{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T14:50:59Z","timestamp":1790952659206,"version":"4.1.0"},"reference-count":53,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,3,11]],"date-time":"2023-03-11T00:00:00Z","timestamp":1678492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>Porcine decellularized conjunctiva (PDC) represents a promising alternative source for conjunctival reconstruction. Methods of its re-epithelialization in vitro with primary human conjunctival epithelial cells (HCEC) have already been established. However, a long-term storage method is required for a simplified clinical use of PDC. This study investigates the influence of several storage variants on PDC. PDC were stored in (1) phosphate-buffered saline solution (PBS) at 4 \u00b0C, (2) in glycerol-containing epithelial cell medium (EM\/gly) at \u221280 \u00b0C and (3) in dimethyl sulfoxide-containing epithelial cell medium (EM\/DMSO) at \u2212196 \u00b0C in liquid nitrogen for two and six months, respectively. Fresh PDC served as control. Histological structure, biomechanical parameters, the content of collagen and elastin and the potential of re-epithelialization with primary HCEC under cultivation for 14 days were compared (n = 4\u201310). In all groups, PDC showed a well-preserved extracellular matrix without structural disruptions and with comparable fiber density (p \u2265 0.74). Collagen and elastin content were not significantly different between the groups (p \u2265 0.18; p \u2265 0.13, respectively). With the exception of the significantly reduced tensile strength of PDC after storage at \u2212196 \u00b0C in EM\/DMSO for six months (0.46 \u00b1 0.21 MPa, p = 0.02), no differences were seen regarding the elastic modulus, tensile strength and extensibility compared to control (0.87 \u00b1 0.25 MPa; p \u2265 0.06). The mean values of the epithelialized PDC surface ranged from 51.9 \u00b1 8.8% (\u2212196 \u00b0C) to 78.3 \u00b1 4.4% (\u221280 \u00b0C) and did not differ significantly (p \u2265 0.35). In conclusion, all examined storage methods were suitable for storing PDC for at least six months. All PDC were able to re-epithelialize, which rules out cytotoxic influences of the storage conditions and suggests preserved biocompatibility for in vivo application.<\/jats:p>","DOI":"10.3390\/bioengineering10030350","type":"journal-article","created":{"date-parts":[[2023,3,13]],"date-time":"2023-03-13T03:28:33Z","timestamp":1678678113000},"page":"350","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Influence of Storage Conditions on Decellularized Porcine Conjunctiva"],"prefix":"10.3390","volume":"10","author":[{"given":"Adam","family":"Skornia","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, University Hospital Duesseldorf, Heinrich Heine University, 40225 Duesseldorf, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2034-3687","authenticated-orcid":false,"given":"Gerd","family":"Geerling","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University Hospital Duesseldorf, Heinrich Heine University, 40225 Duesseldorf, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristina","family":"Spaniol","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University Hospital Duesseldorf, Heinrich Heine University, 40225 Duesseldorf, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7243-7493","authenticated-orcid":false,"given":"Joana","family":"Witt","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University Hospital Duesseldorf, Heinrich Heine University, 40225 Duesseldorf, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1016\/S0161-6420(85)33967-2","article-title":"Staging of conjunctival squamous metaplasia by impression cytology","volume":"92","author":"Tseng","year":"1985","journal-title":"Ophthalmology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"913","DOI":"10.3109\/02713680903198045","article-title":"Tissue engineering for conjunctival reconstruction: Established methods and future outlooks","volume":"34","author":"Schrader","year":"2009","journal-title":"Curr. Eye Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1080\/02713683.2020.1712423","article-title":"Eye Socket Regeneration and Reconstruction","volume":"45","author":"Borrelli","year":"2020","journal-title":"Curr. Eye Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1007\/s00347-016-0419-1","article-title":"New approaches to ocular surface reconstruction beyond the cornea","volume":"114","author":"Spaniol","year":"2017","journal-title":"Ophthalmologe"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Reinhard, T., and Larkin, D. (2006). Cornea and External Eye Disease, Springer.","DOI":"10.1007\/3-540-31226-9"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1084","DOI":"10.1089\/ten.tea.2018.0190","article-title":"Evaluation of Plastic-Compressed Collagen for Conjunctival Repair in a Rabbit Model","volume":"25","author":"Witt","year":"2019","journal-title":"Tissue Eng. Part A"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"109356","DOI":"10.1016\/j.exer.2022.109356","article-title":"Keratin films for ocular surface reconstruction: Wound healing in an in-vivo model","volume":"227","author":"Borrelli","year":"2022","journal-title":"Exp. Eye Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.jtos.2021.06.003","article-title":"Biological tissues and components, and synthetic substrates for conjunctival cell transplantation","volume":"22","author":"Makuloluwa","year":"2021","journal-title":"Ocul. Surf."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1021\/acsbiomaterials.6b00506","article-title":"Overview of the Development, Applications, and Future Perspectives of Decellularized Tissues and Organs","volume":"3","author":"Nakamura","year":"2017","journal-title":"ACS Biomater. Sci. Eng."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e757","DOI":"10.1111\/aos.13649","article-title":"Plastic compressed collagen transplantation\u2014A new option for corneal surface reconstruction?","volume":"96","author":"Schrader","year":"2018","journal-title":"Acta Ophthalmol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.actbio.2019.11.006","article-title":"Ocular surface repair using decellularized porcine conjunctiva","volume":"101","author":"Zhao","year":"2020","journal-title":"Acta Biomater."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1016\/j.ajo.2013.03.017","article-title":"Correction of lower lid retraction using tarSys bioengineered grafts for graves ophthalmopathy","volume":"156","author":"Liao","year":"2013","journal-title":"Am. J. Ophthalmol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Makuloluwa, A.K., Hamill, K.J., Rauz, S., Bosworth, L., Haneef, A., Romano, V., Williams, R.L., Dartt, D.A., and Kaye, S.B. (2021). The conjunctival extracellular matrix, related disorders and development of substrates for conjunctival restoration. Ocul. Surf., in press.","DOI":"10.1016\/j.jtos.2021.05.011"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Bosworth, L.A., Doherty, K.G., Hsuan, J.D., Cray, S.P., D\u2019Sa, R.A., Pineda Molina, C., Badylak, S.F., and Williams, R.L. (2021). Material Characterisation and Stratification of Conjunctival Epithelial Cells on Electrospun Poly(\u03b5-Caprolactone) Fibres Loaded with Decellularised Tissue Matrices. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13030318"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e973","DOI":"10.1002\/term.2419","article-title":"Development of decellularized conjunctiva as a substrate for the ex vivo expansion of conjunctival epithelium","volume":"12","author":"Kasbekar","year":"2018","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1038\/nrm3112","article-title":"Balancing forces: Architectural control of mechanotransduction","volume":"12","author":"DuFort","year":"2011","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2132","DOI":"10.1007\/s10439-019-02408-9","article-title":"Extracellular Matrix-Based Biomaterials and Their Influence Upon Cell Behavior","volume":"48","author":"Cramer","year":"2020","journal-title":"Ann. Biomed. Eng."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/s41578-018-0023-x","article-title":"Extracellular matrix-based materials for regenerative medicine","volume":"3","author":"Hussey","year":"2018","journal-title":"Nat. Rev. Mater."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3233","DOI":"10.1016\/j.biomaterials.2011.01.057","article-title":"An overview of tissue and whole organ decellularization processes","volume":"32","author":"Crapo","year":"2011","journal-title":"Biomaterials"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.bioactmat.2021.09.014","article-title":"Decellularized extracellular matrix scaffolds: Recent trends and emerging strategies in tissue engineering","volume":"10","author":"Zhang","year":"2022","journal-title":"Bioact. Mater."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.actbio.2018.04.044","article-title":"Decellularized matrices in regenerative medicine","volume":"74","author":"Taylor","year":"2018","journal-title":"Acta Biomater."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.actbio.2017.11.054","article-title":"Decellularised conjunctiva for ocular surface reconstruction","volume":"67","author":"Witt","year":"2018","journal-title":"Acta Biomater."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1016\/j.jtos.2020.08.009","article-title":"Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva","volume":"18","author":"Witt","year":"2020","journal-title":"Ocul. Surf."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1111\/aor.14126","article-title":"Recent advancement of decellularization extracellular matrix for tissue engineering and biomedical application","volume":"46","author":"Yang","year":"2022","journal-title":"Artif. Organs"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"119","DOI":"10.4161\/org.5.3.9583","article-title":"The cryopreservation of composite tissues: Principles and recent advancement on cryopreservation of different type of tissues","volume":"5","author":"Bakhach","year":"2009","journal-title":"Organogenesis"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Urbani, L., Maghsoudlou, P., Milan, A., Menikou, M., Hagen, C.K., Totonelli, G., Camilli, C., Eaton, S., Burns, A., and Olivo, A. (2017). Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0179341"},{"key":"ref_27","first-page":"321","article-title":"Comparison of two methods for prolong storage of decellularized mouse whole testis for tissue engineering application: An experimental study","volume":"19","author":"Gharenaz","year":"2021","journal-title":"Int. J. Reprod. Biomed."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.actbio.2018.10.026","article-title":"Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability","volume":"84","author":"Zouhair","year":"2019","journal-title":"Acta Biomater."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1016\/S0002-9394(14)70125-4","article-title":"Amniotic membrane transplantation for persistent epithelial defects with ulceration","volume":"123","author":"Lee","year":"1997","journal-title":"Am. J. Ophthalmol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1097\/00003226-199509000-00006","article-title":"Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas","volume":"14","author":"Kim","year":"1995","journal-title":"Cornea"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3662","DOI":"10.1016\/j.biomaterials.2012.01.064","article-title":"Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues","volume":"33","author":"Baiguera","year":"2012","journal-title":"Biomaterials"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3231","DOI":"10.1016\/j.biomaterials.2013.01.031","article-title":"The effects of storage and sterilization on de-cellularized and re-cellularized whole lung","volume":"34","author":"Bonenfant","year":"2013","journal-title":"Biomaterials"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1016\/j.jtcvs.2008.09.085","article-title":"Structural and morphologic evaluation of a novel detergent-enzymatic tissue-engineered tracheal tubular matrix","volume":"138","author":"Jungebluth","year":"2009","journal-title":"J. Thorac. Cardiovasc. Surg."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1028","DOI":"10.1007\/s00167-010-1278-y","article-title":"Effect of repeated freezing-thawing on the Achilles tendon of rabbits","volume":"19","author":"Chen","year":"2011","journal-title":"Knee Surg. Sports Traumatol. Arthrosc."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s00264-006-0297-2","article-title":"Effects of freezing on the biomechanical and structural properties of human posterior tibial tendons","volume":"32","author":"Giannini","year":"2008","journal-title":"Int. Orthop."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Chani, B., Puri, V., Sobti, R.C., Jha, V., and Puri, S. (2017). Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0173040"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1089\/bio.2019.0106","article-title":"Histological and Biomechanical Characteristics of Human Decellularized Allograft Heart Valves After Eighteen Months of Storage in Saline Solution","volume":"18","author":"Wollmann","year":"2020","journal-title":"Biopreserv. Biobank."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1080\/15476278.2015.1022009","article-title":"Freezing\/Thawing without Cryoprotectant Damages Native but not Decellularized Porcine Renal Tissue","volume":"11","author":"Poornejad","year":"2015","journal-title":"Organogenesis"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1007\/s00417-018-3973-1","article-title":"Cryopreservation of amniotic membrane with and without glycerol additive","volume":"256","author":"Wagner","year":"2018","journal-title":"Graefes Arch. Clin. Exp. Ophthalmol."},{"key":"ref_41","first-page":"523","article-title":"Rabbit conjunctival and corneal epithelial cells belong to two separate lineages","volume":"37","author":"Wei","year":"1996","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1038\/sj.eye.6700575","article-title":"Epithelial stem cells: The eye provides a vision","volume":"17","author":"Lavker","year":"2003","journal-title":"Eye"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1016\/j.ajo.2004.10.056","article-title":"Autologous cultivated conjunctival transplantation for pterygium surgery","volume":"139","author":"Ang","year":"2005","journal-title":"Am. J. Ophthalmol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1002\/term.1991","article-title":"Development of a conjunctival tissue substitute on the basis of plastic compressed collagen","volume":"11","author":"Drechsler","year":"2017","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"9798374","DOI":"10.1155\/2016\/9798374","article-title":"Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies","volume":"2016","author":"Haagdorens","year":"2016","journal-title":"Stem Cells Int."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.preteyeres.2016.04.005","article-title":"Goblet cells of the conjunctiva: A review of recent findings","volume":"54","author":"Gipson","year":"2016","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1007\/s13770-020-00243-x","article-title":"Vitrified Human Umbilical Arteries as Potential Grafts for Vascular Tissue Engineering","volume":"17","author":"Mallis","year":"2020","journal-title":"Tissue Eng. Regen Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1016\/j.dental.2009.03.007","article-title":"Freeze-dried acellular dermal matrix graft: Effects of rehydration on physical, chemical, and mechanical properties","volume":"25","author":"Bottino","year":"2009","journal-title":"Dent. Mater."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1002\/jbm.b.33861","article-title":"Evaluation of freeze-drying and cryopreservation protocols for long-term storage of biomaterials based on decellularized intestine","volume":"106","author":"Carriel","year":"2018","journal-title":"J. Biomed. Mater. Res. Part B Appl. Biomater."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1493","DOI":"10.1590\/S1516-89132009000600021","article-title":"The influence of freezing rates on bovine pericardium tissue Freeze-drying","volume":"52","author":"Borgognoni","year":"2009","journal-title":"Braz. Arch. Biol. Technol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.cryobiol.2011.05.001","article-title":"Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: A comparative study","volume":"63","author":"Polak","year":"2011","journal-title":"Cryobiology"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1089\/ten.tec.2014.0681","article-title":"Sucrose Diffusion in Decellularized Heart Valves for Freeze-Drying","volume":"21","author":"Wang","year":"2015","journal-title":"Tissue Eng. Part C Methods"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1007\/s10561-022-10002-y","article-title":"Cryopreservation of human amniotic membrane for ocular surface reconstruction: A comparison between protocols","volume":"23","author":"Lamon","year":"2022","journal-title":"Cell Tissue Bank."}],"container-title":["Bioengineering"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-5354\/10\/3\/350\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:52:57Z","timestamp":1760122377000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-5354\/10\/3\/350"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,11]]},"references-count":53,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["bioengineering10030350"],"URL":"https:\/\/doi.org\/10.3390\/bioengineering10030350","relation":{},"ISSN":["2306-5354"],"issn-type":[{"value":"2306-5354","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,11]]}}}