Abstract
The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.
Highlights
Corneal epithelial stem cells are located in the basal layer of the limbus, a corrugated and pigmented structure called the palisades of Vogt [1,2,3,4]
The corneal epithelial cells maintained in serum replacement (SR) medium + 3T3 feeder layer displayed a morphology with small size and high nuclei/cytoplasm ratio, which was typical undifferentiated epithelial cells morphology, and the large differentiating flat squamous-like cells were rarely observed (Figure 1(c))
The result shows that epithelial cell yield from SR medium is close to that of cells cultured in FAD medium, as shown in Figure 2(a), and these data are similar to previous reports [4, 10,11,12]
Summary
Corneal epithelial stem cells are located in the basal layer of the limbus, a corrugated and pigmented structure called the palisades of Vogt [1,2,3,4]. Limbal stem cell deficiency (LSCD) and associated ocular surface diseases can be treated successfully using cultured limbal epithelial autograft [7, 8] The success of these surgical treatments depends on efficient expansion of limbal epithelial stem cells which involved 3T3 feeder layer and fetal bovine serum (FBS) in most cases. FBS contains potentially harmful xenogeneic components, which may stimulate immunological reactions and transmit animal diseases and pathogens [18] With all these concerns, there is an increasing need to develop well-defined culture medium to replace the traditional FBS supplemented medium. There are certain serum-free alternative media for the growth of epithelial cells, such as defined Keratinocyte Serum-Free Medium (KSFM6, Invitrogen, USA), keratinocyte growth medium (KGM6, Clontech, USA), Epilife (Invitrogen, USA), and Progenitor Cell Targeted (PCT) media (CellnTEC6, Switzerland) These products have been shown to support the expansion of corneal epithelial cells. Given the similarities in the culture methods of epithelial cells and ES cells, and in light of the fact of KnockOut SR replacing FBS in ES cell culture, here it is hypothesized that the KnockOut SR could be used to replace FBS in limbal epithelial cell culture
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