Naslov
Clinical application of cultured keratinocytes as advanced therapy medicinal products: a twenty-year experience in Croatia

Autori
Zekušić, Marija ; Bujić Mihica, Marina ; Dolenec, Tamara ; Skoko, Marija ; Jularić, Anamarija ; Puljić, Dominik ; Vrgoč Zimić, Ivana ; Ramić, Snježana ; Bendelja, Krešo ; Boranić, Milivoje ; Tomičić, Hrvoje ; Kljenak, Antun ; Batarilo, Ivanka ; Vidović, Dinko ; Vučemilo, Tiha

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of abstract / - , 2022, 245-245

Skup
12th ISABS Conference on Forensic and Anthropologic Genetics and Mayo Clinic Lectures in Individidualized Medicine

Mjesto i datum
Dubrovnik, Hrvatska, 22.06.2022. - 27.06.2022

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Keratinociti, Lijek za napredne terapije
(Keratinocytes, Advanced therapy medicinal products)

Sažetak
The aim of this study is to present development of tissue engineering and clinical application of cultured epidermal autografts (CEA). Advanced therapy medicinal products (ATMPs) are medicines for humans that include gene, somatic cell and tissue-engineered therapeutic products. Cultured keratinocytes regenerate epithelium and belong to the category of ATMP as tissue-engineered products. The development of ATMPs in Croatia began during 2002 in collaboration with the Ruđer Bošković Institute, the Clinic of Traumatology and the Children's Hospital Zagreb on the project Production of skin grafts in vitro. Engineering Laboratory was built in May 2005 in accordance with Good Manufacturing Practice and clean room technology. Tissue and Cell Bank (TCB) was established during 2007. The procedure includes isolation of keratinocytes from the skin biopsy (about 4-6 cm2/0.3 mm thick) after which they are seeded onto a feeder layer of 3T3 cells and incubated at 37 °C, 5 % CO2. Preparation of the optimal number of grafts is accomplished within 3- 4 weeks depending on the area of the injury. Immunocytochemistry (ICC) combines histological, immunological and biochemical techniques in the identification of specific tissue components. Using flow cytometry enabled high-throughput analysis of keratinocytes and residual feeder cells. Detection of bacterial endotoxins was determined using a Lysate of Amebocyte Limulus test. According to the EMA guidance the recommended endotoxin limit for release testing is ≤ 0.5 EU/mL (endotoxin units). Cell growth was monitored on a daily basis on microscope and analyzed on 14th day for evaluation of colony- forming efficiency (CFE). CFE was calculated as the number of colonies divided by the total number of seeded cells (1000 or 1500) and multiplied by 100 to express the result as a percentage. Quality control involves potency (yield, viability, CFE), purity (ΔNp63, CK AE1/AE3, CK5/6, CK14, CK19, vimentin), impurity (remaining 3T3 cells) and safety (sterility, mycoplasma, bacterial endotoxins).The first successful production of epidermal grafts began in the Clinic of Traumatology in September 2002 with 10 epidermal grafts of 700 cm2. This retrospective analysis spans a period from February 2002 to October 2003. The project included donors (n=15), from 2 to 66 years old with total 92 CEA. Microbiological control has proven the sterility of all keratinocytes, cell media as well as epidermal transplants. From July 2007 to March 2022 in TCB, donors (n=62) were from 1 to 74 years old with total 2235 CEA from which 89.2 % were transplanted and 10.8 % discarded. The most common reasons for discardment were patient’s death, initial microbiological contamination (Acinetobacter baumannii, Cutibacterium acnes, Micrococcus spp., Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococus epidermidis and other coagulase negative staphylococcus, etc.) and technical reasons (problem with feeder layer and decontamination of skin biopsy). Growth media from CEA was monitored for bacterial endotoxins prior to clinical application. Our results were < 0.125 EU/mL. CFE for 1000 cells was ≈ 9, 3 % and for 1500 cells was 8.3-15.1 %. ICC analysis showed 100 % epithelial cell marker CKAE1/AE3 positive cells (A), 100 % CK14 expressed in mitotically active basal layer cells (B), without CK19 positive differentiated keratinocytes (C), with 22.5 % proliferation marker Ki-67 positive cells (D) and 32 % vimentin positive cells represent pool of good quality of keratinocytes. Flow cytometry analysis shows high percentage of ΔNp63 (99.6 %) positive cells and low percentage of anti-feeder cells (1.66 %) positive cells ensure a good quality of CEA for the transplantation. Keratinocytes prepared as epidermal grafts or suspension with fibrin glue contributed to the survival of severely burned patients.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti, Kliničke medicinske znanosti

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