The culture of human corneal endothelial cells (CECs) is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs) that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS) technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs. 1. Introduction The inner monolayer of the human cornea—the corneal endothelium (CE)—functions both as a barrier and a pump and plays a critical role in the regulation of corneal hydration. The CE layer prevents excessive fluids from entering the glycosaminoglycan-rich stromal layer while actively pumping fluid out to prevent corneal edema [1–3]. This maintains corneal thickness and keeps the corneal transparent [3, 4]. Cells of the CE do not have the capacity to undergo functional regeneration in vivo [5–7]. Hence, when corneal endothelial cell loss occurs, the existing cells spread out to maintain functional integrity of the CE. However, a critical threshold must be maintained to preserve corneal clarity. If endothelial dysfunction develops, there will be an inability to efficiently pump fluid out of the stroma, resulting in stromal and epithelial edema, loss of corneal clarity, and visual acuity [4], which will eventually lead to corneal blindness—a situation where the retina is normal but the cornea becomes edematous. This is the second leading cause of visual blindness worldwide [8]. Restoration of vision in these situations is only possible by replacing the dysfunctional CE layer with healthy donor CE through corneal transplantation. There is a global shortage of transplant-grade donor corneal tissues, and this greatly restricts the number of corneal transplantation performed yearly [9, 10]. Hence, significant efforts have
References
[1]
D. M. Maurice, “The location of the fluid pump in the cornea,” Journal of Physiology, vol. 221, no. 1, pp. 43–54, 1972.
[2]
J. Fischbarg and J. J. Lim, “Role of cations, anions and carbonic anhydrase in fluid transport across rabbit corneal endothelium,” Journal of Physiology, vol. 241, no. 3, pp. 647–675, 1974.
[3]
W. M. Bourne, “Clinical estimation of corneal endothelial pump function,” Transactions of the American Ophthalmological Society, vol. 96, pp. 229–242, 1998.
[4]
K. H. Carlson, W. M. Bourne, J. W. McLaren, and R. F. Brubaker, “Variations in human corneal endothelial cell morphology and permeability to fluorescein with age,” Experimental Eye Research, vol. 47, no. 1, pp. 27–41, 1988.
[5]
W. M. Bourne, L. I. L. Nelson, and D. O. Hodge, “Central corneal endothelial cell changes over a ten-year period,” Investigative Ophthalmology and Visual Science, vol. 38, no. 3, pp. 779–782, 1997.
[6]
C. Murphy, J. Alvarado, R. Juster, and M. Maglio, “Prenatal and postnatal cellularity of the human corneal endothelium. A quantitative histologic study,” Investigative Ophthalmology and Visual Science, vol. 25, no. 3, pp. 312–322, 1984.
[7]
K. K. Williams, R. L. Noe, H. E. Grossniklaus, C. Drews-Botsch, and H. F. Edelhauser, “Correlation of histologic corneal endothelial cell counts with specular microscopic cell density,” Archives of Ophthalmology, vol. 110, no. 8, pp. 1146–1149, 1992.
[8]
G. S.L. Peh, R. W. Beuerman, A. Colman, D. T. Tan, and J. S. Mehta, “Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview,” Transplantation, vol. 91, no. 8, pp. 811–819, 2011.
[9]
W. J. Armitage, S. J. Moss, D. L. Easty, and B. A. Bradley, “Supply of corneal tissue in the United Kingdom,” British Journal of Ophthalmology, vol. 74, no. 11, pp. 685–687, 1990.
[10]
K. McColgan, “Corneal transplant surgery,” Journal of perioperative practice, vol. 19, no. 2, pp. 51–54, 2009.
[11]
J. W. Ruberti and J. D. Zieske, “Prelude to corneal tissue engineering—Gaining control of collagen organization,” Progress in Retinal and Eye Research, vol. 27, no. 5, pp. 549–577, 2008.
[12]
K. Engelmann, J. Bednarz, and M. Valtink, “Prospects for endothelial transplantation,” Experimental Eye Research, vol. 78, no. 3, pp. 573–578, 2004.
[13]
N. C. Joyce and C. C. Zhu, “Human corneal endothelial cell proliferation: potential for use in regenerative medicine,” Cornea, vol. 23, no. 8, pp. S8–S19, 2004.
[14]
J. L. Baum, R. Niedra, C. Davis, and B. T. Yue, “Mass culture of human corneal endothelial cells,” Archives of Ophthalmology, vol. 97, no. 6, pp. 1136–1140, 1979.
[15]
J. Bednarz, V. Doubilei, P. C. M. Wollnik, and K. Engelmann, “Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells,” British Journal of Ophthalmology, vol. 85, no. 12, pp. 1416–1420, 2001.
[16]
K. H. Chen, D. Azar, and N. C. Joyce, “Transplantation of adult human corneal endothelium ex vivo: a morphologic study,” Cornea, vol. 20, no. 7, pp. 731–737, 2001.
[17]
R. N. Fabricant, A. J. Alpar, Y. M. Centifanto, and H. E. Kaufman, “Epidermal growth factor receptors on corneal endothelium,” Archives of Ophthalmology, vol. 99, no. 2, pp. 305–308, 1981.
[18]
Y. Ishino, Y. Sano, T. Nakamura et al., “Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation,” Investigative Ophthalmology and Visual Science, vol. 45, no. 3, pp. 800–806, 2004.
[19]
R. C. Tripathi and B. J. Tripathi, “Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture. A comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy,” Experimental Eye Research, vol. 35, no. 6, pp. 611–624, 1982.
[20]
B. Y. Yue, J. Sugar, J. E. Gilboy, and J. L. Elvart, “Growth of human corneal endothelial cells in culture,” Investigative Ophthalmology and Visual Science, vol. 30, no. 2, pp. 248–253, 1989.
[21]
W. Li, A. L. Sabater, Y. T. Chen et al., “A novel method of isolation, preservation, and expansion of human corneal endothelial cells,” Investigative Ophthalmology and Visual Science, vol. 48, no. 2, pp. 614–620, 2007.
[22]
J. A. West-Mays and D. J. Dwivedi, “The keratocyte: corneal stromal cell with variable repair phenotypes,” International Journal of Biochemistry and Cell Biology, vol. 38, no. 10, pp. 1625–1631, 2006.
[23]
K. Engelmann, M. Bohnke, and P. Friedl, “Isolation and long-term cultivation of human corneal endothelial cells,” Investigative Ophthalmology and Visual Science, vol. 29, no. 11, pp. 1656–1662, 1988.
[24]
H. F. Edelhauser, “The resiliency of the corneal endothelium to refractive and intraocular surgery,” Cornea, vol. 19, no. 3, pp. 263–273, 2000.
[25]
A. Grützkau and A. Radbruch, “Small but mighty: how the MACS1-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years,” Cytometry A, vol. 77, no. 7, pp. 643–647, 2010.
[26]
S. Miltenyi, W. Muller, W. Weichel, and A. Radbruch, “High gradient magnetic cell separation with MACS,” Cytometry, vol. 11, no. 2, pp. 231–238, 1990.
[27]
C. Y. Fong, G. Peh, A. Subramanian, K. Gauthaman, and A. Bongso, “The use of discontinuous density gradients in stem cell research and application,” Stem Cell Reviews and Reports, vol. 5, no. 4, pp. 428–434, 2010.
[28]
S. D. Klyce and R. W. Beuerman, “Structure and function of the cornea,” in The Cornea, H. E. Kaufman, B. A. Barron, M. B. McDonald, and S. R. Waltman, Eds., pp. 3–28, Churchill Livingstone, New York, NY, USA, 1988.
[29]
R. W. Yee, M. Matsuda, R. O. Schultz, and H. F. Edelhauser, “Changes in the normal corneal endothelial cellular pattern as a function of age,” Current Eye Research, vol. 4, no. 6, pp. 671–678, 1985.
[30]
K. Engelmann and P. Friedl, “Growth of human corneal endothelial cells in a serum-reduced medium,” Cornea, vol. 14, no. 1, pp. 62–70, 1995.
[31]
N. C. Joyce, “Proliferative capacity of the corneal endothelium,” Progress in Retinal and Eye Research, vol. 22, no. 3, pp. 359–389, 2003.
[32]
J. Bednarz, “Different characteristics of endothelial cells from central and peripheral human cornea in primary culture and after subculture,” In Vitro Cellular and Developmental Biology—Animal, vol. 34, no. 2, pp. 149–153, 1998.
[33]
T. Mimura and N. C. Joyce, “Replication competence and senescence in central and peripheral human corneal endothelium,” Investigative Ophthalmology and Visual Science, vol. 47, no. 4, pp. 1387–1396, 2006.
[34]
S. F. Gilbert and B. R. Migeon, “D valine as a selective agent for normal human and rodent epithelial cells in culture,” Cell, vol. 5, no. 1, pp. 11–17, 1975.
[35]
C. Zhu and N. C. Joyce, “Proliferative response of corneal endothelial cells from young and older donors,” Investigative Ophthalmology and Visual Science, vol. 45, no. 6, pp. 1743–1751, 2004.
[36]
S. Zahler, C. Kowalski, A. Brosig, C. Kupatt, B. F. Becker, and E. Gerlach, “The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method,” Journal of Immunological Methods, vol. 200, no. 1-2, pp. 173–179, 1997.
[37]
D. H. Geroski, M. Matsuda, R. W. Yee, and H. F. Edelhauser, “Pump function of the human corneal endothelium. Effects of age and cornea guttata,” Ophthalmology, vol. 92, no. 6, pp. 759–763, 1985.
[38]
M. D. McCartney, T. O. Wood, and B. J. McLaughlin, “Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium,” Current Eye Research, vol. 6, no. 12, pp. 1479–1488, 1987.
