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Kunal Suri, Hwee Gong, Ching Yuan, Stephen C Kaufman; Human Platelet Lysate Supplemented Medium Maintains Growth and Stem Cell Phenotype in Limbal Explant Cultures. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3469.
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© ARVO (1962-2015); The Authors (2016-present)
Recent interest in regenerative medicine has led to ex-vivo expansion of limbal stem cells (LSC) using various protocols, fetal bovine serum (FBS) being a common component of the growth medium. However, a xenobiotic-free medium is clinically important for use in humans. Our preliminary results showed human platelet lysate (HPL) supplemented medium can be used as a replacement for FBS. In the present study, we further evaluate the use of HPL supplemented medium for ex-vivo expansion of LSCs.
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, or FBS (Sigma-Aldrich, Saint Louis, MO). Limbal tissues from donor corneo-scleral rims, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic culture dish or on denuded amniotic membrane (AM). Immunoflourescence staining was performed for stem cell markers ABCG 2 and p63α and corneal differentiation marker K3. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG 2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors (EGF, VEGF, TGFβ and PDGF) in HPL and pHPL was compared to that in human serum (HS) (Sigma-Aldrich, Saint Louis, MO).
Immunoflourescence staining of explants on AM showed expression of ABCG 2, p 63 and weak expression of K3 in HPL and pHPL supplemented medium. qRT-PCR showed 1.7 fold higher expression of ABCG 2 in pHPL supplemented medium, and similar expression of p63 in HPL and pHPL supplemented medium compared to FBS medium. BrdU assay showed that LSCs retained the proliferative potential in HPL supplemented medium. Higher concentration of PDGF, EGF, VEGF and TGFβ was found in platelet lysate than HS.
Human platelet lysate had higher concentration of grown factors and was effective in maintaining growth and stem cell phenotype of limbal explant cultures.
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