With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats. Mammalian cell-based cultured meat has mostly been unstructured, leaving a demand for artificial steak-like meat. Here the authors present an assembled steak-like tissue of bovine skeletal muscle, adipose tissue, and blood capillary tissue fabricated by tendon-gel integrated printing technology.

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Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting

Tissue engineering is an emerging means for resolving the problems of tissue repair and organ replacement in regenerative medicine. Insufficient supply of nutrients and oxygen to cells in large-scale tissues has led to the demand to prepare blood vessels. Scaffold-based tissue engineering approaches are effective methods to form new blood vessel tissues. The demand for blood vessels prompts systematic research on fabrication strategies of vascular scaffolds for tissue engineering. Recent advances in 3D printing have facilitated fabrication of vascular scaffolds, contributing to broad prospects for tissue vascularization. This review presents state of the art on modeling methods, print materials and preparation processes for fabrication of vascular scaffolds, and discusses the advantages and application fields of each method. Specially, significance and importance of scaffold-based tissue engineering for vascular regeneration are emphasized. Print materials and preparation processes are discussed in detail. And a focus is placed on preparation processes based on 3D printing technologies and traditional manufacturing technologies including casting, electrospinning, and Lego-like construction. And related studies are exemplified. Transformation of vascular scaffolds to clinical application is discussed. Also, four trends of 3D printing of tissue engineering vascular scaffolds are presented, including machine learning, near-infrared photopolymerization, 4D printing, and combination of self-assembly and 3D printing-based methods.

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3D printing of tissue engineering scaffolds: a focus on vascular regeneration

Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high-quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties.

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges.

3D-printable plant protein-enriched scaffolds for cultivated meat development.

Dual encapsulation of β-carotene by β-cyclodextrin and chitosan for 3D printing application

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large-sized tissues. Herein, a method based on high water-dispersible collagen microfibers (CMF) to fabricate capillary orientation-controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers-long CMF (CMF-20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers-long CMF (CMF-200). One of the advantages is the prevention of shrinkage on the z-axis of hydrogel-based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF-free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin-induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering.

Collagen Microfibers Induce Blood Capillary Orientation and Open Vascular Lumen

The development of soft tissue regeneration has recently gained importance due to safety concerns about artificial breast implants. Current autologous fat graft implantations can result in up to 90% of volume loss in long-term outcomes due to their limited revascularization. Adipose tissue has a highly vascularized structure which enables its proper homeostasis as well as its endocrine function. Mature adipocytes surrounded by a dense vascular network are the specific features required for efficient regeneration of the adipose tissue to perform host anastomosis after its implantation. Recently, bioprinting has been introduced as a promising solution to recreate in vitro this architecture in large-scale tissues. However, the in vitro induction of both the angiogenesis and adipogenesis differentiations from stem cells yields limited maturation states for these two pathways. To overcome these issues, we report a novel method for obtaining a fully vascularized adipose tissue reconstruction using supporting bath bioprinting. For the first time, directly isolated mature adipocytes encapsulated in a bioink containing physiological collagen microfibers (CMF) were bioprinted in a gellan gum supporting bath. These multilayered bioprinted tissues retained high viability even after 7 days of culture. Moreover, the functionality was also confirmed by the maintenance of fatty acid uptake from mature adipocytes. Therefore, this method of constructing fully functional adipose tissue regeneration holds promise for future clinical applications.

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Bioprinted Vascularized Mature Adipose Tissue with Collagen Microfibers for Soft Tissue Regeneration

Vascular invasion of cancer is a critical step in cancer progression, but no drug has been developed to inhibit vascular invasion. To achieve the eradication of cancer metastasis, elucidation of the mechanism for vascular invasion and the development of innovative treatment methods are required. Here, a simple and reproducible vascular invasion model is established using a vascular organoid culture in a fibrin gel with collagen microfibers. Using this model, it was possible to observe and evaluate the cell dynamics and histological positional relationship of invasive cancer cells in four dimensions. Cancer-derived exosomes promoted the vascular invasion of cancer cells and loosened tight junctions in the vascular endothelium. As a new evaluation method, research using this vascular invasion mimic model will be advanced, and applications to the evaluation of the vascular invasion suppression effect of a drug are expected.

https://www.mdpi.com/2079-7737/9/11/361/pdf

A Four-Dimensional Organoid System to Visualize Cancer Cell Vascular Invasion

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 diff

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Hao Liu

Papers

Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting

Collagen Microfibers Induce Blood Capillary Orientation and Open Vascular Lumen

Bioprinted Vascularized Mature Adipose Tissue with Collagen Microfibers for Soft Tissue Regeneration

A Four-Dimensional Organoid System to Visualize Cancer Cell Vascular Invasion

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