3D Bio-printed Inner Ear Organ Of Corti Organoids Induce Hair Cell Regeneration

Sensorineural hearing loss(SNHL)is caused by damage and irreversible degeneration of inner ear hair cells or spiral neuron ganglia,and may be caused by a variety of factors,such as ototoxic drugs,noise,aging,etc.Cochlear hair cells,as highly differentiated cells,are able to transmit sound signals to the brain centers by converting them into electrical signals.It is difficult to regenerate the hair cells in mammals after they are damaged,so hearing aid or electronic cochlear implant to help people with hearing disability to improve their hearing is far from achieving the effect of restoring natural hearing.Therefore,how to realize the regeneration of cochlear hair cells after damage is the ideal way to solve the sensorineural hearing impairment and realize the hearing function reconstruction.Inner ear stem cells can be isolated from the organ of Corti as well as from the vestibular sensory epithelium.A characteristic of these pluripotent stem cells is their ability to form spheres.These spheroids can pass on and give rise to functionally characterized hair cell-like cells that closely resemble neonatal hair cells.Currently,most of the research on hair cell regeneration and related studies are limited to two-dimensional culture systems,and there is no in-depth study on how to simulate the inner ear hair cell growth microenvironment at the three-dimensional level to achieve hair cell regeneration.In this study,we aim to construct organ of Corti organoids that are closer to the hair cell growth microenvironment to promote inner ear stem cell differentiation for hair cell regeneration.3D bioprinting technology can print bioink containing cells,biomaterials and growth factors,and the constructed organ of Corti organoids can better mimic the Corti microenvironment and induce hair cell regeneration.In this project,we constructed a organ of Corti organoids with multiple mimics in mechanical properties,spatial structure and composition by 3D bioprinting technology,and added induction factors to regulate it,so as to achieve a comprehensive simulation of the microenvironment of cell growth in the inner ear Corti organ,and investigate the proliferation and differentiation of inner ear stem cells,so as to better study the regeneration of inner ear hair cells.First,preparation of Gelma/SA 3D scaffolds and their performance characterization.The 3D bionic scaffolds with different concentration ratios of methacrylate-based gelatin/sodium alginate(Gelma/SA)were prepared by 3D printing technology,and the concentration ratios with high printability and good printing performance were selected.Through experiments and literature references,Gelma/SA was selected as 7%(W/V)/1%(W/V),8%(W/V)/2%(W/V)Two ratios of Gelma/SA were selected,and the two printing scaffolds were characterized in various aspects,and it was found that the Gelma/SA concentration of 7%/1%had the best characterization performance and was more suitable for cell growth,which could be used as the ideal concentration ratio of bioink.Second,the isolation,culture and identification of inner ear stem cells.First,ICR mice were dissected at just three days old to obtain their cochlea,then organ of Corti was isolated from them,and inner ear stem cells were obtained after digestion and culture,and the inner ear stem cells were subjected to primary culture and proliferation ability assay.In order to obtain a large number of inner ear stem cells for 3D bioprinting to construct organ of Corti organoids,the inner ear stem cells were passaged and cultured to obtain P2 and P3 generation inner ear stem cells.The inner ear stem cells were subsequently identified using immunofluorescence staining,and the expression of specific marker proteins of auditory cells(Myosin7a,Tuj1,P27kip1)in inner ear stem cells was analyzed,and it was found that the walled inner ear stem cells still maintained the ability to differentiate into various auditory cells.Third,the organ of Corti organoids induced hair cell regeneration.Firstly,we evaluated the growth adhesion of inner ear stem cells on Gelma/SA 3D bionic scaffold and the differentiation ability to neural cells,and found that the constructed 3D bionic scaffold could better promote the growth and differentiation of inner ear stem cells.The effect of organ of Corti organoids constructed by 3D bioprinting on the differentiation of inner ear stem cells to hair cells was then investigated.Cells and materials were mixed to make a bioink,bioprinted to obtain cell-containing organ of Corti organoids,and cultured for 7 days with a culture medium containing a concentration of 5 μM DAPT(an inhibitor of the Notch pathway)and 1 μM retinoic acid(RA),followed by sectioning and immunofluorescence staining to observe the expression of hair cell-specific marker proteins.The differentiation rate of inner ear stem cells to hair cells was significantly higher in organ of Corti organoids compared to controls.Finally,we compared the expression of Notch signaling pathway-related genes in organ of Corti organoids by RT-PCR and analyzed the role of Notch signaling pathway in hair cell regeneration.In summary,the organ of Corti organoids was constructed by 3D bioprinting technology to study inner ear stem cell differentiation and hair cell regeneration.The experimental results showed that the biomimetic organ of Corti organoids could promote the growth and differentiation of inner ear stem cells.Meanwhile,the organ of Corti organoids can promote the regeneration of hair cells under the induced culture conditions.In conclusion,this project provides a preliminary exploration of the mechanism of action of hair cell regeneration in organ of Corti organoids,which may contribute an idea for future research on hearing disorders and may serve as a possible solution for the treatment of sensorineural hearing loss.