AU2016244319A1 - Cell free preparation and uses thereof - Google Patents
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
CELL FREE PREPARATION AND USES THEREOF Abstract Provided herein is a method for treating a joint, tendon or ligament injury by locally administering a cell free composition generated by exposure of a medium to a population of cells. Also provided is use of cell free composition generated by exposure of a medium to a population of cells for the preparation of a medicament for treatment of a joint, tendon or ligament injury. (11872253 11:GGG
Description
- 1 - 2016244319 14 Oct 2016
CELL FREE PREPARATION AND USES THEREOF
Field
The present invention relates to compositions for treating joint disease or injury, and methods for the generation of such compositions.
Cross-reference to related applications
This application is a divisional of Australian Patent Application No. 2010347212, the entire contents of which is incorporated herein by reference.
Background
Kits for the preparation of autologous conditioned serum are available for use in the treatment of degenerative joint disease or injury in mammals, such as horses. Autologous conditioned serum is considered to be effective in treating joint disease in horses and other animals, including humans, at least in part due to the therapeutic effect of IL1 Ra which is present in the conditioned serum. IL1Ra antagonises IL1, which is an inflammatory cytokine that is thought to be a key antagonistic factor in the development of early stage joint disease and arthritis in horses. Intra-articular injections of IL1Ra have been used to reduce lameness and other symptoms of joint disease in horses.
As some subjects may be become refractory to autologous conditioned serum treatments there is a need for alternative treatments for joint diseases.
Summary
Provided herein is the use of a cell free composition for the treatment of a joint, tendon or ligament injury in a subject, wherein the cell free composition is administered locally to the affected joint, tendon or ligament of the subject, and wherein the cell free composition is produced by a method which comprises: culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition
Also provided herein is a method of treatment of a joint, tendon or ligament injury in a subject, the method comprising administering a cell free composition locally to an injured joint, tendon or ligament of the subject, wherein the cell free composition is produced by a method which comprises: culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, -2- 2016244319 14 Oct 2016 exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition
Also provided is a method for generating a cell free composition for use in the treatment of a joint, tendon or ligament injury in a subject, the method comprising culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition
In certain embodiments of the use or the method the tissue stem cells are mesenchymal stem cells obtained from adipose tissue.
In certain embodiments of the use or the method the population of cells comprising tissue stem cells is substantially free of adipocytes.
In certain embodiments the step of culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells is carried out at the same time as exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro.
Detailed Description
In the context of this specification, the term “comprising” means including but not necessarily solely including. Furthermore, variations of the word “comprising” such as “comprise” and “comprises” have correspondingly varied meanings.
Throughout this specification, reference to “a” or “one” element does not exclude the plural, unless context determines otherwise.
The cell free composition described herein is produced by culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition.
The term “tissue stem cells” as used herein refers to cells which may also be termed “adult” or “somatic” stem cells, which are cells having the ability to self renew and to differentiate into multiple cell types which may be found in the organ from which the stem cells originate (multipotency). -3- 2016244319 14 Oct 2016
The term “tissue stem cells” is intended to encompass any one or both of mesenchymal stem cells and hematopoietic stem cells. Mesenchymal stem cells may be obtained from a variety of tissues. Mesenchymal stem cells may, for example, be obtained from the placenta or cord blood, muscle or adipose tissue, lung, bone marrow and blood and the dentate pulp and periodontal ligament of teeth. Hematopoietic stem cells may, for example, be obtained from bone marrow, blood, adipose tissue, lung, kidney, liver, chord blood or placenta.
In particular embodiments the tissue stem cells are mesenchymal stem cells.
The population of cells comprising tissue stem cells may consist of a substantially pure population of tissue stem cells. The population of cells comprising tissue stem cells may be a mixed cell population which comprises tissue stem cells and one or more other cell types. The population of cells comprising tissue stem cells may comprise two or more different types of tissue stem cells, for example mesenchymal stem cells and hematopoietic stem cells. In certain embodiments the population of cells comprising tissue stem cells is a “stromal vascular cell” population which is produced following a partial or complete digestion of tissue, for example, adipose tissue, lung tissue, kidney tissue or muscle tissue.
In particular embodiments, where the population of cells comprising tissue stem cells is obtained from adipose tissue, the population of cells is substantially free of adipocytes. The population of cells comprising tissue stem cells may, for example, be a cell line. The population of cells comprising tissue stem cells may, for example, be freshly isolated from a tissue. The population of cells comprising tissue stem cells may, for example, be isolated from a tissue and cultured for a period before exposing the population of cells comprising tissue stem cells in vitro to the aqueous medium for at least three days, for example to allow cells to attach to a culture substratum or to provide an opportunity to increase the number and/or proportion of particular cell types within the cell population. The population of cells comprising tissue stem cells may be obtained from a variety of sources. The population of cells comprising tissue stem cells may, for example, be obtained from the same individual subject to which the cell free composition will be administered, in which case the population of cells comprising tissue stem cells is autologous. The population of cells comprising tissue stem cells may, for example, originate from one or more different individuals of the same species to the subject to which the cell free composition will be administered, in which case the population of cells comprising tissue stem cells is allogeneic. The population of cells comprising tissue stem cells may for example be obtained from one or more individuals of a different species to the subject in which the cell free composition will be administered, in which case the population of cells comprising tissue stem cells is xenogeneic. -4- 2016244319 14 Oct 2016
Typically the population of cells comprising tissue stem cells is obtained from a mammal.
It will be understood, however, that tissue stem cells may also be found in non-mammal tissues and that the treatment described herein may also be applicable to non-mammalian animals, and consequently in certain embodiments the population of cells comprising tissue stem cells is obtained from a reptile or a bird. The mammal may be a human. The mammal may be a non-human mammal. The population of cells comprising tissue stem cells may be obtained from a companion animal, such as a canine or a feline domestic animal, or a working animal. The population of cells comprising tissue stem cells may be obtained from a farm animal, stud animal, or racing animal such as equines (including horses, donkeys, asses), bovines (including cattle and buffaloes), ovines, caprines, porcines, and camelids (including camels, llamas, alpacas and the like). The population of cells comprising tissue stem cells may be obtained from a research animal, such as a rodent, or a zoo animal, such as a member of the family Felidae, a member of the family Canidae, a member of the order Rodentia, or a member of the one of the orders of Cetacea, Perissodactyla, Artiodactyla, Tubulidentata, Hyracoidea, Sirenia, or Proboscidea.
The population of cells comprising tissue stem cells is cultured so that there is cell replication in the population of cells comprising tissue stem cells. Culturing of the population of cells comprises the manipulation of the population of cells in vitro for a period and under such conditions as to allow cell replication. Typically culturing will involve providing the population of cells comprising tissue stem cells and a tissue culture medium to a culture chamber or culture vessel which provides a culture substrate and incubating the culture chamber or vessel containing the cell population. Cell replication typically requires adhesion of the cells to a substratum. If a cell population comprising tissue stem cells is introduced as a cell suspension to a culture vessel or chamber, cell adhesion to the culture substrate typically takes place between 12 and 72 hours after introduction, and cell replication may commence shortly thereafter. Typically a tissue culture medium is used to provide conditions suitable for cell replication. The tissue culture medium may be a serum free medium or may be a serum containing medium. A variety of tissue culture media suitable for the culture of mammalian cells are known in the art. Non-limiting examples of suitable culture medium include Modified Eagles Medium (MEM), Dulbeccos Modified Eagles Medium (DMEM), and Roswell Park Memorial Institute medium (RPMI).
Cell replication may be detected using a wide variety of techniques readily available in the art, such as by counting cell numbers manually by microscopy (optionally using methods for distinguishing between live and dead cells, such as a Trypan blue dye exclusion -5- 2016244319 14 Oct 2016 method, so that dead cells are excluded from the cell count) or by counting representative cell samples using flow cytometry.
The population of cells comprising tissue stem cells when cultured may, for example, be present initially as a suspension of isolated cells, as small cellular aggregates, as pieces of tissue ranging in size from less than ten mm in maximum diameter, less than five mm in maximum diameter, less than one mm in maximum diameter, less than 500 pm in maximum diameter or less than 250 pm in maximum diameter or as combinations of two or more of isolated individual cells, small cellular aggregates and pieces of tissue. A cell population may be obtained by variety of methods. For example, a cell population comprising tissue stem cells may be obtained by the dissociation of a previously cultured cell population which comprises tissue stem cells from a culture substrate, using for example the mechanical removal of cultured cells from a cell substrata, trypsin, EDTA or a combination of techniques. A cell population may be obtained by mechanically dissociating a tissue isolate using techniques which are readily available in the art, for example by mincing tissue with blades, or with scissors, or by forcing tissue through screens or meshes with a pore size sufficient to break the tissue into isolated cells and/or small pieces of tissue. A combination of suitable techniques may be used. Small aggregates of tissue may form when dissociated cells associate into larger assemblies, for example on standing in a medium. In certain embodiments, a mechanical dissociation technique is used without using one or more proteolytic enzymes.
In certain embodiments, proteolytic enzymes are used to promote the dissociation of tissue into a cell population comprising adult stem cells. Enzymes which are suitable for such a use are well known in the art, and include but are not limited to one or both of trypsin and collagenase. It is usual to remove and/or otherwise inactivate the proteolytic enzymes, as these enzymes may not be compatible with a desired in vivo use of the cell free composition. In certain embodiments, proteolytic enzymes in combination with techniques for the mechanical dissociation of tissue are used to generate a population of cells comprising adult stem cells.
In culture, the population of cells may for example be adherent to a solid culture substrate, may be attached to a matrix which supports cell replication, may be encapsulated, for example by being positionally constrained in a biocompatible material such as collagen or alginate while at the same time being exposed to the aqueous medium, or may be suspended as cell aggregates or small pieces of tissue in the tissue culture medium. The culture substrate may, for example, be the floor of a culture flask, the culture surface of a flow-through culture reactor, or micro-carrier beads which are suspended in a medium.
In certain embodiments, the cell population comprising tissue stem cells are cultured as confluent cells adhering to a culture substrate. -6- 2016244319 14 Oct 2016 ln culture, the population of cells may for example be not attached to a culture substrate, for example where the population of cells comprising tissue stem cells are cultured in a stirrer culture or a hanging drop culture.
The population of cells comprising tissue stem cells is exposed to an aqueous medium in vitro. In certain embodiments the exposure of the population of cells comprising tissue stem cells to an aqueous medium may take place while the population of cells comprising tissue stem cells is cultured so that there is cell replication in the population of cells comprising tissue stem cells, and so the tissue culture medium which supports the cell replication in the population of cells is also the aqueous medium.
In certain embodiments the exposure of the population of cells comprising tissue stem cells to an aqueous medium may take place following the culture of the population of cells comprising tissue stem cells, and so the tissue culture medium which supports the cell replication in the population of cells may also be the aqueous medium or may be different to the aqueous medium. For example, exposure of the population of cells comprising tissue stem cells to the aqueous medium may be achieved by replacing the tissue culture medium with the aqueous medium.
The aqueous medium may be a medium which supports cell survival and proliferation in vitro, such as a tissue culture medium. Non-limiting examples of suitable culture medium include Modified Eagles Medium (MEM), Dulbeccos Modified Eagles Medium (DMEM), and Roswell Park Memorial Institute medium (RPMI).
In certain embodiments the aqueous medium is a serum free medium, such as a defined medium. In other embodiments the aqueous medium may comprise serum or serum components which support or extend cell survival in the cell population. The serum or serum components may be autologous serum or serum components. The serum or serum components may be allogeneic serum or serum components from a single individual or pooled from multiple individuals.
The aqueous medium may be a solution which is not toxic to the population of cells but which does not itself support cell proliferation and which therefore may result in the death of cells within the cell population over a period of days of exposure, such as an isotonic saline or isotonic buffered saline solution which is suitable for bathing cultured cells. As the cell free composition will be administered to a subject after exposure of the aqueous medium to the cell population, the aqueous medium typically will be suitable for administration in a pharmaceutical or veterinary composition. The properties of an aqueous medium which make it suitable for administration include that the aqueous medium is substantially free of pyrogens or toxic materials, and that the concentration of one or more substituent ions may be adjusted if required prior to administration to the -7- 2016244319 14 Oct 2016 subject such that adverse reactions caused by the constituents of the aqueous medium are minimized.
The method described herein comprises the step of exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro. The exposure of the population of cells comprising tissue stem cells to the aqueous medium is intended to also encompass the exposure of the aqueous medium to the population of cells comprising tissue stem cells. Typically the population of cells comprising tissue stem cells is exposed to the aqueous medium at an initial density of approximately 1 x 104 to 5 x 105 cells per ml of aqueous medium.
The population of cells comprising tissue stem cells is exposed to the aqueous medium for a period sufficient to condition the medium with factors secreted or released from the population of cells comprising tissue stem cells. The exposure may be for a period of at least 12 hours. Typical periods of exposure of the population of cells comprising tissue stem cells to the aqueous medium are selected from 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours.
The exposure of the cell population which comprises tissue stem cells to a medium in vitro may be at a temperature of from approximately 4°C to 50°C, more typically at a temperature of from 10°C to 40°C and most typically at a temperature of from 20°C to 37°C.
Where the population of cells comprising tissue stem cells is adherent to a culture substrate, in certain embodiments the population of cells is a confluent population of cells comprising tissue stem cells. In certain embodiments the population of cells comprising tissue stem cells becomes a confluent population of cells comprising tissue stem cells during the exposure to the aqueous medium in vitro.
In certain embodiments the population of cells comprising tissue stem cells is exposed to an aqueous medium in vitro, wherein the exposure to an aqueous medium in vitro is under hypoxic conditions for at least 12 hours. As described herein, hypoxic conditions are conditions in which the concentration of oxygen in the atmosphere to which the aqueous medium is exposed is less than 20%, less than 15%, less than 10% or less than 5% oxygen v/v. In certain embodiments the population of cells comprising tissue stem cells is exposed to an aqueous culture medium in vitro, wherein the exposure to an aqueous medium is under anaerobic conditions for at least 12 hours.
The production of the cell free composition comprises the step of isolating the aqueous medium which has been exposed to the population of cells from the population of cells comprising tissue stem cells to produce a cell free composition. The cell free composition may be completely cell free or may be substantially free of cells. A composition which is -8- 2016244319 14 Oct 2016 substantially free of cells will have no more than about 103 cells per ml. Where the cell free composition is allogeneic or xenogeneic it may be advantageous to minimize the number of cells present in the cell free composition to minimize the possibility of host vs graft-type immune responses to cellular antigens present in the cell free composition. Where the population of cells comprising tissue stem cells is immobilized or encapsulated and where there is not a substantial number of cells suspended in the aqueous medium, for example where the cell population is adherent to a culture substrate, the isolation may involve simply collecting the aqueous solution from a culture of the cell population which comprises tissue stem cells. The isolation may involve one or more steps to remove cells from the aqueous solution, such as centrifugation and/or filtration.
In order to maintain sterility or to remove possible bacterial or viral contamination, the aqueous solution may be subjected to ultra-filtration after exposure to the cell population comprising tissue stem cells.
In certain embodiments the subject to be treated is a mammalian subject. For example, the subject may be a human subject. The subject may be a non-human mammal. The subject may, for example, be a companion animal, such as a canine or a feline domestic animal, or a working animal. The subject may be a farm animal, stud animal, a racing animal such as an equine (including horses, donkeys, asses), a bovine (including cattle and buffaloes), an ovine, a caprine, a porcine, or a camelid (including camels, llamas, alpacas and the like). The subject may, for example, be a research animal such as a rodent, or a zoo animal such as a member of the family Felidae, or a member of the family Canidae, a member of the order Rodentia, or a member of any one of the orders of Cetacea, Perissodactyla, Artiodactyla, Tubulidentata, Hyracoidea, Sirenia, or Proboscidea.
The cell free composition is used for the treatment of a joint injury, a ligament injury or a tendon injury in a subject, or a combination of any two or more thereof. A variety of injuries are treatable by administration of the cell free composition. For example, the injury may be an injury arising from physical insult to the joint, ligament or tendon, or it may be damage to the joint, ligament or tendon arising from a disease.
The terms “treating”, “treatment”, “therapy” and the like in the context of the present specification refer to alleviation of one or more symptoms and/or alleviation of one or more underlying causes of injury. In certain embodiments a treatment will slow, delay or halt the progression of an injury or the symptoms of the injury, or reverse the progression of the injury at least temporarily. The “treatment” of an injury in a subject may take place at the time the subject exhibits detectable symptoms associated with the injury, or in a subject suspected of suffering from an injury. -9- 2016244319 14 Oct 2016
Where the injury is due to a disease the disease may, for example, be a degenerative joint disease. In particular embodiments the disease is osteoarthritis or pre-osteoarthritis. The disease may, for example, exhibit symptoms of inflammation of an articular cartilage, a joint, a tendon or a ligament. The disease may, for example, exhibit joint effusion. The disease may, for example, be a bone cyst involving a joint. Where the subject is a hoofed animal, the disease may be acute or chronic laminitis.
Treatment may involve one or more of a variety of joints. The affected joint may, for example, be any one or more of an articulation of the hand, an elbow joint, a wrist joint, an acromioclavicular joint, a glenohumeral joint, a sternoclavicular joint, a vertebral articulation, a temporomandibular joint, a sacroiliac joint, a hip joint, a knee joint or an articulation of the foot. Typically, joint-related injuries in humans involve at least one joint in one or both hips, knees, ankles, elbows, shoulders, wrists, the metacarpo-phalangeal articulations or the phalangeal articulations, the metatarso-tarsal articulations or the tarsal articulations or between two or more vertebrae. The tendon or ligament may be a tendon or ligament associated with any one or more of the above mentioned joints. For veterinary joint-related disorders or injuries the corresponding joints are involved in mammalian animals, and these include the stifle and hock joints. Examples of equine joints include coffin, pastern, fetlock, knee, elbow, shoulder, hock, stifle and hip joints. Examples of equine tendons and ligaments include digital flexor tendons, suspensory ligament, common and lateral extensor tendons, peroneus tertius, check ligaments and stifle ligaments. In laminitis inflammation of the epidermal and dermal laminae and possible involvement of the coffin bone are observed.
The cell free composition may be administered in a variety of forms. For example the cell free composition may be administered in liquid form. The cell fee composition may be administered as a solid, for example a freeze dried solid produced after freeze drying the aqueous solution. The cell free composition may be administered alone, or in admixture with agents which, for example, prolong the residency of the cell free composition at the site of administration.
The cell free composition may be administered by a variety of methods, such as by injection, by surgical, microsurgical or keyhole surgical methods, or by implantation of a depot containing a supply of cell free composition which is released over a period of time. Typically local administration is made by direct injection, although local delivery via a slow release pump or a depot is also contemplated.
As described herein, the cell free composition is administered locally. Local administration may be administration directly to the site of joint, tendon or ligament injury. For example, the cell free composition may be administered directly into the articular cartilage, into the synovial fluid and/or into or around the joint capsule or synovial lining, - 10- 2016244319 14 Oct 2016 intra-tendon or peri-tendon, intraligament or peri-ligament, and/or into the musculature overlying or surrounding the joint, and/or subcutaneously to the tissue overlying the joint.
In certain embodiments, for example where the joint, tendon or ligament is difficult to access directly, for example in laminitis. local administration may be achieved by local perfusion via arteries which directly supply the joint, tendon or ligament or hoof.
The cell free composition may be administered alone, or in admixture with one or more suitable pharmaceutical or veterinary carriers, diluents or excipients. Although the examples provided herein demonstrate that some joint, tendon or ligament injuries respond to a single local administration of cell free composition, it is also contemplated that the administration of cell free composition may be repeated over several days or weeks.
The quantity of cell free composition which may be administered will depend on the size and location of the joint, the site of administration or the type of injury. Where administration is by injection into the synovial fluid of a joint, for example, the volume may be constrained by the volume of the synovial fluid which is held at the joint.
The examples which follow are intended to serve to illustrate this invention and should not be construed as limiting the general nature of the disclosure of the description throughout this specification.
Examples
Example 1. Preparation of a cell free solution by culturing partially digested adipose tissue.
Preparation of adipose tissue A 10 g sample of adipose tissue was collected by excision from the tail-base of a horse. The adipose tissue was rinsed with saline and then minced finely using scissors and mixed with 20 ml of Dulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase (Sigma) was added to produce a final concentration of 0.05% w/v and the sample was incubated at 37°C for 30 minutes. At the end of 30 minutes the adipose tissue was partially digested and consisted of a mixture of partially digested fat particles, liberated stromal vascular cells (SVCs) and liberated adipocytes.
The sample was then centrifuged at 500g for 15 minutes. Four distinct layers were visible within the centrifuged sample: a small (2 mm thick) layer of free lipid on the surface, below which was a white 20 mm thick layer of adipose tissue and adipocytes and then a large clear layer of DMEM and then a pellet of adipose tissue-derived non-adipocyte cells. The small layer of lipid was carefully removed with a pasteur pipette. A fresh pasteur pipette was then carefully inserted through the adipocytes and the clear DMEM was removed without disturbing the floating adipose tissue, adipocytes or the pelleted cells. This - 11 - 2016244319 14 Oct 2016 resulted in a sample that contained only the floating pieces of adipose tissue and adipocytes suspended in a small volume of DMEM and the pelleted cells. The pieces of adipose tissue and adipocytes and the pelleted cells were gently mixed with a pasteur pipette and transferred to a 15 ml centrifuge tube.
The pieces of adipose tissue and cells were then washed in DMEM to remove collagenase as follows. DMEM was added to a final volume of 14 ml and the sample centrifuged at 500g for 10 minutes. This resulted in three distinct layers: floating pieces of adipose tissue and adipocytes, DMEM and pelleted adipose tissue-derived non-adipocyte cells. The DMEM was carefully removed by inserting a pasteur pipette through the adipocytes taking care not to disturb the pieces of adipose tissue, adipocytes or the pelleted cells.
Tissue culture
The floating and the pelleted cells were gently resuspended in 10 ml of DMEM and transferred to a 300 ml tissue culture flask. A 30 ml volume of DMEM and 10 mis of autologous sterile serum were added and the flask was then incubated at 37°C with 5% C02.
The flask was examined daily by microscopy. Cells became attached and fibroblast-like in appearance between days 3 and 6. The attached cells became confluent between days 5 and 10.
Harvesting cell free cell secretions
Once cells were confluent on the base of the flask the supernatant was harvested and the suspended adipose tissue and cells were removed by filtration through a 20 micron mesh. The solution was filter sterilised through a 0.22 micron filter and then aseptically dispensed into 10 ml vials and stored frozen at -20°.
Administration of cell secretions to horses with early stage degenerative joint disease A cell free preparation prepared as described above from adipose tissue from a single horse was injected into affected joints of six race horses which had developed early stage degenerative joint disease diagnosed by veterinary examination. Each of the subject horses had previously been treated with locally administered autologous conditioned serum and had shown an initial improvement as assessed by veterinary examination, but each horse had subsequently stopped responding to the autologous conditioned serum. A summary of each subject is presented in Table 1. Each horse was assessed by a vet for joint swelling, pain on joint flexion and lameness prior to local administration of cell free composition by injection to the affected joint.
Subject 1 was a 3 year old unraced horse which had become lame one year prior to treatment with cell free composition. The horse had undergone arthroscopic surgery of both front fetlocks and radiocarpal joints. - 12- 2016244319 14 Oct 2016
The subject was injected in both front fetlocks and radiocarpal joints with cell free composition. This subject received a second administration of cell free composition 7 days after the initial administration and a third administration 14 days after the initial administration. A total of 30 ml of cell free composition was administered to this subject over the three days of administration. The subject is currently undergoing 5 months paddock rest. No adverse reactions to the cell free composition were detected.
Subject 2 was a 6 year old horse which had a history of more than six months of fetlock lameness. Treatment for lameness had included cortisone administration in the first month following diagnosis. The subject received a total of 30 ml of cell free composition over three administrations. A second administration was provided 13 days after the initial administration, and a third administration 26 days after the initial administration. The subject exhibited mild pericapsular swelling in the offside foreleg (O/F) fetlock after the third administration which resolved without treatment. The subject’s trainer noted an improvement in the subject’s gait. The subject has subsequently been raced 6 times, with a result of being placed second twice and third once.
Subject 3 was a 6 year old horse which suffered an O/F superficial digital flexor tendon (SDFT) injury at age 4. Although this injury was treated and the horse was given time off from racing, the injury reoccurred and an ultrasound examination identified a grade 2 lesion of the SDFT O/F. The subject was given a single administration of 3 ml of cell free composition into the tendon under ultrasound guidance. The subject is now having a 1 month break from racing. Following administration the subject’s owner has commented that the subject’s tendon now appears normal.
Subject 4 was a three year old cutting horse which had not been performing as anticipated. A clinical examination identified severe degenerative changes in both tarsal-metatarsal (TMT) joints and distal intertarsal (DIT) joints, together with severe intercarpal joint effusion.
The subject was provided with three administrations of cell free composition to each of the affected TMT, DIT and intercarpal joints. The second administration was provided 9 days after the initial administration, and the third administration was provided 16 days after the initial administration. A total of 60 ml of cell free composition was administered to the subject over the three administrations. The subject has demonstrated a reduction of joint swelling and pain on flexion, and a reduction in lameness over the period in which treatment took place.
Subject 5 was a five year old race horse with a long history of offside front limb lameness, which was isolated to the high suspensory ligament. Lameness returned after the horse returned from 6 months away from racing. Numerous attempts at treatment of the suspensory ligament were made with cortisone. The subject had not raced in 9 months. 2016244319 14 Oct 2016 - 13-
The horse received a single injection of 5 ml of cell free composition into the origin of the suspensory ligament. 16 days following administration the horse was fit to race, and has now competed in several races. The horse’s owner has been pleased with the improvement in the subject’s lameness.
Subject 6 was a 5 year old race horse. This subject had a history of more than a year of foot problems, including laminitis, which had required nerve blocking and a variety of farrier interventions and medications. The horse had never been sufficiently well to race. The subject received 3 administrations of cell free composition via distal limb perfusion into the medial and lateral digital arteries in both front feet. The second administration was made 19 days after the initial administration, and the third administration 55 days after the initial administration. A total of 60 ml of cell free composition was administered over the three separate administration days.
Following each treatment the jockey noted that the horse’s gait and “feel” was improved. Four days after receiving the third administration of cell free composition the horse was raced. 2016244319 14 Oct 2016
Joint T reated Joint Swelling Score 0=normal,5=severe effusion Joint/ Ligament/ Foot Pain on Flexion 0=normal, 5= severe pain Lameness Score at time of injection (0=normal, 5=very lame) Performance - 0 is no improvement, 5 excellent improvement as assessed by owners in the days subsequent. Time Point of scoring 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Subject 1 N/F Radiocarpa I 4 3 2 3 3 2 5 4 3 4 3 2 4 2 2 3 3 2 5 4 3 4 3 2 N/F Fetlock O/F Radiocarpa I O/F Fetlock AVERAGE 4.5 3.25 2.5 3.5 3 2 Subject 2 N/F Fetlock 3 2 2 2 2 1 3 3 3 3 2 1 O/F Fetlock AVERAGE 3 2.5 2.5 2.5 2 1 2 1 0 4 5 5 Subject 3 O/F SDFT 2 3 0 Subject 4 N/F Intercarpal 4 3 3 0 0 0 2 11 2 2 1 0 0 0 2 2 1 4 3 3 0 0 0 2 11 2 2 1 4 0 0 2 2 1 N/H DIT N/H TMT O/F Intercarpal O/H DIT O/H TMT AVERAGE 2.67 1.33 1.33 1.33 1.33 0.67 2 1 0 Subject 5 O/F Limb 4 2 5 Subject 6 O/F Digital arteries 3 1 1 2 1 1 N/F Digital arteries AVERAGE 2.5 1 1 2 1 1 4 4 5 15 2016244319 14 Oct 2016
Example 2. Preparation of a cell free solution by culturing adipose tissue in ceiling culture
Equine adipose tissue was collected, minced, digested and washed as described in Example 1 to produce a cell mixture containing partially digested adipose tissue, adipocytes and stromal vascular cells (SVCs). The cell mixture was cultured in a multilayer tissue culture flask (Millicell, Millipore Ltd). The sample was carefully loaded into the flask so that all floating material was contained underneath one of the layers of the flask, and such that all the suspended cellular material was held submerged beneath the surface of the tissue culture medium. The tissue culture medium was as described in Example 1.
The flasks were incubated as described in Example 1 and when cells were confluent the liquid was harvested, filtered and aseptically dispensed as described in Example 1.
Example 3. Preparation of a cell free solution by culturing adipose derived stromal vascular cells A 10 g sample of adipose tissue was collected by excision from the tail-base of a horse. The adipose tissue was rinsed with saline, minced finely using scissors and mixed with 20 ml of Dulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase (Sigma) was added to produce a final concentration of 0.05% w/v and the sample was incubated at 37°C for 60 minutes. At the end of 60 minutes the adipose tissue appeared to be well digested.
The sample was aseptically filtered through a 300 micron mesh then centrifuged at 500g for 15 minutes. The supernatant was discarded and the pellet was resuspended in 15 ml of DMEM. The sample was centrifuged once again at 500g for 10 minutes. The supernatant was discarded, the pellet resuspended in 40 ml of DMEM plus 10 ml of autologous sterile serum, and then transferred to a tissue culture flask and incubated at 37°C with 5% C02.
The flask was examined daily by microscopy. Cells became attached and fibroblastlike in appearance between days 3 and 6. The attached cells became confluent between days 5 and 10.
Harvesting cell secretions
Once cells were confluent on the base of the flask the supernatant was harvested and the adipose tissue and cells in the medium were removed by filtration through a 20 micron mesh. The cell free solution was filter sterilised through a 0.22 micron filter and then aseptically dispensed into 10 ml vials and stored frozen at -20°C. 2016244319 14 Oct 2016 16
Example 4. Preparation of a cell free solution by culturing plastic adherent cells from adipose tissue A suspension of adipose derived stromal vascular cells was prepared as described in Example 3. After 24 hours in tissue culture non-adherent cells were removed by discarding the tissue culture medium with the non-adherent cells and adding fresh tissue culture medium. The adherent cells were cultured for a further 5 days. Secretions were harvested, filter sterilized and asceptically dispensed and stored as described in Example 3.
Example 5. Preparation of a cell free solution by culturing adipose derived cells in hypoxic conditions.
Adipose derived stromal vascular cells and adipocytes are prepared as described in Example 1. The cell mixture and the partially digested adipose tissue is digested as described in Example 1. Once the cells were confluent the media is replaced with fresh media and the flask was incubated in 2% oxygen at 37°C for 3 days. The supernatant is then harvested, filtered, aliquoted and stored as in Example 1.
Example 6. Preparation of a cell free solution by culturing bone marrow derived cells.
Bone marrow (20 ml) was collected from the sternum of a horse and mixed with 10 mM EDTA. The nucleated cells were purified by density gradient centrifugation with Ficol.
The nucleated cells were cultured as in Example 3. The cells became confluent after 7 days and the supernatant was harvested, filtered and stored frozen.
Example 7. Preparation of a cell free solution by culturing cord blood derived cells.
Bovine cord blood was collected from the umbilical cord by needle aspiration. The nucleated cells were purified by density gradient centrifugation with Ficol.
The nucleated cells were cultured as in Example 3. The cells became confluent after 7 days and the supernatant was harvested, filtered and stored frozen.
Example 8. Preparation of a cell free solution by culturing cells from a human liposuction aspirate. A 50ml volume of human liposuction aspirate was partially digested with collagenase as in Example 1. The floating cells and the stromal vascular cells were cultured as in Example 1. The cells became confluent after 7 days and the supernatant was harvested, filtered and stored frozen.
Claims (5)
- CLAIMS:1. Use of a cell free composition for the treatment of a joint, tendon or ligament injury in a subject, wherein the cell free composition is administered locally to the affected joint, tendon or ligament of the subject, and wherein the cell free composition is produced by a method which comprises: culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition.
- 2. A method of treatment of a joint, tendon or ligament injury in a subject, the method comprising administering a cell free composition locally to an injured joint, tendon or ligament of the subject, wherein the cell free composition is produced by a method which comprises: culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition.
- 3. A method for generating a cell free composition for use in the treatment of a joint, tendon or ligament injury in a subject, the method comprising culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells, exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro and then isolating the aqueous medium from the population of cells to produce a cell free composition.
- 4. The use according to claim 1 or the method according to claim 2 or claim 3, wherein the tissue stem cells are mesenchymal stem cells obtained from adipose tissue.
- 5. The use according to claim 1 or the method according to claim 2 or claim 3, wherein the step of culturing a population of cells comprising tissue stem cells so that there is cell replication in the population of cells comprising tissue stem cells takes place at the same time as the step of exposing the population of cells comprising tissue stem cells to an aqueous medium in vitro.
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