CN116240166A - Self-assembly method and application of osteochondral organoids - Google Patents
Self-assembly method and application of osteochondral organoids Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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Abstract
The invention relates to the field of biological materials, in particular to a self-assembly method of an osteochondral organoid and application thereof. The invention provides a strategy for personalized modification based on a common microcarrier, which utilizes the characteristic of inducing stem cells to conduct cartilage forming differentiation by hyaluronic acid and the characteristic of inducing stem cells to conduct bone forming differentiation by hydroxyapatite to prepare the microcarrier with stronger cartilage forming and bone forming induction differentiation capacity. The cartilage microcarrier and the bone microcarrier have good biocompatibility, can promote cell proliferation and adhesion, and compared with the common microcarrier, the bidirectional self-assembled microcarrier has stronger cartilage forming and osteogenic induction differentiation capacity, and the microcarrier obtained by the invention plays an important role and has wide application prospect in the field of bone cartilage injury repair.
Description
Technical Field
The invention relates to the field of biological materials, in particular to a self-assembly method for osteochondral organoids and application thereof.
Background
The three-dimensional porous cell culture carrier can simulate the three-dimensional cell growth environment, and is beneficial to preserving the physiological characteristics of cells; the three-dimensional porous structure can provide huge space for cell adhesion, proliferation and growth, can better simulate the microenvironment of cells in vivo, and can have the potential of expanding cells in a large quantity. The porous microcarrier has larger porosity, can maintain cell activity at a higher level, promotes cell function, has stronger mechanical elasticity, and can well protect cultured cells from being damaged by stronger mechanical shearing force in a bioreactor, thereby improving the survival rate of the cells.
Articular cartilage is difficult to repair by itself after injury due to the absence of blood supply, innervation and specific structures of the lymphatic circulation. Articular cartilage damage is often accompanied by subchondral bone damage, and the regeneration difficulty is high due to the difference of microenvironment of the articular cartilage damage and the subchondral bone damage. Therefore, in addition to the advantages of good biocompatibility and injectability, the regenerated repair material needs to have stronger capacity of cartilage formation and osteogenic induced differentiation.
At present, development of a bidirectional self-assembled microcarrier for integrated regeneration and repair of bone and cartilage is needed.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent.
To this end, a first aspect of the present invention provides a method for the self-assembly of an osteochondral organoid, said method comprising the steps of:
(1) Placing the cartilage microcarrier and the stem cells in a cartilage forming differentiation medium for pre-differentiation co-culture to obtain cartilage microcarrier-pre-differentiated cartilage cells;
(2) Placing the bone microcarrier and the stem cells in an osteogenic differentiation culture medium for pre-differentiation co-culture to obtain bone microcarrier-pre-differentiated osteoblasts;
(3) Contacting the cartilage microcarrier-preassigned chondrocyte with the bone microcarrier-preassigned osteoblast, placing the cartilage microcarrier-preassigned chondrocyte in a mixed culture medium for mixed co-culture so as to obtain a self-assembled osteochondral organoid,
Wherein the cartilage microcarrier contains hyaluronic acid;
the bone microcarrier contains hydroxyapatite;
the mixed culture medium contains a cartilage forming differentiation culture medium and an osteogenic differentiation culture medium.
The hyaluronic acid can promote the differentiation of stem cells to chondrocytes, and the hydroxyapatite can promote the differentiation of stem cells to osteogenesis after being mixed into biological materials.
The modified bone microcarrier provided by the invention has the following advantages: firstly, the preparation method is relatively simple and convenient, can be widely obtained, can be stored for a long time at low temperature, and is economical and cheap; secondly, the product has stronger stability and long-term storage characteristics by introducing ingredients such as hydroxyapatite and the like without adding exogenous growth factors or active molecules and the like; thirdly, the modified microcarrier has good biocompatibility with the common microcarrier, but the bone microcarrier has stronger osteogenic differentiation characteristics.
In some embodiments of the invention, the stem cells are derived from adult stem cells.
In some embodiments of the invention, the stem cells are derived from human adult stem cells including at least one selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, and bone marrow stem cells.
In some embodiments of the invention, the co-cultivation time in steps (1) and (2) is from 4 to 8 days.
In some embodiments of the invention, the pre-differentiation co-culture is within the range of 4-8 days, enabling the stem cells to be initially differentiated into immature chondrocytes or immature osteoblasts in the corresponding induction medium, preferably for a period of 7 days.
In some embodiments of the invention, the co-cultivation time in steps (1) and (2) is from 6 to 8 days.
In some embodiments of the invention, the co-cultivation time in steps (1) and (2) is 7 days.
In some embodiments of the invention, the contacting is by way of surface contact.
In some embodiments of the invention, in step (3), the ratio by volume of the chondrogenic differentiation medium and osteogenic differentiation medium in the mixed medium is 1: (0.5-3).
In some embodiments of the invention, the volume ratio of the chondrogenic differentiation medium to the osteogenic differentiation medium in the mixed medium is 1: (1-2).
In some embodiments of the invention, the volume ratio of the chondrogenic differentiation medium to the osteogenic differentiation medium in the mixed medium is 1:1.
In some embodiments of the invention, the volume ratio of the chondrogenic differentiation medium to osteogenic differentiation medium is 1: (0.5-3), in which self-assembly of the osteochondral organoids can be better achieved, preferably the volume ratio is 1:1.
In some embodiments of the invention, the mixed co-cultivation time is 4-8 days.
In some embodiments of the invention, the mixed co-cultivation time is from 6 to 8 days.
In some embodiments of the invention, the mixed co-cultivation time is 7 days.
In some embodiments of the invention, the mixed co-culture time is in the range of 4-8 days, such that the immature chondrocytes and the immature osteoblasts are further formed into mature chondrocytes and mature osteoblasts, respectively, preferably the mixed co-culture time is 7 days.
In some embodiments of the invention, the method of preparing a cartilage microcarrier comprises:
(a) Mixing gelatin with hyaluronic acid to prepare a cartilage precursor solution;
(b) Preparing a cross-linking agent solution;
(c) Mixing the cartilage precursor solution with the cross-linking agent solution, pouring the mixed solution into a mould for freezing treatment to obtain the cartilage microcarrier,
wherein the mass ratio of the gelatin to the hyaluronic acid is (2-20): (0.01-1).
In some embodiments of the invention, the mass ratio of the gelatin to the hyaluronic acid is between (2-20): in the range of (0.01-1), the obtained cartilage microcarrier can maintain good biocompatibility, and has the capability of promoting differentiation of stem cells to chondrocytes and promoting formation of mature chondrocytes from immature chondrocytes.
In some embodiments of the invention, the hyaluronic acid is capable of promoting differentiation of stem cells into chondrocytes, promoting formation of mature chondrocytes from immature chondrocytes.
In some embodiments of the invention, the gelatin solution has a mass volume concentration of 2% to 20%.
In some embodiments of the invention, the gelatin solution has a mass volume concentration of 5-10%.
In some embodiments of the invention, the gelatin solution has a mass volume concentration of 8%.
In some embodiments of the invention, the hyaluronic acid is present in a mass volume concentration of 0.01% to 1%.
In some embodiments of the invention, the hyaluronic acid is present in a mass-volume concentration of 0.1% to 0.5%.
In some embodiments of the invention, the hyaluronic acid is at a mass-volume concentration of 0.25%.
In some embodiments of the invention, the crosslinker in the crosslinker solution comprises at least one selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, tetramethyl ethylenediamine, ammonium sulfate, genipin, divinylbenzene, diisocyanate, transglutaminase.
In some embodiments of the invention, the crosslinker solution is an aqueous 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution, and the aqueous 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution has a mass volume concentration of 2% to 25%.
In some embodiments of the invention, the aqueous 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution has a mass volume concentration of 5% to 15%.
In some embodiments of the invention, the aqueous 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution has a mass volume concentration of 10%.
In some embodiments of the invention, the cartilage precursor solution is mixed with the cross-linker solution in a volume ratio of 10:1.
In some embodiments of the invention, in step (c), the temperature of the freezing treatment is from-10 ℃ to-30 ℃.
In some embodiments of the invention, step (c) further comprises washing the microcarriers obtained after the freezing process.
In some embodiments of the invention, the cleaning solution includes ddH 2 O。
In some embodiments of the invention, the method of preparing a bone microcarrier comprises:
(A) Mixing gelatin with hydroxyapatite to prepare a bone precursor solution;
(B) Mixing the cross-linking agent, the pore-forming agent and the bone precursor solution, pouring the mixed solution into a mould for freezing treatment to obtain the bone microcarrier,
wherein, the liquid crystal display device comprises a liquid crystal display device,
the mass ratio of the gelatin to the hydroxyapatite is (2-20): (1-20).
In some embodiments of the invention, the mass ratio of the gelatin to the hydroxyapatite is in the range of (2-20): in the range of (0.01-1), the obtained bone microcarrier can maintain good biocompatibility, and has the capability of promoting differentiation of stem cells to osteoblasts and promoting formation of immature osteoblasts to mature osteoblasts.
In some embodiments of the invention, the hydroxyapatite is capable of promoting differentiation of stem cells into osteogenic directions, promoting formation of immature osteoblasts into mature osteoblasts.
In some embodiments of the invention, the gelatin solution has a mass volume concentration of 2% to 20%.
In some embodiments of the invention, the gelatin solution has a mass volume concentration of 5-10%.
In some embodiments of the invention, the gelatin solution has a mass volume concentration of 8%.
In some embodiments of the invention, the hydroxyapatite is present in a mass volume concentration of 1% to 20%.
In some embodiments of the invention, the hydroxyapatite mass volume concentration is 2% to 10%.
In some embodiments of the invention, the hydroxyapatite mass volume concentration is 6%.
In some embodiments of the invention, the cross-linking agent comprises at least one selected from the group consisting of 1, 5-glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysulfosuccinimide.
In some embodiments of the invention, the cross-linking agent is 1, 5-glutaraldehyde, and the 1, 5-glutaraldehyde is present at a mass volume concentration of 0.5% to 2%.
In some embodiments of the invention, the 1, 5-glutaraldehyde is present at a mass volume concentration of 1%.
In some embodiments of the invention, the porogen comprises at least one selected from the group consisting of dimethyl sulfoxide solution, sucrose, sodium chloride, polyethylene glycol (PEG).
In some embodiments of the invention, step (B) further comprises washing the microcarriers after the freezing.
In some embodiments of the invention, the cleaning agent comprises a compound selected from the group consisting of sodium borohydride solution, dd H 2 At least one of O and potassium borohydride.
In some embodiments of the invention, the cleaning agent is a sodium borohydride solution having a mass volume concentration of 0.05% to 0.4%.
In some embodiments of the invention, the sodium borohydride solution has a mass volume concentration of 0.1% to 0.2%.
In some embodiments of the invention, in step (B), the temperature of the freezing treatment is from-10 ℃ to-30 ℃.
In another aspect, the present invention provides an osteochondral organoid made by the method described above.
In a further aspect the invention provides the use of an osteochondral organoid as described above for the preparation of a model of a disease associated with osteochondral.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows the preparation process of cartilage microcarrier in example 2 of the present invention;
FIG. 2 shows a process for preparing bone microcarriers according to example 3 of the present invention;
FIG. 3 is a general view showing a general microcarrier, a cartilage microcarrier and a bone microcarrier according to example 4 of the present invention, wherein (A) is a general microcarrier and shows a white opaque powdery structure; (B) is a cartilage microcarrier, and presents a white powdery structure; (C) Is bone microcarrier, and presents a gray yellow opaque powdery structure;
FIG. 4 shows Scanning Electron Microscope (SEM) images of general microcarriers, cartilage microcarriers and bone microcarriers according to example 4 of the invention;
FIG. 5 is a graph showing the results of dead/living staining confocal of ordinary microcarriers, cartilage microcarriers and bone microcarriers according to example 5 of the present invention, wherein the scale length is 100. Mu.m, wherein green fluorescence represents living cells, indicated by thin arrows, red fluorescence represents dead cells, indicated by thick arrows;
FIG. 6 shows cytoskeletal confocal maps of ordinary microcarriers, cartilage microcarriers and bone microcarriers according to example 6 of the invention, wherein the scale length is 50 micrometers;
FIG. 7 is a graph showing the results of culturing the human umbilical cord mesenchymal stem cells loaded with the ordinary microcarrier, the cartilage microcarrier and the bone microcarrier for 14 days in example 7 of the present invention;
FIG. 8 shows a staining chart of cytoskeleton of the ordinary microcarrier, cartilage microcarrier and bone microcarrier of example 8 after 14 days of cartilage and bone induced differentiation, wherein thin arrow represents fiber network structure and thick arrow represents nucleus;
FIG. 9 shows the staining pattern of alisxin blue and toluidine blue after 14 days of cartilage-induced differentiation of ordinary microcarriers and cartilage microcarriers according to example 9 of the invention;
FIG. 10 shows alizarin red staining patterns of the ordinary microcarriers and bone microcarriers of example 10 of the present invention after 14 days of osteoinductive differentiation;
FIG. 11 is a schematic diagram showing self-assembly of osteochondral organoids in example 11 of the present invention, wherein A represents a cartilage microcarrier and B represents a bone microcarrier;
FIG. 12 is a diagram showing the general outline of an osteochondral organoid according to example 12 of the present invention;
FIG. 13 is a scanning electron microscope image of an osteochondral organoid according to example 12 of the present invention;
FIG. 14 is a graph showing the results of osteochondral organoid cell tracker in example 13 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
As used herein, the term "microcarrier" refers to a microbead having a diameter of 60-250 μm and which is suitable for adherent cell growth. Is generally composed of natural dextran or various synthetic polymers, including liquid microcarrier, macroporous gelatin microcarrier, polystyrene microcarrier, PHEMA microcarrier, chitin microcarrier, polyurethane foam microcarrier, alginate gel microcarrier, magnetic microcarrier, etc.
In this context, the term "crosslinker" is often a substance containing multiple functional groups in the molecule, such as organic diacids, polyols, etc.; or compounds containing multiple unsaturated double bonds in the molecule, such as divinylbenzene and diisocyanate, N, N-Methylenebisacrylamide (MBA), and the like. Can be fed together with the monomer to be condensed (or polymerized) to a certain degree to crosslink, so that the product becomes insoluble crosslinked polymer; a certain number of functional groups (or double bonds) can be reserved in the linear molecule, and specific substances are added to crosslink, such as curing of phenolic resin, vulcanization of rubber and the like.
In this context, the term "hydroxyapatite" is also known as hydroxyapatite, basic calcium phosphate, which is calciumApatite (Ca) 5 (PO 4 ) 3 (OH)) but is often written as (Ca) 10 (PO 4 ) 6 (OH) 2 ) In a form highlighting that it is composed of two parts: hydroxyl and apatite. OH-can be replaced by fluoride, chloride and carbonate ions to generate fluorite apatite or chlorapatite, wherein calcium ions can be replaced by a plurality of metal ions through ion exchange reaction to form M apatite (M represents metal ions replacing calcium ions) corresponding to the metal ions. Hydroxyapatite is a major inorganic component of human and animal bones. It can realize chemical bond combination with organism tissue on interface, has certain solubility in vivo, can release ions harmless to organism, can participate in metabolism in vivo, has stimulation or induction effect on hyperostosis, can promote repair of defective tissue, and shows biological activity.
In the text, the term "umbilical cord mesenchymal stem cells" (Mesenchymal Stem Cells, MSCs) refers to a multifunctional stem cell existing in neonatal umbilical cord tissues, can differentiate into a plurality of tissue cells, has wide clinical application prospect, can successfully expand human umbilical cord mesenchymal stem cells by using an inactivated umbilical cord serum culture system, and the cultured cells have the basic characteristics of the mesenchymal stem cells, thereby providing theoretical basis for establishing a mesenchymal stem cell library and clinical application.
In the text, the term "DMSO" refers to dimethyl sulfoxide, a sulfur-containing organic compound of formula C 2 H 6 OS is colorless odorless transparent liquid at normal temperature, and is a hygroscopic flammable liquid. The organic solvent has the characteristics of high polarity, high boiling point, good thermal stability, aprotic property and water miscibility, and can be dissolved in most organic matters such as ethanol, propanol, benzene, chloroform and the like, and is known as a universal solvent. Heating in the presence of an acid produces small amounts of methyl mercaptan, formaldehyde, dimethyl sulfide, methanesulfonic acid, and the like. The decomposition phenomenon occurs at high temperature, and the chlorine can react violently to burn in the air to generate light blue flame. Can be used as organic solvent, reaction medium and organic synthesis intermediate. Can also be used as dyeing solvent, stripping agent, dyeing carrier for synthetic fiber, and recovery of acetylene and dioxideSulfur absorbent.
In the text, the term "GA solution" refers to glutaraldehyde, an organic compound of the formula C 5 H 8 O 2 Is colorless or light yellow transparent liquid, is dissolved in water, is easily dissolved in organic solvents such as ethanol, diethyl ether and the like, and is commonly used as bactericide, food industry processing aid, disinfectant, tanning agent, wood preservative, medicine, polymer synthetic raw materials and the like.
In the text, the term "mass-to-volume concentration" refers to the ratio of mass to volume, expressed in percent (%), in g/ml or kg/L.
Preparation method of cartilage microcarrier
According to some embodiments of the invention, the invention provides a method of preparing a cartilage microcarrier, comprising:
(a) Mixing gelatin with hyaluronic acid to prepare a cartilage precursor solution;
(b) Preparing a cross-linking agent solution;
(c) Mixing the cartilage precursor solution with the cross-linking agent solution, pouring the mixed solution into a mould for freezing treatment to obtain the cartilage microcarrier,
wherein the mass ratio of the gelatin to the hyaluronic acid is (2-20): (0.01-1).
According to some embodiments of the invention, the cross-linking agent is any agent known in the art that can cross-link gelatin with hyaluronic acid, including EDC, and any agent that can cross-link gelatin with hyaluronic acid is within the scope of the invention.
According to some more specific embodiments of the present invention, there is provided a method of preparing a cartilage microcarrier, comprising:
(1) 160mg gelatin (cold water fish skin) and 5mg hyaluronic acid powder were weighed into 2ml ddH 2 Placing in O water, placing in a 60 ℃ oven for incubation for 1-2h to thoroughly dissolve, placing on ice, cooling to room temperature, and preparing a cartilage precursor solution with the concentration of 8% for later use;
(2) 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) powder is weighed and added into 1ml of water, vortex vibration is carried out for dissolution, and EDC solution with the concentration of 10% is prepared for standby;
(3) Uniformly mixing 200 μl of 10% EDC solution with 1.8ml of 8% gelatin solution, rapidly taking 425 μl of the mixed solution, spreading, and adding into a mold, wherein the four sides are firstly loaded with the solution, then the middle is used to avoid bubble generation;
(4) Placing the plate in a lunch box, covering, rapidly placing in a refrigerator at-20deg.C, and pre-cooling overnight;
(5) Taking out the gel after the freezing time is over, and filling ddH in a round dish mold 2 O, melting ice;
(6) Ejecting the glue from the mould along the periphery by using a white gun head, and uniformly transferring the glue to the mould filled with dd H 2 In the new dish of O, the shaking table is washed on ice, water is changed once every 20min for 2-3 times, and then the dish is placed on the shaking table for overnight cleaning, wherein the cleaning time is not less than 16 hours;
(7) After overnight cleaning, the water in the dish is replaced and washed for 1 time, about 500ul of water just goes beyond the microcarrier, the bottom and the cover of the dish are wiped clean by paper, the dish is frozen for more than 2 hours in a refrigerator at the temperature of minus 20 ℃, and then the dish is transferred into a freeze dryer for more than 4 hours, and the dish is taken out for preservation in a vacuum tank.
Bone microcarrier preparation method
According to some embodiments of the invention, the invention provides a method of preparing a bone microcarrier, comprising:
(A) Mixing gelatin with hydroxyapatite to prepare a bone precursor solution;
(B) Mixing the cross-linking agent, the pore-forming agent and the bone precursor solution, pouring the mixed solution into a mould for freezing treatment to obtain the bone microcarrier,
wherein the mass ratio of the gelatin to the hydroxyapatite is (2-20): (1-20).
According to some more specific embodiments of the present invention, there is provided a method of preparing a bone microcarrier, comprising:
(1) 160mg of gelatin (cold water fish skin) was weighed out in 2ml ddH 2 Placing in O water, incubating in oven at 60deg.C for 1-2 hr for complete dissolution, and placingCooling to room temperature on ice, and preparing gelatin solution with concentration of 8% for standby;
(2) Weighing 120mg of hydroxyapatite powder, adding the hydroxyapatite powder into 2ml of 8% gelatin solution, vortex oscillating, adding 200ul of pure DMSO, and uniformly mixing again;
(3) Taking 40ul of 50% 1, 5-Glutaraldehyde (GA) solution (50X), adding into the solution, quickly mixing, and transferring into a mold;
(4) Placing the plate in a lunch box, covering, rapidly placing in a refrigerator at-20deg.C, and pre-cooling overnight;
(5) Sodium borohydride solution was prepared, 0.1g (0.2% for high hardness)/0.05 g (0.1% for medium or soft) dissolved in 50ml dd H 2 O (the volume is determined by the number of samples, and is about 2-3mL of a round dish), then the round dish is placed in a refrigerator at 4 ℃ for precooling, and the cover is loosened and easy to give off air;
(6) Taking out the gel after the freezing time is over, and filling ddH in a round dish mold 2 O, melting ice;
(7) Preparing a batch of new small dishes, adding sodium borohydride into each new dish respectively, then placing the new dishes into a lunch box, and placing the lunch box on ice;
(8) Ejecting the glue from the mould along the periphery by using a white gun head, uniformly transferring the glue into a new dish containing sodium borohydride, and washing for 10min by using a shaking table on ice;
(9) Then change to dd H 2 O cleaning, namely, changing water once every 20min, changing the water for 2 to 3 times, and then placing the water on a shaking table for cleaning overnight, wherein the cleaning time is not less than 16 hours;
(10) After overnight cleaning, the water in the dish is replaced and washed for 1 time, about 500ul of water just goes beyond the microcarrier, the bottom and the cover of the dish are wiped clean by paper, the dish is frozen for more than 2 hours in a refrigerator at the temperature of minus 20 ℃, and then the dish is transferred into a freeze dryer for more than 4 hours, and the dish is taken out for preservation in a vacuum tank.
According to some embodiments of the invention, the cross-linking agent comprises any agent known in the art capable of cross-linking gelatin with hydroxyapatite, and any agent capable of cross-linking gelatin with hydroxyapatite, including 1, 5-Glutaraldehyde (GA), is within the scope of the invention.
According to some embodiments of the invention, the porogen comprises any material known in the art capable of porogen-ing the bone precursor, and any material capable of porogen-ing the bone precursor, including DMSO, is within the scope of the invention.
According to some embodiments of the present invention, the sodium borohydride solution is a detergent, and when the crosslinking agent is an aldehyde compound, the sodium borohydride can elute the remaining aldehyde of the reaction, and any substances known in the art that are easily soluble in aldehydes and can be dissolved in water can be used as the detergent of the present invention; when the cross-linking agent is not an aldehyde, the cleaning agent is also adapted to the cross-linking agent, and the principle is that the cleaning agent is easily soluble in the cross-linking agent and water-soluble.
Self-assembly method of osteochondral organoids
According to some embodiments of the invention, the invention provides a method of osteochondral organoid self-assembly, the method comprising:
(1) Placing the cartilage microcarrier and the stem cells in a cartilage forming differentiation medium for co-culture to obtain cartilage microcarrier-preassigned cartilage cells;
(2) Placing the bone microcarrier and stem cells in an osteogenic differentiation medium for co-culture to obtain bone microcarrier-preassigned osteoblasts;
(3) Contacting the cartilage microcarrier-preassigned chondrocytes with the bone microcarrier-preassigned osteoblasts, placing in a mixed culture medium for co-culture so as to obtain self-assembled osteochondral organoids,
wherein the cartilage microcarrier contains hyaluronic acid;
the bone microcarrier contains hydroxyapatite;
the mixed culture medium contains a cartilage forming differentiation culture medium and an osteogenic differentiation culture medium.
According to some embodiments of the invention, the stem cells are derived from adult stem cells.
According to some embodiments of the invention, the stem cells are derived from human adult stem cells comprising at least one selected from mesenchymal stem cells, induced pluripotent stem cells, bone marrow stem cells.
According to some embodiments of the invention, the chondrogenic induced differentiation medium comprises any medium known in the art that is capable of differentiating the stem cells specifically in the direction of chondrocytes, such as DMEM serum-free medium, comprising: dexamethasone 0.1. Mu. Mol/L, bovine serum albumin (1.25 mg/m 1), vitamin C (37.5. Mu.g/m 1), sodium pyruvate (1 mmol/L), TGF-B (10 ng/ml), beta-FGF (1 ng/m 1), ITS (6.25 pg/ml bovine insulin, 6.25. Mu.g/ml transferrin, etc., and all media capable of differentiating the stem cells specifically into cartilage in the art are within the scope of the present invention.
According to some embodiments of the invention, the osteogenic differentiation medium comprises any medium known in the art that is capable of differentiating the stem cells specifically towards osteoblasts, such as DMEM low sugar medium, comprising 10% fbs, 100nmol/L dexamethasone, 0.2mmol/L ascorbic acid, 1% PS (Penicillin-Streptomycin Solution), 10mmol/L sodium beta-glycerophosphate, it being noted that all media known in the art that are capable of differentiating the stem cells specifically towards osteoblasts are within the scope of the invention.
According to some embodiments of the invention, the medium composition is capable of initially determining the differentiation direction of the stem cells, attaching the stem cells to a cartilage microcarrier for differentiation induction using a differentiation medium for cartilage induction, attaching the stem cells to a bone microcarrier for differentiation induction using a differentiation medium for bone induction, obtaining cartilage microcarrier-preassigned chondrocytes and bone microcarriers-preassigned osteoblasts, wherein the chondrocytes and osteoblasts are undifferentiated mature cells. And then the cartilage microcarrier-preassigned chondrocyte is contacted with the bone microcarrier-preassigned osteoblast, the chondrogenic differentiation medium and the osteogenic differentiation medium are used for mixed co-culture, and the components in the mixed co-culture and microcarrier do not change the differentiation direction of the undifferentiated mature cells, but can further promote the further differentiation of the chondrocyte and the osteoblast to form the differentiated mature cells.
According to some embodiments of the invention, the cartilage microcarrier comprises hyaluronic acid, so that immature chondrocytes formed by the primary differentiation of stem cells can be continuously formed into mature chondrocytes, and the bone microcarrier comprises hydroxyapatite, so that immature osteoblasts formed by the primary differentiation of stem cells can be continuously formed into mature osteoblasts.
According to some embodiments of the invention, after mixed culture of the two media, the immature chondrocytes attached to the cartilage microcarrier further form mature chondrocytes, and the immature osteoblasts attached to the bone microcarrier further form mature osteoblasts.
According to some embodiments of the invention, the contacting is by way of surface contact.
According to some embodiments of the invention, the surface contact comprises a top-to-bottom lay on top of each other between maximum cross-sections.
According to some embodiments of the invention, the stacking comprises layering cartilage microcarriers on top of bone microcarriers, and the stacking is more in line with the morphological demands of human body.
The hyaluronic acid can promote the differentiation of stem cells to chondrocytes, and the hydroxyapatite can promote the differentiation of stem cells to osteogenesis after being mixed into biological materials.
The modified cartilage microcarrier and the bone microcarrier provided by the invention have the following advantages: (1) The preparation method is relatively simple and convenient, can be widely obtained, can be stored for a long time at low temperature, and is economical and cheap; (2) The product has stronger stability and long-term preservation characteristics by introducing components such as hyaluronic acid, hydroxyapatite and the like, and no exogenous growth factors or active molecules and the like are required to be added; (3) The modified microcarrier has good biocompatibility with common microcarrier, but the cartilage microcarrier has stronger chondrogenic differentiation characteristics, and the bone microcarrier has stronger osteogenic differentiation characteristics.
The aspects of the present disclosure will be explained below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of ordinary microcarriers
(1) 160mg of gelatin (cold water fish skin) was weighed out in 2ml ddH 2 Placing in O water, placing in a 60 ℃ oven for incubation for 1-2h to dissolve thoroughly, placing on ice, cooling to room temperature, and preparing gelatin solution with concentration of 8% for standby;
(2) 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) powder is weighed and added into 1ml of water, vortex vibration is carried out for dissolution, and EDC solution with the concentration of 10% is prepared for standby;
(3) Uniformly mixing 200 μl of 10% EDC solution with 1.8ml of 8% gelatin solution, rapidly taking 425 μl of the mixed solution, spreading, and adding into a mold, wherein the four sides are firstly loaded with the solution, then the middle is used to avoid bubble generation;
(4) Placing the plate in a lunch box, covering, rapidly placing in a refrigerator at-20deg.C, and pre-cooling overnight;
(5) Taking out the gel after the freezing time is over, and filling ddH in a round dish mold 2 O, melting ice;
(6) Ejecting the glue from the mould along the periphery by using a white gun head, and uniformly transferring the glue to the mould filled with dd H 2 In the new dish of O, the shaking table is washed on ice, water is changed once every 20min for 2-3 times, and then the dish is placed on the shaking table for overnight cleaning, wherein the cleaning time is not less than 16 hours;
(7) After overnight cleaning, the water in the dish is replaced and washed for 1 time, about 500ul of water just goes beyond the microcarrier, the bottom and the cover of the dish are wiped clean by paper, the dish is frozen for more than 2 hours in a refrigerator at the temperature of minus 20 ℃, and then the dish is transferred into a freeze dryer for more than 4 hours, and the dish is taken out for preservation in a vacuum tank.
EXAMPLE 2 cartilage microcarrier preparation
The specific preparation process of the cartilage microcarrier is shown in figure 1:
(1) 160mg gelatin (cold water fish skin) and 5mg hyaluronic acid powder were weighed into 2ml ddH 2 Placing in O water, placing in a 60 ℃ oven for incubation for 1-2h to dissolve thoroughly, placing on ice, cooling to room temperature, and preparing gelatin solution with concentration of 8% for standby;
(2) 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) powder is weighed and added into 1ml of water, vortex vibration is carried out for dissolution, and EDC solution with the concentration of 10% is prepared for standby;
(3) Uniformly mixing 200 μl of 10% EDC solution with 1.8ml of 8% gelatin solution, rapidly taking 425 μl of the mixed solution, spreading, and adding into a mold, wherein the four sides are firstly loaded with the solution, then the middle is used to avoid bubble generation;
(4) Placing the plate in a lunch box, covering, rapidly placing in a refrigerator at-20deg.C, and pre-cooling overnight;
(5) Taking out the gel after the freezing time is over, and filling ddH in a round dish mold 2 O, melting ice;
(6) Ejecting the glue from the mould along the periphery by using a white gun head, and uniformly transferring the glue to the mould filled with dd H 2 Cleaning a new dish of O by a shaking table on ice, changing water once every 20min for 2-3 times, and then cleaning the new dish by placing the new dish on the shaking table overnight for not less than 16 hours;
(7) After overnight cleaning, the water in the dish is replaced and washed for 1 time, about 500ul of water just goes beyond the microcarrier, the bottom and the cover of the dish are wiped clean by paper, the dish is frozen for more than 2 hours in a refrigerator at the temperature of minus 20 ℃, and then the dish is transferred into a freeze dryer for more than 4 hours, and the dish is taken out for preservation in a vacuum tank.
EXAMPLE 3 bone microcarrier preparation
The specific preparation process of the bone microcarrier is shown in figure 2:
(1) 160mg of gelatin (cold water fish skin) was weighed out in 2ml ddH 2 Placing in O water, incubating in oven at 60deg.C for 1-2 hr for complete dissolution, placing on ice, cooling to room temperature, and preparing gelatin solution with concentration of 8%Using;
(2) Weighing 120mg of hydroxyapatite powder, adding the hydroxyapatite powder into 2ml of 8% gelatin solution, vortex oscillating, adding 200ul of pure DMSO, and uniformly mixing again;
(3) Taking 40ul of 50% 1, 5-Glutaraldehyde (GA) solution (50X), adding into the solution, quickly mixing, and transferring into a mold;
(4) Placing the plate in a lunch box, covering, rapidly placing in a refrigerator at-20deg.C, and pre-cooling overnight;
(5) Sodium borohydride solution was prepared, 0.1g (0.2% for high hardness)/0.05 g (0.1% for medium or soft) dissolved in 50ml dd H 2 O (the volume is determined by the number of samples, and is about 2-3mL of a round dish), then the round dish is placed in a refrigerator at 4 ℃ for precooling, and the cover is loosened and easy to give off air;
(6) Taking out the gel after the freezing time is over, and filling ddH in a round dish mold 2 O, melting ice;
(7) Preparing a batch of new small dishes, adding sodium borohydride into each new dish respectively, then placing the new dishes into a lunch box, and placing the lunch box on ice;
(8) Ejecting the glue from the mould along the periphery by using a white gun head, uniformly transferring the glue into a new dish containing sodium borohydride, and washing for 10min by using a shaking table on ice;
(9) Then change to dd H 2 O cleaning, namely, changing water once every 20min, changing the water for 2 to 3 times, and then placing the water on a shaking table for cleaning overnight, wherein the cleaning time is not less than 16 hours;
(10) After overnight cleaning, the water in the dish is replaced and washed for 1 time, about 500ul of water just goes beyond the microcarrier, the bottom and the cover of the dish are wiped clean by paper, the dish is frozen for more than 2 hours in a refrigerator at the temperature of minus 20 ℃, and then the dish is transferred into a freeze dryer for more than 4 hours, and the dish is taken out for preservation in a vacuum tank.
EXAMPLE 4 morphology observations of three microcarriers
The microcarriers in examples 1-3, which are generally seen in FIG. 3, were placed on top of a silicon wafer, the surface was sprayed with gold under vacuum, and the microscopic morphology was observed by scanning electron microscopy.
Figure 3 shows three microcarrier physical plots: (A) Is a common microcarrier and presents a white opaque powdery structure; (B) is a cartilage microcarrier, and presents a white powdery structure; (C) Is bone microcarrier, and presents a gray-yellow opaque powdery structure.
The results of fig. 4 show three microcarrier microstructures: (A) The micro-structure of the common micro-carrier has the diameter of 250-450 mu m, the surface of the micro-carrier presents an obvious loose and porous structure, and different pores are mutually communicated. (B) The micro-structure of the cartilage microcarrier is directly about 150-400 mu m, which shows that the typical loose and porous structure is shown, the diameter of micropores is about 10-30 mu m, the micropores are mutually communicated, and the surface morphology of the microcarrier is not obviously changed along with the addition of hyaluronic acid. The graph (C) shows the microscopic morphology of the bone microcarrier, the diameter of the microcarrier is 50-200 mu m, the surface of the microcarrier presents a bump-shaped structure with irregular fluctuation, no obvious loose porous structure is seen, and the microcarriers are mutually fused.
EXAMPLE 5 three microcarrier cell death and viability staining assays
The microcarrier after ultraviolet sterilization is co-cultured with human umbilical cord mesenchymal stem cells (Beijing Hua niche biotechnology Co., ltd.) and the cells are loaded on a bracket for in vitro culture for 7 days and then subjected to dead-living staining. 1 mu L of calcein-AM and 4 mu L of ethidium dimer-1 are added into 2ml of sterile PBS solution, the cell-microcarrier complex is immersed in the solution for 30min at room temperature, the solution is continuously gently washed for 3 times by using sterile PBS, the PBS solution is sucked, and the solution is photographed and observed under a laser confocal microscope.
The dead/alive staining results (fig. 5) are shown, wherein green fluorescence represents living cells, indicated by thin arrows, red fluorescence represents dead cells, indicated by thick arrows; the vast majority of human umbilical cord mesenchymal stem cells in the common microcarrier and cartilage microcarrier groups show green fluorescence (living cells, thin arrows) with only a small amount of red fluorescence (dead cells, thick arrows); in the bone microcarrier group, the material nonspecifically adsorbed ethidium dimer-1, thus showing a spherical microcarrier red morphology, but not considered dead cells. Since the number of cells carried by the three microcarriers is the same, the cells in the bone microcarrier can be identified as a large number of living cells by comparing with the green fluorescence of the common microcarrier and the cartilage microcarrier to show stronger green signals. It is shown that all three microcarriers have good biocompatibility.
EXAMPLE 6 cytoskeletal staining observations of three microcarriers
The microcarrier after ultraviolet sterilization is co-cultured with human umbilical cord mesenchymal stem cells, and the cells are loaded on a bracket for in vitro culture for 7 days and then are subjected to cytoskeletal staining. After the cells and microcarrier complexes after 7 days of culture are fixed for 20 minutes by using 4% paraformaldehyde, the complexes are stained for 30 minutes by using a green rhodamine dye and a DAPI dye after repeated PBS flushing, 0.1% Triton 100 membrane rupture and other steps, and the complexes are photographed and observed under a laser confocal microscope after PBS flushing for 3 times.
As shown in the figure (FIG. 6), cells on a common microcarrier are more densely adhered to the surface of the microcarrier, and have a typical hemispherical structure. The cartilage microcarrier has obvious cell adhesion on the surface, good cell ductility and relatively spread cell morphology. The bone microcarrier surface exhibited a more dense cell adhesion morphology, exhibiting a typical hemispherical structure. The above results all demonstrate that cells adhere well to microcarriers and are ductile.
EXAMPLE 7 microscopic morphological detection after 14 days of culture of three microcarrier-supported MSCs
After the microcarrier after ultraviolet sterilization and the human umbilical cord mesenchymal stem cells are co-cultured for 14 days, the microcarrier is subjected to pretreatment such as fixation by 2.5% glutaraldehyde solution, and the cell adhesion and growth conditions are observed under a scanning electron microscope so as to evaluate the biocompatibility.
The cell adhesion results are shown in figure 7, and the three microcarriers have the advantages of large cell adhesion on the surfaces, good growth state, changeable cell morphology, large amount of extracellular matrixes, high cell survival rate and more obvious proliferation performance, and the microcarriers have good cell compatibility and certain cell adhesion and growth promoting performance.
EXAMPLE 8 cytoskeletal staining of three microcarriers 14 days after chondrogenic and osteogenic differentiation
In order to more clearly observe morphological differences among various groups of cells after differentiation induction, the microcarrier after ultraviolet sterilization and human umbilical cord mesenchymal stem cells are induced to form cartilage and osteoblast differentiation for 14 days, then the cells are digested to a culture plate and then attached overnight, and F-actin and DAPI are used for respectively carrying out dye observation on cytoskeleton and cell nuclei.
As shown in FIG. 8, after the common microcarrier and the cartilage microcarrier are cultured for 14 days in a cartilage induced differentiation mode, the outline of the cell morphology of the common microcarrier and the cartilage microcarrier is hidden and visible, wherein the thin arrow represents a fiber network structure, the thick arrow represents a cell nucleus, and as shown in FIG. 8, the inside of the cell presents a fiber network structure with different thickness, a plurality of cells are mutually fused, cell fibers are mutually interwoven, and the cell ductility is good. After 14 days of osteogenic differentiation induction of the common microcarrier and the bone microcarrier, the cell morphology of the common microcarrier and the bone microcarrier is similar, the cell edge fiber is obvious, the fiber signal inside the cell is slightly weak, and the fibers between adjacent cells are interwoven.
EXAMPLE 9 common microcarrier and cartilage microcarrier African blue and toluidine blue staining after 14 days of cartilage differentiation
In order to investigate whether the modified microcarrier has stronger chondrogenic differentiation induction capacity, the microcarrier subjected to ultraviolet sterilization and human umbilical cord mesenchymal stem cells were induced to chondrogenic differentiation for 14 days, and then the cells were digested to a culture plate and attached overnight, and were stained with aliskiren blue and toluidine blue. The method comprises the following steps of: after washing with PBS solution, the alisxin blue staining solution (Soy Co.) was immersed for 5 minutes, washed with distilled water for 2 minutes, and observed under a light microscope. Toluidine blue staining: the toluidine blue dye solution (Soy Bao Co.) was dip-dyed for 5 minutes, washed with distilled water for 2 minutes, and observed under a microscope.
As can be seen from the alisxin blue staining results of fig. 9, the degree of staining was shallower in the normal microcarriers than in the cartilage microcarriers, and the cartilage microcarrier group exhibited a multi-point distribution of deep stained areas, indicating that it had more acidic mucopolysaccharide production. As can be seen from the toluidine blue staining results, the cells of the cartilage microcarrier group showed a distinct deep-stained area, and the deep-stained area was much larger than that of the microcarrier group. Both results indicate that the cartilage microcarrier has a stronger capacity to induce differentiation of stem cells into chondrocytes.
EXAMPLE 10 ordinary microcarrier and bone microcarrier alizing alizarin red staining 14 days after differentiation
In order to investigate whether the modified microcarrier has stronger osteogenic differentiation induction capability, the ultraviolet sterilized microcarrier and human umbilical cord mesenchymal stem cells are subjected to osteogenic differentiation induction for 14 days, and then the cells are digested to a culture plate and then attached to the wall overnight, and alizarin red staining is performed.
As can be seen from fig. 10, the microcarrier group cells showed spot stained areas of spots, while the bone microcarrier group showed more areas of calcium nodule stained areas, indicating that the bone microcarrier has a stronger ability to induce differentiation of stem cells to osteoblasts.
EXAMPLE 11 in vitro self-Assembly of osteochondral organoids
In vitro studies were conducted on whether cartilage and bone microcarriers could self-assemble into osteochondral organoids. Firstly, the cartilaginous microcarrier after sterilization and the human umbilical cord mesenchymal stem cells are subjected to chondrogenic differentiation induction for 7 days, and meanwhile, the cartilaginous microcarrier after sterilization and the human umbilical cord mesenchymal stem cells are subjected to osteogenic differentiation induction for 7 days, and then according to the graph shown in fig. 11, wherein A represents the cartilaginous microcarrier, B represents the cartilaginous microcarrier, the cartilaginous microcarrier is placed on the lower layer of a die, the cartilaginous microcarrier is placed on the upper layer of the die, and the culture medium is a chondrogenic differentiation culture medium (Wohunorace biotechnology Co.): osteogenic differentiation medium (Siro biosciences Co., ltd.) =1:1, and after further differentiation induction for 7 days, the self-assembled osteochondral organoids were obtained.
Example 12 scanning electron microscope detection after differentiation of osteochondral organoids
The above osteochondral organoids were observed under a general observation and a scanning electron microscope. The specific steps are that the organoid is cut along the longitudinal axis of the organoid, after the organoid is washed for 3 times by PBS solution, the organoid is fixed for 4 hours by using 2.5% glutaraldehyde solution, the surface of the organoid is sprayed with gold after freeze-drying, and the microscopic morphology is observed by a scanning electron microscope.
The self-assembled osteochondral organoids are shown in FIG. 12 to be generally white and opaque, and the boundary between the bone and cartilage layers is hidden and visible, and the scanning electron microscope result in FIG. 13 shows that the upper left side is the cartilage layer, the lower right corner is the bone layer, and the microcarriers are hidden and visible, and are wrapped by a large amount of extracellular matrix to form compact tissues. The cartilage layer and the bone layer are well fused, and the broken line is the boundary.
Example 13Cell tracker traces of Cell behavior in cartilage and bone microcarriers
To study Cell behavior in different microcarriers, cell tracker was chosen to differentiate cells. Prior to cell seeding, cells were incubated with 1x dye prepared in diluent C provided in kit (SigmaAldrich, MO, USA) according to the manufacturer's instructions and MSCs were labeled with PKH26 red fluorescent cells (for cartilage microcarriers) and PKH67 green fluorescent cell connectors (for bone microcarriers), respectively. Cells were then inoculated into different microcarriers and self-assembled according to the procedure in example 11. After self-assembly was completed, osteochondral organoids were imaged using TCS-SP8 confocal microscope (Leica, germany) under excitation at 490nm (PKH 67) and 551nm (PKH 26).
As shown in fig. 14, the upper red layer is the cartilage layer and the lower green layer is the bone layer, which show a clear demarcation, indicating that the two microcarriers did not mix during self-assembly, but differentiated at their respective locations.
The results show that the modified cartilage microcarrier and the bone microcarrier have good biocompatibility, can promote cell proliferation and adhesion, have stronger chondrogenic differentiation and osteogenic differentiation induction capacity compared with the common microcarrier, can spontaneously assemble into bone cartilage organoids in vitro, and are expected to provide a new idea for solving the integrated damage of bone cartilage.
In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," "some embodiments," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method of osteochondral organoid self-assembly, the method comprising:
(1) Placing the cartilage microcarrier and the stem cells in a cartilage forming differentiation medium for pre-differentiation co-culture to obtain cartilage microcarrier-pre-differentiated cartilage cells;
(2) Placing the bone microcarrier and the stem cells in an osteogenic differentiation culture medium for pre-differentiation co-culture to obtain bone microcarrier-pre-differentiated osteoblasts;
(3) Contacting the cartilage microcarrier-preassigned chondrocyte with the bone microcarrier-preassigned osteoblast, placing the cartilage microcarrier-preassigned chondrocyte in a mixed culture medium for mixed co-culture so as to obtain a self-assembled osteochondral organoid,
wherein the cartilage microcarrier contains hyaluronic acid;
the bone microcarrier contains hydroxyapatite;
the mixed culture medium contains a cartilage forming differentiation culture medium and an osteogenic differentiation culture medium.
2. The method of claim 1, wherein the stem cells are derived from adult stem cells;
optionally, the stem cells are derived from human adult stem cells, including at least one selected from mesenchymal stem cells, induced pluripotent stem cells, bone marrow stem cells;
optionally, the pre-differentiation co-culture time in steps (1) and (2) is 4-8 days;
Optionally, the pre-differentiation co-culture time in steps (1) and (2) is from 6 to 8 days;
optionally, the pre-differentiation co-culture time in steps (1) and (2) is 7 days.
3. The method of claim 1, wherein the contacting is by way of surface contact;
optionally, in step (3), the volume ratio of the chondrogenic differentiation medium and the osteogenic differentiation medium in the mixed medium is 1: (0.5-3);
optionally, the volume ratio of the chondrogenic differentiation medium to the osteogenic differentiation medium in the mixed medium is 1: (1-2);
optionally, the volume ratio of the chondrogenic differentiation medium to the osteogenic differentiation medium in the mixed medium is 1:1;
optionally, the mixed co-cultivation time is 4-8 days;
optionally, the mixed co-cultivation time is 6-8 days;
optionally, the mixed co-cultivation time is 7 days.
4. The method of claim 1, wherein the method of preparing the cartilage microcarrier comprises:
(a) Mixing gelatin with hyaluronic acid to prepare a cartilage precursor solution;
(b) Preparing a cross-linking agent solution;
(c) Mixing the cartilage precursor solution with the cross-linking agent solution, pouring the mixed solution into a mould for freezing treatment to obtain the cartilage microcarrier,
Wherein the mass ratio of the gelatin to the hyaluronic acid is (2-20): (0.01-1).
5. The method of claim 4, wherein the gelatin solution has a mass-to-volume concentration of 2% to 20%;
optionally, the gelatin solution has a mass volume concentration of 5-10%;
optionally, the gelatin solution has a mass volume concentration of 8%;
optionally, the hyaluronic acid has a mass-volume concentration of 0.01% -1%;
optionally, the hyaluronic acid has a mass-to-volume concentration of 0.1% -0.5%;
optionally, the hyaluronic acid has a mass-to-volume concentration of 0.25%;
optionally, the crosslinker in the crosslinker solution comprises at least one selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, tetramethyl ethylenediamine, ammonium sulfate, genipin, divinylbenzene, diisocyanate, transglutaminase;
optionally, the crosslinker solution is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, the mass volume concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution is 2% -25%;
optionally, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution has a mass volume concentration of 5% to 15%;
Optionally, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution has a mass volume concentration of 10%;
optionally, the cartilage precursor solution is mixed with the cross-linker solution in a volume ratio of 10:1.
6. The method according to claim 4, wherein in the step (c), the temperature of the freezing treatment is-10 to-30 ℃;
optionally, step (c) further comprises washing the microcarriers obtained after the freezing process;
optionally, the washing liquid used in the washing includes ddH 2 O。
7. The method of claim 1, wherein the bone microcarrier is prepared by a process comprising:
(A) Mixing gelatin with hydroxyapatite to prepare a bone precursor solution;
(B) Mixing the cross-linking agent, the pore-forming agent and the bone precursor solution, pouring the mixed solution into a mould for freezing treatment to obtain the bone microcarrier,
wherein, the liquid crystal display device comprises a liquid crystal display device,
the mass ratio of the gelatin to the hydroxyapatite is (2-20): (1-20).
8. The method of claim 7, wherein the gelatin solution has a mass volume concentration of 2% to 20%;
optionally, the gelatin solution has a mass volume concentration of 5-10%;
optionally, the gelatin solution has a mass volume concentration of 8%;
Optionally, the hydroxyapatite mass volume concentration is 1% -20%;
optionally, the hydroxyapatite mass volume concentration is 2% -10%;
optionally, the hydroxyapatite mass volume concentration is 6%;
optionally, the cross-linking agent comprises at least one selected from 1, 5-glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysulfosuccinimide;
optionally, the cross-linking agent is 1, 5-glutaraldehyde, and the mass and volume concentration of the 1, 5-glutaraldehyde is 0.5% -2%;
optionally, the 1, 5-glutaraldehyde mass volume concentration is 1%;
optionally, the porogen comprises at least one selected from dimethyl sulfoxide solution, sucrose, sodium chloride and polyethylene glycol;
optionally, step (B) further comprises washing the frozen microcarriers with a detergent;
optionally, the cleaning agent comprises a solvent selected from sodium borohydride solution, ddH 2 At least one of O and potassium borohydride;
optionally, the cleaning agent is sodium borohydride solution, and the mass volume concentration of the sodium borohydride solution is 0.05-0.4%;
optionally, the sodium borohydride solution has a mass volume concentration of 0.1% -0.2%;
Optionally, in step (B), the temperature of the freezing treatment is from-10 ℃ to-30 ℃.
9. An osteochondral organoid, characterized in that it is produced by the method according to any one of claims 1-8.
10. Use of an osteochondral organoid according to claim 9 for the preparation of a model of a disease associated with osteochondral.
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