To generate enough BMSCs for clinical studies, cells must be expanded over several passages in vitro, in addition to testing for functional capacity at multiple stages in the expansion process. Three major challenges in the expansion process include:
- Functional variability brought on by batches obtained from different donors
- Decreases in functional capacity after successive passages
- Lack of rapid and quantitative assays that can simultaneously provide functional characterization and assess the products’ quality parameters including viability, purity, and potency
Benefits of the xCELLigence Real Time Cell Analysis platform for QC and Functional Assessment of MSCs :
- Continuously measure integrated changes in cell number, attachment, and morphology under physiologically relevant conditions to ensure consistency between batches
- Maximize production yields by using less cells, which can be analyzed at any point during differentiation
- More effectively predict BMSC functional capacity (i.e. differentiation potential) compared to traditional methods
Overview of quantitative parameters derived from xCELLigence RTCA growth curves.
Figure 1 Representative growth profiles comparing cell line A and cell line B. The graph represents how maximum CI (CImax) and 50% maximum CI (GT50) are calculated (blue lines). Cell Index Doubling (CIDT) represents the time it takes for the CI to double (solid purple) and is calculated for the entire log growth phase.
Quality Control Assessment of Donor-to-Donor Variability
In the example below, xCELLigence RTCA was used to assess donor-to-donor variability and determine if the xCELLigence RTCA assay can predict loss of function of BMCSs obtained from different sources. To this end,
BMSCs obtained from different donors were propagated in XFM for at least 12 passages and analyzed by RTCA.
Consistent and Functional Assessment
The biologically relevant parameters derived from the kinetic proliferation assay are consistent across multiple passages, while cells continue to proliferate and differentiate into their respective lineages. Failure to meet the quantitative standards of this set of functional values indicates loss of function (i.e. differentiation potential), as observed in our donor-to-donor variability experiments (see Figure 3).
With xCELLigence RTCA, results show a consistent and functional assessment of MSCs. Additionally, only a small subset of cells is needed for the RTCA assay (helping to maximize production yields), and cells can be analyzed at any point during their propagation/differentiation, or can be cryopreserved and analyzed at a later date.
Figure 2 Real-Time kinetic profiles of BMSCs from three different donors over multiple passages depict significant variation in growth potential. BMSCs from different donors were propagated over 12 passages in xeno-free media and a subset of cell samples from each passage were seeded on E-plates and monitored over 10 days. Representative growth profiles from passage 2 (A), passage 6 (B), and passage 10 (C) comparing all three donors.
Figure 3 BMSCs from donor 2 (A-F) and donor 3 (G-L) from Figure 2 were propagated over 12 passages in Xeno-Free serum-free media and a subset of cell samples from different passages were analyzed for quantitative parameters on xCELLigence. (A and G) Growth profiles comparing early and late passage under each growth condition. RTCA software was used to calculate Cell Index Doubling (B and H) and Relative Maximum Cell Index (C and I) as described in the protocol. The dotted lines represent the threshold set for functional status. (D and J) GT50 was calculated for successive passages and plotted for two seeding densities. Slope (S) was calculated as a change over time for each seeding density. (E and K) Phenotypic analysis of negative and positive MSC markers at the indicated passage where loss of function is observed. * represents a value that is over the set threshold outlined in Table 1. (F and L) Differentiated staining at indicated passages with LipidTox Green Lipid Stain, following 14 days of growth in adipogenesis differentiation medium.