WO2017041133A1 - Cell expansion methods and therapeutic compositions - Google Patents
Cell expansion methods and therapeutic compositions Download PDFInfo
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- WO2017041133A1 WO2017041133A1 PCT/AU2016/000316 AU2016000316W WO2017041133A1 WO 2017041133 A1 WO2017041133 A1 WO 2017041133A1 AU 2016000316 W AU2016000316 W AU 2016000316W WO 2017041133 A1 WO2017041133 A1 WO 2017041133A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
Definitions
- the present invention relates to methods for the production of mesenchymal stem cells (MSCs), in particular to methods for the large scale production of MSCs for use in treating various diseases in humans and other animals.
- the methods permit efficient large scale production of allogeneic MSCs for use in therapy.
- the invention also relates to methods which permit the selection of preferred donor cells suitable for large scale production of MSCs.
- the invention also relates to purified MSCs prepared by the methods of the invention.
- the invention also relates to the use of platelet lysate in methods for preparing cultures of MSCs and to the preparation of extracellular matrix-enriched secretions.
- the present invention also relates to methods for the preparation of compositions comprising one or more component(s) secreted from cultured MSCs, such as vascular endothelial growth factor (VEGF), having improved stability characteristics.
- VEGF vascular endothelial growth factor
- the present invention also relates to methods for treating inflammatory conditions, including alleviating the pain thereof, by administering high molecular mass glycoconjugate-enriched conditioned media.
- the present invention also relates to methods for treating neuropathic pain by administering high molecular mass glycoconjugate-enriched conditioned media.
- MSCs allogeneic mesenchymal stem cells
- diseases including osteoarthritis, myocardial infarction, stroke, and others with clear involvement of the immune system, such as graft-versus-host disease, Crohn's disease, rheumatoid arthritis and diabetes.
- MSCs are being used as cell therapy to treat defects in bone and cartilage and to help in wound healing, or in combination with biomaterials in tissue engineering development.
- MSCs mesenchymal stem cells
- secretions may take various forms, such as described in the applicant's co-pending application PCT International Publication No. WO2013/040649 entitled "Therapeutic methods and compositions", the contents of which are incorporated herein by reference.
- the invention addresses the need for improved methods of producing MSCs, and MSC-based products, in particular methods which ameliorate one or more of the limitations of known methods when appl ied to large scale production of MSCs, such as for the production of large numbers of doses of allogeneic cells.
- the instant invention addresses the need for improved methods of producing compositions comprising MSC-secreted cytokines and growth factors, and for such compositions, the improved methods or compositions which may alleviate one or more limitations of known methods and
- compositions such as ease of use, stability of compositions on storage, or therapeutic potential.
- the instant invention addresses a need for improved or alternative methods and agents for treating inflammatory conditions, including alleviating the pain thereof. In embodiments the instant invention addresses a need for improved or alternative methods and agents for treating neuropathic pain.
- the inventors have developed methods that enable the production of very large numbers of doses of MSCs from a single donor.
- the method involves firstly harvesting a large volume of adipose tissue from the donor.
- the lipoaspirate is then digested to isolate the stromal vascular fraction (SVF), which is then placed in to tissue culture and expanded.
- SVF stromal vascular fraction
- the inventors have also developed innovative methods for the production of MSCs in a medium comprising platelet lysate.
- the inventors have identified that growth of MSCs in medium comprising platelet lysate, such as under conditions described herein, has the surprising advantage that the cells secrete high levels of high molecular mass glycoconjugates in to the tissue culture media.
- This allows production of high molecular mass glycoconjugate-enriched conditioned media which provides additional therapeutic advantages for the treatment of, or the alleviation of pain of, inflammatory conditions, including osteoarthritis, or for alleviation of neuropathic pain.
- the conditioned media generated in that manner has a higher viscosity than conditioned media generated by growth of MSCs in the absence of platelet lysate.
- the viscosity can thereby act as an indicator of an appropri ate time to harvest cells and or conditioned media from the culture.
- MSCs cultured in media containing fibroblast growth factor (FGF) and epidermal growth factor (EGF) also resulted in conditioned media that was viscous.
- FGF fibroblast growth factor
- EGF epidermal growth factor
- the inventors have also developed methods for screening cells or tissue samples from different donors, or cells or tissue samples from a single donor, to select cells that are suitable for large-scale manufacturing.
- a method for treating an inflammatory condition in a subject comprising administering to said subject a therapeutically effective amount of a high molecular mass glycoconjugate-enriched conditioned media.
- the inflammatory condition is osteoarthritis.
- the method further comprises administering a therapeutically effecti ve amount of culture-expanded MSCs.
- the method comprises administering a composition comprising culture-expanded MSCs and high molecular mass glycoconjugate-enriched conditioned media.
- the high molecular mass glycoconjugate-enriched conditioned media is prepared by culturing MSCs in media comprising platelet lysate.
- the invention provides use of platelet lysate for the preparation of high molecular mass glycoconjugate-enriched conditioned media.
- the platelet lysate is human platelet lysate.
- the invention provides a method for the preparation of high molecular mass glycoconjugate-enriched conditioned media, the method comprising culturing
- mesenchymal stem cells in media comprising platelet lysate.
- the invention provides a method for the preparation of high molecular mass glycoconjugate-enriched conditioned media, the method comprising culturing mesenchymal stem cells (MSCs) in media comprising FGF and or EGF.
- the cells are cultured to greater than about 80% confluence.
- the cells are cultured for between one to ten days post-confluence.
- the cells are cultured for between one to six days post-confluence.
- the cells are cultured for about one day post-confluence, or for about two days post- confluence, or for about three days post-confluence, or for about four days post-confluence, or for about five days post-confluence, or for about six days post-confluence, or for about seven days post-confluence, or for about eight days post-confluence.
- the conditioned media has a viscosity of at least about 1.5 centistokes. In an embodiment the conditioned media has a viscosity of at least about 1.6 centistokes. In an embodiment the conditioned media has a viscosity of at least about 1.7 centistokes.
- the conditioned media has a viscosity of at least about 1.8 centistokes. In an embodiment the conditioned media has a viscosity of at least about 1.9 centistokes. In an embodiment the conditioned media has a viscosity of at least about 2 centistokes. In an embodiment the conditioned media has a viscosity of at least about 2.1 centistokes. In an embodiment the conditioned media has a viscosity of at least about 2.2 centistokes. In an embodiment the conditioned media has a viscosity of at least about 2.3 centistokes. In an embodiment the conditioned media has a viscosity of greater than 1.5 centistokes. In an embodiment the conditioned media has a viscosity of greater than 1.7 centistokes. In an embodiment the conditioned media has a viscosity of greater than 2
- the conditioned media has a viscosity of greater than 2.5 centistokes.
- the MSCs are adipose tissue-derived MSCs.
- the platelet lysate is human platelet lysate.
- the media comprises about 5% to about 10% v/v platelet lysate.
- the enriched media comprises one or more of a proteoglycan, a glycosaminoglycan and a mucin.
- the enriched media comprises keratan sulphate, chondroitin sulphate or aggrecan.
- the method further comprises the step of removing the cell s from the media.
- the conditioned media, comprising secretions from the cultured cells comprises one or more MSC-secreted growth factor(s) or cytokine(s) having improved stability compared to that MSC-secreted growth factor(s) or cytokine(s) prepared by culturing MSCs in the absence of platelet lysate.
- the conditioned media, comprising secretions from the cultured cells comprises one or more MSC-secreted growth factor(s) or cytokine(s) having improved stability compared to that MSC-secreted growth factor(s) or
- cytokine(s) prepared by culturing MSCs in the absence of FGF and EGF.
- the one or more MSC-secreted growth factor(s) or cytokine(s) is selected from the group consisting of IFN- ⁇ , IL-8, lL-9, IL-12, IL-15, TNF-a, IL-10, MCP-1, RANTES, GM-CSF, lP-10, PDGF- bb, VEGF, IL-6.
- the one or more MSC-secreted growth factor(s) or cytokine(s) is VEGF.
- the improved stability comprises retention of at least 60% activity after one month at room temperature. In an embodiment the improved stability comprises retention of at least 70% activity after one month at room temperature. In an embodiment the improved stability comprises retention of at least 80%) activity after one month at room temperature. In an embodiment the improved stability comprises retention of at least 90% activity after one month at room temperature.
- the improved stability comprises retention of at least 60% acti vity after three months at room temperature. In an embodiment the improved stability comprises retention of at least 70% acti vity after three months at room temperature. In an embodiment the improved stability comprises retention of at least 80% activity after three months at room temperature. In an embodiment the improved stability comprises retention of at least 90%) activity after three months at room temperature.
- the improved stability comprises retention of at least 60%, activity after six months at room temperature. In an embodiment the improved stability comprises retention of at least 70% acti vity after six months at room temperature. In an embodiment the improved stability comprises retention of at least 80% activity after six months at room temperature. In an embodiment the improved stability comprises retention of at least 90%) activity after six months at room temperature. [00020] In a further aspect the invention provides a composition comprising high molecular mass glycoconjugate-enriched conditioned media.
- composition comprising high molecular mass glycoconjugate-enriched conditioned media comprises one or more MSC-secreted growth factor(s) or cytokine(s) having improved stability compared to that MSC-secreted growth factor(s) or cytokine(s)prepared by culturing MSCs in the absence of platelet lysate or in the absence of FGF and EGF.
- the one or more MSC- secreted growth factor(s) or cytokine(s) is selected from the group consisting of IFN- ⁇ , IL-8, IL- 9, IL-12, 1L-15, TNF-a, IL-10, CP-1 , RANTES, GM-CSF, IP-10, PDGF-bb, VEGF, IL-6.
- the one or more MSC-secreted growth factor(s) or cytokine(s) is VEGF.
- the one or more MSC-secreted growth factor(s) or cytokine(s) in said composition has improved stability compared to a composition comprising the one or more MSC-secreted growth factor(s) or cytokine(s) in the absence of said high molecular mass glycoconjugate- enriched conditioned media.
- composition is prepared by culturing MSCs in media comprising platelet lysate. In an embodiment the composition is prepared by culturing MSCs in media comprising FGF and/or EGF.
- the invention provides a method for preparing a composition comprising stable one or more MSC-secreted growth factor(s) or cytokine(s), wherein said stability comprises retention of at least 60% activity after one month at room temperature, the method comprising culturing mesenchymal stem cells (MSCs) in culture media comprising platelet lysate for a time sufficient to permit secretion of the one or more MSC-secreted growth factor(s) or cytokine(s) into said culture medium.
- MSCs mesenchymal stem cells
- the platelet lysate is at a concentration of 5% v/v to 10% v/v.
- the platelet lysate is at a concentration of 10% v/v.
- the invention provides a method for preparing a composition comprising stable one or more MSC-secreted growth factor(s) or cytokine(s), wherein said stability comprises retention of at least 60% activity after one month at room temperature, the method comprising culturing mesenchymal stem cells (MSCs) in culture media comprising FGF and/or EGF for a time sufficient to permit secretion of the one or more MSC-secreted growth factor(s) or cytokine(s) into said culture medium.
- MSCs mesenchymal stem cells
- EGF mesenchymal stem cells
- the FGF and/or the EGF is at a concentration of between l Ong/ml and 30 ng/ml.
- the FGF is at a concentration of 20 ng/ml.
- the EGF is at a concentration of 20 ng/ml.
- the one or more MSC-secreted growth factor(s) or cytokine(s) is selected from the group consisting of IFN- ⁇ , IL-8, IL-9, IL-12, IL-15, TNF-a, IL-10, MCP-1 , RANTES, GM-CSF, IP- 10, PDGF-bb, VEGF, IL-6.
- the one or more MSC-secreted growth factor(s) or cytokine(s) is VEGF.
- the MSCs are human adipose derived MSCs.
- said method comprises culturing the MSCs in media comprising platelet lysate to a cell density of greater than about 80% confluence. In an embodiment said method comprises culturing the MSCs in media comprising platelet lysate to a cell density of greater than about 85% confluence. In an embodiment said method comprises culturing the MSCs in media comprising platelet lysate to a cell density of greater than about 90% confluence. In an embodiment said method comprises culturing the MSCs in media comprising platelet lysate to a cell density of greater than about 95% confluence.
- said method comprises culturing the MSCs in media comprising platelet lysate to a cell density of greater than about 100% confluence.
- said culturing is for a time sufficient to permit preparation of a conditioned media having a viscosity of at least about 1.5 centistokes (cSt).
- said culturing is for a time sufficient to permit preparation of a conditioned media having a viscosity of at least about 1.6 centistokes.
- said culturing is for a time sufficient to permit preparation of a conditioned media having a viscosity of at least about 1.7 centistokes.
- the method further comprises collecting conditioned media from said culture. In an embodiment the method further comprises collecting conditioned media and culture-expanded MSCs from said culture.
- said stability comprises, optionally, retention of at least 70% activity of said one or more MSC-secreted growth factor(s) or cytokine(s) after one month at room temperature, retention of at least 80% activity of said one or more MSC-secreted growth factor(s) or cytokine(s) after one month at room temperature, retention of at least 90% acti vity of said one or more MSC-secreted growth factor(s) or cytokine(s) after one month at room temperature, retention of at least 70% activity of said one or more MSC-secreted growth factor(s) or cytokine(s) after three months at room temperature, retention of at least 80% activity of said one or more MSC-secreted growth factor(s) or cytokine(s) after three months at room temperature, retention of at least 90% activity of said one or more MSC-secreted growth factor(s) or cytokine(s) after three months at room temperature, retention of at least 60% activity of said one or more MSC
- the invention provides a composition comprising culture-expanded MSCs and high molecular mass glycoconjugate-enriched conditioned media.
- a composition according to the invention is a pharmaceutical composition.
- the phamiaceutical composition is an injectable composition.
- the pharmaceutical composition is a composition for topical application, such as to the skin, gum, or mucous membrane of a subject.
- the pharmaceutical composition is a cream, gel, liquid or lotion.
- the pharmaceutical composition comprises high molecular mass glycoconjugate-enriched conditioned media formulated in a gel or cream for topical application.
- the composition does not comprise a surfactant.
- composition comprising culture-expanded MSCs and high molecular mass glycoconjugate-enriched conditioned media the cells are not adhered in a container containing said composition.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising (i) high molecular mass glycoconjugate-enriched conditioned media, or (ii) culture-expanded MSCs and high molecular mass glycoconjugate-enriched conditioned media, and a pharmaceutically acceptable carrier, excipient or adjuvant.
- the invention provides a method of screening a sample of MSCs for suitability for use in large scale production of cultured MSCs, the method comprising the steps of (i) culturing cells of said sample in a culture medium comprising platelet lysate or FCS for 1 , 2, or 3 passages and (ii) culturing said cells or an aliquot thereof after step (i) in a culture medium comprising allogeneic serum, wherein continued proliferation of cells in said culture medium comprising allogeneic serum is indicative of a sample suitable for use in the production of large scale numbers of cultured MSCs.
- suitability for use in large scale production of cultured MSCs comprises the capacity for at least 25 population doublings before senescence. In an embodiment suitability for use in large scale production of cultured MSCs comprises the capacity for at least 30 population doublings before senescence. In an embodiment suitability for use in large scale production of cultured MSCs comprises the capacity for at least 35 population doublings before senescence. In an embodiment suitability for use in large scale production of cultured MSCs comprises the capacity for at least 40 population doublings before senescence. In an embodiment suitability for use in large scale production of cultured MSCs comprises the capacity for at least 45 population doublings before senescence.
- the sample of MSCs is an adipose tissue-derived sample of MSCs.
- the sample of MSCs is selected from human, canine, equine and feline MSCs.
- the invention provides a method of screening a sample of MSCs for unsuitability for use in large scale production of cultured MSCs, the method comprising the steps of (i) culturing cells of said sample in a culture medium comprising platelet lysate or FCS for 1 , 2, or 3 passages and (ii) culturing said cells or a portion thereof from step (i) in a culture medium comprising all ogeneic serum, wherein failure of the cell s to proliferate in said culture medium comprising allogeneic serum is indicative of a sample unsuitable for use in the production of large scale numbers of cultured MSCs.
- failure of the cells to reach confluence is indicative of failure of the cells to proliferate.
- the invention provides a method for large scale production of cultured MSCs, the method comprising the steps of (i) obtaining a cell sample or tissue sample comprising MSCs, (ii) culturing at least a portion of said sample in a culture medium comprising platelet lysate or FCS for 1, 2, or 3 passages to provide a cultured cell population, (iii) culturing a portion of said cultured cell population from step (ii) in a culture medium comprising allogeneic serum, wherein upon continued proliferation of cells in said culture medium comprising allogeneic serum (iv) culturing at least a portion of said cultured cell population from step (i) or step (ii) in a culture medium comprising platelet lysate or FCS for additional passages to provide a large scale preparation of cultured MSCs.
- the cell sample is a cell suspension comprising stromal vascular fraction of adipose tissue.
- the cell sample comprises isolated MSCs.
- the cell sample comprises culture expanded MSCs.
- the cells of at least one of steps (i) to (iv) are cells that have been frozen.
- step (iv) comprises culturing said cell population for 10 or more additional population doublings; or for 15 or more additional population doublings; or for 20 or more additional population doublings; or for 25 or more additional population doublings; or for 30 or more additional population doublings; or for 35 or more additional population doublings.
- the method further comprises harvesting culture expanded MSCs after said additional passages and, optionally, ali quoting said harvested cells into indi vidual containers constituting a therapeutic dose of MSCs.
- a therapeutic dose of MSCs comprises between about 2 million and about 10 million MSCs. In an embodiment a therapeutic dose of MSCs comprises about 5 million MSCs.
- the collagenase concentration is 0.05% wt/vol.
- the calcium is at a concentration between 300 mg/L to 400 mg/L. In an embodiment the calcium is at a concentration of 330mg/L.
- said conditions comprise mixing on an orbital rotor.
- the seeding density is between about 5,000 and about 15,000 cells per cm " of tissue culture flask.
- the tissue culture comprises culturing said cells on microcarrier beads or discs.
- the serum-supplemented media comprises FCS or platelet lysate.
- the FCS is at a concentration of between about 5% and 10%.
- the platelet lysate is at a concentration of between about 5% and 10%).
- the platelet lysate is at a concentration of 10% v/v.
- the method further comprises, prior to the cells having undergone more than three passages, an aliquot of said cells is cultured in a culture medium comprising allogeneic serum to identify cells suitable for continued use in the production of large scale numbers of cultured MSCs.
- the method further comprises continued passaging, in media comprising FCS or platelet lysate, of cells identified as suitable for continued use in the production of large scale numbers of cultured MSCs.
- the method comprises passaging harvested MSCs or progeny thereof for at least 25 population doublings; or for at least 30 population doublings; or for at least 35 population doublings; or for at least 40 population doublings; or for at least 45 population doublings, to obtain culture-expanded MSCs.
- DMEM Dulbecco's Modified Eagles Medium [00040] SVCs stromal vascular cells. [00041 ] SVF stromal vascular fraction, [00042] MSC mesenchymal stem cell(s).
- FCS fetal calf serum may also be abbreviated herein to FBS, being fetal bovine serum).
- FCS fetal calf serum may also be abbreviated herein to FBS, being fetal bovine serum).
- ABC ammonium bicarbonate buffer may also be abbreviated herein to FBS, being fetal bovine serum.
- VEGF vascular endothelial growth factor
- FIG. 1 Comparison of the viscosity of culture media of MSCs grown in fetal calf serum and in platelet lysate. MSCs were cultured in DMEM supplemented with either 10% FCS (squares) or 10% platelet lysate (circles) and the viscosity of the respective medium detennmed after 0, 1, 3, and 6 days post confluence. Kinematic viscosity was determined as described in Example 5 and is stated in centistokes (cSt).
- Figure 2 Stability of VEGF.
- the amount of VEGF in supernatant harvested from culture of human adipose-derived MSCs cultured in DMEM plus 5% platelet lysate for 10 days was measured using an ELISA, with assays being conducted at various times over six months storage of the supernatant composition at room temperature (approximately 23°C).
- Figure 4 Comparison of the viscosity of conditioned media from cultures that are less than or greater than 80% confluent.
- compositions, methods, etc. that is described as comprising integer A, or comprising integer A and B, etc.
- reference herein to use of the inventive methods and compositions in treatment or therapy, or in the obtaining of tissue or cells from donors will be understood to be applicable to human and non-human, such as veterinary, applications.
- reference to a donor, patient, subject or individual means a human or a non-human, such as an individual of any species of social, economic, agricultural or research importance including but not limited to members of the classifications of ovine, bovine, equine, porcine, feline, canine, primates, rodents, especially domesticated or farmed members of those classifications, such as sheep, cattle, horses, pigs, cats and dogs.
- the pharmaceutical composition referred to herein may also be referred to as a medicament, such as when intended for therapeutic use.
- a medicament such as when intended for therapeutic use.
- the invention is described as including the use of a composition of described components for the preparation of a pharmaceutical composition for an intended therapeutic purpose, that description equally means use for the preparation of a medicament for that intended therapeutic purpose, unless the context indicates otherwise.
- a "dose” is an aliquot of culture-expanded MSCs in sufficient numbers which may be administered to an individual for the purposes of providing a therapeutic benefit to the individual.
- reference herein to a "dose” may or may not include additional therapeutic components, such as high molecular mass
- glycoconjugate-enriched conditioned media The actual number of cells in the "dose” is typically in the range of about 2 million to about 20 million cells, preferably such as about 4 million to about 10 million cells, or preferably about 5 million cells. It will be understood that a “dose” may be administered in one or more than one injections. A “dose” may also be seen as a unit of suitable storage of culture-expanded MSCs.
- treating refers to the alleviation of the symptoms and/or the underlying cause of the condition or disease, such as inflammatory disorder.
- a treatment will slow, delay or halt the progression of a disorder or the symptoms of the disorder or injury, or reverse the progression of the disorder or injury, at least temporarily.
- the word “treatment” or derivations thereof such as “treating” when used in relation to a therapeutic application includes all aspects of a therapy, such as the alleviation of pain associated with the condition being treated, alleviation of the severity of the condition being treated, improvement in one or more symptoms of the condition being treated, etc.
- Use of the word “treatment” or derivatives thereof will be understood to mean that the subject being “treated” may experience any one or more of the aforementioned benefits.
- the terai "preventing” and the like in the context of the "prevention” of disease, refers to hindrance of the progression of the symptoms or the underlying cause of the disease. It will be understood that complete prevention of a disease may occur, such that the disease does not occur in a treated animal or subject. Equally, it will be understood that the term includes partial prevention, such as the failure of a disease to progress to the typical state observed in an animal or subject left untreated.
- the invention relates to improved methods for the large scale preparation of culture expanded mesenchymal stem cells (MSCs), such as for use in therapeutic methods.
- MSCs culture expanded mesenchymal stem cells
- the methods allow the production of millions of therapeutic doses of MSCs from a single donor preparation of adipose tissue.
- MSCs Mesenchymal stem cells
- MSCs Mesenchymal stem cells
- tissue culture Prior to use.
- the instant invention provides improved methods for the large scale production of culture expanded cells, including methods for the selection of donor cells that have preferred potential for use in such large scale production.
- the mesenchymal stem cells preferably originate from adipose tissue.
- tissue is meant the tissue type that the MSCs are isolated from for use in the methods or compositions of the present invention.
- the MSCs may be isolated from a tissue specifically for the purposes of the methods and compositions of the invention, or the MSCs may have previously been isolated from a tissue source in a procedure unrelated to the methods or compositions of the invention.
- Adipose tissue may be human adipose tissue or mammalian animal adipose tissue, such as canine, equine, or feline. Typically the source of the adipose tissue will be of the same species as the intended recipient of the MSCs.
- the adipose tissue may comprise "white” adipose tissue, or "brown” adipose tissue.
- adipose tissue Along with an abundance of MSCs, adipose tissue also comprises immune cells, vascular smooth muscle cells, endothelial cells, and pericytes, which together with the MSCs collectively are termed the stromal vascular fraction (SVF).
- SVF stromal vascular fraction
- Adipose tissue is collected from donors by liposuction or by excision.
- a large amount about 200 grams to 2000 grams
- lipoaspirate is collected, or made available, as starting material, as this will influence the number of cells that can be manufactured.
- adipose tissue is typically collected by excision from the falciform, the inguinal fat pads or the shoulder.
- adipose tissue is typically collected by excision from the tail-base, the chest or the abdomen.
- adipose tissue is collected from the abdomen, thighs or buttocks.
- the adipose tissue may initially be processed by mechanically dissociating adipose tissue using techniques which are readily available in the art. Any suitable method for the mechanical dissociation of adipose tissue may be used, for example by mincing adipose tissue with blades, or with scissors, or by forcing adipose tissue through screens or meshes with a pore size sufficient to break the tissue into isolated cells or small pieces of adipose tissue, or a combination of these techniques. In preferred methods, where mechanical dissoci ation is used, the adipose tissue is finely minced with scissors prior to digestion.
- Adipose tissue is digested by incubation with collagenase in the presence of an appropriate buffer.
- collagenase is added to a final concentration of between about 0.2% weight/volume (wt/vol) and about 0.02%> wt/vol, such as about 0.2%> wt/vol, or about 0.15% wt/vol, or about 0.1 % wt/vol, or about 0.9% wt/vol, or about 0.08% wt/vol, or about 0.07% wt/vol, or about 0.06% wt/vol, or about 0.05% wt/vol, or about 0.04% wt/vol, or about 0.03%) wt/vol, or about 0.02% wt/vol.
- the collagenase in the digestion step is added to a final concentration of about 0.05%) wt/vol.
- the concentration of calcium during the digestion step is in the range of about 50 mg/L to about 500 mg/L, such as about 50 mg/L, 100 mg/L, 125 mg/L, 150 mg/L, 175 mg/L, 200 mg/L, 225 mg/L, 250 mg/L, 300 mg/L, 325 mg/L, 330mg/L, 350 mg/L, 375 mg/L, 400 mg/L, 425 mg/L, 450 mg/L, 475 mg/L, or 500 mg/L.
- the concentration of calcium in the digestion is about 330 mg/L.
- the buffer is calcium containing Ringers buffer or calcium containing phosphate buffered saline (PBS).
- the adipose tissue is incubated in the presence of the collagenase at an appropriate temperature, such as about 37°C, for an appropriate time, such as about 30 to about 120 minutes. In a preferred embodiment the incubation is for about 90 minutes.
- the inventors have identified that improved cell yield and viability is also aided by careful mixing during the digestion step to release cells, as too much mixing, or too rigorous mixing, will affect cell viability.
- Mixing may be conducted by hand, such as by gentle inversion of the material at intervals during the incubation, for example about every 15 minutes.
- Mixing may be conducted by mechanical means, such as mixing in the range of about 50 rpm to about 200 rpm, for example on an orbital rotator. In a preferred embodiment the mixing is either by hand or is at about 100 rpm on an orbital rotator.
- the preparation of an adipose tissue-derived cell suspension may comprise a centrifugation step.
- the centrifugation of isolated cells or small aggregates or pieces of adipose tissue suspended in a liquid, such as a medium is at approximately 500 g for 10 minutes, or for sufficient time and at a sufficient g-force to generate a cell pellet which comprises adipose- derived non-adipocyte cells, above which is a layer of medium, floating above which in turn is a layer which comprises the viable adipocytes, and floating at the top is a layer of lipid which is derived from ruptured adipocytes.
- the lipid layer, the adipocytes and the medium layer will preferably be discarded and the pelleted material, referred to as the SVF and comprising MSCs, is retained.
- the pelleted cells may also be referred to as adipose-derived non-adipocyte cells or as SVF cells.
- the SVF cells are placed in to tissue culture.
- the SVF contains a mixed population of cell types, including MSCs.
- Red blood cells (RBCs) are typically also present in the SVF, as may be cell clusters.
- the RBCs are typically present as a contaminant at low numbers, the majority of the RBCs having been removed by washing the adipose tissue pieces prior to digestion with collagenase.
- Other researchers typically purify the MSCs from the other cell types prior to performing tissue culture, such as by fluorescence activated cell sorting (FACS) or immune-magnetic cell separation (IMS) or partially purified by density gradient centrifugation, or the lysis or removal of red blood cells, or removal of cell clusters.
- FACS fluorescence activated cell sorting
- IMS immune-magnetic cell separation
- the SVF cells are seeded at a level of between about 5,000 and about 15,000 cells per cm " of tissue culture flask.
- the MSCs are the most rapidly growing cells present in the SVF and out-grow the other cell types resulting in a pure population of MSCs.
- this approach to expansion gives higher cell yields than purifying a starting population of MSCs prior to tissue culture.
- Methods for culturing MSCs include, for example, culturing the cells to form an adherent cell culture, such as a confluent adherent cell culture. At any appropriate time during, or after, culturing of the cells supernatant is harvested, such as from an adherent cell culture, which may be a confluent adherent cell culture, and optionally, removing cells from said supernatant to form a composition comprising adipose tissue-derived secretions. Any appropriate medium may be used for culture of the cells. Appropriate media include, for example, DMEM, RPMI and minimal essential media. In an embodiment the cells are cultured in DMEM .
- the cell culture is preferably in the presence of sterile serum.
- concentration of the serum in the culture may be any suitable concentration which assists culturing of adipose tissue- derived cells, such as for example in the range of about 1 % volume/volume (v/v) to about 30% v/v, such as about 10% v/v, or about 15% v/v or about 20% v/v.
- the serum may be any appropriate serum for the culturing of adipose tissue-derived cells, such as a commercial fetal calf serum, or a serum prepared in house, such as by methods known in the art.
- the serum is autologous, having been prepared from the same individual from which the adipose tissue was obtained, or allogeneic.
- the cells are cultured at 37°C with 5% C0 2 .
- the serum is from a different species.
- cells may be canine, equine or human and may be cultured in media containing a serum which is other than canine, equine or human, respectively.
- the cells are cultured in media supplemented with fetal calf serum.
- the media comprises 10% FCS.
- Cells may be switched from FCS to allogeneic serum at a later passage, such as the final passage to remove the FCS from the final product.
- the cells are typically grown to at least 85% flask confluence (method of confluence determination is via a microscope) in culture media and serum for 4-21 days in a C0 2 incubator at 37°C.
- the invention also provides for MSCs cultured on microcarriers, for example in a stirred bioreactor.
- the MSCs can be cultured on microcarrier beads (90 - 400um) which may be uncoated, or coated with particular proteins such as collagen and may be treated to have specific charge distribution on the surface.
- the microcarriers may be used for the growth of cells in a number of vessels such as spinner flask, microbioreactors, stirred tank, rocking platforms or microgravity.
- Cells may also be cultured on Fibra-Cel® discs (New Brunswick Scientific, Edison, NJ), which may be maintained in a bioreactor as a suspended stirred system or as a static packed bed.
- the cells in culture secrete components into the culture medium such that the liquid phase obtained from culture will contain components secreted from the cells. These secreted components may collectively be referred to herein as the secretome.
- the liquid phase generated during the cell culturing may be referred to herein as secretions or, where the cells in culture are adipose tissue-derivednon-adipocyte cells, as secretions from adipose tissue- derived non-adipocyte cells.
- the liquid phase generated during culturing and optionally obtained from culturing may also be referred to herein as conditioned media.
- MSCs cultured in platelet lysate and production of high molecular mass glycocon jugate- enriched media
- the cells are cultured in platelet lysate.
- platelet lysate is preferred as the serum source for tissue culture of human cells.
- Platelet lysate is used at a concentration of about 1% volume/volume (v/v) to about 20% v/v, such as about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 1 1 %, or about 12%, or about 13%, or about 14%, or about 15%, or about 16%, or about 17%, or about 18%, or about 19%, or about 20%), preferably at a concentration in the range of about 2% to about 7.5%, such as about 5% v/v, or more preferably at a concentration range of about 7.5% to 15%, such as about 10%.
- Platelet lysate gives a high cell yield of cells and by the growth of the cells in platelet lysate-supplemented media the inventors have surprisingly enabled the production of conditioned medium that contains high levels of extracellular matrix (ECM) components.
- ECM extracellular matrix
- cells grown in platelet lysate secrete high concentrations of extracellular matrix (ECM) components, which may include proteoglycans, glycosaminoglycans and mucins.
- ECM extracellular matrix
- the inventors have also shown that a similarly very viscous conditioned media can be achieved by growing the cells in media containing epidermal growth factor (EGF) and/or basic fibroblast growth factor (FGF).
- EGF extracellular matrix
- the concentration of EGF in the culture media may be in the range of about l Ong/ml to about 30ng/ml, such as about 20 ng/ml.
- the concentration of FGF in the culture media may be in the range of about l Ong/ml to about 30ng/ml, such as about 20 ng/ml.
- the enriched conditioned media will generally be referred to herein as high molecular mass glycoconjugate-enriched media or conditioned media comprising ECM components.
- Preliminary results of kinematic viscosity supported the deduction that the viscosity component is a GAG, a proteoglycan, aggrecan, versican, lumican, mucin or other high molecular weight molecule or complex of molecules.
- the invention thus also provides methods for production of high molecular mass glycoconjugate-enriched media.
- This media provides additional therapeutic advantages for the treatment of inflammatory conditions, including osteoarthritis. It is relevant to note that although the production of high molecular mass glycoconjugate-enriched media by culturing MSCs in platelet lysate-supplemented media was initially developed by the inventors using MSCs obtained from adipose tissue by the methods of the invention described h erein, having made that invention, the inventors consider that the MSCs for use in preparing the enriched media are not limited only to those obtained from adipose tissue by the collection and digestion methods described herein.
- the SVF is preferably not further fractionated or purified to isolate MSCs prior to initial tissue culture steps.
- the MSCs may be prepared according to that method or they may be prepared or obtained according to any other suitable method which provides MSCs that may be used in tissue culture.
- M SCs for use in the methods of preparing high molecular mass glycoconjugate-enriched media by culturing MSCs in platelet lysate-supplemented media may be further purified by any suitable means after initial isolation of the SVF and before placing in media comprising platelet lysate.
- the MSCs for use in the methods of preparing high molecular mass glycoconjugate-enriched media may be purified and stored MSCs, such as cells having previously been purified and or culture expanded by cell culture, or may be cells retrieved from frozen storage.
- MSCs for use in the methods of preparing high molecular mass glycoconjugate-enriched media are not limited to adipose tissue-derived MSCs.
- MSCs for use in the methods of preparing high molecular mass glycoconjugate-enriched media may therefore be sourced from any appropriate source of MSCs, for example from bone marrow, dental pulp, adipose tissue, chord tissue, chord blood and circulating blood.
- the invention thus also provides a combination MSCs and conditioned media comprising extracellular matrix (ECM) components, such media may also be referred to herein as high molecular mass glycoconjugate-enriched media.
- ECM extracellular matrix
- the inventors envisage that a pharmaceutical composition of such a combination of cells and conditioned media comprising ECM may provide additional therapeutic advantages over the use of MSCs alone or conditioned media from culture of MSCs alone.
- the methods of the invention thus allow for a composition comprising MSCs and conditioned media comprising ECM components, in which the conditioned media comprising ECM components may be that in which the cells of the composition were expanded or may be that from expansion of MSCs not comprised in the composition.
- the use of a composition comprising MSCs and conditioned medium comprising ECM components in which those MSCs were expanded may provide additional advantages.
- the MSCs or the high molecular mass glycoconjugate-enriched media or a combination thereof may be in the form of a pharmaceutical composition.
- the composition may typically include a pharmaceutically acceptable carrier, excipient or adjuvant or at least will typically not include a component which is incompatible with therapeutic use in an intended subject.
- the pharmaceutical composition may be in a form for use by injection or in a form for use by topical application. Where the composition comprises MSCs the composition will typically be in a form suitable for injection. Where the composition is for topical application the composition will typically comprise a gel, cream, liquid or lotion formulation.
- the platelet lysate may be obtained from any appropriate source.
- a suitable commercial source is PLT Max from Mill Creek Life Sciences (Rochester, Minnesota, USA).
- the platelet lysate may be derived from the same or a different species as the MSCs being cultured. In preferred embodiments the platelet lysate is derived from the same species as the MSCs being cultured.
- platelet lysate "derived from” describes platelet lysate that has been prepared from a blood sample, such as by isolation of platelets from the blood sample followed by lysing of the isolated platelets.
- the blood sample from which the platelet lysate is derived may or may not be from the same individual as the MSCs, or where MSCs are prepared from adipose tissue, the adipose tissue for preparation of the MSCs.
- the blood sample is from a different individual from that from whom the MSCs or the adipose tissue is or was obtained.
- the platelet lysate may be prepared from fresh whole blood or from stored whole blood using methods or kits known to the skilled addressee.
- the platelet lysate may be from a single donor or may be from pooled blood or cells.
- the platelet lysate may be prepared from expired transfusable whole blood or platelets, such as about 5 to 7 days post-collection.
- the platelet lysate may be prepared from blood using a commercially available kit such as the platelet lysate kit from MacoPharma (France).
- the platelet lysate is prepared from blood collected in the presence of an anticoagulant, such as citrate.
- the blood is centrifuged under appropriate conditions, such as at 200g for about 20 minutes, followed by collection of the platelets (top layer) which are then subjected to freeze-thawing to lyse the cells.
- appropriate conditions such as at 200g for about 20 minutes
- multiple rounds of freeze-thawing are conducted, such as two, three, four, or more rounds.
- the lysed platelets are centrifuged to allow the pelleted cell fragments to be discarded, for example at 4000g for about 10 minutes.
- the platelet lysate may be sterilised, such as by filtering through a suitable matrix, such as a 0.22 micron filter, and stored under appropriate conditions, such as - 80C, until use.
- Heparin when cells are cultured in platelet lysate, heparin is typically also added to the cell culture media to prevent clotting.
- Heparin may be included in the cell culture media at a range of about 0.6 IU/mL to about 5 IU/mL.
- heparin may be included at a concentration of about 0.6 IU/mL, about 0.8 IU/mL, about 1 IU/mL, about 1.5 IU/mL, about 2 IU/mL, about 2.5 IU/mL, about 3 IU/mL, about 2.5 IU/mL, about 4 IU/mL, about 4.5 IU/mL, or about 5 IU/mL.
- heparin is included at about 2 IU/mL.
- the enriched conditioned media was demonstrated to have a higher viscosity than conditioned media generated by culturing MSCs in media supplemented with FCS (in the absence of platelet lysate, FGF and EOF as exemplified herein). The viscosity was shown to increase with increasing cell density of the M SCs.
- the enriched conditioned media has a viscosity of at least about 1.5 centistokes, or at least about 1.6 centistokes, or at least about 1.7 centistokes.
- viscosity may be measured by an alternative means, such as by measuring dynamic viscosity rather than by measuring kinematic viscosity as was done in the Examples herein. It will therefore be understood that in
- viscosity is a descriptive feature of the invention
- a composition which includes the described features of the invention herein but which is said to have a viscosity described in alternative units, for example in centipoise (cP), as may occur if the viscosity was measured by dynamic viscosity, will still lie within the scope of this invention if the viscosity, when measured or described in units of centistokes, is at least about 1.5 centistokes, or at least about 1.6 centistokes, or at least about 1.7 centistokes, or other value as defined herein.
- centipoise centipoise
- the enriched conditioned media of the invention provides for improved stability of secreted cell components, exemplified by vascular endothelial growth factor (VEGF) and multiple other cytokines and growth factors.
- VEGF vascular endothelial growth factor
- the improved stability is characterised by determining the stability of VEGF and other cytokines and growth factors during storage at room temperature over an extended period of time.
- storage of the enriched conditioned media at room temperature (which was approximately 23°C) for a period of six months did not result in appreciable loss of VEGF, as measured using an ELISA.
- the results also show that multiple different cytokines and growth factors were more stable in the viscous material than in the non- viscous when stored at room temperature over a period of fi ve months.
- the inventors have identified that the number of doses of culture-expanded MSCs that can be obtained by expanding cells from a single donor varies greatly between cells from different donors. The cells from some donors stop growing after a limited number of cell doublings whereas cells from other donors continue to grow to a greater number of cell doublings. The inventors have also identified that there may be variation in the number of doses that can be obtained by expanding cells obtained from different locations of a given individual donor animal. The reasons for these differences are not understood and there are no satisfactory methods currently available to analyse cells at a relatively early stage to predict how many doses can be produced from the expansion of particular cells, being cells obtained from a parti cular individual donor or a particular location on a donor.
- the inventors have developed and herein describe methods for screening cells from different donors or locations to rapidly identify which donor's cells will be suitable for manufacturing large numbers of cells.
- the inventors have developed and herein describe methods for screening cells from different donors or locations to rapidly identify which donor's cells will be suitable for manufacturing large numbers of cells, which method invol ves changing the tissue culture medium in which the cells are being cultured from platelet lysate or FBS-supplemented medium to allogeneic serum-supplemented medium. As a short-hand terminology this method of screening may herein be referred to as a 'serum switch' method. Cells from donors that are not suitable for the production of large numbers of cells do not cope well with this change in serum. They stop replicating and fail to reach confluence.
- the methods of screening based on changing the tissue culture medium typically cells are cultured in platelet lysate or FBS for one, two, or three passages, preferably for two passages, and then the platelet lysate or FBS is changed for allogeneic serum.
- concentration of allogeneic serum used in this aspect of the invention is typically about 10% v/v to about 20% v/v.
- Cells that are suitable for large-scale manufacturing will continue to grow and reach confluence in the allogeneic serum. The majority of donor cell samples do not continue to grow in the allogeneic serum, in that they fail to reach confluence.
- a portion of the cells from a donor sample is taken at an early passage, for example after two passages, as a "test" sample, which may also be referred to as a sacrificial test sample as it and its progeny may be discarded after testing.
- the test sample of cells is subjected to the described 'serum switch'. If the cells cope with the change to allogeneic serum-supplemented media, in that they continue to grow and reach confluence, then the user returns to the sample from which the 'test' cells were drawn and continues with the large scale culturing of those cells or a portion thereof in the preferred growth medium, typically being medium containing FBS or platelet lysate.
- the preferred media for culturing of human MSCs is platelet lysate-supplemented media.
- the methods of screening donor cells or donor samples described herein thus permit a user to discriminate between donor cells or samples which are suitable for or conducive to large scale production of MSCs and those which are less suitable. In this manner cells or samples which are unsuitable or less suitable for the intended use can be discarded before extensive efforts are made to use those cells.
- This method of selecting suitable material allows the user to concentrate efforts on cells and samples which do have capability of being cultured for sufficient population doublings to generate the desired large scale production of MSCs.
- cells would typically need to be capable of greater than about 10 population doublings before senescence, more preferably greater than about 15 population doublings before senescence, or greater than about 20 population doublings before senescence, or greater than about 25 population doublings before senescence, or greater than about 30 population doublings before senescence, or greater than about 35 population doublings before senescence.
- a cell line arising from a donor sample becomes senescent after less than about 10 population doublings, more so if senescent after less than about 8 or 7 population doublings, it will typically not be suitable for large scale production of MSCs.
- the 'serum switch' method allows an operator to identify a cell sample as being suitable for or not suitable for very large scale production of MSCs at a relatively early stage, that is without the need for more than about one, two, three or four passages. This ability to discriminate between suitable and unsuitable cell samples is
- MSCs grown in platelet lysate secrete high concentrations of ECM components
- glycoconjugates were present in the media in large amounts (see Examples 4 and 5 herein).
- the glycoconjugates may include proteoglycans, glycosaminoglycans and/or mucins.
- the increase in glycoconjugates occurred at the same time as the increase in the viscosity of the media. Further analysis of the media identified that the viscosity of the media was contributed by GAG, proteoglycan, mucin or other high molecular weight molecule or complex of molecules and that the major proteoglycan present may be aggrecan, versican, lumican or biglycan.
- the invention thus also provides a method for the preparation of high molecular mass glycoconjugate-enriched conditioned media, the method comprising culture expanding MSCs in a growth medium comprising platelet lysate. Methods for the growth or expansion of MSCs in media comprising platelet lysate are described herein.
- the culture medium is DMEM.
- the platelet lysate is human platelet lysate.
- the MSCs are MSCs derived from bone marrow, or MSCs derived from dental pulp, or MSCs derived from bone marrow, or MSCs derived from chord tissue, or MSCs derived from chord blood, or MSCs derived from circulating blood, or adipose tissue-derived MSCs.
- the MSCs are adipose tissue-derived MSCs.
- the MSCs are human adipose tissue-derived MSCs.
- the growth (proliferation) of the cells will be permitted to continue until greater than about 80% confluence, or greater than about 85% confluence, or greater than about 90% confluence, or greater than about 95% confluence, or about 100%) confluence or beyond, so that the media is more concentrated with ECM components.
- the invention thus provides a method for the preparation of high molecular mass glycoconjugate-enriched conditioned media and cultured-expanded MSCs, the method comprising culture expanding MSCs in a growth medium comprising platelet lysate.
- the invention thus provides a combination or composition comprising high molecular mass glycoconjugate-enriched conditioned media and cultured-expanded MSCs.
- This combination which may also be referred to as a composition, may be used for therapeutic purposes in the treatment of a disease, which may herein also be referred to as a disorder or condition, such as an inflammatory disease, disorder or condition.
- the composition may be used for the manufacture of a medicament for the treatment of such conditions.
- a combination of MSCs and high molecular mass glycoconjugate-enriched conditioned media may also be prepared by harvesting the cells and medium from the container after a suitable period of incubation under conditions suitable for the growth of the MSCs.
- conditioned media may also be prepared by combining in a suitable container isolated MSCs and high molecular mass glycoconjugate-enriched conditioned media substantially free of MSCs. In this manner the combination comprises cells in high molecular mass glycoconjugate-enriched conditioned media different to that which the cells were generated (culture expanded).
- isolated MSCs and high molecular mass glycoconjugate-enriched conditioned media substantially free of MSCs.
- the operator is able prepare a combination having a desired number of cells per volume of media. In this manner, for example, a combination having a higher cell density, or number of cells per volume of media, may be generated than would typically be achieved in a combination solely comprised of MSCs in the media in which those same cells were generated.
- a combination having a lower cell density, or number of cells per volume of media may be generated than would typically be achieved in a combination solely comprised of MSCs in the media in which those same cells were generated.
- the ability to control the number of cells per volume of media may be advantageous in situations where, for example, a therapeutic advantage may be achieved by administering to a subject a dose having a chosen cell to media ratio.
- Isolated MSCs may added to a combination of high molecular mass glycoconjugate-enriched conditioned media and MSCs or may be added to high molecular mass glycoconjugate-enriched conditioned media
- culture of MSCs in culture media comprising platelet lysate or EGF and/or FGF provides a viscous conditioned media containing high molecular mass glycoconjugate, generally referred to herein as a high molecular mass glycoconjugate-enriched conditioned media, and that this material has beneficial characteristics as described herein, may also be used by an operator to assess the appropriate stage, or assist in assessment of the appropriate stage, at which to harvest cells or conditioned media from culture, such as from a bioreactor.
- conditioned media having a viscosity of about 1.5 cSt or more is enriched in high molecular mass glycoconjugate and provides for improved stability of components secreted from the MSCs, such as VEGF.
- the Examples demonstrate that the enriched material is beneficial in the treatment of disease, such as inflammatory conditions and neuropathic pain.
- disease such as inflammatory conditions and neuropathic pain.
- a viscosity of about 1.5 cSt or more is typically achieved in a culture of MSCs grown in the presence of platelet lysate at greater than about 80% confluence.
- the results herein therefore allow for an operator to use the viscosity of the media in culture, such as in a bioreactor or other culture vessel or container, particularly in culture conditions where the cell density cannot easily or feasibly be determined, as an indication of the appropriate stage at which to harvest the cells and or the conditioned media, depending on the attributes of the media or the cells desired or required by the operator. This may be achieved, for example, by determining the viscosity of a sample of the liquid phase of the culture at a given time and, on the basis of the determined viscosity, harvesting cells or media or continuing the culture, depending on the operator's requirements.
- composition comprising high molecular mass
- glycoconjugate-enriched conditioned media and culture-expanded MSCs are native to each other, meaning that the cells in the composition are cells which have been grown in the high molecular mass glycoconjugate-enriched conditioned media with which they reside in the composition.
- the method further comprises separating the MSCs from the culture medium.
- the cells may be separated or harvested from the culture medium by methods known to the skilled addressee.
- the invention thus provides for the preparation of high molecular mass glycoconjugate-enriched conditioned media substantially free of MSCs.
- a preparation of high molecular mass glycoconjugate- enriched conditioned media would be considered to be "substantially free" of MSCs if it comprises no MSCs or merely remnant contaminant MSCs that may remain after routine steps to harvest or remove adherent cells from cell culture.
- the media may be considered to be
- the harvested high molecular mass glycoconjugate-enriched conditioned media may be subjected to one or more further processing steps, for example to remove or reduce the presence of contaminant material.
- a contaminant material may be any undesirable component of the media, such as cell fragments or debris.
- a contaminant will also include any component that is incompatible with a pharmaceutical use of the media, for example a component that may be toxic to a recipient animal or which may reduce the therapeutic efficacy of the media or which may reduce the ability of the media to be stored.
- An example of further processing in this context may include centrifugation or filtering the media.
- the harvested high molecular mass glycoconjugate-enriched conditioned media, with or without MSCs, may be stored under any appropriate conditions. Storage is typically frozen at -20°C, -10°C or -80°C. Alternatively storage may be at 4°C.
- the storage may be in separate aliquots or vials such as may be suitable for use in a therapeutic dose in the treatment of a subject.
- the media may be stored in an injectable-ready form, such that an operator merely needs to retrieve the material from storage and thaw or warm it to the required temperature before therapeutic use.
- the storage may be "bulk" storage which may require further dilution or aliquotting for example prior to use, or for example, which may be more suitable for research purposes.
- the invention also provides for MSCs cultured on microcarriers, for example in a stirred bioreactor.
- the MSCs can be cultured on microcarrier beads (90 - 400um) which may be uncoated, or coated with particular proteins such as collagen and may be treated to have specific charge distribution on the surface.
- the microcarriers may be used for the growth of cells in a number of vessels such as spinner flask, microbioreactors, stirred tank, rocking platforms or microgravity. Cells may also be cultured on fibra-cel discs, which may be maintained in a bioreactor as a suspended stirred system or as a static packed bed.
- compositions and Pharmaceutical compositions
- compositions in particular of pharmaceutical compositions.
- the composition may comprise high molecular mass glycoconjugate-enriched conditioned media substantially free of MSCs.
- the composition may comprise culture expanded MSCs prepared by a method of the invention.
- the composition may comprise high molecular mass glycoconjugate-enriched conditioned media and MSCs.
- the composition may be any of the
- a composition of the invention may be used for the preparation of a pharmaceutical composition.
- the composition may comprise one or more of a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
- a composition of the invention such as a pharmaceutical composition, may be supplied to the user as a frozen solution.
- high molecular mass glycoconjugate- enriched conditioned media, culture-expanded MSCs, or a combination thereof can be stored, at approximately -20°C until required for use.
- high molecular mass glycoconjugate- enriched conditioned media, culture-expanded MSCs, or a combination thereof may be stored at a lower temperature, for example in a freezer at -70°C to -90°C, or in liquid nitrogen storage, either in the vapour phase or in the liquid phase, until required for use.
- Compositions comprising cells will typically be stored in liquid nitrogen.
- the composition comprising high molecular mass glycoconjugate-enriched conditioned media, or the culture- expanded MSCs, or a combination of high molecular mass glycoconjugate-enriched conditioned media and culture-expanded MSCs is stored in the liquid phase of liquid nitrogen storage.
- a composition comprising adipose tissue-derived culture-expanded MSCs cells is stored in combination with high molecular mass glycoconjugate-enriched conditioned media.
- a cell free composition of the invention such as a pharmaceutical composition, may be supplied to the user as a freeze-dried preparation.
- high molecular mass glycoconjugate-enriched conditioned media may be freeze dried and stored, at approximately 4°C, -20°C or at room temperature until required for use.
- the composition is typically administered to the subject or animal as soon as possible after thawing.
- the pharmaceutical composition may alternatively be stored, for example on ice or in a refrigerator or in a cool pack, preferably at approximately 2°C to 5°C for a short time between thawing and administration.
- a short time would typically be no more than several hours, such as no more than about half an hour, or no is more than about one hour, or no more than about two hours.
- cryoprotectants are typically toxic to the cells and can cause loss of viability if kept thawed
- the composition particularly where it comprises viable cells, is typically injected to the recipient animal as soon as possible after thawing.
- a pharmaceutical composition of the invention may be supplied in a "ready-to-use" form.
- the user typically requires only thawing to an acceptable temperature for administration before the composition is administered.
- the composition may be supplied in pre-measured doses, such as a premeasured or pre-determined dose suitable for a given recipient subject or animal, for example pre-determined on the basis of the recipient species, or on the basis of the recipient individual, such as a small breed of dog, compared to a large breed of dog, or a juvenile animal compared to an adult animal.
- the pre-measured dose may alternatively or additionally be on the basis of the disease or condition being treated or intended to be prevented.
- a ready-to-use form of the composition may comprise the composition supplied with or in an injectable device, such as a syringe.
- the injectable device may be capable of delivering a single application to an individual recipient or may be capable of delivering single or multiple applications to multiple recipients.
- the injectable device may be adjustable, for example to permit delivery of a range of different doses.
- the composition may be supplied to the user as a combination or as separate compositions for combination by the user.
- a "user” in this context means the individual who actually administers the therapeutic composition to the recipient subject or animal and also means a member of the team or group who is undertaking that administration.
- the user may be any individual who is assisting in the application of the methods of the invention such as a clinician, a doctor, a veterinarian, a farmer, a clinical nurse, a veterinary nurse, a technical assistant, or a farmhand.
- high molecular mass glycoconjugate-enriched conditioned media demonstrates advantageous stability of components, such as VEGF and other molecules secreted from the cultured MSCs, for example when the media is stored at room temperature.
- the invention also provides for compositions that may optionally be stored without freezing or without refrigeration.
- a composition stored without freezing or without refrigeration will comprise high molecular mass glycoconjugate-enriched conditioned media but will not comprise MSCs.
- a pharmaceutical composition comprising high molecular mass glycoconjugate- enriched conditioned media may thus be provided as a ready to use composition which is suitable for storage at room temperature, which would typically be in the range of about 20°C to 25°C.
- the composition may be a composition for topical application, such as to the skin of a subject in need of such application.
- a composition for topical application may be in any appropriate form, including for example, in the form of a liquid, a gel, a cream or a lotion.
- compositions for topical application When provided in the form of a composition for topical application and which can be stored at room temperature the invention provides for ease of use by a subject, such as ease of self- administration compared to, for example, an injectable composition or a composition that is optimally stored refrigerated or frozen.
- a pharmaceutical composition comprising high molecular mass glycoconjugate- enriched conditioned media may be formulated with any appropriate carrier material, such as is suitable for formulation of a therapeutically active ingredient or ingredients for topical application.
- any appropriate carrier material such as is suitable for formulation of a therapeutically active ingredient or ingredients for topical application.
- the use of a carrier material that does not contain a surfactant allows for hi gher levels of VEGF to be detected than does the use of a carrier materi al that does contain a surfactant. Without wishing to be bound by theory the inventors consider this may be due to binding of the VEGF by the surfactant, thereby reducing the detectable VEGF in the composition.
- the carrier material does not include a surfactant.
- the pharmaceutical composition does not include a surfactant.
- the carrier material comprises saline and optionally propylene glycol.
- the carrier material is Solugel or a formulation similar thereto.
- composition comprises one or more thickeners, such as a cosmetic or pharmaceutical thickener(s), for example hydroxyl ethyl cellulose.
- thickeners such as a cosmetic or pharmaceutical thickener(s), for example hydroxyl ethyl cellulose.
- the invention also provides a kit comprising (a) a pharmaceutical composition selected from the group consisting of (i) high molecular mass glycoconjugate-enriched conditioned media, (ii) a composition comprising culture expanded MSCs, and (iii) a combination of (i) and (ii); and (b) instructions for use of said kit in treatment of an inflammatory disorder, or alleviating pain associated with an inflammatory disorder.
- a pharmaceutical composition selected from the group consisting of (i) high molecular mass glycoconjugate-enriched conditioned media, (ii) a composition comprising culture expanded MSCs, and (iii) a combination of (i) and (ii); and (b) instructions for use of said kit in treatment of an inflammatory disorder, or alleviating pain associated with an inflammatory disorder.
- the kit comprises one or more frozen compositions.
- the kit comprises instructions for combining a composition comprising high molecular mass glycoconjugate-enriched conditioned media and a composition comprising culture expanded MSCs, prior to administration of a combined composition.
- the kit further comprises one or more injection devices, such as one or more syringes.
- the injection device contains a composition of the kit.
- a composition of the invention may be used in the treatment or prevention of a medical condition conducive to treatment by MSCs or by proteoglycan-enriched conditioned media.
- a composition of the invention may be used for the treatment or prevention of an inflammatory disorder or of osteoarthritis in a subject requiring said treatment.
- a composition of the invention may be used for the alleviation of pain of an inflammatory disorder or of osteoarthritis in a subject requiring said alleviation.
- composition of the invention was also beneficial to the patient when administered for treating tennis elbow, a tendon injury, chilblains, tendonitis, golfers wrist, bursitis, muscle or calf tear.
- the subject may be any animal.
- the subject is selected from the group consisting of a cat, a dog and a horse.
- the subject is a human.
- the pharmaceutical composition may be administered for the treatment of an inflammatory disorder and/or for alleviating pain associated with an inflammatory disorder in a subject.
- Inflammation may arise as a response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent.
- An inflammation reaction may include the local reactions and resulting morphologic changes, destruction or removal of injurious material such as an infective organism, and responses that lead to repair and healing.
- the term "inflammatory" when used in reference to a disorder refers to a pathological process which is caused by, resulting from, or resulting in inflammation that is inappropriate or which does not resolve in the normal manner. Inflammatory disorders may be systemic or localized to particular tissues or organs.
- Inflammation is known to occur in many disorders which include, but are not limited to: Systemic Inflammatory Response (SIRS); Alzheimer's Disease (and associated conditions and symptoms including: chronic neuroinflammation, glial activation; increased microglia; neuritic plaque formation; Amyotrophic Lateral Sclerosis (ALS), arthritis (and associated conditions and symptoms including, but not limited to: acute joint inflammation, antigen-induced arthritis, arthritis associated with chronic lymphocytic thyroiditis, collagen-induced arthritis, juvenile arthritis, rheumatoid arthritis, osteoarthritis, prognosis and streptococcus-induced arthritis, spondyloarthropathies, and gouty arthritis), asthma (and associated conditions and symptoms, including: bronchial asthma; chronic obstructive airway disease, chronic obstructive pulmonary disease, juvenile asthma and occupational asthma); cardiovascular diseases (and associated conditions and symptoms, including atherosclerosis, autoimmune myocarditis, chronic cardiac hypoxia, congestive heart failure, coronary artery disease,
- neurodegenerative diseases or neuropathological conditions and associated diseases and conditions, including acute neurodegeneration, induction of IL-I in aging and neurodegenerative disease, IL-I induced plasticity of hypothalami c neurons and chronic stress hyperresponsi veness, myelopathy
- ophthalmopathies and associated diseases and conditions, including diabetic retinopathy, Graves' ophthalmopathy, inflammation associated with corneal injury or infection including corneal ulceration, and uveitis
- osteoporosis and associated diseases and conditions, including alveolar, femoral, radial, vertebral or wrist bone loss or fracture incidence
- otitis media adult or paediatric
- pancreatitis or pancreatic acinitis periodontal disease (and associated diseases and conditions, including adult, early onset and diabetic); pulmonary diseases, including chronic lung disease, chronic sinusitis, hyaline membrane disease, hypoxia and pulmonary disease in SIDS; restenosis of coronary or other vascular grafts; rheumatism including rheumatoid arthritis, rheumatic Aschoff bodies, rheumatic diseases and rheumatic myocarditis; thyroiditis including chronic lymphocytic thyroiditis; urinary tract infections including chronic prostatitis, chronic pelvic pain syndrome and urolithiasis; immunological disorders, including autoimmune diseases, such as alopecia aerata, autoimmune myocarditis, Graves' disease, Graves ophthalmopathy, lichen sclerosis, multiple sclerosis, psorias
- goitre and struma lymphomatosa Hashimoto's thyroiditis, lymphadenoid goitre
- lung injury acute hemorrhagic lung injury, Goodpasture's syndrome, acute ischemic reperfusion
- myocardial dysfunction caused by occupational and environmental pollutants (e.g. susceptibility to toxic oil syndrome silicosis), radiation trauma, and efficiency of wound healing responses (e.g. bum or thermal wounds, chronic wounds, surgical wounds and spinal cord injuries), septicaemia, acute phase response (e.g.
- febrile response general inflammatory response
- acute respiratory distress response acute systemic inflammatory response
- wound healing acute systemic inflammatory response
- adhesion immuno-inflammatory response
- neuroendocrine response fever development and resistance
- acute-phase response stress response
- disease susceptibility repetitive motion stress, tennis elbow, and pain management and response.
- the inflammatory disorder is selected from joint-related inflammatory disorders, corneal inflammation, skin inflammation or wound healing.
- the joint-related inflammatory disorder is arthritis, such as osteoarthritis.
- compositions of the invention are for the treatment of tennis elbow or for the treatment of a tendon injury, or for the treatment of chilblains or for the treatment of tendonitis, such as of the foot, of for the treatment of golfer's wrist, or for the treatment of bursitis, such as trochanteric bursitis, or for the treatment of Achilles tendonitis, or for the treatment of a muscle tear, such as a calf tear.
- the pharmaceutical composition may be administered for the treatment of neuropathic pain in a subject.
- the inventor has identified that compositions of the invention are useful in the treatment of subjects having pain for which there is no discernable clinical cause, such as some forms of neuropathic pain.
- Neuropathic pain refers to a group of painful disorders characterized by pain due to dysfunction or disease of the nervous system at a peripheral level, a central level, or both. It is a complex entity with many symptoms and signs that fluctuate in number and intensity over time.
- the three common components of neuropathic pain are steady and neuralgic pain; paroxysmal spontaneous attacks; and hypersensitivity.
- Neuropathic pain can be very disabling, severe and intractable, causing distress and suffering for individuals, including dysaesthesia and panaesthesia. Sensory deficits, such as partial or complex loss of sensation, are also commonly seen. In addition, there are significant psychological and social consequences linked to chronic neuropathic pain, which contribute to a reduction in quality of life.
- Neuropathic pain is quite common in general medical practice.
- the neuropathic pain is not associated with any discemable clinical causative condition.
- the compositions of the invention are effective in alleviating chronic tennis elbow which is considered to be neuropathic pain.
- the neuropathic pain is associated with a discemable clinical condition.
- the prevalence of trigeminal neuralgia is 2.1 to 4.7 persons per 100,000 of the population, and of painful diabetic neuropathy occurs in 1 1% to 16% of Type 1 diabetics as well as Type II diabetics and post -herpetic neuralgia is found in approximately 34 persons per 100,000 of the population. Treatment of neuropathic pain is not easy.
- neuropathic pain patients with neuropathic pain do not always respond to standard analgesics such as non-steroidal anti- inflammatory drugs (NSAIDs) and to some extent neuropathic pain is resistant to opiates.
- NSAIDs non-steroidal anti- inflammatory drugs
- the pharmacologic agents best studied and longest used for the treatment of neuropathic pain are antidepressants and anticonvulsants is both of which may have serious side effects.
- a composition of the invention may be administered to a subject for treatment of such pain at any appropriate site.
- Administration may typically be using an appropriate type of injection or it may be by topical application.
- an injection may be subcutaneous, intramuscular, or directly into an accessible site at or near a site of the pain.
- this type of pain may manifest in multiple areas of the subject's body, for example jaw pain and limb or shoulder pain, the administration may be at or near to one site of the pain and remote from another site affli cted by pain.
- the administration is at or near a site identified as an original or primary site of the pain.
- the examples herein show treatment of golfers wrist and chronic tennis elbow by topical application of a cream or gel comprising high molecular mass glycoconjugate-enriched conditioned media.
- the topical treatment may involve rubbing the gel or cream on to the affected area, alternatively it may involve applying the cream or gel to a dressing or patch which is then applied to the affected area.
- a subject being treated may be administered a single application of a composition of the invention, or may preferably be administered multiple applications.
- Example 1 Screening of canine cells from different donors.
- Adipose tissue was rinsed with saline and then minced finely using scissors and mixed with 20mls of Dulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase (Sigma) was added to a final concentration of 0.05% wt/vol and the sample was incubated at 37°C for 90 minutes. During the incubation the samples was gently inverted by hand every 15 minutes.
- DMEM Dulbecco's Modified Eagle's Medium
- Blood was collected in to blood collection bags with citrate used as the anticoagulant, according to methods known in the art.
- the blood was dispensed into centrifuge tube and centrifuged at 200 g for 20 min.
- the top layer containing the platelets was collected and subjected to 4 cycles of freeze thawing from liquid nitrogen in to a 37°C waterbath.
- the lysed platelets were then serum converted by the addition of thrombin and calcium chloride, then centrifuged for 10 min at 4000g and the pelleted cell fragments discarded.
- the platelet lysate was then filter sterilised (0.22 micron) and stored at -80°C until required.
- Liposuction was used to collect approximately 200 grams of adipose tissue from the abdomen and or thighs of each patient.
- the lipoaspirate was processed immediately after collection by washing with warmed (37°C) sterile Ringers Solution (Baxter) and then digesting by adding sterile collagenase to a final concentration of 0.05% wt/vol.
- the sample was incubated at 37°C for 20 minutes with gentle mixing at 100 rpm on an orbital mixer, filtered through a 800 micron mesh, transferred to centrifuge tubes, and centrifuged at 500g for 15 minutes.
- the pelleted cell samples were resuspended in CryoStor CS 10 (Biolife Solutions, USA) and transferred to cryovials in 2 ml aliquots and the cryovials were frozen in a controlled rate freezing device at approximately 1°C per minute in a -80°C freezer for 24 hours and then transferred to a liquid nitrogen dewar for long tenn storage.
- Example 5 To investigate the gradual increase in viscosity of the cell culture medium: evidence for tissue culture supernatant from mesenchymal stem cells grown in platelet lysate containing extracellular matrix component(s) and or mucins.
- Viscosity was measured using a Cannon-Fenske Routine capillary viscometer (Cannon Instrument Company, serial number T209), which measures kinematic viscosity, as per the manufacturer instructions. Briefly, the viscometer was filled with approximately 5 ml of sample (culture medium or re-suspended precipitate) by suction and allowed to equilibrate to 40° C for at least 10 mins in a water bath. Once at temperature, the sample was pulled up to the first marked point by suction and the time taken for the meniscus to move between the first and second marked points was measured (the effiux time).
- Acetone precipitation (80%, -30°C) precipitated the viscosity component from the culture medium indicating that it is of a high molecular weight, which was estimated to have the similar viscosity of hyaluronic acid at a concentration of about 0.2-0.3 mg/ml.
- HPAEC-PAD Monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection
- Table 1 Digestion conditions optimized for digestion of Chondroitin sulphate, keratan sulphate (component of Aggrecan) and Hyaluronic acid.
- the GAG disaccharide digestion products were detected using either LC-MS or direct MS and their composition confirmed by MS " fragmentation from components separated using a porous graphitized carbon column eluting with ammonium bicarbonate in acetonitrile and negative ion mass spectrometry with a Thenno Velos ion trap mass spectrometer.
- the cell secretion samples were produced from cells grown in alpha MEM media plus 10% platelet lysate (serum converted) (Stemulate, Cook Regentec, Bloomington, IN, USA) and 2. DMEM media plus 5% platelet lysate (PLTMax, Millcreek, Rochester, MN, USA) plus 2 IU/mL heparin.
- Chondroitin sulphate is a GAG component of Aggrecan and also of other
- proteoglycans such as Versican.
- Chondroitin sulphate confirms the presence of one or a mixture of such proteoglycans.
- the glycan mass spectrometry data was determined on the sugars digested from a precipitate taken from an acetone precipitation and the levels of Chondroitin sulphate were lower than expected.
- samples (50 ⁇ ) were dot blotted under vacuum onto PVDF membrane using a Bio-Rad dot blotting apparatus and stained with Alcian blue (0.1 % Alcian blue in 0.1% acetic acid) before and after acetone precipitation and staining intensity compared, the staining intensity (darker blue staining colouration) was higher in the cell secretion samples before acetone precipitation (results not shown) suggesting not all charged macromolecules (i.e. proteoglycans) were precipitated by acetone.
- the column was equilibrated in 100% solvent A (0.1%) (v/v) formic acid and the peptides separated by a two-step gradient of 0%o - 30%o solvent B (0.1 % (v/v) formic acid in acetonitrile), followed by 30%) - 60%) solvent B.
- the peptide mixtures were reconstituted in 15 ⁇ of deionized water and 5 ⁇ was injected onto the column.
- the extracellular matrix proteoglycans that were detected were Lumican, Versican and Biglycan. Chondroitin sulphate is a GAG chain component of Versican as well as Aggrecan. Aggrecan was not detected in the proteomic analysis, however the proteomic analysis was sub- optimal for proteoglycans and the extensi ve sugar chains on Aggrecan could have inhibited the detection of the protein components.
- An ELISA kit with Aggrecan specific antibodies confirmed varying levels of 50 - 900 pg/mL of Aggrecan in conditioned media from both media types (with and without heparin) and across passages 1 -10 (results not shown). Seven different adipose tissue donors were tested and aggrecan was detected from conditioned media from all 7 donors.
- Chondroitin sulphate is a component of both proteoglycans Versican and Aggrecan. Proteomic analysis detected Versican, lumican and biglycan, but did not confirm the presence of aggrecan, potentially the proteomic analysis was inhibited by the extensive sugar chains on Aggrecan. Aggrecan was confirmed to be present by an Aggrecan antibody ELISA assay in conditioned media of both media types and was not present in matching blank growth media.
- Example 6 Industrial scale production of human allogeneic adipose derived MSCs.
- Adipose tissue was collected from human donors as described in Example 3. Cells from each donor were screened according to Example 1. Cells that showed the appropriate properties, such as acceptable performance in the 'serum switch' screening method, were selected for large scale expansion.
- Table 2 shows that over 332 million vials each containing 5 million cells can be produced from the one donor using the methods of the invention. Table 2: Calculated number of vials that can be produced from the one donor.
- Example 7 An alternative tissue culture media for the expansion of MSCs
- Human cells were isolated from adipose tissue and cultured as described in Example 3. After two passages the media was replaced with High Glucose DMEM, 10% fetal calf, 20ng/ml epidermal growth factor (EGF), 20ng/ml basic fibroblast growth factor and 2% B27 Supplement (Life Technologies). Once the cells became confluent the conditioned media was viscous.
- High Glucose DMEM 10% fetal calf
- EGF epidermal growth factor
- B27 Supplement Life Technologies
- Example 8 Treatment of a human patient with osteoarthritis with allogeneic cells and high molecular mass glycoconjugate-enriched conditioned media
- Liposuction was used to collect 832 grams of adipose tissue from the abdomen and or thighs of a patient.
- the lipoaspirate was processed immediately after collection by washing with warmed (37°C) sterile Ringers Solution (Baxter) and then digesting by adding sterile collagenase to a final concentration of 0.05% wt/vol.
- the sample was incubated at 37°C for 20 minutes with gentle mixing at 100 rpm on an orbital mixer, filtered through a 800 micron mesh, transferred to centrifuge tubes, and centrifuged at 500g for 15 minutes.
- the floating cells and the supernatant were discarded and the pelleted cells were gently mixed with a pasteur pipette and transferred to a 15ml centrifuge tube. [000193]
- the cells were then washed in DMEM to remove collagenase. DMEM was added to a final volume of 14 mis and the sample centrifuged at 500g for 10 minutes. The supernatant was discarded and the pelleted SVF cells were was pooled and divided into 8 separate freezing bags.
- the cells were cryopreserved in Cryostor 10 in volumes of 1 1.5-12.5ml per bag.
- a bag of cells was thawed and seeded in a T175 tissue culture flask containing Alpha MEM and 10% human platelet lysate.
- the platelet lysate had been treated by the addition of thrombin and calcium chloride, prior to centrifugation to remove fibrinogen.
- Cells were incubated in a C0 2 incubator at 37°C until a confluent cell monolayer was present (7 to 10 days). Cells were passaged 4 times as described in Example 3. The passaged cells were then stripped and centrifuged. The conditioned media from the final passage was collected and used for cryospreservation of the cells.
- pelleted cell samples were resuspended in 1.8 mL of conditioned media and 0.2 mL of bloodstor- 100 cryopreservative fluid (Biolife Solutions, USA) and transferred to cryovials in 2 ml aliquots and the cryovials were frozen in a controlled rate freezing device at
- a 52 year old patent with grade 3 osteoarthritis of the knee was treated with a single intra-articular injection of expanded allogeneic cells and high molecular mass glycoconjugate- enriched conditioned media.
- the vial containing the cells and conditioned media was thawed at room temperature and immediately injected in to the knee joint.
- the injection consisted of 3.9 million cells in 1.8 mL of high molecular mass glycoconjugate-enriched conditioned media and 0.2 mL of bloodstor-100 cryopreservative fluid.
- Human adipose derived MSCs obtained as described in Example 8) were cultured in 10 layer cell factory flasks in either alphaMEM plus 10% platelet lysate (serum converted) (Stemulate, Cook Regentec, Bloomington, IN, USA) or in alphaMEM plus 10% fetal calf serum. The media was not changed and the cells were kept in culture for 4-7 days until the cells were 80 to 100% confluent. The supernatant was harvested and was observed to be viscous from the platelet lysate media but was not viscous from the fetal calf serum media. The supernatant was not filtered as filtering was observed to reduce the viscosity.
- VEGF Vascular endothelial growth factor
- Samples of the secretions were stored in test tubes at 22°C and tested at regular intervals. Samples were also stored at -80°C as a control. Samples stored at 22°C were compared to the -80°C controls.
- VEGF vascular endothelial growth factor
- Adipose derived cells were prepared as described in Example 8. Cells were cultured in either alphaMEM plus 10% platelet lysate (serum converted) (Stemulate, Cook Regentec, Bloomington, IN, USA) or in DMEM plus 10% fetal calf serum. Cells were cultured through to passage 4 or 5 and the conditioned media was harvested when the cells were between 50% and 100%) confluent. The viscosity of the conditioned media was measured using a cannon-feske U viscometer.
- Example 11 Treatment of tennis elbow with a composition comprising secretions from passaged human adipose derived adherent cells
- Example 12 Treatment of tendon injuries with a composition comprising secretions from passaged human adipose derived adherent cells
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WO2018213795A1 (en) * | 2017-05-18 | 2018-11-22 | The Board Of Trustees Of The Leland Stanford Junior University | Targeted in situ therapeutic delivery of secreted factors from stem cells for treatment of damaged tissue |
CN109022360A (en) * | 2018-07-03 | 2018-12-18 | 湖南未名三胞转化医学科技有限公司 | Autologous fat stem cell, which co-cultures, promotes peripheral blood hematopoietic stem cells enrichment procedure |
WO2019238952A1 (en) | 2018-06-15 | 2019-12-19 | Marinas Pardo Luis | Pharmaceutical composition for dermatology and uses thereof |
WO2024036387A1 (en) * | 2022-08-16 | 2024-02-22 | Omics Biotecnologia Animal Ltda. | Concentrate of proteins and peptides derived from mesenchymal stromal cells, production method and use of the concentrate |
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CN110106142B (en) * | 2019-05-30 | 2020-05-12 | 深圳阿尔法生物科技有限公司 | Production process for preparing billion-grade fat source regenerative cells |
CN111073857A (en) * | 2019-12-30 | 2020-04-28 | 深圳爱生再生医学科技有限公司 | Stem cell biological product for treating arthritis and preparation method and application thereof |
KR102193175B1 (en) * | 2020-04-07 | 2020-12-18 | 주식회사 엑소스템텍 | Stem cell-derived exosomes with pain control factors and uses thereof |
CO2020012570A1 (en) | 2020-10-08 | 2022-04-08 | Inst Distrital De Ciencia Biotecnologia E Innovacion En Salud Idcbis | Mesenchymal cell stimulation method to induce expression of immunomodulatory factors |
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WO2019238952A1 (en) | 2018-06-15 | 2019-12-19 | Marinas Pardo Luis | Pharmaceutical composition for dermatology and uses thereof |
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US20210260129A1 (en) | 2021-08-26 |
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