FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION:
"BIOINK FORMULATION FOR CULTIVATED MEAT DEVELOPMENT
USING 3D BIOPRINTING TECHNIQUE"
APPLICANT(S)
NAME I NATIONALITY I ADDRESS
BIOKRAFT INDIAN D-813, Sentosa
FOODS PRIVATE Heights, Althan,
LIMITED Surat-395017,
Gujarat, India
PREAMBLE TO THE DESCRIPTION
COMPLETE SPECIFICATION
The following specification describes the invention and the manner in which it is to be performed

FIELD OF INVENTION
0001 The present invention generally relates to multi-component bioink for
bioprinting of cultivated meat constructs.
BACKGROUND AND PRIOR ARTS OF THE INVENTION
2 By presenting the first hamburger made from CM in 2013, Mark Post helped pave the way for studying alternative proteins [1]. CM is based on tissue engineering techniques and is developed using animal/avian stem cells and. It is also known as "lab-based meat", "clean meat," or "cultured meat" [2], Cell culture media, bioprocessing conditions, scaffolding and cell line development make up the four main technological components of CM development (GFI India, 2021). Functional skeletal muscle development is necessary for synthesising CM, which is often accomplished by co-culturing various cell types, as we mentioned in the section ahead [4],
3 For extrusion-based printing, the bioink should exhibit notable viscoelastic properties and, most importantly, be correctly characterised to preserve repeatability [5], In essence, bioink refers to a mixture of biomaterials with embedded cells utilized for bioprinting [6]. For printing biomaterials similar to skeletal muscle-like tissue in the CM, the bioink contains muscle satellite cells that initiates myogenesis [4]. Three biomaterials were used: hydrogels, melt-cure polymers, and decellularized extracellular matrix, by researchers for extrusion-based bioprinting [7]. Muscle stem cells are developed in vitro in a healthy physical environment provided by collagen, fibronectin, and laminin [8]. Polymers such as polyurethane, polylactic acid, and polycaprolactone have been researched in tissue engineering to provide structural support and control the proliferation of muscle cells after 3DP [7]. All kinds of biomaterials have used hydrogels extensively for bioink formation because they provide a suitable environment for cell growth and development [5].
4 Although the 3DP technique is often used for biomedical applications, its use in other disciplines has not yet been substantially explored. As opposed to creating an object all at once, 3DP creates the object layer by layer [9-11]. The

three main subtypes of additive manufacturing, or 3DP, are selective laser sintering, stereolithography, and fused deposition modelling [12]. Integrated additive manufacturing systems and design software are typically included with 3D printers, which is a great advantage when producing intricate geometrical designs [13]. Computer-aided design (CAD) software is typically used to create a 3D model for constructing the object [14-16]. The 3D shape is built and exported as an STL file to a tool called a slicer, where it is broken up into layers and fed as a g-code file before being fed as digital data into the printer [15,17]. Additionally, 3D printers' z-axis orientation can be used to create 3D models [13,18]. When designing any 3D food product, printability, process parameters, and post¬processing parameters are given priority [19,20]. These elements influence the printing strategy. A patent for additive layering of meat-based ingestible material using 3DFP, including injection treatment assembly and food printing assembly for product preparation, was granted to Paul Holman and his colleagues [21].
5 The most popular 3DP method for food and other bioprinting applications, such as tissue engineering and the creation of artificial skin, is extrusion-based or fused deposition modelling [5,22-27]. As these printers are fitted with multiple printing heads, which allows regulation of customised shapes, infill density, scaffold designing, and simultaneous use of multiple bioinks, all significant investigations in 3DP of meat and its analogues have been carried out using this technique [28-30]. The extrusion techniques are syringe-based, screw-driven, and air-based [20,24,31].
6 One of the challenges faced by the bioink or meat slurry used as the primary component of 3DP during the extrusion process is viscosity force [32]. Consequently, there is a lot of shear experienced by the cells inside the bioink [33]. Therefore, flow enhancers like biomaterials or hydrocolloids are used for smooth extrusion due to their shear-thinning characteristic and to give the printed structure shape stability [34]. In addition, hydrocolloids have the necessary viscosity for the printed structures' precision and texture [32,34]. Animals, plants, and even microorganisms can all provide these, among other sources [31]. Adding

hydrocolloids is necessary for the smooth extrusion of non-native foods with fibrous characters, such as meat [35-38]. According to C. Liu et al. (2018) [39], an important characteristic for extrusion is the viscosity of the material being used. High-viscosity materials are challenging to extrude, whilst low-viscosity materials may distort throughout the process.
7 Utilising a variety of biomaterials, 3DFP has been studied such as gelatin [40], transglutaminase [41], guar gum [42], xanthan gum [29], chitosan [43], potato starch [44], carrageenan gel [45-47], coconut oil and soyabean oil [48]. Gelatin and other gelling agents have a high water-holding capacity, facilitating simple extrusion [49]. Even transglutaminase [50] contributes to the form stability of the printed product [19,31,35]. Xanthan gum is one of the best enhancers because of its pseudoplastic behaviour in biomaterials like polysaccharides, which has also improved the printability of pork [51]. Refined wheat flour and uncooked ground chicken were used to create 3D-printed chicken nuggets with improved printability [38]. Similar results were obtained when adding transglutaminase and bacon fat [31,49]. Potato starch (PS) demonstrated encouraging results in printability and stability to the printed structures because it provided the binding force required for stability and compatibility with other materials [44]. Additionally, starch made materials containing a lot of oil easier to print [48].
8 Among biopolymers, gelatin is considered the most common biodegradable polymer used in hydrogels and scaffold development. Gelatin is extracted from various natural sources such as porcine skin, cattle bones and fish skin being the primary ones. Its amphipathic nature allows it to undergo gelation and form hydrogels in the presence of chemical crosslinkers. The simple dissolving of gelatin will give a less stable physical gel, making its tuning easy with other polymers. However, it is also a major drawback that can be addressed by using crosslinkers to form a strong covalent network [52]. For crosslinking gelatin-based hydrogels, the most important strategy is to use crosslinkers such as glutaraldehyde involving aldehydes and condensation reactions. Gelatin contains the Arg-Gly-Asp (RGD) sequences that help in cell adhesion and migration,

making its use a promising strategy [53]. Also, gelatin allows the formation of stable polyelectrolyte complexes by getting easily blended with other biopolymers [54].
9 As discussed above, gelatin's unstable network structure results in poor mechanical and thermal properties. Crosslinkers including glutaraldehyde, formaldehyde, and genipin have therefore been employed to enhance the structural and physical properties of gelatin. Nevertheless, these crosslinkers come with their own set of limitations [55], Since we are focusing on edible meat development, the use of toxic chemical crosslinkers will not be preferred. So, this is where green biopolymers come into the picture, which has caught the attention of several researchers [56]. Starch is a promising biopolymer with wide availability and good biocompatibility characteristics. Natural starch does not possess an aldehyde group, so it is chemically modified into dialdehyde starch (DS) by periodic acid oxidation. This dialdehyde structure will react with free amino groups of gelatin to form an imine bond (C=N) [55]. This crosslinking provides higher strength and better mechanical stability as compared to hydrogen bonds. Using this approach, we can eliminate the requirement of toxic crosslinkers in edible hydrogels [56].
10 It is to be noted that after cellulose, alginate is the most abundant biopolymer available, with its sodium salt being the major source. Sodium alginate, an anionic biopolymer, is highly viscous, and divalent cations give it good gelling capability [57]. Properties such as biodegradability, economical, low toxicity, good gelation and hydrogel-forming capability make alginate an ideal biopolymer for tissue engineering. Its shear-thinning behaviour in bioink blends makes it a preferred choice in scaffold development for 3DP [58]. Alginate possesses limited cell adhesion properties, which sometimes acts as a limitation in cell attachment and proliferation due to a lack of RGD sequences [54]. On crosslinking with CaCl2, alginate gives a stable crosslinked structure, and so a blend of alginate-gelatin is a promising strategy [53],

OBJECTIVES OF THE INVENTION
11 The primary objective of the present invention is to propose bioink formulation for cultivated meat development using 3D Bioprinting.
12 Another object of the invention aims to overcome the limitations of existing bioinks and bioprinting techniques by offering a formulation that promotes enhanced cell viability, proliferation, and maturation within printed tissue constructs.
13 Yet another object of bioink formulation is to maximize cell biocompatibility, enabling a high percentage of viable cells to remain functional within the bioprinted constructs for an extended period.
14 Further object of the invention seeks to establish that the bioprinted constructs using the developed bioink maintain their structural integrity over an extended timeframe.
15 These and other objects along with advantages of invention will be understood from the given description alongside the accompanying drawings.
SUMMARY OF THE INVENTION
16 According to this invention is to propose bioink formulation for cultivated meat development using 3D Bioprinting.
17 The biopolymer-based bioink formulation comprising of three components, wherein the first component is a cell supporting material possessing Arg-Gly-Asp (RGD) peptides consisting of arginine, glycine and aspartate, a second component as the crosslinker for cell supporting materal and the third component includes a structural material which also acts as a viscosity enhancer.
18 The first component is cell supporting material having RGD peptides like gelatin in 1-5%.

19 The second component interacting with other components includes starch and its derivatives, preferably dialdehyde starch in 4-8%.
20 The third component includes structural material that also acts as viscosity enhancer such as sodium alginate in 1-5%.
21 The process of preparation of a printable bioink comprises the steps of preparing 1-5% of gelatin in phosphate buffer saline (PBS) with constant stirring and heating; also preparing 4-8% of dialdehyde starch in PBS separately with constant stirring and heating; adding the dissolved gelatin solution by filtering it in the dissolved dialdehyde starch starch solution uder constant stirring; adding sodium alginate in 1-5% to the blend with constant stirring and heating; adding cells in 0.5E6 to 100E6/mL; loading the final bioink in a cartridge for bioprinting.
22 The process of bioprinting of bioink as used in steps, comprising loading of bioink which is Gelatin-Dialdehyde Starch-Sodium Alginate in a cartridge with a nozzle of 25 gauge; keeping the pressure from 0.030 MPa TO 0.15 MPa for extrusion of the bioink on to the well-plate or petriplate; and crosslinking the construct with a divalent cation solution; where the divalent cation solution is calcium chloride in 5-15%.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS/FIGURES
23 Fig. 1: 3D Bioprinted gelatin, dialdehyde starch and sodium alginate multilayer bioink
24 Fig. 2: FTIR analysis of the formulated bioink
25 Fig. 3: Representative plot the formulated bioink (a) Temperature Sweep Test(b) Frequency Sweep Test (c) Compression Test
26 Fig. 4: Swelling analysis of the bioprinted bioink
27 Fig. 5: In-vitro degradation of bioprinted bioink

28 Fig. 6: SEM images of bioink showing the microporosity of the hydrogel with pore size analysis at different time intervals (a) Day 1, (b) Day 7, (c) Day 14, (d) Day 21 and (e) Pore size analysis graph
29 Fig. 7: Z-stack analysis of live and dead cells on bioprinted bioink (a)Top view, (b)side view and (c) diagonal view
30 Fig. 8: Representative images show the live (green in colour), dead (red in colour) and overlap (both live and dead) cells of the bioprinted construct at respective time intervals (a) Day 1, (b) Day 7, (c) Day 14 and (d) Day 21
31 Fig. 9: Quantitative analysis of live cells at respective times of incubation of the bioprinted tissue constructs
DETAILED DESCRIPTION OF THE INVENTION
32 All the materials used in the study were commercially sourced. Gelatin and Calcium Chloride from HiMedia, Dialdehyde Starch from Dynarx India and Sodium Alginate from Loba Chemie.
33 The following disclosure provides materials and methodology for the preparation of bioink in developing cultivated meat tissue constructs by bioprinting.
34 This bioink formulation is designed to enhance cell viability, proliferation, and tissue maturation within printed constructs, ultimately improving the production of cultivated meat products.
35 The invention involves a carefully engineered bioink composition combining gelatin, dialdehyde starch, and sodium alginate. This bioink is specifically designed to support the encapsulation, printing, and growth of cells. Its unique formulation promotes optimal cell biocompatibility, structural integrity, and mechanical properties, making it suitable for creating tissue models that accurately emulate real meat.

36 Compared to existing methods, the proposed bioink offers several significant advantages. It enhances cell viability and tissue integrity, allowing for extended culture periods. It addresses the limitations of conventional bioinks by promoting better cell compatibility and more accurate replication of meat-like textures and structures. This innovation also provides scalability, customization, and control over cellular arrangements, catering to consumer preferences.
37 The invention addresses the limitations of existing bioinks and bioprinting methods, including suboptimal cell viability, limited structural integrity, and challenges in replicating the characteristics of real meat. It also addresses the need for scalable and customizable meat production methods that align with ethical and sustainable practices. The invention overcomes these limitations through a meticulously formulated bioink composition. The combination of gelatin, dialdehyde starch, and sodium alginate in an optimized ratio enhances cell viability, stability, and mechanical properties. Prototypes of bioprinted tissue constructs using the novel bioink have been successfully developed and tested. These prototypes have demonstrated extended cell viability, structural integrity, and mechanical stability, validating the effectiveness of the invention. The proposed bioink's composition utilizes readily available biomaterials, ensuring cost-effectiveness in production. The potential for scaled-up production of cultivated meat using this bioink aligns with industry demands and offers promising economic viability.
38 The invention will now be explained with the help of examples. The examples provided are just illustrative of the uses, methods, and products claimed in this invention and are not intended to serve as limitations on or limitations of the invention itself.

Example 1:
Pre-sterilization of biopolymers:
0039 Pre-sterilization of biopolymers is carried out using Ultraviolet (UV) rays
to eliminate any contamination with 3 repeat cycles of UV of 40 min each with in-
between mixing.
Example 2: Bioink formulation:
40 At 60°C and 200 rpm, 1-5% (w/v) of gelatin was dissolved in phosphate-buffered saline (PBS, pH 7.4). Separately, 4-8% (w/v) of dialdehyde starch (DS) powder was dissolved in PBS at 90°C and 300 rpm for 30 minutes. Both the powder, once completely dissolved was cooled to 40°C. After that, the gelatin solution was filter-sterilized using a 022μ filter into the DS solution and was kept to mix at 40 °C and 300 rpm for 30 mins. Following thorough mixing, 1-5% (w/v) sodium alginate powder has to be dissolved in the mixture at 60°C and 300 rpm.
41 Cells were separated from culture dishes using trypsin-EDTA solution (0.25%). For counting, the cells were resuspended in cell culture medium. After counting, a final concentration of 0.5E6 to 100 E6/mL of bioink was obtained by gently combining the cell suspension with the bioink!s optimised composition.
Example 3:
3D Bioprinting setup and process:
0042 The production of both cell-laden and cell-free 3D constructions is carried
out using a dual-nozzle extruder 3D bioprinter (Mito plus, Avay Biosciences,
Tvasta). The needle size used for bioprinting was 25 gauge. The 3D bioprinter
was housed in a sterile biosafety cabinet, and the bioink rheology does not
necessitate maintaining any particular environmental temperature for the

bioprinter. The software Mito Slicer was used to set up the printing parameters after the intended 3D shape was exported as an STL file, and the bioprinter was then fed the g-code file.
0043 A 5 mL cartridge containing the bioink was inserted into the extruder
situated on the X-carriage. We created constructions as shown in Figure 1 with a
cuboidal geometry, 3.5 mm x 3.5 mm cross-section, and 0.2 mm (3 layers)
thickness using the aforementioned 3D bioprinter. The layer height was held
constant at 0.1 mm, the grid infill density was set at 5% to 15%, and the printing
speed was maintained at 5 to 15 mm/s. After printing, the constructs were treated
with a sterile 5% to 15% (w/v) CaCl2 solution made in distilled water for 5 to 20
minutes to allow the sodium alginate to be chemically cross-linked. The cross-
linked tissue constructions were carefully cleaned with PBS a couple of times
before being incubated at 37°C with 5% C02.
Example 4:
Characterization of bioink and bioprinted construct:
0044 An FT-IR spectrometer (Jasco FT/IR-6600) was used to determine the
chemical functional groups of the bioink formulation. Figure 2 displays the FT-IR
spectra of gelatin, DS, sodium alginate, and Gelatin-DS-Alginate (GDA). Pure
gelatin's FT-IR spectra displayed the following distinguishing bands at 1041 cm"1
(primary amine) and 1077 cm"1 (primary amine). The large absorption band at
3300 cm"1 was attributable to the presence of intra-hydrogen bonds and -OH
stretching in all samples, which indicated the development of hydrogen bonds
with one another. The typical peak of-NH2 in gelatin at 1077 cm-1 was red-shifted
to 1080 cm"1 after the creation of hydrogels, demonstrating the role of amine
groups in bond formation. In addition to showing >C=0 stretching, the wide peak
at 1636 cm"1 also shows the creation of an imine structure band as a result of
higher transmittance, supporting the Schiff-based reaction between gelatin and
DS.

45 The Theological properties of GDA bioink formulation were analyzed with a rheometer (TA Instruments, DHR-20, PP-25 Probe). The instrument was prior calibrated at different temperatures before data acquisition. The temperature and shear rate frequency ranges used to evaluate the bioink samples were 20°C to 40°C and 0.1 s"1 to 100 s"1. GDA showed optimum viscoelastic properties and gave good shape stability. Temperature change had a minimal effect on the viscosity of the GDA formulation as can be seen in Figure 3(a). At shear rates ranging from 1 s'1 to 10 s'1, the bioink viscosity at 25°C dropped from 35 Pa.s to 12 Pa.s as can be seen in Figure 3(b). The bioink blend exhibits shear-thinning behaviour, which aids in smooth extrusion during bioprinting without becoming blocked, by lowering viscosity at a fixed temperature on rising shear rates.
46 The swelling rate of the bioprinted construct over time is shown in Figure 4. The crosslinked construct was immersed in PBS and weighed was recorded at equal intervals till it reached equilibrium. Initially, the construct swelled rapidly due to high water intake but after 24 h, a decrease in swelling rate was observed leading to saturation. For tissue engineering applications, water uptake capacity is an important parameter for hydrogels. According to reports, DS increases the chemical and hydrogen bonds between gelatin and DS chains, which reduces the quantity of water absorption and creates a stable structure. The construct remained stable after reaching swelling equilibrium indicating good mechanical properties and stable volume, which is crucial for tissue engineering and 3D Bioprinting.
47 The percentage weight loss of the bioprinted construct was calculated by comparing the degradation rates of the scaffold in PBS and DMEM as shown in Figure 5, and this information was used to determine the scaffold's in-vitro degradation. The percentage weight reduction in the DMEM sample was 13.52 ± 0.28%, 14.63 ± 0.47%, 15.11 ± 0.14% and 15.79 ± 0.15% on days 1, 7, 14 and 21, respectively. Similarly, the percentage weight reduction in the PBS sample was 14.36 ± 0.15%, 15.60 ± 0.26%, 15.93 ± 0.25% and 16.26 ± 0.15% on days 1, 7, 14 and 21, respectively. The results indicated lower degradation in the DMEM sample than in the PBS sample. The reason behind the low degradation of the

sample in DMEM could be the presence of a significant amount of proteins and amino acids that act like molecular binders. These proteins limit the fiber motion and dissolution rate by filling the void spaces within the construct. Whereas PBS contains only salts, thus having a higher dissolution. Overall, the construct is stable and has a low degradation rate, making it stable for tissue engineering applications.
0048 The microporosity of the constructs has an important role in determining
their mechanical properties and cell distribution across them. Figure 6 (a to d)
shows the microstructure of scaffolds at different intervals. All the constructs
showed optimal microporous network structure. The pore size of the construct
was observed to increase with time slightly, and the average pore size ranged
between 80 |μm and 120 μm. The small pore size indicates a dense surface
network and higher mechanical properties consistent across the scaffold. It can
also be due to increased imine and hydrogen bond formation in the hydrogel,
indicating a higher crosslinking degree. The average pore size was calculated as
84.3 ± 4.2 μm, 109.2 ± 2.6 μm, 110.2 ± 3.5 μm and 115.1 ± 3.8 μm on day 1, 7,
14 and 21 respectively as shown in figure 6(e). The live/dead assay analysis
shows high cell viability and mass distribution of cells across the construct, thus
favouring small pore size.
Example 5:
Cell Viability Study:
0049 To assess the biocompatibility nature of cells within the construct, a
live/dead assay was done on cells-laden constructs. The viability of embedded
cells after one day of incubation was greater than 85%, which progressed to 90%
and above overtime after 7, 14 and 21 days of culture as can be seen in Figure 8.
The z-stack (Figure 7) also showed that the cells were dispersed throughout all of
the constructs layers and that they had started to multiply from the very first day.
Also, after assessing the cell viability for the first day, we have validated our

bioprinting parameters and process that has given consistent good cell viability in the cells-laden constructs. The cells proliferated well between the layers through the pores of the construct suggesting good adherence of cells on its surfaces due to its optimal mechanical properties. Overall, the viability studies indicate favourable biocompatibility properties of the construct by promoting the attachment and proliferation of cells and their distribution throughout the construct as can be seen in Figure 9.

WE CLAIM:
1. A bioink formulation comprising 0.5E6 to 100E6/mL of a cell, 1% to 5% w/v of a
cell supporting material, 1% to 5% w/v of structural material and 4% to 8%
crosslinker for cell supporting material.
2. The bioink formulation of claim 1, wherein the cell-supporting material possesses
Arg-Gly-Asp (RGD) peptides, the crosslinker used for cell-supporting material is a chemically modified biopolymer and the structural material also acts as a viscosity enhancer.
3. The bioink formulation of any of claims 1 to 2, wherein the bioink composition is
one or more animal cells as individual or a co-culture selected from the group consisting of a fibroblast, a myoblast, an adipocyte, a skeletal muscle cell, a smooth muscle cell and an endothelial cell.
4. The bioink formulation of any of claims 1 to 3, wherein the cell-supporting material is gelatin, the structural material is sodium alginate and the crosslinker for cell-supporting material is dialdehyde starch.
5. The bioink formulation of any of claims 1 to 4, wherein it shows shear-thinning
behaviour at room temperature with viscosity dropping from 35 Pa.s to 12 Pa.s with an increase in shear rate from 1 s"1 to 10 s"1.
6. The bioink formulation of any of claims 1 to 5, shows an increase in stress from 3
Pa to 10 Pa on applying strain up to 30% with a compressive modulus between 20 to 30 kPa.
7. The bioink formulation of any of claim 1 to 6, wherein the cell supporting material,
the structural material and the crosslinker is pre-sterilized with Ultraviolet (UV) radiation or Ethylene Dioxide or Gamma irradiation.

8. The bioink formulation according to claims 1 to 7, further comprises the ability to
maintain a homogenous, sterile, and biocompatible composition suitable for encapsulating living cells.
9. A method for preparing cultivated or cultured or cell-based or lab-grown meat-like
tissue construct using bioink formulation according to claims 1 to 8, the method comprising;
(a) Feeding the bioink formulation of any of claims 1 to 8 into a three-dimensional extrusion-based bio-printer.
(b) three-dimensional extrusion-based bioprinting of a desired tissue construct; and
(c) cross-linking the three-dimensional bioprinted tissue construct made of claim 1 to 8 bioink formulation with calcium chloride comprising 5% to 15% w/v for 5 to 20 minutes.
(d) Culturing the crosslinked bioprinted tissue construct further in a culture medium for 1 to 7 days.
10. A cultivated or cultured or cell-based or lab-grown meat-like tissue construct
prepared according to the method of claim.