WO2016019168A1

WO2016019168A1 - Cell expansion methods and systems

Info

Publication number: WO2016019168A1
Application number: PCT/US2015/042957
Authority: WO
Inventors: Neil Kizer; Nicole M. BERGMANN; Michael Maloney; Ulises ALVAREZ; Sophia FINCKBONE
Original assignee: Isto Technologies, Inc.
Priority date: 2014-08-01
Filing date: 2015-07-30
Publication date: 2016-02-04

Abstract

The present disclosure provides a method for expanding a population of chondrocytes, the method involving suspending the chondrocytes in a culture medium in a culture vessel, the culture medium comprising at least one polysaccharide in solution and at least one growth factor; and intermittently agitating the chondrocytes in the culture vessel for a period of time sufficient for the population to undergo at least about one doubling to produce an expanded population, wherein at least a portion of the expanded population of chondrocytes maintains a chondrocyte phenotype.

Description

CELL EXPANSION METHODS AND SYSTEMS

Field of the Invention

[0001 ] The present disclosure relates to methods and systems for expansion of cells in culture.

Summary of the Invention

[0002] In one aspect, the present disclosure provides a method for expanding a population of chondrocytes, the method comprising: suspending the chondrocytes in a culture medium in a culture vessel, the culture medium comprising at least one polysaccharide in solution and at least one growth factor; and intermittently agitating the chondrocytes in the culture vessel for a period of time sufficient for the population to undergo at least about one doubling to produce an expanded population, wherein at least a portion of the expanded population of chondrocytes maintains a chondrocyte phenotype. In the method, the chondrocyte phenotype can be indicated by at least one of rounded morphology and hyaline cartilage gene expression. For example, the chondrocyte phenotype can be indicated by at least 50% of the expanded population of chondrocytes exhibiting rounded morphology and hyaline cartilage gene expression.

[0003] In still another aspect, the present disclosure provides a cell culture system for expanding a population of cells, the system comprising: the population of cells suspended in a culture medium in a culture vessel, wherein the culture medium comprises at least one polysaccharide and at least one growth factor; and wherein the culture vessel comprises an agitation element configured to intermittently agitate the cells in the culture vessel.

[0004] In another aspect, the present disclosure provides a cartilage replacement composition prepared by any of the methods described herein.

[0005] In still another aspect, the present disclosure provides an improvement in a method for expanding a population of human chondrocytes including the steps of (a) obtaining a population of articular chondrocytes from an animal donor; (b) suspending the chondrocytes from step (a) in a serum-free culture medium comprising at least one polysaccharide and at least one growth factor, and culturing the suspended cells for a period of at least one doubling, wherein the improvement increases the percentage of chondrocytes in the expanded culture that retain a chondrocyte phenotype, as evidenced by a rounded morphology and hyaline cartilage gene expression, to at least 50% over a culture period sufficient to allow at least two doublings of the original chondrocyte population, the improvement comprising: adjusting the culture medium to contain between about 0.001 to about 10 weight percent of the polysaccharide; and during said culturing, subjecting the suspended chondrocytes to periodic agitation that is sufficient to inhibit cell clumping, and includes no more than a total of about 1 hour of agitation for every 6 hours of culture time. The frequency of agitation can be at least about once every 12 hours, and the duration of agitation up to about one hour. The chondrocytes can be cultured in a spinner flask, and the agitation step can involve spinning a stirring element at speed of at least about 5 RPM.

[0006] In still another aspect, the present disclosure provides an improvement in a cell culture system for expanding a population of chondrocytes comprising a culture vessel containing a serum free medium, and means for agitating the chondrocytes during culture, the improvement for increasing the percentage of chondrocytes in the expanded culture that retain a chondrocyte phenotype, as evidenced by a rounded morphology and hyaline cartilage gene expression, to at least 50%, over a culture period sufficient to allow at least two doublings of the original chondrocyte population, the improvement comprising between at least one growth factor and about 0.001 to about 10 weight percent of a polysaccharide in the culture medium, and a controller operatively connected to the agitation means for subjecting chondrocytes suspended in the culture vessel to periodic agitation that is sufficient to inhibit cell clumping, and includes no more than a total of about 1 hour of agitation for every 6 hours of culture time. The frequency of agitation can be at least about once every 12 hours, and the duration of agitation up to about one hour. The chondrocytes can be cultured in a spinner flask, and the agitation step can involve spinning a stirring element at speed of at least about 5 RPM. [0007] In any of the methods, method improvements or culture systems, the culture medium can be a serum-free culture medium. In any of the methods, method improvements or culture systems, the cells can be chondrocytes, including for example articular chondrocytes. Chondrocytes can be isolated from a human or animal donor, or from a human or animal juvenile donor. Chondrocytes can be isolated from a human or animal cadaver.

[0008] In any of the methods, method improvements or culture systems, the at least one growth factor can be selected for example from: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs) including BMP-2, BMP-4 and BMP-7, Brain-derived neurotrophic factor (BDNF), Chondromodulin-I (ChM-l), Chondromodulin-ll (ChM-ll) Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), FGF-2, FGF-9, FGF-18, Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-5 (GDF5), Growth differentiation factor-9 (GDF9), Healing factor, Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Keratinocyte growth factor (KGF), 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-a), Transforming growth factor beta (TGF-β), TGF-βΙ , TGF- 2, TGF- 3, Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (PGF), PDGF-bb, Pleotrophin, and SDF-1 .

[0009] In any of the methods, method improvements or culture systems, the culture medium can comprise at least one culture supplement selected from the group consisting of: soluble Type II collagen, glucose, insulin, transferrin, selenium, ascorbic acid, a bioactive nanomaterial, and dexamethasone (SF3D). In any of the methods, method improvements or culture systems, the at least one polysaccharide can be selected from the group consisting of: hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, a bacterially-derived polysaccharide, a synthetic polysaccharide, a sulfated saccharide, and a non-sulfated saccharide. The polysaccharide can be in solution in the culture medium at a concentration of at least about 0.001 % to about 10%. The culture vessel can comprise a culture surface comprising a low attachment material or having a coating comprising a low attachment molecule. A low attachment material or low attachment molecule can comprises at least one of poly-HEMA, hyaluronic acid, N- acteylglucosamine, fluoro-silicate, polydimethylsiloxane (PDMS), polyethylene glycol (PEG), a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, surface- immobilized dextran, or any self-assembled-monolayer (SAM) surface presenting terminal hydrophobic moieties.

[0010] In any of the methods, method improvements or culture systems, intermittently agitating the chondrocytes can comprise for example agitating the chondrocytes at a frequency of about once per minute to about once per week, wherein the duration of agitation is at least about 10 seconds. The frequency of agitation can be at least about once every 12 hours, and the duration of agitation is up to about one hour. Chondrocytes can be maintained in culture for a period of at least about 5 days. Chondrocytes can be maintained under culture conditions comprising a normoxic or a hypoxic atmosphere, and/or a physiologic temperature. Chondrocytes can be maintained under culture conditions comprising a temperature of about 25 °C to about 40 °C, or about 37 °C.

[001 1 ] In any of the methods, method improvements or culture systems, the culture vessel can comprise a vessel selected from a spinner flask and an airlift container. A spinner flask may have for example a stirring element capable of a rotational speed of about 1 RPM. An airlift container can be capable of providing a flow rate of at least about 1 cc per minute. A culture vessel can comprise for example any material selected from polystyrene, polycarbonate, polyethylene, polypropylene, glass and stainless steel.

[0012] In any of the methods, method improvements or culture systems, the culture medium can be replenished at a rate of at least about 50% once per week. Detailed Description of the Disclosure

[0013] The present disclosure provides methods, method improvements and cell culture systems for expansion of a cell population in culture, which provides for convenient scale-up of the expansion process while at the same time maintaining retention of cell phenotype during expansion. The methods, method improvements and cell culture systems are useful for example in expanding an initial population of chondrocytes for preparation of cartilage repair compositions and implants.

[0014] As used herein, the term "subject" refers to an animal, including but not limited to a mammal including a human and a non-human primate (for example, a monkey or great ape), a cow, a pig, a cat, a dog, a rat, a mouse, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig). Preferably, the subject is a human.

[0015] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms as used herein and in the claims shall include pluralities and plural terms shall include the singular.

[0016] The use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0017] The terms "chondrocyte" and "chondrocytes" as used herein refer to cartilage-specific cells that give rise to normal cartilage tissue growth in vivo; these cells synthesize and deposit the supportive matrix (composed principally of collagen and proteoglycan) of cartilage.

[0018] The term "phenotype" refers to the observable characteristics at any level— physical, morphologic, biochemical or molecular— of a cell or tissue. [0019] The term "hyaluronic acid" as used herein is interchangeable with the terms "hyaluronate" and ΉΑ", and refers to a polysaccharide composed of repeating disaccharide units of N-acetylglucosamine and glucuronic acid. Commercial HA is commonly its sodium salt form. HA may be a natural or synthetic hyaluronate, such as sodium hyaluronate purified either from rooster combs or from bacterial fermentation.

[0020] The term "cytokine" as used herein is interchangeable with the terms "growth factor", and refers to a broad range of relatively low molecular weight, pharmacologically active proteins that are secreted by one cell for the purpose of altering either its own function(s) (autocrine effect) or those of adjacent cells (paracrine effect). Individual cytokines can have multiple biological activities. Different cytokines can also have redundant activity.

[0021 ] The term "low cell attachment" refers to a characteristic of cell culture conditions, for example chondrocyte culture conditions, under which a majority of cells seeded onto a tissue substrate do not adhere to the tissue substrate. The terms "low attachment material" and "low attachment molecule" refer respectively to a material and a molecule capable of reducing or preventing cell adhesion to the tissue substrate.

[0022] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Any chemical, enzymatic or staining reactions, or purification techniques are performed according to manufacturer's specifications and protocols, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are also well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, diagnosis and treatment of all subjects, human and animal.

A. Cell Expansion Methods and Improvements

[0023] Basic cell isolation, culture and expansion methods, particularly those for isolating, culturing and expanding chondrocytes, are well known in the art and commonly used. Unless otherwise indicated herein, methods and procedures are performed according to conventional methods well known in the art and as described for example in U.S. Pat. Nos. 6,645,764; 6,235,316; 7,087,227; 7,273,756; and 8,017,394, the entire disclosures of each of which are herein incorporated by reference.

[0024] The methods and systems described herein are based in part on the surprising discovery that native cell phenotype can be preserved during batch, i.e. scaled-up expansion of a cell population according to the methods and systems described herein. The inventors have succeeded in demonstrating that cells can be successfully expanded in a culture vessel having a volume larger than a standard tissue culture plate or other smaller, standard culture surface, wherein the cells maintain phenotype despite the relatively large volume. The methods provide reduction of cell attachment and subsequent spreading within the culture vessel, as indicated by observing that majority of cells in the starting population do not attach to the culture vessel or any culture surface therein. Without wishing to be bound by theory, it is believed that intermittent agitation combined with a serum-free, chemically defined medium provides sufficient exposure of the cells to needed nutrients and other elements in the culture medium, while at the same time not subjecting the cells to potentially damaging shear forces. The result is successful expansion of the cells in relatively large numbers, while the cells still maintain phenotype.

[0025] Methods for isolation of chondrocytes from donor cartilage tissue and basic handling of the chondrocytes are generally as described previously in U.S. Pat. Nos. 6,645,764; 6,235,316; 7,087,227; 7,273,756; and 8,017,394.

[0026] The present disclosure improves on basic ex vivo expansion methods by expanding the dissociated chondrocytes or chondroprogenitor cells under conditions that provide both low attachment of the cells to a culture surface of a culture vessel, limit cell attachment and spreading, and do not require a small culture plate or surface but rather do so in the context of a batch culture in a larger culture vessel. Surprisingly, the culture conditions as described herein, including but not limited to use of intermittent agitation or stirring, have been found to support maintenance of chondrocyte morphology, phenotype and function during the process of expansion in a batch culture, i.e., cells maintained in at least about 100 ml, 200 ml, 300 ml, 400 ml or 500 ml or more of culture medium. Use of the methods and systems described herein can provide a microenvironment within the larger culture vessel, i.e., within a larger volume of culture medium than used in standard expansion culture techniques. The result is that cells expanded in such a batch culture, like cells expanded in standard culture plates of much lower volume, can closely mimic the phenotype of native articular cartilage, including retention of rounded cell shape and hyaline cartilage gene expression once the expanded cells exit the cell cycle. The expanded population of cells is well-suited for preparation of cartilage repair compositions including implants, and for transplantation, and particularly for transplantation by injection.

[0027] Chondrocytes can be isolated for example from cartilage derived from a human or animal donor. An exemplary donor is an immature or juvenile donor, including a neonatal, infant, or juvenile donor. A donor can be a deceased individual, including a deceased immature or juvenile donor, such that the donor tissue is cadaver tissue. A juvenile donor may be for example an individual aged 14 years or less, 12 years or less, 10 years or less, or 7 years or less. Chondrocytes can be avian or mammalian chondrocytes, preferably human chondrocytes. Chondrocytes can be derived from transgenic animals that have been genetically engineered to prevent immune-mediated xenograft rejection.

[0028] Cartilage can be obtained from any tissue containing hyaline, elastic or fibro-cartilage. In a non-limiting example, the donor tissue is articular cartilage. Chondrocytes can be isolated by methods known in the art such as by sequential enzymatic digestion techniques. (See, e.g., U.S. Pat. No. 6,235,316; Adkisson et al., Clin. Orthop., vol. 391 S, pp. S280-S294 (2001 )). [0029] Isolated chondrocytes are then placed into a culture vessel configured to contain a volume of culture medium of at least about 100 ml, 200 ml, 300 ml, 400 ml or 500 ml or more. Although scaffold material is unnecessary, it can be used. Any scaffold as known to those of skill in the art can be used. A non-limiting example of a suitable scaffold is polystyrene microcarrier beads. The culture vessel, and/or a culture vessel liner which provides a culture surface, can comprise any material suitable for tissue culture, such as polystyrene, polycarbonate, polyethylene, polypropylene, glass or stainless steel. A culture vessel can be for example a spinner flask or an airlift container as known in the art. A culture vessel can have a stirring, rotational or flow element configured to provide agitation or stirring. For example, a vessel can have a rotational element configured to provide a rotational speed of about 1 RPM to about 100 RPM, or flow rate of at least about 1 cc/min.

[0030] The culture vessel can comprise a culture surface comprising a low attachment material or having a coating comprising a low attachment molecule. A low attachment material or low attachment molecule can for example comprise at least one of poly-HEMA, hyaluronic acid, N-acteylglucosamine, fluoro-silicate, polydimethylsiloxane (PDMS), polyethylene glycol (PEG), a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, surface-immobilized dextran, or any self-assembled- monolayer (SAM) surface presenting terminal hydrophobic moieties.

[0031 ] An expansion culture of the present disclosure can be produced by isolating immature chondrocytes, e.g., chondrocytes obtained from donor articular cartilage obtained from a neonatal, infant or juvenile donor, and plating the dissociated cells in culture medium in the culture vessel. The culture medium is preferably a substantially serum-free or serum-free expansion medium. The culture medium can for example be a chemically defined medium which is a serum-free medium in which all chemical components are known. For example, the culture medium is preferably a chemically defined medium. Chemically defined media generally include nutrients, growth factors, hormones, attachment factors, and lipids in combinations and amounts adapted for the cell type being cultured. Chemically defined media for chondrocytes is at least substantially serum-free medium comprising at least one growth factor as described herein. An exemplary substantially serum-free growth medium is HL-1®, a serum-free medium containing insulin-transferrin-selenium-complex as its only source of protein. HL-1™ is available from BioWhittaker, Walkersville, MD. Other suitable serum- free growth media will be readily apparent to those skilled in the art.

[0032] Accordingly, the present disclosure encompasses methods for expanding a population of chondrocytes comprising: suspending the previously isolated chondrocytes in a culture medium in the culture vessel. The culture medium contains at least one polysaccharide such as but not limited to hyaluronic acid in solution, and at least one growth factor. In the culture vessel, the chondrocytes are intermittently agitated for a period of time sufficient for the population to undergo at least about one population doubling to produce an expanded population, such that at least a portion of the expanded population of chondrocytes maintains a chondrocyte phenotype. The chondrocyte phenotype can be readily identified by observing at least one of rounded morphology and hyaline cartilage gene expression. For example, the chondrocyte phenotype can be identified by observing that at least 50% of the expanded population of chondrocytes exhibits rounded morphology and/or hyaline cartilage gene expression.

[0033] Methods for assessment of chondrocyte phenotype, biochemistry, morphology, and gene expression are well known to those of skill in the art and have been described in detail in the literature. For example, the differentiation potential of expanded chondrocytes can be assessed by measuring the total proteoglycan and collagen content of newly synthesized neocartilage matrix. Morphological characterization can for example be achieved on visual inspection of viable cells using light microscopy. PCR techniques, including quantitative and semi-quantitative RT-PCR, can be used to determine gene expression profiles of genes of interest such as but not limited to human collagen 1A1 , 2A1 , 9A1 , 1 1A1 , aggrecan core protein, N-cadherin, SOX-9, B7-H1 and GAPDH.

[0034] In any of the methods, method improvements or culture systems, the at least one polysaccharide can be selected from the group consisting of: hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, a bacterially-derived polysaccharide, a synthetic polysaccharide, a sulfated saccharide, and a non-sulfated saccharide. It should be understood that the at least one polysaccharide may comprise any combination of any two, three, four or more polysaccharides. The polysaccharide(s) can be in solution in the culture medium at a concentration of at least about 0.001 % to about 10%.

[0035] In any of the methods, method improvements or culture systems, the at least one growth factor can be selected for example from: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs) including BMP-2, BMP-4 and BMP-7, Brain-derived neurotrophic factor (BDNF), Chondromodulin-I (ChM-l), Chondromodulin-ll (ChM-ll) Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), FGF-2, FGF-9, FGF-18, Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-5 (GDF5), Growth differentiation factor-9 (GDF9), Healing factor, Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Keratinocyte growth factor (KGF), 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-a), Transforming growth factor beta (TGF-β), TGF-βΙ , TGF- 2, TGF- 3, Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (PGF), PDGF-bb, Pleotrophin, and SDF-1 .

[0036] In any of the methods, method improvements or culture systems described herein, the culture medium can further comprise at least one culture supplement selected from the group consisting of: soluble Type II collagen, glucose, insulin, transferrin, selenium, ascorbic acid, a bioactive nanomaterial, and dexamethasone (SF3D). It will be further understood that chemically defined media may contain other additives such as: vitamins such as choline, folic acid, nicotinamide, pantothenate, pyridoxal, riboflavin, and thiamine; lipids; and/or any one or more of the amino acids, essential (Arg, Cys, Gin, His, lie, Leu, Lys, Met, Phe, Thr, Trp, Tyr, Val) or non-essential (Ala, Asn, Asp, Glu, Gly, Pro, Ser); glutamine, typically as L-glutamine. [0037] The amount(s) of any growth factor(s) and/or culture supplement(s) in the medium can vary according to ranges and factors readily ascertainable to the person of average skill in the art with reference to product guidelines and the literature pertaining to use of such components in cell culture, including the many detailed descriptions of chemically defined media adapted for growth of cells, and particularly chondrocytes in vitro. {See, e.g., D. Brunner et al. ALTEX 27: 53-62 (2009) for a review).

[0038] In any of the methods, the cells are maintained in the culture vessel for a culture period of at least about 5 days to at least about 50 days, through 1 , 2, 3 4 or 5 passages. The total polysaccharide concentration in the culture medium can range from about 0.001 % to about 10%. For example, a serum-free culture medium can contain about 0.001 % to 10% HA in solution. The culture medium can be replaced and/or supplemented continuously or intermittently at approximately regular intervals, at a frequency of 1 , 2, 3, 4, 5, 6 or 7 times per week, i.e., at any frequency from daily to weekly. Cells in the culture vessel are maintained at a temperature of about 25°C to about 40°C. For example, cells in the culture vessel can be maintained at a temperature of about 37°C. Cells are maintained in a suitably humidified and normoxic or hypoxic atmosphere. Those of skill in the art can readily determine appropriate cell densities. By way of non-limiting example, cells can be maintained at a density of about 1 .0x10⁵ cells/liter to about 1 .0x10⁹ cells/liter.

[0039] The cells are intermittently agitated or stirred for brief periods, wherein an agitation or stirring period can last from about 10 seconds to about 1 hour, and the intermittent agitation or stirring period, including the agitation or stirring periods interspersed with rest or quiet periods of no agitation, can last from about 1 min to 1week.

[0040] Surprisingly, the neocartilage produced by the substantially serum-free cell culture can be grown and maintained for extended periods of time without dedifferentiating. For example, the neocartilage can be grown and maintained for at least about 30 days, at least about 60 days, at least about 100 days up to at least about 300 days. For example the neocartilage can be grown and maintained for at at least about 120 days, 150 days, 200 days, 250 days or at least about 300 days. Surprisingly, even after about 120 days in culture, the chondrocytes do not dedifferentiate and fail to synthesize collagen types I, III, and X.

[0041 ] The present disclosure also provides a cell culture system for expanding a population of cells, the system comprising: the population of cells suspended in a culture medium in a culture vessel, wherein the culture medium comprises at least one polysaccharide and at least one growth factor; and wherein the culture vessel comprises an agitation element configured to intermittently agitate the cells in the culture vessel.

[0042] The present disclosure also provides a cartilage replacement composition, i.e. neocartilage, which comprises any composition prepared by any of the methods described herein. The cartilage replacement composition can be used in any procedure in which such cartilage replacement tissue is needed, for example repair of damaged cartilage such as in the knee, hip or elbow.

[0043] The present disclosure also provides an improvement in a method for expanding a population of human chondrocytes including the steps of (a) obtaining a population of articular chondrocytes from an animal donor; (b) suspending the chondrocytes from step (a) in a serum-free culture medium comprising at least one polysaccharide and at least one growth factor, and culturing the suspended cells for a period of at least one doubling, wherein the improvement increases the percentage of chondrocytes in the expanded culture that retain a chondrocyte phenotype, as evidenced by a rounded morphology and hyaline cartilage gene expression, to at least 50% over a culture period sufficient to allow at least two doublings of the original chondrocyte population, the improvement comprising: adjusting the culture medium to contain between about 0.001 to about 10 weight percent of the at least one polysaccharide; and during said culturing, subjecting the suspended chondrocytes to periodic agitation that is sufficient to inhibit cell clumping, and includes no more than a total of about 1 hour of agitation for every 6 hours of culture time. The frequency of agitation can be at least about once every 12 hours, and the duration of agitation up to about one hour. The chondrocytes can be cultured for example in a spinner flask as described elsewhere herein, and the agitation step can involve spinning a stirring element at speed of at least about 5 RPM.

[0044] The present disclosure also provides an improvement in a cell culture system for expanding a population of chondrocytes comprising a culture vessel containing a serum free medium, and means for agitating the chondrocytes during culture, the improvement for increasing the percentage of chondrocytes in the expanded culture that retain a chondrocyte phenotype, as evidenced by a rounded morphology and hyaline cartilage gene expression, to at least 50%, over a culture period sufficient to allow at least two doublings of the original chondrocyte population, the improvement comprising between including at least one growth factor and about 0.001 to about 10 weight percent of at least one polysaccharide in the culture medium, and a controller operatively connected to the agitation means for subjecting chondrocytes suspended in the culture vessel to periodic agitation that is sufficient to inhibit cell clumping, and includes no more than a total of about 1 hour of agitation for every 6 hours of culture time. The frequency of agitation can be at least about once every 12 hours, and the duration of agitation up to about one hour. Again, the chondrocytes can be cultured in a spinner flask, and the agitation step can involve spinning a stirring element at speed of at least about 5 RPM.

[0045] In any of the methods, method improvements or culture systems, intermittently agitating the chondrocytes can comprise for example agitating the chondrocytes at a frequency of anywhere from about once per minute to about once per week, i.e., about once per minute, about 2x, 3x or 4x daily, about once daily, about once every other day, about twice a week, or about once per week, wherein at any such frequency the duration of the agitation is at least about 10 seconds. For example, the frequency of agitation can be at least about 2x daily or once every 12 hours, and the duration of agitation at least 10 seconds up to about one hour. Chondrocytes can be thus maintained in culture for a period of at least about 5 days, or for much extended periods as detailed herein above.

[0046] Suitable culture conditions can be readily known to those of skill in the art. In non-limiting example, suitable culture conditions can include 37 °C, a humidified atmosphere with the addition of 2-10% CO2, preferably 5% CO2. Chondrocytes can be maintained under culture conditions comprising a normoxic or a hypoxic atmosphere, and/or at an approximate physiologic temperature, or at a slightly hyperthermic or slightly hypothermic temperature. For example, chondrocytes can be maintained under culture conditions comprising a temperature of any temperature in the range of about 25 °C to about 40 °C. It will be understood that a physiologic temperature for mammals is about 37 °C.

[0047] In any of the methods, method improvements or culture systems, the culture vessel can comprise a vessel selected from a spinner flask and an airlift container. A spinner flask may have for example a stirring element capable of a rotational speed of about 1 RPM. An airlift container can be capable of providing a flow rate of at least about 1 cc per minute. A culture vessel can comprise for example any material selected from polystyrene, polycarbonate, polyethylene, polypropylene, glass and stainless steel.

[0048] In any of the methods, method improvements or culture systems, the culture medium can be replenished at a rate of at least about 50% once per week.

B. Examples

[0049] The following example is included to demonstrate the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes could be made in the disclosure and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense.

Example 1 : Spinner Flask Expansion

[0050] An initial population of chondrocytes isolated from a juvenile donor is placed into a polystyrene spinner flask containing 500 ml serum-free HL-1™ medium to which is added about 0.1 % HA, and maintained in a normoxic atmosphere at about 37°C. The cells are maintained for a culture period of 21 days x 2 passages, with 3+ doublings per passage. The cells are agitated for a period of 4 minutes every 12 hours, using a slow rotational speed setting on the flask. Fifty percent of the culture medium is replenished once per week.

Example 2: Spinner Flask Expansion

[0051 ] An initial population of chondrocytes isolated from a juvenile donor is placed into a polystyrene spinner flask containing 500 ml serum-free HL-1™ medium to which is added about 0.5%, or about 1 .0% HA, and maintained in a normoxic atmosphere at about 37°C. The cells are maintained for a culture period of 21 days x 2 passages, with 3+ doublings per passage. The cells are agitated for a period of about 5 minutes every 12 hours, or a period of about 5 minutes every 24 hours using a slow rotational speed setting of no more than 5 rpm on the flask. Fifty percent of the culture medium is replenished once per week.

[0052] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1 . A method for expanding a population of chondrocytes, comprising: suspending the chondrocytes in a culture medium in a culture vessel, the culture medium comprising at least one polysaccharide in solution and at least one growth factor; and intermittently agitating the chondrocytes in the culture vessel for a period of time sufficient for the population to undergo at least about one doubling to produce an expanded population, wherein at least a portion of the expanded population of chondrocytes maintains a chondrocyte phenotype.

2. The method of claim 1 , wherein the chondrocyte phenotype is indicated by at least one of rounded morphology and hyaline cartilage gene expression.

3. The method of claim 1 , wherein at least 50% of the expanded population of

chondrocytes exhibits rounded morphology and hyaline cartilage gene

expression.

4. The method of claim 1 , wherein the culture medium is a serum-free culture

medium.

5. The method of claim 4, wherein the at least one growth factor is selected from the group consisting of: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs) including BMP-2, BMP-4 and BMP-7, Brain-derived neurotrophic factor (BDNF), Chondromodulin-I (ChM- I), Chondromodulin-ll (ChM-ll) Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), FGF-2, FGF-9, FGF-18, Glial cell line- derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G- CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-5 (GDF5), Growth differentiation factor-9 (GDF9), Healing factor, Hepatocyte growth factor (HGF), Hepatoma-derived growth factor

(HDGF), Insulin-like growth factor (IGF), Keratinocyte growth factor (KGF), 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-a), Transforming growth factor beta (TGF-β), TGF-βΙ , TGF- 2, TGF- 3, Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (PGF), PDGF-bb, Pleotrophin, and SDF-1 .

6. The method of claim 4, wherein the culture medium comprises at least one culture supplement selected from the group consisting of: soluble Type II collagen, glucose, insulin, transferrin, selenium, ascorbic acid, a bioactive nanomaterial, and dexamethasone (SF3D).

7. The method of claim 1 , wherein the population of chondrocytes comprises articular chondrocytes.

8. The method of claim 1 , wherein the population of chondrocytes comprises chondrocytes isolated from a human or animal donor.

9. The method of claim 1 , wherein the population of chondrocytes comprises chondrocytes isolated from a juvenile donor.

10. The method of claim 1 , wherein the population of chondrocytes comprises chondrocytes isolated from a cadaver.

1 1 . The method of claim 1 , wherein the at least one polysaccharide is selected from the group consisting of: hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, a bacterially-derived polysaccharide, a synthetic polysaccharide, a sulfated saccharide, and a non-sulfated saccharide .

12. The method of claim 1 , wherein the polysaccharide is in solution in the culture medium at a concentration of at least about 0.001 % to about 10%.

13. The method of claim 1 , wherein the culture vessel comprises a culture surface comprising a low attachment material or having a coating comprising a low attachment molecule.

14. The method of claim 13, wherein the low attachment material or the low

attachment molecule comprises at least one of poly-HEMA, hyaluronic acid, N- acteylglucosamine, fluoro-silicate, polydimethylsiloxane (PDMS), polyethylene glycol (PEG), a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, surface-immobilized dextran, or any self-assembled-monolayer (SAM) surface presenting terminal hydrophobic moieties.

15. The method of claim 1 , wherein intermittently agitating the chondrocytes

comprises agitating the chondrocytes at a frequency of about once per minute to about once per week, wherein the duration of agitation is at least about 10 seconds.

16. The method of claim 15, wherein the frequency of agitation is at least about once every 12 hours, and the duration of agitation is up to about one hour.

17. The method of claim 1 , wherein the chondrocytes are maintained in culture for a period of at least about 5 days.

18. The method of claim 1 , wherein the chondrocytes are maintained under culture conditions comprising a normoxic or a hypoxic atmosphere.

19. The method of claim 1 , wherein the chondrocytes are maintained under culture conditions comprising a temperature of about 25 °C to about 40 °C.

20. The method of claim 1 , wherein the chondrocytes are maintained under culture conditions comprising a temperature of about 37 °C.

21 . The method of claim 1 , wherein the culture vessel comprises a vessel selected from a spinner flask and an airlift container.

22. The method of claim 21 , wherein the culture vessel is a spinner flask having a stirring element capable of a rotational speed of about 1 RPM.

23. The method of claim 21 , wherein the culture vessel is an airlift container capable of providing a flow rate of at least about 1 cc per minute.

24. The method of claim 1 , wherein the culture medium is replenished at a rate of at least about 50% once per week.

25. The method of claim 1 , wherein the culture vessel comprises a material selected from the group consisting of: polystyrene, polycarbonate, polyethylene, polypropylene, glass and stainless steel.

26. A cartilage replacement composition prepared by a method of any of claims 1 -25.

27. A cell culture system for expanding a population of cells, said system

comprising: the population of cells suspended in a culture medium in a culture vessel, wherein the culture medium comprises at least one polysaccharide and at least one growth factor; and wherein the culture vessel comprises an agitation element configured to intermittently agitate the cells in the culture vessel.

28. The cell culture system of claim 27, wherein the cells comprise chondrocytes.

29. The cell culture system of claim 27, wherein the at least one polysaccharide is selected from the group consisting of: hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, a bacterially-derived polysaccharide, a synthetic

polysaccharide, a sulfated saccharide, and a non-sulfated saccharide.

30. The cell culture system of claim 27, wherein the at least one polysaccharide is in solution in the culture medium at a concentration of at least about 0.001 % to about 10%.

31 . The cell culture system of claim 27, wherein the culture vessel a culture surface comprising a low attachment material or having a coating comprising a low attachment molecule.

32. The method of claim 31 , wherein the low attachment material or the low

33. The cell culture system of claim 27, wherein the culture medium is a serum-free culture medium.

34. The cell culture system of claim 27, wherein the at least one growth factor is selected from the group consisting of: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs) including BMP-2, BMP-4 and BMP-7, Brain-derived neurotrophic factor (BDNF), Chondromodulin-I (ChM-l), Chondromodulin-ll (ChM-ll) Epidermal growth factor (EGF),

Erythropoietin (EPO), Fibroblast growth factor (FGF), FGF-2, FGF-9, FGF-18, Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-5 (GDF5), Growth differentiation factor-9 (GDF9), Healing factor, Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Keratinocyte growth factor (KGF), 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-a), Transforming growth factor beta (TGF-β), TGF-βΙ , TGF- 2, TGF- 3, Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (PGF), PDGF-bb, Pleotrophin, and SDF-1 .

35. The cell culture system of claim 27, wherein the culture medium comprises at least one culture supplement selected from the group consisting of: soluble Type II collagen, glucose, insulin, transferrin, selenium, ascorbic acid, a bioactive nanomaterial, and dexamethasone (SF3D)

36. The cell culture system of claim 27, wherein the population of cells comprises articular chondrocytes.

37. The cell culture system of claim 27, wherein the population of chondrocytes comprises chondrocytes isolated from a human or animal donor.

38. The cell culture system of claim 27, wherein the population of chondrocytes

comprises chondrocytes isolated from a juvenile donor.

39. The cell culture system of claim 27, wherein the population of chondrocytes

comprises chondrocytes isolated from a cadaver.

40. The cell culture system of claim 27, wherein the culture vessel comprises a

vessel selected from a spinner flask and an airlift container.

41 . The cell culture system of claim 40, wherein the culture vessel is a spinner flask having a stirring element capable of a rotational speed of about 1 RPM.

42. The cell culture system of claim 40, wherein the culture vessel is an airlift

container capable of providing a flow rate of at least about 1 cc per minute.

43. The cell culture system of claim 40, wherein the culture vessel comprises a

material selected from the group consisting of: polystyrene, polycarbonate, polyethylene, polypropylene, glass and stainless steel.

44. In a method for expanding a population of human chondrocytes, by the steps of (a) obtaining a population of articular chondrocytes from an animal donor; (b) suspending the chondrocytes from step (a) in a serum-free culture medium comprising at least one polysaccharide and at least one growth factor, and culturing the suspended cells for a period of at least one doubling, an

improvement for increasing the percentage of chondrocytes in the expanded culture that retain a chondrocyte phenotype, as evidenced by a rounded morphology and hyaline cartilage gene expression, to at least 50% over a culture period sufficient to allow at least two doublings of the original chondrocyte population, said improvement comprising: adjusting the culture medium to contain between about 0.001 to about 10 weight percent of the polysaccharide; and during said culturing, subjecting the suspended chondrocytes to periodic agitation that is sufficient to inhibit cell clumping, and includes no more than a total of about 1 hour of agitation for every 6 hours of culture time.

The improvement of claim 44, wherein the culture medium comprises at least one growth factor is selected from the group consisting of: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs) including BMP-2, BMP-4 and BMP-7, Brain-derived neurotrophic factor (BDNF), Chondromodulin-I (ChM-l), Chondromodulin-ll (ChM-ll) Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), FGF- 2, FGF-9, FGF-18, Glial cell line-derived neurotrophic factor (GDNF),

Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony- stimulating factor (GM-CSF), Growth differentiation factor-5 (GDF5), Growth differentiation factor-9 (GDF9), Healing factor, Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Keratinocyte growth factor (KGF), 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- a), Transforming growth factor beta (TGF-β), TGF-βΙ , TGF- 2, TGF- 3, Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (PGF), PDGF-bb, Pleotrophin, and SDF-1 .

46. The improvement of claim 44, wherein hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, a bacterially-derived polysaccharide, a synthetic

polysaccharide, a sulfated saccharide, and a non-sulfated saccharide.

47. The improvement of claim 44, wherein the culture medium comprises at least one culture supplement selected from the group consisting of: soluble Type II collagen, glucose, insulin, transferrin, selenium, ascorbic acid, a bioactive nanomaterial, and dexamethasone (SF3D).

48. The improvement of claim 44, wherein the frequency of agitation is at least about once every 12 hours, and the duration of agitation is up to about one hour.

49. The improvement of claim 44, wherein the chondrocytes are cultured in a

spinner flask, and the agitation step involves spinning a stirring element at speed of at least about 5 RPM.

50. In a cell culture system for expanding a population of chondrocytes comprising a culture vessel containing a serum free medium, and means for agitating the chondrocytes during culture, an improvement for increasing the percentage of chondrocytes in the expanded culture that retain a chondrocyte phenotype, as evidenced by a rounded morphology and hyaline cartilage gene expression, to at least 50%, over a culture period sufficient to allow at least two doublings of the original chondrocyte population, said improvement comprising between at least one growth factor and about 0.001 to about 10 weight percent of a

polysaccharide in the culture medium, and a controller operatively connected to the agitation means for subjecting chondrocytes suspended in the culture vessel to periodic agitation that is sufficient to inhibit cell clumping, and includes no more than a total of about 1 hour of agitation for every 6 hours of culture time.

51 . The improvement of claim 50, wherein the culture medium comprises at least one growth factor is selected from the group consisting of: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs) including BMP-2, BMP-4 and BMP-7, Brain-derived neurotrophic factor (BDNF), Chondromodulin-I (ChM-l), Chondromodulin-ll (ChM-ll) Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), FGF- 2, FGF-9, FGF-18, Glial cell line-derived neurotrophic factor (GDNF),

52. The improvement of claim 50, wherein the at least one polysaccharide is

selected from the group consisting of: hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, a bacterially-derived polysaccharide, a synthetic

polysaccharide, a sulfated saccharide, and a non-sulfated saccharide.

53. The improvement of claim 50, wherein the culture medium comprises at least one culture supplement selected from the group consisting of: soluble Type II collagen, glucose, insulin, transferrin, selenium, ascorbic acid, a bioactive nanomaterial, and dexamethasone (SF3D).

54. The improvement of claim 50, wherein the frequency of agitation is at least about once every 12 hours, and the duration of agitation is up to about one hour. The improvement of claim 50, wherein the chondrocytes are cultured in a spinner flask, and the agitation step involves spinning a stirring element at speed of at least about 5 RPM.