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Kunal Suri, Ching Yuan, Stephen C Kaufman; Platelet Lysate as Replacement for Fetal Bovine Serum in Limbal Stem Cell Cultures: Preliminary Results. Invest. Ophthalmol. Vis. Sci. 2014;55(13):511.
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Limbal stem cell deficiency (LSCD) can result in blindness. Ex vivo expansion and transplantation of limbal epithelial stem cells has been shown to restore the integrity of the corneal surface. Fetal bovine serum (FBS) is a common component of the growth medium used for stem cell expansion. In this study, we evaluate the use of human platelet lysate (HPL) supplemented culture medium for corneal limbal stem cell explant culture and compare it to the conventional medium containing FBS or human serum (HS).
Culture media were prepared using 10% pooled HPL (Zen-Bio Inc. North Carolina), single donor HPL (Innovative research, Novi, MI) prepared by repeated freeze-thaw cycles, HS (Sigma-Aldrich, Saint Louis, MO) or FBS (Sigma-Aldrich, Saint Louis, MO). Limbal tissues from donor corneoscleral rims, obtained from the Minnesota Lions Eye Bank, were cultured in each medium. Immunoflourescence staining was performed for stem cell markers ABCG 2 and p63 and corneal differentiation marker K3. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG 2. Human corneal epithelial cells (HCE-2) were grown in each medium and labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium.
The growth and morphology of limbal explant cultures grown in HPL and pooled HPL were similar to those grown in FBS or HS. Immunoflourescence staining showed expression of ABCG 2, p 63 and weak expression of K 3 in HPL or pooled HPL supplemented medium that was comparable to FBS and HS supplemented medium. qRT-PCR showed 60% higher expression of ABCG 2 in HPL medium than FBS medium. Bromodeoxyuridine assay showed that HPL and pooled HPL showed increased incorporation, with HPL significantly different than FBS (2 folds higher; p=0.04 using t-test).
Human platelet lysate supplemented cultures were effective in supporting growth of limbal stem cell cultures. The use of platelet lysate is of clinical importance in developing xenobiotic free culture medium for culturing limbal stem cells and transplanting it to human eyes.
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