Characterization of human platelet lysate (HPLs) for dedicated clinical uses, and assessment of the neuroprotective and neuro-regenerative therapeutic functions of nanofiltered virus-safe heated-HPL and isolated platelet-extracellular vesicles

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Title:	Characterization of human platelet lysate (HPLs) for dedicated clinical uses, and assessment of the neuroprotective and neuro-regenerative therapeutic functions of nanofiltered virus-safe heated-HPL and isolated platelet-extracellular vesicles
Authors:	DELILA, LILING
Contributors:	生醫材料暨組織工程研究所博士班 BURNOUF, THIERRY DEVOS, DAVID
Keywords:	Human platelet lysates;Regenerative medicine;Virus removal step;Extracellular vesicles;Neuroprotection
Date:	2023-06-21
Issue Date:	2023-12-07 09:39:18 (UTC+8)
Abstract:	Background: Human platelet lysates (HPLs)-based biotherapies, manufactured from clinical grade platelet concentrates (PCs) are emerging as a novel and translational therapeutic strategy in regenerative medicine. HPLs are rich in bioactive molecules and healing factors and their content of platelet-extracellular vesicles (EVs) is gaining attention. Tailored HPL manufacturing methods such as Heat-Treated Human Platelet Lysate (HPPL), was shown to provide robust neuroprotection and neuro-restoration in Parkinson's disease (PD) and traumatic brain injury (TBI) models in-vitro and in-vivo. However, the impact of different HPL preparation methods on composition and functionality is not well-understood, particularly for specific therapeutic purposes. The virus safety is also a concern especially when HPL is prepared by pooling PC donations. Nanofiltration is a known effective technique to remove a viruses from plasma products but has never been evaluated on HPL. Aims: to assess (a) the composition and functional activity contributed by proteins and EVs of various HPLs intended for specific clinical indications; (b) the virus removal efficacy and impact of 19-nm nanofiltration on HPPL compositions and neuroprotective and neuro-regenerative functions; and (c) the bio-physical characterization, biological functions, and neuroprotective and neuroregenerative roles of selected PEVs preparations. Methods: PCs from Taipei Blood Center, Taiwan were processed into five different HPLs. HPL characteristics, including protein content, growth factors, biochemical composition, thrombin activity, PEVs functional activity, and anti-oxidative properties were determined. Furthermore, HPPL was nanofiltratered using 19-nm pore size Planova 20N (Asahi Kasei, Japan), and virus reduction value of mock Murine Virus of Mice, (MMV) and impact of on HPPL proteins and EVs compositions, determined. The functional activity of the nanofiltered HPPL was evaluated in vitro and in vivo. We also prepared platelet-EVs from PC supernatant (PCS-EVs). EVs were pelletized by high-speed centrifugation, and size, number, growth factors, and protein compositions characterized. In vitro assessment of neuroprotective, neurorestorative and anti-inflammatory activities of PCS-EVs were done using LUHMES dopaminergic neuron, differentiated neuroblastoma SH-SY5Y and BV-2 microglia cells, respectively. The functional activity of intranasally administered PCS-EVs was assessed in controlled cortical impact (CCI)-TBI and MPTP-PD mouse models. Results: All five HPL preparations had supraphysiological levels of growth factors, apart IGF-1, present only in plasma-containing HPLs. Significant differences among HPLs were found in protein content, biochemical properties, coagulant activity, and antioxidative capacity, indicating that careful design and evaluation of HPLs based on intended therapeutic use are crucial. HPPL was confirmed to be suitable for brain administration due to its enriched growth factors, depleted plasma proteins, and low pro-coagulant activity. Nanofiltration effectively removed the mock MMV, while preserving neurotrophic factors. Despite some proteins and EVs removal, HPPL neuroprotective and anti-inflammatory functions were preserved. PCS-EVs contained a range of growth factors, cytokines, and antioxidants. They exhibited neuroprotective activity in LUHMES, supported neuronal cell restoration in SH-SY5Y, and showed anti-inflammatory activity in BV-2. Intranasal PCS-EVs ameliorated inflammation in CCI-TBI mice and exerted neuroprotective in the PD mice model. Conclusion: Our study highlights the importance of considering HPL profile differences for clinical purposes. Nanofiltration can safely remove viruses from HPPL without compromising its functional activities. PCS-EVs show robust neuroprotective activity, offering a potential for biotherapies.
Description:	博士指導教授:BURNOUF, THIERRY 共同指導教授:DEVOS, DAVID 委員:BURNOUF, THIERRY 委員:DEVOS, DAVID 委員:曾靖孋委員:周思怡委員:BLUM, DAVID
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Biomedical Materials and Tissue Engineering] Dissertation/Thesis

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