Pooling of Patient-Derived Mesenchymal Stromal Cells Reduces Inter-Individual Confounder-Associated Variation without Negative Impact on Cell Viability, Proliferation and Osteogenic Differentiation

Abstract

Patient-derived mesenchymal stromal cells (MSCs) play a key role in bone tissue engineering. Various donor-specific factors were identified causing significant variability in the biological properties of MSCs impairing quality of data and inter-study comparability. These limitations might be overcome by pooling cells of different donors. However, the effects of pooling on osteogenic differentiation, proliferation and vitality remain unknown and have, therefore, been evaluated in this study. MSCs of 10 donors were cultivated and differentiated into osteogenic lineage individually and in a pooled setting, containing MSCs of each donor in equal parts. Proliferation was evaluated in expansion (assessment of generation time) and differentiation (quantification of dsDNA content) conditions. Vitality was visualized by a fluorescence-microscopy-based live/dead assay. Osteogenic differentiation was assessed by quantification of alkaline phosphatase (ALP) activity and extracellular calcium deposition. Compared to the individual setting, generation time of pooled MSCs was shorter and proliferation was increased during differentiation with significantly lower variances. Calcium deposition was comparable, while variances were significantly higher in the individual setting. ALP activity showed high variance in both groups, but increased comparably during the incubation period. In conclusion, MSC pooling helps to compensate donor-dependent variability and does not negatively influence MSC vitality, proliferation and osteogenic differentiation.

Primary osteoblasts, osteoblast precursor cells or osteoblast‐like cell lines: Which human cell types are (most) suitable for characterizing 45S5‐bioactive glass?

“…Cell line derived from a 15-year-old female Caucasian osteosarcoma patient (Rousseau, Lamoureux et al 2010). Furthermore, it was demonstrated that these cell lines show significant differences in proliferation patterns (Lauvrak et al, 2013 (Bragdon et al, 2015;Friedl et al, 2007;Friedman, Long, & Hankenson, 2006;Widholz et al, 2019). Furthermore, it was demonstrated that these cell lines show significant differences in proliferation patterns (Lauvrak et al, 2013 (Bragdon et al, 2015;Friedl et al, 2007;Friedman, Long, & Hankenson, 2006;Widholz et al, 2019).…”

“…However, since primary cells are prone to variance caused by the donor patient (comorbidities, sex, age, etc. ), transferability of study results is limited, thus the donor patients' individual characteristics have to be considered (Bragdon et al, 2015;Friedl et al, 2007;Friedman, Long, & Hankenson, 2006;Widholz et al, 2019).…”

“…Values were normalized to the corresponding control group.2.6 | Qualitative evaluation of cell viability and cell morphology by fluorescence microscopyCell morphology and viability were qualitatively visualized by a fluorescence-microcopy-based live/dead assay using a previously published protocol containing FDA and propidium iodide (PI)(Widholz et al, 2019). Cells were added by mixing 1 × 10 4 cells into the prepared culture plates.…”

“…mg/ml in 96-well culture plates (Sarstedt, Nümbrecht, Germany). In contrast to FDA, the DNA intercalating and red fluorescent PI cannot penetrate membranes of intact cells and therefore only stains DNA of dead cells with altered membrane integrityWidholz et al, 2019). Cells without the addition of 45S5-BG served as controls.…”

“…Cells without the addition of 45S5-BG served as controls. This assay is based on an ALP mediated hydrolysis of p-NPP to para-nitrophenol resulting in a color change from transparent to yellow that correlates with the enzymatic activity of ALP(Reible et al, 2017;Reible et al, 2018;Widholz et al, 2019). The nonfluorescent FDA can freely pass cell membranes and is actively converted by living cells to the greenfluorescent compound fluorescein that can be used to visualize cell morphology and to measure cell viability.…”