Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation

Piede, Natascha; Bremm, Melanie; Farken, Anne; Pfeffermann, Lisa-Marie; Cappel, Claudia; Bönig, Halvard; Fingerhut, Theres; Puth, Laura; Vogelsang, Kathrin; Peinelt, Andreas; Marschalek, Rolf; Müller, Matthias J.; Bader, Peter; Kuçi, Zyrafete; Huenecke, Sabine

doi:10.3390/cells12060850

Cited by 6 publications

(4 citation statements)

References 65 publications

(126 reference statements)

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“…For example, to assess the linearity of the inhibitory potential of MSCs in T-cell proliferation, different MSC concentrations were tested while keeping the number of peripheral blood mononuclear cells constant. T-cell proliferation, the formation of daughter generations, and the inhibition of T-cell proliferation by MSCs were investigated [51]. For potency assays, a range of 80 to 120 percent of the expected test concentration should be minimally considered.…”

Section: Development Of Potency Assays For Msc Secretome-based Produc...mentioning

confidence: 99%

“…For both active substances as well as medicinal products (if it is impossible to obtain samples of all product components), accuracy can be measured by using an alternative well-characterized procedure that incorporates stated and/or defined accuracy by adding known quantities of the analyte to the product [49]. In the aforementioned assessment of the inhibitory potential of MSCs on T-cell proliferation, the accuracy of the studied assay was measured compared to the well-defined test that had been used as the performance criterion of the MSC qualification [51]. It is noteworthy that for both active substances as well as medicinal products, accuracy may be inferred once precision, linearity, and specificity have been established [49].…”

Section: Development Of Potency Assays For Msc Secretome-based Produc...mentioning

confidence: 99%

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Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration

Sagaradze

Monakova

Efimenko

2023

IJMS

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Adult stem cells maintaining tissue homeostasis and regeneration are tightly regulated by their specific microenvironments or stem cell niches. The dysfunction of niche components may alter the activity of stem cells and ultimately lead to intractable chronic or acute disorders. To overcome this dysfunction, niche-targeting regenerative medicine treatments such as gene, cell, and tissue therapy are actively investigated. Here, multipotent mesenchymal stromal cells (MSCs), and particularly their secretomes, are of high interest due to their potency to recover and reactivate damaged or lost stem cell niches. However, a workflow for the development of MSC secretome-based products is not fully covered by regulatory authorities, and and this issue significantly complicates their clinical translation and has possibly been expressed in a huge number of failed clinical trials. One of the most critical issues in this regard relates to the development of potency assays. In this review, guidelines for biologicals and cell therapies are considered to be applied for the development of potency assays for the MSC secretome-based products that aim for tissue regeneration. Specific attention is paid to their possible effects on stem cell niches and to a spermatogonial stem cell niche in particular.

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Section: Development Of Potency Assays For Msc Secretome-based Produc...mentioning

confidence: 99%

Section: Development Of Potency Assays For Msc Secretome-based Produc...mentioning

confidence: 99%

Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration

Sagaradze

Monakova

Efimenko

2023

IJMS

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“…Next, the cells were stained with Propidium Iodide (50 µg/ml of cell suspension) for 15 minutes at room temperature. Finally, the cell cycle analysis was conducted using ow cytometry, speci cally the BD FACSLyric instrument manufactured by BD Biosciences [15].…”

Section: Scratch Wound Healing Assaymentioning

confidence: 99%

In vitro Wound Healing Efficacy of Silver Nanoparticles Synthesized from Aqueous Extract of Turbinaria conoides.

Suresh,

Poonguzhali,

Anuradha

et al. 2023

Preprint

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The wound healing potentials of brown algae Turbinaria conoides aqueous extract (TCAe) and silver nanoparticles synthesized utilizing T. conoides aqueous extract (TCAgNPs) were investigated in this study. TCAgNPs and TCAe were tested for cytotoxicity on human dermal fibroblast cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed that TCAgNPs and TCAe were not cytotoxic and may be tested for medicinal qualities. TCAgNPs and TCAe were tested for wound healing efficacy using a wound scratch assay on human dermal fibroblast cells. The damaged cells were subjected to TCAgNPs and TCAe, which demonstrated stronger wound repair activities than the control (Untreated). The cell cycle study of human dermal fibroblast primary cell lines treated with TCAgNPs and TCAe, as well as those not treated, was performed using flow cytometry to determine the DNA content of the nuclei. These findings show that TCAgNPs-treated cells proliferated more than TCAe and control-treated cells, implying that cell proliferation is boosted, which aids the wound-healing process. During immunoblot analysis, the TCAgNPs-treated group showed higher collagen and fibronectin expression than the TCAe-treated group. Our findings imply that TCAgNPs and TCAe can repair wounds in vitro and could be used as a source of wound healing agents.

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“…Assays based on the suppression of PBMC proliferation routinely take from 4 to 7 days depending on whether a mixed lymphocyte response (7 days) or a mitogen response (4 days) is followed (34)(35)(36)(37)(38)(39). Readouts of these assays range from dilution of fluorescently labeled cell constituents (e.g., using CFSE or Cell Violate Trace) to monitoring DNA replication with incorporation of bromodeoxyuridine (40) or tritiated nucleotides (41) to simple DNA quantitation by Hoechst assay (42). Surrogate markers for peripheral blood mononuclear cell (PBMC) proliferation include nuclear accumulation of Ki67 in another flow cytometry-based proliferation assay (43), increases in mitochondria with tetrazolium-based dyes in absorbance assays (44), or increases in ATP content proportional to cell number in a luminescent-based assay (45).…”

Section: Background and Introductionmentioning

confidence: 99%

Short-term assays for mesenchymal stromal cell immunosuppression of T-lymphocytes

Herzig,

Christy,

Montgomery

et al. 2023

Front. Immunol.

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IntroductionTrauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3–7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less.MethodsThree potential assays were examined—assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2–72 h.ResultsSuppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h.DiscussionTakeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2–6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.

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Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation

Cited by 6 publications

References 65 publications

Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration

Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration

In vitro Wound Healing Efficacy of Silver Nanoparticles Synthesized from Aqueous Extract of Turbinaria conoides.

Short-term assays for mesenchymal stromal cell immunosuppression of T-lymphocytes

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