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.2016 Mar;5(3):366-78.
doi: 10.5966/sctm.2015-0208. Epub 2016 Jan 29.

A Detailed Assessment of Varying Ejection Rate on Delivery Efficiency of Mesenchymal Stem Cells Using Narrow-Bore Needles

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A Detailed Assessment of Varying Ejection Rate on Delivery Efficiency of Mesenchymal Stem Cells Using Narrow-Bore Needles

Mahetab H Amer et al. Stem Cells Transl Med.2016 Mar.

Abstract

As the number of clinical trials exploring cell therapy rises, a thorough understanding of the limits of cell delivery is essential. We used an extensive toolset comprising various standard and multiplex assays for the assessment of cell delivery postejection. Primary human mesenchymal stem cell (hMSC) suspensions were drawn up into 100-µl Hamilton syringes with 30- and 34-gauge needles attached, before being ejected at rates ranging from 10 to 300 µl/minute. Effects of ejection rate, including changes in viability, apoptosis, senescence, and other key aspects of cellular health, were evaluated. Ejections at slower flow rates resulted in a lower percentage of the cell dose being delivered, and apoptosis measurements of samples ejected at 10 µl/minute were significantly higher than control samples. Immunophenotyping also revealed significant downregulation of CD105 expression in samples ejected at 10 µl/minute (p < .05). Differentiation of ejected hMSCs was investigated using qualitative markers of adipogenesis, osteogenesis, and chondrogenesis, which revealed that slower ejection rates exerted a considerable effect upon the differentiation capacity of ejected cells, thereby possibly influencing the success of cell-based therapies. The findings of this study demonstrate that ejection rate has substantial impact on the percentage of cell dose delivered and cellular health postejection.

Keywords: Cell therapy; Differentiation; Mesenchymal stem cell injection; Mesenchymal stem cells; Needles.

©AlphaMed Press.

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Figures

Figure 1.
Figure 1.
Viability of human mesenchymal stem cells (hMSCs) and percentage of cell dosedelivered via 30G needles.(A): Percentage of hMSCs delivered,determined using CyQuant, following ejection through a 30G 20-mm needle. Datawere normalized against a control value of directly plated cells. Datarepresent averages from 3 donors (n = 5 each) in 5 independentexperiments (mean ± SD). ∗,p ≤ .01 fornumber of ejected cells compared with control (one-way analysis of variance[ANOVA] with Dunnett’s post hoc test).(B): Percentage ofhMSCs delivered as viable cells, measured using PrestoBlue, following ejectionvia a 30G needle (mean ± SD). Data are combined from 3 independentdonors and 3 independent experiments (n = 3 each), eachmeasured in triplicate. ∗,p ≤ .05 for number ofviable ejected cells and control sample (one-way ANOVA with Dunnett’spost hoc test).(C): Representative graph showing proliferation ofhMSCs (because of variants in proliferation rate between donors), given as foldchange in number from day 0 of each sample, measured using PrestoBlue (mean± SD;n = 3; measured in triplicate).(D):Representative Live/Dead-stained fluorescence images of hMSCs 48 hoursfollowing ejection at various flow rates. Scale bar = 50 μm.(E): Graph shows flow cytometric analysis of ejected hMSCs(three independent experiments), illustrating high viable cell proportions atall flow rates under investigation. Abbreviation: Ctrl, control.
Figure 2.
Figure 2.
Cytotoxicity and apoptosis levels of human mesenchymal stem cells (hMSCs)ejected via 30G needles.(A): Cytotoxicity and apoptosismeasurements in hMSCs from 2 donors 4 hours postejection (analyzed byApoTox-Glo Triplex Assay). Cytotoxicity and caspase-3/7 activity measurementswere normalized to viability within the same well. 10 μM staurosporinetreatment was used as positive control (n = 5; mean±SD). ∗,p ≤ .05; ∗∗,p < .01; ∗∗∗,p < .0001.(B): Percentages ofapoptotic and necrotic cells 24 hours postinjection, measured using Alexa Fluor488 Annexin V/PI (3 donors in 6 independent experiments;n =15). ∗,p ≤ .05 for comparison of samples andcontrol using analysis of variance with Dunnett’s post hoc test.Abbreviation: Ctrl, control.
Figure 3.
Figure 3.
Viability and apoptosis levels of human mesenchymal stem cells (hMSCs)delivered via 34G needles.(A): Percentage of hMSCs delivered 24hours postejection via 34G 20-mm needles. Results are normalized mean values tocontrol ± SD (averages from 2 donors and 2 independent experiments, eachn = 5). ∗∗,p ≤ .01one-way analysis of variance (ANOVA).(B): Representativefluorescence images depicting Live/Dead-stained hMSCs ejected at several flowrates 48 hours postejection. Scale bar = 100 μm.(C): Meanpercentages (± SD) of apoptotic and necrotic cells 24 hourspostinjection via 34G needles, measured using Alexa Fluor 488 Annexin V/PI (3donors in 5 independent experiments;n = 11). ∗,p ≤ .05 for comparison of samples and control (ANOVAand Dunnett’s post hoc test). Abbreviation: Ctrl, control.
Figure 4.
Figure 4.
Trilineage differentiation potential of cultured human mesenchymal stem cells(hMSCs) ejected via 30G needles. To verify the differentiation capacity ofejected hMSCs via 30G needles, adipogenic, osteogenic, and chondrogenicdifferentiation was carried out on ejected and control samples following 21days of differentiation. Adipogenic differentiation was assessed by Oil Red Ostaining. Osteogenic differentiation was assessed by Alizarin Red staining ofcalcified matrix. Phase-contrast microscopy of micromass cultures showsdifferentiation down the chondrocyte lineage, as confirmed by Alcian Bluestaining. In Von Kossa-stained samples, calcified deposits are indicated bywhite arrows. Scale bar = 50 µm. Cells cultured in mesenchymal stem cellgrowth medium without induction served as a negative control.
Figure 5.
Figure 5.
Investigation of cell recovery and retention in the delivery device followingejection via 30G needles.(A): To investigate poor cell recoveryat slow flow rates, each ejection was followed by phosphate-buffered saline(PBS) washes (5 × 100 μl at 300 μl/minute), and the numberof cells recovered was quantified using PrestoBlue (mean ± SEM; 2donors; eachn = 3).(B): A representativedissection microscope image of a syringe barrel postejection at 10μl/minute, depicting cells that have been retained in the syringeaggregating during PBS wash. Scale bar = 1 mm.(C): Confocalmicroscopy was used to image cells retained in the syringe postejection at 10μl/minute. Scale bar = 200 µm.(D): To investigatewhere cells were being retained in the delivery device, its components (syringebarrel and needle) were separated after ejection at 10 μl/minute, andeach was separately washed with PBS. The number of cells recovered was measuredusing PrestoBlue (mean ± SEM;n = 3).(E):Adhesion of cells to a borosilicate glass surface was assessed, comparing thenumber of cells adhering with no wash following removal of cell suspensionsfrom wells and after gentle PBS washes (100 μl;n = 3;3 independent experiments using cells from 3 donors).(F): Acomparison of cell-dose recovery between viable and apoptotic cells (inducedusing 10 µM staurosporine) ejected at 10 µl/minute(n = 4). ∗,p ≤ .05 for thedifference between the 2 groups, using the Mann-Whitney test. Abbreviations:Ctrl, control; Inj., injected.
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