CN116948954A - Method for constructing aging cartilage organoids - Google Patents
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Abstract
The application mainly relates to a method for constructing an aging cartilage organoid, which comprises the steps of separating and culturing mouse cartilage to obtain chondrocytes, fully and uniformly mixing the chondrocytes with natural polymer hydrogel, constructing the cartilage organoid by using a 3D biological printing technology, and finally inducing the cartilage organoid into the aging cartilage organoid. The method can realize high-throughput culture and rapid prototyping, has uniform morphological structure, retains the characteristics of the aging cartilage from gene molecules to cells to tissue physiology and pathology, and can be used for research of the pathogenesis of the aging cartilage and evaluation of screening medicines and therapies.
Description
Technical Field
The application mainly relates to the technical field of in-vitro bone organoid construction, in particular to a method for constructing an aging cartilage organoid.
Background
Osteoarthritis (OA) is the most common joint disease worldwide, with nearly 2.5 million people worldwide suffering from osteoarthritis, bringing tremendous mental and physical burden to the elderly. Cell senescence is one of the key factors in the pathogenesis of osteoarthritis and is characterized by permanent cell cycle arrest, resistance to apoptosis, and sustained secretion of secreted phenotype associated with aging (SASP) factors. Aged chondrocytes are found in the tissues of osteoarthritis patients, and accumulate in articular cartilage with age, and at present, the mechanism and treatment of cartilage aging are still unclear. Therefore, research on cartilage aging has become one of the important research directions in the current medical field.
In the past few decades, many models, such as 2D cell culture models, animal models, and 3D cell culture models, have been constructed for studying cartilage aging. However, these models have limitations such as the inability of monolayer cell culture models to fully mimic the real physiological environment in humans, such as cell-to-surrounding tissue, cell-to-cell interactions, cell-to-matrix interactions, etc.; the similarity of animal models cannot be clinically consistent and even animal models similar to human physiology cannot fully mimic human physiological responses. Moreover, the representativeness of animal models is not completely satisfactory, and individual differences exist even in the same animal; most current systems of 3D cell culture models are not capable of completely simulating in vivo biomechanical dynamic characteristics, and animal-or human-derived support materials have a certain pathogenic risk. These limitations not only restrict the understanding and understanding of cartilage tissue by researchers, but also restrict the treatment and prevention of cartilage tissue related diseases.
Disadvantages of the prior art:
cell phenotype of monolayer cell culture technology is easy to change, and research on aging factors is not convinced.
The number of tissue explant technologies is limited and the variability is high, and the preparation of anti-aging drug models has certain limitations.
The bioreactor technology is easy to control, the aging genetic mark is unstable and the expression rate is not high, and certain limitation is generated for the construction of an aging model. The following drawbacks exist as in conventional organoid culture techniques: 1. the epigenetic signature is formed inefficiently and will eliminate a portion of the aging. 2. Lack of standardized preparation procedures results in lower reproducibility. 3. Failing to maintain for a longer period of time, cells exhibit accelerated senescence after being cultured for a period of time. 4. Microenvironments are often absent, and thus, the cellular interstitial interactions are absent, which does not better mimic the complexity of the tissue under study. 5. Organoid culture is expensive and the time-consuming generation of an organoid of a patient's complexity, which hampers the promise of organoids in clinical and application transformations.
The foregoing background knowledge is intended to assist those of ordinary skill in the art in understanding the prior art that is closer to the present application and to facilitate an understanding of the inventive concepts and aspects, and it should be understood that the foregoing background art should not be used to assess the novelty of the inventive concepts that lie in the absence of explicit evidence that such disclosure is already disclosed at the time of filing of this patent application.
Disclosure of Invention
In order to solve at least one technical problem mentioned in the background art, the application aims to provide a method for constructing an aging cartilage organoid by using a 3D biological printing technology, wherein the 3D biological printing technology is utilized to construct the cartilage organoid and induce the cartilage organoid into the aging cartilage organoid, the method can be used for high-throughput culture and rapid prototyping, has uniform morphological structure, retains the characteristics of aging cartilage from gene molecules to cells to tissue physiology and pathology, and can be used for research of pathogenesis of the aging cartilage and evaluation of screening drugs and therapies.
A method for constructing a senescent cartilage organoid comprises the steps of firstly, separating and culturing mouse cartilage to obtain chondrocytes, then fully and uniformly mixing the chondrocytes with gelatin-sodium alginate hydrogel, constructing the cartilage organoid by using a 3D biological printing technology, and finally, inducing the cartilage organoid into the senescent cartilage organoid.
As a preference for the technical solution of the application, the method specifically comprises the following steps:
s1, taking mouse cartilage, digesting the mouse cartilage by collagenase, and culturing the mouse cartilage for later steps;
s2, preparing gelatin-sodium alginate hydrogel as biological ink;
s3, uniformly mixing the biological ink with the cells obtained in the step S1, transferring the mixture into a printing material cylinder of a 3D biological printer, printing according to preset parameters and paths to obtain cartilage organoids, and using CaCl (CaCl) 2 After crosslinking the solution, washing with PBS;
s4, culturing the cartilage organoids obtained in the step S3 in a culture medium;
s5, performing intervention and aging induction on the cartilage organoids after the culture is completed, transferring the cartilage organoids into a culture medium after the induction is completed, and then incubating the cartilage organoids to obtain the aged cartilage organoids.
As a preferable aspect of the present application, the step S1 specifically includes: cartilage is taken off from the joint surface of the male rat, soaked in 0.1% type II collagenase diluent, continuously digested for 8-12 hours, filtered and centrifuged, and chondrocytes are planted in a culture solution and used for subsequent experiments when the culture is carried out for the third generation.
As the preference of the technical scheme of the application, in the step S2, the weight ratio of the gelatin to the sodium alginate in the gelatin-sodium alginate hydrogel is 5-9:5-1.
As a preference for the technical scheme of the application, in the step S2, before preparing the gelatin-sodium alginate hydrogel, the gelatin and the sodium alginate are irradiated by ultraviolet rays for at least 30min.
As the preferable choice of the technical scheme of the application, in the step S3, the uniform mixing is specifically realized by stirring to be uniform by using a Pasteur pipette, and the air bubbles are removed by centrifugation by using an ultracentrifuge at 600-1000rpm for 3-10 min.
As a preferable aspect of the present application, in step S3, the preset parameters include: the temperature of the spray head is controlled to be 10-20 ℃, the temperature of the platform is controlled to be 1-4 ℃, the number of printing layers is 3-20, the thickness of each layer is 0.1-0.5mm, the printing air pressure is 0.1-0.5MPa, and the printing speed is 1-20mm/s.
As a preferred embodiment of the present application, in the step S3, the CaCl is 2 The mass fraction of the solution is 1-5wt%, and CaCl is used 2 The time for crosslinking the solution is 1-20min.
As a preferred embodiment of the present application, in the step S3, the washing with PBS is performed at least three times.
As a preferable aspect of the present application, in the step S4, the time of the culturing is 1-7d.
As a preference for the solution of the application, in said step S5, said intervention induces senescence to be selected from H 2 O 2 One of the ways of inducing senescence, IL-1 beta-induced senescence and doxorubicin-induced senescence.
As a preference for the technical scheme of the application, the H 2 O 2 The aging induction comprises transferring cultured cartilage organoid to H with concentration of 10-500 μm 2 O 2 Inducing in the solution for at least 2h.
As a preference for the technical scheme of the application, in the step S5, the re-incubation is performed in an incubator at 37 ℃ for at least 70 hours.
Use of the foregoing method for constructing a senescent cartilage organoid.
The application comprises the application of the aged cartilage organoids obtained by the method in drug screening and/or therapy screening.
The beneficial effects of the application are as follows:
according to the application, the cartilage organoids are constructed by utilizing a 3D biological printing technology after fully and uniformly mixing the murine chondrocytes and the gelatin-sodium alginate hydrogel, and then the aged cartilage organoids are obtained by intervention and aging induction, the aged cartilage organoids obtained by the 3D biological printing technology are closer to natural tissues, can be cultured in a high-throughput manner and are rapidly formed, have uniform morphological structures, retain the characteristics of aged cartilage from gene molecules to cells to tissue physiology and pathology, can be used for research of pathogenesis of aged cartilage, evaluation of screening of medicines and therapies, are used for individuation accurate treatment, and provide excellent technical platforms and disease models for research and development of regenerative medicine.
Drawings
To make the above and/or other objects, features, advantages and examples of the present application more comprehensible, the accompanying drawings which are needed in the detailed description of the present application are simply illustrative of the present application and other drawings can be obtained without inventive effort for those skilled in the art.
FIG. 1 is a flow chart of a method of constructing a senescent cartilage organoid using 3D bioprinting techniques;
FIG. 2 is a technical roadmap for isolated culture and identification of chondrocytes;
FIG. 3 is a technical roadmap for the preparation and characterization of bio-ink;
FIG. 4 is a technical roadmap for constructing cartilage organoids and performance assessment using 3D bioprinting techniques;
FIG. 5 is a technical roadmap for the construction and performance assessment of senescent cartilage organoids.
Detailed Description
Suitable substitutions and/or modifications of the process parameters will be apparent to those skilled in the art from the disclosure herein, however, it is to be expressly pointed out that all such substitutions and/or modifications are intended to be encompassed by the present application. While the products and methods of preparation of the present application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the products and methods of preparation described herein without departing from the spirit and scope of the application.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The present application uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Unless specifically stated otherwise, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described herein.
In recent years, 3D bioprinting technology has been increasingly applied to the medical field. One of the significant studies is to use 3D bioprinting technology to make tissue/organ models in order to study the occurrence mechanism of related diseases in depth, explore therapeutic methods, and provide assistance for clinic. Currently, more and more researchers are exploring the possibility of constructing cartilage organoids in vitro using 3D bioprinting techniques. This technique can simulate real cartilage tissue by stacking biological materials into a 3D structure. Researchers have successfully used this technique to make cartilage organoids such as ears, nose and ribs. Manufacturing cartilage organoid studies using 3D bioprinting techniques have the following advantages:
firstly, the 3D biological printing technology can build a model according to real human knee joint cartilage data, and can restore the structure and the form of cartilage more truly. Therefore, the mechanism of the cartilage cell aging can be analyzed more accurately, and more reliable data support is provided for researching related diseases.
Secondly, the 3D biological printing technology can adjust parameters of the model according to the needs, including size, shape, hardness and the like, and can simulate the change condition of cartilage at different stages. Thus, the occurrence mechanism of different diseases can be deeply studied, and effective treatment methods can be searched.
Finally, the cartilage research model manufactured by using the 3D biological printing technology can be used for manufacturing a plurality of models in a short time, so that the research efficiency can be greatly improved, and the research period can be shortened.
In summary, the use of 3D bioprinting techniques to manufacture cartilage organoids has important research implications and application value. Therefore, the present study aims to construct aged cartilage organoids by 3D bioprinting technology, providing new exploration and practice for the application of 3D bioprinting technology in biomedical field.
The application provides a method for constructing an aging cartilage organoid by a 3D biological printing technology, which takes cells cultured by joint cartilage of a 4-week-old SD rat as seed cells, combines natural polymer hydrogel with the seed cells, and constructs the aging cartilage organoid in vitro by the 3D biological printing technology. Compared with other models, the aging cartilage organoid constructed by the 3D bioprinting technology is closer to natural tissues, the morphology and structure of the aging cartilage organoid are uniform, the characteristics of physiological pathology of the aging cartilage from gene molecules to cells to tissues are reserved, the model can be rapidly formed, is a high-throughput culture platform, and provides an excellent technical platform and a disease model for research of aging cartilage pathogenesis, evaluation of screening of medicines and therapies, personalized accurate treatment and research and development of regenerative medicine.
The culture medium comprises: 500mL of basal medium plus 50mL of 10% fetal bovine serum plus 5mL of 1% diabody.
The present application is described in detail below.
Example 1:
the method for constructing the aging cartilage organoid by using the 3D biological printing technology is provided, cartilage cells are obtained after the rat cartilage is separated and cultured, then the cartilage cells are fully and uniformly mixed with gelatin-sodium alginate hydrogel, the cartilage organoid is constructed by using the 3D biological printing technology, and finally the cartilage organoid is induced to be the aging cartilage organoid.
Specifically, as shown in fig. 1, the method for constructing the aged cartilage organoids by using the 3D bioprinting technology specifically comprises the following steps.
Isolated culture of S1 chondrocytes
Cartilage is taken off from joint surface of 4-week-old male SD rat, soaked in 0.1% type II collagenase diluent, continuously digested for 8 hours, filtered and centrifuged, chondrocytes are planted in a 100mm culture dish, and cultured to the third generation for subsequent experiments, and a detailed isolated culture and identification technical route diagram is shown in FIG. 2.
Identification of chondrocytes
(1) Observing by an inverted microscope: when the chondrocytes are observed under an inverted microscope, the just inoculated chondrocytes are in a spherical shape, the chondrocytes begin to grow in an adherent way for 10-12 hours, the chondrocytes complete spreading for 24 hours, and the chondrocytes often take a long fusiform, a triangular shape and a polygonal shape, and after 72 hours, the chondrocytes begin to proliferate and grow in a single layer.
(2) HE staining: after HE staining, chondrocytes are seen to be long fusiform, triangular and polygonal, nuclei are seen to be purple blue, obvious double-nucleolus or polynuclear kernel forms are seen, cell matrixes are red stained, and purplish red or red metastaining occurs in cytoplasm and around cells.
(3) Type ii collagen characterization: the cytoplasm and the envelope are clearly green fluorescent after the immunofluorescent staining of the type II collagen, and the nucleus area does not have obvious green fluorescent, so that the characteristic type II collagen of the chondrocyte is mainly distributed on the cytoplasm and the cell membrane.
(4) Proteoglycan characterization: the blue staining of toluidine can be seen that the chondrocytes are purple blue, the cell matrix is blue, and blue-purple metachromatic particles are arranged in and around the chondrocytes.
S2 preparation of biological ink
Gelatin (Gt) -sodium alginate (Alg) hydrogel with concentration ratio of 7:3 is set as the biological ink for subsequent experiments. The specific preparation steps of the biological ink are as follows: firstly, weighing 3.5g of 7% gelatin and 1.5g of 3% sodium alginate according to mass fraction, pouring gelatin and sodium alginate into a 250mL beaker sterilized at high temperature and high pressure for half an hour according to ultraviolet rays, then using sterilized ultrapure water to fix the volume to 50mL, preparing 7Gt-3Alg mixed hydrogel, and placing the mixed hydrogel on a magnetic stirrer at 50 ℃ and 400rpm for stirring for 5 hours to obtain biological ink; the technical route diagram of the preparation and characterization of the biological ink is shown in fig. 3.
Characterization of biological ink
(1) Morphology and structural characterization: the surface morphology and microstructure of the scaffold after freeze-drying can be observed by using an SEM scanning electron microscope.
(2) Tensile/compressive property test of 3D printed hydrogels: the experiment adopts a universal tester to test the tensile/compression performance of the 3D printing hydrogel.
(3) Swelling and Water absorption testing of 3D printed hydrogels: the swelling and water absorption of each layer of 3D printed hydrogel was determined by a weighing method.
(4) Porosity test of 3D printed hydrogels: the experiment adopts an ethanol substitution method to test the porosity of each layer of 3D printing hydrogel.
(5) 3D printing hydrogel degradation rate test: the in vitro degradation rate of each layer of 3D printing hydrogel is tested by adopting a weighing method.
S3 constructing cartilage organoids by using 3D biological printing technology
Using a Pasteur pipette, 5mL of bio-ink and 10 mL of bio-ink were separately aspirated 5 Mixing cells/mL and 500 mu L of chondrocytes, stirring to be uniform by using a Pasteur pipette, centrifuging by using an ultracentrifuge at 1000rpm for 3min to remove bubbles to obtain mixed sol, transferring the mixed sol into a 3D biological printing barrel by using a syringe, ultrasonically removing bubbles, and storing in a refrigerator at 4 ℃.
Printing of cartilage organoids was performed by using a 3D bioprinter Bio-Architect PX manufactured by kuwanofi technologies, inc. And finally realizing biological 3D printing of each layer of cartilage organoids according to a cuboid reticular structure model (21 mm multiplied by 2 mm) designed by CAD/CAM software and a printing mode of alternately superposing 0-90 degrees. Placing the prepared mixed hydrogel in a 3D biological printer in a freezing box, starting the 3D biological printer, opening software, connecting equipment, introducing a 3D cuboid reticular structure model into control software of the 3D printer, controlling the temperature of a spray head to 15 ℃, controlling the temperature of a platform to 4 ℃, and placing a charging basket in the spray head for 15min. The 35mm culture dish is placed behind a platform to measure height, printing parameters such as air pressure 0.4MPa, speed 10mm/s, filling interval 2.0mm, size 21mm multiplied by 2mm, layer height 0.25mm, layer number 8, and after a print task is started, the printer builds a model layer by layer according to the set parameters and paths. After printing, the cartilage organoids were soaked in 2wt% CaCl 2 The solution is physically crosslinked for 10min, and CaCl is removed 2 The PBS was washed three times.
S4 culturing cartilage organoids
Culturing the cartilage organoid obtained in the step S3 in a culture medium for 3d.
A technical roadmap for constructing cartilage organoids and performance assessment using 3D bioprinting techniques is shown in FIG. 4 to assess the performance of 3D bioprinting to construct cartilage organoids
(1) The printed scaffolds and cells were cultured in complete medium, and live/dead staining and CCK8 experiments were performed at 1, 3, and 7d, and the biocompatibility of the hydrogel scaffolds was evaluated according to the experimental results.
(2) HE staining to observe the proliferation of chondrocyte; safranin-O staining observed secretion of extracellular matrix.
(3) RT-PCR detects type II collagen (COL 2A 1) and proteoglycan (ACAN).
S5 construction of aging cartilage organoids
Transfer of 3D bioprinted cartilage organoids to Medium diluted H 2 O 2 In a subsequent intervention-induced senescence experiment, in particular to a transfer to H at a concentration of 300. Mu.M 2 O 2 In solution; the cartilage organoids were induced for 2h, after which they were transferred to fresh medium in an incubator at 37 ℃ and incubated for a further 70h for subsequent evaluation. The construction and performance evaluation roadmap of the senescent cartilage organoids is shown in figure 5.
Assessment of the Properties of aged cartilage organoids
(1) SA-beta-gal stabilizing detects the percentage of senescent chondrocytes;
(2) Semi-quantitatively detecting the expression condition of p16INK4a, p21 and GAPDH proteins by a Western blot method;
(3) The RT-PCR method detects the expression of p16INK4a, p21 and GAPDH genes.
And taking the cells obtained after the separation and culture of the articular cartilage of the SD rat with the age of 4 weeks as seed cells, combining natural polymer hydrogel with the seed cells, constructing the aging cartilage organoids in vitro by utilizing a 3D biological printing technology, and finally obtaining the aging cartilage organoids through aging induction. The organoid constructed by the 3D bioprinting technology is closer to natural tissues, has uniform morphological structure, retains the characteristics of aging cartilages from gene molecules to cells to tissue physiology and pathology, can be rapidly formed, is a high-throughput culture platform, and provides an excellent technical platform and a disease model for research on aging cartilages pathogenesis, evaluation of screening of medicines and therapies, individuation accurate treatment and research and development of regenerative medicine.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the application. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the application pertains or may be substituted in a similar manner without departing from the spirit of the application or beyond the scope of the appended claims.
While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. While the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the application extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Therefore, the present application is not intended to be limited by the specific disclosure of the preferred embodiments herein.
The application is a well-known technique.
Claims (10)
1. A method of constructing a senescent cartilage organoid, comprising: the cartilage of the rat is separated and cultured to obtain chondrocytes, then the chondrocytes and gelatin-sodium alginate hydrogel are fully and uniformly mixed, a 3D biological printing technology is utilized to construct cartilage organoids, and finally the cartilage organoids are induced to be aged cartilage organoids.
2. The method of constructing a senescent cartilage organoid according to claim 1, wherein: the method specifically comprises the following steps:
s1, taking mouse cartilage, digesting the mouse cartilage by collagenase, and culturing the mouse cartilage for later steps;
s2, preparing gelatin-sodium alginate hydrogel as biological ink;
s3, uniformly mixing the biological ink with the cells obtained in the step S1, transferring the mixture into a 3D biological printer, printing according to preset parameters and paths to obtain cartilage organoids, and using CaCl 2 After crosslinking the solution, washing with PBS;
s4, culturing the cartilage organoids obtained in the step S3 in a culture medium;
s5, performing intervention and aging induction on the cartilage organoids after the culture is completed, transferring the cartilage organoids into a culture medium after the induction is completed, and then incubating the cartilage organoids to obtain the aged cartilage organoids.
3. The method of constructing a senescent cartilage organoid according to claim 2, wherein: the step S1 specifically includes: cartilage is taken off from the joint surface of the male rat, soaked in 0.1% type II collagenase diluent, continuously digested for 8-12 hours, filtered and centrifuged, and chondrocytes are planted in a culture solution and used for subsequent experiments when the culture is carried out for the third generation.
4. A method of constructing a senescent cartilage organoid according to claim 2 or 3, wherein: in the step S2, the weight ratio of the gelatin to the sodium alginate in the gelatin-sodium alginate hydrogel is 5-9:5-1.
5. A method of constructing a senescent cartilage organoid according to claim 2 or 3, wherein: in the step S3, the preset parameters include: the temperature of the spray head is controlled to be 10-20 ℃, the temperature of the platform is controlled to be 1-4 ℃, the number of printing layers is 3-20, the thickness of each layer is 0.1-0.5mm, the printing air pressure is 0.1-0.5MPa, and the printing speed is 1-20mm/s.
6. A method of constructing a senescent cartilage organoid according to claim 2 or 3, wherein: in said step S5, said intervention induces senescence to be selected from H 2 O 2 One of the ways of inducing senescence, IL-1 beta-induced senescence and doxorubicin-induced senescence.
7. The method of constructing a senescent cartilage organoid according to claim 6, wherein: the H is 2 O 2 The aging induction comprises transferring cultured cartilage organoid to H with concentration of 10-500 μm 2 O 2 Inducing in the solution for at least 2h.
8. A method of constructing a senescent cartilage organoid according to claim 2 or 3, wherein: in the step S5, the re-incubation is performed in an incubator at 37 ℃ for at least 70 hours.
9. Use of the method of any one of claims 1-8 for constructing a senescent cartilage organoid.
10. The use according to claim 9, characterized in that: the application comprises the application of the aged cartilage organoids obtained by the method in drug screening and/or therapy screening.
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