Comparative study of different centrifugation protocols for a density gradient separation media in isolation of osteoprogenitors from bone marrow aspirate

Naung, Noel Ye; Suttapreyasri, Srisurang; Kamolmatyakul, Suttatip; Nuntanaranont, Thongchai

doi:10.1016/j.jobcr.2014.11.004

Cited by 15 publications

(9 citation statements)

References 23 publications

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“…The study will be parameterized using a study reporting the multipassage expansion of bone marrow mesenchymal stem/stromal cells (BM-MSCs) with DMEM + 10% FBS and with DMEM + 10% hPL using several donors (Table 1) [36,54]. To estimate growth rates and isolation yields, we used studies related to the yield of MSCs per seeded MNCs obtained from the isolation of bone marrow aspirates using the Ficoll-Paque density method [20,21]. Simulations will consider up to five passages and a seeding density of 5000 cells/cm 2 .…”

Section: Case Study Definitionmentioning

confidence: 99%

“…• flasks undergo simulated media changes until reaching confluence, as specified by confluence times and yields of MSCs per MNCs seeded [21,22]. After obtaining the initial MSCs population, expansion starts in order to multiply the cell number until the target number of cells is reached.…”

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confidence: 99%

“…Finally, release testing of the batch is performed. Biological variability is input in the model at the level of the number of MNCs per bone marrow aspirate volume [21], the yield of MSCs per MNCs seeded [22] and the multipassage harvesting densities at confluence [53]. The published experimental data were used to derive probabilistic distributions (Supplementary Tables 4 & 5) from which these parameters are sampled for each donor.…”

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confidence: 99%

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Modeling Biological and Economic Uncertainty on Cell Therapy Manufacturing: the Choice of Culture Media Supplementation

2018

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To evaluate the cost-effectiveness of autologous cell therapy manufacturing in xeno-free conditions. Materials & methods: Published data on the isolation and expansion of mesenchymal stem/stromal cells introduced donor, multipassage and culture media variability on cell yields and process times on adherent culture flasks to drive cost simulation of a scale-out campaign of 1000 doses of 75 million cells each in a 400 square meter Good Manufacturing Practices facility. Results & conclusion: Passage numbers in the expansion step are strongly associated with isolation cell yield and drive cost increases per donor of $1970 and 2802 for fetal bovine serum and human platelet lysate. Human platelet lysate decreases passage numbers and process costs in 94.5 and 97% of donors through lower facility and labor costs. Cost savings are maintained with full equipment depreciation and higher numbers of cells per dose, highlighting the number of cells per passage step as the key cost driver.

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Section: Case Study Definitionmentioning

confidence: 99%

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Modeling Biological and Economic Uncertainty on Cell Therapy Manufacturing: the Choice of Culture Media Supplementation

2018

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“…The PBMCs were separated by density gradient, as previously described [17], and cultured in standard media (RPMI1640, supplemented with FBS 10%, penicillin 100 IU/mL and streptomycin 100 μg/mL). To evaluate the IL12RB1 expression by fluorescence-activated cell sorting (FACS), cells were stimulated by 20 µg phytohemagglutinin (Becton Dickinson) for 72 h to generate phytohemagglutinin (PHA)-activated T cells.…”

Section: Methodsmentioning

confidence: 99%

IL-12Rβ1 deficiency corresponding to concurrency of two diseases, mendelian susceptibility to mycobacterial disease and Crohn's disease

Khoshnevisan

Nekooei-marnany

Klein³

et al. 2019

Journal of Clinical Tuberculosis and Other Mycobacterial Diseas

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BackgroundThe interleukin-12 receptor β1 (IL-12Rβ1) deficiency is a primary immunodeficiency (PID), affecting the immunological pathway of interleukin 12/interferon- γ (IL12/IFN-γ) axis and interleukin 23 receptor (IL23R). Defect in this pathway is mainly affecting the cellular immunity-related disorders. IL-12Rβ1 is a receptor chain of both the IL-12 and the IL-23 receptors and thus, deficiency of IL-12Rβ1 abolishes both IL-12 and IL-23 signaling.Material and methodsIn this study, we performed whole exon sequencing and confirmatory Sanger sequencing in IL-12Rβ1. Evaluation of the IL12/IFN-γ axis was performed by assessment of patients’ whole blood cell to IL12/IFN-γ responding. Total and surface IL-12Rβ1expression was evaluated, in peripheral blood mononuclear cells (PBMCs) and T cell- derived PBMCs, and Th17 count was assessed.ResultsIn the present study, we described a c.1791 + 2T > G mutation at a splicing site position in IL-12Rβ1, using whole exome sequencing, and confirmed with targeted Sanger sequencing in a 26- year-old patient with Mendelian susceptibility to mycobacterial disease (MSMD) and Crohn's disease (CD). Complete lack of IL-12Rβ1 protein expression was detected in patient's PBMCs, compared to the healthy control. Furthermore, no IL-12Rβ1 protein was expressed on the cell surface. Interestingly, IL-12Rβ1-mutant cells showed an impaired response to IL12, and Bacillus Calmette–Guérin stimulation, confirming that the mutation is causative in this patient.ConclusionA 3′splicing site mutation in IL12Rβ1, can be corresponding to the abolished expression of IL12Rβ1 in patients' cells, and associated with an impaired IL12-mediated signaling, which may lead not only to MSMD, but also to inflammatory bowel disease (IBD).

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“…Density gradient centrifugation also has disadvantages such as the loss of smaller cell populations including high proliferative hMSCs. Moreover, there are varying protocols for density gradient centrifugation provided in the literature which does not guarantee reproducibility (89). Furthermore, there is strong evidence in the literature that freshly isolated hMSCs differ from isolated and cultivated hMSCs in their transcriptome and secretome indicating that conclusion from in vitro studies should be translated carefully towards in vivo assumptions (90).…”

Section: Limitationsmentioning

confidence: 99%

HIF-stabilization prevents delayed fracture healing

Lang

Helfmeier

Stefanowski

et al. 2020

Preprint

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AbstractThe initial phase of fracture healing decides on success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis, smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy of fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer – deferoxamine (DFO) – and a less known HIF-enhancer – macrophage migration inhibitory factor (MIF) – to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, the application of DFO with or without MIF during the initial healing phase accelerated callus mineralization and vessel formation in a clinically relevant mouse-osteotomy-model in a compromised healing setting. Our findings provide support for a promising preventive strategy towards bone healing disorders in patients with a higher risk due to e.g. delayed neovascularization by accelerating fracture healing using DFO and MIF to stabilize HIF-1α.

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Comparative study of different centrifugation protocols for a density gradient separation media in isolation of osteoprogenitors from bone marrow aspirate

Abstract: Both protocols could isolate competent and functional osteoprogenitors, while a lower centrifugal force (400 g) with 1:1 dilution produced recovery of more osteoprogenitors.

Cited by 15 publications

References 23 publications

Modeling Biological and Economic Uncertainty on Cell Therapy Manufacturing: the Choice of Culture Media Supplementation

Modeling Biological and Economic Uncertainty on Cell Therapy Manufacturing: the Choice of Culture Media Supplementation

IL-12Rβ1 deficiency corresponding to concurrency of two diseases, mendelian susceptibility to mycobacterial disease and Crohn's disease

HIF-stabilization prevents delayed fracture healing

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