Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement

Shahdadfar, Aboulghassem; Haug, Kristiane; Pathak, Meeta; Drolsum, Liv; Olstad, Ole Kristoffer; Johnsen, Erik O.; Petrovski, Goran; Moe, Morten C.; Nicolaissen, Bjørn

doi:10.1016/j.exer.2012.01.013

Cited by 49 publications

(38 citation statements)

References 46 publications

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“…From a clinical point of view, the content of non-human additives and animal cells in a culture system designed for ex vivo generation of tissues for transplantation to human patients may increase the risk of immune rejection and transfer of animal pathogens [19]. The above mentioned experimental studies may indicate, that such a complex culture system is needed in order to support repair of damage inflicted upon essential cellular molecules during the dissociation procedure.…”

Section: Page -02mentioning

confidence: 99%

“…Here the limbal samples are positioned in medium on a suitable substrate without initial dissociation of the epithelial cells. In recent studies, using this approach, we have demonstrated that transplantable grafts may be generated in a culture system without animal feeder cells and FBS using autologous serum as the single growth promoting supplement [18,19].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

2013

J Ocul Biol

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Section: Page -02mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

2013

J Ocul Biol

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“…Outgrowths from explants of the contralateral limbus are a preferred method to generate autologous corneal epithelial sheets for the regeneration of a corneal surface in the treatment of limbal stem cell deficiency (Tsai etal, 1990; Ramesh and Dhillon, 2003; Shahdadfar etal, 2012; Basu etal, 2012; Satake etal, 2014). These cultures require the presence of serum.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions

Zamudio

Wang

Chung

et al. 2016

Experimental Eye Research

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Outgrowths of limbal epithelium by explant culture are used to treat limbal stem cell deficiency (LSCD). The explant culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these cultures. Limbal biopsies were set on explant culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5 % FBS, henceforth SHEM or a) SHEMSB = SHEM plus SB431542 an inhibitor of TGFβ signaling; b) sfSHEM = SHEM with FBS replaced by 0.05 % Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFβ inhibitor. After the initial outgrowths reached 2-3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p >0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6 % (p < 0.05). Removal of Albumax and ChT to establish a xeno-free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum endogenously generated TGFβ act as an autocrine cytostatic agent and that TGFβ inhibitors allow explant culture in xeno-free, chemically defined medium. Furthermore, the pro-growth effect of serum in limbal explant cultures may result exclusively from neutralization of the TGFβ cytostatic effect.

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“…Fetal calf serum (FCS) is required for limbal stem cell preservation. In an effort to prevent putative xenogenic contaminants, human autologous serum has been proposed as a potential substitute for FCS [48]. However, variability of hormones and growth factors content due to individual genetic background could be detrimental for the reproducibility of the culture.…”

Section: Biological Parametersmentioning

confidence: 99%

Concise Review: Hurdles in a Successful Example of Limbal Stem Cell-based Regenerative Medicine

et al. 2014

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Recent breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. This review is focused on recent developments in cell-based therapy of limbal stem cell deficiency. All findings can provide support for improvement and standardization of the cure for this disabling disease. STEM CELLS 2014;32:26-34

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Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement

Cited by 49 publications

References 46 publications

Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions

Concise Review: Hurdles in a Successful Example of Limbal Stem Cell-based Regenerative Medicine

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