| 摘要: | Background: Human platelet lysates (HPL) can replace fetal bovine serum (FBS) for clinical-grade ex vivo expansion of cells, including mesenchymal stem cells (MSC). HPL contains platelet-derived growth factors that support cell growth and proliferation. HPL avoids the immunological and infectious risks associated with xenogenic FBS materials. However, in spite of the safety nets in place for donors screening and donation testing, infectious agents, including viruses and prions, can contaminate HPL. Therefore, HPL manufacturing processes should preferably undergo virus and prion reduction steps to ensure optimal pathogen safety. Recently, a new anion-exchange hollow-fiber chromatographic filtration membrane (QyuSpeed D, QSD) was shown to remove prions from growth media containing 10% FBS. We hypothesized that such QSD device could be used for removing pathogens from HPL.
Aims: Determine whether growth media supplemented with HPL could be filtered through QSD filtration membrane under conditions (a) not affecting ex vivo cell expansion capacity and (b) removing blood-borne pathogens.
Materials and Methods: HPL batches were prepared from various types of platelet concentrates subjected to 3 freeze/thaw cycles, “serum-converted” by calcium chloride, and centrifuged. Growth media were supplemented with 10% HPL and ca. 50 mL, or more, filtered through 0.6 mL QSD hollow fiber. HPL-supplemented growth media composition was determined by total protein assay, SDS-PAGE, growth factors ELISA assay and chemical analysis. Capacity of HPL-growth media, prior to or after QSD filtration, to support cell growth was compared using Chinese hamster ovary cells, gingival fibroblasts and periodontal ligament primary cells, and Wharton jelly cord blood (WJ) MSC. Cell expansion was evaluated by microscopy, cell counting, and MTT viability assays. Membrane markers were studied by flow cytometry. Doubling time and differentiation capacity of WJMSCs into adipogenic, osteogenic and chondrogenic lineages were determined. QSD removal capacity for prions and viruses was tested (duplicate runs each) with 97.4 ± 1.9 mL of growth medium containing 10% HPL after spiking with strain 263K hamster adapted scrapie (microsomal/cytosolic fraction) or Porcine Parvovirus (PPV), respectively. A Western blot assay was used for detecting PrPSc as a surrogate marker for infectivity. PPV infectivity was assessed by TCID50 infectivity assay on PK13 cells.
Results: QSD filtration did not induce detectable adverse effect on the chemical, protein and growth factor composition of culture media supplemented with 10% HPL, nor on the growth and viability of the four cells evaluated. Mean reduction factors (logs) achieved by QSD were ≥ 5.42 ± 0.44 for 2% PPV and ≥ 3.47 for 2% 263K for the whole flow-through volume (100 mL).
Summary/conclusions: HPL-supplemented growth media can be filtered through QSD anion-exchange hollow fiber membrane to remove prions and PPV under conditions that do not affect the capacity of HPL-growth media to stimulate ex vivo expansion of various cells, including MSC. This study opens the way to novel HPL-supplemented growth media with preserved capacity to support cell expansion, while also improving the level of pathogen safety for applications in cell therapy or in the production of therapeutic protein products made by recombinant technologies using mammalian cells. |
