Feeder-Free Cell Culture of Labial Oral Mucosal Epithelium for Tissue-Engineering and Regenerative Medicine

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Abstract

Background. The cultured cheek mucosa epithelium (buccal epithelium, BE) is used for autologous transplants generation and tissue engineering. An alternative source of cells for these purposes may be the lip mucosa, covered, like BE, with non-keratinized stratified squamous epithelium, but with some histological distinctions. Aims — to characterize the human lip mucosa as a promising source of epithelial cells for autologous transplantation and tissue engineering. Methods. Scrapings of the lip, cheek, and gum mucosa from five healthy volunteers were analyzed by cytofluorimetry to determine the level of desquamation and cytokeratin (CK) 10 and 13 expression. The lip mucosa of two patients was characterized using routine histological staining and fluorescence immunohistochemistry for CK 3, 4, 10, 13, and p63 marker. 35 samples of full-thickness strips of the patient’s lip mucosa were used to set the explant (n = 18) and enzymatic (n = 17) techniques for expansion epithelium. Culture systems with 1.05 and 0.06 mM Calcium contained 5% fetal bovine serum, 5 μg/ml human insulin, 5 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor. Stable cultures were stained for p63, vimentin, zonula occludens-1 (ZO-1), and CK10. Software tools determined levels of their expression. Results. The number of cells in the lip and gum samples was significantly lower than from the cheek. The median number of CK13 positive cells was significantly different for the gum (6.4%) and cheek (64.8%, p = 0.0089). Significant differences for CK10 positive cells were not observed. The epithelium of the lip mucosa was 72.1 ± 3.6 μm thick, relatively flat, and without keratinization sites. Samples were positively stained for CK 4 and 13, in the absence of expression of CK 3 and 10. The primary culture of epithelial cells obtained by explant technique was significantly more effective (p = 0.001) in comparison with the enzymatic method. Stable cultures had a “cobble-stone” morphology in both culture systems. The levels of vimentin and p63 expression in both culture systems was not significantly differ. ZO-1 expression was 3.6-fold higher for 1.05-mM Ca++ medium (p = 0.0006). Conclusions. Epithelium cell culture from the lip mucosa can be obtained by culturing explants without a feeder layer. Quality control steps have been developed for cultured cells and biopsy site.

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About the authors

Sergey A. Borzenok

The S. Fyodorov Eye Microsurgery Federal State Institution; The A.I. Yevdokimov Moscow State University of Medicine and Dentistry

Email: mdborzenok@yandex.ru
ORCID iD: 0000-0001-9160-6240
SPIN-code: 1054-0164

MD, PhD, Professor

Russian Federation, Moscow

Boris E. Malyugin

The S. Fyodorov Eye Microsurgery Federal State Institution; The A.I. Yevdokimov Moscow State University of Medicine and Dentistry

Email: boris.malyugin@gmail.com
ORCID iD: 0000-0001-5666-3493
SPIN-code: 8906-2787

MD, PhD, Professor

Russian Federation, Moscow

Maxim Y. Gerasimov

The S. Fyodorov Eye Microsurgery Federal State Institution

Author for correspondence.
Email: gerasimovmy@mntk.ru
ORCID iD: 0000-0003-3433-8352
SPIN-code: 7539-1262

MD

Russian Federation, Moscow

Dmitriy S. Ostrovskiy

The S. Fyodorov Eye Microsurgery Federal State Institution

Email: dmitriy.ostrovskiy@gmail.com
ORCID iD: 0000-0002-2817-7102
SPIN-code: 9947-6481

PhD in Biology

Russian Federation, Moscow

Anna V. Shatskikh

The S. Fyodorov Eye Microsurgery Federal State Institution

Email: avsatik07@yandex.ru
ORCID iD: 0000-0002-3437-8162
SPIN-code: 1751-9815

MD, PhD

Russian Federation, Moscow

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Supplementary files

Supplementary Files
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1. Figure: 1. Results of cytofluorometry of scrapings from three zones of the oral mucosa

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2. Figure: 2. Histological characteristics of the epithelium of the mucous membrane of the lip

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3. Figure: 3. Morphology of lip epithelial cell culture in culture systems

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4. Figure: 4. Immunophenotype of the primary culture of the epithelium of the mucous membrane of the lip

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5. Figure: 5. The level of expression of markers according to the data of fluorescent immunocytochemistry in culture systems

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