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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.

SUBMITTER: Christensen C

PROVIDER: S-EPMC7159197 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Christensen Christian C Jonsdottir-Buch Sandra Mjoll SM Sigurjonsson Olafur Eysteinn OE

PloS one 20200415 4

<h4>Background</h4>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 transf ...[more]

PMID: 32294080

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Project description:BACKGROUND:Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson's disease models. METHODS:Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (- 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot. RESULTS:Platelet lysates contained the expected level of total proteins (ca. 7-14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death. CONCLUSION:Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.

Project description:Patients affected by severe coronavirus induced disease-2019 (Covid-19) often experience hypoxemia due to alveolar involvement and endothelial dysfunction, which leads to the formation of micro thrombi in the pulmonary capillary vessels. Both hypoxemia and a prothrombotic diathesis have been associated with more severe disease and increased risk of death. To date, specific indications to treat this condition are lacking. This was a single center, investigator initiated, compassionate use, proof of concept, case control, phase IIb study (NCT04368377) conducted in the Intermediate Respiratory Care Unit of L. Sacco University Hospital in Milano, Italy. Our objective was to explore the effects of the administration of anti-platelet therapy on arterial oxygenation and clinical outcomes in patients with severe Covid-19 with hypercoagulability. We enrolled five consecutive patients with laboratory confirmed SARS-CoV-2 infection, severe respiratory failure requiring helmet continuous positive airway pressure (CPAP), bilateral pulmonary infiltrates and a pro-thrombotic state identified as a D-dimer > 3 times the upper limit of normal. Five patients matched for age, D-dimer value and SOFA score formed the control group. Beyond standard of care, treated patients received 25 μg/Kg/body weight tirofiban as bolus infusion, followed by a continuous infusion of 0.15 μg/Kg/body weight per minute for 48 hours. Before tirofiban, patients received acetylsalicylic acid 250 mg infusion and oral clopidogrel 300 mg; both were continued at a dose of 75 mg daily for 30 days. Fondaparinux2.5 mg/day sub-cutaneous was given for the duration of the hospital stay. All controls were receiving prophylactic or therapeutic dose heparin, according to local standard operating procedures. Treated patients consistently experienced a mean (SD) reduction in A-a O2 gradient of -32.6 mmHg (61.9, P = 0.154), -52.4 mmHg (59.4, P = 0.016) and -151.1 mmHg (56.6, P = 0.011; P = 0.047 vs. controls) at 24, 48 hours and 7 days after treatment. PaO2/FiO2 ratio increased by 52 mmHg (50, P = 0.172), 64 mmHg (47, P = 0.040) and 112 mmHg (51, P = 0.036) after 24, 48 hours and 7 days, respectively. All patients but one were successfully weaned from CPAP after 3 days. This was not true for the control group. No major adverse events were observed. Antiplatelet therapy might be effective in improving the ventilation/perfusion ratio in Covid-19 patients with severe respiratory failure. The effects might be sustained by the prevention and interference on forming clots in lung capillary vessels and by modulating megakaryocytes' function and platelet adhesion. Randomized clinical trials are urgently needed to confirm these results.

Dataset Information

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

Publications

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

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