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Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. In this study, we explore the feasibility of bioprinting the corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Corneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before the bioprinting process, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded rat and porcine corneas as well as on denuded human Descemet membrane. Subsequently, the bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells were verified with live\/dead stainings. Histological and immunofluorescence stainings involving ZO1, Na\n                      <jats:sup>+<\/jats:sup>\n                      \/K\n                      <jats:sup>+<\/jats:sup>\n                      -ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments. Additionally, STEM121 marker was used to identify human cells from the ex vivo rat and porcine corneas.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The bioink, modified for human pluripotent stem cell derived corneal endothelial cells successfully supported both the viability and printability of the cells. Following up to 10\u00a0days of ex vivo transplantations, STEM121 positive cells were confirmed on the Descemet membrane of rat and porcine cornea demonstrating the biocompatibility of the bioink. Furthermore, biocompatibility was validated on denuded human Descemet membrane showing corneal endothelial -like characteristics. Seven days post bioprinting, the corneal endothelial -like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na\n                      <jats:sup>+<\/jats:sup>\n                      \/K\n                      <jats:sup>+<\/jats:sup>\n                      -ATPase and CD166. However, mesenchymal-like cells were observed in certain areas of the cultures, spreading beneath the corneal endothelial-like cell layer.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Our results demonstrate the successful printing of human pluripotent stem cell derived corneal endothelial cells using covalently crosslinked hyaluronic acid bioink. This approach not only holds promise for a corneal endothelium transplants but also presents potential applications in the broader mission of bioprinting the full-thickness human cornea.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13287-024-03672-w","type":"journal-article","created":{"date-parts":[[2024,3,14]],"date-time":"2024-03-14T10:02:32Z","timestamp":1710410552000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":31,"title":["Bioprinting of human pluripotent stem cell derived corneal endothelial cells with hydrazone crosslinked hyaluronic acid bioink"],"prefix":"10.1186","volume":"15","author":[{"given":"Pyry","family":"Gr\u00f6nroos","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anni","family":"M\u00f6r\u00f6","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paula","family":"Puistola","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karoliina","family":"Hopia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maija","family":"Huuskonen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taina","family":"Viheri\u00e4l\u00e4","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanja","family":"Ilmarinen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4127-8792","authenticated-orcid":false,"given":"Heli","family":"Skottman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,3,14]]},"reference":[{"issue":"2","key":"3672_CR1","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1001\/jamaophthalmol.2015.4776","volume":"134","author":"P Gain","year":"2016","unstructured":"Gain P, et al. 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The research group also had supportive statements from the Regional Ethics Committee of the Expert Responsibility area of Tampere University Hospital to derive, culture and differentiate hESC lines (R05116), to establish and use hiPSC lines in ophthalmic research (R16116), and to use human donor corneas unsuitable for transplantation for research purposes (R11134). No new hESC lines were derived for the research conducted here. The rat corneas used in this study were acquired from medical teaching lab and they were already sacrificed for medical teaching purposes performed in Tampere University, Faculty of Medicine and Health Technology under appropriate licenses from the National Project Authorization Board. Euthanasia was performed separate from animal housing in dedicated rooms with automated CO<sup>2<\/sup> chambers designed for rats. The porcine corneas used in this study were acquired from the local abattoir as a side product and were thus not the sole reason for animal sacrifice.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"PG, TI and HS are co-inventors of a pending patent transferred from Tampere University (Tampere, Finland) to StemSight Ltd (Tampere, Finland) regarding the cell production technology used herein. AM is an inventor of a patent application related to the bioink described in this study. Based on the Act on the Right in Inventions made at Higher Education Institutions in Finland, all authors employed by Tampere University have given all rights to the University and thus have declared no competing interests. AM, TI and HS are co-founders, shareholders and employees in StemSight Ltd.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"81"}}