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Dispensing of 3D capillary tissue with open vascular lumen. a) Schematic illustration of 3D capillary tissues fabricated by dispensing machine using CMF-20. b) Photographs and confocal laser scanning microscopic (CLSM) images of HUVEC in gellan gum (GG) gel or fibrin (FB) gel 3D tissues after 30 min gelation and subsequent 7 days culture. Scale bars in photographs and fluorescent images are 1 mm and 100 µm respectively. c) Fluorescence area of blood capillary in 3D tissues based on FB gel, stained by anti-CD31 antibody after different culture times (n = 3, **p < 0.01). d) Distribution of blood capillary lumen diameter after 5, 7, and 14 days culture measured by the anti-CD31 immunofluorescence staining images (n = 100). The numbers in (d) indicated the mean vascular lumen diameter.
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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 metabol...
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... the FB gels and GG gels were selected as hydrogels to embed CMF and endothelial cell, to fabricate the 3D capillary tissues (Figure 2a). First, simple droplet-like tissues were prepared using a dispensing machine (Figure 2b). ...
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... the FB gels and GG gels were selected as hydrogels to embed CMF and endothelial cell, to fabricate the 3D capillary tissues (Figure 2a). First, simple droplet-like tissues were prepared using a dispensing machine (Figure 2b). The gelation of 3D tissues using GG gel or FB gel was confirmed after 30 min incubation at 37 °C. ...
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... difference was attributed to the limited ability of cells to adhere onto the pristine GG backbone, [27] which resulted in cells to interact and exert tension forces prevalently with the collagen fiber, shrinking and condensing them within the gel droplet. In contrast, we observed an interconnected blood capillary network formation in the tissues fabricated from FB gel (Figure 2b; Video S1, Supporting Information). FB gel was selected for the subsequent study, due to its ability to support the formation of a homogeneous blood capillary network. ...
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... gel was selected for the subsequent study, due to its ability to support the formation of a homogeneous blood capillary network. For a more detailed understanding of tissue structure stability using FB gel and the confirmation of capillary network formation throughout the culture time, capillary formation at days 5, 7, and 14 were measured from 2D projection images (Figure 2c,d). As shown in the results, the capillary network area is increased from 33% to 58% after 2 weeks of culture. ...
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... compared the tissue thickness with different CMF-20 concentrations from 0 to 2 wt% after 7 days of culture. More than twofold increment in tissue thickness between CMF-free controls and a 0.5 wt% CMF-20 were found after 7 days, even there is no significant difference in thickness just after tissue preparation (Figure 3b,c; Figure S2a, Supporting Information). These differences in thickness shown that increasing CMF-20 concentration can effectively reduce the shrinkage of the tissues and maintain its 3D structure. ...
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... though commercially available collagen gels or Matrigel can support the formation of interconnected capillary networks ( Figure S2b, Supporting Information), most vascular lumens have an inner diameter of less than 10 µm. However, in the 3D capillary tissue fabricated from a mixture of Matrigel and CMF-20, an increase in open vascular lumen was found in histological cross-section images ( Figure S2c, Supporting Information). ...
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... though commercially available collagen gels or Matrigel can support the formation of interconnected capillary networks ( Figure S2b, Supporting Information), most vascular lumens have an inner diameter of less than 10 µm. However, in the 3D capillary tissue fabricated from a mixture of Matrigel and CMF-20, an increase in open vascular lumen was found in histological cross-section images ( Figure S2c, Supporting Information). Accordingly, we hypothesized that the embedded CMF-20 act as a microscaffold that could provide spaces for migration of endothelial cells, and support capillary maturation to form an open vascular lumen. ...
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... lyophilized collagen in an aqueous solution (Fig. 1A). This CMF does not dissolve in the culture medium and can be mixed with cells in suspension to create 3D tissues with as much as 20-30 % dense collagen [19,20]. This proprietary material was applied to gingival models with collagen-dense dermal layers and to adipose tissue models with mature fat and vascular networks, which are difficult to achieve with conventional 3D culture [21,22]. ...
Cell properties generally change when the culture condition is changed. However, mesenchymal stem cells cultured on a hard material surface maintain their differentiation characteristics even after being cultured on a soft material surface. This phenomenon suggests the possibility of a cell culture material to memorize stem cell function even in changing cell culture conditions. However, there are no reports about cell memory function in three-dimensional (3D) culture. In this study, colon cancer cells were cultured with collagen microfibers (CMF) in 3D to evaluate their resistance to reactive oxygen species (ROS) in comparison with a monolayer (2D) culture condition and to understand the effect of 3D-culture on cell memory function. The ratio of ROS-negative cancer cells in 3D culture increased with increasing amounts of CMF and the highest amount of CMF was revealed to be 35-fold higher than that of the 2D condition. The ROS-negative cells ratio was maintained for 7 days after re-seeding in a 2D culture condition, suggesting a 3D-memory function of ROS resistance. The findings of this study will open up new opportunities for 3D culture to induce cell memory function.
... Liu et al. used shear stress generated by the dispensing process to align collagen microfibrils, which combined with integrin-induced adhesion to guide cell alignment. Consequently, directional growth of capillaries was achieved [159]. Furthermore, various types of ECs exposed to the same orientation demonstrated analogous responses under high shear stress. ...
Tissue engineering and regenerative medicine have made great progress in recent decades, as the fields of bioengineering, materials science, and stem cell biology have converged, allowing tissue engineers to replicate the structure and function of various levels of the vascular tree. Nonetheless, the lack of a fully functional vascular system to efficiently supply oxygen and nutrients has hindered the clinical application of bioengineered tissues for transplantation. To investigate vascular biology, drug transport, disease progression, and vascularization of engineered tissues for regenerative medicine, we have analyzed different approaches for designing microvascular networks to create models. This review discusses recent advances in the field of microvascular tissue engineering, explores potential future challenges, and offers methodological recommendations.
... Laminin and collagen type IV, two extracellular matrix (ECM) molecules that are part of the endothelium basal lamina and also indicate vessel maturation, 27,28 were present and located adjacent to the vessel wall, forming the basement membrane ( Figure 2C, right column). The formed vessels were 3D and open inside ( Figure 2D), with an average lumen diameter of 5.9 μm, which is slightly smaller than typically reported values, 29 but within the range for capillaries (5−10 μm). 30 Formation of a 3D Vascular Platform with HMVECs and SCs. ...
Nerves and blood vessels are present in most organs and are indispensable for their function and homeostasis. Within these organs, neurovascular (NV) tissue forms congruent patterns and establishes vital interactions. Several human pathologies, including diabetes type II, produce NV disruptions with serious consequences that are complicated to study using animal models. Complex in vitro organ platforms, with neural and vascular supply, allow the investigation of such interactions, whether in a normal or pathological context, in an affordable, simple, and direct manner. To date, a few in vitro models contain NV tissue, and most strategies report models with nonbiomimetic representations of the native environment. To this end, we have established here an NV platform that contains mature vasculature and neural tissue, composed of human microvascular endothelial cells (HMVECs), induced pluripotent stem cell (iPSCs)-derived sensory neurons, and primary rat Schwann cells (SCs) within a fibrin-embedded polymeric scaffold. First, we show that SCs can induce the formation of and stabilize vascular networks to the same degree as the traditional and more thoroughly studied human dermal fibroblasts (HDFs). We also show that through SC prepatterning, we are able to control vessel orientation. Using our NV platform, we demonstrate the concomitant formation of three-dimensional neural and vascular tissue, and the influence of different medium formulations and cell types on the NV tissue outcome. Finally, we propose a protocol to form mature NV tissue, via the integration of independent neural and vascular constituents. The platform described here provides a versatile and advanced model for in vitro research of the NV axis.
... The recreation of capillaries in vitro has been widely studied. Endothelial cells can self-assemble into a capillary-like network, when cultured in a 3D environment with specific growth factors and mural cells [5][6][7]. Previous studies have shown how such microvessels can be guided and matured, by culturing the abovementioned cells in presence of type I collagen (col-1) microfibers (MFs). With these MFs, capillaries showed more open lumen and larger capillary diameters, contributing to the ability to recreate a more biomimetic in vitro engineered capillary network [6]. ...
... Previous studies have shown how such microvessels can be guided and matured, by culturing the abovementioned cells in presence of type I collagen (col-1) microfibers (MFs). With these MFs, capillaries showed more open lumen and larger capillary diameters, contributing to the ability to recreate a more biomimetic in vitro engineered capillary network [6]. Such collagen MFs are promising candidates as bioink components to carry instructive signals for endothelial cells, as these can be easily resuspended in hydrogels or aqueous formulations without the need for chemical modifications. ...
... It was expected that the addition of MFs would not influence the bioink printability in a relevant way, since gelatin primarily defines the bioink's viscosity, as the sole fibrinogen solution, with and without MFs, behaves as a non-viscous fluid, and is therefore not printable. Also, it was shown before that the addition of 0.5% and 1% w/v col-1 MFs to a fibrin-based bioink resulted in similar rheological properties as compared to the bioink without MFs [6]. ...
Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material 3D bioprinting can potentially resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge. Here, we developed endothelial cell(EC)-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM). EC-driven capillary network formation started two days after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29% after 14 days, compared to col-1 MFs-laden bioinks. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a microgel suspension bath. The constructs were cultured up to 14 days, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications including in vitro models, cancer progression, and testing anti-angiogenic therapies.
... Specifically, the mechanical properties of these tissues depend not only on their biochemical composition, but also on the tissue's microstructure (e.g. anisotropy) [3,[5][6][7][8][9][10]. Beyond the ECM, cell alignment also plays a critical role in a number of processes, such as cellular differentiation, proliferation and matrix deposition, as well as in the organization of subcellular structures such as cytoskeleton and adhesion complexes. ...
... These studies supported subsequent cell alignment in the direction of aligned fibers over time, in contrast to traditional bioprinting methods with non-fibrous bioinks, which typically result in random cell organization and spreading within printed filaments [10,15,[22][23][24][25][26]. Extrusion bioprinting leverages shear stresses during the printing process to induce both cell and fiber alignment [6,22,[27][28][29][30][31]. While these collagen-based examples have shown promising results, collagen has various biophysical properties (such as hydrogel stiffness, fiber density, and pore size) that are challenging to tune independently [15]. ...
The extracellular matrix (ECM) is composed of biochemical and biophysical cues that control cell behaviors and bulk mechanical properties. For example, anisotropy of the ECM and cell alignment are essential in the directional properties of tissues such as myocardium, tendon, and the knee meniscus. Technologies are needed to introduce anisotropic behavior into biomaterial constructs that can be used for the engineering of tissues as models and towards translational therapies. To address this, we developed an approach to align hydrogel fibers within cell-degradable bioink filaments with extrusion printing, where shear stresses during printing align fibers and photocrosslinking stabilizes the fiber orientation. Suspensions of hydrogel fibers were produced through the mechanical fragmentation of electrospun scaffolds of norbornene-modified hyaluronic acid, which were then encapsulated with meniscal fibrochondrocytes, mesenchymal stromal cells, or cardiac fibroblasts within gelatin-methacrylamide bioinks during extrusion printing into agarose suspension baths. Bioprinting parameters such as the needle diameter and the bioink flow rate influenced shear profiles, whereas the suspension bath properties and needle translation speed influenced filament diameters and uniformity. When optimized, filaments were formed with high levels of fiber alignment, which resulted in directional cell spreading during culture over one week. Controls that included bioprinted filaments without fibers or non-printed hydrogels of the same compositions either without or with fibers resulted in random cell spreading during culture. Further, constructs were printed with variable fiber and resulting cell alignment by varying print direction or using multi-material printing with and without fibers. This biofabrication technology advances our ability to fabricate constructs containing aligned cells towards tissue repair and the development of physiological tissue models.
... The crosslinked collagen sponge was mixed with ultra-pure water at a concentration of 10 mg/mL (pH = 7.4, 25°C) and homogenized for 6 min at 30,000 rpm. Then, the solution was ultrasonicated (Ultrasonic processor VC50, SONICS) in an ice bath for 100 cycles (one cycle comprised 20 s of ultrasonication and 10 s of cooling), to make smaller fragments that are able to induce better vascularization and filtrated (40-µm filter, microsyringe 25-mm filter holder, Merck), before being freeze-dried for 48 h (FDU-2200, EYELA) 50 . The obtained CMF was kept in a desiccator at RT. ...
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.
... For more reproducibility, immortalized human cell lines were chosen. A fibrin-collagen type I microfiber ECM was specifically optimized, based on previous works in our laboratory for less sensitive vascular tissues [16,17]. Collagen is the main structural protein of the ECM and represents 30% of all the protein in the human body [18]. ...
... The hydrogel, used as an ECM, has been developed based on previous work in the laboratory [16,17,27,28]. Porcine collagen type I microfibers (CMF) were chosen to support the cell organization. ...
... Type I CMF have already been proved to enhance tissue differentiation and functionalization in 3D gels [27]. Here, the CMF were further processed with an ultrasonic supplementary step allowed debulking to obtain a more homogenous gel, following previously-described protocols [16,17]. The use of fibrin, a fibrous protein involved in blood vessel repair, ensured gel cohesion and avoided shrinkage. ...
Tissue vascularization is essential for its oxygenation and the homogenous diffusion of nutrients. Cutting-edge studies are focusing on the vascularization of three-dimensional (3D) in vitro models of human tissues. The reproduction of the brain vasculature is particularly challenging as numerous cell types are involved. Moreover, the blood-brain barrier, which acts as a selective filter between the vascular system and the brain, is a complex structure to replicate. Nevertheless, tremendous advances have been made in recent years, and several works have proposed promising 3D in vitro models of the brain microvasculature. They incorporate cell co-cultures organized in 3D scaffolds, often consisting of components of the native extracellular matrix (ECM), to obtain a micro-environment similar to the in vivo physiological state. These models are particularly useful for studying adverse effects on the healthy brain vasculature. They provide insights into the molecular and cellular events involved in the pathological evolutions of this vasculature, such as those supporting the appearance of brain cancers. Glioblastoma multiform (GBM) is the most common form of brain cancer and one of the most vascularized solid tumors. It is characterized by a high aggressiveness and therapy resistance. Current conventional therapies are unable to prevent the high risk of recurrence of the disease. Most of the new drug candidates fail to pass clinical trials, despite the promising results shown in vitro. The conventional in vitro models are unable to efficiently reproduce the specific features of GBM tumors. Recent studies have indeed suggested a high heterogeneity of the tumor brain vasculature, with the coexistence of intact and leaky regions resulting from the constant remodeling of the ECM by glioma cells. In this review paper, after summarizing the advances in 3D in vitro brain vasculature models, we focus on the latest achievements in vascularized GBM modeling, and the potential applications for both healthy and pathological models as platforms for drug screening and toxicological assays. Particular attention will be paid to discuss the relevance of these models in terms of cell-cell, cell-ECM interactions, vascularization and permeability properties, which are crucial parameters for improving in vitro testing accuracy.
... Recent studies have reported the effects of microsegment materials of bioscaffolds on cell behaviors [155]. This is synergistic regulation of cell behaviors by biophysical and biochemical stimulations. ...
The demand for artificial organs has greatly increased because of various aging-associated diseases and the wide need for organ transplants. A recent trend in tissue engineering is the precise reconstruction of tissues by the growth of cells adhering to bioscaffolds, which are three-dimensional (3D) structures that guide tissue and organ formation. Bioscaffolds used to fabricate bionic tissues should be able to not only guide cell growth but also regulate cell behaviors. Common regulation methods include biophysical and biochemical stimulations. Biophysical stimulation cues include matrix hardness, external stress and strain, surface topology, and electromagnetic field and concentration, whereas biochemical stimulation cues include growth factors, proteins, kinases, and magnetic nanoparticles. This review discusses bioink preparation, 3D bioprinting (including extrusion-based, inkjet, and ultraviolet-assisted 3D bioprinting), and regulation of cell behaviors. In particular, it provides an overview of state-of-the-art methods and devices for regulating cell growth and tissue formation and the effects of biophysical and biochemical stimulations on cell behaviors. In addition, the fabrication of bioscaffolds embedded with regulatory modules for biomimetic tissue preparation is explained. Finally, challenges in cell growth regulation and future research directions are presented.
... To address all these needs, we recently developed collagen microfibers (CMF) from collagen type I, the most prevalent in adipose tissue, which improved the maintenance of mature adipocyte viability and function [45], while providing a physiological environment and biological signals for the cells. This microenvironment limits the shear stress (i.e. the frictional force that acts on cell surface) around the fragile mature adipocytes, allows the nutrient and oxygen diffusion and help to guide the endothelial cells vasculature construction through their integrin-induced adhesion on the fibers [35,39,46]. The importance of ADSC for the formation of endothelial tubular structures, in addition to mature adipocytes, was confirmed in our previous model [47], showing the development of an in vitro vascularized adipose tissue. ...
... For the future approach, the addition of adipose macrophages in the vascularized drops tissue model should hold promises for a better understanding of their association between the blood vessels and adipocytes, being linked to the inflammatory responses observed in a broad range of obesity-associated diseases, like in diet-induced type 2 diabetes and insulin resistance [77,78]. Collagen microfibers preparation: Based on our previous studies [45,46,[79][80][81], the collagen microfibers (CMF) were first prepared from a collagen type I sponge after dehydration condensation at 200 • C for 24 h crosslinking. The crosslinked collagen sponge was mixed with ultra-pure water at a concentration of 10 mg/mL (pH = 7.4, 25 • C) and homogenized for 6 min at 30 000 rpm (Violamo VH-10 homogenizer, S10N-10G diameter of 10 mm and 115 mm length). ...
Obesity is a complex and incompletely understood disease, but current drug screening strategies mostly rely on immature in vitro adipose models which cannot recapitulate it properly. To address this issue, we developed a statistically validated high-throughput screening model by seeding human mature adipocytes from patients, encapsulated in physiological collagen microfibers. These drop tissues ensured the maintenance of adipocyte viability and functionality for controlling glucose and fatty acids uptake, as well as glycerol release. As such, patients’ BMI and insulin sensitivity displayed a strong inverse correlation: the healthy adipocytes were associated with the highest insulin-induced glucose uptake, while insulin resistance was confirmed in the underweight and severely obese adipocytes. Insulin sensitivity recovery was possible with two type 2 diabetes treatments, rosiglitazone and melatonin. Finally, the addition of blood vasculature to the model seemed to more accurately recapitulate the in vivo physiology, with particular respect to leptin secretion metabolism.
... The techniques are often based on the direct microfabrication of channels and vessellike structures, but it does not fit the complex vascular environment, lacking active vascular remodeling which is essential after implantation. Another way is to induce the self-cellular interactions to spontaneously generate capillary networks in the engineered tissues [36,37], also called prevascularization. Upon implantation, anastomosis of these capillaries with the neighboring host vasculature occurs and ensures adequate blood perfusion to enable the graft survival. ...
... For more physiological maintenance of the mature adipocytes, collagen microfibers (CMF) were applied for their crucial biophysical and bioinductive characteristics [38], allowing the fragile mature adipocytes to be protected from mechanical shrinkage stress during the culture. Moreover, the CMF also provides a scaffold to induce the formation of the vascular network by endothelial cells [37]. Altogether, this method enables the fabrication of fully vascularized reconstructed adipose tissue showing host anastomosis following implantation for a higher graft survival rate [39]. 2 Cyborg and Bionic Systems Using our previous findings, the purpose of this study was thus to show for the first time the possibility of bioprinting viable and functional vascularized adipose tissues, including the fragile mature adipocytes, for a ready-to-use, high-benefit soft tissue regeneration. ...
... Based on our previous studies [36][37][38], the collagen microfibers (CMF) were made from the porcine collagen type I sponge, known for its good tolerance after implantation [42,43]. It was first thermally cross-linked by dehydration condensation at 200°C for 24 h. ...
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.
