| Autor: |
Marija Zekušić, Marina Bujić Mihica, Marija Skoko, Kruno Vukušić, Patrik Risteski, Jelena Martinčić, Iva M. Tolić, Krešo Bendelja, Snježana Ramić, Tamara Dolenec, Ivana Vrgoč Zimić, Dominik Puljić, Ivanka Petric Vicković, Renata Iveković, Ivanka Batarilo, Uršula Prosenc Zmrzljak, Alan Hoffmeister, Tiha Vučemilo |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
Stem Cell Research & Therapy, Vol 14, Iss 1, Pp 1-26 (2023) |
| Druh dokumentu: |
article |
| ISSN: |
1757-6512 |
| DOI: |
10.1186/s13287-023-03586-z |
| Popis: |
Abstract Background Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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