CN105796600B - Methods and compositions for treating osteoarthritis using stem cells - Google Patents
Methods and compositions for treating osteoarthritis using stem cells Download PDFInfo
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Abstract
The present invention relates to methods and compositions for treating osteoarthritis using stem cells. In particular, one aspect of the invention relates to a cell therapy composition for the treatment of osteoarthritis and cartilage defects, the composition comprising: mesenchymal stem cells, hyaluronic acid or a pharmaceutically acceptable salt thereof, sodium chloride, and water. The invention also relates to a method for preparing said composition. The cell therapy composition of the present invention has excellent properties.
Description
Technical Field
The present invention is in the field of pharmacy and relates to pharmaceutical compositions for the treatment and/or prevention of acute and/or chronic osteoarthropathy. In particular, to methods and compositions for treating osteoarthritis using stem cells.
Background
Osteoarthritis is the most common form of arthritis, characterized by a slow degenerative process of articular cartilage, subchondral bone, with marginal osteophyte formation and low levels of inflammation. It is believed that chronic forms of osteoarthritis affect 15% of the population. Of these, one quarter is severely disabled. The cause of most cases of osteoarthritis is unknown and is called "primary osteoarthritis". When the cause of osteoarthritis is known, it is called "secondary osteoarthritis". Secondary osteoarthritis results from other diseases or conditions. Conditions that may lead to secondary osteoarthritis include repeated damage or surgery to joint structures, joint abnormalities at birth (congenital abnormalities), gout, diabetes, and other hormonal disorders. Other forms of arthritis include systemic diseases such as rheumatoid arthritis and Systemic Lupus Erythematosus (SLE).
Osteoarthritis is primarily related to the hip, knee, spine and interphalangeal joints. The most common symptom of osteoarthritis is pain in the affected joints after repeated use. Joint pain is often more severe later in the day. The affected joint can swell, heat, and produce squeaking. The joints can also experience pain and stiffness after prolonged inactivity. In severe osteoarthritis, complete loss of cartilage pads results in friction between bones, causing pain at rest or pain with limited motion.
Osteoarthritis is characterized by a slow degradation of cartilage over several years. In normal cartilage, there is a delicate balance between synthesis and degradation of the matrix; however, in osteoarthritis, cartilage is degraded over synthesized. The balance between synthesis and degradation is affected by age and is regulated by several factors produced by synovium and chondrocytes, including cytokines, growth factors, aggrecanase (aggrecanase) and matrix metalloproteinases. In addition to water, the extracellular matrix contains proteoglycans, which are formed of glycosaminoglycans attached to a backbone formed by hyaluronic acid or a pharmaceutically acceptable salt thereof, and encapsulated (entrap) in a collagen framework or fibril matrix (fibrillary matrix). One important proteoglycan in articular cartilage is aggrecan (aggrecan), which binds to hyaluronic acid or its pharmaceutically acceptable salts and helps to impart compressibility and elasticity to cartilage. Aggrecan is cleaved by aggrecanase, which results in its degradation and subsequent cartilage erosion. Loss of aggrecan in the cartilage matrix is one of the initial pathophysiological changes observed in Osteoarthritis (OA).
In addition, experiments using rabbit synovial cells have shown that proinflammatory cytokines IL-1 β and TNF- α stimulate the expression of hyaluronic acid or a pharmaceutically acceptable salt thereof, which can cause the hyaluronic acid or a pharmaceutically acceptable salt thereof to become green sheets and segment under inflammatory conditions.
Hyaluronic acid or a pharmaceutically acceptable salt thereof (a glycosaminoglycan) is widely used for the treatment of knee osteoarthritis. Two general investigations in the uk have shown that about 15% of patients suffering from osteoarthritis have received intra-articular treatment with a preparation of hyaluronic acid or a pharmaceutically acceptable salt thereof. It can replace synovial fluid due to its viscoelasticity. In addition, it may also reduce the perception of pain. Beneficial molecular and cellular effects have also been reported. Hyaluronic acid or its pharmaceutically acceptable salts are often administered by intra-articular injection, however there is contradiction in the evidence of clinical relevance. A recent systematic review and integrated analysis (meta-analysis) was published in the prior art, the authors concluded that intra-articular hyaluronic acid or its pharmaceutically acceptable salts had at best a minimal effect, whereas the clinically significant effect was an increase in the visual analogue score of pain of at least 15 mm. These data form the basis for intra-articular administration of hyaluronic acid or a pharmaceutically acceptable salt thereof to osteoarthritis patients. Sometimes, efficacy is observed only after one year of injection, and in some experiments 3-5 injections per week are necessary.
Mesenchymal Stem Cells (MSCs) are a heterogeneous group of cells derived from a matrix, and can be obtained from most tissues of the human body. A large number of experimental researches show that the mesenchymal stem cells have epidermal cell differentiation potential and can promote the healing of the wounded skin. The bone marrow derived mesenchymal stem cells are not suitable for batch preparation because of the hidden danger of virus pollution, and the cell number, the amplification and differentiation capacity of the bone marrow derived mesenchymal stem cells are obviously reduced along with the age increase of donors. The mesenchymal stem cells derived from human umbilical cord, placenta or amnion successfully avoid a plurality of limitations of embryonic stem cell source deficiency, variant rejection, moral ethics and the like, and become a good substitute of the bone marrow-derived mesenchymal stem cells; they have self-renewal, tissue repair, immunoregulatory ability, and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, chondrocytes, etc., and also into other mesodermal lineage cells, such as epidermal cells, vascular endothelial cells; and the cells are easy to expand in vitro, and the differentiation capacity and the proliferation capacity are kept stable after expansion, so that the method is suitable for large-scale preparation.
There are known literature reports of the use of mesenchymal stem cells or hyaluronic acid or pharmaceutically acceptable salts thereof for the treatment of osteoarthritis. However, when the two are combined and successfully applied to clinics, a plurality of technical problems need to be faced. Accordingly, it would be highly desirable to provide a method and composition comprising mesenchymal stem cells and hyaluronic acid or a pharmaceutically acceptable salt thereof and useful for the treatment of osteoarthritis.
Disclosure of Invention
It is another object of the present invention to provide a method and composition for treating osteoarthritis using stem cells, and in particular, the cell therapy composition can be used for the treatment of osteoarthritis and cartilage defects by intra-articular cavity injection. It has been surprisingly found that the compositions of the present invention have excellent properties.
The invention is realized by the following scheme.
In a first aspect of the invention, there is provided a cell therapy composition for the treatment of osteoarthritis and cartilage defects, the composition comprising: mesenchymal stem cells (may be abbreviated as MSC, the source of which may be, for example, umbilical cord or placenta), hyaluronic acid or a pharmaceutically acceptable salt thereof, sodium chloride, and water.
The cell therapy composition according to the present invention, wherein the mass volume percentage of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 3% to 5%.
The cell therapy composition provided by the invention is characterized in that the mass volume percentage of the hyaluronic acid or the medicinal salt thereof is 3-4%.
The cell therapy composition according to the present invention, wherein the mass volume percentage of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 3%.
The cell therapy composition according to the present invention, wherein the mesenchymal stem cells have a density of 2 x 107one/mL to 5X 107one/mL.
The cell therapy composition according to the present invention, wherein the mesenchymal stem cells have a density of 3 x 107one/mL.
The cell therapy composition according to the present invention, wherein the mass volume percentage of the sodium chloride is 0.8% to 1.0%.
The cell therapy composition according to the present invention, wherein the mass volume percentage of the sodium chloride is 0.9%.
The cell therapy composition according to the present invention, wherein the mass volume percentage of hyaluronic acid or a pharmaceutically acceptable salt thereof is 3%, and the density of mesenchymal stem cells is 3 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
The cell therapy composition according to the present invention, wherein the mass volume percentage of hyaluronic acid or a pharmaceutically acceptable salt thereof is 4%, and the density of mesenchymal stem cells is 5 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
The cell therapy composition according to the present invention, wherein the mass volume percentage of hyaluronic acid or a pharmaceutically acceptable salt thereof is 5%, and the density of mesenchymal stem cells is 5 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
In the cell therapy composition of the present invention, the survival rate is greatly reduced due to insufficient nutrition if the density of the stem cells is too high, the survival rate is not maintained due to too low density of the stem cells, and the amount of the preparation required is increased due to too low density of the stem cells. The invention proves that the density of the stem cells is 2 multiplied by 10 through experiments7one/mL to 5X 107The survival rate of stem cells is highest when the cells are cultured per mL.
The compositions of the present invention are aqueous suspensions comprising sodium chloride at a concentration substantially equivalent to physiological saline. The normal saline is sodium chloride solution with osmotic pressure equal to that of animal or human plasma, which is commonly used in physiological experiments or clinic. Therefore, the mesenchymal stem cells in the cell therapy composition of the present invention are basically dispersed in physiological saline, i.e., sodium chloride solution with a mass volume percentage of 0.9%, and the osmotic pressure of the composition is consistent with that of human tissues, and the mesenchymal stem cells are not damaged by using the physiological saline as a solvent.
Hyaluronic acid or its medicinal salt is also called hyaluronic acid and hyaluronic acid, and is a polymer. Is a high-grade polysaccharide composed of units of D-glucuronic acid and N-acetylglucosamine. Hyaluronic acid or its pharmaceutical salt is a main component constituting connective tissues such as human intercellular substance, ocular vitreous body, joint synovial fluid, etc., and plays important physiological functions of water retention, extracellular space maintenance, osmotic pressure regulation, lubrication, and cell repair promotion in vivo. Hyaluronic acid or its pharmaceutically acceptable salts can be classified into: high molecular weight hyaluronic acid or a pharmaceutically acceptable salt thereof, medium molecular weight hyaluronic acid or a pharmaceutically acceptable salt thereof, and low molecular weight hyaluronic acid or a pharmaceutically acceptable salt thereof. Wherein: the molecular weight of the high molecular weight hyaluronic acid or the medicinal salt thereof is 1800000-2200000, and the high molecular weight hyaluronic acid or the medicinal salt thereof is mainly used for preventing dehydration, regenerating tissue cells and tightening skin; the molecular weight of the hyaluronic acid or the medicinal salt thereof is 1000000-1800000, and the hyaluronic acid or the medicinal salt thereof is mainly used for tightening skin and keeping moisture for a long time; the molecular weight of the hyaluronic acid or the medicinal salt thereof with the small molecular weight is 400000-1000000, and the hyaluronic acid or the medicinal salt thereof is mainly used for quickly hydrating cells and keeping the water of the cells. The addition of hyaluronic acid or its medicinal salt in the preparation of mesenchymal stem cells can provide proper matrix environment for adipose-derived stem cells, maintain the survival rate of the mesenchymal stem cells, and facilitate the differentiation of the mesenchymal stem cells into cartilage tissues in the process of treating joint diseases.
The cell therapy composition according to the present invention, wherein the molecular weight of the hyaluronic acid or a pharmaceutically acceptable salt thereof is 400000 to 1000000.
The cell therapy composition according to the present invention, wherein the mesenchymal stem cell is a mesenchymal stem cell of primary to tenth generations.
The cell therapeutic composition according to the present invention, wherein the mesenchymal stem cell may be derived from various known routes, for example, it may be derived from bone marrow, umbilical cord or placenta.
The cell therapy composition according to the present invention, wherein the method for obtaining mesenchymal stem cells is well known, for example, the method described in CN102586184A (2012100446386) for obtaining mesenchymal stem cells from placenta. For example, the method described in CN102660497A (2012101599162) is a method for obtaining mesenchymal stem cells from umbilical cord.
The cell therapeutic composition according to the present invention, wherein said mesenchymal stem cells are obtained by a method comprising the steps of (placenta-derived):
(a) washing the placental lobules sufficiently with PBS buffer to remove residual blood from the placenta;
(b) cutting placenta lobule into pieces, adding PBS buffer solution containing tissue digestive enzyme, and incubating and digesting at 37 deg.C;
(c) filtering the tissue mass with a copper mesh, grinding if necessary to facilitate filtration;
(d) centrifuging the collected filtrate, separating mononuclear cells, suspending the obtained cells in MSC medium, and then adding 5% CO at 37 deg.C2Culturing in an incubator;
(e) after the cells are scattered to form clones, selecting each clone cell, respectively culturing by using an MSC culture medium, and after the cells are fused, digesting and passaging by using pancreatin to obtain the placenta mesenchymal stem cell. Passaging was performed if necessary and/or frozen in liquid nitrogen for future use.
The cell therapy composition according to the present invention, wherein the mesenchymal stem cell is obtained by a method comprising the steps of (umbilical cord-derived):
(1) umbilical cord tissue adherence treatment: taking cell culture plates, spreading the tissue blocks in the plates, and keeping the number of the tissue blocks in each plate at 5-20, so that the tissue blocks are air-dried for 2-50 minutes until the tissue is attached to the plates;
(2) umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; placing the plate in CO2Culturing in a 5% culture box at 37 deg.C, taking out the plate from the culture box when culturing for 3-7 days, and supplementing with appropriate amount of mesenchymal stem cell culture medium; removing the culture medium in the plate on days 9-11, adding a proper amount of fresh mesenchymal stem cell culture medium, and continuously culturing; removing all umbilical cord tissue pieces and continuing culturing on days 11-13; then, the liquid is changed once every 1 to 3 days;
(3) cell passage: when the fusion rate of adherent cells in the dish reaches 50-70%, the adherent cells are separated from the bottom of the dish by using trypLEexpress; centrifuging, removing supernatant, adding mesenchymal stem cell culture medium to resuspend cells, inoculating to a T25 cell culture bottle, and performing passage and amplification culture; and then, changing the liquid every 1 to 3 days until the fusion rate reaches 70 to 90 percent, thus obtaining the umbilical cord mesenchymal stem cells. Passaging was performed if necessary and/or frozen in liquid nitrogen for future use. Detecting one or more of the following items of the obtained mesenchymal stem cells when necessary: cell viability, cell contamination, genetic disease, HLA-ABC/DR match.
The cell therapy composition according to the present invention is prepared by a method comprising the steps of:
dissolving hyaluronic acid or its pharmaceutically acceptable salt and sodium chloride in water, optionally sterilizing the solution to obtain matrix of the preparation;
suspending the prepared mesenchymal stem cells in the matrix, and packaging.
The cell therapy composition according to the present invention is prepared under aseptic conditions.
The cell therapy composition according to the present invention, during the preparation thereof, the temperature of the matrix before mixing with the mesenchymal stem cells is less than 25 ℃.
The cell therapy composition according to the present invention is prepared by mixing the matrix with the mesenchymal stem cells at a temperature of less than 25 ℃.
The preparation method of the cell therapy composition of the mesenchymal stem cells provided by the invention is simple and mild, and the activity of the stem cells can not be damaged, so that the survival rate of the stem cells in the mesenchymal stem cell preparation is improved.
According to a second aspect of the present invention there is provided a method of preparing a cell therapeutic composition useful for cell therapeutic compositions for the treatment of osteoarthritis and cartilage defects, the composition comprising: mesenchymal stem cells (which may be derived from, for example, umbilical cord or placenta), hyaluronic acid or a pharmaceutically acceptable salt thereof, sodium chloride, and water.
The method according to the invention comprises the following steps:
dissolving hyaluronic acid or its pharmaceutically acceptable salt and sodium chloride in water, optionally sterilizing the solution to obtain matrix of the preparation;
suspending the prepared mesenchymal stem cells in the matrix, and packaging.
According to the method of the present invention, the mass volume percentage of the hyaluronic acid or a pharmaceutically acceptable salt thereof in the cell therapy composition is 3% to 5%.
According to the method, the mass volume percentage of the hyaluronic acid or the medicinal salt thereof in the cell therapy composition is 3-4%.
According to the method of the present invention, the mass volume percentage of the hyaluronic acid or a pharmaceutically acceptable salt thereof in the cell therapy composition is 3%.
According to the method of the present invention, in the cell therapy composition, the density of the mesenchymal stem cells is 2 x 107one/mL to 5X 107one/mL.
According to the method of the present invention, in the cell therapy composition, the density of the mesenchymal stem cells is 3 x 107one/mL.
According to the method of the present invention, the mass volume percentage of the sodium chloride in the cell therapy composition is 0.8% to 1.0%.
According to the method of the present invention, the mass volume percentage of the sodium chloride in the cell therapy composition is 0.9%.
According to the method of the invention, in the cell therapy composition, the mass volume percentage of the hyaluronic acid or the medicinal salt thereof is 3%, and the density of the mesenchymal stem cells is 3 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
According to the method of the invention, in the cell therapy composition, the mass volume percentage of the hyaluronic acid or the medicinal salt thereof is 4%, and the density of the mesenchymal stem cells is 5 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
According to the method of the invention, in the cell therapy composition, the mass volume percentage of the hyaluronic acid or the medicinal salt thereof is 5%, and the density of the mesenchymal stem cells is 5 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
According to the method of the present invention, the molecular weight of the hyaluronic acid or a pharmaceutically acceptable salt thereof in the cell therapy composition is 400000 to 1000000.
According to the method of the present invention, in the cell therapy composition, the mesenchymal stem cell is a mesenchymal stem cell of primary to tenth generations.
In the cell therapy composition according to the method of the present invention, the mesenchymal stem cell may be derived from various known sources, for example, it may be derived from bone marrow, umbilical cord or placenta.
According to the method of the present invention, the method for obtaining mesenchymal stem cells in the cell therapy composition is well known, and for example, the method described in CN102586184A (2012100446386) is a method for obtaining mesenchymal stem cells from placenta. For example, the method described in CN102660497A (2012101599162) is a method for obtaining mesenchymal stem cells from umbilical cord.
According to the method of the present invention, in the cell therapy composition, the mesenchymal stem cells are obtained by a method comprising the steps of (placenta-derived):
(a) washing the placental lobules sufficiently with PBS buffer to remove residual blood from the placenta;
(b) cutting placenta lobule into pieces, adding PBS buffer solution containing tissue digestive enzyme, and incubating and digesting at 37 deg.C;
(c) filtering the tissue mass with a copper mesh, grinding if necessary to facilitate filtration;
(d) centrifuging the collected filtrate, separating mononuclear cells, suspending the obtained cells in MSC medium, and then adding 5% CO at 37 deg.C2Culturing in an incubator;
(e) after the cells are scattered to form clones, selecting each clone cell, respectively culturing by using an MSC culture medium, and after the cells are fused, digesting and passaging by using pancreatin to obtain the placenta mesenchymal stem cell. Passaging was performed if necessary and/or frozen in liquid nitrogen for future use.
According to the method of the present invention, in the cell therapy composition, the mesenchymal stem cells are obtained by a method comprising the steps of (umbilical cord-derived):
(1) umbilical cord tissue adherence treatment: taking cell culture plates, spreading the tissue blocks in the plates, and keeping the number of the tissue blocks in each plate at 5-20, so that the tissue blocks are air-dried for 2-50 minutes until the tissue is attached to the plates;
(2) umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; placing the plate in CO2Culturing in a 5% culture box at 37 deg.C, taking out the plate from the culture box when culturing for 3-7 days, and supplementing with appropriate amount of mesenchymal stem cell culture medium; removing the culture medium in the plate on days 9-11, adding a proper amount of fresh mesenchymal stem cell culture medium, and continuously culturing; removing all umbilical cord tissue pieces and continuing culturing on days 11-13; then, the liquid is changed once every 1 to 3 days;
(3) cell passage: when the fusion rate of adherent cells in the dish reaches 50-70%, the adherent cells are separated from the bottom of the dish by using trypLEexpress; centrifuging, removing supernatant, adding mesenchymal stem cell culture medium to resuspend cells, inoculating to a T25 cell culture bottle, and performing passage and amplification culture; and then, changing the liquid every 1 to 3 days until the fusion rate reaches 70 to 90 percent, thus obtaining the umbilical cord mesenchymal stem cells. Passaging was performed if necessary and/or frozen in liquid nitrogen for future use. Detecting one or more of the following items of the obtained mesenchymal stem cells when necessary: cell viability, cell contamination, genetic disease, HLA-ABC/DR match.
According to the process of the present invention, the preparation thereof is carried out under aseptic conditions.
According to the method of the present invention, the temperature of the matrix is less than 25 ℃ before mixing with the mesenchymal stem cells during the preparation process.
According to the method of the present invention, the matrix is mixed with the mesenchymal stem cells at a temperature of less than 25 ℃ during the preparation process.
The preparation method of the cell therapy composition of the mesenchymal stem cells provided by the invention is simple and mild, and the activity of the stem cells can not be damaged, so that the survival rate of the stem cells in the mesenchymal stem cell preparation is improved.
According to any aspect of the invention, wherein the pharmaceutically acceptable salt of hyaluronic acid is sodium hyaluronate.
According to any one of the aspects of the present invention, wherein in the cell therapy composition, maltitol is further included. In one embodiment, the mass volume percentage of maltitol in the cell therapy composition is between 0.2% and 0.5%. In one embodiment, the mass volume percentage of maltitol in the cell therapy composition is between 0.3% and 0.5%. The cell therapy compositions of the present invention can be pre-filled into disposable syringes, typically after formulation, and then cryopreserved to keep the cells viable; the cell therapy composition is then thawed and returned to room temperature when needed for use. However, the present inventors found that the cell therapeutic composition obtained using the existing method, after being freeze-thawed, the viscosity of the composition is significantly increased compared to that before freezing, and caused a problem that smooth injection cannot be performed. The present inventors have surprisingly found that this problem of viscosity change can be overcome when adding a small amount of maltitol to the cell therapy composition, and this excellent technical effect is completely unexpected. For example, assaying the cell therapeutic compositions of the present invention
Viscosity before freezing in a refrigerator at-80 ℃ (M0); then, the cytotherapeutic composition was frozen in a refrigerator at-80 ℃ for 30 days, and then thawed at room temperature, and the viscosity after freezing for 30 days was measured (M1), and the relative viscosity after freezing was calculated by the equation (M1/M0). times.100%, and the results showed that the relative viscosity of all the compositions of examples 1 to 8 of the present invention was in the range of 186 to 203%, the relative viscosity of the compositions obtained by the conventional method (example 3 of CN 105106238A, example 3 of CN 104771414A, example 1 of CN 102670654A) was in the range of 182 to 217%, and the relative viscosity of all the compositions of example 9 of the present invention was in the range of 96 to 113%. The composition to which hyaluronic acid or a pharmaceutically acceptable salt thereof is added generally has a high viscosity, and requires a slow injection due to the high viscosity at the time of injection, and it is apparent that the whole composition of examples 1 to 8 of the present invention and the composition of the prior art show an increase in viscosity after undergoing a freezing process that the cell therapy composition generally needs to undergo, which is disadvantageous for its clinical use; it was also found that these compositions have a tendency to continue to increase in viscosity with prolonged freezing time, but this tendency is not seen with the overall composition of example 9 of the present invention.
The cell therapy composition of mesenchymal stem cells provided by the present invention is preferably an injectable composition, which is in an injection liquid state at normal temperature, the injection solution being suitable for intra-articular administration (percutaneous injection) to the intra-articular of a mammalian subject, preferably a human patient. For example, the cell therapeutic compositions of mesenchymal stem cells provided by the present invention are also suitable for topical administration (transdermal injection into or near an inflamed joint of a mammalian subject, preferably a human patient), wherein the topical administration injection involves the epidermis, muscle or any deep organ.
The present invention provides a cell therapeutic composition of mesenchymal stem cells which, upon intraarticular administration of the pharmaceutical composition to a mammalian subject, particularly a human patient, is capable of ameliorating osteoarthropathy-related symptoms, such as relief of osteoarticular pain, improved joint mobility and/or function, reduced inflammatory fluid accumulation in the joint, reduced time for the first component to separately exert therapeutic activity, increased duration of action of the first component, and reduced joint degeneration or progression thereof which may be caused by the above-mentioned disease or condition.
The present invention also relates to a method for the treatment and/or prevention of a condition selected from the above-mentioned diseases or symptoms, in particular osteoarthritis, comprising the steps of: a sufficient amount of a cell therapeutic composition of mesenchymal stem cells of the invention is administered to a mammalian subject (preferably a human patient) by intra-articular administration, particularly in the knee, hip and spine of the mammalian subject (preferably a human patient).
In the present invention, hyaluronic acid or a pharmaceutically acceptable salt thereof is used as a carrier in the cell therapy composition of mesenchymal stem cells provided by the present invention. Different forms of commercially available hyaluronic acid, such as non-animal stabilized hyaluronic acid and hyaluronan, may be used for the purposes of the present invention. In an exemplary embodiment, those produced by Q-med, Seminariegatan, Uppsala may be usedAnd
non-animal stabilized hyaluronic acid. Hyaluronic acid is in the form of a gel in injections. Hyaluronic acid gels, e.g.
And
the gel particles, are slowly resorbed at the injection site. As the gel breaks down by hydrolysis, water replaces its position. The less concentrated the gel becomes, the more water it can bind. When fully absorbed, the gel disappears unnoticed from the body. With different hyaluronic acid concentrations and gel particle sizes, the absorption rate of the gel at the injection site may be different, and thus the release rate of the growth factor in the injected composition may be different. In this respect, therefore, the hyaluronic acid gel acts as a delayed release carrier or delivery vehicle for the growth factors in the composition for the instantaneous injection, which gradually releases the growth factors over time into the dermis or subcutaneous tissue receiving the injection.
In the above-described steps of the preparation method of the present invention, although the specific steps described therein are distinguished in some detail or in language description from the steps described in the preparation examples of the detailed embodiments below, those skilled in the art can fully summarize the above-described method steps in light of the detailed disclosure throughout the present disclosure.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The cell therapy composition of the mesenchymal stem cells provided by the invention has excellent properties.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it. In the following, the molecular weight of hyaluronic acid/salt is 500000, unless otherwise specified. Hereinafter, the mesenchymal stem cell is a mesenchymal stem cell of the fifth generation, unless particularly stated.
Example 1 compositions of placental MSCs
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cell, 4 × 107Per mL;
4% of sodium hyaluronate;
0.9 percent of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from placenta by the method described in CN102586184A (2012100446386).
Weighing sodium hyaluronate and sodium chloride, adding appropriate amount of water for injection to dissolve, sterilizing at 121 deg.C under high pressure for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 2 compositions of placental MSCs
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cell, 2X 107Per mL;
sodium hyaluronate (molecular weight 400000), 5%;
0.8 percent of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from placenta by the method described in CN102586184A (2012100446386).
Weighing sodium hyaluronate and sodium chloride, adding appropriate amount of water for injection to dissolve, sterilizing at 121 deg.C under high pressure for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 3 placental MSC compositions
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cells, 5 × 107Per mL;
sodium hyaluronate (molecular weight 1000000), 3%;
1.0% of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from placenta by the method described in CN102586184A (2012100446386).
Weighing sodium hyaluronate and sodium chloride, adding appropriate amount of water for injection to dissolve, sterilizing at 121 deg.C under high pressure for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 4 placental MSC compositions
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cells (third generation), 3 × 107Per mL;
hyaluronic acid, 4%;
0.9 percent of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from placenta by the method described in CN102586184A (2012100446386).
Weighing hyaluronic acid and sodium chloride, dissolving with appropriate amount of water for injection, autoclaving at 121 deg.C for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 5 composition of umbilical cord MSCs
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cells (seventh generation), 4X 107Per mL;
4% of sodium hyaluronate;
0.9 percent of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from umbilical cord by the method described in CN102660497A (2012101599162).
Weighing sodium hyaluronate and sodium chloride, adding appropriate amount of water for injection to dissolve, sterilizing at 121 deg.C under high pressure for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 6 composition of umbilical cord MSCs
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cells (tenth generation), 2X 107Per mL;
5% of sodium hyaluronate;
0.8 percent of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from umbilical cord by the method described in CN102660497A (2012101599162).
Weighing sodium hyaluronate and sodium chloride, adding appropriate amount of water for injection to dissolve, sterilizing at 121 deg.C under high pressure for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 7 composition of umbilical cord MSC
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cells (primary), 5 × 107Per mL;
3% of sodium hyaluronate;
1.0% of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from umbilical cord by the method described in CN102660497A (2012101599162).
Weighing sodium hyaluronate and sodium chloride, adding appropriate amount of water for injection to dissolve, sterilizing at 121 deg.C under high pressure for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 8 composition of umbilical cord MSCs
The proportion of the ingredients of the final product is as follows:
mesenchymal stem cell, 4 × 107Per mL;
hyaluronic acid, 4%;
0.9 percent of sodium chloride;
water for injection and the rest.
The preparation method comprises the following steps:
mesenchymal stem cells were obtained from umbilical cord by the method described in CN102660497A (2012101599162).
Weighing hyaluronic acid and sodium chloride, dissolving with appropriate amount of water for injection, autoclaving at 121 deg.C for 15 min, and cooling; mixing the mesenchymal stem cell suspension with the aqueous solution of sodium hyaluronate and sodium chloride, adding water to full amount, mixing uniformly, subpackaging into pre-filled syringes, and freezing in a refrigerator at-80 deg.C.
Example 9: 9 compositions were obtained according to the formulation and method of examples 1-8, respectively, except that maltitol was added simultaneously with the addition of sodium chloride, so that the mass volume percentage of maltitol in the final composition was 0.2% to 0.5% (0.4% according to examples 1, 5, 0.3% according to examples 2, 6, 0.5% according to examples 3, 7, and 0.2% according to examples 4, 8).
The total compositions of examples 1-9 above, which had a cell viability in the range of 93-96% at 6 hours and 83-87% at 24 hours, before cryopreservation, showed excellent cell viability.
The above-mentioned total compositions of examples 1 to 9, which were frozen in a refrigerator at-80 ℃ for 10 days and thawed at ordinary temperature to have a cell viability within the range of 86 to 91%, were determined to have a cell viability within the range of 78 to 81% for 6 hours, showed excellent cell viability.
All cells of examples 1-9 above were tested for cell surface markers by flow cytometry and met The following requirements in marker assays according to The guidelines for assaying mesenchymal stem cells published in 2006 in The U.S. "The International Society for Cellular Therapy (ISCT)": positive for expression of markers CD73, CD90, CD 105; markers CD11b, CD34, CD45, CD19, HLA-DR expression negative. The results of each marker detection were determined to meet the standard requirements for all cells of examples 1-9 above.
Claims (20)
1. A cell therapy composition for the treatment of osteoarthritis and cartilage defects, the composition comprising: mesenchymal stem cellCell, hyaluronic acid or a pharmaceutically acceptable salt thereof, sodium chloride, maltitol and water; wherein the density of the mesenchymal stem cells is 2 × 107one/mL to 5X 107The hyaluronic acid or the medicinal salt thereof accounts for 3 to 5 percent of the total volume of the hyaluronic acid or the medicinal salt thereof, 0.8 to 1.0 percent of the mass volume of the sodium chloride and 0.2 to 0.5 percent of the mass volume of the maltitol.
2. The cytotherapeutic composition according to claim 1, wherein the mass volume percentage of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 3-4%.
3. The cytotherapeutic composition according to claim 1, wherein the mass volume percentage of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 3%.
4. The cell therapy composition according to claim 1, the mesenchymal stem cells having a density of 3 x 107one/mL.
5. The cytotherapeutic composition according to claim 1, wherein the mass volume percentage of sodium chloride is 0.9%.
6. The cell therapy composition according to claim 1, wherein the mass volume percentage of hyaluronic acid or a pharmaceutically acceptable salt thereof is 3%, and the density of mesenchymal stem cells is 3 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
7. The cell therapy composition according to claim 1, wherein the mass volume percentage of hyaluronic acid or a pharmaceutically acceptable salt thereof is 4% and the density of mesenchymal stem cells is 5 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
8. The cell therapy composition according to claim 1, wherein the mass volume percentage of hyaluronic acid or a pharmaceutically acceptable salt thereof is 5%, and the density of mesenchymal stem cells is 5 x 107The mass volume percentage of sodium chloride per mL is 0.9%.
9. The cell therapy composition according to claim 1, wherein the molecular weight of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 400000 to 1000000.
10. The cell therapy composition according to claim 1, the mesenchymal stem cells being primary to tenth generation mesenchymal stem cells.
11. The cell therapy composition according to claim 1, wherein said mesenchymal stem cells are derived from bone marrow, umbilical cord or placenta.
12. The cytotherapeutic composition according to claim 1, which is prepared by a method comprising the steps of:
dissolving hyaluronic acid or its pharmaceutically acceptable salt, sodium chloride and maltitol in water, optionally sterilizing the solution to obtain matrix of the preparation;
suspending the prepared mesenchymal stem cells in the matrix, and packaging.
13. The cell therapy composition according to claim 12, which is prepared under aseptic conditions.
14. The cell therapy composition according to claim 12, wherein the temperature of the matrix is less than 25 ℃ prior to mixing with the mesenchymal stem cells during preparation thereof.
15. The cell therapy composition according to claim 12, wherein the matrix is mixed with the mesenchymal stem cells at a temperature of less than 25 ℃ during the preparation process.
16. The cell therapy composition according to any one of claims 1 to 15, said pharmaceutically acceptable salt of hyaluronic acid being sodium hyaluronate.
17. A method of preparing a cell therapy composition according to any one of claims 1 to 16, comprising the steps of:
dissolving hyaluronic acid or its pharmaceutically acceptable salt, sodium chloride and maltitol in water, optionally sterilizing the solution to obtain matrix of the preparation;
suspending the prepared mesenchymal stem cells in the matrix, and packaging.
18. The method according to claim 17, wherein the preparation is carried out under aseptic conditions.
19. The method of claim 17, wherein the temperature of the matrix is less than 25 ℃ prior to mixing with the mesenchymal stem cells during preparation.
20. The method of claim 17, wherein the matrix is mixed with the mesenchymal stem cells at a temperature of less than 25 ℃.
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| CN107693844A (en) * | 2016-08-07 | 2018-02-16 | 李媚 | A kind of composition gels and application |
| CN106421917A (en) * | 2016-11-08 | 2017-02-22 | 华南生物医药研究院 | Method for preparing composition for repairing cartilage injuries |
| CN107375329A (en) * | 2017-06-12 | 2017-11-24 | 佛山科学技术学院 | One kind treats arthritic stem cell medicine of dog and preparation method thereof |
| CN108126187A (en) * | 2018-01-19 | 2018-06-08 | 深圳光彩生命工程技术有限公司 | A kind of composition and preparation method |
| KR20210008873A (en) * | 2018-06-05 | 2021-01-25 | 메디포스트(주) | Pharmaceutical composition for preventing or treating inflammatory diseases comprising mesenchymal stem cells as an active ingredient |
| CN112237589A (en) * | 2019-07-19 | 2021-01-19 | 丰泽康生物医药（深圳）有限公司 | Umbilical cord mesenchymal stem cell active matter and preparation method and application thereof |
| CN110755452B (en) * | 2019-11-23 | 2021-07-06 | 博雅干细胞科技有限公司 | Use of stem cells for treating osteoarthritis |
| CN110840914B (en) * | 2019-11-23 | 2021-07-06 | 博雅干细胞科技有限公司 | Method for alleviating or improving vascular disorders using cell therapeutic agent |
| CN110755451B (en) * | 2019-11-23 | 2021-07-06 | 博雅干细胞科技有限公司 | Mesenchymal stem cell composition for treating osteoarthritis and application thereof |
| CN110882276B (en) * | 2019-11-23 | 2021-07-06 | 博雅干细胞科技有限公司 | Cell therapy compositions and methods for treating vascular disorders |
| CN110974847B (en) * | 2019-12-25 | 2021-07-06 | 博雅干细胞科技有限公司 | Method for treating lower limb ischemic diseases by using stem cells |
| CN111012801B (en) * | 2019-12-25 | 2021-07-06 | 博雅干细胞科技有限公司 | Stem cell therapeutic agent for treating ischemic diseases of lower limbs and use thereof |
| CN112451482A (en) * | 2020-11-30 | 2021-03-09 | 深圳博雅感知药业有限公司 | Method of treating osteoarthritis |
| CN112386611A (en) * | 2020-11-30 | 2021-02-23 | 深圳博雅感知药业有限公司 | Mesenchymal stem cell composition for treating osteoarthritis and preparation method and application thereof |
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