Stability enhancement of clinical grade multipotent mesenchymal stromal cell-based products

Abstract

Background: Successful delivery of cell-based therapeutics into patients is compromised by their short shelf-life upon release from production facilities due to the living nature of the active component that rapidly loses viability, and therefore its properties. In this context, the use of appropriate additives may contribute to the stabilisation of the cellular component within specifications for a longer time until administration.

Results: In the present study, we evaluated the effect of different formulations on the stability of viability, identity, and potency of clinical grade multipotent mesenchymal stromal cells in suspension, both electrolyte solution and protein content were found to impact on their shelf-life. Particularly cryopreservation of cells in a Plasmalyte 148 supplemented with 2% (w/v) AlbIX (a yeast-derived recombinant albumin) and 10% (v/v) dimethyl sulfoxide, and final formulation post-thawing in Plasmalyte 148 supplemented with 2% (w/v) AlbIX enabling prolonged stability from 24 h up to 72 h in optimal conditions. Further investigation on the mechanisms of action involved revealed a delay of apoptosis progression into late stage when AlbIX was present.

Conclusions: The use of optimal formulations for each cell type of interest is crucial to extend the shelf life of cell-based pharmaceuticals and contribute to solve logistical challenges. We demonstrated that the use of Plasmalyte 148 supplemented with 2% (w/v) AlbIX resulted in superior stability of multipotent mesenchymal stromal cells without affecting their identity and multipotency.

Keywords: Apoptosis; Cell culture; Cellular therapy; Logistics; Multipotent mesenchymal stromal cell; Potency assay; Quality compliance; Stability assessment.

Fig. 1

Effect of different electrolyte solutions on the stability of MSC in a syringe. Evolution of cell viability (%) (a) and cell recovery (b) along time at room temperature using 5% glucoside solution, Ringer lactate solution, and Viaflo plasmalyte 148 supplemented with 2% (w/v) HSA. One sample per condition and time point was tested in duplicates. HSA, human serum albumin; MSC, multipotent mesenchymal stromal cells

Fig. 2

Effect of albumin type and concentration on the viability of cryopreserved MSC. Cryopreserved MSC were thawed using either one of the albumins at the concentrations (w/v) stated in the image. One cell line was used for all conditions tested. HSA, human serum albumin; MSC, multipotent mesenchymal stromal cells; PI, propidium iodide

Fig. 3

Retention of viability, phenotypic profile and multipotentiality of MSC after cryopreservation in the presence of either HSA or recombinant albumin. a Post-thawing stability assessed by flow cytometry showing the mean of the percentage of viability (%) along the stability follow up time using HSA (in black) or AlbIX (in grey) additives (n = 6 cell lines), percentage of reduction and results of ANOVA Tukey Multiple comparison test are showed; Phenotypic characterisation of MSC at times 0 and 72 h. Finally, in b, in vitro differentiation assays were performed to confirm osteogenic, adipogenic and chondrogenic potential of MSC and the outcome of each differentiation were assessed by specific stainings: alkaline phosphatase (ALP) and Alizarin Red (AR) for bone tissue, Oil Red O for fat; and Safranin O, for cartilage

Fig. 4

Impact of albumin supplementation when used as cryoprotectant, or additive, or both. Cryopreservation of MSC was performed using either AlbIX or HSA and followed by thawing and conditioning with either one of the two albumins generating the 4 possible combinations (HSA:HSA, HSA:AlbIX, AlbIX:AlbIX, and AlbIX:HSA). Viability pre- and post-cryopreservation shows higher viability rates in samples supplemented with AlbIX (a). Viability of all four conditions were followed up for 72 h (b). Data resulting from these experiments have been grouped according to the cryoprotectant used, either HSA (c) or AlbIX (d). In e, viability reduction is presented for all conditions along time, namely: control (HSA:HSA), as a cryoprotectant (AlbIX:HSA), as a stabilizer post-thawing (HSA:AlbIX), or both (AlbIX:AlbIX)

Fig. 5

Analyses of apoptosis of cryopreserved MSC. Conditions tested were HSA:HSA and AlbIX:AlbIX and demonstrated that cryopreservation and post-thawing conditioning with Plasmalyte 148 supplemented with AlbIX extended the shelf-life of hMSC by preserving cells in the early apoptotic stage. Bars represent the percentage of early (a) and late (b) apoptotic cells for HSA and AlbIX cryopreservation conditions (n = 3 cell lines), ANOVA parametric Sidak’s multiple comparison tests were performed