Culture of human cell lines by a pathogen-inactivated human platelet lysate

Fazzina, Raffaella; Iudicone, Paola; Mariotti, Andrea; Fioravanti, Daniela; Procoli, Annabella; Cicchetti, Enrico Mazzeo; Scambia, Giovanni; Bonanno, Giuseppina; Pierelli, Luca

doi:10.1007/s10616-015-9878-5

Cited by 32 publications

(29 citation statements)

References 34 publications

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“…Recently, our group has developed a protocol for the production of a standardized pathogen-free human PL for clinical-grade expansion of BM-MSCs [56]. Moreover, we have also confirmed that PL is a feasible FBS substitute for supporting growth and propagation of UCT-MSCs and human cell lines [57, 58]. The availability of defined culture conditions, optimized by a very standardized growth stimulus such as our PL, is a unique opportunity to provide a qualified and reliable growth assay for each relevant tissue with mesodermal capacity.…”

Section: Introductionmentioning

confidence: 64%

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues

et al. 2016

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BackgroundMesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus.MethodsMSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics.ResultsThe use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro.ConclusionsThe adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

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Section: Introductionmentioning

confidence: 64%

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues

et al. 2016

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“…For example, human pathogens that escape screening by the providers may contaminate HPL samples. One possible way to address this risk would be to inactivate pathogens in HPL [ 33 ]. We did not inactivate pathogens in our pooled HPL, but the repeated freeze-thaw process followed by the filtration through a 0.2 um filter should remove all potential bacteria and parasites.…”

Section: Discussionmentioning

confidence: 99%

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP‐Compliant Medium and a Simplified Isolation Method

Smith

Pfeifer

Petry

et al. 2016

Stem Cells International

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Umbilical cord derived mesenchymal stromal cells (UC-MSCs) are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP) for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed-vessel dissociation following enzymatic digestion which reduces contamination risk and manipulation time. The new method produced >10 times more cells per cm of UC than our previous method. When biographical variables were compared, more UC-MSCs per gram were isolated after vaginal birth compared to Caesarian-section births, an unexpected result. UC-MSCs were expanded in medium enriched with 2%, 5%, or 10% pooled human platelet lysate (HPL) eliminating the xenogeneic serum components. When the HPL concentrations were compared, media supplemented with 10% HPL had the highest growth rate, smallest cells, and the most viable cells at passage. UC-MSCs grown in 10% HPL had surface marker expression typical of MSCs, high colony forming efficiency, and could undergo trilineage differentiation. The new protocol standardizes manufacturing of UC-MSCs and enables clinical translation.

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“…It is achievable to virally inactivate HPL (obtained from non–pathogen‐reduced PCs) using solvent/detergent (S/D) technology . Recently, pathogen reduction/virus inactivation treatments of PC based on photoinactivation have been licensed, making this type of material available for HPL preparation and suitable for propagation of MSCs . In this study, we evaluated for the first time the possibility to apply a S/D treatment to a HPL made from psoralen‐ultraviolet (UV)A (Intercept)‐treated PCs.…”

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A double‐virally‐inactivated (Intercept–solvent/detergent) human platelet lysate for in vitro expansion of human mesenchymal stromal cells

Barro

Nebie

et al. 2019

Transfusion

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BACKGROUND: Pooled human platelet lysate (HPL) can replace fetal bovine serum (FBS) as xeno-free supplement for ex vivo expansion of mesenchymal stromal cells (MSCs). We evaluate here whether a double-virallyinactivated HPL (DVI-HPL) prepared from expired Intercepttreated platelet concentrates (PCs) and treated by solvent/detergent (S/D) can be used for MSC expansion.STUDY DESIGN AND METHODS: Expired Intercepttreated PCs in 65% platelet (PLT) additive solution were pooled and subjected to a 1% tri-n-butyl phosphate/1% Triton X-45 treatment followed by soybean oil, hydrophobic interaction chromatography purification, and sterile filtration. Bone marrow-derived MSCs (BM-MSCs) were expanded for four passages in growth medium containing 10% DVI-HPL, I-HPL (from Intercept-PC only), untreated HPL, and FBS. MSC morphology, doubling time, immunophenotype, immunosuppressive activity, and differentiation capacity were compared. RESULTS:Expanded cells had typical spindle morphology and showed higher viability in all HPL conditions than in FBS. The DVI-HPL and FBS-expanded cells were morphologically larger than in I-HPL and HPL supplements. The cumulative population doubling was lower using DVI-HPL than with HPL and I-HPL, but significantly higher than using FBS. Immunophenotype was not affected by the supplements used. Immunosuppressive activity was maintained with all supplements. Differentiation capacity into chondrocytes and osteocytes was more effective in DVI-HPL but less toward adipocytes compared to other supplements. CONCLUSIONS: Human PLT lysate made fromIntercept-PCs subjected to S/D treatment may be an alternative to untreated HPL and to I-HPL for BM-MSC expansion. This finding reinforces the potential of HPL as a virally safe alternative to FBS for clinical grade MSC expansion protocols. ABBREVIATIONS: BDNF = brain-derived neurotrophic factor; BM-MSC(s) = bone marrow-derived mesenchymal stromal cells; BrdU = bromodeoxyuridine/5-bromo-2 0 -deoxyuridine; CCK-8 = cell counting kit-8; DVI = double-virally-inactivated; DVI-HPL = doublevirally-inactivated (intercept-solvent/detergent) human platelet lysate; ECM = extracellular matrix; EGF = epidermal growth factor FBS = fetal bovine serum; HGF = hepatocyte growth factor HLA-DR = human leukocyte antigen DR; HPL = human platelet lysate; IGF = insulin-like growth factor-1; I-HPL = Intercept human platelet lysate; MSC(s) = mesenchymal stromal cells; PAS = platelet additive solution; PB19V = parvovirus B19; PC(s) = platelet concentrate(s); PD (s) = population doubling(s); PDGF-AB = platelet-derived growth factor-AB; PF4 = platelet factor 4; PHA = phytohemagglutinin; PPARG = peroxisome proliferator-activated receptor gamma; SOX9 = SRY (sex-determining region Y)-box 9; SPP1 = secreted phosphoprotein 1. From the

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Culture of human cell lines by a pathogen-inactivated human platelet lysate

Cited by 32 publications

References 34 publications

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP‐Compliant Medium and a Simplified Isolation Method

A double‐virally‐inactivated (Intercept–solvent/detergent) human platelet lysate for in vitro expansion of human mesenchymal stromal cells

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