Effects of amotosalen treatment on human platelet lysate bioactivity: A proof-of-concept study

Abstract

Background: Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation.

Methodology/principal findings: Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates.

Conclusion/significance: These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing.

Fig 1. Long-term proliferation of MSCs, expressed as cumulative population doublings.

Proliferation of MSCs from two donors (D6 and D13) was evaluated in cell culture media supplemented with hPL-PIPC or hPL-PIPL after passage 3 (indicated by the vertical dotted line). Points represent mean ± SEM at the end of each passage (n = 6 cell cultures per passage, assessed in two independent experiments). Asterisks (* p < 0.05) indicate statistical significance between hPL-PIPC and hPL-PIPL for an individual donor, evaluated via a two-way ANOVA with Tukey’s post hoc test.

Fig 2. Alkaline phosphatase activity during osteogenic differentiation.

MSCs were differentiated in osteogenic media supplemented with hPL-PIPL or hPL-PIPC, with MSCs grown in expansion media included as a control. Points represent means ± SEM (n = 6 cell cultures per timepoint, assessed in two independent experiments). Asterisks (* p < 0.05) indicate statistical significance versus the control, evaluated via a two-way ANOVA with Tukey’s post hoc test.

Fig 3. Tri-lineage differentiation of MSCs.

MSCs differentiated using hPL-PIPC are shown in A, C, and E, while MSCs differentiated using hPL-PIPL are shown in B, D, and F. A and B show Alizarin Red S staining, used to demonstrate mineralization (black arrows) after 28 days of stimulation in osteogenic medium. C and D show Oil Red O staining, used to demonstrate accumulation of lipid droplets (black arrows) after 14 days of stimulation in adipogenic medium. E and F show Masson’s trichrome staining, used to demonstrate collagen fibers (black arrows) and lacunae formation (yellow arrows) after 35 days of chondrogenic stimulation.

Fig 4. Concentration of glycosaminoglycans.

GAG concentration was measured in MSC pellets stimulated with chondrogenic media supplemented with either hPL-PIPC or hPL-PIPL. Pellets grown in expansion media were included as a control. Points represent mean ± SEM (n = 2 pellets per timepoint, assessed in two individual experiments). Asterisks (* p < 0.05) indicate statistical significance versus the control, evaluated via a two-way ANOVA with Tukey’s post hoc test.