Patent Application
20250009939
This non-provisional application claims priority under 35 U.S.C. § 119 (a) on Patent Application No(s). 112125512 filed in Taiwan on Jul. 7, 2023, the entire contents of which are hereby incorporated by reference.
The present invention relates to cell culture technology, specifically to a vascular bone organoid and its compression-perfusion Fabricator system.
The concept and technology of organoids have been gradually developing in recent years. Organoids are primarily generated using stem cells to create 3D structures that mimic the functions of human organs and tissues. Compared with traditional 2D cell cultures or animal models, organoids represent the physiological model of human organs more accurately. Organoids can serve as models for studying organ development, disease mechanisms, drug screening, and personalized medicine. In the fields of drug development and pathology research, organoids provide a powerful tool for investigating the molecular and cellular mechanisms of diseases and developing new treatments and therapies. Moreover, organoids offer realistic models for drug efficacy and toxicity testing and can be used in personalized medicine allowing clinicians to test potential treatments on the patient's cells before administering them. This technology enables faster and more accurate diagnoses, as well as the development of more effective treatments with fewer side effects.
Bone organoids with a similar structure to real bone tissue are fabricated through tissue engineering and stem cell technology. Bone organoids can simulate the structure, function, and physiological characteristics of bones, aiding in studying skeletal development, disease progression, and related treatment methods. For example, osteoporosis and its associated fractures have always been a significant global health issue, yet there are currently no ideal treatments for osteoporosis, with most developed drugs having cardiovascular side effects. One major reason for the above problem is the lack of a good research model. Most of the existing drug development models overly focus on the effects on bone tissue while neglecting the effects on other tissues. Osteogenesis and angiogenesis are inseparable, and the development of osteoporosis drugs must consider both the impact on bone tissue and vasculature. Bone organoids, created using tissue engineering technology and biological principles, represent a new in vitro concept capable of simulating complex in vivo organ functions and have the potential to become important models for pathology or drug development in the future.
However, most of the relevant researches are currently difficult to make progress due to bone organoids requiring mineralization to increase the hardness and strength. Additionally, the complex distribution of vascular networks within the bone marrow cavity poses a significant obstacle to integrating bone cells with vascular endothelial cells to mimic the actual organ structure within the human body remains an intractable problem in this field.
Furthermore, in the physiological environment within the body, actual bone organs will be affected by various solid and fluid physical forces such as pressure, tension, torsion, and shear stress. Multiple studies have indicated that mechanical stimuli can alter the shape of bone organs and enhance their hardness and strength. However, the mechanisms by which physical forces are translated into molecular and biological signals remain unclear. Therefore, to develop effective and physiologically relevant models for pathological or drug development studies, overcoming the challenge of accurately simulating the complex bone tissue environment ex vivo, including mechanical forces, fluid shear stress, and tissue architecture, is urgently needed for the development of organoids.
The present invention provides a compression-perfusion fabricator system, comprising:
In some embodiments, the system further comprises at least one support bracket for fixing the compression device, the perfusion chamber, and the perfusion device.
In some embodiments, the microcomputer driver further comprises a flow sensor.
In some embodiments, the system is used for culturing and differentiating at least one stem cell, wherein the stem cell is selected from the group consisting of osteoprogenitor cells (OPCs), human umbilical vein endothelial cells (HUVEC), mesenchymal stem cells (MSC), and hematopoietic stem cells.
In some embodiments, the perfusion device applies pressure through the peristaltic pump to transport fresh culture medium from the culture medium bottle into the perfusion chamber via tubing, and to transport the culture medium from the perfusion chamber into the culture medium recycling bottle via tubing.
In some embodiments, the microcomputer driver controlled the peristaltic pump by adjusting control parameters displayed on the control panel.
In some embodiments, the control parameters comprise liquid flow rate, liquid flow volume, and perfusion time.
In some embodiments, the compression device shortens the distance between the first electric gripper jaw and the second electric gripper jaw through the micromotor to generate pressing force on the cell growth area.
In some embodiments, the microcomputer driver controlled the micromotor by adjusting control parameters displayed on the control panel.
In some embodiments, the control parameters comprise the position of the first electric gripper jaw, the position of the second electric gripper jaw, the compression time of the first and second electric gripper jaws, the compression rate of the first and second electric gripper jaws, the amount of the compressive force, the duration of the compressive force, the frequency of the compressive force, and the amount of deformation.
In some embodiments, the compression device secures an organoid in the cell growth area with the first and second electric gripper jaws and immerses the organoid in the perfusion chamber.
In some embodiments, the organoid is a vascular bone organoid, comprising:
In some embodiments, the compression-perfusion fabricator system is used for culturing an organoid.
In one aspect, the present invention provides a vascular bone organoid, comprising:
In some embodiments, the first osteogenic mesh scaffold, the second osteogenic mesh scaffold, and the vascular mesh scaffold are made by 3D printing.
In some embodiments, the first, second, and third biocompatible materials are independently selected from the group consisting of polycaprolactone, gelatin methacryloyl (GelMa), Pluronic F127, chitosan, collagen, and alginate.
In some embodiments, the vascular bone organoid is cultured through the compression-perfusion fabricator system as described in the present invention, wherein the vascular bone organoid is cultured in the cell growth area.
In another aspect, the present invention provides a method for culturing organoids, comprising:
In some embodiments, the stem cell is selected from the group consisting of osteoprogenitor cells (OPCs), human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells (MSCs), and hematopoietic stem cells.
In some embodiments, the method is used for culturing the vascular bone organoid as described in the present invention, wherein the vascular bone organoid is cultured in the cell growth area.
Compared with traditional 2D cell culture, the vascular bone organoid disclosed in the present invention combines 3D bioprinting technology and stem cell generation to simulate the 3D structure and functions of human bone organs, which more accurately reflect the actual physiological conditions of human organs.
Furthermore, compared with traditional static culture, the compression-perfusion fabricator system disclosed in the present invention allows for precise simulation of the physiological environment of human bone tissue, including mechanical forces and fluid shear stress. This is achieved through a microcomputer driver that adjusts parameters such as compression force, compression duration, compression frequency, perfusion flow rate, and perfusion velocity. By utilizing the compression-perfusion fabricator system, researchers can conduct more accurate and effective studies on bone tissue disease models, drug screening, and molecular mechanisms.
The present invention can further utilize a patient's bone marrow cells and cooperate with the scaffold materials, to fabricate vascular bone organoids through 3D bioprinting. Under the stimulation of mechanical compression and perfusion fluid systems, this approach can rapidly promote vascularization within bone tissue and enhance extracellular matrix mineralization (increasing bone tissue hardness). Additionally, the compression-perfusion fabricator system serves as an in vitro research instrument that simulates the normal living environment of bone cells in vivo. This is advantageous for solving the problem of detailed mechanisms by which mechanical and fluid forces stimulate bone and vascular formation, thereby contributing to the development of more effective and safer treatments for osteoporosis and related fractures.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In view of the problems and shortcomings of traditional techniques, the present invention utilizes 3D bioprinting and cell culture technologies to fabricate a vascular bone organoid. The vascular bone orgnoid combined with the compression-perfusion fabricator system, which provides mechanical stimulation through compression and perfusion forces simulates the effects of the real in vivo environment, offering an effective model for drug screening and pathological research.
It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory but are not restrictive of the invention as claimed. Certain details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the non-exhaustive list of representative examples that follows, and also from the appending claims.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.
As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients. Unless explicitly stated otherwise in the context, the terms “or” and “and/or” can be used interchangeably.
As used interchangeably herein, “around”, “about” and “approximately” shall generally mean plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 1% means in the range of 0.9% to 1.1%. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of” is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.
Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, medicine, pharmacy, genetics, and protein and nucleic acid chemistry, hybridization, and 3D printing described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.
To facilitate the understanding of the technical features, content, advantages, and efficacy of the present invention, the invention is described in detail below with reference to the accompanying drawings and in the form of embodiments. The purpose of the drawings is only for illustration and auxiliary description, and may not necessarily represent the actual proportions and precise configurations of the invention as implemented. Therefore, the proportions and spatial relationships shown in the drawings should not be interpreted as limiting the scope of the present invention in actual implementation.
The present invention provides a compression-perfusion fabricator system, comprising:
In some embodiments, the system further comprises at least one support bracket for fixing the compression device, the perfusion chamber, and the perfusion device.
In some embodiments, the microcomputer driver further comprises a flow sensor.
In some embodiments, the system is used for culturing and differentiating at least one stem cell, wherein the stem cell is selected from the group consisting of osteoprogenitor cells (OPCs), human umbilical vein endothelial cells (HUVEC), mesenchymal stem cells (MSC), and hematopoietic stem cells.
In some embodiments, the perfusion device applies pressure through the peristaltic pump to transport fresh culture medium from the culture medium bottle into the perfusion chamber via tubing, and to transport the culture medium from the perfusion chamber into the culture medium recycling bottle via tubing.
In some embodiments, the microcomputer driver controlled the peristaltic pump by adjusting control parameters displayed on the control panel.
In some embodiments, the control parameters comprise liquid flow rate, liquid flow volume, and perfusion time.
In some embodiments, the compression device shortens the distance between the first electric gripper jaw and the second electric gripper jaw through the micromotor to generate pressing force on the cell growth area.
In some embodiments, the microcomputer driver controlled the micromotor by adjusting control parameters displayed on the control panel.
In some embodiments, the control parameters comprise the position of the first electric gripper jaw, the position of the second electric gripper jaw, the compression time of the first and second electric gripper jaws, the compression rate of the first and second electric gripper jaws, the amount of the compressive force, the duration of the compressive force, the frequency of the compressive force, and the amount of deformation.
Please refer to
The microcomputer driver 13 is operated through the control panel 130 to control the parameters of the micromotor 103 and the peristaltic pump 122, thereby regulating the compression mechanics and perfusion mechanics stimulation in the compression-perfusion fabricator system of the present invention, and simulating the real dynamics physiological environment in the body. The parameters that the microcomputer driver 13 can control in the micromotor 103 include but are not limited to the position of the first electric gripper jaw, the position of the second electric gripper jaw, the compression time of the first and second electric gripper jaws, the compression rate of the first and second electric gripper jaws, the amount of the compressive force, the duration of the compressive force, the frequency of the compressive force, and the amount of deformation. The parameters that the microcomputer driver 13 can control in the peristaltic pump 122 include but are not limited to liquid flow rate, liquid flow volume, and perfusion time. In some embodiments, the microcomputer driver 13 further comprises a flow sensor to detect parameters such as the flow rate and flow rate of the liquid.
Please refer to
Please refer to
In some embodiments, the compression device secures an organoid in the cell growth area with the first and second electric gripper jaws and immerses the organoid in the perfusion chamber.
In some embodiments, the organoid is a vascular bone organoid, comprising:
In some embodiments, the compression-perfusion fabricator system is used for culturing an organoid.
In one aspect, the present invention provides a vascular bone organoid, comprising:
In some embodiments, the first osteogenic mesh scaffold, the second osteogenic mesh scaffold, and the vascular mesh scaffold are made by 3D printing.
In some embodiments, the first, second, and third biocompatible materials are independently selected from the group consisting of polycaprolactone, gelatin methacryloyl (GelMa), Pluronic F127, chitosan, collagen, and alginate.
Please refer to
In some embodiments, the vascular bone organoid is cultured through the compression-perfusion fabricator system as described in the present invention, wherein the vascular bone organoid is cultured in the cell growth area.
In another aspect, the present invention provides a method for culturing organoids, comprising:
In some embodiments, the stem cell is selected from the group consisting of osteoprogenitor cells (OPCs), human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells (MSCs), and hematopoietic stem cells.
In some embodiments, the method for culturing organoids is used for culturing the vascular bone organoid as described in the present invention, wherein the vascular bone organoid is cultured in the cell growth area.
As used herein, the term “dynamic microenvironment” refers to the constantly changing and interacting local environments that surround cells and tissues. They are influenced by various factors such as biochemical signals, physical forces, cellular interactions, and the presence of extracellular matrix components.
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
In this example, the producing process of the vascular bone organoid is divided into preparing the osteogenic mesh scaffold and prepareing the vascular mesh scaffold.
In this embodiment, the first osteogenic mesh scaffold and the second osteogenic mesh scaffold of the vascular bone organoid are made through 3D scaffold printing technology, wherein the osteoprogenitor cells (OPCs) are cultured on the scaffold through 3D cell culture technology. The specific steps comprise: Designing the scaffold model using 3D computer-aided design software (Tinkercad) for subsequent 3D printing.
Filling the hot-melt extruder with polycaprolactone (PCL) pellets and installing the extruder onto the BioX bioprinter (Cellink, Sweden).
Using a 27G stainless steel nozzle for printing with the following parameters: 25% density, a temperature of 180° C., a pressure of 200 kPa, and a speed of 1.2 mm/s. After printing, allow the PCL to cool and solidify, changing from transparent to white, then remove it from the culture dish.
Sterilizing the printed PCL scaffold by soaking it in 75% ethanol for 10 minutes. Then, soak the PCL scaffold in a 1:1000 Poly-L-Lysine solution and place it in an incubator at 37° C. and 5% CO2 for one hour to enhance cell adhesion. Wash the scaffold three times with PBS.
Seeding 50 μL of cell suspension containing 2×105 OPCs onto the PCL scaffold. After one hour, adding osteoblast medium (ObM) and incubating overnight. Sandwiching a GelMA scaffold without cells between two PCL scaffolds containing OPCs, and adding 1 mL of ObM for incubating. Change the medium every three days.
In this embodiment, the vascular mesh scaffold of vascular bone organoid is fabricated using 3D scaffold printing technology. Subsequently, human umbilical vein endothelial cells (HUVECs) were seeded and cultured using 3D cell culture technology. The specific steps comprise:
Designing the scaffold model using 3D computer-aided design software (Tinkercad) for subsequent 3D printing.
Preparing gelatin methacryloyl (GelMA) bio-ink at a concentration of 3 mL containing 1×107 HUVECs. Mix the GelMA and HUVEC cell suspension in a 10:1 ratio using a syringe and a specialized adapter to ensure uniform mixing. Injecting the mixture into a sterilized specialized ink cartridge to complete the preparation of the bio-ink. Installing the temperature-controlled nozzle to the BioX bioprinter and loading the ink cartridge containing the GelMA bio-ink described above. Using a 27G plastic needle for printing with the following parameters: 40% density, temperature range of 20 to 25° C., pressure range of 85 to 110 kPa, and a speed of 2 mm/s. After printing, expose to 405 nm UV light for 30 seconds to crosslink and solidify the GelMA scaffold.
After UV cross-linking, the GelMA scaffold can be easily removed from the culture dish by adding phosphate buffered saline (PBS). Transferring the scaffold to a 24-well plate and adding endothelial cell medium (ECM) for 37° C. incubating with 5% CO2. The next day, sandwiched a GelMA scaffold containing HUVECs between two sterilized PCL cell-free scaffolds and added 1 mL of ECM for incubating. Change the medium every three days.
Following the aforementioned process, taking one vascular mesh scaffold (GelMA scaffold containing HUVECs) and sandwiching it between two osteogenic mesh scaffolds (PCL scaffolds containing OPCs), forming a structure as shown in
In this embodiment, cell numbers of HUVECs (on GelMA scaffold) and OPCs (on PCL scaffold) growing on the vascular bone organoid are counted through Cell Counting Kit-8 (CCK8, Dojindo, Japan, catalog number No. CK04-11) on day 1, 4, 7, 14, 21 and 28. Each sample is performed in triplicate (n=3). Please refer to
In this example, human umbilical vein endothelial cells (HUVECs) were seeded onto the gelatin methacryloyl (GelMA) vascular mesh scaffold by the process described in Example 1. The growth of the HUVECs on the vascular mesh scaffold was observed. The vascular bone organoid described in the present invention is cultured using the compression-perfusion fabricator system of the present invention to apply compressive force and perfusion stimulation, thereby simulating the dynamic microenvironment in vivo. The cell viability of the HUVECs was observed after receiving the stimulation.
2.1 The cell growth of HUVECs
In this embodiment, Human umbilical vein endothelial cells (HUVECs) were seeded onto the Gelatin Methacryloyl (GelMA) vascular mesh scaffold as described in Example 1.2 and cultured for 21 days. Immunofluorescence (IF) staining using a CD31 antibody was performed to label the HUVECs within the GelMA vascular mesh scaffold, combined with DAPI nuclear staining to observe cell growth. The specific experimental steps comprise:
Removing the culture medium from the GelMA vascular mesh scaffold and wash with PBS. Adding 4% paraformaldehyde and shaking for 40 minutes to immobilize the cells in the scaffold. Wash three times with PBS and shake for 10 minutes each time to remove the paraformaldehyde.
Covering the scaffold with primary antibody (Mouse/rat CD31/PECAM-1 antibody, Bio Basic, Canada, Catalog No. PB0684), diluted in blocking buffer at a ratio of 1:200, and incubate at 4° C. for three days.
Wash three times with PBS and shake for 10 minutes each time. Adding the secondary antibody (Donkey anti-goat IgG FITC, Jackson, USA, Catalog No. 705-095-003), diluted in blocking buffer at a ratio of 1:200. Covering with aluminum foil paper to protect from light, and incubating at room temperature for three days. Wash three times with PBS and shake for 10 minutes each time.
Staining the cell nuclei with DAPI (BioLegend, USA, Catalog No. 422801), diluted 1:10000 in PBS, covering the scaffold and shaking for 5 minutes. Subsequently, wash three times with PBS and shake for 5 minutes each time. Transfering the scaffold to a glass-bottom culture dish and adding fluorescent mounting medium to observe the results.
Please refer to
2.2 The survival rate of HUVECs
In this embodiment, the vascular bone organoid was cultured using the compression-perfusion fabricator system for 2 days. Subsequently, compressive force stimulation was applied, with daily stimulation for 2 hours over periods of 2, 5, and 7 days. Cell number was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan, Catalog No. CK04-11) to observe the survival rate of HUVECs following the application of compressive force via the compression-perfusion fabricator system. The specific experimental steps comprise:
Adding culture medium mixed with ECM and ObM to the perfusion chamber of the compression-perfusion fabricator system described in the present invention. Adjusting the compression parameters to a deformation amount of 10%, a voltage of 10 V (frequency 0.2 Hz) to provide the compressive stimulation, and placing the compression-perfusion fabricator system in a 37° C. incubator with 5% CO2.
Before cell counting, prepare a standard concentration curve. Prepare six 5 ml centrifuge tubes, adding 4 ml of medium to the first tube and 2 ml of medium to each of the remaining tubes. Adding 2×106 cells to the first tube, mix well, then transfer 2 ml of the cell suspension to the second tube. Repeat the process sequentially up to the fifth tube. The sixth tube does not receive any cell suspension. The dilution results in standard samples with cell concentrations of 2×106 cells/4 ml, 1×106 cells/4 ml, 5×105 cells/4 ml, 2.5×105 cells/4 ml, 1.25×105 cells/4 ml, and 0 cells/4 ml.
Loading the standard samples into a 24-well plate with duplicates of 1 ml per well, resulting in cell concentrations of 5×105 cells/ml, 2.5×105 cells/ml, 1.25×105 cells/ml, 6.25×104 cells/ml, 3.125×104 cells/ml, and 0 cells/ml. Incubate the plate at 37° C. with 5% CO2 for one day to allow cells to adhere to the wells, forming the standard concentration curve.
After culturing the vascular bone organoid, remove the medium from the scaffold, wash it with PBS, and transfer the scaffold to a 24-well plate. Mix 500 μL of medium with 50 μL of CCK-8 reagent (medium to CCK-8 reagent ratio of 10:1). Cover the scaffold with the CCK-8 mixture and incubate at 37° C. with 5% CO2 for three hours.
Loading 100 μL of the well-mixed CCK-8 solution incubated for 3 hours into each well of a 96-well plate, with three replicates for each concentration. Measure the absorbance at 450 nm.
Based on the linear regression analysis of the absorbance results against the standard concentration curve, the number of cells on or within the scaffold can be extrapolated through the absorbance result of the sample.
Please refer to
2.3 The function of HUVECs
In this embodiment, the vascular bone organoid was cultured using the compression-perfusion fabricator system for 2 days. Immunofluorescence (IF) staining using a CD31 (green) antibody was performed to label the HUVECs, combined with DAPI (blue) nuclear staining. CD31 represents the functional marker of HUVECs.
Please refer to
In this example, osteoprogenitor cells (OPCs) were seeded onto a polycaprolactone (PCL) osteogenic mesh scaffold using the process described in Example 1.1. Confocal microscope analysis was performed to observe the growth and differentiation of OPCs.
In this embodiment, OPCs were seeded onto a polycaprolactone (PCL) osteogenic mesh scaffold and cultured for 21 days. Immunofluorescence (IF) staining with an actin antibody was used to label the cytoskeleton of osteoblasts within the PCL osteogenic mesh scaffold, combined with DAPI nuclear staining. The specific immunofluorescence staining protocol follows the steps described in Example 2.1. The primary antibody used was an HRP-conjugated beta-actin mouse monoclonal antibody, diluted at a ratio of 1:2500.
Please refer to
In this example, the effectiveness of the compression-perfusion fabricator system described in the present invention is confirmed by analyzing the expression of PIEZO1 and endomucin (EMCN) in human umbilical vein endothelial cells (HUVECs). Furthermore, the compression-perfusion fabricator system was used to study the phosphorylation mechanism of yes-associated protein (YAP) in HUVECs. This study aims to demonstrate that the compression perfusion culture system described in the present invention can serve as an effective experimental research platform.
In this embodiment, a vascular mesh scaffold containing human umbilical vein endothelial cells (HUVECs) was prepared using the process described in Example 1.2. The vascular mesh scaffold, containing HUVECs, was then cultured in the compression-perfusion fabricator system described in the present invention. Compressive force stimulation was applied for 0 minute (NC), 30 minutes (C30), 60 minutes (C60), and 120 minutes (C120), respectively, to observe the change of expression of PIEZO1, EMCN, and the dephosphorylation of YAP protein in the cells. The specific experimental steps comprise:
After compression cultivation, remove the medium and wash the vascular mesh scaffold with PBS. Then, cut the vascular mesh scaffold into small pieces and homogenize it using a tissue grinder. Adding 200 μl of 2X RIPA buffer (Bio Basic, Canada, Catalog No. RB4475) and 2 μl of protease inhibitor cocktail (FUTURE, Taiwan, Catalog No. F1PICool). Rotating and mixing at 4° C. for 30 minutes, and centrifuge at 13,200 rpm for 30 minutes. The supernatant obtained is the protein sample.
Performing Western blotting analysis. Using anti-PIEZO1 antibody (Proteintech, USA, Cat. No. 28511-1-AP), anti-endomucin antibody (R&D Systems, USA, Cat. No. AF7206), or anti-phospho-YAP (Ser127) antibody (Cell Signaling Technology, USA, Catalog No. #4911) as the primary antibody, diluted 1:1000. Incubate at 4° C. with gentle shaking for one day. Wash away the unbound primary antibody and add the secondary antibody (Goat anti-rabbit HRP, Jackson ImmunoResearch, USA, Catalog No. 111-005-003), diluted 1:2500. Incubate at room temperature with gentle shaking for 90 minutes. After removing the secondary antibody and washing, proceed with signal detection. The signal detection method involves using chemiluminescence to detect the target protein bands. Applying electrochemiluminescence (ECL) substrate evenly over the entire transfer membrane. Placing the membrane in a chemiluminescence imaging system, adjusting the exposure time for image analysis, and using software to quantitatively analyze the protein expression levels.
While facing compressive stimulation, HUVECs will express the expression of PIEZO1 and EMCN protein. Please refer to
Please refer to
In this example, HUVECs were cultured with or without OPCs to verify the influence of cell co-culture (HUVECs and OPCs) in the vascular bone organoid described in the present invention.
In this embodiment, the term “mono-culture” represents a vascular mesh scaffold cultured without osteogenic mesh scaffold, wherein “mono-culture” can be also regarded as using the GelMa containing HUVEC inserted into two PCL plate (without OPCs) for an experiment. In this embodiment, the term “co-culture” represents a vascular mesh scaffold cultured with osteogenic mesh scaffold, wherein “co-culture” can be also regarded as using the vascular bone organoid described in the present invention for an experiment.
In this embodiment, the vascular mesh scaffold containing HUVECs was prepared using the process described in Example 1.2. The vascular bone organoid was prepared using the process described in Example 1.3. All of the samples were cultured in the compression-perfusion fabricator system described in the present invention. Compressive force stimulation was applied for 0 minute (NC), 30 minutes (C30), 60 minutes (C60), and 120 minutes (C120), respectively, to observe the change in the RNA expression level of PIEZO1 and EMCN. RNA of the samples are extracted by Total RNA purification kit (GeneMark, USA, Cat. No. TR01), and the RNA expression level is detected by RT-PCR.
Please refer to
After culture and experiments, it has been confirmed that the vascular bone organoid described in the present invention supports the survival of osteoprogenitor cells (OPCs) seeded on the first and second osteogenic mesh scaffolds, as well as human umbilical vein endothelial cells (HUVECs) seeded on the vascular mesh scaffold. These cells exhibit normal differentiation and functional responses to external stimuli. Furthermore, the compression-perfusion fabricator system described in the present invention has been experimentally validated to provide mechanical and fluid stress stimulation, simulating the real tissue environment in vivo. Cells cultured using the compression-perfusion fabricator system maintain high survival rates and normal cellular functions, demonstrating that the system can serve as an effective platform for drug development and related research. It has extensive application potential in the field of cell culture.
In summary, the present invention utilizes 3D bioprinting technology to culture human osteoprogenitor cells (OPCs) on a hollow cubic mesh polycaprolactone (PCL) osteogenic mesh scaffold. Additionally, a mixture of human umbilical vein endothelial cells (HUVECs) and gelatin methacryloyl (GelMA) hydrogel is combined with the aforementioned PCL osteogenic mesh scaffold, successfully creating vascular bone organoid capable of normal growth and functionality. Furthermore, the invention provides a compression-perfusion fabricator system that integrates mechanical compression and fluid flow stimulation into cell scaffolds. This system effectively simulates real physiological dynamic environments, enhancing cell vitality, promoting angiogenesis, and reinforcing extracellular matrix mineralization. Consequently, it increases the strength and success rate of artificial organ fabrication. This system can serve as an effective experimental animal replacement research platform or personalized drug screening platform. Moreover, it may contribute to the future development of personalized autologous organ transplantation.
Many changes and modifications in the above described embodiment of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 112125512 | Jul 2023 | TW | national |
