multi-stage shaking culture with such characteristics, cell spheroids can build up multiple layered improvements.

  For example, when the knee cartilage tissue is observed from the viewpoints of anatomy, histology, and embryology, the shape of the chondrocytes constituting the knee cartilage tissue changes as it reaches the deep part from the surface toward the bone marrow. It is considered that the chondrocytes having different shapes have different properties and functions. By creating a cell spheroid structure with different cells for each layer, it is possible to construct a very similar environment in the living body. Evaluation of the pharmacological effects and toxic physiological effects and toxicity of compounds, drugs, toxicants and the like by the above-described system using the cell spheroid structure produced in this way can be more accurately evaluated and predicted.

  The present invention will be described more specifically by the following examples, but the scope of the present invention is not limited by these examples.

Example 1: Preparation of cell spheroids for cell transplantation treatment using a mixture of rabbit knee articular chondrocytes and membrane-derived cells (1-A) Isolation and culture of chondrocytes and synovial cells Rabbit (Japanese White Rabbit, After collecting cartilage tissue and synovial tissue from 1 kg ± 200 g (female) knee and removing skin tissue, subcutaneous tissue, muscle tissue, subchondral bone, ligament, meniscus, and other connective tissue, It was usually placed inside a commercially available 50 ml capacity centrifuge container, chopped using a surgical blade, and chopped. Then, 1.25% trypsin (Invitrogen Co.) or 0.5% collagenase I (collagenase class I: Worthington, Biochemical Co.) is added to D-MEM / F-12 medium (Gibco. Co.), 1% antibacterial. Dissolved in a medium prepared with an agent / antifungal mixture (10,000 units / ml penicillin G, 10,000 μg / ml streptomycin sulfate and 25 μg / ml amphotericin B: Invitrogen Co.) and 50 μg / ml ascorbic acid The digested solution was adjusted to prepare a primary digestive enzyme solution and a secondary digestive enzyme solution. Cartilage tissue and synovial tissue are treated with each of the digestive solutions described above and received in a sterile glass bottle, and at the same time a sterile magnetic bar for a magnetic stirrer is placed. Thereafter, digestion is performed with stirring at 60 rpm using an electromagnetic stirrer (model name: Magnetic Stirrer RCN-3D, EYELA Co.) under conditions of 37 ° C. and 5% CO _{2 in} a normal cell culture incubator. At this time, digestion for 1 hour with the primary digestive enzyme solution and digestion for 3 hours with the secondary digestive enzyme solution were performed to separate the cartilage tissue and synovial tissue into single cells. The cell suspension thus obtained was continuously passed through a 70 μm and 40 μm nylon cell-strainer (BD Falcon ^™ : BD Biosciences), and then washed twice using a phosphate buffer.

Single cells obtained by such a process were seeded and cultured in a growth medium in order to increase the number of chondrocytes and synovial cells. Cell density of the first time (2.0 ± 0.4) × 10 ⁴ in / cm ² seeded, followed cell density in subculture is (2.0 ± 0.4) × 10 ⁴ / cm ² seeding did. Also, 10% FBS (fetal bovine serum), 1% antibacterial / antifungal mixture (10,000 units / ml) inactivated by heat in D-MEM / F-12 medium (Gibco. Co.) Of penicillin G, 10,000 μg / ml streptomycin sulfate and 25 μg / ml amphotericin B: Invitrogen Co.) and 50 μg / ml ascorbic acid, and reaching 90% confluency, Chondrocytes grown by treatment with trypsin / EDTA were detached from the culture dish.

  As a cell material for producing a spheroid for cell transplantation treatment of a cell mixture, a chondrocyte is subcultured twice in a subculture period of 12 to 13 days, and a synovial cell is subcultured up to 4 times. 3rd passage chondrocytes and 4th to 5th passage synovial cells were used.

(1-B) Tagging of fluorescent reagent to isolated cultured chondrocytes and synovial cell-derived cells Each isolated cultured chondrocyte and synovial cell recovered by (1-A) above is D-MEM / F-12. Medium (Gibco. Co.) was mixed with 1% antibacterial / antifungal mixture (10,000 units / ml penicillin G, 10,000 μg / ml streptomycin sulfate and 25 μg / ml amphotericin B: Invitrogen Co.) and Resuspend in a medium adjusted with 50 μg / ml ascorbic acid and centrifuge (1,800 rpm / 5 min, 24 ° C.) to target the number of chondrocytes and synovial cell chondrocytes. Preparations for tagging of photoreagents were made using a PKH staining kit (Sigma Co.) for tracking over time under a confocal laser microscope. Chondrocytes are performed according to the protocol using red fluorescent tag MINI26, and synovial cells using green fluorescent tag MINI67KIT (Fluorescent Cell Linker Mini Kit for General Cell), so that chondrocytes and synovial cells can be compared with each other. I made it. By this operation, cell suspensions of chondrocytes and synovial cells derived from fluorescent reagents were prepared.

(1-C) Preparation of cell mixture by shaking culture Chondrocytes and synovial cells derived from the appropriate number of passages and tagged with a fluorescent reagent, adjusted according to the above (1-A) and (1-B) Of 10% FBS (fetal bovine serum), 1% antibacterial / antimycotic mixture (10%) in which D-MEM / F-12 medium (Gibco. Co.) was inactivated by heat. , 000 units / ml penicillin G, 10,000 μg / ml streptomycin sulfate and 25 μg / ml amphotericin B: Invitrogen Co.) and 50 μg / ml ascorbic acid, and resuspended in 5 ml. the number of cells and synovial-derived cells were adjusted 600 × 10 ⁴ / 5ml. Such chondrocytes and synovial cell-derived cells are further used alone or in a ratio determined by chondrocytes and synovial cell-derived cells (that is, (1) a cell suspension of 100% synovial cell and 0% chondrocytes, ( 2) 75% synovial cell / 25% chondrocyte cell suspension, (3) 50% synovial cell / 50% chondrocyte cell suspension, (4) 25% synovial cell / 75 Cell suspension containing (5) cell suspension of% chondrocytes, (5) cell suspension of 0% synovial cell and 100% chondrocytes). As a culture vessel, a HydroCell ^™ culture dish (CellSeed. Co.) having a diameter of 60 mm was used, and a shaker for shaking culture (model name: 37 ° C., 5% CO ₂ ) in a normal cell culture incubator. Using a double shaker NR-3, TAITEK Co.), the cells were cultured at 37 ° C. for 1 to 5 days while applying a vibration of 70 rpm while performing planar circular movement. The total production process of the spheroids for cell transplantation treatment including the subculture period and the shaking culture period was about 14 days. Here, the schedule is shown in FIG. 5 by taking as an example a mode of using an auxiliary sheet-like structure (chondrocyte sheet) in order to satisfactorily fix the cell mixture to the damaged area.

  Fluorescent filter that selectively passes only a suitable range of wavelengths through the phase contrast microscope when observing the cell spheroids collected during each culture period under the phase contrast microscope and the chondrocytes and synovial cells derived from the fluorescent reagent. Additional observations were made and the results are shown in FIGS. 6A shows the ratio of chondrocytes and synovial cell-derived cells (2) Immediately after the start of shaking culture with a cell suspension of 75% synovial cell-derived cells and 25% chondrocytes, B shows 12 hours later, C is a phase contrast microscope image after 24 hours and D is 36 hours.

  FIG. 7A shows the ratio of chondrocytes and synovial cell-derived cells (4) immediately after the start of shaking culture for a cell suspension of 25% synovial cell-derived cells and 75% chondrocytes, and B shows 12 hours later. C is an observation image after adding a fluorescent filter that selectively passes only a wavelength in an appropriate range through a phase contrast microscope after 24 hours and D after 36 hours.

  Further, in the shaking culture, the above-mentioned chondrocytes and synovial cell-derived cells are further used alone or the ratio of chondrocytes and synovial cell-derived cells is determined as (5) 0% synovial cell-derived cell suspension / 100% chondrocyte cell suspension. It carried out for 4 days, moving a liquid to a plane circular shape or a plane left-right type | mold, and the result was shown to FIG. 8A and B. FIG.

  FIG. 9 is suitable for a phase-contrast microscope after 36 hours from the start of shaking culture of a cell suspension of chondrocytes and synovial cell-derived cells under the ratios (1) to (5) above. An observation image is obtained by adding a fluorescent filter that selectively passes only a wavelength in a certain range. Under confocal laser microscope, the ratio of chondrocytes and synovial cells was observed by (3) shaking culture of 50% synovial cells and 50% chondrocytes for 5 days. The resulting image is shown in FIG.

  Over time, each cell material that makes up a spheroid for cell transplantation does not stay at the bottom of the culture vessel due to the flow of the culture fluid resulting from the continuous repetitive shaking motion resulting from human manipulation. Many other cellular materials can be maintained in a suspension state floating in the culture solution during the shaking culture period, mainly in small-sized mixed cell complexes that have become cell spheroids at the beginning of shaking. It quickly became attached and spheroids for cell transplantation grew. During this period, the cell mixture could be observed macroscopically from 12 hours after the start of the shaking culture, and was observable during the entire shaking culture period. At this time, the obtained cell spheroids were visually observed, and the results are shown in FIG.

  The cell mixture was observed to gradually smooth the contour over time. Microparticles that had passed 125 hours after the start of culture were observed under a phase contrast microscope, and the results are shown in FIG. 6 and FIG. In the case of a cell spheroid having a small size, the diameter was about 250 ± 100 μm, and in the case of a cell spheroid having a large size, the diameter was about 700 ± 250 μm.

  A spheroid for cell transplantation treatment comprising a cell mixture expressed as a cartilage-like tissue according to the present invention is prepared by separating each cell material constituting the cell mixture, and subcultured each of the separated cells to proliferate. Then, one or two or more types of cells are singly or mixed in a culture solution to prepare a cell material suspension, and the cells are cultured by shaking to adhere the cell materials to each other. It is obtained by producing a mixture and separating the produced cell spheroids. The spheroid for cell transplantation treatment of the present invention can be used for cell transplantation treatment by moving to the affected area, and can also be used as a system for evaluating physiological effects such as the efficacy and toxicity of compounds, drugs, and toxicants. Useful. In the spheroid for cell transplantation treatment of the present invention, each cell constituting the spheroid shows not only a single use of chondrocytes but also one or two or more advanced proliferative ability and easy differentiation ability into cartilage tissue. Any stem cell or cartilage progenitor cell or the like that possesses can be used, and a large amount of spheroids for cell transplantation treatment expressed as cartilage-like tissue can be prepared. As a result, the spheroid for cell transplantation treatment of the present invention is extremely useful in establishing a treatment technique for a wide range of osteochondral damage that frequently occurs in the elderly, and partial defects in which the degree of damage does not reach the subchondral bone from the cartilage surface layer. It is.

Claims (24)

A spheroid for cell transplantation treatment comprising a mixture of cells expressed as cartilage-like tissue. The cell mixture is chondrocyte, cartilage progenitor cell, synovial cell, synovial stem cell, osteoblast, bone marrow derived cell, living stem cell or mesenchymal stem cell, adipose derived cell, adipose derived stem cell, embryonic stem cell (ES cell) Or a mixture of two or more types of cells selected from induced pluripotent stem cells (iPS cells) obtained by reprogramming differentiated cells. The spheroid for cell transplantation treatment according to claim 1 or 2, wherein the cell mixture is a mixture of cells derived from autologous, allogeneic or heterogeneous tissues. The spheroid for cell transplantation according to any one of claims 1 to 3, wherein the size of the cell mixture is 50 to 999 µm. The spheroid for cell transplantation according to any one of claims 1 to 4, which is used for transplanting a part or all of a cartilage tissue to an affected part damaged or missing, or a part of a bone tissue damaged or missing. . The spheroid for cell transplantation treatment according to claim 5, wherein the cartilage tissue is articular cartilage, meniscus, intervertebral disc, costal cartilage, nasal septum, or auricular cartilage. The spheroid for cell transplantation treatment according to any one of claims 1 to 6, which is used for the treatment of arthritis, arthropathy, cartilage injury, osteochondral injury, meniscus injury, intervertebral disc degeneration, or osteoarthritis. The spheroid for cell transplantation treatment according to claim 1, which is used for evaluating the efficacy or toxicity of a test substance. A method for producing a spheroid for cell transplantation treatment, comprising the following steps (1) to (4).
(1) A step of preparing for separation of one or more cells constituting the cell mixture;
(2) a step of subculturing and proliferating the cells isolated in step (1);
(3) One or two or more types of cells grown in step (2) are singly or mixed in a culture solution to prepare a cell suspension, and the cells are maintained in a high-density floating state and cultured. A step of producing a cell mixture in which cells are adhered to each other; and (4) a step of separating the cell spheroids produced in step (3): Cells prepared for separation in step (1) are chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, bone marrow derived cells, living stem cells or mesenchymal stem cells, adipose derived cells, adipose derived cells The cell transplant according to claim 9, which is one or more cells selected from stem cells, embryonic stem cells (ES cells), or induced pluripotent stem cells (iPS cells) obtained by reprogramming differentiated cells. A method for producing therapeutic spheroids. The method for producing a spheroid for cell transplantation treatment according to claim 9 or 10, wherein in the step (1), the target tissue is chopped, chopped and pulverized, and then treated with a proteolytic enzyme to isolate cells. The target tissue has a concave bottom, and when cut during shredding, the target tissue is in a container with a side wall that is long enough to scatter away from the receiving equipment where the target tissue is located. The method for producing a spheroid for cell transplantation according to claim 11, wherein the spheroid is chopped by using a curved blade for surgery which is positioned in the bottom of the container and is longer than the long axis of the side surface of the container. The method for producing a spheroid for cell transplantation treatment according to claim 11 or 12, wherein the target tissue is digested while stirring with an electromagnetic stirrer under conditions of 37 ° C and 5% CO ₂ when it is treated with a proteolytic enzyme. . In the step (2), when the cells are chondrocytes and cartilage progenitor cells, the production of the spheroid for cell transplantation according to any one of claims 9 to 13, wherein the number of subcultures is 3 or less. Method. In the step (3), the culture in which the cells are maintained in a high-density floating state is caused by the flow of the culture solution resulting from the continuous repetitive motion resulting from the artificial manipulation, so that each cell does not stay at the bottom of the culture vessel, The method for producing a spheroid for cell transplantation according to any one of claims 9 to 14, which is a shaking culture capable of maintaining a suspended state floating in a culture solution during a shaking culture period. The method for producing a spheroid for cell transplantation according to claim 15, wherein the shaking culture is performed for 1 to 7 days. 17. The method for producing a spheroid for cell transplantation according to claim 15 or 16, wherein the cell density of the cell suspension in the shaking culture is 1 × 10 ⁴ cells / ml to 3,000 × 10 ⁴ cells / ml. In the step (3), when the cell suspension is subjected to shaking culture, the cells can be successfully adhered to each other, the efficiency of cell spheroid formation can be improved, and the therapeutic effect can be achieved by enhancing the expression of the phenotype to a cartilage-like tissue suitable for transplantation. The method for producing a spheroid for cell transplantation according to any one of claims 9 to 17, comprising one or two or more microcarriers for maximization of the cell transplantation. The cell according to claim 18, wherein the microcarrier is a molecule for promoting mutual adhesion between cells in a cell suspension, and good adhesion to each other and the microcarrier is maintained. A method for producing a spheroid for transplantation treatment. Microcarriers are collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, lubricin, mucin, chitosan, chitosan derivatives, polyrotaxane, polyrotaxane derivatives, chitin, chitin derivatives, urethane, cellulose, agarose, gelatin, fibronectin , Fibrin, platelet-rich plasma, heparin, heparin derivatives, Fragmin (generic name: dalteparin sodium), protamine sulfate, Avidin, Streptavidin, Biotin, laminin, 2-Octyl Cyanoacryleate, calcium alginate , Polylactic acid, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polypropylene, polyglycolic acid, polycaprolactone, polylactic acid polyglycolic acid copolymer, polylactic acid poly The material according to claim 18 or 19, wherein the material is formed of one or more biocompatible materials or bioabsorbable materials selected from the group consisting of a licaprolactone copolymer and a polyglycolic acid polycaprolactone copolymer. A method for producing a spheroid for cell transplantation treatment. In the step (3), primary cells spheroids in which the cells are adhered to each other are produced by culturing while maintaining the cells in a high-density floating state, and then further adding 1 around the primary cell spheroids. A secondary cell spheroid obtained by adhering the primary cell spheroid and the additional cells to each other is prepared by mixing seeds or two or more cells in a culture solution to prepare a cell suspension and subsequently culturing. The method for producing a spheroid for cell transplantation treatment according to any one of claims 9 to 20, which is produced. The method for producing a spheroid for cell transplantation according to any one of claims 9 to 21, wherein the culture solution used in step (3) is a cartilage differentiation induction medium. The method for producing a spheroid for cell transplantation according to any one of claims 9 to 22, wherein in the step (4), the cell spheroid is shaped to be usable for an endoscope or an arthroscopic operation. The method for producing a spheroid for cell transplantation according to any one of claims 9 to 23, wherein the process for producing the spheroid for cell transplantation is within 14 days.