How to establish a stable
in vitro culture system, including location of corneal limbal epithelial stem cells,
in vitro sample harvest,
in vitro culture, vector selection, as well as identification methods, play a key role in culture of corneal limbal epithelial stem cells. Here, more living cells were harvested by using direct culture of corneal limbal tissues and shorten trypsin digestion duration, thus, reduced damage to cells, which is prone to cell’s migration. The corneal limbal epithelial cells spread the bottom of 6-well plate at 8-14 days after culture. In the experiment, we found that few fibroblasts could be found grew with epithelial cells in the cultured corneal limbal tissues at 3-4 days after culture with DMEM: F12 medium (containing 10% FCS), which gradually decreased with time prolonged, and corneal limbal epithelial cells with high purity could be obtained at 8 days after removing tissue blocks. It is supposed that, this result is related to disparity of differentiating potential between two kinds of cells, that is, the corneal limbal epithelial cells has stem cell characteristics, which differentiating potential is superior to fibroblasts, according, the corneal limbal epithelial stem cells was purified with culture time prolonged.
As a skelemin in horny cells, cytokeratin is not only an important structure in anterior segment, but also plays a protective role in maintaining anterior segment epithelial integrity. Specific expression of keratin is closely associated with epithelial tissue types and differentiating levels[6]. AE5 monoclonal antibody can specific bind to CK3 alkaline keratin. When using CK3 expression to detect corneal limbal stem cells, Schermer et al[7] found that CK3 was positive expressed in corneal limbal surface layer and epithelial cells at the corneal center, but negative expressed in basilar part of corneal limbus, it thought that CK3 is a molecular markers of corneal epithelium[8]. Accordingly, if cells in the basilar part of corneal limbus can not express CK3, they would be corneal limbal stem cells. Immunohistochemical staining showed that, the primary cultured cells were 5% positive for cytokeratin AE5 monoclonal antibody, which was gradually increased with passage increasing, and up to 75% at the 4th generation. On the basis of CK3 expression, we presumed that, the obtained corneal limbal epithelial cell in the tissue block culture was stem cells, which occupied different proliferation period and gradually positive for CK3. Nuclear factor P63 was widely but selectively expressed in basal cells, which intimately correlated to differentiating potential[9]. In 2003, Wang et al[10] first found there were P63 protein expression in basal lamina of corneal limbal epithelial cells. Arpitha et al[11] found that limbal epithelial basal cells were highly expressed P63 protein, thus, cells with positive P63 expression and had small nucleus/cytoplasm ratio were limbal stem cells[12]. In this experiment, the immunohistochemical staining showed, the P63 positive rate was up to 90% in primary culture, which gradually decreased with generation increasing. The primary cultured cells were high expressed P63 but low expressed CK3, however, the P63 positive rate was declined and CK3 rate increased in passaged cells. Immunohistochemical staining demonstrated that plenty of limbal stem cells could be harvested and maintained highly biological characteristics of stem cells using limbal tissue culture, but, how to elevate in vitro passage number and to maintain the stem cell features during serial subcultivation need further exploration.
Amniotic membrane is preferred in culture corneal limbal epithelial cells
in vitro due to non-antigenicity and degradation
[13]. Kim
et al [14] reconstruct ocular surface using amniotic membrane transplantation successfully and rapid spread the amniotic membrane transplantation. More studies demonstrated that amniotic membrane transplantation can suppress inflammation, promote corneal epithelialization and mitigate fibration. Many growth factors, such as EGF, HGF, and TGF, which possess specific roles in amniotic membrane transplantation, can be detected in amniotic membrane
[15]. During experiment, we found that, corneal limbal epithelial stem cells migrate and grow faster on amniotic membrane than that
of 6-well plate. Additionally, CK3 and P63 immunohistochemistry confirmed corneal limbal epithelial cells have biological characteristics of stem cells, thus, primary and passaged corneal limbal epithelial cells amniotic membrane grafts provide a new approach for the treatment of ocular surface diseases resulted from corneal limbal stem cell deficiency. In the experiment, few corneal limbal epithelial tissues were used, so it had small damage to the sampling eyes, simultaneously, it can form a stable culture system for corneal limbal epithelial cells, which can maintain the high proliferation ability of cells. All of these provide a support for corneal epithelium transplantation. However, the biological and histological variations of amniotic membrane grafts after transplantation still need further investigation.
