РАЗРАБОТКА БИОИНЖЕНЕРНОЙ КОНСТРУКЦИИ ИСКУССТВЕННОЙ РОГОВИЦЫ НА ОСНОВЕ ПЛЕНОЧНОГО МАТРИКСА ИЗ СПИДРОИНА И КУЛЬТИВИРОВАННЫХ КЛЕТОК ЛИМБАЛЬНОЙ ЗОНЫ ГЛАЗНОГО ЯБЛОКА

Oftal mokhirurgiya

Б. Э. Малюгин¹  Ю. А. Комах¹  А. А. Желтоножко¹  И. А. Попов¹  С. А. Борзенок²  Т. Д. Колокольцова³  В. С. Репин³  И. М. Зурина⁴  Н. В. Кошелева⁵  И. Н. Сабурина⁶  И. И. Агапов⁷  В. Г. Богуш⁸  Л. И. Давыдова⁸ 

[1] ФГБУ «МНТК «Микрохирургия глаза» им. акад. С.Н. Федорова» Минздрава России, Москва;ФГБУ «МНТК «Микрохирургия глаза» им. акад. С.Н. Федорова» Минздрава России, МоскваГБОУ ВПО «Московский государственный медико-стоматологический университет им. А.И. Евдокимова» Минздрава России, Москва;ФГБУ «МНТК «Микрохирургия глаза» им. акад. С.Н. Федорова» Минздрава России, МоскваФГБУ «Научно-исследовательский институт общей патологии и патофизиологии» РАМН, МоскваГБОУ ДПО «Российская медицинская академия последипломного образования» Минздрава России, Москва;ФГБУ «Научно-исследовательский институт общей патологии и патофизиологии» РАМН, Москва;ФГБУ «Научно-исследовательский институт общей патологии и патофизиологии» РАМН, МоскваГБОУ ВПО «Московский государственный университет им. М.В. Ломоносова», Москва;ФГБУ «Научно-исследовательский институт общей патологии и патофизиологии» РАМН, МоскваГБОУ ДПО «Российская медицинская академия последипломного образования» Минздрава России, Москва;ФГБУ «Федеральный научный центр трансплантологии и искусственных органов им. акад. В.И. Шумакова» Минздрава России, МоскваГБОУ ВПО «Московский государственный университет им. М.В. Ломоносова», Москва;ФГУП «Государственный научно-исследовательский институт генетикии селекции промышленных микроорганизмов», Москва

关键词: bioengineering;    artificial cornea;    cell spheroids;    2D and 3D cultivation;    biopolymer matrix;    spidroin;    биоинжиниринг;    искусственная роговица;    клеточные сфероиды;    2D- и 3D-культивирование;    биоплимерный матрикс;    спидроин;   

DOI  :  10.25276/0235-4160-2013-4-89-97

学科分类：眼科学

来源: M H T K Mikrokhirurgiya Glaza

【 摘 要 】

Purpose. To study prerequisites for a development of artificial cornea bioengineered design based on recombinant spidroin tissue matrix by behavior evaluation of 2D (planar) and 3D cell (threedimensional) cultures on its surface.Material and methods. We studied epithelioid and stromal primary cell cultures (MSC-L) received from the limbal zone of post-mortem donor eyes. Cells were seeded on Petri dishes and on cavities of cultural trays (Corning, USA). To get spheroid structures the cells after the second passage underwent the centrifuge and were seeded on agarous trays then were cultivated in thermostatic chamber (Cell-IQ, Chip Man Technologies, Finland) under standard conditions (37° C, 5% CO2). Control over cell growth and morphology in trays was conducted under inverted microscope CKX41 (Olympus, Japan). To count the cell quantity and their viability the automatic cell counter Countess (Invitrogen, USA) was used, to analyze the surface proteins expression the flow cytofluorimetry was applied. For matrices colonization we used the 3rd passage MSC-L and 7-day spheroids of MSC-L origin. To evaluate 2D and 3D cell cultures growth on the surface of membranous matrices of recombinant spidroin, to estimate its non-toxicity and adhesiveness the immunohistochemistry, light time-lapse microscopy (Cell-IQ, Chip Man Technologies, Finland), laser scanning confocal microscopy (FluoView FV10i, Olympus, Japan) and raster electronic microscopy (CamScan, Japan) were incorporated.Results. Few hours after cell seeding there was active cells’ attachment to the substrate. Attached cells were characterized by rounded, oval or polygonal ordonnance. 24 hours later bipolar elongated cells and islets of migrating epithelioid cells appearance were observed. In the incubation process under gravity force the spheroids were accumulated predominantly in the central zone of the matrix, 2 hours later an active migration of spheroids surface zone epithelioid cells was registered on the membrane. After 24 hours of incubation all seeded on the surface of membranous matrix cells possessed a mesenchyme-like phenotype. Spheroids had an ability to merge limitlessly, later we observed a new microtissue formation with epithelioid cells on the surface and mesenchyme-like cells in the central area. Both solitary spheroids and merger-derived microtissue contained epithelial and mesenchymal components as well as regularly organized fibrils of extracellular matrix.Conclusions. According to aforementioned data the development of artificial cornea bioengineered cell-tissue constructions based on the technology of 3D cell spheroids cultivation derived from multipotent stem cells of the limbus and spidroin matrix presents a promising prospect requiring a further profound investigation.

【 授权许可】

CC BY