Engineered Whole Cut Meats Assembled of Cell Fibers Constructed by 1 Tendon-Gel Integrated Bioprinting 2

1 With the current interest in artificial meat, mammalian cell-based cultured meat has mostly 2 been in minced form. There is thus still a high demand for artificial steak-like meat. Herein, 3 we demonstrate in vitro construction of engineered steak-like meat assembled of three types of 4 edible bovine cell fibers, such as skeletal muscle, adipose, and blood capillary fabricated by 5 tendon-gel integrated printing (TIP) technology. Because actual meat is an anisotropically 6 aligned assembly of the fibers connected to tendon for the actions of contraction and relaxation, 7 TIP was discovered to construct the fiber assembly connecting tendon gels with engineered 8 structures. In this study, a total of 72 fibers comprising 42 muscle, 28 adipose, and 2 blood 9 capillary were constructed by TIP and subsequently assembled to fabricate a steak-like meat 10 with a diameter of 5 mm and a length of 10 mm by consulting histological images of actual 11 Wagyu beef steak. The TIP discovered here could be a powerful manufacturing technology for 12 fabrication of the desired types of steak-like cultured meats. 13 Introduction 1 Over the past decade, cultured meat has drawn tremendous attention from standpoints of 2 ethics, economics, the environment, and public health. More recently, meat analogs that taste 3 like meat but are based on plant proteins have been released commercially1,2. Although 4 challenges remain unlike with meat analogs, cultured meat is highly sought after due to the 5 possibility of imitating real meat through the manipulation of flavor, muscle/adipose cells 6 ratio, and texture3,4. Bovine cells for cultured meat can currently be secured by two 7 approaches5,6. One is that after obtaining edible tissues from cattle, they are separated into 8 each cell type such as muscle satellite cells, adult stem cells, and multipotent stem cells etc. 9 which are then cultured to increase the number of cells. The other is to transform somatic 10 cells into induced pluripotent stem cells (iPSCs) and differentiate to each cell type. Even 11 though primary cultured stem cells, particularly muscle satellite cells that maintain the 12 differentiation capability within 10 passage7, have a limited number of divisions, they would 13 still be safe and acceptable for consumption. Edible forms can also be constructed by the 14 assembly of acquired bovine cells. Since Post and co-workers unveiled minced meat 15 composed of lab-grown bovine cells, various types of cultured meat have been demonstrated. 16 However, cultured steak with a compositional and structural similarity to real steak, 17 comprising mostly muscle and adipose cells with muscle cells in alignment, is still 18 challenging4,8,9. 19 To realize the structural characteristics of steak, various tissue engineering techniques have 20 been considered such as cell sheet engineering10,11, cell fiber engineering12, cell culture on a 21 3D printed scaffold13, and 3D cell printing14,15. It is noteworthy that in the 3D cell printing 22 field some researchers have adopted a supporting bath assisted 3D printing (SBP) technique 23 where ink is dispensed inside the gel or suspensions with thixotropy. Since the SBP is able to 24 overcome the shortcomings of restricted available range of viscosity in ink and drying for 25 prolonged printing in extrusion-based 3D printing on the air-interfaced environment, several 1 studies over the past 5 years have shown evidence of its potential in a complex tissue 2 fabrication16–22. 3 Steak meat is an aligned assembly of skeletal muscle fascicles with a diameter from around 4 900 μm to 2.3 mm23 depending on age and animal parts, which are the assembly of skeletal 5 muscle fibers, connecting to tendon for the movements of its shrinkage and relaxation. The 6 muscle fibers are covered with basement membrane and the muscle fascicles are surrounded 7 by fat together with blood capillaries (Fig. 1a). The component ratio and location of the 8 muscle, adipose, and blood capillary tissues are significantly different according to meat type 9 and original country of origin. For example, red meat in the rump of Japanese Wagyu has 10 only 10.7% adipose tissues, whereas the sirloin of the Wagyu has 47.5%24. Accordingly, 11 development of a novel methodology for assembling the three types of fibers with desired 12 location, ratio, and amount will be a key manufacturing technology of cultured steak. 13 Here, we demonstrate a three-step strategy for the construction of engineered steak-like 14 meat: (1) Collection of edible bovine satellite cells (bSCs) and adipose-derived stem cells 15 (bADSCs) from approved block beef meats and subsequent expansion, (2) development of 16 tendon-gel integrated bioprinting (TIP) technology for the fabrication of pre-cell fibers and 17 subsequent differentiation to skeletal muscle, adipose, and blood capillary fibers, (3) 18 assembly of the differentiated cell fibers to construct engineered steak-like meat by 19 mimicking the histological structures of actual beef steak (Fig. 1b). Since tendon is a key 20 tissue for anisotropic alignment and maturation of muscle fibers, we fabricated tendon gels by 21 TIP for consecutive connection between muscle cell fibers and tendon gels to obtain scalable 22 anisotropically aligned matured muscle fibers. In this study, a total of 72 fibers comprising 42 23 muscle, 28 adipose, and 2 blood capillary were constructed by TIP and subsequently 24 assembled to fabricate a steak-like meat with a diameter of 5 mm and a length of 10 mm by 25 consulting histological images of actual Wagyu beef steak. TIP is expected to become a 1 powerful technology for constructing engineered steak-like meat with desired location, 2 component ratio, and amount of the three types of fibers. 3 4 Results and Discussion 5 Verification of Differentiation Conditions in Extracted bSCs and bADSCs 6 The bSCs were isolated from the masseter muscle of a 27-month-old Japanese black cow 7 obtained from a slaughterhouse using a method modified from a previously reported one7. The 8 crude cell fraction separated from the approved beef meat by collagenase treatment was 9 cultured until passage (P) 3 for cell sorting. The CD31−, CD45−, CD56+, and CD29+ cells were 10 isolated by FACS, in which Pax7+ bSCs were around 80%. 2D culture of the isolated bSCs was 11 performed to evaluate the capability of proliferation and differentiation into muscle cells with 12 prolonged passaging. After seeding the bSCs the passage was counted after every 2 days of 13 culture. The media for proliferation contains not only fetal bovine serum (FBS) and basic 14 fibroblast growth factor but also a p38 inhibitor to maintain the differentiation capacity of 15 proliferating bSCs7. The number of seeded bSCs doubled around once a day until P8, and 16 around once every 2 days thereafter (Fig. 2a). The differentiation after 2 days of seeding was 17 induced by changing the basic media to a differentiation media containing 2% horse serum 18 (HS), which is a well-known differentiation induction method for muscle cells. The cells were 19 immunostained with the antibody of myosin II heavy chain (MHC) after 5 days of 20 differentiation induction. We quantified the differentiation capacity on passage number of the 21 seeded bSCs by calculating the ratio of DAPI fluorescence intensity between MHC+ and MHC22 cells from fluorescence images (Supplementary Fig. 1). The bSCs from P3 to P7 expressed a 23 comparable differentiation level, but the differentiation capability of bSCs above P8 24 significantly decreased (Figs. 2b and 2c). Therefore, we conducted experiments using cells 1 prior to P8. 2 Next, 3D encapsulated culture in collagen microfibers (CMF)/fibrin gel was performed for 3 assessment of the adipogenic differentiation potential of bADSCs with a variety of media 4 condition since it is known that the adipogenesis of adipose-derived stem cells (ADSCs) in 3D 5 culture is higher than in 2D culture25 and suitable differentiation factors rely on species26. 6 Conventional human adipogenic factors like insulin, rosiglitazone, or troglitazone were thus 7 first found with limited adipogenic induction potential (Supplementary Fig. 4), leading to the 8 direct addition of free fatty acids (pristanic acid, phytanic acid, erucic acid, elaidic acid, oleic 9 acid, palmitoleic acid, and myristoleic acid) to the culture medium27. The different 10 combinations of the seven aforementioned free fatty acids were thus compared and the results 11 showed significantly higher adipogenesis by lipids storage in vesicles in the cytoplasm of the 12 bovine preadipocytes for all seven free fatty acids contained media (from 1.8 to 2.7 times more 13 at day 13 of differentiation) (Fig. 2d and Supplementary Figs. 2 and 3). To further increase the 14 lipogenesis until reaching a matured bovine adipocyte state, the transforming growth factor 15 (TGF) type I receptor activin-like kinase 5 inhibitor (ALK5i) effect was evaluated because this 16 factor is an inhibitor of the TGF-β receptor ALK5 and TGF‐β family ligands, contained in the 17 10% FBS of the culture medium, which are known to inhibit both adipogenesis and adipocyte 18 hypertrophy28. The TGF‐β family also includes myostatin, which is expressed by the myocytes 19 to impair adipogenesis29. In the context of future co-culture between bovine myoblasts and 20 adipocytes, ALK5i appeared relevant for further inducing the adipogenic potential of the 21 culture medium containing the seven free fatty acids. Several concentrations were thus assessed 22 from 1 to 10 μM. The results showed a tendency to a higher lipogenesis by lipids storage with 23 5 μM ALK5i (Fig. 2e). The adipogenic maturation of the bADSCs then increased progressively 24 between 3 and 7 days of differentiation (Fig. 2f). 25 Recently, ADSCs have been considered to be a useful cell source for angiogenesis in tissue 1 engineering, but unlike human ADSCs there are no reports on endothelial differentiation of 2 bADSCs30,31. Knowing that they lose their differentiation potentials during ADSCs culture 3 expansion32, bADSCs were thus used at P1 to evaluate their endothelial differentiation in the 4 different conditions. HS was surprisingly found to be a significant inducer of its endothelial 5 differentiation, even at low concentration (6.4 and 10 times more for 1 and 10% HS, compared 6 to the 10% FBS condition), independently of the medium used, DMEM or F12K (Figs. 2g-h 7 and Supplementary Fig. 5). Human serum also provided an enhanced endothelial 8 differentiation, compared to the FBS condition, but was impaired by the low cell proliferation 9 observed (Supplementary Fig. 5). The DMEM + 10% HS was then used for the endothelial 10 differentiation from bADSCs in this study. 11 12 Bovine Muscle Fiber Fabrication by SBP 13 To organize the isolated bSCs into a cell fiber, we utilized an SBP in which a bioink is 14 dispensed inside a supporting bath that is usually composed of hydrogel particles (or high15 viscous polymer melt) with thixotropy. Several studies have demonstrated its promise in cell 16 printing for structural controllability and stable printing during prolonged operation18,19,21,22. 17 We selected gelatin and gellan gum as supporting bath materials, respectively, due to their 18 edible, removable, and cell compatible properties. Gelatin is a gel at room temperature (RT) 19 and a solution at 37°C, therefore it is easy to remove after printing by incubation at 37°C17. 20 Gellan gum hydrogel is also known to dissolve in Tris-HCl buffer at pH 7.4 and at 37°C33. The 21 gellan gum and gelatin were fabricated in bulk hydrogel and homogenized into granular 22 particles for implementation in a supporting bath, and their thixotropy was confirmed 23 (Supplementary Fig. 6). Firstly, we tried to print the bioink containing bSCs, fibrinogen, and 24 Matrigel solution in media into the supporting bath mixed with granular particles of gelatin (G25 Gel) or gellan gum (G-GG) and thrombin for the fabrication of a fibrous muscle fiber 1 mimicking the bundle of muscle fiber in steak (Supplementary Movies 1 and 2). With the 2 confirmation of the gel formation followed by the removal of supporting baths, high cell 3 viability was observed for 3 days after printing in both the G-Gel and G-GG by live/dead 4 staining (Figs. 3a, 3b and Supplementary Fig. 7). 5 When the printed cell fiber was cultured in suspension, it transformed from a fibrous to a 6 globular form (Fig. 3c). Studies related to muscle tissue engineering have implied that an 7 anchor structure enables 3D muscle tissue to not only maintain its initial shape but also improve 8 the cell alignment, fusion, and differentiation against the muscle fiber’s contraction15,34–38. We 9 placed a printed cell fiber onto a silicone rubber and anchored it with needles to fasten both 10 ends to withstand cell contractions (Fig. 3d, left). With the needle fixed culture, the cell fibers 11 printed inside G-GG and G-Gel retained the fibrous structure, but the diameter had shrunk by 12 around 60% in G-GG and 80% in G-Gel at day 9 of culture (Fig. 3d, right). It would be 13 reasonable to suppose that the size decrease was caused by alignment and fusion of bSCs with 14 an enzymatic decomposition of fibrin gel by proteases secreted from cells39,40. We also took 15 immunofluorescence images inside the cell fibers to examine the cellular behavior w/ and w/o 16 needle anchoring after 7 days of differentiation. To quantify the improvement in terms of 17 muscle maturation, the cell alignment, i.e. the angle setting for the straight line between needles, 18 was measured from the immunofluorescence images (Fig 3e, left). The results showed that the 19 cells in the cell fiber of suspension culture randomly oriented regardless of the type of 20 supporting baths and in needle fixed culture the cells in the cell fiber printed inside G-Gel were 21 highly anisotropically oriented compared to those of G-GG (Fig 3e, right). We postulate that 22 the difference in the degree of alignment between G-GG and G-Gel arises from the hindrance 23 of cell behaviors by residual substances that might exist inside or on the printed cell fibers. 24 That is, the residual G-GG in the cell fiber may not be degraded or dissolved, in which case the 25 cells rarely remodel the ECM around them and are not able to migrate to fuse other cells 1 (Supplementary Fig. 8). On the other hand, G-Gel is easily dissolved at 37°C and may be 2 degraded by proteases, enabling active cell behavior although there are residues inside the 3 printed cell fiber. Printing bSCs inside G-Gel and anchoring them are essential steps for 4 fabrication of the muscle cell fiber, but the anchoring method may not be appropriate for scale5 up. Therefore, we developed a modified SBP to include a part to simultaneously anchor the 6 printed cell fiber. 7 8 Fabrication of Muscle, Fat, and Vascular Cell Fibers by TIP 9 The important feature in the modified SBP, which we have named TIP, is the introduction of 10 tendon-gels to anchor the printed cell fibers for culture. Fig. 4a illustrates the process of the 11 TIP in which the printing bath is divided into three parts; bottom tendon-gel, supporting bath, 12 and upper tendon-gel. G-Gel is used as a supporting bath as described in the above section and 13 the volume of tendon-gels is filled with 4 wt% collagen nanofiber solution (CNFs) which 14 shows the reversible sol-gel transition from 4°C to 37°C (Supplementary Fig. 9). To separate 15 the layers and maintain the structure we fabricated polydimethylsiloxane (PDMS) wells 16 (Supplementary Fig. 10). After bSCs fiber formation inside the PDMS well (Supplementary 17 Movie 3), incubation for 2 h at 37°C induced the supporting bath and tendon-gels to become a 18 solution and gel, respectively, followed by placement of a PDMS well itself in the media for 19 culture. 20 On day 3, we could confirm that the printed cell fiber maintained its fibrous shape with 21 reduced size (data not shown) and that there was a connection between two tendon-gels in 22 phase contrast and H&E staining images (Figs. 4b and 4c). The bSCs fiber by TIP also showed 23 a high alignment of cells after 3 days of differentiation, which is comparable with that of the 24 needle fixed culture (Figs. 4d and 4e and Supplementary Movie 4). Interestingly, the sarcomere 1 structure of matured muscle fibers was shown in some of the TIP derived bSCs fibers (Fig. 4f), 2 but we could not show any cell fibers by the needle fixed culture after 14 day of differentiation. 3 Even though we did not investigate thoroughly here, it may have been caused by cell adhesion 4 of bSCs to the collagen gel at anchorage regions in the TIP whereas there is no cell adhesion 5 in the needle fixed culture (Supplementary Fig. 11). TIP is a promising method for muscle fiber 6 fabrication, but it still has a problem in that the bSCs fiber become detached from the tendon7 gels, especially the bottom tendon-gels, in a prolonged culture due to its strong contraction. 8 Increasing concentration of CNFs or additional crosslinking will hopefully provide a solution 9 to this problem. Moreover, double printing, after fabrication of a cell fiber by general TIP, then 10 rotating the PDMS well 180° so that it reversed and then printing one more time around the 11 first formed cell fiber, may be another way of solving the problem. When double printing, two 12 printed cell fibers close to each other are fused into one thicker cell fiber (Supplementary Fig. 13 13) and it seems to be more resilient to strong contraction than by simply printing once (data 14


Introduction 1
Over the past decade, cultured meat has drawn tremendous attention from standpoints of 2 ethics, economics, the environment, and public health. More recently, meat analogs that taste 3 like meat but are based on plant proteins have been released commercially 1,2 . Although 4 challenges remain unlike with meat analogs, cultured meat is highly sought after due to the 5 possibility of imitating real meat through the manipulation of flavor, muscle/adipose cells 6 ratio, and texture 3,4 . Bovine cells for cultured meat can currently be secured by two 7 approaches 5, 6 . One is that after obtaining edible tissues from cattle, they are separated into 8 each cell type such as muscle satellite cells, adult stem cells, and multipotent stem cells etc. 9 which are then cultured to increase the number of cells. The other is to transform somatic 10 cells into induced pluripotent stem cells (iPSCs) and differentiate to each cell type. Even 11 though primary cultured stem cells, particularly muscle satellite cells that maintain the 12 differentiation capability within 10 passage 7 , have a limited number of divisions, they would 13 still be safe and acceptable for consumption. Edible forms can also be constructed by the 14 assembly of acquired bovine cells. Since Post and co-workers unveiled minced meat 15 composed of lab-grown bovine cells, various types of cultured meat have been demonstrated. 16 However, cultured steak with a compositional and structural similarity to real steak, 17 comprising mostly muscle and adipose cells with muscle cells in alignment, is still 18 challenging 4,8,9 . 19 To realize the structural characteristics of steak, various tissue engineering techniques have 20 been considered such as cell sheet engineering 10,11 , cell fiber engineering 12 , cell culture on a 21 3D printed scaffold 13 , and 3D cell printing 14,15 . It is noteworthy that in the 3D cell printing 22 field some researchers have adopted a supporting bath assisted 3D printing (SBP) technique 23 where ink is dispensed inside the gel or suspensions with thixotropy. Since the SBP is able to 24 overcome the shortcomings of restricted available range of viscosity in ink and drying for Gel) or gellan gum (G-GG) and thrombin for the fabrication of a fibrous muscle fiber 1 mimicking the bundle of muscle fiber in steak (Supplementary Movies 1 and 2). With the 2 confirmation of the gel formation followed by the removal of supporting baths, high cell 3 viability was observed for 3 days after printing in both the G-Gel and G-GG by live/dead 4 staining (Figs. 3a, 3b and Supplementary Fig. 7). 5 When the printed cell fiber was cultured in suspension, it transformed from a fibrous to a 6 globular form (Fig. 3c). Studies related to muscle tissue engineering have implied that an 7 anchor structure enables 3D muscle tissue to not only maintain its initial shape but also improve 8 the cell alignment, fusion, and differentiation against the muscle fiber's contraction 15,34-38 . We 9 placed a printed cell fiber onto a silicone rubber and anchored it with needles to fasten both 10 ends to withstand cell contractions (Fig. 3d, left). With the needle fixed culture, the cell fibers 11 printed inside G-GG and G-Gel retained the fibrous structure, but the diameter had shrunk by 12 around 60% in G-GG and 80% in G-Gel at day 9 of culture ( Fig. 3d, right). It would be 13 reasonable to suppose that the size decrease was caused by alignment and fusion of bSCs with 14 an enzymatic decomposition of fibrin gel by proteases secreted from cells 39,40 . We also took 15 immunofluorescence images inside the cell fibers to examine the cellular behavior w/ and w/o 16 needle anchoring after 7 days of differentiation. To quantify the improvement in terms of 17 muscle maturation, the cell alignment, i.e. the angle setting for the straight line between needles, 18 was measured from the immunofluorescence images (Fig 3e, left). The results showed that the 19 cells in the cell fiber of suspension culture randomly oriented regardless of the type of 20 supporting baths and in needle fixed culture the cells in the cell fiber printed inside G-Gel were 21 highly anisotropically oriented compared to those of G-GG (Fig 3e, right). We postulate that 22 the difference in the degree of alignment between G-GG and G-Gel arises from the hindrance 23 of cell behaviors by residual substances that might exist inside or on the printed cell fibers. 24 That is, the residual G-GG in the cell fiber may not be degraded or dissolved, in which case the cells rarely remodel the ECM around them and are not able to migrate to fuse other cells 1 ( Supplementary Fig. 8). On the other hand, G-Gel is easily dissolved at 37℃ and may be 2 degraded by proteases, enabling active cell behavior although there are residues inside the 3 printed cell fiber. Printing bSCs inside G-Gel and anchoring them are essential steps for 4 fabrication of the muscle cell fiber, but the anchoring method may not be appropriate for scale-5 up. Therefore, we developed a modified SBP to include a part to simultaneously anchor the 6 printed cell fiber. 7 8

Fabrication of Muscle, Fat, and Vascular Cell Fibers by TIP 9
The important feature in the modified SBP, which we have named TIP, is the introduction of 10 tendon-gels to anchor the printed cell fibers for culture. Fig. 4a illustrates the process of the 11 TIP in which the printing bath is divided into three parts; bottom tendon-gel, supporting bath, 12 and upper tendon-gel. G-Gel is used as a supporting bath as described in the above section and 13 the volume of tendon-gels is filled with 4 wt% collagen nanofiber solution (CNFs) which 14 shows the reversible sol-gel transition from 4℃ to 37℃ ( Supplementary Fig. 9). To separate 15 the layers and maintain the structure we fabricated polydimethylsiloxane (PDMS) wells 16 ( Supplementary Fig. 10). After bSCs fiber formation inside the PDMS well (Supplementary 17 Movie 3), incubation for 2 h at 37℃ induced the supporting bath and tendon-gels to become a 18 solution and gel, respectively, followed by placement of a PDMS well itself in the media for 19 culture. 20 On day 3, we could confirm that the printed cell fiber maintained its fibrous shape with 21 reduced size (data not shown) and that there was a connection between two tendon-gels in 22 phase contrast and H&E staining images (Figs. 4b and 4c). The bSCs fiber by TIP also showed 23 a high alignment of cells after 3 days of differentiation, which is comparable with that of the needle fixed culture (Figs. 4d and 4e and Supplementary Movie 4). Interestingly, the sarcomere 1 structure of matured muscle fibers was shown in some of the TIP derived bSCs fibers (Fig. 4f), 2 but we could not show any cell fibers by the needle fixed culture after 14 day of differentiation. 3 Even though we did not investigate thoroughly here, it may have been caused by cell adhesion 4 of bSCs to the collagen gel at anchorage regions in the TIP whereas there is no cell adhesion 5 in the needle fixed culture ( Supplementary Fig. 11). TIP is a promising method for muscle fiber 6 fabrication, but it still has a problem in that the bSCs fiber become detached from the tendon-7 gels, especially the bottom tendon-gels, in a prolonged culture due to its strong contraction. 8 Increasing concentration of CNFs or additional crosslinking will hopefully provide a solution 9 to this problem. Moreover, double printing, after fabrication of a cell fiber by general TIP, then 10 rotating the PDMS well 180° so that it reversed and then printing one more time around the 11 first formed cell fiber, may be another way of solving the problem. When double printing, two 12 printed cell fibers close to each other are fused into one thicker cell fiber ( Supplementary Fig.  13 13) and it seems to be more resilient to strong contraction than by simply printing once (data 14 are not shown). 15 Multiple printing for 25 bSCs cell fiber fabrication in one large PDMS well was also 16 performed, demonstrating the possibility of mass production of TIP ( Fig. 4g and  17 Supplementary Movie 5). We expected that large tissue composed of various types of cell fibers 18 could be fabricated in one PDMS well, but we fabricated muscle, fat, and vascular cell fibers 19 individually in this study because the differentiation should be induced in the media 20 corresponding to each cell fiber based on the information discussed in the first section. After 21 the success in optimization of the differentiated media for all three types of cell fibers at the 22 same time, programed printing of them in desired locations will be feasible. Fig. 4g and 23 Supplementary Movies 6-8 show whole muscle, adipose, and blood capillary cell fibers 24 independently fabricated by TIP. Fat and vascular fibers were obtained by inducing the adipogenesis of bADSCs fiber for 7 days and by culturing the cell fiber of endothelial-1 differentiated bADSCs for 7 days, respectively. The adipose fibers showed a high density of 2 bovine adipocytes in a mature state, displaying a cytoplasm full of lipids vesicles. The blood 3 capillary fibers were fully covered by CD31 expressing differentiated bovine endothelial cells 4 ( Fig. 4h and Supplementary Fig. 12). 5 The characteristics of cell concentration, compressive modulus, and water contents of muscle 6 and adipose cell fibers by TIP were compared with the fibers extracted from commercial beef. 7 Cell concentration of muscle and fat cell fibers of commercial beef were calculated from the 8 DNA content of one tissue with a similar volume to TIP-derived cell fibers (Supplementary 9 Fig. 14). While the cell density of the TIP-derived muscle and fat cell fibers were calculated to 10 be 3x10 6 and 3x10 5 cells/fiber, respectively, the cell densities of muscle and fat cell fibers of 11 commercial beef were 8.61x10 5 and 1.89x10 5 cells/fiber (Fig. 4i). The reason for the 12 approximately fourfold higher cell density of TIP muscle fibers seems to be lower maturation 13 (fusion) of differentiated satellite cells because higher maturation induces lower cell numbers 14 because of cell fusion. Although water content showed the disparity between commercial beef 15 and TIP-derived cell fibers ( Supplementary Fig. 16), the compressive modulus in all cell fibers 16 showed a similar value, which was within one order of kPa ( Fig. 4j and Supplementary Fig.  17 15). Since the TIP-derived cell fibers were not controlled for tenderness, flavor, and additional 18 nutrient components in this study, these factors will need to be addressed to produce customer-19 oriented cultured meat. 20 21

Engineered Steak Construction by Assembly of Muscle, Fat, and Vascular Cell Fibers 22
The assembly of TIP-derived cell fibers was attempted to demonstrate the construction of 23 cultured steak. To mimic the structure of commercial beef, we first took a cross-sectional image of Wagyu by sarcomeric α-actinin and laminin staining, which denote muscle in double-1 positive and adipose in laminin only positive, respectively (Fig. 5a, left). We tried to produce 2 cultured steak with dimensions of approximately 5 mm x 10 mm x 5 mm (WxLxH), and from 3 Wagyu's image we made the model image showing the required number of muscle, adipose, 4 and blood capillary cell fibers and arrangement (Fig. 5a, right). The diameters of the cell fibers 5 obtained by TIP were estimated to be approximately 500, 760, and 600 µm, which means the 6 required number of each cell fiber were 42, 28, and 2, respectively. To distinguish each cell 7 fiber, muscle and vascular cell fibers were stained in red using food coloring, leaving fat cell 8 fiber as a white color. After physically stacking the cell fibers like the model image, it was 9 treated with transglutaminase which is a common food crosslinking enzyme to accelerate the 10 assembly for 2 days at 4℃. The final product is shown in Fig. 5b and the cross-sectional image 11 was taken to verify that the structure was analogous to Wagyu (Fig. 5c). which implied the 12 feasibility of TIP-based engineered steak fabrication. 13 14

Conclusion 15
In this research, we reported a new technology for constructing cultured steak-like meat with 16 muscle, adipose, and blood capillary cell fibers composed of edible bovine cells. After isolation 17 and purification of bSCs and bADSCs, we verified cell behaviors; the proliferation and 18 differentiation of bSCs and adipogenesis and vasculogenesis from bADSCs depending on 19 media conditions. It was shown that resistance to the contraction force during culture of bSCs 20 derived cell fiber was essential to realize highly aligned muscle fibrils. A modified supporting 21 bath assisted cell printing method, TIP, was developed, in which the collagen gel-based tendon 22 tissues withstands cell traction force during bSCs differentiation, leading to a well-maintained 23 fibrous structure and anisotropic cell alignment during bSCs differentiation. Comparison of cell density, compressive modulus, and water content showed the gap between TIP derived and 1 commercial muscle and fat cell fibers. Further elaboration will therefore be required with 2 consideration of texture and flavor in future. We demonstrated engineered meat analogous to 3 the structure of commercial beef through the assembly of muscle, adipose, and blood capillary 4 cell fibers due to TIP's multiple cell fiber printing, which could be beneficial for the scale-up 5 of not only cultured meat but also muscle tissue engineering in the future. 6

Methods 1
Isolation and purification of bSCs and bADSCs. bSC were isolated from 160 g fresh masseter muscle 2 samples (within 6 h of euthanasia) of 27-month-old Japanese black cattle obtained at a slaughterhouse as 3 previously described 7 with some modifications. The freshly harvested bovine muscle was kept on ice, transferred 4 to a clean bench, and washed with cold 70% ethanol for 1 min, followed by cold PBS1x 2 times. Then, the fat 5 tissues were removed, cut into small pieces with a knife, and minced with a food processor mechanically. The  TIP & culture. 4 wt% CNFs was produced from collagen sponge (Nipponham, Type Ⅰ & Ⅲ mixture) based on 1 the previous method. After cutting a small area of PDMS wells' side to make the media flow channel, it was 2 sterilized with 70% ethanol and UV treatment, then put it on the slide glass. The PDMS well was filled with 4 wt% 3 CNFs, G-Gel, and 4 wt% CNFs at the bottom, middle, and top layers, respectively. Cell printing was conducted 4 the same as that of SBP. After printing cells, printed area at the top layer of PDMS well was covered one more 5 time with 4 wt% CNFs, incubated at RT for 1 h, and then it was incubated at 37℃ for 2 h to dissolves G-Gel and 6 induce CNFs gelation, and finally placed in a culture container. The bioinks were prepared the same as in SBP 7 for muscle cell fiber, by mixing 5x10 6    Water content measurement. The water content is calculated according to the mass before and after freeze 49 drying. Briefly, the printed fibers in PBS1x was taken out, the surface liquid was removed, and the wet weight 50 (Wwet) of the fibers was measured by a balance. The dry weight (Wdry) of the fibers was measured after freeze 51 drying (24 h). The water content is given by the following formula: 1 DNA content measurement. Commercial beef was bought from supermarket and intramuscular fat tissues as 2 wells as muscle tissues parts were cut in small fibers of the same size than the printed fibers (Fig. S12). One fiber 3 was put per microcentrifuge tube and the tissues were lysed following the DNeasy Blood & Tissue Kit (QIAGEN, 4 69504) to extract their DNA content, which was quantified by the NanodropTM N1000 device (Thermo Fisher 5 Scientific). Then to estimate the cell number from the DNA amount, it was considered that one mammalian cell 6 contains around 6 pg of DNA 42 , which allows the evaluation of the total cell number per fiber. b, Schematic of the construction process for cultured steak. The first step is cell purification of 4 tissue from cattle to obtain bovine satellite cells (bSCs) and bovine adipose-derived stem cells 5 (bADSCs). Second is supporting bath assisted printing (SBP) of bSCs and bADSCs to fabricate 6 the muscle, fat, and vascular tissue with a fibrous structure. The third is the assembly of cell 7 fibers to mimic the commercial steak's structure. *SVF: stromal vascular fraction 8 9 Fig. 2    The schematic of TIP for cell printing. b, Optical (upper) and phase-contrast (lower) images 2 of the bSCs tissue printed by TIP, keeping the fibrous structure on day 3. The images were 3 taken after fixation. Scale bar, 1 mm. c, The H&E stained image of half of collagen gel 4 (dotted black line) -fibrous bSCs tissue (dotted red line) and a magnified image of the fibrous 5 bSCs tissue (right). Scale bars, 2 mm (left) and 50 µm (right). d, 3D fluorescence image (left) 6 and cell alignment measurement (right) of the TIP-derived bSCs tissue stained with actin 7 (red) and MHC (green) on 3 day of differentiation. Scale bar, 50 µm. e, SEM images of TIP-8 derived bSCs tissue on 3 day of differentiation. Scale bars, 10 µm & 100 µm (inset). f, 9 Fluorescence image of TIP-derived bSCs tissue stained with actin (red), MHC (green), and 10 nucleus (blue) on 14 day of differentiation. A scale bar, 50 µm.  were stained with carmine (red color), but fat tissue was not. 27