Abstract:The increasing use of cultured human cells in clinical trials is highlighting the need for alternatives to media containing animal sera that are typically used to support these cultures. Perfused cultures of BM mononuclear cells (MNC) were used to evaluate animal sera alternatives with respect to the output of primitive, progenitor, and stromal cells. A serum level of 20% was optimal, and this could be provided by FBS alone or by a mixture of horse serum (HoS) and FBS, but not by HoS alone. Allogeneic human pl… Show more
“…Many studies have been performed to try and substitute the animal serum with human autologous or allogeneic serum or with synthetic media, but have not usually produced satisfactory results. [13][14][15] The addition of the PL has been suggested previously, [16][17][18] but the exact determination of hMSC yield starting from a reduced number of nucleated cells to reach clinically relevant numbers has never been shown previously.…”
We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P ¼ 0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 Â 10 6 to 365 Â 10 6 hMSCs in PL versus a variable number from 2.7 Â 10 6 to 31 Â 10 6 in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.
“…Many studies have been performed to try and substitute the animal serum with human autologous or allogeneic serum or with synthetic media, but have not usually produced satisfactory results. [13][14][15] The addition of the PL has been suggested previously, [16][17][18] but the exact determination of hMSC yield starting from a reduced number of nucleated cells to reach clinically relevant numbers has never been shown previously.…”
We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P ¼ 0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 Â 10 6 to 365 Â 10 6 hMSCs in PL versus a variable number from 2.7 Â 10 6 to 31 Â 10 6 in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.
“…Replacement of FCS in expansion of BMSCs has been discussed controversially. Positive effects on the formation of fibroblast colony-forming units and proliferation rates have been reported when human serum was used with and without additional mitogenic growth factors [Anselme et al, 2002;Stute et al, 2004], whereas Koller et al [1998] found decreased support for expansion of human MSCs when both autogenous and allogenic human serum were used. Koller et al [1998] and Kuznetsov et al [2000] reported a reduction in bone formation by human MSCs cultivated using human serum.…”
The aim of the present study was to analyze the effect of 3 different expansion media on the expression of marker genes of mesenchymal differentiation (bone, cartilage, fat) as well as apoptosis and senescence during repeated passaging in human bone marrow stromal cells (hBMSCs) in order to identify potential expansion strategies for the use of these cells into tissue-engineered growth of bone. Medium 1 (EGF, PDGF, low Glc, 2% FCS) was associated with the highest proliferation rate compared to medium 2 (β-mercaptoethanol, high Glc DMEM, 15% FCS) and medium 3 (low Glc DMEM, 10% FCS). Real time RT-PCR indicated the lowest levels of expression of osteonectin, core binding factor-α 1, lipoprotein lipase and cartilage oligo matrix protein in medium-1 cultures as compared to media 2 and 3. Early passages expressed higher levels of peroxisome proliferator-activator receptor-γ2 in medium 1 than in media 2 and 3, whereas no difference of Sox-9 expression was noticed among the 3 media. Expression of apoptosis- and senescence-related genes (Bax, BCL-2 and P16INK4a) exhibited the lowest level of Bax/BCL-2 ratio and P16INK4a gene expression of hBMSC in medium 1. In conclusion, the replacement of FCS by recombinant EGF and PDGF promoted rapid proliferation of hBMSCs without inducing differentiation of hBMSCs. It also inhibited expression of apoptosis-related genes and limited replicative senescence during repeated passaging. Media with the lowest possible FCS content and replacement by EGF and PDGF thus should be used for 2D culturing during expansion of hBMSCs, whereas β-mercaptoethanol and high concentrations of FCS can help to commence osteogenic differentiation.
“…Shigeno ve Ashton [29] OS'un FBS'dan daha üstün oldu unu söylerken, Anselme ve Yamamoto [30,31] OS ve FBS ile kültüre ettikleri MKH' lerde benzer ekilde sonuçlar elde ettiler. Koller, Kuznetsov ve Yamaguchi [32][33][34] gibi di er baz ara t r c lar ise FBS ile daha iyi sonuçlar rapor ettiler. Y lmaz ve ark.…”
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