Extracellular vesicles (EVs or exosome) are nano-sized subcellar particles involved in intercellular communication. Because they reflect their parental cell phenotype , EVs produced by mesenchymal stem cells (MSC) have regenerative properties and are promising as cell-free therapy to repair damaged tissues .
However, lack of large-scale GMP compatible EVs production processes limits clinical translation. EV production yield can be improved by mechanical stress induced by turbulence  but a large number of cells is still required.
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Scaling up cell culture in 3D culture systems
Conventional 2-Dimensional systems are time consuming and allows insufficient control of cell culture parameters.
Development of 3D culture systems tend to give interesting solutions to these issues. Cell bead-to-bead transfer (Btb, i.e. adding fresh microcarrier in the media) is a method which use the ability of MSCs to migrate from confluent microcarrier to new one.
So, adding new microcarriers increase specific area for cell growth without using enzyme for cell detachment  . Here, we designed a microcarrier-based (MC) cell culture process in stirred tank bioreactors using Btb as a scale-up method for EV production.
Cell bead-to-bead transfer as a cell culture process
Human adipose derived stem cells (hADSC) were cultivated at passage 5 in human platelet lysate supplemented media, on microcarriers in 100 mL spinner flasks with and without microcarriers addition (Figure 1). After each addition, 24 hours of intermittent agitation was applied to promote cell migration . Samplings were taken daily for cell counting by DAPI nuclei staining and images analysis from Leica fluorescence microscope and to monitor metabolites with a Gallery Discrete Analyzer.
ADSCh cultivated in spinner flasks with and without microcarrier addition
Figure 1: Scale up in spinner flasks from 40 mL to 100 mL by using bead to bead transfer
Results showed that 24 hours of intermittent agitation allow efficient cell migration with 97,5 percent of microcarriers occupied by at least one cell. Btb led to prolong cell expansion, and increase final harvest compared to control (8.1 vs 3.8 million of cells) (Figure2). Fresh microcarrier addition permit to reduce number of overpopulated microcarriers (>12 cells per bead) from 36 to 6 percent, which limit cell growth inhibition by confluence and delay aggregate apparition.
Figure 2: Effect of microcarriers addition on hADSC growth in spinner flasks. Orange circles: spinner batch control with 40mL of culture; blue squares: spinners with 40 mL of culture with addition at 74 hours of 60 mL of media with fresh microcarriers.
Nevertheless, this technique does not permit to dissociate them once they have appeared. Timing of addition is crucial to obtain best culture performance and cannot be define only by a critical mean cell per bead ratio, because the cell population on microcarriers is heterogeneous and differs over time and between experiments.
BtB is a scale-up method compatible with MSC large-scale production, but the optimal timing addition needs to be precised, because insufficient addition could lead to confluence or excessive addition could drop cell density bellow a critical level which cause cell growth inhibition in both cases.
Bead to bead is efficient method to maintain culture during a long term  and limit cell aggregation which is harmful for cell growth and production of EVs by mechanical stress stimulation. This scale-up method is promising to obtain large number of cells required for the development EV based therapies. However, process parameters like timing, frequency and quantity added must be precised to maintain high expansion rate.
Finally, it could be considered to automated microcarriers in addition with on-line solutions like permittivity measurement to have more control on process and ensure high quality of cells produced.
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