Tissue Services

Liverpool, United Kingdom

Tissue Services

Liverpool, United Kingdom
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Figueiredo G.S.,Northumbria University | Bojic S.,Northumbria University | Rooney P.,Tissue Services | Wilshaw S.-P.,University of Bradford | And 7 more authors.
Acta Biomaterialia | Year: 2017

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). Y(hooktop)-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 Y(hooktop)-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 Y(hooktop)-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. Statement of Significance: 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. © 2017 Acta Materialia Inc.

Warwick R.M.,National Health Service Blood and Transplant NHSBT | Armitage W.J.,University of Bristol | Chandrasekar A.,Tissue Services | Mallinson G.,International Blood Group Reference Laboratory | And 2 more authors.
Cell and Tissue Banking | Year: 2012

Transplanted tissues have transmitted transmissible spongiform encephalopathies and in the UK there have been more cases of variant Creutzfeldt-Jakob disease (vCJD) than elsewhere in the world. A pilot study was undertaken to look at the feasibility of testing for vCJD in deceased donors using tonsillar tissue. This pilot showed that obtaining consent for removal and testing tonsil tissue was feasible. Donor eligibility for inclusion in the pilot was limited to tissue donors from the National Health Service Blood and Transplant, Tissue Services and to donors shared with the Corneal Transplant Service Eye Banks. Obtaining tonsillar tissue in the immediate post-mortem period was limited by the presence of rigor mortis. Tonsillar tissue was suitable for routine analysis for the presence of prion associated with vCJD in deceased tissue donors. Production and processing of tissue was straightforward and a low assay background was obtained from most samples. Since palatine and lingual tonsil tissue can be obtained in pairs it was possible, in the majority of cases, to set aside an intact sample for confirmatory testing if required. In one instance a sample was reactive by Western blot. However, the pattern of reactivity was not typical for that obtained from vCJD patients. Unfortunately the sample was not of sufficient quality for the confirmatory test to provide a conclusive result. © 2010 Springer Science+Business Media B.V.

Kitchen A.D.,National Transfusion Microbiology Reference Laboratory | Gillan H.L.,Tissue Services
Vox Sanguinis | Year: 2010

Background and Objectives The overall effectiveness of the NHSBT screening programme for infectious agents in deceased tissue donors is examined and evaluated in terms of current outcomes and how to improve upon these outcomes. Materials and Methods The screening results and any subsequent confirmatory results from a total of 1659 samples from NHSBT deceased donors referred to NTMRL for screening for infectious agents were included in the analysis. Results Overall 1566/1659 (94·4%) of the samples were screen negative. A total of 93 were repeat reactive on screening for one or more of the mandatory markers screened for, of which only 12 (13%) were subsequently confirmed to be positive on confirmatory testing. The majority of the repeat reactive samples were demonstrating non-specific reactivity with the screening assays in use. Conclusion Overall, the NHSBT screening programme for infectious agents in deceased tissue donors is very effective with a relatively low overall loss of donors because of non-specific reactivity. However, unnecessary loss of tissue products is not acceptable, and although this programme compares favourably with the outcomes of other such programmes, the confirmatory results obtained demonstrate both the need and the potential for improving the outcomes. This is particularly important as one donor may donate more than one product, and can be achieved very easily with a change to the screening algorithm followed, using the confirmatory data obtained to support and validate this change. Contents Summary Critical analysis of the NHSBT screening programme for infectious agents in deceased tissue donors and a strategy involving the design and use of a different screening algorithm to improve these outcomes. © 2009 International Society of Blood Transfusion.

Kitchen A.D.,National Transfusion Microbiology Reference Laboratory | Newham J.A.,National Transfusion Microbiology Reference Laboratory | Gillan H.L.,Tissue Services
Cell and Tissue Banking | Year: 2013

A comprehensive and effective screening programme is essential to support the banking of tissues from deceased donors. However, the overall quality of the samples obtained from deceased donors, quantity and condition, is often not ideal, and this may lead to problems in achieving accurate and reliable results. Additionally a significant percentage of referrals are still rejected upon receipt as unsuitable for screening. We are actively involved in improving the overall quality of deceased donor screening outcomes, and have specifically evaluated and validated both serological and molecular assays for this purpose, as well as developing a specific screening strategy to minimise the specificity issues associated with serological screening. Here we review the nature and effectiveness of the deceased donor screening programme implemented by National Health Service Blood and Transplant (NHSBT), the organisation with overall responsibility for the supply of tissue products within England. Deceased donor screening data, serological and molecular, from August 2007 until May 2012 have been collated and analysed. Of 10,225 samples referred for serology screening, 5.5 % were reported as reactive; of 2,862 samples referred for molecular screening, 0.1 % were reported as reactive/inhibitory. Overall 20 % of the serological and 100 % of the molecular screen reactivity was confirmed as reflecting true infection. The use of a sequential serology screening algorithm has resulted in a marked reduction of tissues lost unnecessarily due to non-specific screen reactivity. The approach taken by NHSBT has resulted in the development of an effective and specific approach to the screening of deceased tissue donors. © 2013 © Crown Copyright as represented by the NHS Blood and Transplant.

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