High-throughput production of functional prototissues capable of producing NO for vasodilation

Abstract

Bottom-up synthesis of prototissues helps us to understand the internal cellular communications in the natural tissues and their functions, as well as to improve or repair the damaged tissues. The existed prototissues are rarely used to improve the function of living tissues. We demonstrate a methodology to produce spatially programmable prototissues based on the magneto-Archimedes effect in a high-throughput manner. More than 2000 prototissues are produced once within 2 h. Two-component and three-component spatial coded prototissues are fabricated by varying the addition giant unilamellar vesicles order/number, and the magnetic field distributions. Two-step and three-step signal communications in the prototissues are realized using cascade enzyme reactions. More importantly, the two-component prototissues capable of producing nitric oxide cause vasodilation of rat blood vessels in the presence of glucose and hydroxyurea. The tension force decreases 2.59 g, meanwhile the blood vessel relaxation is of 31.2%. Our works pave the path to fabricate complicated programmable prototissues, and hold great potential in the biomedical field.

Fig. 1. Generation of prototissues using Magneto-Archimedes effect.

a Schematic of the device with a woven nickel mesh (NM) at the center of a circular magnet. Cyan and blue arrows represented the strong and weak magnetic field, respectively. b Simulation results of the magnetic field of the bottom layer and top layer, which indicated the distribution of the magnetic field inside and above the NM, respectively. Dark blue areas indicated the weak magnetic field regions. c Schematic and fluorescence images (from at least five independent samples) of the prototissues assembled in a vertical magnetic field. With the number of added green giant unilamellar vesicles (gGUVs) increasing, the prototissues changed from single layer (I, II) to double layers (III, IV). The vertical purple arrow indicated the vertical magnetic field. d 3D reconstructed confocal fluorescence images of the prototissue array with protruded structure from top (left) and bottom (right) views. e A differential interference contrast (DIC) image (from at least three independent samples) of the 3D prototissue array. The lighter region in the image indicated the protruding top layer, which was marked by the yellow dashed box in the right image. f Schematic of the NM at the edge region of the circular magnet. g The simulation result of the magnetic field of the NM in f. h A fluorescence image (from three independent samples) of gGUVs prototissues in the magnetic field (g). Red line in the inset image corresponded to dashed line intensity analysis. The scale bars were 100 μm.

Fig. 2. Diverse multicomponent prototissues.

a Purple arrow, yellow arrow, and cyan line indicated the vertical, inclined, and no magnetic field, respectively. Red, green, and gray rings indicated the red, green, and non-labeled GUVs, respectively. b Schematic and fluorescence images of a GUVs prototissue of eye structures with rGUVs inside gGUVs under a vertical magnetic field with the addition of gGUVs (3 × 10⁵/mL) and rGUVs (2 × 10⁵/mL) successively. c Schematic and fluorescence images of a GUVs prototissues of modified eye structures by trapping successively gGUVs (3 × 10⁵/mL) under a vertical magnetic field and rGUVs (1 × 10⁵/mL) under an inclined magnetic field. d Schematic and fluorescence images of prototissues by trapping successively gGUVs (1 × 10⁵/mL) and rGUVs (1 × 10⁵/mL) under an inclined magnetic field. e Schematic and fluorescence images of binary prototissues of protruded structures by trapping the mixture of gGUVs (6 × 10⁵/mL) and rGUVs (6 × 10⁵/mL) under a vertical magnetic field. f Schematic and fluorescence images of prototissues of protruded structures with gGUVs at the bottom and rGUVs at the top by trapping successively gGUVs (6 × 10⁵/mL) and rGUVs (4 × 10⁵/mL) under vertical magnetic field. g Schematic and fluorescence images of prototissues with two-layered structures by trapping successively gGUVs (1.2 × 10⁶/mL) under a vertical magnetic field and rGUVs (4 × 10⁵/mL) in the absence of magnetic field. h Schematic and fluorescence images of prototissues composed of three components by trapping successively non-labeled GUVs (6 × 10⁵/mL) and gGUVs (6 × 10⁵/mL) under vertical magnetic field to form protruded structrues, and subsequently rGUVs (2 × 10⁵/mL) in the absence of magnetic field. All the prototissues were assembled on the top of a circular magnet with 0.3 T magnetic flux density. After one type of GUVs were trapped, the time intervals were 1 h before adding another type of GUVs. The representative fluorescence images were from at least three independent samples. The scale bars were 100 μm.

Fig. 3. Signal communication between two-component spatial coded prototissues.

a, b Schematic illustration of signal communication in the prototissues composed of GOx-gGUVs with melittin pores and non-labeled HRP-GUVs. GOx and HRP indicated glucose oxidase and horseradish peroxidase, respectively. c Fluorescence image of the GOx-gGUVs populations (left), the merged image of the fluorescence and bright-field images of the GOx-gGUVs and non-labeled HRP-GUVs populations from three independent samples. d Confocal fluorescence images (from three independent samples) of the prototissues as a function of time after the addition of 30 mM glucose and 0.05 μM Amplex Red. e The mean fluorescence intensities in the HRP-GUVs regions in the images in d (red line), and the control samples using gGUVs to replace GOx-gGUV_S (black line). The error bars were the standard deviation (SD, n = 5). The scale bars were 100 μm.

Fig. 4. Signal communications in hybrid three-component prototissues composed GUVs and C6 glioma cells.

a The cascade reaction formulas for the ternary signal communications among prototissues. Glu, GOx, NO and L-A indicated glucose, glucose oxidase, nitric oxide, and L-Arginine, respectively. b Schematic illustration of signal communications among these three components in the prototissues with GOx-GUVs at the bottom, C6 glioma cells at the protruded top layer, and Arginine-rGUVs (containing 20 mM L-Arginine (L-A), pH = 6) at the rest regions of top layer. c Fluorescence images (from three independent samples) of the prototissues as a function of time triggered by the addition of glucose (30 mM) in the solution. The red and green regions indicated the Arginine-rGUVs and the cell populations, respectively. d The corresponding green fluorescence intensities of prototissues in c (green line), same prototissues but using Arginine-free GUVs to replace Arginine-rGUVs (blue line), and the same prototissues but using GUVs with no GOx to replace GOx-GVUs (red line). The error bars were the standard deviation (SD, n = 5). e A confocal fluorescence image with projected images of the hybrid prototissues composed of three components at 6 h after the addition of glucose. The gray, red, and green regions indicated the non-labeled GOx-GUVs at bottom layer, the Arginine-rGUVs at top edge layer, and the protruded cell at top layer. The scale bars were 100 μm.

Fig. 5. Prototissues capable of producing NO.

a The 3D reconstructed confocal image of the GUVs prototissues detached from the NM. The red box indicated an enlarged GUVs prototissue. b Fluorescence images of the detached prototissue in Rhodamine-dextran (20 kDa) solution. The yellow dashed line in the fluorescence image correspond to line intensity analysis. c Schematic illustration of signal communcations among NO-prototissue composed of GOx-GUVs with melittin pores and HRP-rGUVs. The addition of glucose and hydroxyurea triggered the reaction to generate NO in HRP-rGUVs. NO was detected by the DAF-2 in the solution to generate green fluorescent DAF-2T. Glu, GOx, HRP, Ha and NO indicated glucose, glucose oxidase, horseradish peroxidase, hydroxyurea, and nitric oxide, respectively. d Schematic illustration of the structure of the NO-prototissue (top). Fluorescence image of the HRP-rGUVs in the NO-prototissue (bottom left), bright-field image (bottom middle) and their merged image (bottom right) of the NO-prototissue. e Time-dependent fluorescence microscopy images of the NO-prototissue in the solution containing DAF-2 (10 μM) after adding glucose (20 mM) and hydroxyurea (10 mM). The green fluorescence channel responded to NO production. f Plots of fluorescence intensity against time for internal (pink box) and external regions (blue box) in e. The representative fluorescence images were from three independent samples. The scale bars were 100 μm.

Fig. 6. NO-prototissues for vasodilation.

a Schematic illustration of vasodilation induced by NO from NO-prototissues. Representative tension curve of vasodilation against time in the presence of NO-prototissues composed with GOx-GUVs and HRP-GUVs (b), NO-free-prototissues composed of GOx-free GUVs and HRP-GUVs (c) and SNP (d). The black arrow indicated the point of the addition of hydroxyurea (Ha, 10 mM) or SNP (0.1 μM). Glu, GOx, HRP, Ha, NO and SNP indicated glucose, glucose oxidase, horseradish peroxidase, hydroxyurea, nitric oxide, and sodium nitroprusside, respectively. e, f Bar charts of the decrease in tension force and relaxation of vascular rings observed in the presence of NO-prototissues (2.59 ±  0.77 g; 31.2 ± 10.1% relaxation, black columns), NO-free-prototissues (0.14 ± 0.12 g; 3.4 ± 2.9% relaxation, red columns), and 0.1 μM SNP (2.84 ± 0.92 g; 43.3 ± 9.0% relaxation, blue columns). Data are presented as mean values ± SD, n = 5. Statistical analyses were carried out by unpaired two-tailed student’s t-test (***p  <  0.001). NO-prototissues: SNP: p = 0.6566 in e and p = 0.0802 in f. p < 0.05 was considered statistically significant. Source data are provided as a Source Data file. g Fluorescence images (from three independent samples) of vascular sections stained with DAF-FM DA (the green fluorescence channel responded to NO production, left column), DAPI (blue fluorescence with nuclei, middle column), and their merge images (right column). The blood vessels were treated using NO-prototissues (top row) or NO-free-prototissues (middle row) in the HEPES solution containing 10 mM hydroxyurea for 20 min, respectively. The blood vessel was treated using 0.1 μM SNP in the HEPES solution for 20 min (bottom row). The scale bars were 100 μm.