hMSCs therapies are of great interest, not just for their multipotent characteristics, but also for their use in treating defense diseases [ 3 ]. In regenerative medicine, there are worries regarding the immune rejection that a patient can suffer in the event that animal proteins become adhered to the in vitro extended human cells [ 4 , 7 ]. Thus, the replacement of the particular commonly used FBS is a necessity, and different strategies are being created. In a previous report, we described that commercial BM-hMSCs could be expanded in SCC-containing medium supplemented with development factors from platelet lysate (PL-Medium) [ 29 ]. Furthermore, hMSCs expanded in SCC-based medium have been shown to protect their immunomodulatory and chemotactic properties [ 30 ]. With this study, we extended the results for non-commercial BM-hMSCs plus commercial AT-hMSCs and UC-hMSCs. Our results indicated the fact that use of platelet lysate as a supplement in SCC-containing moderate is optional. In addition , we showed that SCC may be used to culture and expand hMSCs under xeno-free conditions, preserving their phenotype, genetic stability and multipotentiality.
Xeno-free media supplemented with human autologous serum has been shown to support hMSCs expansion; however , it would be difficult to obtain an adequate amount of serum to expand hMSCs sufficiently, as well as the age of the donor would influence its properties [ 8 , 32 ]. There is controversy on the use of this medium, plus contradictory results have been published [ 33 – 41 ]. There are simply no established protocols for the isolation and culture of hMSCs; thus, different laboratories use different strategies, making it hard to compare the outcomes of different studies that try to develop a xeno-free medium. Usually, the strategies consist of growing hMSCs within the media in the pipeline and checking the International Society intended for Cellular Therapy hMSCs defining characteristics (hMSCs adherence in order to plastic, phenotype, and differentiation into osteoblasts, chondroblasts plus adipocytes) [ 42 ].
SCC is an industrial GMP product, obtained from a large quantity of lcd sourced from more than 1000 donors, which considerably decreases the lot-to-lot variability. Moreover, no supply problems are required. In our study, all hMSCs tested were able to adhere and become expanded in XF-Medium and PL-Medium. The proliferation price of the commercial BM- and AT- hMSCs was greater than in the Reference Medium, as observed in the previous studies [ 42 ]. As a high proliferation rate could induce chromosomic aberrations, the genetic stability of the cultures was evaluated. hMSCs were passaged for 3 and 4 operates in XF-Medium, PL-Medium and Reference Medium. Our outcomes showed that hMSCs expanded using the different media had been genetically stable, showing a normal diploid karyotype. Other research also showed a higher proliferation of hMSCs in other xeno-free and serum-free media than in FBS-supplemented media; however , generally the genetic stability was not studied and only hMSCs from origin were used [ 12 , 13 , 41 ].
After verifying that the cells could be expanded within the xeno-free media, we tested whether the defining hMSCs features were maintained. All hMSCs used presented the typical hMSC phenotype, being positive for CD29, CD44, CD73, CD90, CD105, CD166 and Stro-1, and negative for CD14, CD19. The multipotentiality of the cells was also studied; all of the cells tested could be differentiated into osteoblasts, chondrocytes plus adipocytes. Although the assays performed were qualitative, some variations in the differentiation levels could be observed when using the different cellular types. Non-commercial BM-hMSCs showed a higher chondrogenic and osteogenic differentiation than the other cell types, an observation which was made in other studies [ 24 ]. On the other hand, UC-hMSCs demonstrated a lower osteogenic differentiation, when compared with the other cells, which was furthermore observed by other researchers [ 43 , 44 ].
Although platelet lysate has shown promising features for hMSCs culture and expansion and is being used in numerous studies, some drawbacks are coming out, such as donor variability [ 45 ], decreased expression of adipogenic and osteogenic differentiation markers [ 46 , 47 ], and interaction with immunomodulatory properties of MSCs [ 48 , 49 ]. Due to these worries, in this study we tested the use of SCC without the add-on of platelet lysate. Our results showed that the inclusion of platelet lysate to SCC-containing Medium is optionally available. All the hMSCs defining characteristics were maintained in XF-Medium without the addition of platelet lysate. The only differences noticed were in the proliferation rate of non-commercial BM-hMSCs, that was higher when introducing platelet lysate to the medium. Therefore, due to the potential negative effects, as mentioned before, we recommend staying away from the supplementation with platelet lysate in SCC-supplemented mass media.
It has been previously shown that will SCC can be used to culture not only hMSCs, but also Chinese hamster ovarian cells, Vero cells, and mouse BALB/c myeloma cells [ 29 ]. All the studies with the commercial cellular lines were done in parallel using different lots of human being plasma fraction. In this study, hMSCs behaved similarly whatever the SCC lot used, which indicates that the batch chosen does not present significant variations regarding the other batches, not surprisingly from its origin (large plasma pools from over multitude of donors).