Aedating v4 1
suspension bioreactors) to that obtained from standard culture conditions (i.e. We report herein that, using PPRF-msc6, we were able to rapidly expand cells in both static cultures as well in our 500 m L suspension bioreactors (dynamic culture) (Fig. Additionally, flow cytometry analysis of static and dynamic culture expanded h BM-MSCs revealed that both types of cells expressed the standard h MSCs markers CD13, CD73, CD90 and CD105 at h BM-MSCs adhered to both the tissue culture flasks and the microcarriers in the suspension bioreactors on day 1, and proliferated up to day 4 (A).FACS analysis for h MSC markers, CD13, CD73, CD90, CD105 were .Conventionally, h MSCs are expanded using static culture flasks in the presence of fetal bovine serum (FBS) or human-sourced supplements.However, these expansion platforms can lead to variable culture conditions (i.e.
After 7 days post-injection, both CM (static and dynamic) were able to increase the (B, C) levels of endogenous proliferating cells (Ki-67After observing that both CM (from the static and dynamic conditions) were able to stimulate cell proliferation in the DG, we next aimed to determine their effects on the differentiation of DG resident cells. 3D–F), we observed an increase in the number of DCX-expressing cells (newborn neurons) in all DG granular layers (F = 0.842), namely in the SGZ (Fig. From the MS/SWATH analysis we observed that the bioreactor-based bioprocess (dynamic condition) modulated the h BM-MSCs secretome to produce a different pattern of protein expression when compared to the secretome collected from the static condition (Fig. Indeed, through the use of Venn diagrams, we were able to identify 120 proteins in the static and 130 proteins in the dynamic condition, in which 102 proteins were common to the two conditions (Fig. From these, when we analyzed the relative protein levels of the two h BM-MSC secretomes for specific proteins with actions in CNS physiology, we were able to find that molecules (according with the Uni Prot KB/Swiss-Prot classification) such as cystatin-C (Cys C (P01034); t = 1.211, p = 0.292), glia-derived nexin (GDN (P07093); t = 0.492, p = 0.648), galectin-1 (Gal-1 (P09382); t = 1.397, p = 0.234), and pigment epithelium-derived factor (PEDF (P36955); t = 0.857, p = 0.439), were upregulated in the dynamic conditions (Fig. Moreover it was also found that some other proteins with important roles in CNS regulations were only found in the CM from h BM-MSCs cultured in the bioreactor, namely Ezrin (P15311), Radixin (P35241), Beta-1,4-galactosyltransferase (P15291) and connective tissue growth factor (CTGF; P29279).
Specific neuroregulatory molecules such as (D) cystatin C (Cys C), (E) glia-derived nexin (GDN), (F) galectin-1 (Gal-1) and (G) pigment epithelium-derived factor (PEDF) were found to be upregulated in the dynamic h BM-MSCs secretome (data are expressed as mean ± SEM, n=3). Trophic factor values were normalized to cell density (i.e. In the present work, we have expanded h BM-MSCs on microcarriers in computer-controlled stirred suspension bioreactor (Fig. This technology allows us to: (1) expand a large number of cells in one vessel, (2) monitor and ensure tight control of process variables such as p H, temperature and dissolved oxygen concentration (Fig.
pg (of each factor)) per 10,000 cells in each condition, respectively. 1C), and (3) develop a process, which allows for scale-up to larger bioreactor systems.
Therefore it is possible to hypothesize that the modulation, and further enrichment of growth factors/vesicles, of their secretome could be achieved by using these dynamic culturing systems.
With this in mind, in the present work we aimed to characterize and analyze the effects of the human bone marrow-derived MSCs (h BM-MSCs) secretome collected from dynamic culture conditions (i.e. during the exponential growth phase) of the h BM-MSCs in static culture was 37.8 ± 6.0 h, which was similar to the doubling time in dynamic culture (36.4 ± 4.9 h).