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Aspiration-assisted bioprinting for precise positioning of biologics

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Science Advances  06 Mar 2020:
Vol. 6, no. 10, eaaw5111
DOI: 10.1126/sciadv.aaw5111
  • Fig. 1 Step-by-step illustration of picking and bioprinting of spheroids.

    In step 1, spheroids are picked from the cell media by a glass pipette, where required back pressure is set to lift spheroids. Afterward, spheroids can be bioprinted into sacrificial hydrogels (scaffold-free bioprinting) or functional hydrogels (scaffold-based bioprinting). In this regard, in step 2, microvalve bioprinting is used to bioprint a gel substrate, which can then be partially cross-linked using various different cross-linking schemes—such as but not limited to enzymatic, photo, and ionic cross-linking—as highlighted in step 3. Next, in step 4, spheroids are bioprinted precisely into designed positions, and spheroid bioprinting is repeated as many times as needed. Steps 2 to 4 can be repeated as needed. In step 5, bioprinted tissues are isolated from the support hydrogel (for scaffold-free bioprinting) or further grown in the functional hydrogel (for scaffold-based bioprinting). UV, ultraviolet.

  • Fig. 2 Picking and lifting spheroids.

    (A) Time-lapse images during spheroid lifting process (at the interface of cell media and air). (B) A schematic showing physical parameters involved in lifting of a spheroid from the cell media. (C) SEM images, (D) surface tension (n = 5), and (E) the normalized collagen content of HUVEC, 3T3, 4T1, HDF, MSC/HUVEC, and MSC spheroids (compared to HUVEC spheroids) at day 2 (n = 4; *P < 0.05, **P < 0.01, and ***P < 0.001). (F) Critical lifting pressure to lift spheroids (in the range of 200 to 600 μm in diameter) (n = 5). The experimental data spread under the theoretical curve, which was determined using the experimental data for 4T1 spheroids with parameters (θd = 64° and s1,2 = 57.4 mN/m). Spheroids made of other cell types had lower theoretical critical lifting pressure values (data are not shown in the paper). For instance, the theoretical critical lifting pressure for HUVEC spheroids was 20% smaller than that of 4T1 spheroids. (G) Viscoelastic behavior of spheroids under aspiration (n = 3). Here, h denotes the advancement of spheroids inside a glass pipette. The aspiration experiment used a similar pipette as that in bioprinting. The aspiration pressure was determined according to the size of spheroids satisfying the condition that spheroids could be lifted from the cell media.

  • Fig. 3 Patterning and bioprinting of a wide range of biologics.

    (A1) A schematic showing critical parameters during bioprinting. (A2) An image from the traveling camera showing spheroid placement onto a gel substrate. Fluorescent images showing (A3) PSU and (A4) matrix patterns. GFP+ MSC spheroids were patterned onto (A5) COL I and (A6) gelatin-methacryloyl (GelMA). (A7 and A8) Images showing that eight fixed spheroids were bioprinted on top of each other in air without any gel support during bioprinting. DAPI, 4′,6-diamidino-2-phenylindole. (B1) Time-lapse images of self-assembly process after bioprinting of 1-day cultured 3T3 spheroids at 0, 24, 48, and 72 hours (B2) and the normalized contact length and intersphere angle of fusing spheroids up to 24 hours. (B3) Cell viability of 3T3 spheroids that were not treated with bioprinting (control), after bioprinting inside the cell media (case 1), and after bioprinting into a gel substrate (case 2) (n = 3; *P < 0.05). CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine; CMFDA, 5-chloromethylfluorescein diacetate. (C1) Hematoxylin and eosin staining from the sagittal plane of a tail segment of electric fish showing stacked electrocytes in series. (C2) The SEM image of a single electrocyte. (C3) Calcein staining of bioprinted electrocytes. (D) A bioprinted cartilage tissue strand between pins. Photo credit: Bugra Ayan, Penn State University.

  • Fig. 4 3D bioprinting of spheroids.

    (A) A schematic illustration of 3D bioprinting of a heterogeneous pyramid construct using different sizes and types of spheroids. (B) A photograph of the bioprinted three-layer heterogeneous pyramid. (C1 to C6) 3D reconstruction of confocal images of bioprinted pyramid of tdTomato+ HUVEC spheroids (first and third layers) and GFP+ MSC spheroids (second layer). (D) A schematic illustration of a diamond construct made of MSC/HUVEC spheroids. (E) A photograph showing 3D bioprinted diamond from the side camera. (F) Fluorescent images of the bioprinted diamond, which were stained with DAPI, CD31, and F-actin. (G1 to G4) Confocal images of the diamond construct (bottom view) (note that 1-day cultured HUVEC and 2-day cultured MSCs were used in these experiments). Photo credit: Bugra Ayan, Penn State University.

  • Fig. 5 Bioprinting of physiologically relevant culture environments to study the angiogenic sprouting behavior of HUVEC spheroids.

    (A1) Epifluorescent images of bioprinted tdTomato+ HUVEC spheroids with varying distances (400 to 3000 μm) apart on day 7. (A2) Graphical representation of various sprouting properties—namely, total vessels length, total number of junctions formed, vessel area, and mean lacunarity—obtained at day 7 for bioprinted HUVEC spheroids (n = 3; ***P < 0.001). (B1) Epifluorescent images of bioprinted GFP+ and tdTomato+ HUVEC spheroids with varying distances (400 to 3000 μm) apart on day 7 along with higher-magnification confocal images of the interface region in XY and YZ planes showing capillaries formed by both GFP+ and tdTomato+ HUVECs (indicated by white arrows). (B2) Directionality analysis demonstrating the direction and percentage of normalized number of sprouts on day 7 (n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001) [note that green and red bars demonstrate the angles of interest (AOIs), which are [60°, −60°] for GFP+ HUVECs and [120°, 240°] for tdTomato+ HUVECs]. (B3) Confocal images at the interface of two spheroids showing capillaries formed by both GFP+ and tdTomato+ HUVECs. (C1) A schematic illustration of the directionality of sprouts from a HUVEC spheroid toward a spheroid of GFP+ HDF and MSC cocultures. (C2) The directionality analysis for different mixing ratios of HDF:MSC, including HDF (control), 2:1, 1:1, and MSC (control), on day 7 (n = 3; ***P < 0.001 shows significance between AOI and “Other” for each group, and #P < 0.05, ##P < 0.01, and ###P < 0.001 show significance among AOIs of different groups) [note that N/A represents the none applicability of the directionality analysis, as no sprouts were observed in the MSC-only group; no directionality analysis was performed for the 3000-μm distance, as sprouting was not observed and HUVECs exhibited spreading only; 1-day cultured HUVECs were used in all experiments; and the critical lifting pressure for coculture HDF/MSC spheroids (2:1 and 1:1 ratio) was determined to be 28.7 and 29.1 mmHg, respectively, through interpolation of the critical lifting pressure values for HDF and MSC spheroids presented in Fig. 2F].

  • Fig. 6 Biofabrication of osteogenic tissues.

    Strategy no. 1: (A) Triangle-shaped tissue complexes were bioprinted using MSC/HUVEC spheroids and cultured for 3 days in GM and 12 days in OM. (B) Time-lapse images showing fusion of GFP+ spheroids up to day 15 (D15) after bioprinting. (C) An optical image showing the assembled tissue at day 15 after bioprinting. (D) Immunofluorescence staining (DAPI, CD31, F-actin, RUNX2, and DAPI + RUNX2) and (E) Alizarin red staining of the sectioned tissue. Strategy no. 2: (F) The final shape of the bioprinted tissue of osteogenic spheroids (cultured for 10 days in OM before bioprinting and 2 days in OM after bioprinting). Immunofluorescent images of (G) the bioprinted tissue and (H) confocal images of its histological sections stained for DAPI, CD31, and F-actin and (I) RUNX2 and DAPI + RUNX2. (J) Alizarin red staining of the tissue section. (K) Quantification of normalized RUNX2 intensity at different regions including the surface of assembled tissue, spheroid-spheroid interface, and core of spheroids (n = 50; **P < 0.01 and ***P < 0.001). (L) A representative heat map figure showing RUNX2/DAPI distribution in the surface of assembled tissue, spheroid-spheroid interface, and core of spheroids for strategy nos. 1 and 2. (M) BSP, COL1, ALP, RUNX2, and CDH2 gene expressions of 2D MSCs cultured in OM (control), 3D bioprinted tissues cultured in GM (control), and 3D bioprinted tissues cultured using strategy nos. 1 and 2 (n = 5; **P < 0.01 and ***P < 0.001).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaw5111/DC1

    Supplementary Materials and Methods

    Fig. S1. Aspiration-assisted bioprinter.

    Fig. S2. Simulation of aspiration.

    Fig. S3. Dimensional and surface tension measurements.

    Fig. S4. Compactness of different spheroid types over time.

    Fig. S5. Calibration of the pneumatic system and positional precision and accuracy for AAB.

    Fig. S6. Bioprinting of tissue spheroids in 2D and 3D.

    Fig. S7. Bioprinting of a representative 3D hollow structure.

    Fig. S8. Printing parameters for microvalve-based bioprinting.

    Fig. S9. A heterogeneous pyramid construct.

    Fig. S10. Effect of distance on angiogenic sprouting.

    Fig. S11. Effect of distance on the directionality of angiogenic sprouting.

    Fig. S12. Directionality analysis of angiogenic sprouting over time.

    Fig. S13. Fluorescent and fluorescent/phase images showing angiogenic sprouts from a tdTomato+ HUVEC spheroid toward a spheroid of GFP+ HDF and MSC coculture in different mixing ratios—including HDF (control), 2:1, 1:1, and MSC (control)—on day 7.

    Fig. S14. Fluorescent and fluorescent/phase images showing angiogenic sprouts from a tdTomato+ HUVEC spheroid toward a spheroid of GFP+ HDF and MSC coculture in different mixing ratios—including HDF (control), 2:1, 1:1, and MSC (control)—on day 7.

    Fig. S15. Fluorescent and fluorescent/phase images showing angiogenic sprouts from a tdTomato+ HUVEC spheroid toward a spheroid of GFP+ HDF and MSC coculture in different mixing ratios—including HDF (control), 2:1, 1:1, and MSC (control)—on day 7.

    Fig. S16. Directionality analysis of angiogenic sprouts toward GFP+ HDF and MSC coculture spheroids on day 2.

    Fig. S17. Directionality analysis of angiogenic sprouts toward GFP+ HDF and MSC coculture spheroids on day 5.

    Fig. S18. Culture strategies: A schematic of osteogenic culture strategies including strategy nos. 1 and 2.

    Fig. S19. Osteogenic tissue biofabrication using strategy nos. 1 and 2.

    Fig. S20. Computer interface of AAB.

    Table S1. Dynamic contact angles of spheroids at a lifting speed of 5 mm/s.

    Table S2. Determination of Fup and Fdown for bioprinting of various spheroids in 1% (w/v) alginate.

    Table S3. Surface tension coefficient of different types of cell media used in our experiments.

    Movie S1. Real-time video of spheroid lifting and bioprinting process.

    Movie S2. Simulation of spheroid aspiration.

    Movie S3. Bioprinting of a tower and a bridge using MSC spheroids in air.

    Movie S4. Bioprinting of a tissue strand.

    Movie S5. 3D printing of fibrinogen and thrombin via microvalves.

    Movie S6. 3D reconstruction of the heterogeneous pyramid construct constituted of tdTomato-labeled HUVEC spheroids on the first and third layers and GFP-labeled MSC spheroids on the second layer.

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Aspiration-assisted bioprinter.
    • Fig. S2. Simulation of aspiration.
    • Fig. S3. Dimensional and surface tension measurements.
    • Fig. S4. Compactness of different spheroid types over time.
    • Fig. S5. Calibration of the pneumatic system and positional precision and accuracy for AAB.
    • Fig. S6. Bioprinting of tissue spheroids in 2D and 3D.
    • Fig. S7. Bioprinting of a representative 3D hollow structure.
    • Fig. S8. Printing parameters for microvalve-based bioprinting.
    • Fig. S9. A heterogeneous pyramid construct.
    • Fig. S10. Effect of distance on angiogenic sprouting.
    • Fig. S11. Effect of distance on the directionality of angiogenic sprouting.
    • Fig. S12. Directionality analysis of angiogenic sprouting over time.
    • Fig. S13. Fluorescent and fluorescent/phase images showing angiogenic sprouts from a tdTomato+ HUVEC spheroid toward a spheroid of GFP+ HDF and MSC coculture in different mixing ratios—including HDF (control), 2:1, 1:1, and MSC (control)—on day 7.
    • Fig. S14. Fluorescent and fluorescent/phase images showing angiogenic sprouts from a tdTomato+ HUVEC spheroid toward a spheroid of GFP+ HDF and MSC coculture in different mixing ratios—including HDF (control), 2:1, 1:1, and MSC (control)—on day 7.
    • Fig. S15. Fluorescent and fluorescent/phase images showing angiogenic sprouts from a tdTomato+ HUVEC spheroid toward a spheroid of GFP+ HDF and MSC coculture in different mixing ratios—including HDF (control), 2:1, 1:1, and MSC (control)—on day 7.
    • Fig. S16. Directionality analysis of angiogenic sprouts toward GFP+ HDF and MSC coculture spheroids on day 2.
    • Fig. S17. Directionality analysis of angiogenic sprouts toward GFP+ HDF and MSC coculture spheroids on day 5.
    • Fig. S18. Culture strategies: A schematic of osteogenic culture strategies including strategy nos. 1 and 2.
    • Fig. S19. Osteogenic tissue biofabrication using strategy nos. 1 and 2.
    • Fig. S20. Computer interface of AAB.
    • Table S1. Dynamic contact angles of spheroids at a lifting speed of 5 mm/s.
    • Table S2. Determination of Fup and Fdown for bioprinting of various spheroids in 1% (w/v) alginate.
    • Table S3. Surface tension coefficient of different types of cell media used in our experiments.
    • Legends for movies S1 to S6

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Real-time video of spheroid lifting and bioprinting process.
    • Movie S2 (.mp4 format). Simulation of spheroid aspiration.
    • Movie S3 (.mp4 format). Bioprinting of a tower and a bridge using MSC spheroids in air.
    • Movie S4 (.mp4 format). Bioprinting of a tissue strand.
    • Movie S5 (.mov format). 3D printing of fibrinogen and thrombin via microvalves.
    • Movie S6 (.mov format). 3D reconstruction of the heterogeneous pyramid construct constituted of tdTomato-labeled HUVEC spheroids on the first and third layers and GFP-labeled MSC spheroids on the second layer.

    Files in this Data Supplement:

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