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- Barro, Lassina, et al.
(författare)
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Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells
- 2020
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Ingår i: Cytotherapy. - : Elsevier BV. - 1465-3249 .- 1477-2566. ; 22:8, s. 458-472
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Tidskriftsartikel (refereegranskat)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 marrow-derived 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, membranemarkers, 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.
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- Barro, Lassina, et al.
(författare)
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Removal of minute virus of mice-mock virus particles by nanofiltration of culture growth medium supplemented with 10% human platelet lysate
- 2021
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Ingår i: Cytotherapy. - : Elsevier. - 1465-3249 .- 1477-2566. ; 23:10, s. 902-907
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Tidskriftsartikel (refereegranskat)abstract
- Declaration of Competing Interest NW and MT are employees of Asahi Kasei Medical. TB is the treasurer of the Working Party on Cellular Therapies of the International Society of Blood Transfusion. Funding This study was partially funded by Asahi Kasei Medical, Tokyo, Japan, through a research agreement with Taipei Medical University. The sponsor played no role in data collection, analysis or interpretation; manuscript writing; or the decision to submit the article for publication. Author Contributions Conception and design of the study: TB, NK, MT, FK. Acquisition of data: LB, ON, LD, YWW. Analysis and interpretation of data: LB, ON, LD, YWW, NK, MT, TB. Drafting or revising the manuscript: LB, TB, FK, NK, MT. All authors have approved the final article. Acknowledgments The authors thank the Uppsala University blood bank, Uppsala, Sweden, and Taipei Blood Center, Guandu, Taiwan, for supplying platelet concentrates.
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- Begum, Setara, 1974, et al.
(författare)
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Characterization and engraftment of long-term serum-free human fetal liver cell cultures.
- 2010
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Ingår i: Cytotherapy. - : Elsevier BV. - 1477-2566 .- 1465-3249. ; 12:2, s. 201-11
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AIMS: Cultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity. METHODS: Using cell culture and molecular techniques, we studied the in vitro and in vivo characteristics of FLC grown long-term in serum-free conditions. RESULTS: Serum-free FLC obtained from 6-10-week-old human fetal livers grew as multiple clusters in suspension and could be subcultured for at least six passages. These cells maintained stable hepatocyte phenotypes and gene expression patterns in culture for up to 6 months. When a cluster of these cells in various passages was placed on collagen-coated plates, they formed a monolayer and morphologically resembled hepatocytes. The cells expressed alpha -fetoprotein, cytokeratin (CK) 8, CK18 and CK19 and albumin (ALB). Hepatocyte nuclear factor 4alpha and 1beta and cytochrome P450 (CYP) 3A4 and CYP3A7 mRNA expression was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells at different passages, when transplanted into nude mice with liver injury, engrafted successfully, as detected by in situ hybridization using a human-specific DNA probe. Colonies of human-specific CK8, CK18, c-Met nuclear antigen (Ag), mitochondrial Ag, hepatocyte-specific Ag and ALB-expressing cells were present in the livers of recipient animals. CONCLUSIONS: Primary human FLC can be kept in culture consistently over a long time period and are potential candidates for cell therapy and in vitro diagnostics.
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- Beljanski, Vladimir, et al.
(författare)
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Pleiotropic roles of autophagy in stem cell-based therapies
- 2019
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Ingår i: Cytotherapy. - : ELSEVIER SCI LTD. - 1465-3249 .- 1477-2566. ; 21:4, s. 380-392
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Forskningsöversikt (refereegranskat)abstract
- Stem cells (SCs) have been proven to possess regenerative and immunomodulatory properties and can be used to treat diseases that involve loss of cells due to tissue damage or inflammation. For this approach to succeed, SCs or their derivatives should be able to engraft in the target tissue at least for a short period of time. Unfortunately, once injected, therapeutic SCs will encounter a hostile environment, including hypoxia, lack of nutrients and stromal support, and cells may also be targeted and rejected by the immune system. Therefore, SC's stress-response mechanisms likely play a significant role in survival of injected cells and possibly contribute to their therapeutic efficacy. Autphagy, a stress-response pathway, is involved in many different cellular processes, such as survival during hypoxia and nutrient deprivation, cellular differentiation and de-differentiation, and it can also contribute to their immunovisibility by regulating antigen presentation and cytokine secretion. Autophagy machinery interacts with many proteins and signaling pathways that regulate SC properties, including PI3K/Akt, mammalian target of rapamycin (mTOR), Wnt, Hedgehog and Notch, and it is also involved in regulating intracellular reactive oxygen species (ROS) levels. In this review, we contend that autophagy is an important therapeutic target that can be used to improve the outcome of SC-based tissue repair and regeneration. Further research should reveal whether inhibition or stimulation of autophagy increases the therapeutic utility of SCs and it should also identify appropriate therapeutic regimens that can be applied in the clinic.
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- Brohlin, Maria, 1966-, et al.
(författare)
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Effects of a defined xeno-free medium on the growth and neurotrophic and angiogenic properties of human adult stem cells
- 2017
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Ingår i: Cytotherapy. - : ELSEVIER SCI LTD. - 1465-3249 .- 1477-2566. ; 19:5, s. 629-639
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Tidskriftsartikel (refereegranskat)abstract
- Background. The growth properties and neurotrophic and angiogenic effects of human mesenchymal stromal cells (MSCs) cultured in a defined xeno-free, serum-free medium (MesenCult-XF) were investigated. Methods. Human MSCs from adipose tissue (ASCs) and bone marrow (BMSCs) were cultured in Minimum Essential Medium-alpha (alpha-MEM) containing fetal calf serum or in MesenCult-XF. Proliferation was measured over 10 passages and the colony-forming unit (CFU) assay and expression of cluster of differentiation (CD) surface markers were determined. Neurite outgrowth and angiogenic activity of the MSCs were determined. Results. At early passage, both ASCs and BMSCs showed better proliferation in MesenCult-XF compared with standard a-MEM containing serum. However, CFUs were significantly lower in MesenCult-XF. ASCs cultured in MesenCult-XF continued to expand at faster rates than cells grown in serum. BMSCs showed morphological changes at late passage in MesenCult-XF and stained positive for senescence beta-galactosidase activity. Expression levels of CD73 and CD90 were similar in both cell types under the various culture conditions but CD105 was significantly reduced at passage 10 in MesenCult-XF. In vitro stimulation of the cells enhanced the expression of brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF-A) and angiopoietin-1. Stimulated ASCs grown in MesenCult-XF evoked the longest neurite outgrowth in a neuron co-culture model. Stimulated BMSCs grown in MesenCult-XF produced the most extensive network of capillary-like tube structures in an in vitro angiogenesis assay. Conclusions. ASCs and BMSCs exhibit high levels of neurotrophic and angiogenic activity when grown in the defined serum free medium indicating their suitability for treatment of various neurological conditions. However, long-term expansion in MesenCult-XF might be restricted to ASCs.
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