1. A bio-ink formulation for 3D bioprinting, comprising: o a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen; o one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin; o a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase; o living cells comprising mammalian cells, plant cells, or bacterial cells; and o a nutrient solution suitable for maintaining the viability of said living cells.

2. The bio-ink formulation of claim 1, wherein said biocompatible polymer base comprises alginate in a concentration range of 2-4% (w/v).

3. The bio-ink formulation of claim 1, wherein said one or more cell adhesion promoters comprises fibronectin in a concentration range of 0.1-1% (w/v).

4. The bio-ink formulation of claim 1, wherein said crosslinking agent comprises calcium chloride in a concentration range of 50-150 mM.

5. The bio-ink formulation of claim 1, wherein said living cells are mammalian cells selected from the group consisting of fibroblasts, stem cells, and chondrocytes.

6. The bio-ink formulation of claim 1, further comprising a viscosity modifier selected from the group consisting of hyaluronic acid, methylcellulose, and xanthan gum.

7. The bio-ink formulation of claim 1, wherein said nutrient solution comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v).

8. The bio-ink formulation of claim 1, further comprising a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).

9. The bio-ink formulation of claim 1, wherein said biocompatible polymer base further comprises gelatin in a concentration range of 5-15% (w/v) to enhance printability.

10. The bio-ink formulation of claim 1, wherein said formulation is prepared under sterile conditions to ensure the viability and sterility of said living cells. Dated 30 December 2024 Pallavi Sinha IN/PA- 4068 Bio-Ink Formulation for 3D Bioprinting Abstract The present disclosure provides a bio-ink formulation for 3D bioprinting, comprising a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen; one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin; a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase; living cells comprising mammalian cells, plant cells, or bacterial cells; and a nutrient solution suitable for maintaining the viability of said living cells. Dated 30 December 2024 Pallavi Sinha IN/PA- 4068 Agent for the Applicant , Claims:Claims :

1. A bio-ink formulation for 3D bioprinting, comprising: o a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen; o one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin; o a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase; o living cells comprising mammalian cells, plant cells, or bacterial cells; and o a nutrient solution suitable for maintaining the viability of said living cells.

2. The bio-ink formulation of claim 1, wherein said biocompatible polymer base comprises alginate in a concentration range of 2-4% (w/v).

3. The bio-ink formulation of claim 1, wherein said one or more cell adhesion promoters comprises fibronectin in a concentration range of 0.1-1% (w/v).

4. The bio-ink formulation of claim 1, wherein said crosslinking agent comprises calcium chloride in a concentration range of 50-150 mM.

5. The bio-ink formulation of claim 1, wherein said living cells are mammalian cells selected from the group consisting of fibroblasts, stem cells, and chondrocytes.

6. The bio-ink formulation of claim 1, further comprising a viscosity modifier selected from the group consisting of hyaluronic acid, methylcellulose, and xanthan gum.

7. The bio-ink formulation of claim 1, wherein said nutrient solution comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v).

8. The bio-ink formulation of claim 1, further comprising a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).

9. The bio-ink formulation of claim 1, wherein said biocompatible polymer base further comprises gelatin in a concentration range of 5-15% (w/v) to enhance printability.

10. The bio-ink formulation of claim 1, wherein said formulation is prepared under sterile conditions to ensure the viability and sterility of said living cells. Dated 30 December 2024 Pallavi Sinha IN/PA- 4068

Description:Bio-Ink Formulation for 3D Bioprinting
Field of the Invention
[0001] The present disclosure generally relates to 3D bioprinting and particularly to a bio-ink formulation.
[0001]
Background
[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] The field of 3D bioprinting has seen significant advancements in recent years. Such technology enables the fabrication of complex biological structures by layering bio-inks containing living cells and biomaterials. This field has garnered attention due to the potential applications in tissue engineering, regenerative medicine, and pharmaceutical testing. 3D bioprinting facilitates the creation of constructs that mimic the intricate architecture and functionality of natural tissues.
[0004] Various bio-ink formulations are employed in 3D bioprinting. One common formulation includes a biocompatible polymer base. Polymers such as alginate, gelatin, fibrin, and collagen are frequently used due to their biocompatibility and ability to form stable gels. These polymers provide the necessary structural support for the embedded living cells and maintain the integrity of the printed constructs.
[0005] The inclusion of cell adhesion promoters in bio-ink formulations enhances the attachment and proliferation of cells within the printed structures. Common cell adhesion promoters include fibronectin, laminin, and vitronectin. These promoters interact with cell surface receptors, facilitating cellular adhesion and spreading, which are crucial for tissue formation and function.
[0006] Crosslinking agents play a vital role in the stabilization of the bioprinted constructs. Agents such as calcium chloride, genipin, and transglutaminase are commonly used for this purpose. Crosslinking agents induce the formation of covalent bonds between polymer chains, resulting in a more robust and resilient structure. The choice of crosslinking agent depends on the type of polymer used and the desired mechanical properties of the final construct.
[0007] Living cells are an essential component of bio-inks for 3D bioprinting. The types of cells incorporated can vary widely, including mammalian cells, plant cells, or bacterial cells. The selection of cell types depends on the intended application of the bioprinted structure. For example, mammalian cells are often used in tissue engineering applications, while plant cells may be employed for biotechnological purposes.
[0008] To maintain the viability of living cells during and after the printing process, bio-inks must include a nutrient solution. Such solutions provide the necessary nutrients and growth factors required for cell survival and proliferation. The composition of the nutrient solution can vary, but it typically includes components such as amino acids, vitamins, minerals, and growth factors.
[0009] In light of the above discussion, there exists an urgent need for solutions that overcome the problems associated with conventional systems and/or techniques for formulating bio-inks for 3D bioprinting.
[00010] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Summary
[00011] Various objects, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.
[00012] The present disclosure generally relates to 3D bioprinting and particularly to a bio-ink formulation.
[00013]
[00014] In an aspect, the present disclosure provides a bio-ink formulation for 3D bioprinting. The formulation comprises a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen; one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin; a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase; living cells comprising mammalian cells, plant cells, or bacterial cells; and a nutrient solution suitable for maintaining the viability of said living cells. Such a formulation enables the creation of bioprinted structures with enhanced cell viability and structural integrity.
[00015] The bio-ink formulation comprises alginate in a concentration range of 2-4% (w/v). Such a concentration ensures optimal viscosity for bioprinting applications.
[00016] The bio-ink formulation comprises fibronectin in a concentration range of 0.1-1% (w/v). Such a concentration range enhances cell adhesion properties of the bioprinted structures.
[00017] The bio-ink formulation comprises calcium chloride in a concentration range of 50-150 mM. Such a concentration facilitates effective crosslinking of the polymer base.
[00018] The bio-ink formulation comprises mammalian cells selected from the group consisting of fibroblasts, stem cells, and chondrocytes. Such cells are critical for various tissue engineering applications.
[00019] The bio-ink formulation further comprises a viscosity modifier selected from the group consisting of hyaluronic acid, methylcellulose, and xanthan gum. Such a modifier optimizes the rheological properties of the bio-ink.
[00020] The bio-ink formulation comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v). Such a nutrient solution supports the viability of the living cells.
[00021] The bio-ink formulation further comprises a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Such growth factors promote cell proliferation and differentiation.
[00022] The bio-ink formulation comprises gelatin in a concentration range of 5-15% (w/v) to enhance printability. Such a concentration improves the structural fidelity of bioprinted constructs.
[00023] The bio-ink formulation is prepared under sterile conditions to ensure the viability and sterility of said living cells. Such preparation ensures contamination-free bioprinting processes.
Brief Description of the Drawings
[00024] The features and advantages of the present disclosure would be more clearly understood from the following description taken in conjunction with the accompanying drawings in which:
[00025] FIG. 1 illustrates a block diagram of a bio-ink formulation for 3D bioprinting, in accordance with the embodiments of the present disclosure. FIG. 2 illustrates a sequence diagram of a bio-ink formulation for 3D bioprinting, in accordance with the embodiments of the present disclosure.
Detailed Description
[00026] The following is a detailed description of exemplary embodiments to illustrate the principles of the invention. The embodiments are provided to illustrate aspects of the invention, but the invention is not limited to any embodiment. The scope of the invention encompasses numerous alternatives, modifications and equivalent; it is limited only by the claims.
[00027] In view of the many possible embodiments to which the principles of the present discussion may be applied, it should be recognized that the embodiments described herein with respect to the drawing figures are meant to be illustrative only and should not be taken as limiting the scope of the claims. Therefore, the techniques as described herein contemplate all such embodiments as may come within the scope of the following claims and equivalents thereof.
[00028] The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The use of the same reference numbers in different instances in the description and the figures may indicate similar or identical items.
[00029] Pursuant to the "Detailed Description" section herein, whenever an element is explicitly associated with a specific numeral for the first time, such association shall be deemed consistent and applicable throughout the entirety of the "Detailed Description" section, unless otherwise expressly stated or contradicted by the context.
[00030] The present disclosure generally relates to 3D bioprinting and particularly to a bio-ink formulation.
[00031]
[00032] Pursuant to the "Detailed Description" section herein, whenever an element is explicitly associated with a specific numeral for the first time, such association shall be deemed consistent and applicable throughout the entirety of the "Detailed Description" section, unless otherwise expressly stated or contradicted by the context.
[00033] The term "bio-ink formulation" as used throughout the present disclosure relates to a composition used in 3D bioprinting that enables the creation of bioprinted structures with enhanced cell viability and structural integrity. Such a bio-ink formulation integrates various components to ensure the successful fabrication and functionality of bioprinted tissues.
[00034] The term "biocompatible polymer base" as used throughout the present disclosure relates to the foundational component of the bio-ink formulation. The biocompatible polymer base is selected from the group consisting of alginate, gelatin, fibrin, and collagen. Alginate provides a hydrogel structure that is conducive to cell encapsulation and is typically used in concentrations of 2-4% (w/v) to balance viscosity and printability. Gelatin, in concentrations of 5-15% (w/v), enhances the printability by providing thermoreversible properties, allowing it to solidify at body temperature. Fibrin supports cell attachment and proliferation due to its natural presence in wound healing processes. Collagen, being the most abundant protein in the extracellular matrix, supports cell adhesion, migration, and differentiation. The biocompatible polymer base provides the structural matrix necessary for bioprinting applications and supports cell growth and proliferation.
[00035] The term "cell adhesion promoters" as used throughout the present disclosure relates to substances that enhance the ability of cells to adhere to the biocompatible polymer base. The cell adhesion promoters are selected from the group consisting of fibronectin, laminin, and vitronectin. Fibronectin, typically used in concentrations of 0.1-1% (w/v), facilitates cellular adhesion, growth, and differentiation by binding to cell surface receptors. Laminin promotes cell attachment, spreading, and migration, playing a critical role in tissue morphogenesis and repair. Vitronectin supports cell adhesion and spreading by interacting with integrins and other cell surface receptors. The cell adhesion promoters improve the overall integrity and functionality of the bioprinted structures by ensuring strong and stable cell attachment to the polymer matrix.
[00036] The term "crosslinking agent" as used throughout the present disclosure relates to a chemical compound that facilitates the formation of a stable, three-dimensional network within the biocompatible polymer base. The crosslinking agent is selected from the group consisting of calcium chloride, genipin, and transglutaminase. Calcium chloride, in concentrations of 50-150 mM, ionically crosslinks alginate to form a gel. Genipin, a natural crosslinker derived from gardenia fruit, covalently bonds with amine groups in proteins, creating a biocompatible and stable network. Transglutaminase enzymatically crosslinks proteins by forming isopeptide bonds between glutamine and lysine residues, enhancing the mechanical properties of the bioprinted structures. The crosslinking agent ensures the mechanical stability and durability of the bioprinted constructs.
[00037] The term "living cells" as used throughout the present disclosure relates to the biological entities incorporated into the bio-ink formulation. The living cells comprise mammalian cells, plant cells, or bacterial cells. Mammalian cells, such as fibroblasts, stem cells, and chondrocytes, are essential for regenerative medicine and tissue engineering applications due to their ability to proliferate, differentiate, and form functional tissues. Plant cells and bacterial cells offer unique properties for specialized applications, such as producing bioactive compounds or forming structural components. The inclusion of living cells is essential for the intended biological function of the bioprinted structures, providing the necessary cellular activity for tissue engineering applications.
[00038] The term "nutrient solution" as used throughout the present disclosure relates to the liquid medium that provides essential nutrients to sustain the living cells during and after the bioprinting process. The nutrient solution is suitable for maintaining the viability of said living cells and typically comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v). DMEM provides a balanced mixture of amino acids, vitamins, glucose, and inorganic salts necessary for cell growth. FBS supplies additional growth factors, hormones, and proteins that support cell proliferation and viability. The nutrient solution ensures the sustained viability and metabolic activity of the cells within the bioprinted constructs.
[00039] The bio-ink formulation described is prepared under sterile conditions to ensure the viability and sterility of said living cells. Such preparation prevents contamination, thereby ensuring the safety and effectiveness of the bioprinted tissues for clinical and research applications.
[00040] In an embodiment, the bio-ink formulation comprises a biocompatible polymer base that includes alginate in a concentration range of 2-4% (w/v). Alginate is a naturally derived polysaccharide known for its biocompatibility and gel-forming capabilities. The concentration range of 2-4% (w/v) optimizes the viscosity of the bio-ink formulation, enabling it to flow smoothly through the bioprinter nozzle while maintaining sufficient structural integrity upon deposition. This concentration range balances the mechanical strength and flexibility of the bioprinted constructs, facilitating cell encapsulation and subsequent release in a controlled manner. The use of alginate in this specific concentration ensures that the bio-ink formulation retains adequate printability and supports cell viability, making it suitable for various tissue engineering applications. The concentration of alginate within this range ensures the formation of a stable hydrogel network that can sustain cell growth and differentiation, contributing to the overall effectiveness of the bioprinted tissue constructs. Such optimization of the alginate concentration enhances the mechanical properties and biological functionality of the final bioprinted structures, ensuring their applicability in regenerative medicine.
[00041] In an embodiment, the bio-ink formulation comprises one or more cell adhesion promoters that include fibronectin in a concentration range of 0.1-1% (w/v). Fibronectin is a high-molecular-weight glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. This concentration range is critical for enhancing cell attachment, spreading, and proliferation within the bioprinted constructs. By incorporating fibronectin in the specified concentration range, the bio-ink formulation significantly improves the cellular interactions with the biocompatible polymer base, thereby promoting better integration and functionality of the bioprinted tissues. The fibronectin concentration ensures that cells within the bio-ink formulation exhibit strong adhesion properties, leading to increased cellular viability and activity. This enhancement is particularly important for applications in tissue engineering and regenerative medicine, where effective cell adhesion is crucial for the formation of functional tissue structures. The presence of fibronectin in the bio-ink formulation fosters a conducive environment for cellular activities, thus improving the overall performance and efficacy of the bioprinted tissues.
[00042] In an embodiment, the bio-ink formulation comprises a crosslinking agent that includes calcium chloride in a concentration range of 50-150 mM. Calcium chloride acts as an ionic crosslinker for alginate, facilitating the formation of a gel matrix upon interaction. The specified concentration range of 50-150 mM ensures optimal crosslinking density, which is essential for maintaining the structural integrity and mechanical strength of the bioprinted constructs. The use of calcium chloride in this concentration range results in a well-defined and stable hydrogel network, capable of supporting cellular growth and function. This concentration is carefully selected to balance the gelation speed and the mechanical properties of the final bioprinted product. The calcium chloride concentration is pivotal in achieving a consistent and reproducible gelation process, which is crucial for the precision and reliability of the 3D bioprinting process. Such a concentration range ensures that the bioprinted constructs possess the necessary mechanical robustness and biological compatibility for successful application in tissue engineering and other biomedical fields.
[00043] In an embodiment, the bio-ink formulation comprises living cells that are mammalian cells selected from the group consisting of fibroblasts, stem cells, and chondrocytes. Mammalian cells are integral to the formulation due to their relevance in tissue engineering and regenerative medicine. Fibroblasts contribute to the formation of connective tissue and play a crucial role in wound healing. Stem cells offer the potential for differentiation into various cell types, providing versatility for creating diverse tissue structures. Chondrocytes are essential for cartilage formation, making them valuable for orthopedic applications. The inclusion of these specific mammalian cells in the bio-ink formulation ensures that the bioprinted constructs can mimic the biological functions of native tissues. These cells are selected for their ability to proliferate, differentiate, and integrate within the bioprinted matrix, thereby enhancing the overall functionality and effectiveness of the bioprinted tissues. The use of mammalian cells in the bio-ink formulation is critical for achieving the desired therapeutic outcomes in various tissue engineering applications.
[00044] In an embodiment, the bio-ink formulation further comprises a viscosity modifier selected from the group consisting of hyaluronic acid, methylcellulose, and xanthan gum. Viscosity modifiers are added to the bio-ink formulation to adjust its rheological properties, thereby enhancing the printability and structural fidelity of the bioprinted constructs. Hyaluronic acid is a naturally occurring polysaccharide that increases the viscosity and provides additional biocompatibility. Methylcellulose, a derivative of cellulose, serves as a thickening agent that improves the mechanical stability of the bio-ink. Xanthan gum, a microbial polysaccharide, enhances the viscosity and flow properties of the formulation. The inclusion of these viscosity modifiers ensures that the bio-ink formulation can be precisely extruded through the bioprinter nozzle, resulting in accurate and reproducible bioprinted structures. Such modifiers are essential for maintaining the shape and integrity of the bioprinted constructs during and after the printing process, thereby contributing to the overall success of the bioprinting application.
[00045] In an embodiment, the bio-ink formulation comprises a nutrient solution that includes Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-

20% (v/v). DMEM is a widely used cell culture medium that provides essential nutrients, vitamins, and amino acids necessary for cell growth and maintenance. The supplementation with FBS enhances the nutrient profile by supplying additional growth factors, hormones, and proteins that support cellular proliferation and viability. The concentration range of 5-20% (v/v) for FBS is critical for maintaining the optimal growth conditions for the living cells within the bio-ink formulation. This nutrient solution ensures that the cells remain viable and metabolically active throughout the bioprinting process and subsequent tissue development. The use of DMEM supplemented with FBS in the specified concentration range fosters a conducive environment for cellular activities, thereby improving the overall performance and efficacy of the bioprinted tissues.
[00046] In an embodiment, the bio-ink formulation further comprises a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Growth factors are critical for promoting cell proliferation, differentiation, and tissue development. VEGF stimulates the formation of new blood vessels, enhancing the vascularization of the bioprinted tissues. bFGF promotes the growth and differentiation of fibroblasts and other cell types, supporting tissue regeneration. EGF stimulates cell growth and proliferation by binding to its receptor on the cell surface. The inclusion of these growth factors in the bio-ink formulation ensures that the bioprinted constructs can develop functional tissue structures with enhanced cellular activities. Such growth factors play a vital role in tissue engineering applications, where the formation of well-vascularized and functionally integrated tissues is essential. The presence of these growth factors in the bio-ink formulation enhances the overall biological performance and therapeutic potential of the bioprinted tissues.
[00047] In an embodiment, the bio-ink formulation comprises a biocompatible polymer base that further includes gelatin in a concentration range of 5-15% (w/v) to enhance printability. Gelatin is a denatured form of collagen that provides thermoreversible properties, allowing it to solidify at body temperature and melt at higher temperatures. The concentration range of 5-15% (w/v) for gelatin ensures that the bio-ink formulation possesses optimal printability characteristics, enabling it to form well-defined structures upon extrusion. The inclusion of gelatin enhances the structural fidelity and mechanical stability of the bioprinted constructs, making them suitable for various tissue engineering applications. This concentration range is carefully selected to balance the viscosity and gelation properties of the bio-ink, ensuring that the bioprinted structures maintain their shape and integrity during and after the printing process. The use of gelatin in the specified concentration range contributes to the overall success of the bioprinting process by improving the printability and functionality of the bio-ink formulation.
[00048] In an embodiment, the bio-ink formulation is prepared under sterile conditions to ensure the viability and sterility of said living cells. Sterile preparation is critical for preventing contamination and ensuring the safety and effectiveness of the bioprinted tissues for clinical and research applications. The sterile conditions involve the use of aseptic techniques and sterile equipment throughout the formulation process. This ensures that the living cells within the bio-ink remain viable and free from microbial contamination, which could otherwise compromise the integrity and functionality of the bioprinted constructs. The sterile preparation of the bio-ink formulation is essential for achieving high-quality bioprinted tissues that are suitable for use in tissue engineering and regenerative medicine applications. Ensuring sterility throughout the formulation process contributes to the overall success and reliability of the bioprinting technology.
[00049] The bio-ink formulation for 3D bioprinting incorporates a biocompatible polymer base selected from alginate, gelatin, fibrin, and collagen, providing a versatile and stable scaffold for cell encapsulation. The inclusion of one or more cell adhesion promoters, such as fibronectin, laminin, and vitronectin, enhances cell attachment and proliferation, which is essential for the formation of functional tissue structures. A crosslinking agent, chosen from calcium chloride, genipin, or transglutaminase, ensures the structural integrity and mechanical stability of the printed constructs by forming a robust three-dimensional network. The integration of living cells, including mammalian, plant, or bacterial cells, into the bio-ink formulation facilitates the creation of biologically active tissues, supporting various applications in tissue engineering and regenerative medicine. The nutrient solution, designed to maintain cell viability, provides essential nutrients and growth factors, ensuring the metabolic activity and long-term survival of the encapsulated cells.
[00050] The biocompatible polymer base, when comprising alginate in a concentration range of 2-4% (w/v), offers optimal viscosity and printability, allowing for precise extrusion and the formation of stable hydrogel structures. This concentration is crucial for balancing the mechanical properties and biological functionality of the printed constructs.
[00051] The inclusion of fibronectin as a cell adhesion promoter in a concentration range of 0.1-1% (w/v) significantly improves the interaction between cells and the polymer matrix, promoting enhanced cell attachment, spreading, and differentiation. This concentration ensures that cells maintain high viability and functionality within the printed tissue.
[00052] The crosslinking agent, calcium chloride, in a concentration range of 50-150 mM, provides effective ionic crosslinking of alginate, resulting in a mechanically robust and stable hydrogel network. This concentration range is critical for achieving consistent gelation, which is necessary for the structural integrity and reproducibility of the printed constructs.
[00053] Utilizing mammalian cells such as fibroblasts, stem cells, and chondrocytes within the bio-ink formulation enables the creation of tissues that closely mimic native biological structures. These cells contribute to tissue regeneration and repair, making the bio-ink suitable for a wide range of medical applications.
[00054] Incorporating viscosity modifiers like hyaluronic acid, methylcellulose, and xanthan gum into the bio-ink formulation optimizes its rheological properties, ensuring smooth extrusion and accurate layer deposition during the printing process. This modification enhances the printability and fidelity of the bioprinted structures.
[00055] The nutrient solution, comprising Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v), provides a rich medium that supports cell growth and viability. This supplementation is essential for maintaining the metabolic activity and overall health of the living cells within the bio-ink.
[00056] The addition of growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) to the bio-ink formulation promotes cell proliferation, differentiation, and tissue maturation. These factors are vital for developing functional and vascularized tissue constructs.
[00057] When the biocompatible polymer base further comprises gelatin in a concentration range of 5-15% (w/v), the printability of the bio-ink is significantly enhanced. Gelatin provides thermoreversible properties, improving the structural fidelity and mechanical strength of the printed constructs.
[00058] Preparing the bio-ink formulation under sterile conditions is essential to ensure the viability and sterility of the living cells. Sterile preparation prevents contamination, ensuring the safety and efficacy of the bioprinted tissues for clinical and research applications.
[00059] FIG. 1 illustrates a block diagram of a bio-ink formulation for 3D bioprinting, in accordance with the embodiments of the present disclosure. The bio-ink formulation consists of several critical components, including a biocompatible polymer base, cell adhesion promoters, a crosslinking agent, living cells, and a nutrient solution. The biocompatible polymer base is selected from alginate, gelatin, fibrin, and collagen, each providing a supportive matrix for cellular structures. Cell adhesion promoters such as fibronectin, laminin, and vitronectin enhance the attachment and proliferation of cells within the matrix. Crosslinking agents like calcium chloride, genipin, and transglutaminase ensure the structural stability and integrity of the bioprinted constructs. The inclusion of living cells, which can be mammalian, plant, or bacterial, adds biological functionality to the bio-ink. Finally, the nutrient solution supports the viability and metabolic activity of the living cells, maintaining their health throughout the bioprinting process and subsequent tissue formation. This comprehensive formulation enables the creation of complex, functional tissue constructs suitable for various biomedical applications.
[00060] FIG. 2 illustrates a sequence diagram of a bio-ink formulation for 3D bioprinting, in accordance with the embodiments of the present disclosure. The process begins with a biocompatible polymer base, which forms the structural matrix necessary for supporting cellular growth. This polymer base can be selected from alginate, gelatin, fibrin, or collagen. Following this, cell adhesion promoters such as fibronectin, laminin, or vitronectin are added to enhance the attachment and proliferation of cells within the matrix. The formulation then incorporates a crosslinking agent, like calcium chloride, genipin, or transglutaminase, to stabilize the three-dimensional structure of the bioprinted construct. Living cells, including mammalian, plant, or bacterial cells, are then integrated into the formulation to provide biological functionality. A nutrient solution, designed to maintain the viability and metabolic activity of the living cells, is subsequently added. The final bio-ink formulation is then utilized in the 3D bioprinting process to create complex tissue structures. This sequence ensures that the bioprinted constructs have the necessary mechanical stability, biological functionality, and cellular viability required for various biomedical applications.Further, while operations are depicted in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In certain circumstances, multitasking and parallel processing may be advantageous. Likewise, while several specific implementation details are contained in the above discussions, these should not be construed as limitations on the scope of the subject matter described herein, but rather as descriptions of features that may be specific to particular embodiments. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination.
[00061] The term “memory,” as used herein relates to a volatile or persistent medium, such as a magnetic disk, or optical disk, in which a computer can store data or software for any duration. Optionally, the memory is non-volatile mass storage such as physical storage media. Furthermore, a single memory may encompass and in a scenario wherein computing system is distributed, the processing, memory and/or storage capability may be distributed as well.
[00062] Throughout the present disclosure, the term ‘server’ relates to a structure and/or module that include programmable and/or non-programmable components configured to store, process and/or share information. Optionally, the server includes any arrangement of physical or virtual computational entities capable of enhancing information to perform various computational tasks.
[00063] Throughout the present disclosure, the term “network” relates to an arrangement of interconnected programmable and/or non-programmable components that are configured to facilitate data communication between one or more electronic devices and/or databases, whether available or known at the time of filing or as later developed. Furthermore, the network may include, but is not limited to, one or more peer-to-peer network, a hybrid peer-to-peer network, local area networks (LANs), radio access networks (RANs), metropolitan area networks (MANS), wide area networks (WANs), all or a portion of a public network such as the global computer network known as the Internet, a private network, a cellular network and any other communication system or systems at one or more locations.
[00064] Throughout the present disclosure, the term “process”* relates to any collection or set of instructions executable by a computer or other digital system so as to configure the computer or the digital system to perform a task that is the intent of the process.
[00065] Throughout the present disclosure, the term ‘Artificial intelligence (AI)’ as used herein relates to any mechanism or computationally intelligent system that combines knowledge, techniques, and methodologies for controlling a bot or other element within a computing environment. Furthermore, the artificial intelligence (AI) is configured to apply knowledge and that can adapt it-self and learn to do better in changing environments. Additionally, employing any computationally intelligent technique, the artificial intelligence (AI) is operable to adapt to unknown or changing environment for better performance. The artificial intelligence (AI) includes fuzzy logic engines, decision-making engines, preset targeting accuracy levels, and/or programmatically intelligent software.

Claims
I/We Claim:
1. A bio-ink formulation for 3D bioprinting, comprising:
o a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen;
o one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin;
o a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase;
o living cells comprising mammalian cells, plant cells, or bacterial cells; and
o a nutrient solution suitable for maintaining the viability of said living cells.
2. The bio-ink formulation of claim 1, wherein said biocompatible polymer base comprises alginate in a concentration range of 2-4% (w/v).
3. The bio-ink formulation of claim 1, wherein said one or more cell adhesion promoters comprises fibronectin in a concentration range of 0.1-1% (w/v).
4. The bio-ink formulation of claim 1, wherein said crosslinking agent comprises calcium chloride in a concentration range of 50-150 mM.
5. The bio-ink formulation of claim 1, wherein said living cells are mammalian cells selected from the group consisting of fibroblasts, stem cells, and chondrocytes.
6. The bio-ink formulation of claim 1, further comprising a viscosity modifier selected from the group consisting of hyaluronic acid, methylcellulose, and xanthan gum.
7. The bio-ink formulation of claim 1, wherein said nutrient solution comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v).
8. The bio-ink formulation of claim 1, further comprising a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).
9. The bio-ink formulation of claim 1, wherein said biocompatible polymer base further comprises gelatin in a concentration range of 5-15% (w/v) to enhance printability.
10. The bio-ink formulation of claim 1, wherein said formulation is prepared under sterile conditions to ensure the viability and sterility of said living cells.
Dated 30 December 2024 Pallavi Sinha
IN/PA- 4068

Bio-Ink Formulation for 3D Bioprinting
Abstract
The present disclosure provides a bio-ink formulation for 3D bioprinting, comprising a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen; one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin; a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase; living cells comprising mammalian cells, plant cells, or bacterial cells; and a nutrient solution suitable for maintaining the viability of said living cells.
Dated 30 December 2024 Pallavi Sinha
IN/PA- 4068
Agent for the Applicant , Claims:Claims
I/We Claim:
1. A bio-ink formulation for 3D bioprinting, comprising:
o a biocompatible polymer base selected from the group consisting of alginate, gelatin, fibrin, and collagen;
o one or more cell adhesion promoters selected from the group consisting of fibronectin, laminin, and vitronectin;
o a crosslinking agent selected from the group consisting of calcium chloride, genipin, and transglutaminase;
o living cells comprising mammalian cells, plant cells, or bacterial cells; and
o a nutrient solution suitable for maintaining the viability of said living cells.
2. The bio-ink formulation of claim 1, wherein said biocompatible polymer base comprises alginate in a concentration range of 2-4% (w/v).
3. The bio-ink formulation of claim 1, wherein said one or more cell adhesion promoters comprises fibronectin in a concentration range of 0.1-1% (w/v).
4. The bio-ink formulation of claim 1, wherein said crosslinking agent comprises calcium chloride in a concentration range of 50-150 mM.
5. The bio-ink formulation of claim 1, wherein said living cells are mammalian cells selected from the group consisting of fibroblasts, stem cells, and chondrocytes.
6. The bio-ink formulation of claim 1, further comprising a viscosity modifier selected from the group consisting of hyaluronic acid, methylcellulose, and xanthan gum.
7. The bio-ink formulation of claim 1, wherein said nutrient solution comprises Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a concentration range of 5-20% (v/v).
8. The bio-ink formulation of claim 1, further comprising a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).
9. The bio-ink formulation of claim 1, wherein said biocompatible polymer base further comprises gelatin in a concentration range of 5-15% (w/v) to enhance printability.
10. The bio-ink formulation of claim 1, wherein said formulation is prepared under sterile conditions to ensure the viability and sterility of said living cells.
Dated 30 December 2024 Pallavi Sinha
IN/PA- 4068