Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T-flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex-1®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50×10(6) cell g(-1) Cytodex-1®). Then, the MSC aggregates were put into contact with additional available surfaces (T-flask, fresh and used Cytodex-1®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T-flasks or fresh Cytodex-1® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex-1® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex-1® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number.

α-Casozepine is a peptide, corresponding to the sequence 91-100 of the bovine α(s1)-casein, displaying anxiolytic activity in the rat. The α(s1)-casein tryptic hydrolysate containing this peptide decreases stress effects after oral administration in various species including man. Therefore, the stability of this peptide toward gastric and pancreatic proteases has been assessed by using pepsin, chymotrypsin/trypsin, Corolase PP, pepsin followed by chymotrypsin/trypsin or pepsin followed by Corolase PP. α-Casozepine was slowly degraded by chymotrypsin, much more sensitive to pepsin and Corolase PP but not completely destroyed after 4 h kinetics. The bonds in the region 91 to 95 of the α-casozepine were totally resistant to hydrolysis by all studied proteases. Surprisingly, a fragment, corresponding to the sequence 91-97 and found in all the hydrolysis media in significant amount, possessed an anxiolytic activity in three behavioral tests measuring this parameter. This peptide could participate in the in vivo activity of α-casozepine.

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α-Casozepine and f91-97, peptides from α(s1)-casein, display anxiolytic activity in rats and may have to cross the intestinal epithelium to exert this central effect. We evaluated their resistance to hydrolysis by the peptidases of Caco-2 cells and their ability to cross the cell monolayer. To mimic physiological conditions, two preparations of bile salts were used in noncytotoxic concentrations: porcine bile extract and an equimolar mixture of taurocholate, cholate, and deoxycholate. The presence and composition of bile salts appeared to modulate the peptidase activities of the Caco-2 cells involved (i) in the hydrolysis of α-casozepine, leading to much higher formation of fragments f91-99, f91-98, and f91-97, and (ii) in the hydrolysis of f91-97, leading to lower degradation of this peptide. Transport of α-casozepine across Caco-2 monolayer increased significantly, in the presence of bile extract, and of fragment f91-97, in the presence of bile salts.

The extensive use of mesenchymal stem cells (MCS) in tissue engineering and cell therapy increases the necessity to improve their expansion. Among these, porcine MCS are valuable models for tissue engineering and are classically expanded in static T-flasks. In this work, different processes of stirred cultures were evaluated and compared. First, the effect of glucose, glutamine, antioxidant, and growth factors concentrations on porcine MSC expansion were analyzed in a suitable medium by performing kinetic studies. Results showed that a lower glucose concentration (5.5 mM) enabled to increase maximal cell concentration by 40 % compared with a higher one (25 mM), while addition of 2 to 6 mM of glutamine increased maximal cell concentration by more than 25 % compared with no glutamine supplementation. Moreover, supplementation with 1 μM thioctic acid increased maximal cell concentration by 40 % compared with no supplementation. Using this adapted medium, microcarriers cultures were performed and compared with T-flasks expansion. Porcine MSC were shown to be able to proliferate on the five types of microcarriers tested. Moreover, cultures on Cytodex 1, Cytopore 2, and Cultispher G exhibited a MSC growth rate more than 40 % higher compared with expansion in T-flasks, while MSC metabolism was similar.