Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells

Abstract

Background aims: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrowÀderived MSCs. Methods: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35-and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. Results: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. Conclusions: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.

Human Platelet Lysate Supports Efficient Expansion and Stability of Wharton’s Jelly Mesenchymal Stromal Cells via Active Uptake and Release of Soluble Regenerative Factors

“…Here we observed that WJ-MSC expanded in hPL enhanced their proliferative capacity, observing an increase of 33.3% in PD and consequent reduction of PDT (by 38.1%), as compared to WJ-MSC expanded in hS or FBS. Several studies described mixed proliferation rates of different MSC types following hPL supplementation [ 13 , 22 , 31 , 33 ]. In particular Kandoi et al evidenced significantly faster cell growth of umbilical cord tissue-derived MSC in hPL-enriched cultures, reaching PDT of 20.95 h vs. PDT in FBS [ 5 ].…”

“…Therefore, the inclusion of techniques that lead to pathogen inactivation without altering the characteristics of hPL is vital to produce GMP grade supplements. Recently, protocols for pathogen inactivation including solvent/detergent, photoinactivation or nanofiltration technology have been tested, proving to be innocuous for the prepared hPL in terms of factors composition and mechanism of action [ 12 , 13 ]. In that regard, one of the limitations of the present study deals with the absence of methodologies for pathogen inactivation and their impact on the stability of soluble factors present in hPL batches and the biological activity of WJ-MSC.…”

“…Importantly, the use of allogeneic donors has become an attractive approach for hPL generation, exhibiting several advantages over autologous preparations such as low batch-to-batch variations via donor pooling, increased volume availability, reproducibility during production and affordable manufacturing and release process [ 4 , 8 ]. However, larger pool sizes might also enhance the risk of transmitting infectious agents, introducing the need of inactivation or removal of viruses during hPL production [ 8 , 12 , 13 ]. More importantly, correct characterization of pooled-hPL batches in terms of potency, stability and content, has not been fully explored in the context of MSC production.…”

Developing a Glyoxal-Crosslinked Chitosan/Gelatin Hydrogel for Sustained Release of Human Platelet Lysate to Promote Tissue Regeneration

“…Therefore, platelet lysates have demonstrated the advantages of replacing fetal bovine serum (FBS) for culturing various types of cells, including stem cells, fibroblasts and endothelial cells [ 1 , 12 ]. The use of HPL in cell culture is desired for clinical use by circumventing the risks of xeno-immunization against bovine antigens and the transmission of zoonotic diseases [ 13 , 14 ]. Moreover, the effect of HPL on directly promoting angiogenesis and tissue regeneration has been demonstrated [ 15 , 16 ], so HPL holds great promise for treating various tissue defects.…”

Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets

“…The aforementioned studies employed fetal bovine serum (FBS) for ASC culture during cell sheet fabrication. However, due to the risks of xeno-immunization against bovine antigens, transmission of zoonotic diseases, and potential limits of availability, it was necessary to find suitable human alternatives for the manufacture of ASC sheets to meet clinical therapeutic purposes (Mackensen et al, 2000;Horwitz et al, 2002;Schallmoser and Strunk, 2013;Barro et al, 2020). Human platelet lysate (HPL) contains a number of mitogenic growth factors, including fibroblast growth factor (FGF), endothelial growth factor (EGF), platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF; Fekete et al, 2012;Chen et al, 2018).…”