Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of biotechnology and regenerative medicine, in specific, relates to a method for three-dimensional (3D) bioprinting of organoids and tissue constructs using menstrual stem cells and biocompatible gels for biomedical and therapeutic applications.
Background of the invention:
[0002] Bioprinting has emerged as a transformative technology in the field of regenerative medicine, thereby offering new possibilities for tissue engineering, organ fabrication, and personalized medical treatments. Traditional methods for growing three-dimensional (3D) cell cultures involve scaffolds and specialized culture environments that aim to mimic in vivo conditions. However, these approaches often suffer from limitations such as poor structural control, difficulty in maintaining cell viability, and high costs associated with specialized growth media and substrates.

[0003] Stem cells play a crucial role in regenerative medicine due to their ability to differentiate into various cell types. Mesenchymal stem cells (MSCs) derived from sources such as bone marrow, adipose tissue, and umbilical cord blood have been extensively studied for their therapeutic potential. However, obtaining these stem cells typically involves invasive procedures, limited donor availability, and ethical concerns in the case of embryonic stem cells. Furthermore, maintaining the viability and differentiation potential of stem cells in a 3D culture environment remains a challenge in the field of bioprinting.

[0004] Recent advancements in hydrogel-based bioprinting have improved the structural integrity of printed organoids and tissues. Hydrogels are biocompatible, water-retaining polymers that provide a suitable microenvironment for cell growth and differentiation. Various biomaterials, such as alginate and Matrigel, as well as agarose and gelatin, have been explored as bio-inks for 3D bioprinting applications. However, many of these materials present challenges, such as limited cell adhesion, mechanical instability, and the inability to support long-term cell survival.

[0005] In organoid research, the ability to generate functional tissue models has significant implications for disease modeling, drug screening, and regenerative therapies. Conventional organoid cultures rely on self-assembly processes that often result in structures with inconsistent sizes and shapes, thereby limiting their applicability in personalized medicine. Additionally, the inability to precisely control the spatial organization of cells within organoids restricts their use in advanced biomedical applications.

[0006] The scalability of 3D bioprinting is another key challenge. While traditional methods allow for the generation of small-scale tissue models, the ability to produce larger, functionally complex constructs suitable for transplantation remains an ongoing area of research. Addressing these challenges requires a bio-ink formulation that supports high cell viability, structural stability, and controlled differentiation, along with a method that enables precision bioprinting for various medical applications.

[0007] Ethical considerations also play an important role in stem cell-based research. The use of embryonic stem cells is associated with ethical and legal concerns, while harvesting adult stem cells from bone marrow or adipose tissue involves invasive procedures. Thus, alternative, non-invasive, and ethically acceptable sources of stem cells are being explored to address these concerns. An ideal stem cell source should be easily accessible, abundant, and capable of differentiating into multiple cell types while maintaining high proliferative potential.

[0008] Additionally, the need for patient-specific tissue models has gained traction in precision medicine. Personalized approaches to regenerative therapy require bioprinting techniques that can tailor organoid structures to an individual’s genetic and physiological profile. This requires a combination of biomaterials and cells that can be easily adapted to different applications without compromising cell viability or function. The ability to customize bioprinted constructs using system-guided techniques presents an exciting opportunity for advancing regenerative medicine.

[0009] By addressing all the above-mentioned problems, there is a need for a method for three-dimensional (3D) bioprinting of organoids and tissue constructs using menstrual stem cells and biocompatible gels for biomedical and therapeutic applications. There is a need for a method that enables the use of menstrual stem cells as a renewable, non-invasive, and ethical source of stem cells for regenerative medicine. There is a need for a method that supports the differentiation of menstrual stem cells into multiple cell lineages, thereby making it suitable for tissue engineering and personalized medicine. There is a need for a method that maintains cell viability, proliferation, and differentiation capabilities within a 3D bioprinted structure.

[0010] Additionally, there is also a need for a method for fabricating 3D bioprinted organoids and tissue constructs with precise control over shape, size, and internal architecture using the 3D bioprinter. There is also a need for a method that allows system-guided customization of 3D bioprinted organoids and tissue constructs for patient-specific applications. There is also a need for a method that can be used for disease modeling, drug screening, and in vitro studies in biomedical research. There is also a need for a formulation and method for developing bioengineered implants, skin grafts, and organ patches for transplantation. Further, there is also a need for a cost-effective and scalable solution for 3D bioprinting applications in regenerative medicine, thereby reducing dependence on traditional organ donors and invasive stem cell extraction methods.
Objectives of the invention:
[0011] The primary objective of the present invention is to provide a method for three-dimensional (3D) bioprinting of organoids and tissue constructs using menstrual stem cells and biocompatible gels for biomedical and therapeutic applications.

[0012] Another objective of the present invention is to provide a method that enables the use of menstrual stem cells as a renewable, non-invasive, and ethical source of stem cells for regenerative medicine.

[0013] Another objective of the present invention is to provide a method that supports the differentiation of menstrual stem cells into multiple cell lineages, making it suitable for tissue engineering and personalized medicine.

[0014] Another objective of the present invention is to provide a method that maintains cell viability, proliferation, and differentiation capabilities within a 3D bioprinted structure.

[0015] Another objective of the present invention is to provide a method for fabricating organoids and tissue constructs with precise control over shape, size, and internal architecture using a bioprinter.

[0016] Another objective of the present invention is to provide a method that allows for system-guided customization of bioprinted organoids and tissue constructs for patient-specific applications.

[0017] Another objective of the present invention is to provide a method that can be used for disease modeling, drug screening, and in-vitro studies in biomedical research.

[0018] Yet another objective of the present invention is to provide a formulation and method for developing bioengineered implants, skin grafts, and organ patches for transplantation.

[0019] Further objective of the present invention is to provide a cost-effective and scalable solution for 3D bioprinting applications in regenerative medicine, thereby reducing dependence on traditional organ donors and invasive stem cell extraction methods.
Summary of the invention:
[0020] The present disclosure proposes a method for 3D bioprinting of organoids using menstrual stem cells and biocompatible materials. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0021] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for three-dimensional (3D) bioprinting of organoids and tissue constructs using menstrual stem cells and biocompatible gels for biomedical and therapeutic applications.

[0022] According to one aspect, the invention provides a method for three-dimensional (3D) bioprinting of organoids. At one step, endometrial stem cells collected from menstrual blood are isolated and purified to remove unwanted cellular debris and contaminants, thereby obtaining isolated endometrial stem cells. In one embodiment herein, the endometrial stem cells isolated from menstrual blood are multipotent and capable of differentiating into epithelial, mesenchymal, endothelial, neurogenic, or cardiomyogenic cell lineages, thereby enabling diverse tissue engineering applications. The endometrial stem cells are considered more potent and genetically stable compared to adult stem cells from other sources. The endometrial stem cells eliminate issues associated with embryonic stem cells, thereby making them more widely acceptable.

[0023] At another step, the isolated endometrial stem cells are expanded and cultured in a nutrient-rich growth medium under optimal temperature and environmental conditions to enhance viability, proliferation, and multipotency of the isolated endometrial stem cells. At another step, a bio-ink is prepared by homogenously mixing the expanded endometrial stem cells with a biocompatible hydrogel selected from at least one or a combination of Matrigel, collagen, gelatin, alginate, and fibrin and supplementing the bio-ink with a nutrient-rich culture medium containing essential growth factors to maintain cellular integrity.

[0024] In one embodiment herein, the nutrient-rich culture medium is supplemented with at least one growth factor, selected from at least one or a combination of fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-β), to enhance cell survival, proliferation, and functional differentiation.

[0025] In one embodiment herein, the biocompatible hydrogel exhibits tunable mechanical properties, thereby allowing precise control over scaffold stiffness, porosity, degradation rate, and viscoelasticity to optimize cellular behavior and tissue formation. In one embodiment herein, the bio-ink is patient-specific, thereby enabling customization of organoids, tissue constructs, and implantable grafts for personalized regenerative medicine and disease modeling applications. In one embodiment herein, the bio-ink formulation is optimized for extrusion-based, inkjet, or laser-assisted 3D bioprinting, thereby ensuring high printing fidelity, cell viability, and structural resolution of the bioprinted organoids and tissue constructs.

[0026] In one embodiment herein, the biocompatible hydrogel mimics a native extracellular matrix (ECM) by providing cell-adhesive ligands, dynamic remodeling properties, and bioactive cues, thereby enhancing cell adhesion, migration, differentiation, and tissue maturation within the 3D bioprinted organoids. The biocompatible hydrogel is configured to be crosslinked chemically, thermally, or enzymatically post-printing to enhance mechanical stability, biofunctionality, and long-term structural integrity of the 3D bioprinted construct.

[0027] At another step, the bio-ink is bioprinted using the extrusion-based, inkjet, or laser-assisted 3D bioprinter to fabricate the organoids or tissue constructs with a predefined architecture, thereby ensuring precise spatial distribution of cells within the printed structure and obtaining 3D bioprinted organoids. At another step, the 3D bioprinted organoids are cross-linked post-printing to enhance mechanical stability, biomimetic extracellular matrix properties, and controlled degradation for improved tissue integration.

[0028] Further, at other step, the 3D bioprinted organoids are cultured in a differentiation-inducing environment to guide cell fate, thereby enabling functional tissue generation for biomedical applications, which include regenerative medicine, disease modeling, and transplantation. In one embodiment herein, the 3D bioprinted organoids are subjected to dynamic bioreactor culture conditions, which include mechanical stimulation, fluid perfusion, or electrical cues, to enhance tissue maturation, vascularization, and functional integration for biomedical applications.

[0029] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0030] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0031] FIG. 1 illustrates a schematic representation of a method for three-dimensional (3D) bioprinting of organoids using menstrual stem cells and biocompatible materials, in accordance to an exemplary embodiment of the invention.

[0032] FIG. 2 illustrates an image depicting an isolation of endometrial stem cells from menstrual blood, in accordance to an exemplary embodiment of the invention.

[0033] FIG. 3 illustrates a microscopic image depicting a qualitative analysis of the endometrial stem cells before and after washing with phosphate-buffered saline (PBS), in accordance to an exemplary embodiment of the invention.

[0034] FIG. 4 illustrates a microscopic image depicting the proliferation and regeneration capacity of the endometrial stem cells, in accordance to an exemplary embodiment of the invention.

[0035] FIGs. 5A-5C illustrate images depicting the viability of the endometrial stem cells under oxidative stress and their response to various natural antioxidant treatments, in accordance to an exemplary embodiment of the invention.

[0036] FIG. 6 illustrates an image depicting the 3D bioprinting of the endometrial stem cells, in accordance to an exemplary embodiment of the invention.

[0037] FIG. 7 illustrates a flowchart of a method for three-dimensional (3D) bioprinting of organoids in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0038] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0039] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for three-dimensional (3D) bioprinting of organoids and tissue constructs using menstrual stem cells and biocompatible gels for biomedical and therapeutic applications.

[0040] According to an exemplary embodiment of the invention, FIG. 1 refers to a schematic representation of a method for three-dimensional (3D) bioprinting of organoids using menstrual stem cells and biocompatible materials. In one embodiment herein, the female reproductive system consists of the uterus, ovaries, fallopian tubes, and vagina. The uterus is lined with an inner mucosal layer known as the endometrium, which undergoes cyclic changes during the menstrual cycle. The endometrial layer contains stem cells with the potential to regenerate and differentiate into various cell lineages.

[0041] Endometrial stem cells (EnSCs) are multipotent adult stem cells found in the menstrual blood. These cells exhibit self-renewal capabilities and can differentiate into epithelial, mesenchymal, endothelial, and neurogenic cells. Due to their high proliferation rate, ease of collection, and immunomodulatory properties, they are ideal for regenerative medicine applications. The endometrial stem cells can be obtained painlessly from menstrual blood, providing a renewable and ethical stem cell source. At step 102, the menstrual blood is collected from healthy volunteers using a sterile menstrual cup or other medical-grade collection methods. Unlike invasive procedures for bone marrow or embryonic stem cells, this method is non-invasive, ethical, and easily accessible. The menstrual blood contains a heterogeneous mixture of blood cells, epithelial cells, stromal cells, and endometrial stem cells. The presence of multipotent stem cells makes it a valuable resource for biomedical research and tissue engineering.

[0042] At step 104, after collection, the menstrual blood undergoes centrifugation and purification to isolate endometrial stem cells. These cells are then cultured in a nutrient-rich growth medium under optimal temperature and pH conditions to enhance their viability, proliferation, and differentiation potential. The nutrient-rich growth medium provides essential amino acids, vitamins, minerals, and growth factors to maintain cell viability. This medium is supplemented with factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) to stimulate cellular growth and function. Once the stem cells reach an optimal growth stage, they are mixed with a biocompatible hydrogel such as Matrigel. This combination forms a bio-ink, which is further used for 3D bioprinting. The biocompatible hydrogels are three-dimensional, water-retaining polymer networks designed to mimic a native extracellular matrix (ECM) of tissues. Common hydrogels include Matrigel, collagen, gelatin, alginate, and fibrin, which facilitate cell adhesion, migration, and differentiation.

[0043] At step 106, the bio-ink, consisting of cultured menstrual stem cells and Matrigel, is loaded into a 3D bioprinter. The 3D bioprinter deposits the bio-ink layer by layer in a predesigned structure, thereby resulting in the formation of 3D bioprinted organoids or tissue constructs. 3D bioprinting is an additive manufacturing technology that enables the precise deposition of bio-inks to create complex tissue structures. It employs different bioprinting techniques, such as extrusion-based bioprinting (dispensing bio-ink through a nozzle), inkjet-based bioprinting (droplet-based printing), and laser-assisted bioprinting (using laser energy to pattern cells). After printing, the 3D bioprinted organoids or tissue constructs undergo crosslinking and further culturing to ensure the development of functional tissue.

[0044] In the present invention, the endometrial stem cells are non-invasively isolated from menstrual blood, which is otherwise considered biological waste. The isolated stem cells are subsequently purified and subjected to a series of characterization assays to evaluate their viability, regenerative potential, and cytotoxic tolerance, particularly against oxidative agents such as hydrogen peroxide. Following successful characterization, the endometrial stem cells are incorporated into biocompatible gels, including but not limited to hydrogels and Matrigel, to form a printable bio-ink. This bio-ink is then utilized in a 3D bioprinter to fabricate simple three-dimensional structures such as spheres, cubes, and spirals. These preliminary constructs serve as foundational models demonstrating the feasibility of developing complex bioprinted tissues and organs in the future, which include vital organs such as the liver, pancreas, and kidneys.

[0045] According to an exemplary embodiment of the invention, FIG. 2 refers to an image 200 depicting an isolation of endometrial stem cells from menstrual blood. The menstrual blood is collected from healthy female volunteers using sterile menstrual collection devices under appropriate ethical approvals, ensuring the anonymity of the donors. This non-invasive and ethically sound method enables the extraction of a renewable source of stem cells from menstrual waste, eliminating the need for invasive tissue harvesting procedures.

[0046] The collected menstrual blood undergoes centrifugation at 1,100 revolutions per minute (RPM) to separate its components based on density. As shown in FIG. 2, the centrifuged sample stratifies into four distinct layers: a golden brown plasma layer at the top, a pinkish-red intermediate layer composed of loosely packed waste from lysed red blood cells (RBCs), a creamish-white buffy coat layer containing white blood cells (WBCs) and endometrial stem cells (EnSCs), and a dense dark red sediment of intact RBCs at the bottom. The upper plasma and waste layers are carefully aspirated, and the buffy coat, which is enriched with EnSCs, is meticulously transferred into a sterile tube for further purification.

[0047] The harvested buffy coat is washed three times using sterile 1x phosphate-buffered saline (PBS) containing antibiotics and antifungal agents to eliminate cellular debris and microbial contaminants. This washing step ensures the removal of non-adherent cells and particulate matter. Upon completion of washing, the purified endometrial stem cells are plated in sterile culture dishes. The viable stem cells adhere to the surface of the culture plate under standard incubation conditions, thereby providing a clean and enriched population of EnSCs for downstream applications, including qualitative analysis, proliferation assays, and 3D bioprinting.

[0048] According to an exemplary embodiment of the invention, FIG. 3 refers to a microscopic image 300 depicting a qualitative analysis of the endometrial stem cells before and after washing with phosphate-buffered saline (PBS). The buffy coat layer, obtained from centrifuged menstrual blood, initially contains a heterogeneous mixture of clumped cells, menstrual debris, and non-adherent impurities. As observed under an inverted microscope, this unwashed fraction shows dense cellular suspensions and aggregates that compromise purity.

[0049] Following triple washing with sterile 1x PBS supplemented with antimicrobial agents, the same buffy coat sample is reanalyzed. The post-wash microscopic field reveals predominantly individual and well-spread adherent cells with minimal clumping or particulate debris. These adhered cells represent the purified endometrial stem cells (EnSCs), which are free of blood contaminants and suitable for further expansion. The transformation from a debris-filled suspension to a clean, adherent cell culture confirms the effectiveness of the washing protocol in isolating viable and pure EnSCs for subsequent experimental procedures.

[0050] According to an exemplary embodiment of the invention, FIG. 4 refers to a microscopic image 400 depicting the proliferation and regeneration capacity of the endometrial stem cells. The regenerative potential of EnSCs is assessed through an in vitro scratch assay conducted in a controlled laboratory setting. The stem cells are seeded into sterile 24-well plates and cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), thereby providing optimal conditions for cell growth.

[0051] Upon reaching approximately 80% confluency, a mechanical scratch is introduced into the cell monolayer using a sterile pipette tip, creating a cellular zone that simulates tissue injury. The cultured plate is then incubated at 37°C under standard atmospheric conditions for 24 hours. As shown in FIG. 4, the endometrial stem cells demonstrate significant migration and proliferation by partially repopulating the scratched area within the incubation period. These results substantiate the intrinsic regenerative capabilities of EnSCs, thereby affirming their potential application in tissue engineering and wound healing therapies.

[0052] According to an exemplary embodiment of the invention, FIGs. 5A-5C refer to images (500, 502, 504) depicting the viability of the endometrial stem cells under oxidative stress and their response to various natural antioxidant treatments. The purified endometrial stem cells (EnSCs) are cultured in 24-well plates using DMEM supplemented with 10% FBS and incubated overnight at 37°C until approximately 80% confluency is achieved. To simulate oxidative stress, the culture medium is removed and replaced with hydrogen peroxide solution, exposing the cells for a duration of one hour.

[0053] Following the oxidative insult, the endometrial stem cells are treated with four different natural antioxidant extracts, such as amla (A), green tea (GT), pomegranate (P), and garlic (GC), along with positive (C+) and negative (C−) controls. These treatments are applied for a time period of six hours at a temperature of at least 37°C, after which the antioxidants are removed and the cells are replenished with fresh growth medium. The treated plates are then incubated overnight. As shown in FIGs. 5A–5C, amla and green tea treatments exhibit no visible color change, indicating a high degree of stem cell viability and protection against oxidative damage. In contrast, pomegranate and garlic show mild color shifts, denoting partial rescue. The positive control reveals a similar moderate change, whereas the negative control demonstrates a complete color transition, thereby confirming extensive cell death due to a lack of antioxidant protection. These findings validate the differential cytoprotective effects of natural antioxidants on EnSCs exposed to oxidative stress.

[0054] According to an exemplary embodiment of the invention, FIG. 6 refers to an image 600 depicting the 3D bioprinting of the endometrial stem cells using a biocompatible matrix, forming simple geometrical constructs. Once fully characterized and expanded, the endometrial stem cells are suspended in a hydrogel-based bio-ink composed of materials such as Matrigel, phytogel, or other hydrophilic polymer gels. This hydrogel provides an extracellular matrix (ECM)-mimicking microenvironment that supports cell viability, differentiation, and spatial organization during and after printing.

[0055] The prepared bio-ink is loaded into a syringe cartridge and mounted onto a 3D bioprinter equipped with a LASER-assisted piston system. As shown in FIG. 6, the bio-ink is extruded layer-by-layer in predefined shapes, including a basic 3D cube and a 3D sphere. These simple structures represent proof-of-concept constructs and serve as foundational models for printing more complex organoids and tissues in the future. The 3D bioprinting process maintains cell viability and allows precise spatial placement of cells, thereby opening avenues for regenerative applications such as organ fabrication and customized tissue engineering.

[0056] According to an exemplary embodiment of the invention, FIG. 7 refers to a flowchart 700 of a method for the 3D bioprinting of organoids using the menstrual stem cells and the biocompatible materials. At step 702, the endometrial stem cells collected from menstrual blood are isolated and purified to remove unwanted cellular debris and contaminants, thereby obtaining isolated endometrial stem cells. In one embodiment herein, the endometrial stem cells isolated from menstrual blood are multipotent and capable of differentiating into epithelial, mesenchymal, endothelial, neurogenic, or cardiomyogenic cell lineages, thereby enabling diverse tissue engineering applications.

[0057] At step 704, the isolated endometrial stem cells are expanded and cultured in the nutrient-rich growth medium under optimal temperature and environmental conditions to enhance viability, proliferation, and multipotency of the isolated endometrial stem cells. At step 706, the bio-ink is prepared by homogenously mixing the expanded endometrial stem cells with the biocompatible hydrogel selected from at least one or a combination of the Matrigel, collagen, gelatin, alginate, and fibrin and supplementing the bio-ink with the nutrient-rich culture medium containing essential growth factors to maintain cellular integrity. In one embodiment herein, the nutrient-rich culture medium is supplemented with at least one growth factor, selected from at least one or a combination of platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β), to enhance cell survival, proliferation, and functional differentiation.

[0058] In one embodiment herein, the biocompatible hydrogel exhibits tunable mechanical properties, thereby allowing precise control over scaffold stiffness, porosity, degradation rate, and viscoelasticity to optimize cellular behavior and tissue formation. In one embodiment herein, the bio-ink is patient-specific, thereby enabling customization of organoids, tissue constructs, and implantable grafts for personalized regenerative medicine and disease modeling applications. In one embodiment herein, the bio-ink formulation is optimized for extrusion-based, inkjet, or LASER-assisted 3D bioprinting, thereby ensuring high printing fidelity, cell viability, and structural resolution of the bioprinted organoids and tissue constructs.

[0059] In one embodiment herein, the biocompatible hydrogel mimics the native extracellular matrix (ECM) by providing cell-adhesive ligands, dynamic remodeling properties, and bioactive cues, thereby enhancing cell adhesion, migration, differentiation, and tissue maturation within the 3D bioprinted organoids. The biocompatible hydrogel is configured to be crosslinked chemically, thermally, or enzymatically post-printing to enhance the mechanical stability, biofunctionality, and long-term structural integrity of the 3D bioprinted construct.

[0060] At step 708, the bio-ink is bioprinted using the extrusion-based, inkjet, or LASER-assisted 3D bioprinter to fabricate the organoids or tissue constructs with a predefined architecture, thereby ensuring precise spatial distribution of cells within the printed structure and obtaining the 3D bioprinted organoids. At step 710, the 3D bioprinted organoids are cross-linked post-printing to enhance mechanical stability, biomimetic extracellular matrix properties, and controlled degradation for improved tissue integration. Further, at step 712, the 3D bioprinted organoids are cultured in a differentiation-inducing environment to guide cell fate, thereby enabling functional tissue generation for biomedical applications, which include regenerative medicine, disease modeling, and transplantation. In one embodiment herein, the 3D bioprinted organoids are subjected to dynamic bioreactor culture conditions, which include mechanical stimulation, fluid perfusion, or electrical cues, to enhance tissue maturation, vascularization, and functional integration for biomedical applications.

[0061] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a method for three-dimensional (3D) bioprinting of organoids and tissue constructs using menstrual stem cells and biocompatible gels for biomedical and therapeutic applications is disclosed. The proposed invention provides a method that enables the use of menstrual stem cells as a renewable, non-invasive, and ethical source of stem cells for regenerative medicine.

[0062] The method supports the differentiation of menstrual stem cells into multiple cell lineages, thereby making it suitable for tissue engineering and personalized medicine. The method maintains cell viability, proliferation, and differentiation capabilities within a 3D bioprinted structure. The method fabricates organoids and tissue constructs with precise control over shape, size, and internal architecture using a bioprinter. The method allows for system-guided customization of bioprinted organoids and tissue constructs for patient-specific applications. The method can be used for disease modeling, drug screening, and in-vitro studies in biomedical research. The method develops bioengineered implants, skin grafts, and organ patches for transplantation. The method provides a cost-effective and scalable solution for 3D bioprinting applications in regenerative medicine, thereby reducing dependence on traditional organ donors and invasive stem cell extraction methods.

[0063] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application. , Claims:CLAIMS:
I/We Claim:
1. A method for formulating three-dimensional (3D) bioprinting of organoids using menstrual stem cells and biocompatible materials, comprising:
isolating and purifying endometrial stem cells collected from menstrual blood to remove unwanted cellular debris and contaminants, thereby obtaining isolated endometrial stem cells;
expanding and culturing the isolated endometrial stem cells in a nutrient-rich growth medium under at an optimal temperature and environmental conditions to enhance viability, proliferation, and multipotency of the isolated endometrial stem cells;
preparing a bio-ink by homogenously mixing the expanded endometrial stem cells with a biocompatible hydrogel selected from at least one or a combination of Matrigel, collagen, gelatin, alginate, fibrin, and supplementing the bio-ink with a nutrient-rich culture medium containing essential growth factors to maintain cellular integrity;
bioprinting the bio-ink using an extrusion-based, inkjet, or laser-assisted 3D bioprinter to fabricate organoids or tissue constructs with a predefined architecture, thereby ensuring precise spatial distribution of cells within the printed structure and obtaining 3D bioprinted organoids;
crosslinking the 3D bioprinted organoids post-printing to enhance mechanical stability, biomimetic extracellular matrix properties, and controlled degradation for improved tissue integration; and
culturing the 3D bioprinted organoids in a differentiation-inducing environment to guide cell fate, thereby enabling functional tissue generation for biomedical applications, which include regenerative medicine, disease modeling, and transplantation.
2. The method as claimed in claim 1, wherein the endometrial stem cells isolated from menstrual blood are multipotent and capable of differentiating into epithelial, mesenchymal, endothelial, neurogenic, or cardiomyogenic cell lineages, thereby enabling diverse tissue engineering applications.
3. The method as claimed in claim 1, wherein the biocompatible hydrogel exhibits tunable mechanical properties, thereby allowing precise control over scaffold stiffness, porosity, degradation rate, and viscoelasticity to optimize cellular behavior and tissue formation.
4. The method as claimed in claim 1, wherein the biocompatible hydrogel is configured to be crosslinked chemically, thermally, or enzymatically post-printing to enhance mechanical stability, biofunctionality, and long-term structural integrity of the 3D bioprinted construct.
5. The method as claimed in claim 1, wherein the bio-ink is patient-specific, thereby enabling customization of organoids, tissue constructs, and implantable grafts for personalized regenerative medicine and disease modeling applications.
6. The method as claimed in claim 1, wherein the biocompatible hydrogel mimics a native extracellular matrix (ECM) by providing cell-adhesive ligands, dynamic remodeling properties, and bioactive cues, thereby enhancing cell adhesion, migration, differentiation, and tissue maturation within the 3D bioprinted organoids.
7. The method as claimed in claim 1, wherein the nutrient-rich culture medium is supplemented with at least one growth factor, selected from at least one or a combination of fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), to enhance cell survival, proliferation, and functional differentiation.
8. The method as claimed in claim 1, wherein the bio-ink formulation is optimized for extrusion-based, inkjet, or laser-assisted 3D bioprinting, thereby ensuring high printing fidelity, cell viability, and structural resolution of the bioprinted organoids and tissue constructs.
9. The method as claimed in claim 1, wherein the 3D bioprinted organoids are subjected to dynamic bioreactor culture conditions, which include mechanical stimulation, fluid perfusion, or electrical cues, to enhance tissue maturation, vascularization, and functional integration for biomedical applications.