scholarly journals Strategy for the Generation of Engineered Bone Constructs Based on Umbilical Cord Mesenchymal Stromal Cells Expanded with Human Platelet Lysate

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Ingrid Silva-Cote ◽  
Mónica Cruz-Barrera ◽  
Mariana Cañas-Arboleda ◽  
Luz Correa-Araujo ◽  
Leidi Méndez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Growth Performance ◽  
Stromal Cells ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Clinical Use ◽  
Lineage Differentiation ◽  
Human Platelet Lysate

Umbilical cord mesenchymal stromal cells (UC-MSC) are promising candidates for cell therapy due to their potent multilineage differentiation, enhanced self-renewal capacity, and immediate availability for clinical use. Clinical experience has demonstrated satisfactory biosafety profiles and feasibility of UC-MSC application in the allogeneic setting. However, the use of UC-MSC for bone regeneration has not been fully established. A major challenge in the generation of successful therapeutic strategies for bone engineering lies on the combination of highly functional proosteogenic MSC populations and bioactive matrix scaffolds. To address that, in this study we proposed a new approach for the generation of bone-like constructs based on UC-MSC expanded in human platelet lysate (hPL) and evaluated its potential to induce bone structures in vivo. In order to obtain UC-MSC for potential clinical use, we first assessed parameters such as the isolation method, growth supplementation, microbiological monitoring, and cryopreservation and performed full characterization of the cell product including phenotype, growth performance, tree-lineage differentiation, and gene expression. Finally, we evaluated bone-like constructs based on the combination of stimulated UC-MSC and collagen microbeads for in vivo bone formation. UC-MSC were successfully cultured from 100% of processed UC donors, and efficient cell derivation was observed at day 14±3 by the explant method. UC-MSC maintained mesenchymal cell morphology, phenotype, high cell growth performance, and probed multipotent differentiation capacity. No striking variations between donors were recorded. As expected, UC-MSC showed tree-lineage differentiation and gene expression profiles similar to bone marrow- and adipose-derived MSC. Importantly, upon osteogenic and endothelial induction, UC-MSC displayed strong proangiogenic and bone formation features. The combination of hPL-expanded MSC and collagen microbeads led to bone/vessel formation following implantation into an immune competent mouse model. Collectively, we developed a high-performance UC-MSC-based cell manufacturing bioprocess that fulfills the requirements for human application and triggers the potency and effectivity of cell-engineered scaffolds for bone regeneration.

Human Platelet Lysate Sustains the Osteogenic/Adipogenic Differentiation Potential of Adipose-Derived Mesenchymal Stromal Cells and Maintains Their DNA Integrity in vitro

2019 ◽  
Vol 207 (3-4) ◽  
pp. 149-164 ◽  
Author(s):  
Yaroslav D. Shansky ◽  
Natalia S. Sergeeva ◽  
Irina K. Sviridova ◽  
Pavel A. Karalkin ◽  
Valentina A. Kirsanova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Mesenchymal Stromal Cells ◽  
Cell Morphology ◽  
Stromal Cells ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Dna Integrity ◽  
Differentiation Potential ◽  
Human Platelet Lysate

Human platelet lysate (HPL) is a promising alternative to fetal calf serum (FCS) for the expansion of adipose tissue mesenchymal stromal cells (AT-MSCs) for translational medicine applications. However, some biological effects of HPL are still to be elucidated. We aimed to compare complex characteristics, such as cell morphology, proliferative activity, differentiation potential, and especially monolayer recovery, DNA integrity, and the gene expression pattern, between AT-MSCs cultured with HPL or FCS. Primary AT-MSC cultures were expanded in medium containing FCS or pooled HPL. Cell growth and proliferation were estimated by cell doubling time and the monolayer formation rate, while migration was assessed by wound-healing assay. The capacity for adipogenic and osteogenic differentiation was evaluated by alkaline phosphatase and Oil Red O staining. DNA integrity was evaluated by comet assay, and analysis of gene expression by real-time PCR. Media supplemented with HPL or FCS provided a similar surface immunophenotype, cell morphology (except some cell dimensions and a bigger colony size in HPL), DNA integrity, and rate of wound healing. Meanwhile, AT-MSC proliferated more intensively in HPL-supplemented media (especially at 5% HPL) and had a reduced doubling population time. AT-MSC in HPL had increased adipogenic potential and similar osteogenic potential in comparison with FCS. Our results indicate the feasibility and evident prospects for the use of pooled HPL as an alternative to FCS and safe non-xenogenic growth supplement for ex vivo expansion of clinical-grade AT-MSCs for regenerative medicine purposes.

Expansion of Adipose Mesenchymal Stromal Cells is Affected by Human Platelet Lysate and Plating Density

2011 ◽  
Vol 20 (9) ◽  
pp. 1409-1422 ◽  
Author(s):  
Dominik Cholewa ◽  
Thomas Stiehl ◽  
Anne Schellenberg ◽  
Gudrun Bokermann ◽  
Sylvia Joussen ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Plating Density ◽  
Human Platelet Lysate

scholarly journals Influence of platelet storage time on human platelet lysates and platelet lysate-expanded mesenchymal stromal cells for bone tissue engineering

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Siddharth Shanbhag ◽  
Samih Mohamed-Ahmed ◽  
Turid Helen Felli Lunde ◽  
Salwa Suliman ◽  
Anne Isine Bolstad ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Bone Tissue Engineering ◽  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Stromal Cells ◽  
Storage Time ◽  
Human Platelet ◽  
Scaling Up ◽  
Platelet Lysate ◽  
Lineage Differentiation

Abstract Background Human platelet lysate (HPL) is emerging as the preferred xeno-free supplement for the expansion of mesenchymal stromal cells (MSCs) for bone tissue engineering (BTE) applications. Due to a growing demand, the need for standardization and scaling-up of HPL has been highlighted. However, the optimal storage time of the source material, i.e., outdated platelet concentrates (PCs), remains to be determined. The present study aimed to determine the optimal storage time of PCs in terms of the cytokine content and biological efficacy of HPL. Methods Donor-matched bone marrow (BMSCs) and adipose-derived MSCs (ASCs) expanded in HPL or fetal bovine serum (FBS) were characterized based on in vitro proliferation, immunophenotype, and multi-lineage differentiation. Osteogenic differentiation was assessed at early (gene expression), intermediate [alkaline phosphatase (ALP) activity], and terminal stages (mineralization). Using a multiplex immunoassay, the cytokine contents of HPLs produced from PCs stored for 1–9 months were screened and a preliminary threshold of 4 months was identified. Next, HPLs were produced from PCs stored for controlled durations of 0, 1, 2, 3, and 4 months, and their efficacy was compared in terms of cytokine content and BMSCs’ proliferation and osteogenic differentiation. Results BMSCs and ASCs in both HPL and FBS demonstrated a characteristic immunophenotype and multi-lineage differentiation; osteogenic differentiation of BMSCs and ASCs was significantly enhanced in HPL vs. FBS. Multiplex network analysis of HPL revealed several interacting growth factors, chemokines, and inflammatory cytokines. Notably, stem cell growth factor (SCGF) was detected in high concentrations. A majority of cytokines were elevated in HPLs produced from PCs stored for ≤ 4 months vs. > 4 months. However, no further differences in PC storage times between 0 and 4 months were identified in terms of HPLs’ cytokine content or their effects on the proliferation, ALP activity, and mineralization of BMSCs from multiple donors. Conclusions MSCs expanded in HPL demonstrate enhanced osteogenic differentiation, albeit with considerable donor variation. HPLs produced from outdated PCs stored for up to 4 months efficiently supported the proliferation and osteogenic differentiation of MSCs. These findings may facilitate the standardization and scaling-up of HPL from outdated PCs for BTE applications.

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