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Process of human platelet lysate production A) A minimum of 3 units of enriched platelets were pooled. Platelets were frozen at -80°C and thawed at 37°C thrice. Two centrifugation steps (2600xg, 20 min) removed larger cell debris. The lysate was filtered through a 0.22 µM filter to remove smaller debris. To avoid coagulation, 2 U/ml heparin for suspension cells and 4 U/ml for adherent cells were added. B) The protein concentrations of three independent stocks of FCS and hPL were compared using the Bradford assay, n = 3; mean ± SD; unpaired, two-tailed Student's t-test, p = 0.0008.
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Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used medium supplement worldwide. FCS contains a variable mixture of growth factors and cytokines that support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesir...
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... 3 weeks of growth in 5% FCS or 5% hPL, all four cell lines were morphologically indistinguishable from cells grown in 10% FCS (Fig. S1A 1 ). Furthermore, K562 cells and HCT116 cells grew equally well under the three different conditions ( Fig. 2A, S1B 1 ). NB4 cells grew best with 10% FCS and slightly slower with 5% FCS and 5% hPL. MV4-11 cells cultured with 5% hPL grew as well as those with 10% FCS, both better than with 5% FCS ( Fig. ...
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... tested whether cells grown in hPL can be frozen and thawed for continuous propagation. Indeed, cells that had been frozen in medium containing 5% hPL started to grow similarly to cells that had been frozen with 10% FCS (Fig. S1B 1 and data not ...
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... HCT116 cells are driven by mutant β-catenin ( Morin et al., 1997), we also assessed β-catenin expression in HCT116 cells cultured with hPL or FCS. We found no differences in β-catenin levels under the two culture conditions (Fig. S1C 1 ). We additionally assessed the levels of the adhesion molecule and epithelial marker protein E-cadherin, which contributes to the regulation of β-catenin, and of RAC1, which controls epithelial phenotypes ( Kimura et al., 2006;Sander and Collard, 1999). These proteins were expressed equally well in HCT116 cells grown with FCS or hPL ...
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... (Fig. S1C 1 ). We additionally assessed the levels of the adhesion molecule and epithelial marker protein E-cadherin, which contributes to the regulation of β-catenin, and of RAC1, which controls epithelial phenotypes ( Kimura et al., 2006;Sander and Collard, 1999). These proteins were expressed equally well in HCT116 cells grown with FCS or hPL (Fig. S1C 1 ). croscope (Nikon Instruments). DNA damage (measured as tail intensities) was evaluated with Comet IV software (Perceptive ...
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... more than two groups, a two-way ANOVA test was performed with Bonferroni's multiple comparisons and 95% confidence interval. Figure 1A shows how we generated hPL stocks (Fig. 1A). When we determined the protein concentrations of batches of FCS and hPL, we found that our hPL batch had a slightly higher protein content than the FCS batch (Fig. 1B). ...
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... results were tested for statistical significance with GraphPad Prism 6. When two groups were analyzed, the unpaired, two-tailed Student's t-test was used. With more than two groups, a two-way ANOVA test was performed with Bonferroni's multiple comparisons and 95% confidence interval. Figure 1A shows how we generated hPL stocks (Fig. 1A). When we determined the protein concentrations of batches of FCS and hPL, we found that our hPL batch had a slightly higher protein content than the FCS batch (Fig. 1B). The FCS batch that we had chosen was used by us in previous experiments and gave immaculate results regarding pathogen-free cell proliferation (data not ...
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... used. With more than two groups, a two-way ANOVA test was performed with Bonferroni's multiple comparisons and 95% confidence interval. Figure 1A shows how we generated hPL stocks (Fig. 1A). When we determined the protein concentrations of batches of FCS and hPL, we found that our hPL batch had a slightly higher protein content than the FCS batch (Fig. 1B). The FCS batch that we had chosen was used by us in previous experiments and gave immaculate results regarding pathogen-free cell proliferation (data not ...
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... corroborate that leukemia cells respond equally to structurally diverse HDACi in FCS and hPL, we used MV4-11 cells as an additional system and treated them with entinostat as a further HDACi. PI-staining showed a comparable cell cycle distribution following treatment, independent of culture conditions in FCS or hPL (Fig. S3A 1 ). As MV4-11 cells react very sensitively to this agent, we had a closer look at the amount of subG1 fractions, which did also not vary significantly under different culture conditions (Fig. S3B 1 ). As expected from our data in K562 cells treated with Romidepsin (Fig. 5B), we could observe a loss of WT1 in MV4-11 cells treated with ...
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... as a further HDACi. PI-staining showed a comparable cell cycle distribution following treatment, independent of culture conditions in FCS or hPL (Fig. S3A 1 ). As MV4-11 cells react very sensitively to this agent, we had a closer look at the amount of subG1 fractions, which did also not vary significantly under different culture conditions (Fig. S3B 1 ). As expected from our data in K562 cells treated with Romidepsin (Fig. 5B), we could observe a loss of WT1 in MV4-11 cells treated with entinostat (data not ...
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... patterns (Fig. 3A). Differences that we noted between the two culture conditions were not significantly higher than differences that we noted among the triplicates. Via a correlation plot, we could show that all samples were at least 98% identical (Fig. 3B). Not a single protein was expressed differentially under the two culture conditions (Fig. S2 1 ...
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... imatinib is a standard drug for the treatment of CML carrying BCR-ABL (t9;21) ( Lamontanara et al., 2013), we assessed how led to an accumulation of acetylated tubulin (Fig. 5D, S3C,D 1 ). The levels of WT1 remained constant under such conditions, which supports our data illustrating that a pharmacological inhibition of HDAC6 does not attenuate WT1 expression (Fig. ...
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... genetic manipulation of cultured eukaryotic cells is a mainstay in the search for molecular mechanisms. To assess whether culture conditions based on FCS or hPL have a differential effect on the transfection of leukemic cells with RNAi molecules, we electroporated siRNAs against HDAC6 in K562 (Fig. 5D), MV4-11, and NB4 cells (Fig. S3C,D 1 ). We could successfully reduce HDAC6 protein in all three cell lines under both culture conditions (Fig. 5D, S3C,D 1 ). As expected from HDAC6 being a tubulin deacetylase ( Krämer et al., 2014), the reduction of HDAC6 ...
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... mechanisms. To assess whether culture conditions based on FCS or hPL have a differential effect on the transfection of leukemic cells with RNAi molecules, we electroporated siRNAs against HDAC6 in K562 (Fig. 5D), MV4-11, and NB4 cells (Fig. S3C,D 1 ). We could successfully reduce HDAC6 protein in all three cell lines under both culture conditions (Fig. 5D, S3C,D 1 ). As expected from HDAC6 being a tubulin deacetylase ( Krämer et al., 2014), the reduction of HDAC6 ...
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Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used media supplement worldwide. FCS contains a variable mixture of growth factors and cytokines, which support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesir...
Citations
... Media were supplemented with 5% fetal calf serum (FCS) (Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco). As previously reported [38], using 5% instead of 10% FCS reduces cell stress during proliferation. Cell density was kept below 400,000 cells per mL. ...
Introduction:
Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers.
Objectives:
We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells.
Methods:
We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells.
Results:
Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p<0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62.
Conclusion:
These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.
... 42 Total protein content shows a notable difference from FCS (p < 0.05), confirming the data from other studies. 43 Samples had higher glucose and albumin content than commercially available bovine serum, possibly influencing cell growth rate. 44 A varying degree of hPL content concentration is perceived when F I G U R E 2 Total protein and growth factor levels variation. ...
Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical‐grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin‐like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications.
... Touch only with gloves, also to avoid contaminating it. Human platelet lysate (hPL) can be used as a better controlled alternative to FBS, and hPL also allows electroporation of cells with siRNAs (Pons et al., 2019). Note: Dissolve Tris in deionized water. ...
... We effectively decrease proteins by electroporation and RNAi in MOLM-13 acute myeloid leukemia cells (voltage: 1,600 V, width: 10 ms, 3 pulses), in RS4-11 B cell precursor leukemia cells (voltage: 1,750 V, width: 20 ms, 1 pulse), and in HEL erythroleukemia cells (voltage: 1,400 V, width: 20 ms, 2 pulses). For K562 chronic myeloid leukemia cells and NB4 promyelocytic leukemia cells, see our recent publications(Pons et al., 2019(Pons et al., , 2021. ...
Genetic silencing of leukemia-associated proteins with small interfering RNAs (siRNAs) is a straightforward way to delineate their functions. It can be very challenging to deliver siRNAs to leukemia-derived cells with high transfection efficiency and without compromising their viability. This protocol describes an efficient approach to silence oncogenic feline McDonough sarcoma (FMS)-like tyrosine kinase-3 in leukemia cells using siRNAs that are delivered by electroporation. The protocol maintains high cell viability and is generally useful to decrease RNAs encoding proteins of interest.
For complete details on the use and execution of this protocol, please refer to Beyer et al. (2022).
... We can exclude that this is a downstream effect of cell death. Thus, like inhibition of HDAC6 in these and other leukemic cells, 4,[30][31][32] inhibition of HDAC10 modulates biological processes. Highly consistent with these data are results from knock-out mice with a deletion of HDAC6 or HDAC10. ...
Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells.
... We found that hydroxyurea and COH29 induced apoptosis and an accumulation of the tumor suppressor p53 and its target p21 in murine PDAC cells. In contrast to this, hydroxyurea increased the transcription of the p21 mRNA p53-dependently, but this did not translate into an accumulation of the p21 protein in human solid tumor-derived and leukemic cancer cells [32,33,51,60]. This can be explained by a CHK1-dependent suppression of p21 mRNA translation [61]. ...
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a dismal prognosis. Here, we show how an inhibition of de novo dNTP synthesis by the ribonucleotide reductase (RNR) inhibitor hydroxyurea and an inhibition of epigenetic modifiers of the histone deacetylase (HDAC) family affect short-term cultured primary murine PDAC cells. We used clinically relevant doses of hydroxyurea and the class 1 HDAC inhibitor entinostat. We analyzed the cells by flow cytometry and immunoblot. Regarding the induction of apoptosis and DNA replication stress, hydroxyurea and the novel RNR inhibitor COH29 are superior to the topoisomerase-1 inhibitor irinotecan which is used to treat PDAC. Entinostat promotes the induction of DNA replication stress by hydroxyurea. This is associated with an increase in the PP2A subunit PR130/PPP2R3A and a reduction of the ribonucleotide reductase subunit RRM2 and the DNA repair protein RAD51. We further show that class 1 HDAC activity promotes the hydroxyurea-induced activation of the checkpoint kinase ataxia-telangiectasia mutated (ATM). Unlike in other cell systems, ATM is pro-apoptotic in hydroxyurea-treated murine PDAC cells. These data reveal novel insights into a cytotoxic, ATM-regulated, and HDAC-dependent replication stress program in PDAC cells.
... Human platelet lysate (hP) is an alternative to FBS for culturing mesenchymal stem cells (Hemeda et al., 2014;Shanbhag et al., 2017;Ren et al., 2018;Saury et al., 2018), monocytes (Barsotti et al., 2013;Svajger, 2017), human umbilical vein endothelial cells (HUVECs) (Barsotti et al., 2013;Hofbauer et al., 2014;Robinson et al., 2016), and cell lines for leukemia, colon cancer (Pons et al., 2018) and hepatocellular carcinoma (Carr et al., 2014). Interestingly, the use of hP has been also reported for tissue engineering. ...
For decades, fetal bovine serum (FBS) has been used routinely for culturing many cell types, based on its empirically demonstrated effects on cell growth, and the lack of suitable non-xenogeneic alternatives. The FBS-based culture media do not represent the human physiological conditions, and can compromise biomimicry of preclinical models. To recapitulate in vitro the features of human bone and bone cancer, we investigated the effects of human serum and human platelet lysate on modeling osteogenesis, osteoclastogenesis, and bone cancer in two-dimensional (2D) and three-dimensional (3D) settings. For monitoring tumor growth within tissue-engineered bone in a non-destructive fashion, we generated cancer cell lines expressing and secreting luciferase. Culture media containing human serum enhanced osteogenesis and osteoclasts differentiation, and provided a more realistic in vitro mimic of human cancer cell proliferation. When human serum was used for building 3D engineered bone, the tissue recapitulated bone homeostasis and response to bisphosphonates observed in native bone. We found disparities in cell behavior and drug responses between the metastatic and primary cancer cells cultured in the bone niche, with the effectiveness of bisphosphonates observed only in metastatic models. Overall, these data support the utility of human serum for bioengineering of bone and bone cancers.
... BCR-ABL1 constitutively activates pro-survival and oncogenic signaling cascades through RAS-RAF-MEK-ERK, the transcription factors WT1 and STAT5, BCL-XL, and other proteins [37][38][39][40]. This may shield K562 cells from pro-apoptotic effects of hydroxyurea and could be a vulnerability to the ABL inhibitor imatinib [13,19,41,42]. Immunoblot revealed that imatinib reduced BCL-XL, pY-STAT5, STAT5, WT1, and p-ERK in untreated and hydroxyurea-treated K562 cells. ...
... Imatinib is a gold standard for the treatment of CML cells, and hydroxyurea can be given as cytoreductive therapy [19,41,42]. Imatinib plus hydroxyurea may achieve better responses in patients that are not successfully treated with imatinib and other TKi. ...
The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis
and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether
cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics
to uncover which pathways control the transition of the hydroxyurea-induced replication stress
into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the
serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant
doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the
sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition
of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with
hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that
causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the
ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic
effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3
that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea
and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant
drug combinations.
... It is therefore clinically tested, xeno-free, i.e., free of animal components, and contains factors that support cell growth and proliferation (van der Valk et al., 2018). A comparative study confirmed that cancer cells cultured in media supplemented with FBS or outdated hPL grow very similarly and have practically identical proteomes (Pons et al., 2019). Furthermore, they responded equally to different drugs and stress conditions that were tested in the study, demonstrating that hPL can substitute for FBS in various experimental settings. ...
Bioprinting is a rapidly developing technology that enables the exact positioning of living cells embedded in biomaterials in precise spatial arrangements to fabricate engineered tissues and organs. While the ultimate goal of bioprinting approaches is to produce organs for transplantation purposes, bioprinted organ models also hold great potential for research purposes to serve as alternatives to animal experiments. By using human cells, humanized organ models can be generated that may produce more relevant results for human (patho-)physiology than animal models. However, standard bioprinting procedures currently use numerous hidden animal components. Virtually all studies published in the field to date make use of cells grown in media with fetal bovine serum (FBS). In addition, Matrigel, the extracellular matrix (ECM) harvested from Engelbreth-Holm-Swarm sarcoma grown in mice, is widely employed to cultivate stem cells and 3D organ models. Finally, most bioinks currently in use contain gelatin or comparable animal components to improve cell viability and adhesion. The present review will give an introduction to the potential of bioprinting to fabricate 3D models that may be substituted for animal experiments and will go on to describe strategies to replace animal components currently included in standard procedures of bioprinting. These approaches comprise the adaptation of cells to FBS-free media, the use of bioinks composed of synthetic or plant material, and the replacement of animal ingredients by materials of human origin. We propose denoting bioprinting strategies devoid of animal components as clean bioprinting.
... Han sido probablemente la ausencia de ellos y de otros componentes que han afectado el crecimiento celular cuando el SFB estuvo ausente. Sin embargo, sería intere-sante investigar cómo sustitutos del SFB, entre ellos, los derivados plaquetarios, son capaces de reemplazar estos elementos y contribuir a condiciones de cultivo compatible con la terapia celular, como lo han demostrado estudios recientes (14)(15)(16)(17)(18). ...
Introducción.
Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación.
Objetivo.
Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos.
Materiales y métodos.
Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias.
Resultados.
Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados.
Conclusión.
Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares.
... However, we were the first study directly comparing two different concentrations of hPL in ODM, therefore further studies should follow in order to approach an optimum hPL concentration in ODM for BMSC. Considering the higher costs of hPL compared to FCS as well as the fact that the differences between the H10 and H1 groups were only detectable in the ALP activity assay, the use of hPL in smaller concentrations seems to be reasonable in order to avoid a significant increase of the budget necessary for cell culture experiments [54]. Based on the findings of this study, hPL should at least be considered as a potential alternative to FCS when analyzing osteogenic differentiation. ...
Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC.
