Human platelet lysate standardization across three independent European blood establishments

Abstract

Human platelet lysate (hPL) is a clinically safe alternative to fetal bovine serum (FBS). However, variability in blood donation practices, platelet concentrate preparation methods, storage and hPL manufacturing complicates standardization across jurisdictions. This study aimed to establish a first multinational hPL manufacturing standardization across three European blood centers to test feasibility and variability. A single batch of hPL production sets was distributed to the participating centers. There, hPL was produced following a single standard operating protocol but starting from each center's unique platelet concentrates. Each center prepared four 'national' hPL batches and four 'international' batches. Researchers conducted blinded quality and variation analyses to ensure unbiased results. All hPL batches exhibited comparable total protein levels, pH, ionic strength, and lactate content. Analysis of twelve growth factors showed minor variations across batches. Endothelial cell outgrowth and wound closure were slower in hPL than FBS but remained consistent across batches. Mesenchymal stem cell (MSC) doubling was significantly faster in hPL than in FBS, with MSC phenotype consistency confirmed via flow cytometry. Differentiation into adipogenic and osteogenic tissue was successful in all hPL samples. The inter-institutional variation across all national batches was higher for all critical outcome parameters compared to the variation in the four international batches. These findings confirm the feasibility of manufacturing standardized hPL across borders and show lower variability when doing so. This supports further efforts to stabilize hPL supply and advance cytotherapy standardization in Europe.

Keywords: Culture media; Human platelet lysate; Manufacturing; Mesenchymal stem cells; Standardization; Tissue culture.

Fig. 1

Study design. Each blood institution prepared 24 hPL products derived from 24 platelet concentrates using one hPL production set per concentrate. hPL was shipped to the central lab where 4 national ‘batches’ were prepared by pooling of 6 individual hPL products per country, symbolized by unicolored spheres per country (middle). In addition, the central lab prepared 4 international ‘batches’, consisting of 2 individual hPL products per country (bottom). From these ‘batches’, blinded samples were prepared and sent back to the participating centers for analysis

Fig. 2

hPL production set efficiency. A The success rate of PC conversion to hPL is shown. B hPL volume yield was the ratio of hPL volume over original PC volume. Data are shown as mean with standard deviation (n = 24), ****P < 0.0001. Two-letter country code is used for Belgium (BE), Iceland (IS) and the Netherlands (NL)

Fig. 3

Biochemical parameters in hPL. A pH, B ionized calcium, C osmolality, D glucose concentration and E lactate concentration were measured in undiluted hPL. The vertical dotted line is used to separate national batches (left) from international ones (right). Individual data are shown and error bars represent median with interquartile range (n = 4), *P < 0.05, **P < 0.01. Two-letter country code is used for Belgium (BE), Iceland (IS) and the Netherlands (NL). The international batch is labeled INT

Fig. 4

Growth factors and cytokines in hPL. A Luminex assay was used to quantify concentrations of A EGF, B FGF2, C PDGF-AA, D PDGF-AB/BB, E TGF-ß1, F TGF-ß2, G TGF-ß3, H VEGF, I IL-1a, J IL-6, K sCD40L and L TNF-α. The vertical dotted line is used to separate national batches (left) from international ones (right). Individual data are shown and error bars represent median with interquartile range (n = 4), *P < 0.05, **P < 0.01. Two-letter country code is used for Belgium (BE), Iceland (IS) and the Netherlands (NL). The international batch is labeled INT

Fig. 5

HUVEC proliferation and scratch assay. A All hPL batches and FBS were used for culturing HUVECs. Proliferation was tracked by measuring absorbance at 590 nm of cell staining with crystal violet on days 1, 2, 3 and 4 post seeding. Negative control was basal medium without supplement. B Scratch assays were performed with confluent HUVEC cultures in 2% hPL and C 10% hPL. The fraction of open wound is expressed as a function of time post scratch. Error bars represent median with interquartile range (n = 4), *P < 0.05. Two-letter country code is used for Belgium (BE), Iceland (IS) and the Netherlands (NL). The international batch is labeled INT

Fig. 6

Expansion of MSC and phenotypic characterization by flow cytometry. MSCs were cultured in hPL or FBS for five consecutive weeks. A Cumulative population doublings were recorded. The horizontal dotted line is set at cPD8, depicting the moment that cells were phenotyped by flow cytometry. B Cell doubling time of first five passages and C the growth kinetics during the linear phase of culture in panel A. The vertical dotted lines are used to separate national batches (left) from international ones (middle) and FBS (right). Error bars represent median with interquartile range (n = 4), ****P < 0.0001. MSCs were phenotyped using flow cytometry at cPD8 for D markers CD13, CD105, CD90 and CD73. E MSC should be negative for markers CD11b, CD19, CD34, CD45 and HLA-DR. Two-letter country code is used for Belgium (BE), Iceland (IS) and the Netherlands (NL). The international batch is labeled INT

Fig. 7

MSC differentiation potential. MSC were cultured in the presence of either hPL or FBS and subsequently differentiated in identical differentiation media. Adipogenic differentiation was analyzed using Oil Red O staining and Osteogenic differentiation by Von Kossa staining. The positive control is a biologically paired cell sampled cultured in FBS, differentiated and stained. The negative control is biologically paired cell sample that was equally stained, but not differentiated. Microscopic pictures were selected representative for each hPL batch group. Scale bar is 500 µm, total magnification 40×