Isolated intervertebral disc cells, methods of use thereof, and methods of preparing same from mammalian tissue - Patents.com

Abstract

To provide a biological treatment for an intervertebral disc that can aid in the repair or replacement of the disc. The present invention relates to intervertebral disc cell populations, methods for deriving same, and methods of use thereof. The intervertebral disc cell populations herein can be used to repair or regenerate a damaged, impaired, or defective intervertebral disc in a subject. The intervertebral disc cell populations of the present invention can be derived from a subject, administered or transplanted into a subject, or derived from an unrelated donor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority pursuant to 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61 / 794,691, filed March 15, 2013, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION FIELD OF THE DISCLOSURE The present disclosure relates to the isolation of intervertebral disc cells and methods of their use in the treatment of degenerative disc disease. [Background technology]

[0003] The mammalian spine serves two basic functions: (1) weight-bearing support for the upper body and (2) protection for the nerves that comprise the spinal column. The spine consists of interlocking vertebrae separated by intervertebral discs. The discs act as shock absorbers, allowing the spine to bend, compress, and twist. Intervertebral discs have two basic parts: an outer fibrous structure (annulus fibrosus) and a gel-like inner structure (nucleus pulposus). A healthy nucleus pulposus in young mammals is approximately 80% water. Over time, the nucleus pulposus loses its high water content and thus its ability to absorb shock. Furthermore, intervertebral discs can be compromised by dehydration, disease, overuse, injury, or trauma, resulting in rupture, bulging, herniation, and the like. Intervertebral discs are also susceptible to other conditions, such as degenerative disc disease.

[0004] In a healthy disc, cells are only a small fraction of the total volume. Most of the disc volume is extracellular matrix (ECM; collagen and proteoglycans) produced by cells, which helps retain large amounts of water. The differences between the nucleus pulposus and the annulus fibrosus are primarily in the water content and composition of the ECM.

[0005] Back pain resulting from degenerative disc disease is a major cause of morbidity, disability, and lost productivity. Back pain is frequently reported as limiting activity for people up to age 45 and is the reason for physician visits, hospitalizations, and surgical procedures. Chronic back symptoms are reported by 15%-45% of the population annually and by 70%-85% of the population at some point in their lifetime. The financial impact is significant in terms of medical expenses and lost work time. More than one million spine surgical procedures are performed each year in the United States. Furthermore, the lumbar fusion portion of the spine surgery market is estimated to generate well over $1 billion in revenue.

[0006] Despite continuing improvements in both surgical and non-surgical treatment options for subjects suffering from back pain and spinal disorders, no solution exists to eliminate or reliably improve this condition. Current treatments for spinal disorders include steroid injections, physical therapy, discectomy, and spinal fusion. Artificial spines have been introduced by several companies. However, these prosthetic devices vary in design, e.g., bearing surface, fixation to bone, number of joints, materials, constraints, and rotational mobility, and appear to have little success in practice.

[0007] Nucleus arthroplasty or nucleus replacement is also an option for treating degenerative disc disease. In some cases, these devices consist of a hydrogel core encased in a polyethylene sleeve, allowing it to contract and expand under normal load and unload conditions. This can partially help restore disc space height and mimic a healthy human disc.

[0008] Disc replacement is not without complications. The most common complications include adjacent level spinal disease, subsidence, and facet joint arthropathy. Furthermore, recent clinical studies have demonstrated the occurrence of infection, vertebral fractures, implant malposition, subsidence, organic failure, and paravertebral heterotopic ossification. More serious complications, such as anterior implant dislocation, have also been reported. Furthermore, the issue of wear particles from total disc replacement and their potential impact on the spinal cord are still unknown. Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for biological treatments for intervertebral discs that can help repair or replace a subject's disc. [Means for solving the problem]

[0010] Disclosed herein are isolated discogenic cell populations, methods of use, and methods of preparation. The discogenic cell populations are used in the repair, regeneration, and replacement of damaged, injured, or disordered discs. In some embodiments, the discogenic cell populations are derived from mammalian disc tissue and grown in vitro under anchorage-independent conditions. In some embodiments, the cell culture comprises a medium containing one or more additives selected from the group consisting of EGF, bFGF, serum, fibroblast-conditioned medium, and a viscous, non-reactive substance. In some cases, the cells are grown in a container with a low-adhesion coating. The disclosed discogenic cell populations can be used for autologous and / or non-autologous treatment of intervertebral discs in subjects in need thereof.

[0011] The disclosed intervertebral disc cells can be used to form in vitro or in vivo artificial disc replacements using non-resorbable or resorbable materials. The materials can form an artificial annulus that functions to house the disc cell population, and may or may not be combined with at least one of the following: a scaffold material, a matrix material, a carrier material, growth factors, and / or other bioactive substances. The artificial outer annulus can include attachment means to allow it to be fixed to one or more vertebral bodies. For example, the artificial annulus can include through-holes, cuffs, tabs, loops, or washers to allow screw fixation to one or more vertebral bodies. The artificial disc can be surgically implanted into a subject to provide a total disc replacement.

[0012] Also disclosed are various methods for obtaining and preparing intervertebral disc cells from autologous and non-autologous donors. A method for deriving an intervertebral disc cell population is disclosed, including isolating one or more cells from tissue, subculturing one or more cells in an anchorage-dependent culture medium, and transferring one or more cells to an anchorage-independent culture medium. A method for using intervertebral disc cells to treat at least one intervertebral disc in a subject in need thereof is also disclosed, including administering a therapeutic amount of the intervertebral disc cell population to the subject, thereby treating the subject. In various embodiments, the tissue is mammalian intervertebral disc tissue, obtained, for example, from a donated organ or spine. In some embodiments, the disclosed method includes subculturing the cell population at least once in an anchorage-independent culture, whereby the cell population produces extracellular matrix. In some embodiments, the cell population expresses one or more cell surface markers selected from the group including CD24, CD34, CD44, CD73, CD90, CD105, CD166, Stro-1, HIF1, nestin, CK8, and HLA proteins, and the percentage of cells in the population expressing the cell surface markers is greater than 70% or less than 40%. In some embodiments of the disclosed methods, the cell population expresses one or more genes or gene products selected from the group including GAPDH, SDHA, HPRT1, B2M, Sox9, aggrecan, Col1, Col2, nestin, CK8, Sox1, CD44, ALPI, PPARG, ADAMTS, MMP, FMOD, IL.

[0013] In another aspect, in the described intervertebral disc cell populations, greater than 40% of the cells produce cell surface markers CD44, CD73, CD90, HLA-A, B, or C, CD24, CD105, CD166, or a combination thereof, and less than about 20% of the cell population produce CD34, HLA-DR or -DQ, or STRO-1. In some embodiments, about 80-100% of the population produce CD73, CD90, CD44, HLA ABC, or a combination thereof. In some embodiments, about 20-75% of the population produce CD105, CD166, CD24, or a combination thereof.

[0014] According to one aspect, the present invention provides a method for treating a subject having damage or injury to at least one intervertebral disc caused by disc injury induced by aging, trauma, toxin exposure, drug exposure, radiation exposure, oxidation, immune complex deposition, or graft rejection.

[0015] According to another aspect, the present invention provides a kit for treating a subject having at least one intervertebral disc disorder or injury, the kit comprising a pharmaceutically acceptable carrier, an amount of intervertebral disc cells effective to treat the disorder or injury, and spinal column tissue, wherein the cells are capable of proliferation and differentiation in culture. In some embodiments, the kit comprises at least one agent. Further aspects of the present invention are described below: [Section 1] At least one cell derived from mammalian intervertebral disc tissue and grown in an anchorage-independent culture in vitro. Intervertebral disc cell populations, including: [Section 2] One or more cells derived from cartilaginous tissue, wherein at least one cell is maintained in anchorage-independent culture. A heterogeneous cell population comprising: [Section 3] The cell population described in item 1 or 2, wherein the culture comprises a medium containing one or more additives selected from the group consisting of EGF, bFGF, serum, fibroblast-conditioned medium, and viscous non-reactive substances. [Section 4] The cell population according to item 3 above, which is passaged in a culture vessel having a low-adhesion coating. [Section 5] 5. The cell population according to any one of items 1 to 4 above, which has been passaged at least once by anchorage-independent culture. [Section 6] 6. The cell population according to any one of items 1 to 5 above, which produces an extracellular matrix. [Section 7] 7. The cell population according to any one of items 1 to 6, which produces one or more cell surface markers selected from the group consisting of CD24, CD34, CD44, CD73, CD90, CD105, CD166, Stro-1, HIF1, FIT-1, nestin, CK8, and HLA proteins. [Section 8] Item 8. The cell population according to item 7, wherein the percentage of cells expressing the cell surface marker is greater than 70% or less than 40%. [Section 9] The cell population described in any one of items 1 to 6 above, which expresses one or more genes or gene products selected from the group including GAPDH, SDHA, HPRT1, B2M, Sox9, aggrecan, Col1, Col2, nestin, CK8, Sox1, CD44, ALPI, and PPARG. [Section 10] 10. The cell population according to any one of items 1 to 9 above, which is obtained from intervertebral disc tissue. [Section 11] isolating one or more cells from the tissue; passaging the one or more cells in an anchorage-dependent culture medium; transferring said one or more cells to an anchorage-independent culture medium. 20. A method for inducing an intervertebral disc cell population, comprising: [Section 12] 1. A method of using intervertebral disc cells to perform at least one intervertebral disc treatment in a subject in need thereof, comprising: A method comprising administering to a subject a therapeutically effective amount of a population of intervertebral disc cells, thereby treating the subject. [Section 13] 1. A method of treating a subject having at least one injured, damaged, or defective intervertebral disc, comprising: A method comprising administering to a subject an amount of a population of intervertebral disc cells effective to treat said disorder or injury. [Section 14] 1. A method of treating an indication selected from the group consisting of degenerative disc disease, herniated disc, and disc injury, comprising: A method comprising administering a therapeutically effective amount of a population of intervertebral disc cells, thereby treating the indication. [Section 15] Item 12. The method according to item 11 above, wherein the tissue is mammalian intervertebral disc tissue. [Section 16] Item 16. The method according to item 15 above, wherein the tissue is donated organ tissue. [Section 17] 15. The method according to any one of items 11 to 14 above, wherein the cell population is passaged at least once by anchorage-independent culture. [Section 18] 16. The method according to any one of items 11 to 15 above, wherein the cell population produces an extracellular matrix. [Section 19] 17. The method of any one of paragraphs 11 to 16, wherein the cell population expresses one or more cell surface markers selected from the group consisting of CD24, CD34, CD44, CD73, CD90, CD105, CD166, Stro-1, HIF1, nestin, CK8, and HLA proteins. [Section 20] 18. The method according to any one of items 11 to 17 above, wherein the percentage of cells in the population that produce the cell surface marker is greater than 70% or less than 40%. [Section 21] 17. The method according to any one of items 11 to 16, wherein the population expresses one or more genes or gene products selected from the group including GAPDH, SDHA, HPRT1, B2M, Sox9, aggrecan, Col1, Col2, nestin, CK8, Sox1, CD44, ALPI, PPARG, ADAMTS, MMP, FMOD, and IL. [Section 22] 1. A device for treating a disordered or damaged intervertebral disc, comprising: a population of intervertebral disc cells derived from cartilaginous tissue, wherein at least one cell is grown in anchorage-independent culture; and A device comprising a scaffold, matrix, or implantable structure. [Section 23] 23. The device according to item 22, further comprising a biologically active substance. [Section 24] artificial outer ring; Intervertebral disc cell populations 1. An artificial disc replacement device, comprising: [Section 25] 25. The artificial disc replacement device according to claim 24, wherein the outer ring comprises a non-absorbable material. [Section 26] 26. The artificial disc replacement device according to item 25, wherein the non-absorbable material is polyurethane. [Section 27] 25. The artificial disc replacement device according to item 24, wherein the outer ring comprises an absorbable material. [Section 28] 28. The artificial disc replacement device according to item 27, wherein the absorbable material is polyglycolic acid or polylactic acid, or a combination thereof. [Section 29] 25. The artificial disc replacement device of paragraph 24, wherein the disc cell population further comprises one or more of a scaffold material, a matrix material, a carrier material, growth factors, and other bioactive substances. [Section 30] 25. An artificial disc replacement device according to paragraph 24, further comprising attachment means for securely fixing the device to one or more vertebral bodies. [Section 31] 31. The artificial disc replacement device of claim 30, further comprising a through-hole, cuff, tab, loop, or washer to allow screw fixation to one or more vertebral bodies. [Section 32] 1. A method of replacing an intervertebral disc, comprising: 24. Producing the artificial intervertebral disc according to item 24 in vitro; and surgically implanting the artificial disc into a subject, thereby replacing the disc. A method comprising: [Brief explanation of the drawings]

[0016] The file of this patent contains at least one color drawing / photograph. Copies of this patent with color drawing / photograph(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0017] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed at the end of this specification. However, the invention, both as to organization and manner of operation, together with objects, features and advantages thereof, may best be understood by reference to the following detailed description taken in conjunction with the accompanying drawings.

[0018] [Figure 1] Figure 1 shows the expression profiles of various surface markers known to identify stem cells and chondrogenic cells. Eight cell types have been investigated: fibroblasts, chondrocytes, mesenchymal stem cells (MSCs), intervertebral disc cells grown in monolayer (adhesion-dependent), and intervertebral disc cells grown in suspension (adhesion-independent). [Figure 2] FIG. 2 shows the cell morphology of intervertebral disc cells grown in suspension culture. [Figure 3] FIG. 3 shows the chondrogenic potential of intervertebral disc cells compared to MSCs. [Figure 4] FIG. 4 shows the adipogenic and osteogenic potential of intervertebral disc cells. [Figure 5] Figure 5 shows the viability of cells after combination with a viscous 1% hyaluronic acid scaffold and after extrusion through a 27-gauge 1.5-inch surgical needle (green, or light if monochrome—live cells; red, or dark if monochrome—dead cells). [Figure 6] Figure 6 shows devices and methods that may be used in the therapeutic use of intervertebral disc cells to treat various forms of disc disease. Treatment may be by injection (top diagram) or implantation (bottom diagram). The cells and carrier / scaffold may be kept together or combined immediately prior to use. [Figure 7] FIG. 7 shows the in vivo efficacy of intervertebral disc cells in combination with a viscous scaffold carrier to repair degenerated intervertebral discs (animal model) in rabbits. [Figure 8]Figure 8 shows a flow diagram illustrating the preparation and transplantation of intervertebral disc cells. Step a shows a diagram of an intervertebral disc. Step b shows a fresh human intervertebral disc isolated from the spine. Step c shows nucleus pulposus cells after dissection and enzymatic digestion. Adherent cells are grown in the presence of EGF and FGF-2. Scale bar = 50 μm. Step d shows a micrograph of cells transferred to a contact-inhibited culture environment containing methylcellulose. Clusters and spheres develop in approximately two weeks. Scale bar = 200 μm. Step e shows the syringe used to inject the cells combined with a non-crosslinked hyaluronic acid scaffold after washing away the methylcellulose-containing medium. Step f shows the syringe used to inject the cell-scaffold mixture into a degenerated rabbit intervertebral disc. Safety and efficacy were then evaluated over a period of approximately one month. [Figure 9] Figures 9A-F show intervertebral disc cells examined for aggrecan and collagen production. Figure 9A is a phase image of hematoxylin and eosin staining. Figure 9B is a phase image of Alcian blue counterstained with Nuclear Fast Red. Note the presence of matrix surrounding a single cell (left panel). Scale bar = 10 μm. Figure 9C is a phase image of Picrosirius Red staining. Figure 9D is a confocal image containing actin (red) and cell nuclei (blue). Figure 9E is a further magnified confocal image of aggrecan, collagen, and actin (without nuclei), where black arrows indicate intracellular aggrecan and white arrows indicate extracellular aggrecan. Figure 9F is a bar graph showing RT-PCR analysis of matrix molecules (aggrecan and collagen 2A) over time in culture, at harvest on day 14, and after chondrogenic differentiation. Fold expression was calculated by crossing threshold normalized to the housekeeping gene HRPT and baseline gene expression at day 0. [Figure 10]Figures 10A-C show flow cytometry analysis of intervertebral disc cells. Figure 10A is a forward and side scatter plot showing the gating applied to all subsequent analyses, which included 89% of the cell population. Figure 10B is a bar graph showing expression levels (compared to isotype controls) of intervertebral disc cells derived from five different human donors. Figure 10C shows a representative histogram of surface marker expression. [Figure 11] Figures 11A-E show the pluripotency of intervertebral disc cells. Figure 11A shows osteogenic differentiation as indicated by Alizarin Red staining. Figure 11B shows adipogenic differentiation as indicated by Oil Red O staining. Figure 11C shows chondrogenic differentiation (Alcian Blue and Nuclear Fast Red) after micromass formation. Scale bar = 100 μm. Figure 11D is a bar graph quantitatively assessing the production of soluble (medium) and insoluble (micromass) GAGs after chondrogenic differentiation for articular chondrocytes (AC), adult fibroblasts (FB), bone marrow-derived MSCs, and intervertebral disc cells (DC). Figure 11E is a bar graph showing total GAG production normalized to DNA content for various cell types. Lines indicate significant differences (p<0.01, one-way ANOVA with Bonferroni's post-hoc test). [Figure 12] Figure 12A shows the safety and efficacy evaluation of treatment in a rabbit model of degenerative disc disease. Figure 12A shows representative radiographs taken every two weeks and used to calculate the disc height index (DHI) based on the 18 bony landmarks shown. Figure 12B shows that the rabbits' weights remained within the normal range throughout the study period (injection treatment on day 14). Figure 12C is a graph of DHI over 6 weeks of treatment, showing that treatment improved DHI compared to control conditions at 4 and 6 weeks, with lower doses being more effective than higher doses. No improvement was observed in scaffold or injury controls, and disc height in uninjured controls remained unchanged from week 0. [Figure 13]Figures 13A-B show histological evaluation of the treatment after a 6-week pilot study. Figure 13A shows cross-sections of healthy, injured, and treated discs (hematoxylin and eosin staining, scale bar = 2 mm). Figure 13B shows the histology of various regions of the IVD after treatment, including the bone marrow, annulus fibrosus (AF), cartilage endplate (CEP), and nucleus pulposus (NP); stained with hematoxylin and eosin (H&E) or Alcian blue (scale bar = 100 μm). [Figure 14] Figures 14A-C show the safety and efficacy of a 12-week pilot study of treatment in pigs. Figure 14A is a graph showing that doses of IDCT consistently improved DHI compared to injury controls at 12 weeks (p<0.05); there was no improvement in scaffold or injury controls, and disc height in uninjured controls remained unchanged from week 0. Figure 14B is a fluoroscopic image of a pig spine undergoing the study (disc space collapse is observed in injured discs). Figure 14C is a histological evaluation of an IDCT-treated disc, including the nucleus pulposus (NP), cartilage endplate (CEP), and annulus fibrosus (AF), stained with hematoxylin and eosin (H&E) and Alcian blue. DETAILED DESCRIPTION OF THE INVENTION

[0019] The disc cells described herein are cells derived from intervertebral disc tissue and can be used to treat and / or repair the disc. In some cases, disc cells can be treated in vitro to provide disc cells that are more potent than other cells at repairing, replacing, or augmenting existing or damaged nucleus pulposus tissue. In various embodiments, disc cells produce extracellular matrix. In some embodiments, disc cells produce proteoglycans. In other embodiments, disc cells produce collagen. In other embodiments, disc cells implanted adjacent to native cells can serve to stimulate the native cells via chemical, mechanical, or other forces. For example, disc cells can release growth factors, cytokines, or other proteins.

[0020] As used herein, "discogenic" (or "discogenic") refers to the ability to generate intervertebral disc tissue in vivo. In some embodiments, disc cells can regenerate damaged or injured disc tissue in vivo, and / or disc tissue that has lost one or more disc tissue characteristics. In some cases, disc cells can generate disc tissue in vitro, e.g., disc cells can be used to create artificial discs for implantation.

[0021] As used herein, "maintained" when referring to cells grown in vitro includes cells growing in culture for more than 24 hours. In some cases, maintained cells are cells that have divided in cell culture.

[0022] A "micromass" is formed by concentrating approximately 10,000 to 1,000,000 cells in a conical vessel that inhibits adhesion, resulting in the cells forming at least one single mass. The micromass may also contain extracellular matrix. It is also called a pellet, based on an assay that determines chondrogenic potential.

[0023] As used herein, "about" when referring to a measurable value such as an amount, time, etc., is meant to encompass a variation of less than about ±20%. In some cases, about may refer to a variation of 10% or less, or ±5% or less. In some cases, about may refer to a variation of ±1% to ±0.1%.

[0024] "Derived" may be used to indicate that a cell has been obtained or isolated from its native or previous biological state or location and maintained, grown or expanded in culture, or immortalized, or otherwise processed in vitro. For example, in some embodiments of the invention, the intervertebral disc populations of the invention may be derived from intervertebral disc tissue or cartilaginous tissue, and in some embodiments, the discospheres may be derived from intervertebral disc cell populations.

[0025] When a cell or molecule is "isolated," it has been removed from or altered with respect to its natural state by human intervention.

[0026] The terms "express," "expressed," or "expression" refer to the biosynthesis of a gene product from a nucleic acid molecule or gene, e.g., the biosynthesis of a polypeptide. A cell surface marker is said to be expressed on a cell surface if it is present to some extent on the cell surface after some event, e.g., in vitro growth.

[0027] "Injury" refers to any damage, injury, degeneration, or trauma to the intervertebral disc, whether due to aging, trauma, or disease.

[0028] A "disease" is any deviation from or impairment of the health, condition, or function of a cell, tissue, organ, system, or entire organism, as measured by any means suitable in the art.

[0029] "Treate," "treating," or "treatment" refers to any attenuation or improvement of a disease, disorder, or condition, including any objective or subjective parameter, such as relief, remission, or reduction of symptoms, or making the disease, disorder, or condition more tolerable to the subject (e.g., by reducing pain), slowing the rate of degeneration or deterioration, lessening the severity of the degenerative endpoint, improving the subject's physical or mental well-being, or prolonging survival. The treatment or amelioration of symptoms may be based on objective and / or subjective parameters, including the results of a physical, radiological, neurological, and / or psychiatric evaluation.

[0030] The terms "effective amount" or "treatment-effective amount" are used interchangeably herein and refer to an amount of a compound, material, or composition described herein that is effective to achieve a particular biological result, such as, but not limited to, a biological result disclosed, described, or exemplified herein. Such a result includes, but is not limited to, the treatment of an intervertebral disc disease or disorder in a subject, as determined by any suitable means in the literature.

[0031] "Pharmaceutically acceptable" refers to properties and / or substances that are acceptable to the subject from a pharmaceutical / toxicological standpoint and to pharmaceutical chemists from a physical / chemical standpoint, with respect to composition, formulation, stability, subject tolerance and bioavailability.

[0032] "Pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. One example of a pharmaceutically acceptable carrier is hyaluronic acid.

[0033] "Discospheres" are described in U.S. Patent No. 8,227,246B2 and PCT Application No. PCT / US2012 / 025066, which are incorporated herein by reference in their entireties.

[0034] Obtaining intervertebral disc cells from intervertebral disc tissue The intervertebral disc cells described herein can be obtained from intervertebral disc tissue. Intervertebral disc tissue can include any of nucleus pulposus tissue, transition zone tissue, and annulus fibrosus tissue. In some cases, the intervertebral disc cells can be obtained from the cartilaginous endplates of the intervertebral disc. In other cases, the intervertebral disc cells can be obtained from other cartilaginous tissues in the body.

[0035] In various embodiments, the intervertebral disc tissue can be obtained from a living or deceased donor. The donor can be a mammal, such as a human. In some cases, the donor is a tissue donor and may not be genetically related to the recipient. The donor can be of any age, including newborns, juveniles, adults, and the elderly.

[0036] In various embodiments, the disc tissue can be healthy disc tissue or diseased or injured disc tissue. Diseased or injured disc tissue that can be used in the disc cells and methods disclosed herein includes, for example, degenerated tissue, herniated tissue, tissue removed from painful discs, and tissue removed from deceased donors.

[0037] In various embodiments, the tissue is used directly to obtain cells. In other embodiments, the tissue is frozen prior to use, for example, by cryopreservation or vitrification, and used at a later date. In other embodiments, the tissue is kept at 4°C in a specialized medium until the cells are extracted. The tissue can be maintained in a medium containing sugars, cryoprotectants, stabilizers, serum, etc.

[0038] Intervertebral disc cell culture Intervertebral disc cells can be grown in mammalian cell culture. In many cases, the cell culture can be scaffolded or non-scaffolded on a substrate. In some cases, the cell culture can include a medium. The cell culture medium can be any medium suitable for growing mammalian cells in culture, such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Modified Eagle's Medium), RPMI, RPMI 1640, etc. In some cases, the medium can further include additives, such as a nutrient medium, for example, Ham's F12 (F12). In some cases, the cell culture medium may or may not include additional additives.

[0039] In various embodiments, serum may or may not be added to the culture medium. Serum may refer to animal serum derived from mammals, such as cows, chickens, goats, horses, humans, sheep, pigs, rabbits, etc. In some cases, serum may be derived from adult, newborn, or fetal animals; for example, fetal bovine serum may be obtained from cows or fetal bovines. In some cases, serum additives such as animal platelet lysate (e.g., human platelet lysate), serum-converted platelet lysate, animal serum albumin (bovine serum albumin), or conditioned medium derived from another cell culture (e.g., neonatal foreskin fibroblast conditioned medium) may be added together with or instead of serum.

[0040] In some cases, the serum or serum supplement concentration in the culture medium may be greater than 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, and 30% by volume, and / or less than about 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% by volume. In some cases, the serum concentration in the medium may be 0%. In some embodiments, the serum concentration is 0-17%, 0-5%, 5-17%, or 0-2.5%.

[0041] In some cases, additional supplements may or may not be added to the culture medium. In some cases, the supplements may be hormones or growth factors. In some cases, the hormones or growth factors may be adrenomedullin (AM), angiopoietin (Ang), autocrine motility stimulating factor, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), basic fibroblast growth factor (bFGF, FGF-2, or FGF-β), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF). , insulin-like growth factor (IGF), migration-stimulating factor, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), placental growth factor (PlGF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, insulin, progesterone, putrescine, transferrin, sodium selenite. Often, the growth factors may be EGF and bFGF.Often, the concentration of the supplement in the cell culture medium is about 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml,In some cases, the supplement concentrations are 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 50nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM, 1100nM, 1200nM, 1300nM, 1400nM, 1500nM, 1600nM, 1700nM, 1800nM, 1900nM, 2100nM, 2200nM, 2300nM, 2400nM, 2500nM, 2600nM, 2700nM, 2800nM, 2900nM, 3000nM, 3100nM, 3200nM, 3300nM, 3400nM, 3500nM, 3600nM, 3700nM, 3800nM, 4000nM, 4100nM, 4200nM, 4300nM, 4400nM, 4500nM, 4600nM, 4700nM, 4800nM, 4900nM, 5000nM, 5100nM, 52 00nM, 800nM, 900nM, 1μM, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM, 200μM, 300μM, 400μM, 500μM, 600μM, 700μM, 800μM, 900μM, and higher than 1mM and / or about 1.1mM, ... μM, 800 μM, 700 μM, 600 μM, 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40μM, 30μM, 20μM, 10μM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, 1μM, 900nM, 800nM, 700nM, 60 The concentration may be less than 0 nM, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, and 1 nM. In some embodiments, the concentration is about 5-110 ng / ml, 5-15 ng / ml, or 90-110 ng / ml.

[0042] In some cases, the cell culture medium may or may not contain a neural supplement. In some cases, the neural supplement may be a commercially available neural supplement, such as B27, N2, or N10. When a neural media supplement is added, the concentration of the supplement in the cell culture medium may be greater than about 0X, 1X, 2X, 3X, 4X, and 5X, and / or less than about 10X, 6X, 5X, 4X, 3X, 2X, and 1X. Other commercially available products include NeuroCult, ANS Neural Media Supplement, Neurobasal Supplement, B28, NS21, G5, N21, NS21, etc.

[0043] In some cases, the cell culture medium may contain other chemicals, molecules, supplements, or additives known in the mammalian cell culture art, such as amino acids, peptides, salts, vitamins, antibiotics, antifungal agents, antimycotics, minerals, pH buffers, pH indicators, and sugars. In many cases, the pH of the cell culture medium may be greater than about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, and 7.9, and / or less than about 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.5, 6.6, 6.4, 6.3, 6.2, 6.1, and 6.0. In various embodiments, the pH is about 6.9 to 7.7, 7.0 to 7.4, or 7.3 to 7.7.

[0044] Intervertebral disc cells can be grown in monolayers or in suspension. In some cases, cells can be grown in cell-free containers, such as plates, dishes, flasks, roller flasks, and reactors for mammalian cell culture, which allow for gas and medium exchange as needed. In various embodiments, cells can be grown in a stationary container or while the container is in motion, for example, by rotation or rolling. In some cases, cell culture medium can be agitated, for example, by rotating, shaking, or rolling the container. Cell culture medium can also be agitated by other methods, for example, by physically moving cell culture medium in a stationary container, for example, with a stir bar, stir bar, or other mechanical agitation mechanism therein. In some cases, the container can include baffles to aid in agitation of the medium.

[0045] In some cases, the container can be treated, for example, to support or inhibit cell adhesion. Various culture methods can be used to grow cells under anchorage-independent conditions. Generally, cells that can grow in suspension can be grown under anchorage-independent conditions. For example, cells that can grow and divide without adhering to a substrate can be anchorage-independent. In some cases, the container can be coated with, for example, gelatin or collagen to support adhesion. In some cases, the container can be coated with, for example, ultra-low attachment surface modification to inhibit cell adhesion or attachment. In some cases, the container can be commercially available, for example, ultra-low attachment container (Corning). Furthermore, viscous non-reactive medium additives such as methylcellulose, poloxamer, or agar / agarose can be used or not to maintain a free-floating suspension of cells.

[0046] In some cases, when an additive is added to the cell culture medium to prevent or inhibit cell adhesion, for example, when a viscous non-reactive substance is added, the concentration of the additive in the cell culture medium may be greater than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 15%, and / or less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, and 0.1%. The final concentration of the viscous additive may depend on the additive used, for example, if methylcellulose is used, the concentration may be about 0.6-0.9%, 0.7-0.8%, or 0.75%, and if agarose is used, the concentration may be about 1-5%, 2-4%, or 3%.

[0047] In some cases, intervertebral disc cells can be grown in an atmosphere with ambient levels of oxygen or higher or lower levels of oxygen. Often, ambient levels of oxygen can be 22-19% oxygen. In some cases, the atmosphere in which the cells are grown is less than 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, and 5% oxygen, and / or greater than about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% oxygen. In some embodiments where hypoxic conditions are desired, the concentration of oxygen can be about 3-7%, 4-6%, 5%, or 6% oxygen.

[0048] Surface marker In some cases, the disc cell population can be characterized by the expression of cell surface markers. In some cases, the disc cell population may or may not express one or more specific cell surface markers and / or differentiation proteins. In various embodiments, the disc cell population may have a higher or lower percentage of cells with a particular marker selected by gating than that of reference cells, such as chondrocytes or adipocytes. In other cases, the disc cell population may contain a certain percentage of cells with a particular cell surface marker selected by gating. In some cases, the percentage of cells with a particular cell surface marker selected by gating is greater than 40%, 50%, 60%, 70%, 80%, and 90%, and / or less than about 100%, 90%, 80%, 70%, 60%, and 50%.

[0049] Cell surface markers that can be useful for characterizing intervertebral disc cell populations include, but are not limited to, CD24, CD34, CD44, CD73, CD90, CD105, CD166, Stro-1, HIF1, nestin, CK8, and HLA proteins (human leukocyte antigens, such as HLA-A, -B, -C, HLA-DQ, and HLA-DR).In some cases, CD24 can be a glycoprotein expressed on the cell surface, which is fixed to the cell surface (e.g., lymphocytes, granulocytes, and neuroblastoma cells) by glycosylphosphatidylinositol (GPI) linkage.CD24 is also referred to as heat-stable antigen (HSA).CD44 can refer to a cell surface glycoprotein involved in cell-cell interaction, cell adhesion, and migration. CD73, also known as 5'-ribonucleotide phosphohydrolase, is expressed on, for example, B cells, T cells, endothelial cells, pericytes, follicular dendritic cells, fibroblasts, epithelial cells, cardiomyocytes, neurons, osteoblasts, trophoblasts, and mesenchymal stem cells (MSCs). CD90 may refer to the glycoprotein Thy-1 thymocyte antigen. CD105 may refer to endoglin, a glycoprotein component of the TGF-β receptor complex. CD166 may refer to activated leukocyte cell adhesion molecule (ALCAM). Stro-1 may refer to a marker for immature mesenchymal stem cells. HIF-1 may refer to hypoxia-inducible factor. Nestin may refer to a neural marker. CK8 may refer to a cytokeratin marker.

[0050] In some cases, the expression of cell surface proteins / markers on intervertebral disc cells can be measured. In various embodiments, cell surface protein expression is measured by using fluorescent antibodies that recognize epitopes of the cell surface protein being measured. In some cases, measurement is performed using flow cytometry, including fluorescence-activated cell sorting (FACS) using standard techniques. When measured by FACS, expression is measured as the percentage of cells within a specific range selected by gating, where gating is set using an IgG control. In some cases, expression is greater than about 70% for HLA-ABC, CD44, CD73, and CD90, and less than 40% for CD24, CD105, and CD106.

[0051] Additionally, in some cases, surface markers can be used to sort, isolate, or enrich for specific subpopulations or cells, for example, cell sorting using magnetic beads, fluorescent markers, or other techniques can be used to select for subpopulations within a population.

[0052] Gene expression In addition to surface markers, genomic and genetic analysis is sometimes used to identify disc cells. Techniques include quantitative polymerase chain reaction, microarray analysis, Western blot, etc. By measuring DNA, mRNA, miRNA, or protein, intervertebral disc cells can be identified using expression of genes (e.g., transcription factor Sox9, extracellular matrix component aggrecan, extracellular matrix components collagen 1 and 2, neural marker nestin, cytokeratin 8, transcription factor Sox1, CD44 (a receptor for hyaluronic acid), ALPI (alkaline phosphatase), PPARG (peroxisome proliferator-activated receptor gamma), MMP (matrix metalloproteinase), ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), FMOD (fibromodulin), interleukins, etc.) measured as an increase or decrease relative to housekeeping genes (e.g., GAPDH-glyceraldehyde 3-phosphate dehydrogenase, SDHA-succinate dehydrogenase complex, subunit A, HPRT1-hypoxanthine phosphoribosyltransferase, B2M-beta2 microglobulin, etc.).

[0053] Cryopreservation In some cases, cells can be cryopreserved. Cells can be mixed with pre-formulated cryopreservation media, such as Cryostor, HyCryo, UltraCruz, or Cyagen. Alternatively, cells can be mixed with formulated cryopreservation media that may or may not contain serum, albumin, dimethyl sulfoxide, trehalose, sucrose, other sugars, ethylene glycol, glycerol, propylene glycol, hyaluronic acid, collagen, Matrigel, or other natural extracellular matrix molecules. Cells can be frozen rapidly (vitrified) or slowly (over a defined time course at various temperatures or with a rate-controlled freezing device). Cells can be frozen at 100,000 to 10 million cells / mL.

[0054] Isolation of intervertebral disc cell populations The present invention describes methods for deriving, obtaining, or isolating intervertebral disc cells from intervertebral disc tissue. In some cases, the isolated intervertebral disc cell population is derived from an autologous or non-autologous donor. An autologous donor can be when the intervertebral disc cell population is derived from the subject to be treated with the cells. A non-autologous donor, also referred to as an allogeneic donor, can be a different subject. Also disclosed are various methods for obtaining and preparing intervertebral disc cells from living and / or deceased donors.

[0055] In many cases, methods for isolating disc cells from intervertebral disc tissue include separating the disc cells from the extracellular matrix. In some cases, the disc tissue can be mechanically, chemically, and / or enzymatically disrupted. In some cases, the disc tissue can be chopped, sliced, or minced. In some cases, the disc tissue is treated with enzymes, such as collagenase. Treatment of the disc tissue can aid in the removal of the extracellular matrix. In some cases, the tissue is placed in a tissue culture-treated dish with media in direct contact with the surface of the dish, allowing cells to migrate from the tissue onto the plate. In other cases, the cells are separated from the tissue using a filter.

[0056] The extracellular matrix may include collagen, proteoglycans, and other molecules. In some cases, collagen may refer to a naturally occurring protein found in animals, such as mammals. Native collagen can form long fibers containing triple helices. Often, the three helices of collagen contain two identical alpha 1 chains (α1) and one alpha 2 chain (α2). Collagen is often post-transcriptionally modified by hydroxylation, cross-linking, glycosylation, cleavage, etc. Collagen can be obtained from animals or animal cells. Collagen can be synthesized from a variety of cells, including mammalian cells and non-mammalian cells such as bacteria, using techniques well known to those skilled in the art. Proteoglycan may refer to glycosylated proteins. Proteoglycans may have one or more sulfated glycosaminoglycan (sGAG) chains (generally the sequence -Ser-Gly-X-Gly-, where X can be any amino acid residue) that may be linked at a Ser residue. Proteoglycan chains are generally long, linear, and negatively charged under physiological conditions. Glycosaminoglycans can be assayed to determine proteoglycan production from cells, for example, using dimethylmethylene blue colorimetric assay or enzyme-linked immunosorbent assay. In many cases, both glycosaminoglycans in the micromass and the replaced medium (by medium change) are assayed. In some cases, for example, intervertebral disc cells grown in micromass culture in prochondrogenic medium containing TGF-B (transforming growth factor beta) or other growth factors known to generate a chondrocyte phenotype may produce more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg of sGAG, and less than about 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 μg. Often, these results can be normalized to cell number, DNA content, or protein content to determine proteoglycan production per cell.When normalized to cell number, the values ​​can be greater than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 ng sGAG / cell and less than 1, 0.09, 0.08, 0.07 ng sGAG / cell. When normalized to its protein content, the values ​​can be greater than 50, 60, 70, 80, 90, 100, 200, 300, 400 ng sGAG / μg protein and less than about 1000, 900, 800, 700, 600, 500 ng sGAG / μg protein. In many cases, disc cells produce more or less soluble proteoglycan than other cells (e.g., fibroblasts, mesenchymal stem cells, or disc cells) grown or not in adhesion-independent conditions for similar periods of time.

[0057] Cells can be differentiated along adipocyte, osteocyte, and neuronal lineages using techniques known to those skilled in the art, such as using the StemPro pluripotency kit from Life Technologies, which uses a special medium for differentiated adherent monolayer cell cultures. After osteocyte differentiation, Alizarin Red dye is used to confirm the presence of mineralized bone. After adipocyte differentiation, Oil Red O dye is used to confirm the presence of fat. After neuronal differentiation, the presence of neuronal morphology is observed. Furthermore, genetic markers can be tested to confirm associated phenotypic changes.

[0058] The cells may be capable of self-renewal, defined as the ability to replicate without changing cell phenotype, a property that can be confirmed in vitro by growth characterization over many passages or in vivo by serial transplantation and extraction.

[0059] In some embodiments, disc cells can produce extracellular matrix molecules. In other embodiments, disc cells can produce proteins. In other embodiments, disc cells can produce growth factors. In other embodiments, disc cells can produce cytokines. In other embodiments, disc cells can produce hormones. In other embodiments, disc cells can produce sugars.

[0060] After removing, reducing or degrading extracellular matrix, cells derived from intervertebral disc tissue can be transferred to adhesion-dependent or adhesion-independent culture system.In many cases, when intervertebral disc tissue is placed in adhesion-dependent system, the container can be treated with gelatin and / or collagen.When intervertebral disc tissue cells are placed in adhesion-independent culture system, the medium can contain a viscous non-reactive material that forms a gel, such as methylcellulose.

[0061] Once attached, the cells are passaged (detached from the vessel, resuspended at a lower density, and reattached to the vessel) up to approximately 10 times. In some cases, the cells never reach confluence in the vessel. In other cases, the cells reach confluence in the vessel. The cells are passaged using standard cell culture techniques. Once a sufficient number of cells are obtained (but before growth "drops off" (i.e., the cells divide at a significantly slower rate)), the cells are transferred to suspension culture using a viscous, non-reactive medium for the desired period of time. Upon completion, the cells are isolated, washed free of other materials, and further processed as needed for cryopreservation or direct therapeutic use.

[0062] Growth of intervertebral disc tissue-derived cells on gelatin- or collagen-coated vessels may allow for growth, proliferation, and / or differentiation of the intervertebral disc cells. Intervertebral disc cells can be grown at 1,000-50,000 cells / cm. 2In such cases, cells can be grown in the presence of serum, EGF, and bFGF. In some cases, serum supplements can be added to the cell culture medium, for example, conditioned medium from fibroblast cultures can be added. In these cases, non-discogenic and hypodiscogenic cells can also be grown, proliferated, and / or differentiated along with the disc cells.

[0063] Growth of cells derived from intervertebral disc tissue under anchorage-independent conditions can aid in the growth, proliferation, and / or selection of intervertebral disc cells. Intervertebral disc cells can be added at 10,000 cells / mL or up to 80,000 cells / mL. In some cases, neural supplements, bFGF, and EGF may be added to the anchorage-independent cell culture medium. In some cases, serum may or may not be added to the medium. In many cases, intervertebral disc cells derived from intervertebral disc cell cultures grown under anchorage-dependent conditions produce less extracellular matrix than cells grown under anchorage-independent conditions.

[0064] In many cases, the cell culture medium can be washed off the cells. In some cases, the cells are washed with PBS, additional medium, cryoprotectant medium, etc. In some cases, the container is cooled to 4°C before washing to aid in dissolving viscous components. Sometimes, repeated centrifugation is used to remove undesired components. Sometimes, the cells are allowed to adhere to a new container, allowing for removal of undesired components. Sometimes, the cells are allowed to adhere to a carrier, allowing for removal of undesired components. Sometimes, chemicals are used to remove undesired components.

[0065] According to standard techniques, the cells may or may not be detached or separated from the container or other cells using enzymes such as trypsin, recombinant trypsin, Accutase, HyQTase, TrypLE, etc. The cells can be mixed with additional medium and centrifuged at various speeds, for example, low speed to allow separation of different cell densities (single cells and clusters), standard speed to form a cell concentrate at the bottom, high speed to form a dense cell pellet, etc.

[0066] In many embodiments, the disc cell population is characterized by the ability to restore, regenerate, and / or grow intervertebral disc tissue in vivo.For example, the disc cell population can restore damaged or damaged discs in subjects with damaged or damaged discs.In many embodiments, introducing the disc cell population into the damaged disc of a subject restores the disc height to approximately the height before injury.

[0067] Cell morphology The disc cells may be mononuclear. The disc cells may be multinuclear. The disc cells may have organelles such as mitochondria, Golgi apparatus, and ribosomes. The viability of the disc cells may be demonstrated by trypan blue, alamar blue, live / dead assay (Life Technologies), or other assays. The disc cells may be capable of proliferation. The disc cells may be capable of producing extracellular matrix.

[0068] Disc cells grown under anchorage-independent conditions may have a different morphology than disc cells grown as a monolayer. For example, disc cells derived from anchorage-independent cell cultures may be isolated round cells. Alternatively, they may form loosely associated cell clusters with other cells and / or aggregates of round cells. Alternatively, they may form tight cell clusters known as discospheres. Disc cell clusters may have at least one dimension greater than about 50 μm after sufficient proliferation. Disc cells may begin as isolated round cells, and over time, some cells may proliferate to form clusters or discospheres. This time period may be 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 21, or 28 days. Disc cells may have associated extracellular matrix, such as proteoglycans and collagen.

[0069] cell population In one embodiment of the disc cell population, the cells may include, but are not limited to, one or more of the following cells: chondrocytes, fibroblasts, nucleus pulposus cells, annulus fibrosus cells, mesenchymal stem cells, stem cells, progenitor cells, and chondrocytes. In another embodiment of the disc cell population, the cells may include, but are not limited to, one or more of the following cells modified ex vivo: chondrocytes, fibroblasts, nucleus pulposus cells, annulus fibrosus cells, mesenchymal stem cells, stem cells, progenitor cells, and chondrocytes. In another embodiment of the disc cell population, the population may include one or more of the following: isolated cells, clusters of aggregated cells, or discospheres. In another embodiment of the disc cell population, the cells may be dissociated to form a population of isolated cells. In another embodiment of the disc cell population, the cells may be aggregated to form at least one micromass. In another embodiment, the disc cells may be more therapeutically effective before the cells are dissociated.

[0070] Therapeutic Uses of Intervertebral Disc Cell Populations Intervertebral disc cells can be delivered directly to damaged tissue. An effective amount of intervertebral disc cells may or may not be mixed with a pharmaceutically acceptable biomaterial scaffold to support implantation. For example, viscous natural materials containing hyaluronic acid, collagen, or other extracellular matrix molecules can be used. Alternatively, solid natural materials can be used. In some cases, additives can be included for stability. In some cases, additives can be included to support cryopreservation.

[0071] It has been discovered in accordance with the present invention that administration of intervertebral disc cells can repair damaged discs and / or regenerate disc structure, thereby restoring or stabilizing degenerative disc disease and other disc injuries. It has also been discovered that administration of such cells to a subject with a damaged disc partially restores pre-injury disc height. Thus, the present invention features a method for isolating and expanding intervertebral disc cells for use in treating a subject with at least one disc injury or damage. The method typically involves administering a therapeutically effective amount of intervertebral disc cells to a subject so that repair and / or regeneration of the damaged disc occurs.

[0072] In a highly preferred embodiment, the method comprises administering cells obtained or isolated from the spinal column or other cartilaginous tissue to a subject in need of treatment for at least one disorder or disordered intervertebral disc, wherein the cells are capable of self-renewal and / or proliferation in culture. Cells isolated from the spinal column and cartilaginous tissue can be propagated or maintained in culture prior to administration.

[0073] In the methods disclosed herein, intervertebral disc cells can be administered in combination with a bioactive substance. The intervertebral disc cells can be administered sequentially or simultaneously with the bioactive substance. Lysates, soluble cell fractions, membrane-enriched cell fractions, proteins, growth factors, hormones, cell culture media (e.g., conditioned media), or extracellular matrices derived from spinal cord, intervertebral disc, or cartilaginous tissue or intervertebral disc cells can also be administered to a subject as needed, for example, intervertebral disc cells and other cells or substances can be used in combination. The particular substance selected can be delivered as part of a kit from a provider. Alternatively, the particular substance may be at the discretion of the medical professional treating the subject and may vary depending on the subject's particular needs or condition. The substance selected can be used for a variety of purposes, including, but not limited to, facilitating cell administration, improving disc repair and / or regeneration, improving the subject's overall health, alleviating pain, and / or enhancing the survival of transplanted cells.

[0074] Cells can be administered to subject by injection.For example, cells can be directly injected into one or more intervertebral discs of subject.In many cases, cells can be injected into the nucleus pulposus, transition zone or annulus fibrosus of intervertebral disc.Intervertebral disc cells can be administered alone or in combination with bioactive substance or treatment active substance, and / or scaffold or matrix agent.

[0075] In some embodiments, the disc cell population can be implanted into a subject. For example, the disc cell population can be surgically implanted into a damaged or disordered disc. In some embodiments, the disc cell population can be surgically implanted into a disc space from which all or part of the disc has been removed. In some embodiments, the disc cell population can be implanted into a disc space as part of an artificial disc or disc replacement.

[0076] Cells can also be administered as scaffold- or matrix-cell complexes. Scaffold and matrix compositions include, but are not limited to, proteins, hydrogels, synthetic polymers, and combinations thereof. Scaffold and matrix compositions may or may not be biodegradable. Such materials are known in the fields of therapeutic treatment, surgical repair, tissue engineering, and wound healing. Scaffold- and matrix-cell compositions can be introduced into a subject's body by any method known in the art, including, but not limited to, implantation, injection, surgical attachment, or implantation with other tissues. In some embodiments, in vivo, or even more preferably, in situ matrix forms, such as in situ polymerizable gels, can be used in accordance with the present invention. Examples of such gels are known in the art.

[0077] Intervertebral disc cells can be mixed with scaffolds and matrices before implantation, or seeded on such compositions in vitro, thereby allowing cells to proliferate and / or establish extracellular matrices.In some cases, the matrix is ​​similar to the structure of mammalian intervertebral discs, and the matrix can replace an entire intervertebral disc in a subject.In some cases, the matrix can contain a therapeutic agent.

[0078] Intervertebral disc cells can be used to fabricate artificial disc replacement devices in vitro or in vivo. In one example, a suitable non-absorbable material, such as polyurethane, is used to fabricate the artificial outer annulus. In another example, an absorbable material, such as polyglycolic acid or polylactic acid, is used. The artificial annulus serves as a container for the disc cells, which may or may not be combined with at least one of a scaffold material, a matrix material, a carrier material, growth factors, and / or other bioactive substances. In many embodiments, the artificial annulus structure can be porous and / or fibrous. The artificial outer annulus can include attachment means to allow it to be fixed to one or more vertebral bodies. For example, the artificial annulus can include through-holes, cuffs, tabs, loops, or washers to allow screw fixation to one or more vertebral bodies. The artificial disc can be surgically implanted into a subject to provide a total disc replacement.

[0079] Artificial disc replacement devices can include the disc cells of the present invention. In various embodiments, the disc cells can be inserted into an artificial annulus structure. The artificial annulus structure can be designed to provide a containment structure for the disc cells and can further include an attachment structure for securely attaching the disc replacement device to one or more vertebral bodies.

[0080] The disc cells are added to the artificial disc replacement device at or near the time of insertion of the disc replacement device. In other embodiments, the disc cells are added to the disc replacement device well before insertion to allow the cells to grow, divide, and provide a matrix or scaffolding material. In many embodiments, scaffolding materials, matrix materials, carrier materials, growth factors, and / or other bioactive substances can be added to the disc replacement device before, after, or together with the addition of the disc cells.

[0081] Artificial disc replacement devices may include resorbable or non-resorbable artificial annulus. In some embodiments, the artificial annulus may include matrix materials, scaffolds, growth factors, or other bioactive substances that help support the proliferation of disc cells. In one embodiment, the artificial annulus supports the proliferation and / or differentiation of annulus fibrosus cells, for example, by providing a local source of growth factors and / or cytokines that can promote disc cell differentiation into annulus fibrosus cells. In many embodiments, artificial disc replacement devices containing disc cells may have a cellular structure similar to that of non-artificial discs, for example, the cellular structure of the annulus fibrosus, nucleus pulposus, and endplate tissues.

[0082] In some cases, the disc cells and scaffolds can be cryopreserved in liquid nitrogen. Alternatively, they can be stored at various subzero temperatures, such as -80°C, -20°C, or -1°C. Additionally, the disc cells can be stored at 4°C or 37°C. The disc cells can be combined with a scaffold before storage. Alternatively, the disc cells may or may not be combined with a scaffold immediately before implantation.

[0083] Thus, effective treatment involves treating a subject with a lesion, abnormality, or trauma to the intervertebral disc with a therapeutic cell composition comprising intervertebral disc cells, with or without a therapeutic agent, matrix, or scaffold. The cells are present in an amount effective to promote, for example, direct proteoglycan production or stimulate natural cell regeneration. The result can be regeneration, repair, or reconstruction of natural tissue architecture, as demonstrated in a rabbit model. This can be determined by medical imaging (X-ray, MRI) or pain relief. For implantation into a human intervertebral disc, the cell dose can range from 1,000 to 10,000,000 cells, including the intervertebral disc cell population or a subpopulation extracted from the intervertebral disc cells. Additionally, the scaffold volume can range from 10 μL to 1,000 μL, or from 10 mg to 10 g, depending on the spatial needs of the subject.

[0084] In some embodiments, one or more subpopulations of cells in the intervertebral disc cells can be transplanted. This subpopulation can be isolated using magnetic beads for sorting, fluorescent markers for sorting, density gradients, fluorescent gene tagging for sorting, physical separation, filtration, etc. This subpopulation can be more effective in treatment compared to the whole population.

[0085] In treating a subject, intervertebral disc cells can provide superior therapeutic effects compared to adhesion-dependent disc cells or other cell populations such as nucleus pulposus cells, fibroblasts, chondrocytes, stem cells, progenitor cells, etc. This effect may or may not be improved by the use of a scaffold, carrier, or other biomaterial.

[0086] The present invention also features kits for carrying out the methods of the present invention. In one embodiment, a kit for treating a subject having at least one intervertebral disc disorder or injury is provided. The kit may include a pharmaceutically acceptable carrier; an amount of intervertebral disc cells effective to treat the disorder or injury; and instructions for using the kit in a method for treating a subject having at least one intervertebral disc disorder or injury. The kit may further include at least one agent and instructions for culturing the cells. The kit may further include at least one biologically active or therapeutically active agent. The kit may further include a vial and a syringe. The kit may further include a discography needle for direct access to the intervertebral disc. The kit may further include a radiopaque material to assist in imaging during the procedure.

[0087] Experiment details material and method Supplier / drug: In some cases, adhesion-dependent cell culture may be referred to as a growth condition. Growth medium can be used for cell growth in a growth condition. Growth medium contained DMEM / F12 with 10% fetal bovine serum. 30% of this medium may optionally be preconditioned in the presence of neonatal foreskin fibroblasts for 3 days. This preconditioned portion was filtered before use. Before adding the growth medium to the cells, bFGF and EGF were added to make the medium "complete" with a final concentration of 10 ng / mL bFGF and 10 ng / mL EGF (obtained from a stored 1000X stock solution).

[0088] In some cases, adhesion-independent cell culture may be referred to as a suspension condition. A suspension medium can be used for cell growth in a suspension condition. The suspension medium contained 1% A4M Premium methylcellulose (Dow Chemical) in DMEM / F12 supplemented with 1X B27 (Life Technologies), 5% fetal bovine serum, 10 ng / ml EGF, and 10 ng / ml bFGF. The suspension feed medium is the same, but without methylcellulose.

[0089] For growth, plates or flasks coated with collagen, gelatin, or other similar matrix proteins were used. Such ready-made vessels were purchased. Alternatively, plates could be coated manually by dissolving 1 g of powdered gelatin (Sigma) in 1 L of ddH2O (double distilled water), or prepared using a ready-made solution (Sigma) diluted to a final concentration of 1%. If coated in the laboratory, the vessels were incubated at room temperature for at least 15 minutes. For suspension, Corning ultra-low attachment vessels were used.

[0090] In some cases, a viscous scaffold carrier was used for in vivo cell implantation. Specifically, a pre-made sterile gel consisting of 1.7% hyaluronic acid (0.8–1.2 MDa) in PBS was diluted with human serum albumin and 30,000 intervertebral disc cells to obtain a 1% hyaluronic acid gel containing 2.5% human serum albumin. This was then placed into a sterile 50 μl glass Hamilton syringe. A 27-gauge Precision Glide needle was attached to the Hamilton syringe via a Luer lock for implantation into animals.

[0091] method: Human adult nucleus pulposus tissue is obtained from consenting donors undergoing surgery using an IRB-approved protocol. In various embodiments, tissue can be obtained from a variety of sources and tissues, including living and deceased non-autologous donors, autologous donors, intervertebral disc tissue, or other cartilaginous tissue. Non-nucleus pulposus tissue, including the annulus fibrosus and cartilage endplates, was manually excised and discarded. 2-7 g of the resulting tissue was mixed with 15 ml of DMEM / F12 containing 300 units / ml of collagenase II in a T75 flask and incubated overnight under standard tissue culture conditions (37°C and 5% CO2). The freed cells were then transferred to a 50 ml tube, pelleted by centrifugation (4 minutes, 1200 rpm), the supernatant removed, and resuspended in DMEM / F12 to a final concentration of approximately 10,000 cells / ml.

[0092] Alternatively, cells were obtained from cryovials (stored in liquid nitrogen) by thawing in a 37°C water bath and immediately transferring to a 15 mL tube containing 10 mL of growth medium. The cell mixture was then centrifuged at 1200 rpm for 4 minutes. The supernatant was aspirated, the cells were resuspended in growth medium, and the cells were counted and viability confirmed.

[0093] For expansion, cells were seeded at 10,000 cells / mL in complete growth medium, with medium changes every 2–3 days. Before reaching confluence, cells were washed with PBS and incubated with 0.25% trypsin for 7 minutes, then removed from the plate using growth medium and transferred to a 50 ml conical tube. The tube was centrifuged as before, and the trypsin-containing supernatant was removed. Cells were then resuspended in growth medium to allow for cell counting and viability checks. At this point, cells could be cryopreserved by mixing 0.5–3 million cells with 90% FBS / 10% DMSO in a cryovial. To freeze the contents, the cryovial was stored at -80°C overnight before being transferred to the vapor phase of liquid nitrogen for long-term storage.

[0094] For suspension culture, cells were mixed with suspension medium at 10,000 cells / ml (15 ml total) and plated into ultra-low attachment 100 mm plates (Corning). Every two days, 300 μl of suspension feed medium was added to the plate. Images were taken at 4X, 10X, and 20X magnifications using a phase-contrast microscope, as described in Figure 2. After two weeks, the plates were incubated at 4°C for 20 minutes, and then the cells were harvested by diluting the gel-like medium with 15 mL of PBS. The contents of the plate were then transferred to a 15 mL tube, the volume was increased to 50 mL with additional PBS, and the tube was centrifuged at 1200 rpm for 4 minutes. The supernatant was then removed. The cells were then washed in this manner two more times to ensure the removal of the methylcellulose. Finally, the intervertebral disc cells were resuspended in growth medium to allow for cell counting and viability checks.

[0095] To determine surface marker expression, disc cells were treated with 0.25% trypsin for 7 minutes to form a single-cell suspension. Additional cell populations tested included pre-discogenic cells derived from expansion cultures, human adult mesenchymal stem cells (CET Company), human neonatal foreskin fibroblasts (AATC), and human adult articular chondrocytes (ScienCell). Cells were analyzed using standard techniques using a Partec CyFlow flow cytometer. Briefly, 70,000 cells and antibodies (one per tube) were diluted in 200 μl of PBS containing 0.5% bovine serum albumin and incubated for 30 minutes at 4°C in the dark. The following antibodies were used: CD73-PE (BD), CD90-PE-Cy5 (BD), CD105-PE (Miltenyi), CD166-PE (BD), HLA-ABC (BD), and controls. This procedure was performed twice, and one representative data set is shown.

[0096] To determine proteoglycan production in a chondrogenic environment, a standard chondrocyte pellet assay was used. Briefly, 200,000 cells (intervertebral disc cells or mesenchymal stem cells) were placed in individual conical wells of a 96-well plate containing 200 μl of chondrogenic medium (StemPro medium, Life Technologies) and briefly centrifuged. Every three days, the medium was removed, collected, and replaced, taking care not to aspirate the formed central micromass. After two weeks (see Figure 3), the cell pellets were harvested, dried, and digested overnight with 250 μl of papain (Sigma) in buffer at 60°C. The digest, as well as the collected medium, were assayed for sGAG content, a component of proteoglycans, using a standard DMMB assay. Specifically, to prepare a DMMB working solution, 1 g of sodium formate was dissolved in 490 mL of deionized water, and 1 mL of formic acid was added. In a separate tube, 8 mg of dimethylmethylene blue (DMMB) powder (Sigma) was dissolved in 2.5 mL of ethanol and the two solutions were mixed. Water was then added to bring the volume to 500 mL. To prepare the chondroitin-6-sulfate standard, 40 mg of chondroitin-6-sulfate (CS) was mixed with 40 mL of water to create a 1 mg / mL standard solution (stock). The stock was then diluted to 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 μg / mL. To assay proteoglycan content, 100 μl of standard or sample was mixed with 100 μl of DMMB working solution in a 96-well clear plate and measured at 525 nm within 5 minutes. The concentration of the sample was determined using the standard, and then normalized to the original volume to determine the amount. Additionally, digests were assayed for protein content using the Pierce protein assay according to the manufacturer's instructions or for DNA content using the Quanti-IT PicoGreen assay (Life Technologies) according to the manufacturer's instructions.

[0097] To determine osteogenic and adipogenic potential (Fig. 4 ), intervertebral disc cells were grown using StemPro Osteogenic and Adipogenic Kits (Life Technologies) according to the manufacturer's instructions.

[0098] To evaluate in vivo efficacy, 30,000 intervertebral disc cells were mixed with 25 μl of 1% hyaluronic acid (0.8–1.2 MDa) in PBS containing 2.5% human serum albumin. The cells and viscous scaffold were placed in a 50 μl glass Hamilton syringe. Using a previously internally validated degenerative disc disease model, the lumbar discs of three New Zealand rabbits were surgically accessed and needle-punctured to induce degeneration (n = 4 discs / animal, approved by local IACUC). After two weeks, the injured discs were injected with cells or acellular scaffold control. In addition, injured and uninjured control lumbar discs were maintained in each animal. Every two weeks for six weeks, disc height was measured by plain radiography and normalized to the value at week 0 to obtain the disc height index (DHI). After six weeks, the rabbits were euthanized. Discs were harvested and processed for histological analysis. Sections were stained with H&E or Alcian blue, and abnormalities were blindly scored; scores of 0 to 2 were assigned for the AF / NP boundary, AF organization, NP extracellular matrix, and NP cellularity (AF - annulus fibrosus; NP - nucleus pulposus). The four results were summed (0 = normal, 8 = abnormal). [Example]

[0099] The following examples detail the preparation and properties of embodiments of the microenvironments of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to construction, materials, and methods, can be practiced without departing from the scope of the present disclosure. [Example]

[0100] Surface marker expression of various cell types by FACS analysis Cell surface marker expression was analyzed for various cells, including intervertebral disc cells. As shown in Figure 1, human cell types tested included adult mesenchymal stem cells, neonatal foreskin fibroblasts (Fibroblasts), articular chondrocytes (Chondroblasts), anchorage-dependent intervertebral disc cells (AD-DCs), and anchorage-independent intervertebral disc cells (AI-DCs). Figure 1A shows the percentage of cells expressing a given marker based on standard IgG gating. DC cells exhibit low CD105 and CD166 expression compared to other cell types. Other surface markers have been investigated to identify a unique phenotypic fingerprint for DCs. Figure 1B shows representative cell marker flow cytometry analysis of MSCs, anterior disc cells, and intervertebral disc cells for CD105 and CD166, demonstrating that disc cells exhibited a unique pattern. [Example]

[0101] Morphology of intervertebral disc cells in suspension culture The morphology of intervertebral disc cells was investigated. Figure 2A is a micrograph showing intervertebral disc cells consisting of dense spheres known as discospheres (*), loose cell aggregates (**), and single cells (***). Figure 2B shows that the diameters of the discospheres and aggregates vary, typically being greater than 50 µm. [Example]

[0102] Chondrogenic potential of mesenchymal stem cells (MSCs) and intervertebral disc cells (DCs) Figure 3 shows an analysis of the chondrogenic potential of mesenchymal stem cells and intervertebral disc cells. Figure 3A shows the morphology of cells after two weeks of growth in a chondrogenic environment. As shown, DCs produced larger micromasses than MSCs. Figure 3B is a graph comparing protein and GAG production by MSCs and DCs. As shown, DCs produced more sGAG (a component of proteoglycans) than MSCs, both in the culture medium and in the digested micromass. Meanwhile, the protein content of each micromass was comparable. [Example]

[0103] Adipogenic and osteogenic potential of intervertebral disc cell populations Figure 4A shows that after differentiation in adipogenic medium according to the manufacturer's instructions (Life Technologies), adipogenesis was confirmed by Oil Red O staining counterstained with hematoxylin, and Figure 4B shows that after differentiation in osteogenic medium according to the manufacturer's instructions (Life Technologies), osteogenesis was confirmed by Alizarin Red staining (scale = 50 μm). [Example]

[0104] Live / dead assay of intervertebral disc cell viability at various stages prior to therapeutic use (green indicates live, red indicates dead). Figure 5A shows that viability is confirmed after 24 hours in a viscous hyaluronic acid scaffold, where the majority of cells are viable (bright cells; some dead cells are indicated by arrows). Figure 5B shows that viability (bright cells) is also confirmed after extrusion through a 27-gauge surgical needle used for surgical implantation into a rabbit. [Example]

[0105] Therapeutic use of intervertebral disc cells using a viscous scaffold carrier Figure 6 is a schematic diagram illustrating an embodiment and process for using disc cells to treat DDD. Figure 6(A) illustrates the use of disc cells by injection. The cells and scaffold can be mixed and pre-loaded into a syringe or vial. The cells and scaffold can be separate and mixed immediately before injection. The preparation can be delivered from a syringe or false bottom vial. The preparation is injected directly into the degenerated disc. Figure 6(B) illustrates the implantation of disc cells. The cells and scaffold can be mixed or delivered separately. Prior to implantation, the material is cut or trimmed to fill the intended implantation area. The material is then implanted into the defect. Cells may be added after implantation (not shown). In the in vivo animal studies described, the cells and scaffold were mixed and shipped in a vial at 4°C, and the contents were placed into a syringe immediately before injection. [Example]

[0106] Efficacy of intervertebral disc cells in a viscous scaffold carrier in a rabbit model of degenerative disc disease Figure 7A is a graph showing that disc cell therapy within a scaffold carrier restored disc height index (DHI) compared to the control group at 4 and 6 weeks. All control groups showed consistency across the 2, 4, and 6 week time points. N=3. * indicates p<0.001 compared to both scaffold and injury controls by two-way ANOVA and Tukey's post-hoc test. Figure 7B is a graph showing histological scores. Blinded disc scores (0-8, 0=normal) at 6 weeks. Cell therapy showed significant improvement compared to injury and scaffold controls. ** indicates p<0.05 compared to scaffold control, and * indicates p<0.05 compared to injury control (by t-test). [Example]

[0107] Generation and evaluation of intervertebral disc cells material and method Generation of intervertebral disc cells The procedure described is shown in the flow diagram in Figure 8. First, human adult nucleus pulposus tissue, waste material from discectomy, was obtained from consenting donors under IRB approval (Baptist Hospital, TN). The annulus fibrosus and other tissue contaminants were removed by dissection. The remaining material was then washed three times with 2X antibiotic-antimycotic (ABAM, HyClone by Thermo Scientific) in PBS and digested overnight with 300 units of recombinant type 2 collagenase (Life Technologies) in DMEM / F12 containing 1X ABAM (Life Technologies). The isolated cells were plated in growth medium (DMEM / F12 containing 10% FBS (HyClone), 10 ng / mL EGF, and 10 ng / mL FGF-2 (Peprotech)) in gelatin-coated flasks. Over time, a subpopulation of cells composed of stem / progenitor cells adhered to the plate. These cells were expanded for up to four passages.

[0108] Cells were then combined with suspension medium (10,000 cells / cm in DMEM / F12 containing 5% FBS, 10 ng / ml EGF, and 10 ng / ml FGF-2) in the presence of 1% methylcellulose (A4M Premium, Dow Chemical) in ultra-low attachment vessels (Corning; Corning, NY). 2 )(1cm 2 (1.5 mL of medium was added per well.) After two weeks, cells were harvested for further use by washing three times with phosphate-buffered saline (PBS, Corning CellGro; Manannas, VA) to remove the methylcellulose. Intervertebral disc cells were generated from five different human donors. Intervertebral disc cell suspensions were fixed in 10% formalin (Sigma-Aldrich; St. Louis, MO) for 15 minutes.

[0109] Histology of intervertebral disc cells The cells were then washed three times with PBS and resuspended in 37°C agarose (approximately 1x10 cells per 0.5 mL in 1% BioReagent low gelling temperature agarose from Sigma).7 Once the agarose solidified, the pellet was kept in PBS until frozen, frozen in OCT medium (Sakura Tissue-Tek; Torrance, CA) on a Leica Cryostat, and sectioned at 6 μm thickness onto charged slides. Samples were then stained with either hematoxylin and eosin, Alcian blue and nuclear fast red counterstain, or picrosirius red according to standard protocols. Tissue from one representative donor is shown.

[0110] Confocal microscopy A suspension of intervertebral disc cells was fixed in 10% formalin for 15 minutes and washed three times with PBST (PBS + 0.1% Triton 100X, Sigma; St. Louis, MO). The fixed cells were then incubated with primary antibodies in PBSTA (PBST + 0.5% human albumin, Baxter Healthcare; Westlake Village, CA) at a dilution of 1:100 for anti-human aggrecan antibody (Santa Cruz Biotechnology; Dallas, TX) or 1:20 for anti-collagen II antibody (Developmental Studies Hybridoma Bank; Iowa City, IA) at room temperature for 2 hours, followed by three washes with PBST. The cells were then bound with Alexa-Fluor 488 for aggrecan or Alexa-Fluor 633 for collagen II (both in PBSTA) for 1 hour at room temperature and washed three times with PBST. Finally, cells were counterstained with DAPI and phalloidin (Molecular Probes, Life Technologies) for nuclei and actin, respectively, and imaged using an Olympus FV1000 confocal microscope. Tissue from one representative donor is shown.

[0111] Flow cytometry Cell surface antigen expression of intervertebral disc cells was analyzed by flow cytometry using the following fluorescently labeled mouse anti-human monoclonal antibodies: CD105-phycoerythrin (PE, Miltenyi Biotec, Inc., Auburn, CA, USA); Stro-1-Alexa Fluor 647 (BioLegend, San Diego, CA, USA); CD166-PE, CD73-APC, and CD90-FITC, CD44-FITC, CD-24-PerCP-Cy5.5, CD34-PE, HLA-DRDP-FITC, and HLA-ABC-FITC (all from BD Biosciences, San Jose, CA, USA). Appropriate isotype controls were also run in parallel. Cells were incubated in PBS containing 50% mouse serum for 30 min at 4°C, then washed and resuspended in PBS containing 1.0% bovine serum albumin. Viable cell mass was measured using DAPI dilactate (Life Technologies, Carlsbad, CA, USA). A minimum of 20,000 events were collected on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) using FlowJo Software for data acquisition and analysis.

[0112] multipotent Osteogenesis and adipogenesis were induced using a kit provided by Life Technologies (Grand Island, NY). Briefly, intervertebral disc cells were dissociated using TrypLE (Gibco, Life Technologies) for 15 minutes to form a single cell suspension and incubated at 20,000 cells / cm. 2 Cells were seeded onto tissue culture-treated dishes at 100°C. Dishes of cells for osteogenic and adipogenic differentiation were maintained in DMEM containing 10% FBS for 3 days and then fed with the appropriate supplemented differentiation medium for 3 weeks. After differentiation, monolayers were stained with Alizarin Red for mineralization or Oil Red O for adipose tissue as indicated. Phase images were captured at various magnifications. Studies were performed on four different donors.

[0113] Chondrogenesis was induced as described in [Johnstone 1998]. Briefly, 250,000 cells were placed in individual conical wells of a 96-well plate with 200 μl of chondrogenic medium (Life Technologies) and briefly centrifuged. Every 3 days, the medium was completely removed, collected, and replaced, taking care not to aspirate the formed central micromass. After 2 weeks, the cell micromass was harvested, dried, and digested overnight at 60°C with 250 μl of papain (Sigma-Aldrich). The digest, as well as the collected medium, was assayed for GAG content as described in [Farnesdale]. Additionally, the digest was assayed for DNA content using the Quant-IT PicoGreen Assay (Invitrogen), and the results were normalized to assess GAG / DNA.

[0114] For comparison, the chondrogenic potential of intervertebral disc cells was evaluated in comparison with other known human adult cell lines. Articular chondrocytes (Sciencell; Carlsbad, CA), bone marrow-derived mesenchymal stem cells (CET, Thermo Scientific; Waltham, MA), and dermal fibroblasts (ATCC; Manassas, VA) were purchased and grown according to the provided instructions.

[0115] result Histology, confocal microscopy and gene expression Intervertebral disc cells were assayed for aggrecan and collagen production. After 2 weeks in non-plastic adherent culture, individual NP-derived stem / progenitor cells proliferated into clusters of various sizes embedded in the extracellular matrix (ECM). The matrix was composed of proteoglycans, the main components of nucleus pulposus tissue, and various collagens (Figures 9A-C). Confocal imaging confirmed aggrecan and collagen 2 in the ECM, indicating that matrix content varied among the clusters (Figure 9D). Furthermore, high-magnification images revealed both intracellular and extracellular aggrecan (Figure 9E), suggesting active transport of ECM molecules out of the cells at the time of imaging.

[0116] As shown in Figure 9F, expression of aggrecan and collagen 2 was assessed relative to the housekeeping gene HPRT over time during culture (days 3 and 7), at cell harvest (day 14), and after chondrogenic differentiation. Expression of extracellular matrix molecules significantly increased during the culture period, with aggrecan expression increasing approximately 20-fold and collagen 2 expression increasing approximately 70-fold compared to plastic-adherent cells. Gene expression further increased during chondrogenic differentiation.

[0117] Flow cytometry Disc cells were dissociated to form a single-cell suspension and analyzed by flow cytometry for various surface markers. The population was homogeneous in terms of size and internal structure, as seen in the forward / side scatter plot shown in Figure 10A. A wide range of surface markers were tested and compared to mesenchymal stem cells (as a control). Expression of these markers was generally uniform across five different human donors (p>0.05), with expression of CD44, CD73, CD90, and HLA-ABC exceeding 80% and expression of CD34, HLA-DR / DQ, and STRO-1 below 10% compared to isotype controls (Figures 10B and 10C). Expression of CD24, CD105, and CD166 was approximately 40%, showing slightly higher variability.

[0118] pluripotency Intervertebral disc cells were tested for their ability to form bone, fat, and cartilage following standard differentiation protocols. Staining of differentiated cell monolayers revealed clear adipogenesis and osteogenesis (Figure 11A-B). Upon differentiation, disc cells formed large, solid cartilage micromasses that stained positive for proteoglycans (Figure 11C).

[0119] After 3 weeks of culture, the medium and micromass were quantitatively assayed for proteoglycan content. Disc cells (DCs) were compared with articular chondrocytes (ACs), fibroblasts (FBs), and mesenchymal stem cells (MSCs). As shown in Figure 11E, the amount of insoluble (micromass) proteoglycan did not differ significantly between cell types, but the measured amounts of soluble proteoglycan varied considerably. When these two matrix types were combined and normalized to DNA content (Figure 11D), disc cells produced more proteoglycan and DNA than fibroblasts (p<0.01) and at levels comparable to MSCs and articular chondrocytes. [Example]

[0120] In vivo pilot study in rabbits Female New Zealand White rabbits (3-4 kg) were used in this study under the approval of a private IACUC. Three rabbits were fasted overnight before surgery. For the first surgery to induce degeneration, the animals were anesthetized intravenously, and the surgical site was prepared for aseptic surgery. An 8-10 cm longitudinal incision was made on the left flank between the iliac crest and the last rib. The lumbar intervertebral disc was accessed via a retroperitoneal approach using blunt dissection. An 18-gauge needle was then inserted at least 5 mm into the target lumbar intervertebral disc to injure the disc. Discs L2-L3, L3-L4, L4-L5, and L5-L6 were injured in this manner. L5-L6 was left intact. The muscle and skin were then closed in two or three layers using sutures, and the animals were monitored during recovery. Prior to this study, six rabbits were injured and evaluated for 8 weeks to ensure that a stable and adequate defect was created (data not shown).

[0121] Two weeks later, the rabbits were again prepared for surgery, anesthetized, and the discs accessed as described above. Using a 27-gauge needle, 25 μl of either cell therapy (L5-L6) containing 30,000 cells or scaffold alone (L4-L5) was injected. For high-dose therapy, 300,000 cells were injected. One disc was left untouched and served as an injury control (L2-L3). The injection was held in place for 5 seconds, and no leakage of material was observed when the needle was removed.

[0122] Animals were monitored for adverse events or health status over a 6-week period. Body weights were measured weekly. Additionally, every 2 weeks, animals were briefly anesthetized to undergo lumbar spine radiographs (FIG. 12A). Distances between bony landmarks on plain radiographs (three measurements of the disc space, three measurements of the adjacent left vertebra) were measured with a micrometer by the same individual and normalized to the distances at week 0 to obtain the percent disc height index (DHI).

[0123] After 6 weeks, the rabbits were euthanized; the intervertebral discs were harvested and prepared in paraffin for histological analysis. Sections (4 μm) were stained with hematoxylin and eosin or a mixture of Alcian blue and eosin.

[0124] statistical analysis All statistical analyses were performed using StatPlus software (AnalystSoft; Vancouver, Canada) utilizing Tukey's post-hoc test. p values ​​for significance were indicated for each test. One-way analysis of variance (ANOVA) was used to compare surface marker expression and proteoglycan production of intervertebral disc cells in vitro. Two-way ANOVA was used to analyze disc height data over time, and one-way ANOVA was used to compare histological scores at week 6. Graphs show the mean, along with error bars representing the standard deviation of the data set.

[0125] result As described above, "degeneration" was induced in the intervertebral discs of three New Zealand White rabbits (n=3 conditions) by surgical puncture. At the time of injury, nucleus pulposus material was observed to emerge from the needle passage after removal. After surgery, the animals did not show any abnormal signs of distress due to the injury. Two weeks later, low dose, high dose, or scaffold control was injected into the intervertebral disc, pressure was held for 5 seconds, and the needle was then removed. After injection, some of the injectate was observed to emerge from the intervertebral disc after the needle was removed.

[0126] No safety issues were observed over the 6-week study period. No significant weight changes were observed after the initial injury (FIG. 12B; average weight change from 3.4 grams before injury to 3.3 grams on day 7) or after cell therapy injection (FIG. 12B; average weight change of 3.2 grams). No health or behavioral issues were reported by animal care staff in any of the animals that received human cell injections into the intervertebral disc.

[0127] Significant changes in disc height were measured by X-ray over a 6-week period (representative X-rays are shown in Figure 12A along with the measurement method). Two weeks after injury, all discs had lost an average of 59% of their original height. At weeks 4 and 6, discs treated with the low and high doses showed significant improvements in disc height compared to both the scaffold and injury controls, from 70% and 64% DHI to 94% and 71% DHI, respectively (p<0.001) (Figure 12C). Scaffold control injections showed slightly better results than no injections (shown by the injury control) (64% DHI and 53% DHI, respectively; p<0.01).

[0128] Histologically, H&E images showed that cell therapy treatment normalized the disc structure. The height of the nucleus pulposus, from endplate to endplate, decreased with injury and increased with cell therapy (Figure 13A). As shown in Figure 13B, no immunoreactivity or abnormal tissue formation was observed in the bone marrow, annulus fibrosus (AF), cartilage endplate (CEP), or nucleus pulposus (NP) after treatment. The nucleus pulposus remained dense in proteoglycans, as shown by Alcian blue staining (Figure 13B). [Example]

[0129] In vivo pilot study in pigs Female Göttingen minipigs (10-15 kg) were used in this study under the approval of a commercial IACUC. Two pigs were fasted overnight before surgery. The injury surgery was performed as described in Example 9, with the additional step of confirming proper needle placement using fluoroscopic imaging. The L2-L3, L3-L4, and L3-L4 intervertebral discs were injured. L5-L6 was left intact. The muscle and skin were then closed in two or three layers with sutures, and the animals were monitored during recovery. Prior to this study, six minipigs were injured and evaluated for 12 weeks to ensure stable and adequate defects were created (data not shown).

[0130] Two weeks later, the pigs were again prepared for surgery, anesthetized, and the intervertebral discs were accessed as described above. Using a 27-gauge needle, 150 μl of either cell therapy containing 100,000 cells (high dose; animal 1: L3-L4, L4-L5), cell therapy containing 500,000 cells (low dose; animal 2: L3-L4, L4-L5), or scaffold alone (L2-L3) was injected. One disc was left untouched and served as an injury control (L2-L3). The injection was held in place for 5 seconds, and no leakage of material was observed when the needle was removed.

[0131] Animals were monitored for adverse events or health status for an additional 10 weeks. Body weights were measured weekly. After 4, 8, and 12 weeks, animals were briefly anesthetized and radiographs of the lumbar spine were taken, and DHI was determined as described above. After 12 weeks, the pigs were euthanized; intervertebral discs were harvested and prepared in paraffin for histological analysis as described above.

[0132] result Injury to porcine intervertebral discs resulted in a 20-30% reduction in disc height. Both low- and high-dose treatments resulted in immediate improvements in disc height that persisted through week 12 and were superior to injury controls (p<0.05). Scaffold and injury controls showed no improvement over time (Figure 14A). Fluoroscopic images aided in accurate needle placement and also demonstrated differences in disc height along the injured vertebrae (Figure 14B). Similar to the rabbit pilot study described above, no immunoreactivity or abnormal tissue formation was observed in the nucleus pulposus, cartilage endplates, annulus fibrosus, or bone marrow (not shown), and nucleus pulposus staining for proteoglycans was similar to untreated tissue (Figure 14C).

[0133] There are alternative ways of implementing the embodiments disclosed herein. While several exemplary aspects and embodiments have been described, those skilled in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. Accordingly, the embodiments of the present invention should be considered illustrative and not limiting. Moreover, the claims are not limited to the details set forth herein, but are entitled to their full scope and equivalents.

Claims

1. It is a population of intervertebral disc cells, The intervertebral disc cell population comprises multiple nucleus pulposus cells derived from mammalian intervertebral disc tissue, which are passaged at least twice in scaffold-dependent culture and then grown in vitro in a scaffold-independent culture medium containing 0.1% to 5% methylcellulose, poloxamer, or agar / agarose, wherein, after 14 days of scaffold-independent culture, the intervertebral disc cell population shows at least a 20-fold increase in aggrecan expression and at least a 70-fold increase in collagen 2a expression, as measured by RT-PCR, compared to the same multiple nucleus pulposus cells grown in scaffold-dependent culture on day 0, and less than 40% of the intervertebral disc cell population expresses the cell surface markers CD24 and CD105, as measured by fluorescence-activated cell sorting. Intervertebral disc cell population.

2. It is a population of intervertebral disc cells, The intervertebral disc cell population comprises multiple cells derived from cartilaginous tissue, which are passaged at least twice in scaffold-dependent culture and then transferred to a scaffold-independent culture medium containing 0.1% to 5% methylcellulose, poloxamer, or agar / agarose, where they are maintained. The intervertebral disc cell population, after 14 days of scaffold-independent culture, shows at least a 20-fold increase in aggrecan expression and at least a 70-fold increase in collagen 2a expression, as measured by RT-PCR, and less than 40% of the intervertebral disc cell population expresses the cell surface markers CD24 and CD105, as measured by fluorescence-activated cell sorting. Intervertebral disc cell population.

3. The intervertebral disc cell population according to claim 1 or 2, wherein the culture medium comprises one or more additives selected from the group consisting of EGF, bFGF, serum, fibroblast-conditioning medium, and viscous nonreactive substances.

4. The intervertebral disc cell population according to claim 3, wherein the plurality of cells are subcultured in a culture vessel having a low-adhesion coating.

5. The intervertebral disc cell population according to any one of claims 1 to 4, wherein the plurality of cells have been passaged at least once in a scaffold-independent culture.

6. A population of intervertebral disc cells according to any one of claims 1 to 5, which generates an extracellular matrix.

7. An intervertebral disc cell population according to any one of claims 1 to 6, expressing one or more cell surface markers selected from the group comprising CD34, CD44, CD73, CD90, CD166, Stro-1, HIF1, FIT-1, nestin, CK8, and HLA proteins.

8. An intervertebral disc cell population according to any one of claims 1 to 6, expressing one or more genes or gene products selected from the group comprising GAPDH, SDHA, HPRT1, B2M, Sox9, Col1, Nestin, CK8, Sox1, CD44, ALPI, and PPARG.

9. The intervertebral disc cell population according to any one of claims 1 to 8, wherein the plurality of cells are derived from intervertebral disc tissue.

10. A pharmaceutical product for treating a disorder selected from the group consisting of degenerative disc disease, herniated disc, and disc injury, Contains a treatment-effective amount of intervertebral disc cell population, Here, the intervertebral disc cell population was passaged at least twice in scaffold-dependent culture and then transferred to a scaffold-independent culture medium containing 0.1% to 5% methylcellulose, poloxamer, or agar / agarose, where it was maintained. Here, after 14 days of scaffold-independent culture, the intervertebral disc cell population showed at least a 20-fold increase in aggrecan expression and at least a 70-fold increase in collagen 2a expression compared to day 0, as measured by RT-PCR, and less than 40% of the population expressed the cell surface markers CD24 and CD105, as measured by fluorescence-activated cell sorting. Pharmaceuticals.

11. The pharmaceutical product according to claim 10, wherein the intervertebral disc cell population is derived from mammalian intervertebral disc tissue.

12. The pharmaceutical product according to claim 11, wherein the provided organ tissue is mammalian intervertebral disc tissue.

13. The pharmaceutical product according to claim 10, wherein intervertebral disc cells are passaged at least once in a scaffold-independent culture.

14. The pharmaceutical product according to claim 10 or 11, wherein the intervertebral disc cell population generates an extracellular matrix.

15. The pharmaceutical product according to any one of claims 10 to 12, wherein the intervertebral disc cell population expresses one or more cell surface markers selected from the group including CD34, CD44, CD73, CD90, CD166, Str-1, HIF1, nestin, CK8, and HLA proteins.

16. The pharmaceutical product according to any one of claims 10 to 12, wherein the intervertebral disc cell population expresses one or more genes or gene products selected from the group including GAPDH, SDHA, HPRT1, B2M, Sox9, Col1, Nestin, CK8, Sox1, CD44, ALPI, PPARG, ADAMTS, MMP, FMOD, and IL.

17. A device for treating a damaged or injured intervertebral disc, Intervertebral disc cell populations derived from chondrogenic tissue cells, grown in scaffold-dependent culture, passed at least twice, and then grown in vitro in scaffold-independent culture medium containing 0.1% to 5% methylcellulose, poloxamer, or agar / agarose; and Scaffolding, matrix, or portable structure The intervertebral disc cell population, after 14 days of scaffold-independent culture, shows at least a 20-fold increase in aggrecan expression and at least a 70-fold increase in collagen 2a expression, as measured by RT-PCR, compared to cells grown in scaffold-dependent culture on day 0, and less than 40% of the population expresses the cell surface markers CD24 and CD105, as measured by fluorescence-activated cell sorting. device.

18. The device according to claim 17, further comprising a bioactive substance.

19. An artificial intervertebral disc replacement device, Artificial outer ring; and Intervertebral disc cell populations derived from nucleus pulposus cells, which were passaged at least twice in scaffold-dependent culture and then grown in vitro in scaffold-independent culture medium containing 0.1%–5% methylcellulose, poloxamer, or agar / agarose. Including, here, After 14 days of scaffold-independent culture, the intervertebral disc cell population showed at least a 20-fold increase in aggrecan expression and at least a 70-fold increase in collagen 2a expression compared to day 0, as measured by RT-PCR, and less than 40% of the population expressed the cell surface markers CD24 and CD105, as measured by fluorescence-activated cell sorting. Artificial intervertebral disc replacement device.

20. The artificial intervertebral disc replacement device according to claim 19, wherein the outer ring is composed of a non-absorbable material.

21. The artificial intervertebral disc replacement device according to claim 20, wherein the non-absorbable material is polyurethane.

22. The artificial intervertebral disc replacement device according to claim 19, wherein the outer ring is composed of an absorbent material.

23. The artificial intervertebral disc replacement device according to claim 22, wherein the absorbent material is polyglycolic acid, polylactic acid, or a combination thereof.

24. The artificial intervertebral disc replacement device according to claim 19, wherein the intervertebral disc cell population is further combined with one or more of the following: a scaffold material, a matrix material, a carrier material, a growth factor, and other bioactive substances.

25. The artificial intervertebral disc replacement device according to claim 19, further comprising attachment means for securely fixing the device to one or more vertebral bodies.

26. The artificial intervertebral disc replacement device according to claim 25, further comprising through-holes, cuffs, tabs, loops, or washers for enabling screw fixation to one or more vertebral bodies.

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