Article pubs.acs.org/Langmuir
Design and Assessment of a Microfluidic Network System for Oxygen Transport in Engineered Tissue Tae-Yun Kang,† Jung Min Hong,† Jin Woo Jung,† James J. Yoo,‡ and Dong-Woo Cho*,† †
Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja dong, Nam-gu, Pohang, Gyungbuk 790-784, Korea ‡ Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, North Carolina 27157, United States S Supporting Information *
ABSTRACT: Oxygen and nutrients cannot be delivered to cells residing in the interior of large-volume scaffolds via diffusion alone. Several efforts have been made to meet the metabolic needs of cells in a scaffold by constructing mass transport channels, particularly in the form of bifurcated networks. In contrast to progress in fabrication technologies, however, an approach to designing an optimal network based on experimental evaluation has not been actively reported. The main objective of this study was to establish a procedure for designing an effective microfluidic network system for a cellseeded scaffold and to develop an experimental model to evaluate the design. We proposed a process to design a microfluidic network by combining an oxygen transport simulation with biomimetic principles governing biological vascular trees. The simulation was performed with the effective diffusion coefficient (De,s), which was experimentally measured in our previous study. Porous scaffolds containing an embedded microfluidic network were fabricated using the lost mold shape-forming process and salt leaching method. The reliability of the procedure was demonstrated by experiments using the scaffolds. This approach established a practical basis for designing an effective microfluidic network in a cell-seeded scaffold.
■
INTRODUCTION Tissue engineering and regenerative medicine research aims to develop artificial tissues to replace defective tissues or organs.1−3 The basic concept is to transplant cells and growth factors seeded on a porous degradable structure, which is known as a scaffold, into the defective site. Tissue can form and eventually replace the scaffold, and the scaffold preserves the tissue volume and provides structural support. Research has been actively conducted on various tissues, and recent advances have offered new therapeutic opportunities in the field of medicine. Although these tissues have shown potential experimental applicability, only a limited number of tissues such as skin4,5 have been successfully engineered for clinical use. One of the challenges that hamper the rapid clinical translation of engineered tissues is an inadequate metabolite supply to the interior parts of a scaffold. The introduction of solid freeform fabrication (SFF) technologies to tissue engineering has allowed for the fabrication of scaffolds with perfectly interconnected pores,1,6 which are advantageous for © 2012 American Chemical Society
metabolite transport. However, cells residing in the interior of large-volume scaffolds lack nutrients and oxygen,7,8 and the metabolites dissolved in the medium are transported only by diffusion from the outer surface of the scaffold. Several studies have been performed to meet this metabolic need by means of perfusion to force flow through preconstructed channels in a scaffold. Simple single-channel arrays have been constructed in scaffolds for such a purpose.9,10 Recent advances in fabrication technology have enabled the construction of microfluidic networks in scaffolds. Microscale technologies such as photolithography, micromachining, and micromolding have been utilized to fabricate 2D microfluidic networks on various biomaterials.11−15 Because of the inherent 2D nature of those technologies, a 3D microfluidic network cannot be constructed unless 2D layers are stacked. However, Received: September 3, 2012 Revised: November 20, 2012 Published: December 12, 2012 701
dx.doi.org/10.1021/la303552m | Langmuir 2013, 29, 701−709
Langmuir
Article
Figure 1. Design of the microfluidic network system. (a) The concepts of 2D static and perfusion culture models were established to evaluate the effect of a microfluidic network system. The pink shading represents steady-state oxygen distribution, (b) the flow chart represents the process of designing an optimal microfluidic network considering the compromise needed between mass-transfer efficiency and volume loss, and (c) the schematic diagram represents the geometric dimensions of the microfluidic network, where H is the diagonal length of a square scaffold (21.2 mm), D0 is the inlet diameter set to be the same size as the blood vessel adjacent to the implant site (1 mm), Dn(n ≥ 1) is the diameter of the nth branch determined by eq 1, l0 is the length of the initial channel determined by eq 4, ln(n ≥ 1) is the length of the nth branch determined by eqs 2 and 3, and θ is the branch-opening half angle (π/4).
capillaries. Recently, thermal extrusion and fiber drawing with a 3D printer have allowed for the rapid casting of patterned vascular networks for three-dimensional hydrogel-containing cells.19 In contrast to such progress in fabrication technology, approaches to designing an optimal microfluidic network for in
microscale technology can achieve submicrometer resolution, which is smaller than the dimensions of a capillary. A 3D network has been fabricated in a porous scaffold by repeated layering/micromachining16,17 and selective laser sintering18 combined with a salt leaching method, even though the channel diameter (1 mm) was much larger than that of the 702
dx.doi.org/10.1021/la303552m | Langmuir 2013, 29, 701−709
Langmuir
Article
basis of Murray’s law. In nature, a biological distribution system requires energy to overcome viscous drag as well as energy to maintain metabolic processes. Murray originally derived the relationship between parent and daughter diameters based on the principle of minimum work. It is now known as Murray’s law and is expressed as follows22,23
vivo application have not been actively reported. In a perfusive system, the transport of metabolites to the scaffold interior proceeds in two steps: advection-driven mass transfer through flow channels and diffusion-driven mass transfer from the channel wall to the interior.10,20 Therefore, channel arrangement is a key factor in determining the overall range and efficiency of metabolite transport into a scaffold. Bifurcated networks have been the focus of a biomimetic design for efficient microfluidic distribution. Similar to the mammalian cardiovascular and respiratory systems, an artificial vascular network has stepwise scaling from a single channel to thinner branched channels. The fascinating feature of this hierarchical structure is that it provides uniformly distributed flow over the region while minimizing the amount of work required to operate and maintain the system, as demonstrated by Murray’s law.21 This study started with the practical question of how many times a microfluidic network needs to branch to maximize cell viability in a cell-seeded scaffold. Although highly branched microfluidic networks are able to cover a region densely, the increase in channel volume is accompanied by a decrease in cell residence area, which is the space in which the cells live around the microfluidic network in a scaffold. Moreover, thinner channels face a higher risk of thrombus formation, resulting in channel clogging when the network is surgically connected to the host vasculature and blood flows through the channel in vivo. Therefore, a design strategy should be devised from the perspective of compromising between mass transfer efficiency and volume loss/thrombogenic potential. The main objectives of this study were to establish a procedure for designing an effective microfluidic network system for a cell-seeded scaffold for in vivo application and to develop an experimental model to evaluate the design. In a previous study, we measured the change in the effective diffusion coefficient (De,s) in a cell-seeded scaffold to investigate oxygen transport within the scaffold. In the present report, we propose a process for designing a microfluidic network by combining oxygen transport simulation using De,s and biomimetic principles governing biological vascular trees. Porous scaffolds containing a microfluidic network were fabricated using the lost mold shape-forming process and the salt-leaching method. The effects of the microfluidic network on oxygen delivery were assessed by culturing the scaffolds in a perfusion culture system, which was constructed to restrict the direction of oxygen diffusion.
■
D0 3 = D13 + D2 3
(1)
where D0 is the diameter of the parent branch and D1 and D2 are the diameters of the daughter branches (Figure 1c). Step C. Morphometric studies have revealed a positive correlation between the diameter and the length of a branch.24−27 In general, small-diameter branches have shorter lengths. In this study, the length of each branch was simply determined by multiplying a scaling factor, α, and the diameter (eq 2). The scaling factor α was adjusted to satisfy eq 3
ln = Dn × α ,
n≥1
∑ ln = ∑ Dn × α=
H − l0 , 2
(2)
n≥1
(3)
where H is the diagonal length of the scaffold and Dn and ln are the diameter and length, respectively, of the nth branch (Figure 1c). The length of the initial channel l0 was set to align the next branch at a proper distance from the scaffold boundary regardless of eq 2. First, the limit of the nonhypoxic area (Lnonhypoxic), which is defined as the distance between the channel lumen and the point at which hypoxia first occurs, was calculated from the oxygen profile of a model in which a single channel is embedded in a scaffold (Bifurcation: 0 in Figure 4a). Then, l0 was determined by eq 4 to align the next branch at a distance of Lnonhypoxic from the scaffold boundary as described in Figure 1c.
l0 =
Lnonhypoxic (4)
sin θ
Step D. When branches are aligned in a space, a large bifurcating angle gives the effect of delivering metabolites in a new direction and covering the space with better uniformity, but a large bifurcating angle is inevitably accompanied by a high-pressure loss.28 In this study, the branch-opening half angle (θ) (Figure 1c) was fixed at π/4, which is the maximum value in the physiologically relevant range of π/25 < θ < π 29 /4. Step E. In this process, the design of a microfluidic network begins with a single channel, and the number of bifurcations increases as the cycle is repeated until it reaches the point of compromise between mass-transfer efficiency and volume loss/thrombogenic potential. In this study, the criterion for this point was set to be whether the decrease in the cell residence area exceeded the increase in the nonhypoxic area. The cell residence area is the space in which the cells live around the microfluidic network in the scaffold. Mathematical Model for Oxygen Transport in a Scaffold. Oxygen Transport from the Surface of the Scaffold. Considering the oxygen consumption of cells according to Michaelis−Menten kinetics, a mathematical model for oxygen transport in a cell-seeded scaffold under a static culture can be expressed by the following governing equation:
MATERIALS AND METHODS
Design of a Microfluidic Network System for a Porous Scaffold. We established a 2D model to evaluate the effect of a microfluidic network system (Figure 1a). Under general static culture conditions, oxygen can be supplied via diffusion from the outer surface of the scaffold. If the scaffold is assumed to be long enough in the y direction, then oxygen molecules that are supplied from the surface perpendicular to the y axis cannot reach the center part. In this crosssectional region, x- and z-directional diffusions from the surface are assumed to be the only oxygen supply source. We considered this static model to be the control and designed a microfluidic network system to cover the same size region based on the process represented in Figure 1b. The details of each step in Figure 1b are explained below. Step A. The size and shape of a scaffold depend on the target tissue/organ to be regenerated. In this study, microfluidic network systems were designed to cover a square scaffold (15 mm × 15 mm). Step B. Considering the surgical connection to the host vasculature in vivo, we set the inlet diameter to be the same size as the blood vessel adjacent to the implant site. The relationship between the diameters of parent and daughter branches was determined on the
⎡ ∂ 2C VO maxCs ∂ 2Cs ⎤ ⎥ − Ncell 2 0 = De,s⎢ 2s + 2 K m + Cs ∂z ⎦ ⎣ ∂x
(5)
where Cs is the oxygen concentration in the scaffold, De,s is the effective diffusion coefficient in the scaffold, Ncell is cell density, VO2max is the maximum oxygen uptake rate, and Km is the Michaelis−Menten constant for NIH-3T3 cells. In particular, De,s and Ncell were set to be a converged value from the experimentally measured data for the same scaffold and cell type used in a previous study.20 Oxygen Transport by the Microfluidic Network System. A mathematical model was employed on the basis of the Krogh cylinder model to investigate enhanced oxygen transport by embedding the 703
dx.doi.org/10.1021/la303552m | Langmuir 2013, 29, 701−709
Langmuir
Article
microfluidic system within a cell-seeded scaffold.10,30,31 Oxygen diffusion from the outer surface of a scaffold is excluded in the model. In the scaffold, the microfluidic network region was defined as the microfluidic domain, and the porous region, in which cells reside, was defined as the tissue domain. In the microfluidic domain, the conservation equation for oxygen in the steady state can be expressed as follows Vz
⎡ 1 ∂ ⎛ ∂C ⎞ ∂ 2C ⎤ ∂Cc c ⎜r c ⎟ + ⎥ = Dc⎢ ∂Z ∂Z2 ⎦ ⎣ r ∂r ⎝ ∂r ⎠
sacrificial mold and sodium chloride crystals, respectively. The fabricated scaffolds were sputter-coated with gold, and the surfaces were observed using scanning electron microscopy (SEM; Hitachi SU6600, Hitachi, Tokyo, Japan). Cell Culture under Static and Perfusion Conditions. Prior to cell seeding, all scaffolds were sterilized overnight under ultraviolet light. Scaffolds were prewet in 70% ethanol for 2 h and washed three times in phosphate-buffered saline (15 min per wash). NIH-3T3 mouse fibroblasts were seeded onto the scaffolds (9 × 105 cells/ scaffold) using a vacuum-aided seeding technique35 that was reported not to have deleterious effects on cells and to distribute the cells homogeneously across the surface and thickness of the scaffold. Briefly, the scaffolds were placed in a well plate, and cell suspensions (150 μL/ scaffold) were seeded onto the scaffold. Then, six vacuum−release cycles were applied using a vacuum desiccator to improve the infiltration of the cells into the scaffolds. The vacuum up to 85 mmHg absolute lasted 30 s for each cycle. Static or perfusive cultures were preceded by shaking and incubating for 3 h. The effects of the microfluidic network on oxygen delivery were assessed by culturing the scaffolds while restricting the direction of oxygen diffusion from the scaffold surface. Parylene-C-coated poly(dimethylsiloxane) (PDMS) chambers, which had different designs for static and perfusion culture, were used to block oxygen diffusion.36 The gas-permeable property of PDMS was reduced by the parylene-C coating, and the effect was confirmed by cell-culturing experiments (Supporting Information). Static Culture. On the basis of the mathematical model for static culture, oxygen can be transported via diffusion from the outer surface of a scaffold in the x and z directions, whereas y-directional diffusion is excluded. To realize this environment, a cell-seeded scaffold was inserted into a parylene-C-coated PDMS chamber prepared for static culturing. As depicted in Figure 3a, the chamber was composed of two plates connected by small blocks at the four corners. The plates prevented oxygen from diffusing from the top and bottom, but the sides were open. The chambers containing scaffolds were placed in well plates with a culture medium, followed by gentle periodic shaking. Perfusion Culture. A perfusion bioreactor composed of a peristaltic pump, medium reservoir, gas exchanger, and chamber was used to force flow through the preconstructed microfluidic network (Figure 3b). Similar to the static culture chamber, the perfusive culture chamber was made of parylene-C-coated PDMS and was designed to meet the conditions described in the mathematical model (Figure 3c). A scaffold was placed in a double-layered chamber to block oxygen diffusion from the outer surface. The top layer had a space for scaffold loading, and tubes were connected to inlet and outlet holes, whereas the bottom layer was a simple flat plate. The double-layered chamber was sandwiched between two acrylic plates and held together with four screws. In this setup, oxygen could only be delivered by the culture medium flowing through the microfluidic network system. Assessment of Proliferation and the Hypoxic Areas. The Pimonidazole conjugation assay was used to map the hypoxic regions across the scaffold. Positive staining indicated that cells were at once alive under hypoxic conditions (oxygen concentration
