Abstract
Background
Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants.
Methods
An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na+,K+-ATPase, and vinculin.
Results
All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na+,K+-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions.
Conclusions
Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.
Similar content being viewed by others
References
Aboalchamat B, Engelmann K, Bohnke M, Eggli P, Bednarz J (1999) Morphological and functional analysis of immortalized human corneal endothelial cells after transplantation. Exp Eye Res 69:547–553, doi:10.1006/exer.1999.0736
Bednarz J, Doubilei V, Wollnik PCM, Engelmann K (2001) Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells. Br J Ophthalmol 85:1416–1420, doi:10.1136/bjo.85.12.1416
Bednarz J, Teifel M, Friedl P, Engelmann K (2000) Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells. Acta Ophthalmol Scand 78:130–136, doi:10.1034/j.1600-0420.2000.078002130.x
Gramm S, Komber H, Schmaljohann D (2005) Copolymerization kinetics of N-isopropylacrylamide and diethylene glycol monomethylether monomethacrylate determined by online NMR spectroscopy. J Polym Sci Part Polym Chem 43:142–148, doi:10.1002/pola.20514
Hadlock T, Singh S, Vacanti JP, Mclaughlin BJ (1999) Ocular cell monolayers cultured on biodegradable substrates. Tissue Eng 5:187–196, doi:10.1089/ten.1999.5.187
Hatakeyama H, Kikuchi A, Yamato M, Okano T (2007) Patterned biofunctional designs of thermoresponsive surfaces for spatiotemporally controlled cell adhesion, growth, and thermally induced detachment. Biomaterials 28:3632–3643, doi:10.1016/j.biomaterials.2007.04.019
Ishino Y, Sano Y, Nakamura T, Connon CJ, Rigby H, Fullwood NJ et al (2004) Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci 45:800–806, doi:10.1167/iovs.03-0016
Koizumi N, Sakamoto Y, Okumura N, Okahara N, Tsuchiya H, Torii R et al (2007) Cultivated corneal endothelial cell sheet transplantation in a primate model. Invest Ophthalmol Vis Sci 48:4519–4526, doi:10.1167/iovs.07-0567
Korinek PM (1994) Amorphous fluoropolymers - a new-generation of products. Macromolecular Symposia 82:61–65, http://www3.interscience.wiley.com/journal/60500249/home
Lai JY, Hsiue GH (2007) Functional biomedical polymers for corneal regenerative medicine. Reactive Funct Polymers 67:1284–1291, doi:10.1016/j.reactfunctpolym.2007.07.060
Lange TM, Wood TO, Mclaughlin BJ (1993) Corneal endothelial-cell transplantation using Descemets-membrane as a carrier. J Cataract Refract Surg 19:232–235
Melles GRJ, Lander F, Rietveld FJR (2002) Transplantation of Descemet’s membrane carrying viable endothelium through a small scleral incision. Cornea 21:415–418, doi:10.1097/00003226-200205000-00016
Mimura T, Yamagami S, Yokoo S, Usui T, Tanaka K, Hattori S et al (2004) Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci 45:2992–2997, doi:10.1167/iovs.03-1174
Mohay J, Lange TM, Soltau JB, Wood TO, Mclaughlin BJ (1994) Transplantation of corneal endothelial-cells using a cell carrier device. Cornea 13:173–182, doi:10.1097/00003226-199403000-00011
Moller-Pedersen T, Hartmann U, Ehlers N, Engelmann K (2001) Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay. Graefes Arch Clin Exp Ophthalmol 239:778–782, doi:10.1007/s004170100354
Nitschke M, Götze T, Gramm S, Werner C (2007) Detachment of human endothelial cell sheets from thermo-responsive poly(NiPAAm-co-DEGMA) carriers. Express Polym Lett 1:660–666, doi:10.3144/expresspolymlett.2007.90
Nitschke M, Gramm S, Götze T, Valtink M, Drichel J, Voit B et al (2007) Thermo-responsive poly(NiPAAm-co-DEGMA) substrates for gentle harvest of human corneal endothelial cell sheets. J Biomed Mater Res A 80:1003–1010, doi:10.1002/jbm.a.31098
Nitschke M, König U, Lappan U, Minko S, Simon F, Zschoche S et al (2007) Low pressure plasma based approaches to fluorocarbon polymer surface modification. J Appl Polym Sci 103:100–109, doi:10.1002/app.24717
Nitschke M, Zschoche S, Baier A, Simon F, Werner C (2004) Low pressure plasma immobilization of thin hydrogel films on polymer surfaces. Surf Coat Tech 185:120–125, doi:10.1016/j.surfcoat.2003.12.006
Richards RG, Stiffanic M, Owen GRH, Riehle M, Gwynn IAP, Curtis ASG (2001) Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy. Cell Biol Int 25:1237–1249, doi:10.1006/cbir.2001.0807
Rzaev ZMO, Dincer S, Piskin E (2007) Functional copolymers of N-isopropylacrylamide for bioengineering applications. Prog Polym Sci 32:534–595, doi:10.1016/j.progpolymsci.2007.01.006
Schild HG (1992) Poly (N-Isopropylacrylamide) - experiment, theory and application. Prog Polym Sci 17:163–249, doi:10.1016/0079-6700(92)90023-R
Schmaljohann D (2005) Thermo-responsive polymers and hydrogels in tissue engineering. e-Polymers 21, http://www.e-polymers.org/journal/abstract.cfm?abstract_Id=811
Schmaljohann D, Beyerlein D, Nitschke M, Werner C (2004) Thermo-reversible swelling of thin hydrogel films immobilized by low-pressure plasma. Langmuir 20:10107–10114, doi:10.1021/la034653f
Schmaljohann D, Oswald J, Jorgensen B, Nitschke M, Beyerlein D, Werner C (2003) Thermo-responsive PNiAAm-g-PEG films for controlled cell detachment. Biomacromolecules 4:1733–1739, doi:10.1021/bm034160p
Tsuda Y, Kikuchi A, Yamato M, Sakurai Y, Umezu M, Okano T (2004) Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces. J Biomed Mater Res A 69:70–78, doi:10.1002/jbm.a.20114
Valtink M, Gruschwitz R, Funk RHW, Engelmann K (2007) Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics. Cells Tissues Organs 187:286–294, doi:10.1159/000113406
van Dooren BTH, Mulder PGH, Nieuwendaal CP, Beekhuis WH, Melles GRJ (2007) Endothelial cell density after posterior lamellar keratoplasty: Five- to seven-year follow-up. Am J Ophthalmol 144:471–473, doi:10.1016/j.ajo.2007.05.015
Wencan W, Mao Y, Wentao Y, Fan L, Jia Q, Qinmei W et al (2007) Using basement membrane of human amniotic membrane as a cell carrier for cultivated cat corneal endothelial cell transplantation. Curr Eye Res 32:199–215, doi:10.1080/02713680601174165
Yamato M, Akiyama Y, Kobayashi H, Yang J, Kikuchi A, Okano T (2007) Temperature-responsive cell culture surfaces for regenerative medicine with cell sheet engineering. Prog Polym Sci 32:1123–1133, doi:10.1016/j.progpolymsci.2007.06.002
Yang J, Yamato M, Nishida K, Ohki T, Kanzaki M, Sekine H et al (2006) Cell delivery in regenerative medicine: The cell sheet engineering approach. J Control Release 116:193–203, doi:10.1016/j.jconrel.2006.06.022
Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M et al (2007) Reconstruction of functional tissues with cell sheet engineering. Biomaterials 28:5033–5043, doi:10.1016/j.biomaterials.2007.07.052
Acknowledgement
The authors thank J. Drichel (IPF Dresden) for excellent assistance with cell culture experiments.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Götze, T., Valtink, M., Nitschke, M. et al. Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers. Graefes Arch Clin Exp Ophthalmol 246, 1575–1583 (2008). https://doi.org/10.1007/s00417-008-0904-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00417-008-0904-6