Jing
Nie‡
ab,
Qing
Gao‡
ab,
Chaoqi
Xie‡
ab,
Shang
Lv
ab,
Jingjiang
Qiu
c,
Yande
Liu
d,
Mengzi
Guo
e,
Rui
Guo
f,
Jianzhong
Fu
ab and
Yong
He
*ab
aState Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China. E-mail: yongqin@zju.edu.cn
bKey Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China
cSchool of Mechanics and Safety Engineering, Zhengzhou University, Zhengzhou 450001, China
dSchool of Mechatronics & Vehicle Engineering, East China Jiaotong University, Nanchang 330013, China
eMcCormick School of Engineering, Northwestern University, USA
fDepartment of Biomedical Engineering, Jinan University, Guangzhou 510632, China
First published on 28th August 2019
Despite the substantial progress in the construction of vascular structures in the past few years, building a whole blood circulatory system model containing both large vessels and capillaries inside cell-laden hydrogels remains a big challenge. Here, we present a flexible and novel process to construct a hydrogel-based microfluidic chip with such a multi-scale network by combining high-resolution three-dimensional (3D) printing and a twice-crosslinking strategy. The whole process includes: (1) vascular system design (from arteries and capillaries to veins), (2) template printing (ultrafine fiber network), (3) hydrogel material casting (formation of partially-crosslinked hydrogel sheets), (4) template peeling off (creation of microgrooves on the surfaces of the hydrogel sheets), (5) hydrogel sheet bonding (formation of a closed channel network) and (6) cell loading (specific cells seeded onto specific positions mimicking in vivo conditions). We demonstrated that it is easy to fabricate the ubiquitous structures of biological vascular systems, highly-branched networks, spiral vessels, stenosis, etc. The endothelial cell (EC) channels exhibit representative vascular functions. As a proof of concept, a bulk breast tumor tissue with a functional vascular network was built. Additionally, a vascular–tumour co-culture concept has been proposed and constructed through the process to investigate the interaction between tumours and blood vessels. The proposed strategy can also be applied to help engineer diverse meaningful in vitro models for extensive biomedical applications, from physiology and disease study to therapy evaluation.
New conceptsA new concept, multi-scale vascular chips, has been proposed for the first time. We pioneer the application of a combination of multi-scale 3D printing technology and a twice-cross-linking strategy to construct this model. Whereas most of the previous studies about the construction of vascular models are limited to a certain scale range, we aim to realize the construction of the whole vascular system containing both vessels and capillaries. This is achieved by a shift of the printing mode between FDM printing and EHD printing. Additionally, ubiquitous structures mimicking biological vascular morphologies are established through the proposed method. Our study further proposed a vascular–tumour co-culture system for modelling the interaction between tumours and blood vessels and we envision that it can pave a new way for cancer study and anti-tumour drug screening. The significance of the built-in vessel has been proven through an initial study of the migration behaviour of the tumour cells towards the endothelial barrier and the introduction of drug candidates through an endothelial barrier. |
Numerous approaches have been proposed for building large blood vessels, including the deformation of cell sheets into tubular structures,13–16 preparation of fibres with cell-embedded hydrogels,17,18 thin wire-based moulding,19–21 use of sacrificial templates,22–26 3D bio-printing,27–33 light-assisted processes,34–36 and layer-by-layer assembly.37–42 However, the major difficulty lies in the construction of a bifurcation structure and capillaries at the end.43 Few studies have been reported about the engineering of capillaries, which are essential components of a vascular system. To remodel organized and stable capillaries, a self-development strategy is applied by extending new capillaries from pre-existing vessels toward angiogenic signals.44–47 However, the use of growth factors on a large scale is cost-prohibitive and inefficient.48,49 An alternative approach, referred to as a physical structure-based strategy, is to engineer ultrafine channels before EC seeding.50 This approach makes it convenient to control the distribution of the capillaries, from direction to range. In addition, the in vivo vascular morphology is more diverse than a single linear structure,25 which is closely related to physiological and pathological conditions. For example, spiral arteries reside in the uterine endometrium,51,52 while stenosis can lead to inflammation and dysfunction.53,54 This complicated organization motivates the development of new techniques featuring excellent flexibility, cost-effectiveness and versatility.
Tumour development is a complicated multi-step process, involving the origin, growth, and metastasis. In particular, the vascular network is the crucial condition for tumour development,63,64 and the interaction between tumours and the vascular network plays an important role in the process. Angiogenic growth factors secreted by tumour cells prompt angiogenesis, while newly-induced capillaries supply nutrition for tumour growth and the necessary pathway for tumour metastasis.55,56 A series of tumour cell-laden constructs have been created.57,58 Multi-cellular tumour sphere models consisting of aggregated cells emerged, showing an excellent structure to mimic solid tumours.59,60 In addition, a set of microfluidic systems have been introduced as vascularized models for the study of tumour development.12,61,62,65,66 However, a microfluidic device is far from a real soft tissue.67 Accordingly, there is an urgent demand for an effective model to study the interaction between tumour tissues and blood vessels, as well as the key processes during metastasis.68–70
This study pioneers the combination of multi-scale 3D printing and a twice-crosslinking strategy to construct multi-scale structures within hydrogel materials. This method has the ability to construct the whole vascular hierarchy (from arteries to veins, as shown in Fig. S1, ESI†) and to emulate the ubiquitous structures of biological vascular systems. As a proof of concept, a vascularized breast tumour model is established. In addition, the strategy can also be applied to cell-laden hydrogel materials for engineering ubiquitous in vitro biological structures for a wide range of biomedical applications. Based on this method, a demonstrative vascular–tumour co-culture model with a parallel-channel structure is designed and constructed as a feasible system for comprehensive and systematic investigation of tumour-related physiological phenomena, including tumour development and anti-tumour drug screening. A meaningful vascular–tumour co-culture model has been proposed, showing great potential in future applications through a series of preliminary phenomena and basic data. The migration ability of the tumour cells and the barrier function of the vessels to anti-cancer drugs are verified. Furthermore, a simple model mimicking tumour development is engineered, showing the processes of primary tumours, angiogenesis, and invasion, which can be further used as an experimental platform to investigate metastasis. The initial feasibility and potential application scenario are displayed through the preliminary phenomena and basic data, ensuring the matching of the materials, structures, and processes with the potential application.
In comparison with other reported in vitro tumour models,66,71–73 this model displays several innovative features and outstanding merits, including: (i) development of a multi-scale vascular network, (ii) 3D culture conditions mimicking the in vivo environment, (iii) convenience to construct varied tumour tissues with diverse vascular networks, (iv) layer-by-layer assembly for the production of heterogeneous tumour tissues, (v) real-time loading of functional materials at reserved vacancies, (vi) diversity in bio-materials for customized models with individual and personal designs and (vii) the endothelial channel which offers a tool to study the invasion process of tumour cells and provides a barrier to drug candidates, mimicking in vivo drug delivery.74,75
Fig. 1 Schematic for the construction of a multi-scale vascular chip and display of the representative models through confocal images. |
Moreover, it is simple to reserve a vacancy of a specific shape at a designated location for post injection and deposition on demand, bringing flexibility to the construction of various heterogeneous structures. Different cell loading conditions are displayed in Fig. 1, including EC cells seeded onto the surface of the channel, tumor cells injected into the vacancy and tumor cells embedded in the surrounding hydrogel bulk.
Several models were constructed to display the versatility of this strategy, including a multi-scale vascular network model, a parallel-channel model, a vascularized tumour model and a tumor invasion model, as shown in Fig. 1.
By adopting the minimal dichotomy, the feasible domains for peeling off and bonding were determined,42 as shown in Fig. 2A. This revealed that the minimal time for peeling off and bonding increased with the increasing thickness of the hydrogel constructs.
Notably, the cross-linking of the hydrogel compositions during the peeling off and bonding processes brought about, to a certain extent, the increase of the diameter of the channel compared with the diameter of the template, showing good repeatability. Also, the subsequent swelling process during the soaking of the hydrogel constructs in medium or buffer caused a certain decrease of the channel diameter, which could reach a stable balance point after 53 hours. This was verified through experiments, as shown in Fig. 2B.
The effects of the printing parameters on the diameters of the filaments were systematically investigated for the FDM and EHD printing, respectively. During the printing process, the diameters of the printed filaments could be adjusted in the corresponding scale range and were affected by several parameters, including extrusion pressure and printing speed, as shown in Fig. 2C. The results indicated that the diameter of the filaments increased with increasing extrusion pressure, whereas it decreased with increasing printing speed. This occurred because a higher extrusion pressure directly increased the extrusion quantity, eventually leading to an increase of the diameter of the filament. Additionally, the filament was stretched and became thinner with the increase of the printing speed. In this study, both the printing speed and extrusion pressure were used to adjust the diameter of the filament. Instructively, by adjusting the printing parameters (i.e., printing speed and extrusion pressure), macro-scale filaments ranging from 253 to 732 μm and micro-scale filaments ranging from 3.16 to 94.19 μm could be obtained during the FDM printing process and EHD printing process, respectively, as shown in Fig. 2C. In conclusion, a final channel network containing macro-scale channels ranging from 233 to 589 μm and micro-scale channels ranging from 4.17 to 102.36 μm could be obtained.
Based on these results, interesting channel patterns could be successfully constructed through the special design of printing paths, such as a snail-like structure, a pentagram-like structure and a flower-like structure, as shown in Fig. 2D. It is conceivable that this process can be used to obtain various intricate and detailed internal channels over a wide scale range within hydrogel materials, such as a multi-scale structure, a superfine patterned structure, and a variable-scale structure, which can be further used as vascular system, capillary network, and vascular disease models.
An in vivo vascular network contains arteries, veins and capillaries with different scales and functions. A new concept, multi-scale vascular chips, has been demonstrated for the first time. Existing research studies about the construction of vascular models are limited to a certain scale range (Section S2, ESI†), while the multi-scale printing technology in this paper overcomes the limitation and can be applied to realizing the construction of the whole vascular system containing both big vessels and capillaries.
Furthermore, Fig. 3F and G display the microscopy morphologies of the ECs seeded onto the channel from both the longitudinal and axial directions through high magnification images, showing a healthy state, indicating the excellent bio-compatibility of the proposed system.
A three-level EC network was visualized through confocal microscopy, showing substantial spreading of ECs (Fig. 3H). Emulating the ubiquitous structures of biological vascular systems in hydrogels is significant for the construction of in vitro models. However, in spite of the lot of progress, the existing methods are restricted by the complicated microfabrication process, introduction of toxic materials, and consumption of disposable molds. In contrast, in this method, a trace amount of PCL materials was printed according to the specific demand for every single unique structure. For example, a spiral EC channel inspired by the spiral artery residing in the uterine endometrium was constructed by this method, as shown in Fig. 3I. As a main cause and risk factor of cardiovascular diseases, stenosis can bring about the reduction of blood flow and ischemia. A stenosis-mimicking EC channel was also constructed as a disease model for further investigation of relevant drugs, as shown in Fig. 3J.
In this paper, a breast cancer model containing tumour stroma (Gel-Gel-MA hydrogel), a functional vascular system (EC channels), and tumour cells (MDA-MB-231) was developed, as shown in Fig. 4B–D. The cytoskeleton staining of the construct confirmed the conceived distribution of specific cell types. Data revealed that the cells maintained high viability and a good proliferation ability, as shown in Fig. 4E and F. The expression of VE-cadherin protein indicated the formation of cell–cell junctions (Fig. 4G), which is significant for the barrier function of the EC channels. The expression of CD31 protein demonstrated the achievement of key endothelial function (Fig. 4H). The expression of vinculin protein further supported the tight adherence between cells and channels (Fig. 4I).42
In order to reconstruct the vascular inflammation model, a similar protocol from previously published work42 was applied to this model and the obtained results confirmed the realization of the simulation of the pathological conditions based on our model (Fig. 4J and K).
Together the above results proved that the engineered in vitro EC network could model endothelial functionality under different physiological conditions to mimic the native vascular system.
It was observed that the MDAs gradually migrated towards the EC channel spontaneously during the culture period, as shown in Fig. 5B. Eventually, the tumour cells reached the EC channels, migrating a total distance of 500 μm (Fig. 5C and D).
Although this model could not be completely generalized to the complex in vivo tumour microenvironment, it greatly shortened the gap between 2D monolayer culture and in vivo conditions.
This model can additionally be used as a preclinical tool for drug screening. We conducted a set of studies to apply the established model in the initial drug dose-reaction to mimic the in vivo endothelial barrier to drug candidates. Paclitaxel solutions of different concentrations were separately introduced into both the empty channels and the EC channels and then, through diffusion, affected the tumor cells in the neighboring parallel channels, as shown in Fig. 5E and F. The data revealed that the viability of the tumor cells decreased with the increase of the drug concentration, which was the same for both the empty channel conditions and the EC channel conditions. Furthermore, the presence of ECs blocked the effect of the drug to a certain degree, indicating a good barrier function, as shown in Fig. 5E–G.
The development of a tumour occurs through a multi-step mechanism, and several critical processes (as shown in Fig. 6A) were reproduced based on the above parallel-channel model containing tumour stroma (Gel-Gel-MA hydrogel), chemical environment (endothelial growth factor), functional vascular system (EC channels) and tumour cells (MDA-MB-231).
The first step is the formation of a primary tumour. This small tumour tissue is far from blood vessels and the nutrients required for tumour cell growth are supplied through penetration from the microenvironment of the tissue and organ. Tumour spheres and tumour strips located far from the EC channels were constructed to mimic the small primary tumour at an early stage, as shown in Fig. 6B.
The second step is angiogenesis and vascularization, which is the crucial requirement for tumor growth, invasion and metastasis. When the diameter of the tumor tissue reaches 1–2 mm, the nutrients provided by penetration cannot satisfy the growth of the tumor tissue. Tumour cells start to secrete angiogenesis factors to stimulate the growth and migration of vascular ECs, and capillaries gradually sprout from the original blood vessels, providing nourishment for tumour growth. Tumor cell-induced capillaries were observed on the established model, as shown in Fig. 6Ci. However, the degree of angiogenesis was limited by the hydrogel matrix properties, which could be well solved through the pre-construction of superfine channel structures, as shown in Fig. 6Cii. Compared with the chemically-induced capillaries, the physical structure-induced ones showed better integrity, continuity and customization. Subsequently, the tumor tissue gradually grew with the steady supply of nutrients from blood vessels.
The third step is tumour invasion. During the growth process, the tumour cells continuously secrete factors to promote the formation of new capillaries. At the same time, these abundant capillaries provide a basic way for tumour cells to enter the circulatory system. Malignant tumour cells are qualified for invasion into the blood vessel as long as they reach a newly-formed capillary. The arrival of the migrated tumour cells to the capillaries was observed in the above model, as shown in Fig. 6D, which is the prerequisite for the realization of the invasion process.
After that, tumour cells spread to a secondary tissue or organ through the vascular system and infiltration occurs before a secondary tumour forms.
During the whole process, the vascular system and the tumour tissue show a mutual improvement. On the one hand, the vascular system provides the necessary conditions for tumour growth and further metastasis. On the other hand, the tumour cells continuously secrete factors to promote the development of new capillaries. The proposed model for simulating tumour development is initially verified through a set of basic data which indicate the realization of the prerequisites for tumour-related studies, including angiogenesis, migration, and endothelial barrier function.
In order to develop an effective tumor therapy, reliable tumor models for preclinical testing and screening are urgently demanded. However, challenges exist in the clinical translation of potential anti-cancer drugs and treatments due to the discrepancy between the existing models and in vivo conditions.86 2D monolayer tumour models87 have difficulty in recapitulating the native microenvironment, and cells are unable to mimic their natural behaviors and responses to anti-cancer drugs.76,88,89 On the other hand, animal experiments sometimes show false effects.90,91 To overcome these obstacles, a promising strategy is to develop preclinical in vitro 3D tumor models based on human tumour cells,79,80,88 which are supposed to be able to physically and chemically recapitulate the tumor physiological environment,92,93 and further be used to develop effective cancer therapy.94 However, the existing 3D in vitro tumor models are far from being able to mimic the complex tumor microenvironment. In the present study, we have demonstrated the versatility of this method through the construction of a series of vascularized tumour models, and they greatly narrow the gap between the 2D monolayer culture and the in vivo environment.88,95–97 We envision that the proposed new strategy and the established in vitro models can pave a new way and provide a new idea for the study of the interaction between tumours and blood vessels.
This vascular–tumour co-culture platform can also be adapted to other biological systems and will be used as a valuable tool to model the interaction between different cells/tissues and to control the microenvironment.
In addition, more types of cells, such as stroma cells, immune cells and stem cells, can further be introduced into the models,84 to conduct research on the interaction between different cells. These models have shown the potential to provide valuable insights into tumour development.
For the multi-scale vascular construction, a set of experimental data showing the feasibility of the proposed method to construct a multi-scale vascular model containing both big blood vessels and capillaries, the directional effect of the capillary channel, and the excellent bio-compatibility of the proposed system have been displayed. Ubiquitous structures of biological vascular systems have been fabricated to demonstrate the versatility of this method.
Based on this method, a vascularized tumour tissue model exhibiting representative vascular functions under both physiological and pathological conditions has been constructed.
In our work, a parallel-channel model has been proposed for better quantitative analysis of the interaction between tumours and blood vessels, as well as the drug effects, which can further be used as a platform to investigate the metastasis process. Based on these data, we anticipate that this method can further be used as a significant preclinical tool for evaluating and screening novel cancer therapies.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9mh01283d |
‡ These authors contributed equally to the work. |
This journal is © The Royal Society of Chemistry 2020 |