Patent Application
20250161364
The present disclosure relates to the fields of tissue engineering and regenerative medicine, and in particular to animal origin-free cell sheet culture supernatant and preparation methods and use thereof.
Cell sheets represent a more efficient method of stem cell transplantation than previous transplantation methods involving the injection of cells with stem cell suspensions. Cell sheets can effectively prevent the loss of stem cells during the transplantation process and improve the efficiency of stem cell transplantation. In addition, the preparation process of cell sheets does not involve using of enzymes and analogues for cell digestion, which effectively avoids the destruction of the extracellular matrix and reduction of cell functions caused by enzyme digestion. Cell sheet transplantation allows stem cells to better perform their functions in vivo.
The preparation process of mesenchymal stem cell sheets usually involves digesting the subcultured mesenchymal stem cells into single cells with biological enzymes, followed by washing to remove the residue of mesenchymal stem cell culture medium, then resuspending the cells in sheet-forming medium, and inoculating the cells into a pre-coated temperature-sensitive culture dish. Under the culture conditions of 37° C., the cells grew adherently in the culture dish and proliferated to confluence. After the culture is completed, the culture temperature is lowered, and the cells will automatically detach from the temperature-sensitive surface in the form of sheets, and the cell sheets can be harvested.
Cell sheets are film-like structures composed of a single layer or multiple layers of cells. The sheet-forming medium and the matrix coated in temperature-sensitive culture dishes used in the preparation of mesenchymal stem cell sheets remain in the intercellular spaces and on the surface, and are difficult to be completely washed away by external force. The sheet-forming medium used in the sheet preparation methods that have been reported so far is divided into three categories: serum-containing medium, commercially available serum-free medium, and self-prepared serum-free medium. In the traditional serum sheet-forming system, serum can promote the attachment of cells to the surface of temperature-sensitive culture dishes and promote adhesion between cells. However, the bovine serum in the serum-containing medium contains bovine serum albumin, a heterologous macromolecule allergic component, which is not safe enough for clinical use. Due to the lack of serum in the serum-free culture system, it is difficult for the cells to adhere to the wall, and are unable to form a sheet but appear in a broken state, or the sheet is easily broken due to insufficient toughness during the sheet obtaining process (
In addition, the current coating matrix in the sheet preparation process is scientific research grade reagents such as fibronectin and laminin. These reagents do not meet GMP standards during the production process.
Therefore, there is an urgent need to develop a method for preparing mesenchymal stem cell sheets with good shape and toughness using a safer sheet-forming medium and/or safer coating matrixes, so as to obtain a mesenchymal stem cell sheet that is safer, easier to store and use.
In addition, the umbilical cord mesenchymal stem cells may secrete a variety of growth factors, cytokines, microRNAs, messenger RNAs, and the like (hereinafter collectively referred to as secretions of umbilical cord mesenchymal stem cells) into the culture medium during culturing. The secretions of umbilical cord mesenchymal stem cells have effects such as inhibiting inflammatory response and promoting tissue self-repair. The umbilical cord mesenchymal stem cell supernatant, which riches in the secretions of umbilical cord mesenchymal stem cells, may be collected and purified for the treatment of an autoimmune system disease, or tissue and organ damage diseases, and repair and anti-aging of skin.
However, the human umbilical cord mesenchymal stem cell culture supernatant currently commercially available or reported has uneven functions, contains bovine serum albumin, exogenous growth factors or insulin and a variety of components that may cause allergic reactions or hypoglycemia risk, and has a limited repair and anti-inflammatory effect.
Compared with the conventional human umbilical cord mesenchymal stem cell supernatant, the human umbilical cord mesenchymal stem cell sheet will secrete more kinds and quantities of growth factors, cytokine, microRNA and messenger RNA into the culture liquid during the culture and preparation. Therefore, the human umbilical cord mesenchymal stem cell sheet culture supernatant will have a better therapeutic and repair effect.
In addition, factors secreted by mesenchymal stem cells are affected by cell state and the composition of the culture medium. Under a stress state, umbilical cord mesenchymal stem cells can secrete more repair factors.
Therefore, there is an urgent need to develop a simple and safe human umbilical cord mesenchymal stem cell sheet culture medium and culture method, wherein the cells are in a stress state and thus secrete more repair factors, thereby improving the safety and effectiveness of the human umbilical cord mesenchymal stem cell sheet culture supernatant.
In order to solve the above technical problems, the present disclosure provides a production method for mesenchymal stem cell sheet products. The sheet-forming medium used in this method has simple ingredients, including a basal medium and a binder (such as human serum albumin (such as pharmaceutical grade)), which does not contain any animal-derived components (such as serum) and exogenous growth factors. In addition, during the preparation of cell sheets, adhesion factors (such as human fibrinogen) can be used as a coating matrix.
The mesenchymal stem cell sheet product prepared by the above method of the present disclosure is safer and does not contain animal-derived components (such as serum) and exogenous growth factor residues (such as bovine serum albumin residues that meet drug standards), and the medium formula used in the sheet formation process is simple. In addition, the mesenchymal stem cell sheet product of the present disclosure has a certain degree of toughness and can be folded and flattened in appropriate culture medium, buffers or preservation solutions. The sheet products can still maintain good sheet shape after being stored in a specific preservation solution at 4° C. for 24 hours, with the cell survival rate as high as over 70%; the cells can secrete high levels of pro-angiogenic factors and anti-inflammatory factors, inhibit lymphocyte proliferation and inflammatory factor secretion, and can be used clinically for a variety of diseases, including autoimmune system diseases, organ damage diseases, rejection and GVHD during organ transplantation, etc.; at the same time, the cells can maintain stem cell properties, have specific surface markers, and have the ability of three-direction differentiation when induced, and can be used clinically to repair damaged tissues.
Accordingly, the present disclosure relates to the following aspects.
In the first aspect, the present disclosure relates to a method of preparing a cell sheet, the method comprising the following steps:
The term “temperature-sensitive culture dish” or “thermosensitive culture dish” as used herein refers to a culture dish whose surface is coated with a layer of temperature-sensitive polymer material. The stretching states of the molecular chain segments of the polymer material are different at different temperatures to show hydrophilicity or hydrophobicity, so that the hydrophilicity and hydrophobicity of the polymer material can change with changes in external temperature. When the surface of the temperature-sensitive culture dish becomes hydrophilic, the adhesion to the cells and the extracellular matrix secreted by the cells become poor, and the cells will fall off in layers. In a specific application, when the temperature is lowered below the low critical dissolution temperature of the polymer substance, the surface of the temperature-sensitive culture dish becomes hydrophilic, so that the cells will fall off in layers.
In some embodiments, the cell sheet is a stem cell sheet, such as a mesenchymal stem cell sheet.
In some embodiments, the matrix is fibronectin, laminin, gelatin, collagen, vitronectin, or human fibrinogen. In some embodiments, the matrix is gelatin. In some embodiments, the concentration of gelatin (w/w) is 0.01-0.5%, such as 0.05-0.2%, such as 0.1%. In some embodiments, the matrix is vitronectin. In some embodiments, the concentration of vitronectin is 1-20 μg/mL, such as 5-15 μg/mL, such as 10 μg/mL. In some embodiments, the matrix is poly-D-lysine (PDL). In some embodiments, the concentration of PDL is 0.01-0.5 mg/mL, such as 0.05-0.2 mg/mL, such as 0.1 mg/mL. Coating a temperature-sensitive culture dish with the above matrix enables mesenchymal stem cells to attach to the culture dish.
In a preferred embodiment, the matrix is human fibrinogen. In some embodiments, the concentration of human fibrinogen is 0.1-10 mg/mL, such as 0.2-5 mg/mL, such as 1-2.5 mg/mL.
In some embodiments, in step b, the basal medium in the sheet-forming medium can be selected from the group consisting of DMEM (high glucose), DMEM (low glucose), RPMI1640, a-MEM, DMEM/F12, and F12. In a preferred embodiment, the basal medium in the sheet-forming medium is a-MEM.
In some embodiments, in step b, the concentration of human serum albumin in the sheet-forming medium is 0.1-10%, preferably 0.1-5%, such as 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.
In some embodiments, in step b, the sheet-forming medium further comprises non-essential amino acids (glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline, L-serine) and/or L-glutamine.
In some embodiments, the concentration of L-glutamine in the sheet-forming medium used in step b is 0.5 mM to 4 mM, preferably about 2 mM.
In some embodiments, the concentration of each of the non-essential amino acids glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline and L-serine is 50 μM to 200 μM, preferably about 100 μM.
In some embodiments, in step a, the mesenchymal stem cells are cultured and passaged with a serum-containing medium (such as a medium containing fetal bovine serum).
In some embodiments, in step a, the mesenchymal stem cells are cultured and passaged using a serum-free medium. In some embodiments, the serum-free culture system includes the use of a basal medium selected from the group consisting of: RPMI1640, DMEM, α-MEM, DMEM/F12, and F12 serum-free medium, and the medium is supplemented with one or more additives selected from the group consisting of: vitamin C, sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin (plant expression), progesterone, putrescine, biotin, sodium pyruvate, ethanolamine, carnitine, amino acids, vitamins, glutathione, linoleic acid and linolenic acid.
In other embodiments, the serum-free culture system includes use of a commercial medium selected from: CTS™ Stem Pro™ MSC SFM, MesenCult™-ACF Medium, MesenCult™-ACF Plus Medium, and MesenCult™-XF medium.
In some embodiments, the method further includes a step of washing the cells after step a and before step b.
In some embodiments of the above methods, the mesenchymal stem cells can be derived from a tissue selected from the group consisting of: amniotic fluid, amnion, chorion, chorionic villi, decidua, placenta, umbilical cord blood, umbilical cord, adult bone marrow, adult peripheral blood and adult adipose tissue.
In some embodiments, the mesenchymal stem cells can be selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells, as well as mesenchymal stem cells from other sources known in the art. In a preferred embodiment, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the method further comprises a step of obtaining mesenchymal stem cells from the umbilical cord before step a).
In some embodiments, obtaining umbilical cord mesenchymal stem cells from the umbilical cord comprises the following steps:
In some embodiments, the coating time of the temperature-sensitive culture dish is from 1 hour to 7 days, preferably from 1 to 36 hours, and most preferably from 2 to 18 hours. In some embodiments, the coating temperature is 2-37° C., preferably 37° C.
In some embodiments, the cells are inoculated into a temperature-sensitive culture dish in an inoculation amount as shown below.
When inoculated into a 100 mm culture dish, the number of inoculated cells is 1×107 to 10×107, such as 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107 or 10×107. The volume of the culture medium in which the inoculated cells are suspended is 15-30 mL, such as 15, 20, 25 or 30 mL.
When inoculated into a 35 mm culture dish, the number of inoculated cells is 1×106 to 30×106, such as 2×106 to 20×106, 5×106 to 15×106, or 8×106 to 12×106, such as 8×106, 9×106, 10×106, 11×106 or 12×106. The volume of the culture medium in which the inoculated cells are suspended is 1.5-5 mL, such as 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mL.
When inoculated into a 60 mm culture dish, the number of inoculated cells is 3×106 to 75×106, such as 5×106 to 60×106, 10×106 to 50×106, 15×106 to 40×106, 20×106 to 30×106 or 20×106 to 25×106, such as 20×106, 20.5×106, 21×106, 21.5×106, 22×106, 22.5×106, 23×106, 23.5×106, 24×106, 24.5×106 or 25×106. The volume of culture medium in which the inoculated cells are suspended is 3-12.5 mL, such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6, 5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 or 12.5 mL.
In the method of the present disclosure, the mesenchymal stem cells are detached from the temperature-sensitive culture dish by lowering the temperature, thereby forming a mesenchymal stem cell sheet. For example, when the culture temperature is about 37° C., the mesenchymal stem cells are detached from the temperature-sensitive culture dish by lowering the temperature to 4-32° C.
In a second aspect, the present disclosure relates to a cell sheet prepared by the methods of the present disclosure.
The residual amount of bovine serum albumin in the cell sheet obtained by the above preparation method complies with the human drug standard, and the content is ≤1 ng/cm2, the residual amount of human serum albumin is 10 ng/cm2-15000 ng/cm2, and the residual amount of human fibrinogen is 0.5 ng/cm2-5 ng/cm2.
The mesenchymal cell sheet obtained by the above preparation method can secrete a large amount of HGF, VEGF and IL-8 pro-angiogenic function-related factors, secrete a large amount of anti-inflammatory related factors such as IL-6 and IL-8; and has the function to inhibit lymphocyte Th1 subtype as well as inhibit lymphocyte proliferation and lymphocyte TNFα secretion.
In a third aspect, the present disclosure relates to the use of the mesenchymal stem cell sheet obtained by the above preparation method for regulating an inflammatory response or treating an autoimmune system disease in a subject, or the use in the manufacture of a medicament for regulating inflammatory response or treating autoimmune system diseases.
In some embodiments, the autoimmune disease is rheumatoid, allergy, lupus erythematosus, etc.
In a fourth aspect, the present disclosure relates to the use of the mesenchymal stem cell sheet obtained by the above preparation method for damage repair of damaged tissue in a subject, or the use in the manufacture of a medicament for repairing damaged tissue in a subject.
In some embodiments, the damaged tissue is a tissue of heart, liver, pancreas, uterus, or another tissue.
The present invention further relates to an animal origin-free human umbilical cord mesenchymal stem cell sheet culture supernatant and a preparation method thereof. The animal origin-free human umbilical cord mesenchymal stem cell sheet medium, i.e. sheet-forming medium used in the method has simple ingredients, including a basal medium and a binder (such as human serum albumin (such as pharmaceutical grade)), which does not contain any animal-derived components and exogenous growth factors (such as insulin). Furthermore, the method is related to preparing in a cellular stress state.
The human umbilical cord mesenchymal stem cell sheet culture supernatant product prepared by the above method of the present disclosure is safer, and does not contain animal-derived ingredients and exogenously added growth factors, such as insulin. The human umbilical cord mesenchymal stem cell sheet culture supernatant of the present invention can contain high levels of pro-angiogenic factors and anti-inflammatory factors, inhibit lymphocyte proliferation and inflammatory factor secretion. The culture supernatant can be used clinically for a variety of diseases, including skin disorders, autoimmune system diseases, etc., and can also be used clinically for damage repair of a damaged tissue. In addition, the human umbilical cord mesenchymal stem cell sheet culture supernatant of the present invention is convenient to use, and is ready to use after thawing without formulation.
Accordingly, the present disclosure further relates to the following aspects.
In a fifth aspect, the disclosure relates to a method of preparing a cell sheet culture supernatant, the method comprising the following steps of:
In some embodiments, the cell sheet culture supernatant may be a stem cell sheet culture supernatant, for example, a mesenchymal stem cell sheet culture supernatant. In some preferred embodiments, the cell sheet culture supernatant is a human umbilical cord mesenchymal stem cell sheet culture supernatant.
As used herein, the term “a human umbilical cord mesenchymal stem cell sheet culture supernatant” refers to a relatively pure supernatant obtained by removing cells, cell debris and other solid materials in the human umbilical cord mesenchymal stem cell sheet medium, i.e. sheet-forming medium by centrifugation or other separation techniques during the preparation of a human umbilical cord mesenchymal stem cell sheet.
As used herein, the term “stress state” refers to a stress state of cells due to high cell seeding density, simple medium composition, and less abundant nutrient content. Under a cellular stress state, umbilical cord mesenchymal stem cells will secrete more repair factors.
In some embodiments, in step b, the cell transferred may be at a density of 0.3-3E6/cm2, preferably 0.5-1.5 E6/cm2, for example 0.5 E6/cm2, 0.6 E6/cm2, 0.7 E6/cm2, 0.8 E6/cm2, 0.9 E6/cm2, 1 E6/cm2, 1.1 E6/cm2, 1.2 E6/cm2, 1.3 E6/cm2, 1.4 E6/cm2 or 1.5 E6/cm2, more preferably 1 E6/cm2.
In some embodiments, in step b, the basal medium in the sheet-forming medium may be selected from DMEM (high glucose), DMEM (low glucose), 1640, α-MEM, DMEM/F12, or F12. In a preferred embodiment, the basal medium in the sheet-forming medium is α-MEM and DMEM/F12. In a more preferred embodiment, the basal medium in the sheet-forming medium is DMEM/F12.
In some embodiments, in step b, the volume of the sheet-forming medium may be 0.2-1 mL/cm2, preferably 0.25-0.75 mL/cm2, for example 0.25 mL/cm2, 0.3 mL/cm2, 0.35 mL/cm2, 0.4 mL/cm2, 0.45 mL/cm2, 0.5 mL/cm2, 0.55 mL/cm2, 0.6 mL/cm2, 0.65 mL/cm2, 0.7 mL/cm2 or 0.75 mL/cm2, more preferably 0.4 mL/cm2.
In some embodiments, in step b, the concentration of human serum albumin in the sheet-forming medium may be 0.5-10%, preferably 0.5-5%, for example 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, more preferably 2%.
In some embodiments, in step b, the sheet-forming medium may further comprise a vitamin, for example, vitamin C.
In some embodiments, in step b, the concentration of the vitamin C in the sheet-forming medium may be 5-500 μg/mL, preferably 20-100 μg/mL, for example 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL or 100 μg/mL, more preferably 60 μg/mL.
In some embodiments, in step b, the sheet-forming medium further comprises non-essential amino acids (glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline, L-serine) and/or L-glutamine.
In some embodiments, the concentration of the L-glutamine in the sheet-forming medium used in step b is from 0.5 mM to 4 mM, preferably about 2 mM.
In some embodiments, the concentration of each of glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline, and L-serine among the non-essential amino acids in the sheet-forming medium used in step b is 5 mM to 20 mM, preferably about 10 mM.
In some embodiments, the method may further comprise a step of washing the cells after step a and before step b.
In some embodiments, the method may further comprise, before step a), a step of obtaining mesenchymal stem cells from an umbilical cord.
In some embodiments, in step c, the collecting may be performed at 8-48 hours, preferably 8-24 hours, such as 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours, more preferably 12 hours after culturing the cells. In some embodiments, the collecting is performed by centrifugation.
In some embodiments, the method further comprises, after step c, a step of subpackaging and/or packaging the supernatant, for example, into a vial or an ampoule.
In a sixth aspect, the present disclosure relates to a cell sheet culture supernatant prepared by the method of the present disclosure.
The human umbilical cord mesenchymal stem cell sheet culture supernatant obtained by the above preparation method contains a larger amount of pro-angiogenic function-related factors such as HGF, VEGF, IL-8; and has the function to better, inhibit lymphocyte Th1 subtype, as well as inhibit lymphocyte proliferation and TNFα secretion by lymphocytes.
In some embodiments, the cell sheet culture supernatant may comprise increased HGF, IL-8, and/or VEGF.
In some embodiments, the concentration of the HGF may be at least 11,000 pg/mL, i.e. at least 11 ng/ml, such as at least 12 ng/ml, at least 15 ng/ml, at least 18 ng/ml, at least 21 ng/mL, at least 24 ng/mL, preferably at least 25 ng/ml, at least 30 ng/ml, at least 35 ng/ml, at least 40 ng/ml, at least 45 ng/ml, more preferably at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/mL.
In some embodiments, the concentration of the IL-8 may be at least 20 ng/ml, such as at least 25 ng/ml, at least 30 ng/mL, at least 35 ng/mL, preferably at least 80 ng/mL, at least 85 ng/mL, more preferably at least 90 ng/ml, at least 92 ng/ml, at least 94 ng/ml, at least 96 ng/ml, at least 98 ng/mL, at least 100 ng/ml.
In some embodiments, the concentration of the VEGF may be at least 40 pg/mL, i.e. at least 0.04 ng/ml, preferably at least 1 ng/mL, at least 1.5 ng/ml, at least 2 ng/mL, at least 2.5 ng/ml, at least 3 ng/ml, at least 3.5 ng/mL, more preferably at least 3 ng/mL, at least 4 ng/ml, at least 5 ng/ml, at least 6 ng/ml, at least 7 ng/ml, at least 8 ng/ml, at least 9 ng/ml, at least 10 ng/mL.
In a seventh aspect, the present disclosure relates to a formulation comprising a mesenchymal stem cell sheet culture supernatant obtained by the preparation method described above.
In some embodiments, the formulation may be applied topically or by injection, for example, to the skin. In some preferred embodiments, the injection may be a microneedle injection.
In some embodiments, the formulation may be a cryopreserved formulation for injection.
In some embodiments, the formulation may be a liquid formulation or a lyophilized formulation. In some embodiments, the liquid formulation may be packaged into a vial or an ampoule. In some embodiments, the lyophilized formulation may be packaged into a vial.
In an eighth aspect, the present disclosure relates to use of the mesenchymal stem cell sheet culture supernatant obtained by the preparation method described above for treating an autoimmune system disease, or in the manufacture of a medicament for modulating an inflammatory response or treating an autoimmune system disease, in a subject. The present disclosure also relates to a method for modulating an inflammatory response or treating an autoimmune system disease in a subject, comprising administrating the cell sheet culture supernatant obtained by the preparation method described above.
In some embodiments, the autoimmune disease is rheumatoid, allergic, lupus erythematosus, and the like.
In a ninth aspect, the present disclosure relates to use of the mesenchymal stem cell sheet culture supernatant obtained by the preparation method described above for performing damage repair of a damaged tissue in a subject, or in the manufacture of a medicament for performing damage repair of a damaged tissue in a subject. The present disclosure also relates to a method for performing damage repair of a damaged tissue in a subject, comprising administrating the cell sheet culture supernatant obtained by the preparation method described above.
In some embodiments, the damaged tissue is a tissue of myocardium, liver, kidney, skin, uterus, or other tissue.
In a tenth aspect, the present disclosure relates to use of the mesenchymal stem cell sheet culture supernatant obtained by the preparation method described above for preventing and/or treating a skin disorder, or in the manufacture of a medicament for preventing and/or treating a skin disorder, in a subject. The present disclosure also relates to a method for preventing and/or treating a skin disorder in a subject, comprising administrating the cell sheet culture supernatant obtained by the preparation method described above.
In some embodiments, the skin disorder is dryness, pigmentation such as spots, wrinkles, acne including acne marks, allergies, aging, and the like.
The invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the invention. The scope of the present invention is defined by the appended claims. A person of ordinary skill in the art can clearly understand the scope defined by the claims based on this description and common knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.
The collected newborn umbilical cord was washed with physiological solution, the arteries, veins, and adventitia were removed, and the Wharton's jelly was isolated, cut into small tissue pieces of 0.1˜2 mm, and the tissue pieces were plated evenly into a culture container coated with matrix, with the distance between tissue blocks being 2˜30 mm. Then the culture container was placed in a cell culture incubator. After 2 to 7 days, an appropriate amount of complete culture medium (α-MEM supplemented with 20 IU/mL bFGF and 10% fetal bovine serum) was added to cover the tissue block. The umbilical cord mesenchymal stem cells migrated out can be observed after 8 to 21 days.
When the cells were to 70˜100% confluence, the tissue pieces were removed and the cells were subcultured. The cells were separated from the culture container by trypsinization and cell scraping. Then, the cells were dispersed in the culture medium by stirring, vortexing, etc., and the cells were seeded into a culture container at a density of 500 to 100,000 cells/cm2. An appropriate amount of α-MEM supplemented with 20 IU/mL bFGF and 10% fetal bovine serum was added. The culture medium was replaced with an appropriate amount of fresh culture medium every 1 to 5 days according to the cell growth status. When the cells were expanded to 70 to 100% confluence, cell passage was repeated. The cultured umbilical cord mesenchymal stem cells grew adherently in a fibrous form with uniform morphology.
Before preparing the umbilical cord mesenchymal stem cell sheets, 0.1-5 mg/mL human fibrinogen was firstly used to coat the temperature-sensitive culture dish, which helps the mesenchymal stem cells adhere to the inner surface of the culture dish. The temperature-sensitive culture dish was coated at 37° C. for 2 hours, and then the coating solution was discarded.
The old culture medium was removed from the umbilical cord mesenchymal stem cell culture medium obtained in Example 1, then the cells were washed with PBS for 1-3 times, and the digestive enzyme TryPLE (purchased from Life technologies, Catalog number 12604021) was added for digestion until the cells were in the single-cell state. PBS was added to stop or reduce the digestion of the enzyme. After centrifugation, the supernatant was discarded to obtain a cell pellet.
The digested cells were washed with PBS buffer for 3 times at a washing density of less than or equal to 5×10{circumflex over ( )}5 cells/mL.
The cells were washed to remove the residue of umbilical cord mesenchymal stem cell culture medium, and then sheet-forming medium (α-MEM, containing 50 μM to 200 μM non-essential amino acids, 0.5 mM to 4 mM L-glutamine, 0.1%-10% human serum albumin) was added to resuspend the cells, then the cells were preheated to 37° C. and inoculated in a temperature-sensitive culture dish that has been coated with a matrix facilitating cell adhesion. The cells grew adherently in the culture dish.
When the culture medium was lowered to room temperature, the cells would automatically detach from the culture dish in the form of single-layer films, and the cell sheets could be harvested. The temperature-sensitive culture dish was taken out from the incubator, the culture medium was removed, and the sheet-forming culture medium pre-cooled at 4° C. was added. After 0.25-1 hour, it was observed that the cell sheets began to detach from the edge of the culture dish.
The results showed that coating human fibrinogen on a temperature-sensitive culture dish in advance can promote cell adhesion and sheet formation. When the coating matrix was not used for coating, the cell sheet detached in advance and the sheet was in a poor condition (
The mesenchymal stem cell sheet obtained above was fixed with 4% paraformaldehyde, and then the cell sheet was prepared into tissue sections by the frozen section method for immunofluorescence staining (
By paraffin embedding and sectioning, the cell sheets were made into tissue sections for H&E staining (
The results show that the cell sheets are composed of simple cells and extracellular matrix components secreted by the cells, with a thickness of about 10 cells stacked. The extracellular matrix components can be stained by fibronectin and integrin-β1.
For the three batches of sheets prepared (sample 1, sample 2, and sample 3) (
The steps for the apoptosis test were as follows: the umbilical cord mesenchymal stem cell sheets were digested into single cells and resuspended to a cell concentration of 2˜5×105/ml. Then the Annexin V/PI apoptosis staining kit was used for staining according to the instructions. The steps were as follows: 1. washing the cells twice with Binding Buffer; 2. resuspending the cells with 195 μL Binding Buffer and adding 5 μL Annexin V staining solution. Incubating the cells in the dark for 30 minutes. 3. washing the cells twice with Binding Buffer and resuspending the cells in 190 μL Binding Buffer. Adding 10 μL PI staining solution. 4. Testing the cells after staining with flow cytometry. Those cells that are negative for Annexin V/PI staining are normal cells without apoptotic.
In addition, the residues of substances introduced during the preparation of cell sheets were tested.
The mesenchymal stem cell sheets were removed from the protective solution, and digested with 1 mL TryPLE at 37° C. for 3 minutes. During the digestion process, 50 μL of 20% human serum albumin injection was added to ensure that bovine serum albumin was not destroyed during the digestion process. Then 1 mL of sheet-forming medium was added to terminate the digestion, and the sheets were pipetted into single cells with a 1 mL pipette, and centrifuged at 300 g for 5 minutes. Afterwards, the supernatant was taken, and the content of bovine serum albumin was tested with an anti-bovine serum albumin antibody by ELISA. See
The mesenchymal stem cell sheets were moved from the protective solution, digested with 3 mL TryPLE at 37° C. for 3 minutes, then the digestion was terminated with 7 mL PBS. The sheets were pipetted into single cells with a 1 mL pipette, and centrifuged at 300 g for 5 minutes. Afterwards, the supernatant was taken and the content of human serum albumin was tested with anti-human serum albumin HSA antibody by ELISA. See
The mesenchymal stem cell sheets were removed from the protective solution, lysed with 5 mL of lysis solution (physiological saline+0.5% Triton+2.5% protease inhibitor) at 4° C. for 20 minutes, centrifuged at 12000 g at 4° C. for 20 minutes. Afterwards, the supernatant was taken and the content of human fibrinogen was tested with an anti-human fibrinogen antibody by ELISA. See
The cell culture medium was obtained as follows: replacing the cell culture medium in culture after subculture and before preparing the sheets with fresh medium (α-MEM supplemented with 20 IU/mL bFGF and 10% FBS), culturing for 24 hours at 37° C. with 5% carbon dioxide.
The sheet culture medium was the culture medium without cells obtained after sheet formation.
The sheet re-attachment culture medium was obtained as follows: washing the obtained sheet (e.g. 3 times with PBS), attaching it to the culture container, and adding umbilical cord mesenchymal stem cell culture medium (supplemented with 10% FBS and 20 IU/mL bFGF α-MEM), culturing for 24 hours at 37° C. with 5% carbon dioxide.
The cytokine content in the cell culture medium, sheet culture medium and sheet re-attachment culture medium were tested according to the instructions of the enzyme-linked immunoassay kit. The cytokines tested were hepatocyte growth factor (HGF) (see
The above results show that after preservation of the sheet, the ability to secrete the cytokine HGF was not decreased, and the ability of the cell sheet to secrete the cytokine HGF was stronger than that of the cells; after preservation of the sheet, the ability to secrete the cytokine VEGF was not decreased, and the ability to secrete the cytokine VEGF was stronger than that of cells.
The obtained sheet was washed three times with PBS, attached to a culture container and then co-cultured with lymphocytes. An experimental group (the sheet+lymphocyte+lymphocyte activator), a negative control group (the sheet+lymphocyte) and a positive control group (lymphocyte+lymphocyte activator) were set up, and the inhibitory effect of the sheet on Th1 lymphocyte subpopulation was tested by flow cytometry. See
The obtained sheet was washed three times with PBS, attached to a culture container and then co-cultured with lymphocytes. An experimental group (the sheet+lymphocyte+lymphocyte stimulating agent), a negative control group (the sheet+lymphocyte) and a positive control group (lymphocyte+lymphocyte stimulating agent) were set up, the lymphocytes were labeled with BrdU and the inhibitory effect of the sheet on BrdU-positive lymphocyte population was tested by flow cytometry. See
The obtained sheet was washed three times with PBS, attached to a culture container and then co-cultured with lymphocytes. An experimental group (the sheet+lymphocyte+lymphocyte stimulating agent), a negative control group (the sheet+lymphocyte) and a positive control group (lymphocyte+lymphocyte stimulating agent) were set up. The culture supernatant was collected, and the secretion of TNFα in each group was tested with anti-TNFα antibody by ELISA method, thereby calculating the inhibitory effect of the sheet on the TNFα secretion by lymphocytes. See
The umbilical cord mesenchymal stem cell sheets were digested into single cells and resuspended to a cell concentration of 1×106/ml. 200 μL of the cells were taken into the flow tube, and the corresponding CD73, CD90, CD105, CD11b, CD19, CD34, CD45, and HLA-DR fluorescent staining antibodies were added to each tube according to the instructions. After incubation in the dark for 30 minutes, the cells were washed twice with PBS buffer solution, and then the stained cells were tested using a flow cytometer.
The flow cytometry results of cell sheet surface markers are shown in
6. The Cells in the Sheets have the Ability of Three-Direction Differentiation by Induction
The umbilical cord mesenchymal stem cell sheets were digested into single cells, and inoculated into a suitable culture vessel according to the ratio in the reagent instructions for the three-direction differentiation by induction. When the cells for osteogenic induction testing grew to about 70% confluence, and the cells for adipogenic induction testing grew to more than 90% confluence, osteogenic and adipogenic induction mediums were added respectively. During chondrogenic induction, a certain number of cells were centrifuged to the bottom of the centrifuge tube, and then a chondrogenic induction medium was added. After the cells were aggregated into pellets, the cell pellets were kept away from the bottom of the tube to ensure complete contact with the induction medium. All cells were tested after induction and culture for more than 21 days. Osteogenesis induction can be stained with Alizarin Red, etc., adipogenesis induction can be stained with Oil Red O, etc., and chondrogenesis induction can be stained with Alcian Blue, etc.
The results of the three-direction differentiation by induction are shown in
The cell sheets were prepared with different combinations of coating matrix and sheet-forming medium according to the preparation process as described above. The results are as follows in Table 1.
The old culture medium was removed from the umbilical cord mesenchymal stem cell culture medium obtained in Example 1, then the cells were washed with PBS for 1 to 3 times, and the digestive enzyme (TryPLE or trypsin) was added for digestion until the cells were in the single-cell state. PBS, cell complete medium, physiological saline, DPBS, Hanks, etc. were added to stop or reduce the digestion of the enzyme. After centrifugation, the supernatant was discarded to obtain a cell pellet.
The digested cells were washed with PBS, physiological saline, DPBS, and Hanks buffer, and washed twice or more at a washing density equal to or less than 5×10{circumflex over ( )}5 cells/mL.
After washing the cells to remove the residue of umbilical cord mesenchymal stem cell culture medium, the cells were resuspended by adding human umbilical cord mesenchymal stem cell sheet medium (sheet-forming medium, containing DMEM/F12 and containing 0.5-10% human serum albumin, 5-500 μg/mL vitamin C, and about 2 mM glutamine, about 10 mM non-essential amino acids). The cells were then seeded in a culture dish, culture plate or culture flask at a seeding density of 0.3-3 E6/cm2, and cultured for 8-48 hours.
The human umbilical cord mesenchymal stem cell sheet culture supernatant was collected into centrifuge tubes for centrifugation at 500-1000 g for 10-20 minutes, and then the supernatant was taken. The centrifuged supernatant was filtered through a filter with 0.22 μm pore size for future use.
The filtered supernatant was subpagaked to a required specification and sealed and packaged sterilely in a Clean Environment Class B environment in a Class A environment. It is then stored and transported in dry ice or a refrigerator below-80° C.
The cell culture supernatant was human umbilical cord mesenchymal stem cell culture supernatant.
The sheet culture supernatant was the human umbilical cord mesenchymal stem cell sheet culture supernatant obtained from Example 6;
The sheet culture supernatant (without Vc) was a human umbilical cord mesenchymal stem cell sheet culture supernatant obtained by a method similar to that in Example 6, wherein the cell sheet medium did not contain vitamin C.
According to the instructions of the enzyme-linked immunosorbent kit, the content of cytokines in the cell culture supernatant, sheet culture supernatant, and sheet culture supernatant (without Vc) was detected. The cytokines were hepatocyte growth factor (HGF) (see
The above results indicate that the sheet culture supernatant collected under a cellular stress state contains a larger amount of HGF, VEGF and IL-8, and the addition of vitamin C in the sheet-forming medium increase the activity of human umbilical cord mesenchymal stem cells, making them secrete more cytokines HGF, VEGF and IL-8 into the supernatant.
The human umbilical cord mesenchymal stem cell sheet culture supernatant was co-cultured with lymphocytes. An experimental group (culture supernatant+lymphocyte+lymphocyte activator), a negative control group (culture supernatant+lymphocyte) and a positive control group (lymphocyte+lymphocyte activator) were set up respectively, and the inhibitory effect of the culture supernatant on lymphocyte subpopulation Th1 was detected by flow cytometry. The results are shown in
The human umbilical cord mesenchymal stem cell sheet culture supernatant was co-cultured with lymphocytes. An experimental group (culture supernatant+lymphocyte+lymphocyte activator), a negative control group (culture supernatant+lymphocyte) and a positive control group (lymphocyte+lymphocyte activator) were set up respectively. Lymphocytes were labeled with CFSE, and the inhibitory effect of culture supernatant on lymphocyte proliferation was detected by flow cytometry. The results are shown in
The human umbilical cord mesenchymal stem cell sheet culture supernatant was co-cultured with lymphocytes. An experimental group (culture supernatant+lymphocyte+lymphocyte activator), a negative control group (culture supernatant+lymphocyte) and a positive control group (lymphocyte+lymphocyte activator) were set up respectively. The culture supernatants of the above groups were collected, and the secretion of TNFα in each group was detected by ELISA using anti-TNFα antibodies, thereby calculating the inhibitory effect of the human umbilical cord mesenchymal stem cell sheet culture supernatant on the TNFα secretion by lymphocytes. The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210475292.9 | Apr 2022 | CN | national |
The present application is a continuation-in-part application of U.S. non-Provisional application Ser. No. 18/842,631 filed on Aug. 29, 2024, which is based on International Patent Application No. PCT/CN2023/091164 filed on Apr. 27, 2023, which claims the priority of the CN application No. 202210475292.9, filed on Apr. 29, 2022. All the aforementioned patent applications are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18842631 | Jan 0001 | US |
| Child | 19021864 | US |
