Fabrication and endothelialization of spiral tubes in PDMS and collagen gels
We exploited subtractive molding techniques to fabricate spiral tubes in both polydimethylsiloxane (PDMS) and collagen hydrogels and tested the fabrication limit and fidelity. In PDMS, stainless steel springs of various dimensions were molded in liquid-phase PDMS (10:1, base:curing agent) and manually removed after cross-linking. Robust perfusable spiral tubes with constant curvature were generated in PDMS with a diameter larger than 200 μm and a pitch greater than 1 mm per turn. The fabrication of smaller spiral tubes in PDMS is less consistent because of the distortion of the channel structure during spring removal. In collagen hydrogels (6 to 7.5 mg/ml), an automatic two-axis motion system was designed to retract the spring from the hydrogel after thermal gelation. Automatic retraction was critical to minimize distortion of the spiral pattern and maximize continuity of the luminal geometry in three dimensions in soft matrices (see Materials and Methods) (Fig. 1A). Spiral tubes of a wide range of wire diameter (dw = 120 to 400 μm), spiral diameter (ds = 1 to 3 mm), and pitch (p ≥ 400 μm) were formed in collagen hydrogels, corresponding to curvature κ in the range of 0.43 to 1.05 mm−1 and torsion τ in the range of 0.32 to 0.72 mm−1 (fig. S1A). Fluorescent beads were perfused to visualize the 3D structure of the spiral lumen [Fig. 1B (a); fig. S1, A and B; and movie S1], where loops of the spiral tubes were periodically spaced with distinct boundaries. Using off-the-shelf springs, we were able to achieve spacing between loops (δ = p − dw) as small as 210 μm. We further modified the spiral mold by adding a cylinder in the center of the spring to generate a second independently perfusable lumen in the hydrogel structure [Fig. 1B (b)]. We fabricated constructs with a central tube concentrically wrapped with a spiral tube and separated by a wall as thin as 200 μm.
Next, we perfused human umbilical vein ECs (HUVECs) into the spiral tubes in either PDMS or collagen to allow cell attachment followed by culture under flow. Both materials supported the growth of a robust endothelium under steady flow for at least 1 week (Q = 1 μl/min; Fig. 1, C to E). PDMS spiral vessels with a lumen diameter of less than 200 μm often had sparse coverage of ECs on the vessel surface after seeding and were not used in experimental conditions. Collagen spiral vessels better supported endothelialization, and HUVECs were seeded and cultured under similar flow conditions for spiral vessels as small as 180 μm with high reproducibility (fig. S1C). ECs in PDMS vessels (lumen diameter > 200 μm) and all sized collagen vessels had robust junctions at cell-cell contacts and localized expression of CD31 to the plasma membrane (Fig. 1, C to E). Together, we successfully generated spiral microvessels with constant curvature and torsion at high fidelity and reproducibility and with robust endothelialization and perfusion.
Spiral vessels for 3D vascularization
The fabrication process for spiral vessels has the flexibility to integrate with existing vascularization approaches to further enhance tissue perfusion. By incorporating ECs into the bulk matrix, the endothelium in spiral tubes was readily anastomosed with self-assembled vessel networks and increased vascular density (fig. S1D). When combined with lithography and injection molding techniques (31), we successfully connected a spiral vessel with a microfabricated rectilinear vessel so that the spiral outflow was connected to the perfusion of microvessels in an orthogonal direction to the spiral. This integration allows the rotation of the spiral flow direction into another plane and mimics the architecture of the spiral artery to vascular bed connection found in vivo (Fig. 1F). We observed a continuous endothelium in the spiral microvessel connection [Fig. 1F (a)]. ECs in the planar microvessels near the spiral vessel outflow showed greater alignment with the direction of flow, likely due to higher flow stresses [Fig. 1F (a); average angle, 13.5° ± 10.2°] compared to cells in regions distant from the spiral microvessel interface [Fig. 1F (b and c); average angle, 39.3° ± 23.7° and 58.13° ± 29.25°, respectively]. These findings illustrate the potential of spiral vessels as a new strategy for rapidly generating long and high surface area vascular structures that may enhance tissue vascularization.
Using the concentric spiral platform in collagen gel, we further demonstrated the potential of spiral vessels in supporting 3D tissue function. By dispensing tumor cells (KG1a, a leukemia cell line; Materials and Methods) in a collagen gel (6 mg/ml) into the spatially defined center cylinder (1.3 mm diameter), we formed an artificial tumor surrounded by spiral vessels and monitored the sprouting of vessels from the spiral. This cell-remodelable system mimics the physiological origins of some tumors, where malignancies begin as an avascular cellular mass surrounded by host vasculature that it must recruit for expansion (32). When cultured under flow (Q = 1 μl/min) in normal growth medium, spiral vessels (dw = 400 μm, ds = 3.0 mm, and p = 1 mm) maintained patency throughout 7 days of culture and sprouted consistently by day 7, but not at day 3 (N = 4 for each time point) (Fig. 1G). These sprouts extended exclusively toward the tumor, with sprouts reaching as far as 220 μm from the vessel wall by day 7. No sprouts were observed when there were no tumor cells in the center.
We also created a thick cardiac chamber supported by a spiral vessel using the same concentric model (1.3-mm-diameter by 6-mm-long chamber surrounded by spiral vessel). GCaMP3-transduced human embryonic stem cell–derived cardiomyocytes (hESC-CMs) and stromal cells (HS27a) were added into the bulk collagen matrix (33) and ECs (HUVECs) into both the bulk matrix and the spiral lumen, while the center of the tissue was kept open (Fig. 1H). By day 12 of culture, organized calcium waves were observed and appeared to propagate in three dimensions along the spiral vessel wall (movie S2). The conduction velocity in engineered cardiac tissues was 2.7 ± 0.97 cm/s, as determined by analysis of the GCaMP3 signal (fig. S2). These proof-of-concept examples show that the spiral vessel platform can be used to support 3D vascularization and perfusion in large tissues, to study the vascular-tissue interaction in a spatially and temporally controlled manner, and to model complex tissue functions.
Flow characteristics of spiral tubes
We next examined the flow characteristics in these spiral microvessels and compared them with straight vessels of the same caliber. We visualized the flow characteristics by perfusing fluorescent bead solutions in two parallel streams through straight and spiral PDMS vessels of the same diameter and length (dvessel = 400 μm, dspiral = 3 mm, pspiral = 1 mm, κspiral = 0.46 mm−1, τspiral = 0.31 mm−1, and L = 6.5 cm) at three steady flow conditions (Q = 1, 50, and 100 μl/min, corresponding to Re = 0.01, 0.76, and 1.52, respectively). The 3D flow images were taken under confocal fluorescence microscopy at set distances (Lv = 5, 30, and 55 mm in straight vessels, or loops 3/4, 3 3/4, and 6 3/4 in spiral vessels) from the vessel inlet. Straight tubes displayed a classical parallel flow profile where the two streams of beads traveled to the outlet and maintained their position over the whole vessel length at both flow rates (Fig. 2, A and C). In spiral tubes, the two bead streams remained distinct and parallel at low flow (Q = 1 μl/min) but rotated over the vessel length without obvious mixing in the bulk [Fig. 2B (a to c)]. The orientation of the two parallel streams inverted after approximately four loops from the inlet [Fig. 2B (b)] and completed a full rotation at approximately loop 7 [Fig. 2B (c)]. At a higher flow rate (Q = 50 μl/min) in the same spiral geometry (De = 2.77), the two bead streams developed obvious bulk mixing with the leading edge of flow rotating 270° after three loops [Fig. 2B (e)] and completed another full rotation by loop 7 [Fig. 2B (f)]. At even higher flow (Q = 100 μl/min), a stronger mixing effect was observed in the same spiral geometry [Fig. 2B (g to i)], whereas the two streams remained parallel and unmixed in straight vessels under the same flow conditions.
Using numerical simulation with COMSOL, we confirmed these flow characteristics: (i) Idealized parallel streamlines were present in fully developed flow in straight vessels [Fig. 2C (a)]; (ii) parallel streamlines in spiral vessels slightly rotate along circumferential direction at low flow (Q = 1 μl/min; fig. S3A); and (iii) streamline rotation was enhanced in spiral vessels and developed twists at higher flow [Q = 50 μl/min; Fig. 2D (a)] and had clear twists at Q = 100 μl/min (fig. S3B). The spiral geometry did not induce a significant change in the primary flow compared to straight vessels but did lead to the emergence of secondary flows with a peak magnitude of around 1% of the primary flow velocity [Q = 50 μl/min; Fig. 2, C (b and c) and D (b and c)]. This also led to the development of a shear stress gradient in 3D space and a change in the wall shear stress (WSS), with a maximum (10% increase over the straight tube) on the surface of the inner curvature and minimum on the outer bend, unlike in a straight tube where the WSS was constant across the lumen cross section with zero gradients [Fig. 2, C (d) and D (d)]. These data demonstrated that spiral vessels induced bulk flow mixing and heterogeneous hemodynamic forces on the endothelium lining the wall due to 3D curvature and torsion.
3D curvature alters endothelial morphology and mRNA expression under flow
To understand how the distinct hemodynamic features of flow in spiral vessels affected ECs, we cultured cells in both geometries under flow. In straight and spiral vessels, ECs formed robust junctions and a stable endothelium in low (Q = 1 μl/min and WSS = 0.1 dyne/cm2 in straight vessels) and high (Q = 50 μl/min and WSS = 4.6 dynes/cm2 in straight vessels) flow conditions. The increased flow appeared to change the EC morphology and enhance EC alignment in the direction of flow (Fig. 3A). Under low flow conditions (Q = 1 μl/min; Fig. 3B), fewer Ki67+ proliferating cells were observed in spiral vessels than in straight vessels. When exposed to higher flow, however, more proliferating cells were observed in the spiral geometry than the straight geometry, suggesting distinct roles for geometry and flow on the ECs. Previous literature has highlighted that very low laminar flows activate ECs, whereas high laminar flow enhances EC quiescence (11). Our data were consistent with this in straight vessels with significantly reduced cell proliferation at higher flow. In spiral vessels, however, the flow rotation in low flow may alter transport and promote quiescence at low flow. Given that the magnitude of flow forces is very low in the low flow conditions, it is also likely that differences in substrate curvature between straight and spiral geometries contribute to these observed differences (34).
We next examined the transcriptional changes in ECs in these conditions via RNA sequencing (RNA-seq) for ECs cultured under both flow conditions in straight and spiral vessels and under static conditions. Principal components analysis (PCA) of gene expression data showed clustering of individual groups, with the largest variance between static and all flow conditions (Fig. 3C). Activation of classical flow-dependent genes was confirmed in all flow conditions compared to static culture (Fig. 3, D and E). Among these genes, KLF2 and KLF4 appeared to only change with the onset of flow but were not sensitive to a further increase in flow, whereas SMAD6, SMAD7, and NOS3 increased further at higher flow conditions. Among the genes differentially expressed in straight vessels due to the increase of flow, 52% (533 of 1012) overlap with genes differentially expressed in the onset of flow (static versus low flow condition) (fig. S4, A and B). The genes unique to the increase of flow include up-regulation of many genes previously reported to regulate vascular development and flow sensing (35), such as Notch ligands JAG1 and JAG2; Notch target HEY2 and other transcription factors such as SNAI2; transmembrane proteins IL21R and EFNB2; transporter GJA5; peptidases MMP10, MMP1, and MT1F; growth factors and cytokines NOG, DKK2, WNT4, CXCL12, and TGFB1; and other molecules such as VCAN and CYP1B1 (fig. S4C). Gene Ontology (GO)–enriched terms for this group of genes showed up-regulation of cellular response to growth factors, vascular development, transmembrane receptor protein tyrosine kinase signaling pathway, blood vessel morphogenesis, cell migration and motility, and others (fig. S4D).
Approximately 66% (722 of 1136) of differentially expressed genes in straight vessels overlap with those in spiral vessels in response to increased flow (Fig. 3D). Almost all overlapping genes are changed in the same direction (99%), suggesting a conserved response to flow in both geometries (fig. S5A). MARC2, PTX3, and STX11 did not follow this trend and were up-regulated in spiral vessels with increased flow but down-regulated in straight vessels. PTX3 has been reported as a biomarker for endothelial dysfunction in preeclampsia, which is a disease caused by spiral artery dysfunction (36). Many genes down-regulated in straight vessels by the increase of flow did not show changes in spiral vessels, such as growth factors CTGF, FGF2, NRG1, and FGF16; transmembrane proteins CAV1, UNC5A, KIT, and SMAD4A; transcription regulators EGR1/2/3, MAF, MYRF, and MZF1; transporters such as LDLR; and cytokines TNFSF18, IL12A, CCL2, CCL16, and CCL28 (fig. S5B). This suggests that the EC response to flow in spiral vessels is a combination of both canonical flow pathways and a distinct response involving a wide range of other transcripts.
Increased flow also led to an additional 1294 genes significantly changed in spiral vessels that were not in straight ones (Fig. 3F). High flow in spiral vessels appeared to activate growth factors such as DKK1, ESM1, BMP2, PDGFA, OSGIN2, and VEGFC; many solute carrier (SLC) and adenosine triphosphate (ATP)–binding cassette (ABC) superfamily transporters; transcriptional regulators such as GLI2; cytokine CXCL1; peptidases TLL1, ADAMTS1, ADAMTS9, and TASP1; and kinases PODXL, EPHA5, HK2, PRKCA, CCT2, and MAP2K1 (Fig. 3G and fig. S6A). In addition, high flow in spiral vessels repressed growth factors such as MST, NRG2, GDF3, GAS6, and IGF2; transmembrane receptors CHRNA1, SELP, LRP1, ITGB3, and ROBO3; transporters including MAL2, ATP2A3, RBP1, and APOL1 and several members of SLC and ABC superfamilies; transcriptional regulators such as NOTCH3, CITED4, CAND2, FOXO4, DACH1, and EBF3; cytokines DKK3, CSF1, and FLT3LG; GPCR (G protein–coupled receptor) group SIPR4, OPRL1, and HTR2B; and kinases PDGFRB, CKB, and SBK1 (Fig. 3F and fig. S6A). GO term analysis showed the up-regulation of primarily ribosome biogenesis, which would be critical for cellular growth and proliferation (fig. S6B). These expression profiles show that spiral vessels share a common set of flow-responsive elements with straight vessels but have an additional response that appeared to promote vascular growth.
PCA analysis showed that the separation of straight and spiral geometries was enhanced under higher flow conditions (Fig. 3E). Under low flow conditions (Re << 1; inertia effect is negligible), ECs in the two geometries were largely similar, with only a handful of significantly regulated transcripts (fig. S7A). These included CYTL1, which is known to up-regulate proangiogenic function, but not proinflammatory pathways (37). HES2, a downstream Notch pathway gene, STK32B (serine/threonine kinase 32B), and CCND1, a cell cycle regulation gene, were also up-regulated in low flow spiral vessels. The up-regulation of these genes was further enhanced in high flow conditions. In addition, many genes that regulate vascular development were up-regulated when comparing spiral to straight vessels at high flow, for example, growth factors HGF, DKK1, ESM1, PGF, PDGFA, GDF6, PDGFB, CTGF, VEGFC, BMP2, and PDGFC; peptidases ADAMTS1, ADAMTS9, MME, and CTSS; kinases EPHA5, MPP4, PODXL, SPRY2, CDK7, and MAP2K1; transmembrane receptors KIT, SELE, ULBP2, PLXNA2, and LRP8; transcriptional regulators GLI2 (a hedgehog pathway mediator), ATF3 (required for endothelial regeneration) (38), and FOSL1 (required for vascular formation) (39); and many SLC and ATP family transporters (Fig. 3G). Ingenuity Pathway Analysis (IPA) showed that ECs in spiral vessels have activated upstream regulators including prosurvival factors HGF, PGF, EGF, VEGF, and HIF-1a. Up-regulated functional pathways included vascular development, angiogenesis, vasculogenesis, cell invasion, and cell survival, whereas cell death and necrosis were decreased compared to the straight vessel in high flow conditions (Fig. 3H and fig. S7B). Spiral vessels also showed the activation of antiapoptotic and proliferative pathways marked by cell cycle and mitotic genes. PDGF (platelet-derived growth factor) family members were relatively more abundant, as were molecules associated with IL-8 (interleukin-8) and HGF (hepatocyte growth factor) signaling (fig. S7B).
Together, the bulk RNA-seq showed that spiral vessels maintain a normal flow response to a certain extent, but curvature and torsion modified the response by up-regulating markers for transporters, cycling, and survival and down-regulating markers of cell death. These data suggest that flow in spiral vessels promoted vascular growth or development rather than inducing a common inflammatory response to disturbed flow.
Single-cell RNA-seq reveals heterogeneity in transcriptional responses to flow based on vessel geometry
We hypothesized that ECs exposed to flow within spiral vessels experienced a spatial variation in hemodynamic forces not present in straight vessels that would result in a heterogeneous transcriptional response to flow. To understand this heterogeneity at the single-cell level, we sequenced the transcriptomes of more than 2000 individual ECs pooled from three to four devices of each geometry cultured at high flow (Q = 50 μl/min). Dimensionality reduction by Uniform Manifold Approximation and Projection (UMAP) and cluster analysis was performed using Monocle (40–42). Projection in the top two UMAP dimensions shows overlapping contributions of ECs from spiral and straight vessels that form mostly contiguous clusters with nearly uniform expression of pan-endothelial markers such as CDH5 (VE-cadherin) (Fig. 4, A and B). Expression of classical flow-dependent genes, including KLF4 and NOS3, is distributed throughout the major clusters of ECs in this projection (Fig. 4C). We identified variation in gene expression across the first UMAP dimension driven largely by cell cycle genes that have been shown to be regulated, in part, by flow. Specifically, a large cluster of cells to the right in UMAP space (cluster 3) express genes such as MKI67, consistent with active cell cycle status, whereas cells clustered to the opposite pole (cluster 1) express genes implicated in cell cycle arrest and arterial phenotype shown to be regulated by the Notch pathway downstream of laminar shear stress (including CDKN1C, EFNB2, HEY1, GJA4, and IL33; Fig. 4C) (43–45).
To evaluate heterogeneity in the transcriptional response of ECs resulting from vessel geometry, we next identified differentially expressed genes on the basis of single-cell RNA-seq (scRNA-seq) data of EC from straight versus spiral vessels. Examination of the third UMAP dimension revealed separation in transcriptional space between EC from straight versus spiral vessels, with many of the identified differentially expressed genes polarized in this dimension (Fig. 4, D to F). Among the genes up-regulated in EC from spiral vessels are many that were also identified as differentially expressed in bulk RNA-seq analysis, including ATF3, SPRY2, IL8, JUN, AKAP12, ANGPTL4, FOSL1, ADAMTS1, and ADAMTS9 (Fig. 4G and fig. S8A). Most of these genes are expressed in a common pattern, with increased expression in cells localized in UMAP space to the lower (spiral) portion of cluster 2. This suggests a distinct transcriptional program among the primarily spiral ECs in this region that may correspond with their transcriptional response to specific hemodynamic conditions unique to spiral vessels under high flow. In further support of this hypothesis, analysis of the scRNA-seq data also identified differentially expressed genes not detected in bulk RNA-seq, including DCN, SLC6A9, GEM, NRG1, RSPO3, BAMBI, TGFBI, and PRRX2, that were highly specific to EC from spiral vessels localized in cluster 2 (Fig. 4G and fig. S8B). These data suggest that flow in spiral vessels induced a population of ECs with unique gene expression profiles that are not present in straight vessels, with potential roles in processes such as angiogenesis, vascular growth, and inflammatory and stress responses.