A comparative analysis of cultivation techniques of epithelial stem cells of cornea and creation of biomedical cell product (biocomposite) on the basis biocompatible matrix and stem cells
- Authors: Kvacheva Z.B1, Vasilevich I.B1, Chekina A.Y2, Marchenko L.N2, Dzhumova M.F2, Pinchuk C.V.1, Fedulov A.S2, Volotovski I.D1
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Affiliations:
- Institute of Biophysics and Cell Engineering of NAS of Belarus
- Belarus State Medical University
- Issue: Vol 14, No 4 (2019)
- Pages: 29-34
- Section: Articles
- URL: https://genescells.ru/2313-1829/article/view/122193
- DOI: https://doi.org/10.23868/201912029
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Abstract
In recent years the autologous stem cells of front cornea epithelium (CSC) are increasingly used in the treatment of degenerative diseases of this transparent part of the outer shell of the eye. Limb is considered as a specialized storage for these cells in eye. A number of techniques for isolation of stem cells from limb epithelium and their cultivation in vitro were developed. Goal of the work: To analyze the possibilities of two methods of cultivation of epithelial stem cells front epithelium of cornea (CSC), explants and suspension ones by criteria of efficiency of proliferation, time consumption and constancy of morphology and function parameters of cell cultures. To study of biocompatibility of CSC with commercial gel preparations for its application as an ingredient of biocomposite to use in clinical practice. The primary multilayer CSC cultures were obtained by two methods (explant and suspension) from biopsies of intact parts of cornea limb of 10 patients at the age from 39 to 73 years suffering different types of ceratopathies. Under comparative analysis of CSC cultures their viability, proliferation potential and immunophenotype were investigated. It was shown that explant method is more effective as compared with suspension one as it allows to gain the significant amount of cells with epithelium like morphology. The cell population was presented primarily by epithelium CSC: 95% of cells synthesized K19+ protein. CSC proliferation index of cells isolated by explant method was by 3.5-4 times higher, the time of doubling of population at cultivation in DMEM/F12 medium with 10 ng/ml of EGF was 48 h. Using explant method from one limb biopsy (1 х2 mm) a biomass of CSC in amount of 4 ± 1,2 mln during 3-4 weeks (1-2 passages) was obtained. It was show under investigation for biocompatibility of cells with a number of commercial gel preparations that preparation Provisk is the best on this indicator and this allows to recommend it as a carrier for cells when biomedical cell product are produced.
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About the authors
Z. B Kvacheva
Institute of Biophysics and Cell Engineering of NAS of Belarus
Email: volotovski@yahoo.com
Minsk, Belarus
I. B Vasilevich
Institute of Biophysics and Cell Engineering of NAS of Belarus
Email: volotovski@yahoo.com
Minsk, Belarus
A. Y Chekina
Belarus State Medical University
Email: volotovski@yahoo.com
Minsk, Belarus
L. N Marchenko
Belarus State Medical University
Email: volotovski@yahoo.com
Minsk, Belarus
M. F Dzhumova
Belarus State Medical University
Email: volotovski@yahoo.com
Minsk, Belarus
C. V. Pinchuk
Institute of Biophysics and Cell Engineering of NAS of Belarus
Email: volotovski@yahoo.com
Minsk, Belarus
A. S Fedulov
Belarus State Medical University
Email: volotovski@yahoo.com
Minsk, Belarus
I. D Volotovski
Institute of Biophysics and Cell Engineering of NAS of Belarus
Email: volotovski@yahoo.com
Minsk, Belarus
References
- Scheffer C.G., Tseng M.D., Hua He. et al. Niche regulation of limbal epithelial stem cells: relationship between inflammation and regeneration. Ocul. Surf. 2016; 14(2): 100-12.
- Ahmad S. Concise review: limbal stem cell deficiency, dysfunction and distress. Stem Cells Transl. Med. 2012; 1(2): 110-5.
- Haagdorens M., Van Acker S.I., Van Gerwen V. et al. Limbal stem cell deficiency: current treatment options and emerging therapies. Stem Cells Int. 2016; 2016: 9798374.
- Branch M.J., Hashmani K., Dhillon P. et al. Mesenchymal stem cells in the human corneal limbal stroma. Invest. Ophthalmol. Vis. Sci. 2012; 53(9): 5109-16.
- Ghoubay-Benallaoua D., de Sousa C., Martos R. et al. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea. PloS One 2017; 12(11): e0188398.
- Truong T.T., Huynh K., Nakatsu M.N. et al. SSEA4 is a potential negative marker for the enrichment of human corneal epithelial stem/progenitor cells. Invest. Ophthalmol. Vis. Sci. 2011; 52: 6315-20.
- Ksander B.R., Kolovou P.E., Wilson B.J. et al. ABCB5 is a limbal stem cell gene required for corneal development and repair. Nature 2014; 511(7509): 353-7.
- Szabo D.J., Noer A., Nagymihaly R. et al. Long-term cultures of human cornea limbal explants form 3D structures ex vivo - implications for tissue engineering and clinical applications. PLoS One 2015; 10: e0143053.
- Vazirani J., Ali M.H., Sharma N. et al. Autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency: multicentre results. Br. J. Ophthalmol. 2016; 100(10): 1416-20.
- Понятовская А.П., Коротченко С.А., Давыденко Д.В. и др. Трансплантат лимбальных эпителиальных стволовых клеток на биорезорбируемом носителе. Современные технологии в медицине 2017; 9(4): 44-50.
- Oie Y., Nishida K. Regenerative medicine for the cornea. Biomed. Res. Int. 2013; 2013: 428247.
- Dziasko M.A., Daniels J.T. Anatomical features and cell-cell interactions in the human limbal epithelial stem cell niche. Ocul. Surf. 2016; 14: 322-30.
- Pellegrini G., Traverso C.E., Franzi A.T. et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997; 349: 990-3.
- Xie H.T., Chen S.Y., Li G.G. et al. Isolation and expansion of human limbal stromal niche cells. Invest. Ophthalmol. Vis. Sci. 2012; 53: 279-86.
- Kethiri A.R., Basu S., Shukla S. et al. Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An in vitro study. PLoS One 2017; 12: e0185623.
- Deshpande P., Notara M., Bullet N. et al. Development of a surface-modified contact lens for the transfer of cultured limbal epithelial cells to the cornea for ocular surface diseases. Tissue Eng. Part A 2009; 15(10): 2889-902.
- Pellegrini G., Rama P., Di Rocco A. et al. Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine. Stem Cells 2014; 32(1): 26-34.
- Shahdadfar A., Haug K., Pathak M. et al. Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement. Exp. Eye Res. 2012; 97(1): 1-9.
- Freshney R. Ian. Culture of animal cells: a manual of basic technique and specialized applications. 7th ed. New Jersey: John Wiley & Sons; 2015.
- Laurenza A., Sutkowski E.M., Seamon K.B. Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action. Trends Pharmacol. Sci. 1989; 10(11): 442-7.
- Menzel-Severing J., Kruse F.E., Scolotzer-Scherardt U. Stem cell-based therapy for corneal epithelial reconstruction: present and future. Can. J. Ophthalmol. 2013; 48(1): 13-21.
- Utheim T.P. Limbal epithelial cell therapy: past, present and future. In: Wright B., Connon C.J., editors. Соrneal Regenerative Medicine. Methods Mol. Biol. 2013; 1014: 3-43.
- Barrandon Y., Green H. Three clonal types of keratinocyte with different capacities for multiplication. PNAS USA 1987; 84(8): 2302-6.
- Ordonez P., Di Girolamo N. Limbal epithelial stem cells: role of the niche microenvironment. Stem Cells 2012; 30(2): 100-7.
- Stasi K., Goings D., Huang J. et al. Optimal isolation and xeno-free culture conditions for limbal stem cell function. Invest. Ophthamol. Vis. Sci. 2014; 55: 375-86.
- Wright B., Connon C.J. Corneal Regenerative Medicine. Methods and Protocols. New York: Springer; 2013.
- Koizumi N., Cooper L., Fullwood N.J. et al. An evaluation of cultivated corneal limbal epithelial cells, using cell suspension culture. Invest. Ophthalmol. Vis. Sci. 2002; 43: 2114-21.
