Saratov JOURNAL of Medical and Scientific Research

On preparation of am-niotic membrane as a scafold for cultivated cells to create corneal bioengineering constructs

Year: 2019, volume 15 Issue: №2 Pages: 409-413
Heading: Ophtalmology Article type: Original article
Authors: Aleksandrova OI, Gavrilyuk IO, Mashel TV, Chernysh VF, Churashov SV, Kulikov AN, Blinova MI.
Organization: Institute of Cytology, Russian Academy of Sciences, Military Medical Academy n.a. S. M. Kirov, St. Petersburg Polytechnic University
Summary:

Aim: to determine the preparation of amniotic membrane (AM) necessary for its use as a scafold for cultured cells to create bioengineered constructions (BEC). Material and Methods. Native AM was placed in a special clamping device and subjected to additional mechanical, thermal and enzymatic treatment: removal of mucus residues from its surface and cryoconservation of AM scafolds was performed at –80°C, — 20°C with subsequent decellularization with a 0.25 % Tripsin-EDTA mixture. The lifetime assessment of the morphology of cells cultivated on AM scafolds was performed using a Nikon Eclipse TS100 inverted microscope equipped with camera. The viability and metabolic activity of AM cells was determined by means of an MTT test using a UNIFLAN AIFR-01 tablet spectrophotometer (Picon, Russia) at a wavelength of 570 nm and a reference wavelength of 620 nm. Results. It has been established that the presence of mucous residueson the surface of native AM, which are not removed during standard mechanical processing, negatively afect the survival of the cell test system. The efects of cryoconservation of AM scafolds revealed the positive efects of this process together with enzymatic decellularization for improving the viability of cells cultured on scafolds. Conclusion. Standard mechanical processing of native AM does not guarantee complete cleaning of its surface from mucus residues that interfere with adhesion and even distribution of cultured cells. It is necessary to reliably control the removal of AM surface mucus before immobilization and further manipulations. Cryopreservation and subsequent decellularization of AM scafolds contributes to the increased viability of the cell test system. AM scafolds, purifed from amniotic mucus, cryopreserved at –80 ° C in a mixture of DMEM-F12 and DMSO (1:1) and enzymatic decellularization after thawing, turned out to be the best of the studied substrates for cell cultivation.

Bibliography:
1 Chernysh VF, Boyko EV. Eye burns: State of the problem and new approaches. Мoscow: GEOTAR-Media, 2017; 184 p.
2 Burman S, Tejwani S, Vemuganti GK, et al. Ophthalmic applications of preserved human amniotic membrane: a review of current indications. Cell and tissue banking 2004; 5 (3): 161–75
3 Lavker RM, Tseng SCG, Sun TT. Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Experimental eye research 2004; 78 (3): 433–46
4 Gavrilyuk IO, Aleksandrova OI, Khorolskaya YuI, et al. To the question of the intake, isolation and cultivation of stem cells of the epithelium of the oral mucosa. Modern technologies in ophthalmology 2018; (4): 60–3
5 Dubovikov AS, Bezushko AV, Gavrilyuk IO, et al. The study of the possibility of using limbal epithelial epithelial stem cells cultured on the amniotic membrane to treat limbal insufciency in the experiment. Modern technology in ophthalmology 2017; (4): 72–75
6 Dubovikov AS, Bezushko AV, Kulikov AN, et al. On using the corneal epithelial stem cells cultured on the amniotic membrane for limbal stem cell defciency treatment in an experiment. Practical medicine 2017; 2 (9 (110)): 67–71
7 Nakamura T, Inatomi T, Sotozono C, et al. Ocular surface reconstruction using stem cell and tissue engineering. Progress in retinal and eye research 2016; 51: 187–207
8 Sitnik GV. Modern cellular biotechnology in ophthalmology: Amniotic membrane as a substrate for the cultivation of stem epithelial cells. Medical Journal 2006; (4): 16– 9
9 Abramova IA, Boyko EV, Chernysh VF. On the use of the amniotic membrane for the purpose of conjunctival plasty in the experiment. Fedorov Journal of Ophthalmic Surgery 2004; (3): 8–12
10 Endo KI, Nakamura T, Kawasaki S, Kinoshita S. Human amniotic membrane, like corneal epithelial basement membrane, manifests the alpha-5 chain of type IV collagen. Investigative Ophthalmol 2004; 45 (6): 1771–4
11 Solomon A, Rosenblatt M, Monroy D, et al. Suppression of interleukin 1alpha and interleukin 1beta in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. Br J Ophthalmol 2001 85 (4): 444–9
12 Murzabekova FA. Double keratoamniokrytiya in the system of surgical treatment of ulcerative processes of the cornea: PhD abstract. Мoscow, 2007; 25 p.
13 Jirsova K, Jones GLA. Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting: a review. Cell and tissue banking 2017, 18 (2): 193– 204
14 Kubo M, Sonoda Y, Muramatsu R, Usui M. Immunogenicity of human amniotic membrane in experimental xenotransplantation. Invest Ophthalmol Vis Sci 2001; 42 (7): 1539–46.

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