Fig. 4: Implantation of printed single-layer cortical tissue into brain explants.

a Steps in the implantation process. Step i, bioink prepared from DNPs or UNPs with Matrigel. Step ii, droplet-printing of cortical tissue. Step iii, post-printing culture of the printed tissue for 1 day. Step iv, preparation of mouse brain slices with a thickness of 300 µm. Step v, brain slices (explants) were kept under condition A or condition B before implantation. Step vi, creating the brain lesion with a biopsy punch. Step vii, implanting printed tissue into the lesion of the explant, which was then further incubated. b Incubation of the implanted explant and drug treatment. Explants were incubated under either condition A or condition B for 1 day, followed by treatment with or without DAPT for 4 days. c Left, tiled fluorescence confocal image of an implanted mouse brain explant at 3 days post implantation (DPI) under condition B. The implant contained RFP-labelled DNs. Right, bright field (top), DAPI staining (upper middle), RFP-labelled implanted tissue (lower middle) and higher magnification view (bottom). The culture media are described in the “Methods” section. d Confocal image of a representative 1 DPI implanted explant (left) and confocal fluorescence images of process outgrowth and neuron migration from an explant with RFP-labelled DNPs at 1 (top right) and 5 (bottom right) DPI. e Profile plots of fluorescence intensity along the white dashed boxes from left to right indicated in d. The vertical dashed lines indicate the margins of the red fluorescence. f Confocal images of implant-to-host process outgrowth and neuron migration at 5 DPI. The implanted explants with RFP-labelled DNPs were cultured under the two conditions and with or without DAPT treatment. Dashed lines indicate the original borders of the implants. g Quantitative analysis of process outgrowth and neuron migration distance visualised in f. (n = 4,4,5,4 biological replicates from left to right; mean ± SEM; two-sided unpaired Student’s t tests). h A tiled confocal image of an explant implanted with RFP-labelled deep-layer cortical tissue that had been cultured for 14 days prior to implantation. The implant is immunostained with the human-specific neural marker, HNCAM. RFP localisation indicates process outgrowth and neuron migration from the implanted tissue. i Quantitative analysis of process outgrowth and neuron migration from deep-layer cortical tissue cultured for 1 day or 14 days prior to implantation (n = 5, 6 biological replicates from left to right; mean ± SEM; two-sided unpaired Student’s t test). j Representative confocal images of an explant with RFP-labelled DNPs at 3 and 5 DPI (top), and explants with RFP-labelled UNPs at 3 and 5 DPI (bottom), revealing the migration of RFP-labelled neurons from implanted cortical tissues into the brain explants. Arrowhead indicates a migrating human neuron. Scale bar, 50 µm. k Quantitative analysis of neuron migration from implanted cortical tissues into host brain explants (n = 3,3,5,3,4,6,4 biological replicates from left to right; mean ± SEM; one-way ANOVA). Field size, 0.1 mm². For g, i and k: ns not significant; *P < 0.05; **P < 0.01. For c, d, f and h: scale bars, 500 µm.