Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.
Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.
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DOI：
10.1016/j.actbio.2017.07.041
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发表时间：
2017-10-01
影响因子：
9.7
通讯作者：
Lako M
中科院分区：
文献类型：
--
作者：
Figueiredo GS;Bojic S;Rooney P;Wilshaw SP;Connon CJ;Gouveia RM;Paterson C;Lepert G;Mudhar HS;Figueiredo FC;Lako M
用于人角膜缘干细胞（LSC）的离体扩增的金标准基质仍然是人羊膜（HAM），但这不是确定的基质，并且受到生物学可变性和传播疾病的可能性的影响。为了更好地定义HAM并减轻疾病传播的风险，我们试图确定去细胞化和/或γ-照射是否对培养物生长和LSC表型具有不利影响。在新鲜HAM、用0.5M NaOH和0.5%（w/v）十二烷基硫酸钠（SDS）脱细胞的HAM上建立离体角膜缘外植体培养物，有或没有γ-辐射。测量外植体生长速率，并通过组织学、免疫染色和qRT-PCR（ABCG 2、Δ Np 63、Ki 67、CK 12和CK 13）表征LSC表型。通过布里渊光谱显微镜测量，经γ-辐照略微硬化的HAM。HAM硬度和γ-照射不显著影响LSC表型，然而LSC在γ-照射的SDS去细胞化HAM上显著更快地扩增（p < 0.05），这也通过Ki 67和推定的LSC标志物ABCG 2的最高表达证实。集落形成效率测定显示，在照射过的SDS去细胞化的HAM上培养的细胞中，全克隆的产率和比例更高。总之，我们的数据表明，SDS脱细胞HAM可能是用于扩增LSC的更有效的底物，并且使用γ-辐照HAM允许用户用无菌底物开始制造过程，可能使其更安全。尽管其缺点，包括其生物学可变性和其转移疾病的能力，但人羊膜（HAM）仍然是角膜缘干细胞（LSC）培养的金标准基质。为了解决这些缺点，我们使用通过γ-照射灭菌的去细胞化HAM用于LSC培养。我们在新鲜HAM、用NaOH脱细胞的HAM、用十二烷基硫酸钠（SDS）脱细胞的HAM和用SDS脱细胞并用γ-辐射灭菌的HAM上培养LSC。我们证明，尽管用SDS去细胞化并用γ-辐射灭菌的HAM明显更硬，但这不影响LSC培养物的生长速率或培养的LSC的表型。因此，我们建议在未来的LSC临床试验中使用SDS脱细胞γ-辐照HAM。
The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.
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影响因子：
2
作者：
Koizumi, N;Inatomi, T;Kinoshita, S
通讯作者：
Kinoshita, S
影响因子：
2.8
作者：
Fukuda, K;Chikama, T;Nishida, T
通讯作者：
Nishida, T
DOI：
10.1007/s11552-009-9195-6
发表时间：
2009-09-01
期刊：
Hand (New York, N.Y.)
影响因子：
--
作者：
Karabekmez, Furkan E;Duymaz, Ahmet;Moran, Steven L
通讯作者：
Moran, Steven L
影响因子：
5.2
作者：
Kolli, Sai;Ahmad, Sajjad;Figueiredo, Francisco
通讯作者：
Figueiredo, Francisco
影响因子：
3.7
作者：
Chierico L;Rizzello L;Guan L;Joseph AS;Lewis A;Battaglia G
通讯作者：
Battaglia G