BACKGROUND
[0001] Unless otherwise indicated herein, the materials described in this section are not prior art in this application and are not admitted to being prior art by inclusion in this section. Field of the invention:
[0002] The subject described herein, in general, relates to a process of three-dimensional (3D) bioprinting. More particularly, but not exclusively, the subject matter relates to a process for 3D bioprinting a tissue and increasing cell viability. Discussion of the related art: [0003] The process of utilization of 3D printing technology to combine cells
with growth factors and biomaterials to develop biomedical parts that imitate natural tissue characteristics is known as three-dimensional (3D) bioprinting. As is the case with conventional 3D printing machine, 3D bioprinters also adopt the layer by layer method of depositing bioink to create tissue-like structures, generally referred to as scaffolds. The scaffolds may be further incubated to obtain
bioengineered products.
[0004] Conventionally, during the layer by layer deposition of bioink, the cells within the bioink are subjected to sudden change in temperature, which may harm or kill the cells. Further, in some instances, the bioink may be relatively highly viscous, thereby requiring relative high pressure to extrude the bioink, which in turn
may damage cells within the bioink and adversely impact cell viability. In other instances, the bioink may be relatively less viscous, due to which the extruded bioink may fail to form a solid structure with desired accuracy or may require excessive use of power to cool the deposited bioink to form a solid structure. [0005] In one of the conventional processes, a first set of layers of bioink are
deposited, followed by crosslinking, and thereafter a second set of layers of bioink are deposited over the first set. It has been observed that, it is challenging to obtain accurate alignment between the first set of layers and the second set of layers. Misalignment has an adverse impact on cell proliferation, and eventually impacts the end product. Further, the instant technique is also found to be relatively time
consuming, since there are processing steps to be carried out in between deposition

of sets of layers.
[0006] In light of the foregoing discussion, there is a need for an improved technique to 3D bioprint tissues with good cell viability.
SUMMARY [0007] A method for fabricating a bioengineered product using three-dimensional
bioprinting is disclosed. The method comprising a) preparing a bioink composition, wherein the bioink is prepared by mixing, cell carrier, serum based nutritional supplement and cryoprotectant. The temperature of the prepared bioink is altered to modify the viscosity of the bioink. In the next step the bioink is deposited onto a print plate to form a scaffold and one or more crosslinker is added to the scaffold.
BRIEF DESCRIPTION OF DRAWINGS
[0008] Embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements and in which: [0009] FIG. 1 is a flowchart explaining the 3D bioprinting process, in
accordance with an embodiment;
[0010] FIG. 2A illustrates a criss-cross pattern of the scaffold, in accordance with an embodiment; and
[0011] FIG. 2B illustrates a lattice pattern of the scaffold, in accordance with an embodiment.
DETAILED DESCRIPTION
[0012] The following detailed description includes references to the accompanying drawings, which form part of the detailed description. The drawings show illustrations in accordance with example embodiments. These example embodiments are described in enough details to enable those skilled in the art to
practice the present subject matter. However, it may be apparent to one with ordinary skill in the art that the present invention may be practised without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to unnecessarily obscure aspects of the embodiments. The embodiments can be combined, other
without

departing from the scope of the invention. The following detailed description is, therefore, not to be taken as a limiting sense.
[0013] In this document, the terms "a" or "an" are used, as is common in patent documents, to include one or more than one. In this document, the term "or" is used to refer to a non-exclusive "or", such that "A or B" includes "A but not B", "B
[0014] It should be understood, that the capabilities of the invention described in the present disclosure and elements shown in the figures may be implemented in various forms of hardware, firmware, software, recordable medium or combinations thereof.
Disclosed is a method of fabricating bioengineered product using three dimensional bioprinting process. The bioengineered product is developed using living cells. The method includes preparing a bioink composition by mixing the cells, serum based nutritional supplement, cell carrier and cryoprotectant. The
temperature of the bioink is altered to modify the viscosity of the bioink. Once the bioink of desired viscosity is obtained, the bioink is deposited onto a print plate to form a scaffold. Further, one or more crosslinker is added to the scaffold to provide structural and chemical integrity to the scaffold. The scaffold is rested to allow crosslinking for a specific period depending on the type of crosslinker used. The
scaffold is then washed with another solution to remove residual crosslinker. The scaffold is then incubated for culturing the cells to eventually form the bioengineered product.
METHOD OF FABRICATING BIOENGINEERED PRODUCT [0016] Referring to FIG. 1, a method is provided for fabricating bioengineered
product using three dimensional bioprinting process. At step 102, living cells may be added to a solution comprising serum based nutritional supplement and cryoprotectant.
[0017] The cells are selected based on the bioengineered product or scaffold that has to be fabricated.
[0018] In an embodiment, the cells may be derived from a group consisting of

an epithelial, muscular, nervous, or connective tissue, or a suitable combination thereof. The cells are obtained from healthy or diseased donor. Further the cells may be genetically engineered cells, including induced pluripotent stem cells (iPSCs) or disease specific model cells. [0019] In an embodiment, the tissue specific cells may be derived from a tissue
selected from a group consisting of liver, gastrointestinal, pancreatic, kidney, lung, tracheal, vascular, skeletal muscle, cardiac, skin, smooth muscle, connective tissue, corneal, genitourinary, breast, reproductive, endothelial, epithelial, fibroblast, neural, Schwann, adipose, bone, bone marrow, pericytes, mesothelial, endocrine, stromal, lymph, and blood.
[0020] The dilution of the solution (comprising serum based nutritional
supplement and cryoprotectant) is based on the cell count. In an embodiment, 1 ml of the solution may be used for a cell count of 1 million. Further, the solution is prepared such that it does not affect the concentration of the cell carrier. When the cells are immersed, the serum based nutritional supplement and cryoprotectant
solution provides the cells a protective coating. This helps in protecting the cells from being exposed to sudden decrease in temperature during the bioprinting process. The absence of a protective coating may kill or harm the cells. In an embodiment, 1 ml of the bioink comprises about 1*10^4 -9*10A6 cells. In an embodiment, the quantity of cell count may depend on the surface area of the
desired scaffold to be printed.
[0021] In an embodiment, 1 ml of the bioink comprises 2 -20 v/v % of serum
based nutritional supplement. The serum based nutritional supplement may be biological fluids like serum (2% - 20% v/v). The serum is for example, but not limited to, bovine serum (cow), chicken serum, caprine (goat), equine (horse),
human, ovine (sheep), porcine (pig) or rabbit sera. In another embodiment, 1 ml of the bioink comprises 0.01- 20 v/v % of chemically defined supplements. The serum-based supplements may be substituted by the chemically defined supplements. The chemically defined supplements may be a tissue extract for example but not limited to bovine pituitary extract (0.1% - 2% v/v) or growth
factors or growth hormones or growth regulating factor for example but not limited

to EGF (0.05 - 100 ng/ml), VEGF (2 - 50 ng/ml), hydrocortisone (0.1 - 20 ug/ml),
insulin (0.5 - 50ug/ml), epinephrine (0.05 - 4ug/ml), transferrin (1 - 25ug/ml),
heparin, non-essential amino acids, PDGF (1 - 50 ng/ml), TGF (0.001 - 20ug/ml).
[0022] In an embodiment, 1 ml of the bioink comprises 2% -10% v/v of
cryoprotectant. In an embodiment, a single cryoprotectant or a combination of
various cryoprotectants may be used to prepare the bioink. The cryoprotectant may be Dimethyl Sulfoxide DMSO (0.5 % -10% v/v), glycerol (1% - 5% v/v), hydroxy ethyl starch (0.1 - 10%), PEG or combinations thereof. The concentration of the various cryoprotectants when used in combination to form the cryoprotectant having a minimum concentration of 2% v/v are for example, but not limited to, a)
DMSO 0.5% and glycerol 1.5%; b) Glycerol 1% and DMSO 1%; c) DMSO 0.5%, hydroxy ethyl starch 0.1% and glycerol 1.4%. Further, when a single cryoprotectant is used for example DMSO or any other cryoprotectant, the concentration of the DMSO or any other cryoprotectant is at least 2% v/v, but when used in combination the range may vary as provided above in the form of examples. Similarly, the
cryoprotectant having the maximum concentration i.e. 10% v/v may be prepared by
using various combination of the cryoprotectants. When a single cryoprotectant is
used to prepare the bioink, the concentration of the single cryoprotectant is
maximum 10% v/v.
[0023] In an embodiment, 1 ml of the bioink comprises 1-12 w/v % of cell
carrier. The cell carrier is prepared at step 104. The cell carrier is prepared by dissolving biomaterials in a solvent. In another embodiment, the cell carrier is prepared by dissolving one or more biomaterials in a solvent. [0024] In an embodiment, the solvent may be a combination of water (0% -75% v/v) and culture media (100% -25% v/v), for example but not limited to, 10%
water and 90% media. In another embodiment, water may be replaced with PBS, HBSS, TBS, HEPES or MOPS, and the media with DMEM. [0025] In yet another embodiment, the solvent may be prepared using the following components NaOH (0.8%), HC1 (1.8%), KOH (0.3%), Nicotinic acid (5mg/L), S02 (1%), Ethanol (2%), Chloroform (1%), Dichloromethane (1%),
Carbon tetrachloride (2.5mM). Benzene (15mM). Toluene (lOmM).Ethvlbenzene

(lOmM), Xylene (5mM), Acetone (5%), Dimethyl formamide (0.5%), Glycerol (5%), Polyethelyene glycol i.e, PEG (2%) , triethylene glycol (1.5%) or combination thereof. The range disclosed for each of the component is the maximum range. The above listed components may be used in combination with media and water (water could be replaced PBS, HBSS, TBS, HEPES or MOPS)
or with media alone. The combinations are for example, but not limited to, a) PEG (2%) and media 98%; b) PEG (2%), water (25%) and media (73%); c) PEG (1%), ethanol (1%), water (24%) and media (73%); d) Ethanol (1%) and media (99%); and e) Acetone (2%) and DMEM (98%). [0026] In an embodiment, the selection of the culture media may depend on the cell
type used. The culture media could be for example, but not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute medium (RPMI), Minimum Essential Media (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Reduced-Serum Medium (Opti - MEM), Minimum Essential Media alpha (a MEM), Mc Coy's 5 A and modifications of these media.
20 [0027] In an embodiment, the biomaterial may be natural polymers, synthetic polymers or a combination of both natural and synthetic polymers. In an embodiment, the biomaterial may be a naturally occurring polymer selected from the group consisting of collagen, fibrin, chitosan, alginate, oxidized alginate, starch, hyaluronic acid, laminin, silk fibroin, agarose, gelatine, glycans, and combinations
thereof. In an embodiment the biomaterial when used in combination, the concentration of each of the biomaterials may vary based on the type of the biomaterial used. A list of various natural biomaterial with the concentration range is provided when the cell carrier is prepared using one or more biomaterials in combination, such as, but not limited to, collagen (0.1- 5% w/v), gelatin (1-12 %
w/v), agarose (0.2-2 % w/v), agar (0.01-1 % w/v), alginate (0.05- 2% w/v), hyaluronic acid (0.01- 0.5% w/v), fibrin (0.5-10%)or combinations thereof. [0028] In other embodiment, the biomaterial may be a synthetic polymer selected from the group consisting of polyphosphazene, polyacrylic acid, polymethacrylic acid, polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid)
(PLGA), polyorthoester (POE), polycaprolactone (PCL), polyamine acid (such as

polylysine), degradable polyurethane, copolymers, and combinations thereof.
[0029] In an embodiment, the choice of the solvent may depend on the polymer used. The biomaterials are mixed in the solvent using a shaker for about 5 minutes to 60 minutes, and for about 1 hour to 24 hours for natural polymers and synthetic polymers respectively. 10 [0030] A change in the viscosity with a variance in the temperature and concentration of the cell carrier is discussed. In order to evaluate the change in viscosity, the cell carrier having the concentration 3% w/v and 12% w/v was used for the analysis. Table 1 represents the results of the experimental study performed with the cell carrier having 3% w/v concentration.
[0031] From the study, it has been observed that the temperature of the cell carrier is maintained between 6.4 degree Celsius and 9.3 degree Celsius to obtain the desired viscosity and the size of the needle gauge used for the bioprinting process ranges from27G-30G. Further, table 2 represents results of the experimental study 0 conducted with the cell carrier having 12% w/v concentration.
Table 2

[0032] The study reveals that the temperature of the cell carrier is maintained between 12 degree Celsius 27.9 degree Celsius to obtain the desired viscosity and the size of the needle gauge used in the bioprinter ranges from 20G-30G. The experimental study performed with different concentration of the cell carrier reveals that lower the concentration of the cell carrier, lower is the temperature to be
maintained to obtain the desired viscosity.
[0033] Once the cell carrier and the cells are prepared, at step 106, the cells (that were immersed in serum based nutritional supplement and cryoprotectant solution) are added to the cell carrier to form bioink. In an embodiment, the quantity of cell mixture (cells that are immersed in the serum based nutritional supplement
and cryoprotectant solution) may be negligible compared to the quantity of cell carrier.
[0034] In another embodiment, the bioink may be prepared by mixing the living cells, the cell carrier, the serum based nutritional supplement and the cryoprotectant.

[0035] In an embodiment, the bioink is processed, for example by regulating or altering the temperature of the bioink that correspond to a range of preferred viscosity. In an embodiment, the temperature of the bioink is altered by cooling. At step 108, the bioink is immersed in ice prior to the bioprinting process for a predetermined time. In an embodiment, the predetermined time may be three
minutes to four minutes. In another embodiment, the predetermined time varies accordingly depending on the concentration of the cell carrier used for the preparation of the bioink (Refer table 1 and 2). For example, the bioink is immersed for about half an hour if the concentration of the cell carrier is 3% w/v and for about 30 seconds if the concentration of the cell carrier is 12% w/v.
[0036] The immersion of bioink in the ice makes the bioink semi gel like. This semi gel composition gives the bioink the desired viscosity that is required for bioprinting process. If the viscosity is not right, then either the bioink may be too thick or too watery. High viscosity of the bioink may result in applying excessive force to extrude the bioink. Excessive force may lead to high shear within the bioink
20 which may end up harming or killing the cells. On the other hand, if the viscosity of the bioink is too less, the scaffold eventually formed may not be structurally stable and may lack accuracy. Thus, keeping the bioink in ice for a predetermined time just before adding it to the extruder, provides the bioink with stable characteristics.
[0037] In an alternate embodiment, the bioink, without the need to be kept in ice for a predetermined time, may be directly placed in the extruder to cool the bioink. The extruder may comprise a cooling jacket, wherein the cooling jacket may provide the extruder with the required cooling. This may keep the bioink within the extruder semi gel like, to provide the bioink the desired viscosity (refer table 1 and
table 2).
[0038] In an embodiment, the inner diameter of the extruder needle (of the extruder) may be varied to extrude bioinks of different viscosities. The inner diameter of the extruder needle may be reduced to extrude the bioink with lower viscosity. The inner diameter of the extruder needle may be increased to extrude
the bioink with higher viscosity.

[0039] At step 110, the bioink (that was kept in the ice for 3-4 minutes) is introduced into the extruder of the 3D bioprinter. In an embodiment, prior to extruding, the print plate is allowed to cool. In an embodiment, the print plate is cooled to about -10 degree Celsius to -25 degree Celsius. In other embodiment, the print plate may be cooled via cooling a mechanism for example but not limited to a
peltier device. In another embodiment, the print plate may be a petri plate, a quartz plate or a glass slide. In yet another embodiment, the print plate may be made of metals for example but not limited to aluminium and stainless steel. Further, the print plate should be sterile, non- corrosive having a smooth flat surface. Once the print plate has reached the required temperature, the bioink is extruded onto the
print plate layer by layer to form a scaffold.
[0040] In an embodiment, the scaffold may be printed in any of the patterns that exists or that may exist in the future. In an example embodiment, the scaffold may be printed in a lattice pattern or a criss-cross pattern or a combination of both lattice pattern and criss-cross pattern. Referring to FIGs.2A-2B illustrates the criss-
cross pattern and lattice pattern of the scaffold. The criss-cross pattern of the scaffold increases contact between the cells. Further, the criss-cross pattern also decreases the cell migration distance between the pores.
[0041] The processes explained so far and the process of adding the crosslinker (which will be explained later) makes it possible to extrude multiple layers required
to form the scaffold in a single extrusion process. It may be noted that, different extruders may be used based on the cell type desired in different layers of the scaffold.
[0042] In an embodiment, the extruder may extrude the bioink using any one of the existing extruder mechanisms such as, pneumatic, hydraulic, mechanical,
electronic, combination of the existing mechanism or the mechanisms that may be made available in future.
[0043] Once the extrusion process is over, the scaffold is allowed to rest on the print plate (without switching off the peltier) for a predetermined time. In an embodiment, the predetermined time may be 2-3 minutes.
[0044] At step 112, the crosslinker is added to the scaffold. The crosslinker is

added to provide structural cohesion to the scaffold. In an embodiment, the crosslinker may be added either manually or mechanically. Manual crosslinking may be done using a pipette. Mechanical or automated crosslinking may be done using a mechanism that may be similar to the extruder mechanism. Once the crosslinking of the scaffold is done, the scaffold is allowed to rest on the print plate
(without switching off the peltier) for a predetermined time. In an embodiment, the predetermined time may be 5-6 minutes.
[0045] In an embodiment, the bioprinted scaffold may undergo physical crosslinking, chemical crosslinking, photo crosslinking or combinations thereof. [0046] In an example embodiment, the chemical cross linking may be
performed by any cationic or anionic or non-ionic cross-linkers.
[0047] In another embodiment, the scaffold may be crosslinked by calcium, magnesium, sodium, chloride, alginate, or any combination thereof. [0048] In yet other embodiment, enzymatic cross-linkers may be used for cross linking the bioprinted scaffold.
[0049] At step 114, the scaffold, after allowing to be rested on the print plate for the predetermined time, is stored at a predetermined temperature for a predetermined time. In an embodiment, the predetermined temperature may range from -10 degree Celsius to -196 degree Celsius. In another embodiment, the predetermined temperature may be in the range of-10 degree Celsius to -40 degree
Celsius and the predetermined time may be 6 to 18 hours. The predetermined time for which the scaffold is kept for crosslinking may depend on the quantity and concentration of the crosslinker and the cell carrier.
[0050] Once the scaffold is kept for crosslinking for the predetermined time, at step 116, the scaffold is washed with a solution to remove any residual crosslinker.
In an embodiment, the solution may be Phosphate Buffered Saline (PBS).
[0051] At step 118, the scaffold is dipped in a media and introduced into an incubator that is maintained at a predetermined temperature. Further, the amount of CO2 within the incubator may be maintained at a predetermined level for culturing the cells. In an embodiment, the predetermined temperature may range from 37
degree Celsius to 39 degree Celsius and the predetermined level of CO2 maintained

may range from 5% to 7% v/v. The tissue thus obtained may have high cell viability. METHOD OF FABRICATING SKIN TISSUE
[0052] An exemplary embodiment for 3D bioprinting a skin tissue is explained. The skin tissue may include keratinocytes, melanocytes and fibroblasts. In an embodiment, skin tissue comprising of only keratinocytes may be 3D bio printed
or skin tissue comprising of only fibroblasts may be 3D bio printed or skin tissue comprising of a combination of both fibroblasts and keratinocytes may be 3D bio printed. Skin cells are prepared and mixed with the solution comprising the serum based nutritional supplement and cryoprotectant. to protect the skin cells from being exposed to sudden decrease in temperature. Meanwhile, the cell carrier is prepared
by dissolving the biomaterial in a combination of water and media. The cells (that were immersed in the solution comprising the serum based nutritional supplement and cryoprotectant.) are added to the cell carrier. The mixture of cells and cell carrier may be called as bioink. The bioink is immersed in ice for 3-4 minutes and placed in the extruder. The peltier device is turned on before printing the bioink so
that the surface of the print plate is cooled enough. The bioink is then deposited in layers. The number of layers of the bioink to be deposited may depend on the nature of the tissue that may be developed.
[0053] After the bioprinting, the scaffold is left on the print plate for about 2-3 minutes, with the print plate kept on. The crosslinker is then added to the scaffold
for crosslinking. Crosslinking helps in giving structural cohesion to the tissue. Once the crosslinker has been added to the scaffold, the scaffold is left on the print plate for 5-6 minutes, with the print plate still cold. Then, the print plate is switched off and the scaffold is removed from the print plate, sealed and stored at 4°C to -20°C for about 6 hours 12 hours. The scaffold is then immediately washed with PBS to
remove any residual crosslinkers if there are any.
[0054] The scaffold is then dipped in the media and sent to the incubator for culturing the skin cells. The incubator is maintained at 37°C and 5% CO2 for the duration of the culture. During the incubation process, after a period of 3 days the media is changed. The volume of new media added is such that the tissue is brought
to Air Liquid Interface (ALI), i.e., so that keratinocytes can proliferate and

differentiate. Thereafter, the media is changed for every 2-3 days for replenishing the required nutrients. The scaffold is kept in the incubator until the tissue is formed. The skin tissue thus obtained has high cell viability.
[0055] The present invention overcomes the drawbacks of the conventional bioprinting processes, by providing an improved way of bioprinting to achieve high
cell viability. The present invention as discussed in this document with respect to different embodiments will be advantageous at least in protecting the cells from being exposed to sudden change in the printing parameters such as temperature and pressure during the bioprinting process. This may prevent the killing or harming of the cells during the bioprinting process. Further, the present invention is
advantageous in providing the bioink with the required viscosity. This helps in providing the bioink the required stability during the bioprinting process. Also, the present invention is advantageous in bioprinting multiple layers in a single extrusion process, thus preventing multiple manual extrusion processes. Further, the present invention reduces the overall cost of developing the tissue, reduces the
amount of materials required, increases the precision during the bioprinting process, provides proper spatial location of the cells. Additional advantages not listed may be understood by a person skilled in the art in light of the embodiments disclosed above. [0056] It shall be noted that the processes described above are described as
sequence of steps; this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, the order of the steps may be re-arranged, or some steps may be performed simultaneously. [0057] Although embodiments have been described with reference to specific example embodiments, it will be evident that various modifications and changes
may be made to these embodiments without departing from the broader spirit and scope of the system and method described herein. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense. [0058] Many alterations and modifications of the present invention will no doubt become apparent to a person of ordinary skill in the art after having read the
foregoing description. It is to be understood that the phraseology or terminology

employed herein is for the purpose of description and not of limitation. It is to be understood that the description above contains many specifications; these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the personally preferred embodiments of this invention. Thus, the scope of the invention should be determined by the appended claims and 10 their legal equivalents rather than by the examples given.

We Claim:
1. A method for fabricating a bioengineered product using three-dimensional
bioprinting, the method comprising:
preparing a bioink composition, the method comprising:
mixing cells, cell carrier, serum based nutritional supplement and
cryoprotectant;
altering the temperature of the prepared bioink to modify the viscosity of the
bioink;
depositing the bioink onto a print plate to form a scaffold; and
adding one or more crosslinker to the scaffold.
2. The method as claimed in claim 1, wherein the cell carrier is selected from
a group consisting of synthetic polymers, natural polymers or a combination
thereof.
3. The method as claimed in claim 1, where the temperature of the bioink is
altered by cooling.
4. The method according to claim 3, wherein the cooling of the bioink is
performed by immersing the bioink in ice for about three minutes to four minutes
to attain desired viscosity for printing.
5. The method according to claim 3, wherein the bioink is cooled in a cooling
jacket of an extruder.
6. The method according to claim 1, wherein the crosslinker is selected from a group consisting of chemical cross linker, physical crosslinker, photo crosslinker or combinations thereof.
7. The method according to claim 1, wherein the print plate is cooled.
8. The method according to claim 1, wherein the print plate is cooled to a
temperature ranging between -10 degree Celsius to -25 degree Celsius.
9. The method according to claim 1, the method further comprising:
storing the scaffold after allowing the scaffold to rest on the print plate at a
predetermined temperature ranging between -10 degree Celsius and -196
degree Celsius for a time period ranging between 6 hours to 18 hours;

removing an excess of the one or more cross linker added to the scaffold on
completion of the crosslinking; and culturing the scaffold in an incubator.
10. The method according to claim 9, wherein the scaffold is stored for the time
period of about 6 hours to 12 hours maintaining the temperature at about 4 degree
to -20 degree Celsius.
11. The method according to claim 9, wherein the scaffold has a criss -cross pattern, a lattice pattern, or a combination of criss cross partem and lattice pattern.
12. The method according to claim 9, wherein the excess of the one or more cross linker added to the scaffold is removed using a Phosphate Buffered Saline
(PBS) solution.
13. The method according to claim 1, wherein the concentration of living cells ranges between 1*10^4 and 9*10A6/ml of the bioink.
14. The method according to claim 1, wherein the concentration of the cell carrier in the bioink ranges from 1 % w/v to 12 % w/v.
15. The method according to claim 1, wherein the concentration of the cryoprotectant in the bioink ranges from 2 % v/v to 10 % v/v.
16. The method according to claim 1, wherein the concentration of the
nutritional supplements in the bioink ranges from 2 % v/v to 20 % v/v.
17. The method according to claim 1, wherein the cell carrier is prepared in a
solvent, wherein the solvent comprises at least one of water and culture media.
18. The method according to claim 17, wherein the concentration of the water
in the solvent ranges from 0% v/v to 75% v/v and the concentration of the culture
media in the solvent ranges from 100% v/v to 25%
19. The method according to claim 1, wherein the viscosity of the bioink ranges
between 1698.1 centinoise and 1057298 centinoise.