Fig 1.
Scheme of the research strategy.
Step 1 shows the cryopreservation process of normal human dermal fibroblast (NHDF) spheroids. Spheroids are prepared in a 96-well plate and frozen. These spheroids can be stored for a long period until clinical application. Step 2 shows the bio-3D printing system. After the spheroids are thawed, they are set up on the bio-3D printer, together with the needle array and 3D design. The spheroids are loaded onto the needle array according to the 3D design. Next, the spheroids are fused for a few days after loading.
Fig 2.
Fabrication of the scaffold-free tubular construct using cryopreserved spheroids.
Bio-3D printing process using cryopreserved spheroids (A). The fabricated tubular construct just after printing (B) was cultured on the needle array for 7 days (C). The cryopreserved tubular construct was set on the plastic catheter after removing the construct from the needle array (D), and maturation occurred for 7 days after culture (E). Scale bar, 1mm in (B, C, D, E).
Fig 3.
Time-lapse photograph of cryopreserved spheroids after thawing.
In the positive control, non-cryopreserved spheroids were fused with each other (A). For the negative control, spheroids were cryopreserved in PBS (B). Cryopreserved spheroids in cryopreservation solution were washed 0, 1, 3, and 5 times after thawing, and fusion activities of the spheroids were analyzed (C). The scale bar is 200 μm.
Fig 4.
Size of cryopreserved spheroids after thawing.
Spheroid sizes before cryopreservation (A), just after thawing (B), and 3 days after thawing (C) were measured with a measuring system on the bio-3D printer. The spheroid diameter comparison among spheroids before cryopreservation, just after thawing and after thawing (D). N = 239 for each group. *P<0.01.
Fig 5.
Roundness of cryopreserved spheroids after thawing.
Spheroid roundness was measured before cryopreservation (A), just after thawing (B), and 3 days after thawing (C). The spheroid roundness comparison among spheroids before cryopreservation, just after thawing and after thawing (D). N = 239 for each group. *P<0.01.
Fig 6.
Histological analysis of spheroids.
Non-cryopreserved spheroids (A), cryopreserved spheroids using PBS solution (B), and cryopreserved spheroids using StemSure® solution (C) were analyzed by hematoxylin and eosin staining. Non-cryopreserved spheroids (D), cryopreserved spheroids using PBS solution (E), and cryopreserved spheroids using StemSure® solution (F) were analyzed by TUNEL staining to detect apoptotic cells in the spheroids. Non-cryopreserved spheroids (G), cryopreserved spheroids using PBS solution (H), and cryopreserved spheroids using StemSure® solution (I) stained for collagen type Ⅰ. The scale bar is 100 μm. The number of dead cells was calculated from TUNEL staining images (K). N = 5 for each group, *P<0.01 The cell proliferation comparison between non-cryopreserved spheroids and cryopreserved spheroids (L). N = 3 for each group, *P<0.01.
Fig 7.
Histological analysis of constructs.
Tubular constructs using non-cryopreserved spheroids (A) or cryopreserved spheroids (B) were analyzed by hematoxylin and eosin staining. These samples were analyzed by TUNEL staining to detect apoptotic cells in the construct (C and D). The scale bar is 100 μm. Cell viabilities in these constructs were measured by counting the number of dead cells (E). N = 5 for each group, **P<0.05.
Fig 8.
Extracellular matrix deposition and tensile strength of constructs.
Constructs using non-cryopreserved spheroids or cryopreserved spheroids were analyzed by Masson’s trichrome staining at 14 days (A or B), 21 days (C or D), and 28 days (E or F). The scale bar is 200 μm. The tensile strength of the constructs was measured and analyzed (G). N = 3 for each group, *P<0.01, **P<0.05.