Concentration Of Fetal Bovine Serum Research Articles

Plasma of Argon Affects the Earliest Biological Response of Different Implant Surfaces

The aim of this in vitro study was to evaluate the early cell response and protein adsorption elicited by the argon plasma treatment of different commercially available titanium surfaces via a chair-side device. Sterile disks made of grade 4 titanium (n = 450, 4-mm diameter) with 3 surface topographies (machined, plasma sprayed, and zirconia blasted and acid etched) were allocated to receive 4 testing treatments (2% and 10% protein adsorption and cell adhesion with MC3T3-E1 and MG-63). Furthermore, the specimens were divided to undergo 1) argon plasma treatment (10 W, 1 bar for 12 min) in a plasma reactor, 2) ultraviolet (UV) light treatment for 2 h (positive control group), or 3) no treatment (control group). Pretreatment surface analyses based on a scanning electron microscope and profilometer images were also performed. Profilometric analysis demonstrated that the evaluated specimens perfectly suit the standard parameters. The use of argon plasma was capable of affecting the quantity of proteins adsorbed on the different surfaces, notwithstanding their roughness or topographic features at a low fetal bovine serum concentration (2%). UV light treatment for 2 h attained similar results. Moreover, both the plasma of argon and the UV light demonstrated a significant increase in the number of osteoblasts adherent at 10 min in all tested surfaces. Within its limitations, this in vitro study highlights the potential biological benefits of treating implant surfaces with plasma of argon or UV, irrespective of the roughness of the titanium surface. However, in vivo experiments are needed to confirm these preliminary data and settle the rationale of a treatment that might be clinically relevant in case of bone-reparative deficiencies.

Development and characterization of a cell line from tilapia head kidney with melanomacrophage characteristics.

A novel cell line THK, derived from the tilapia head kidney, was developed and characterized. The THK cell line comprised fibroblastoid cells that markedly proliferated in Leibovitz L-15 medium containing 2%-15% fetal bovine serum (FBS) at 20°C-35°C. Cell proliferation was dependent on the FBS concentration, and the optimal temperature for proliferation ranged between 25°C and 30°C. THK cells were characterized for the presence of phagocytic activity, acid phosphatase, alkaline phosphatase, α-naphthyl acetate esterase, lipofuscin, and tyrosinase. Transcripts of CD33, CD53, CD82, CD205, macrophage colony stimulating factor receptor, GATA2, and GATA3 that are specific for leucocytes or monocytes/macrophages or both were detected in the THK cells through PCR. However, THK cells lacked for CD83, a specific marker for dendritic cells. The results indicated that the fibroblastoid THK cells were melanomacrophage-related progenitors. PCR revealed that the THK cells exhibited the transcripts of toll-like receptor 1 (TLR1), TLR2, TLR3, and CD200, of which concern with immunity as well as the transcripts of vascular endothelial growth factor receptor 3, angiomotin, and angiopoietin-like protein 2 that associate with angiogenesis regulation and macrophage proliferation. THK cells were subcultured more than 90 times and can be useful for investigating the development and functioning of the teleostean innate immune system.

Effect of fetal bovine serum in culture media on MS analysis of mesenchymal stromal cells secretome

The analysis of mesenchymal stromal cells secretome is fundamental to identify key players of processes involving these cells. Truly secreted proteins may be difficult to detect in MS based analysis of conditioned media (CM) due to proteins supplemented with fetal bovine serum (FBS). We compared different growth conditions to determine the effect of varying FBS concentration on the number and quantity of truly secreted human proteins vs contaminating bovine proteins. The results suggest that to minimize interference cells should be grown in presence of FBS until confluence and transferred into a serum-free medium prior to secretome collection.

Internalization of bevacizumab by retinal endothelial cells and its intracellular fate: Evidence for an involvement of the neonatal Fc receptor

Bevacizumab is one of the VEGF-binding proteins that are established in clinical practice to treat various ocular diseases. In view of therapeutic long-term application, potential accumulation of the antibody in retinal cells gave reason for safety concerns. Internalization of considerable amounts of bevacizumab by retinal endothelial (REC) and pigment epithelial cells has been observed which may affect their important functions. Therefore we investigated the transport and intracellular localization of bevacizumab in immortalized bovine REC (iBREC) in detail, considering possible roles of vesicles and receptors mediating uptake and intracellular transport.By performing transcytosis assays with iBREC monolayers cultivated on porous membrane inserts, we demonstrated that bevacizumab was transported efficiently through a tight monolayer from the lower to the upper chamber or vice versa. When added to the lower chamber in excess, the internalized antibody was transported through the cells, but it was also recycled to be set free at the same side of the cell into a bevacizumab-free environment. The rates of both processes strongly depended on the concentration of fetal bovine serum (FBS) in the environment. This observation is important because in vivo REC might be exposed to varying amounts of serum, e.g. in patients with macular edema. FBS also affected the intracellular localization of bevacizumab as shown by analyses of subcellular fractions and direct immunofluorescence staining. When iBREC were cultivated in low-serum medium, most of the antibody was found in the fraction of cytoskeleton proteins and spots of high intensity of bevacizumab-specific staining close to the nuclei were observed. Cultivation in medium with FBS resulted in internalized bevacizumab predominately found in the membrane/organelle fraction in addition to its weaker association with proteins from the cytoskeleton and uniform staining of the cell. Bevacizumab-specific staining close to the cytoskeleton proteins α-tubulin or vimentin was also observed. Accumulation and association of the antibody with the cytoskeleton induced by serum reduction could be reversed by subsequent FBS addition. In uptake and transport of bevacizumab vesicles and binding to a receptor seems to be involved: Internalization was strongly temperature-dependent which ruled out paracellular passage and a fraction of the internalized bevacizumab was associated with early endosomes. Protein A inhibited transcytosis and affected intracellular localization suggesting a key role of the neonatal Fc receptor (FcRn). Interestingly, FcRn expression was decreased when iBREC were cultivated without FBS.Our results suggest this pathway of bevacizumab uptake and transition through iBREC: Independent of serum, bevacizumab is taken up through a nonspecific mechanism. The subsequent sorting into transport vesicles depends on the presence of serum as regulator of FcRn expression. Without sufficient amounts of the receptor being expressed, a likely obstructed exocytosis results in intracellular accumulation and an increased association with cytoskeleton proteins. Interaction of substantial amounts of bevacizumab with the cytoskeleton may be the reason for under these conditions suppressed migration of iBREC. If long-term therapies by intravitreal injection lead to accumulation of bevacizumab in REC in vivo and potentially harmful consequences, will have to be revealed by future investigations.

Comparison of Proliferative Effect of Human Lactoferrin and Its Proteolytic Peptide on Normal and Transformed Epithelial Cells

Human lactoferrin (hLF) is an iron-binding glycoprotein with a variety of functions. hLF undergoes proteolytic cleavage to smaller peptides in the stomach following ingestion. In the present study, we evaluated the effects of hLF and its proteolytic product, human lactoferrin peptide (hLFP), on the proliferation of two epithelial cells, HEK293 normal cells and KATO III gastric carcinoma cells, using an MTT assay and expression of proliferative nuclear cell antigen (PCNA), a notable proliferation marker. When the two epithelial cells were stimulated with hLF and hLFP in the presence of fetal bovine serum (FBS), hLFP stimulated proliferation of both cell types at lower concentrations than hLF by two orders of magnitude. The cancer cells exhibited proliferative responses to both hLF and hLFP at lower concentrations by 2∼3 orders of magnitude than the normal cells. Either hLF or hLFP alone did not support appreciable proliferation of these cell lines in the absence or low concentrations of FBS. Bovine serum albumin or its proteolytic product failed to promote cellular proliferation even in the presence of 10 % FBS, indicating the specificity of the proliferative activity of hLF and hLFP. These data highlight feasibility of hLF and its peptide for adjuvants for tissue culture medium.

Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells.

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

Monoolein-based nanocarriers for enhanced folate receptor-mediated RNA delivery to cancer cells

We report the development and characterization of a novel nanometric system for specific delivery of therapeutic siRNA for cancer treatment. This vector is based on a binary mixture of the cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and the helper lipid monoolein (MO). These liposomes were previously validated by our research group as promising non-viral vectors for nucleic acid delivery. In this work, the DODAC:MO vesicles were for the first time functionalized with polyethylene glycol and PEG-folate conjugates to achieve both maximal stability in biological fluids and increase selectivity toward folate receptor α expressing cells. The produced DODAC:MO:PEG liposomes were highly effective in RNA complexation (close to 100%), and the resulting lipoplexes also demonstrated high stability in conditions simulating their administration by intravenous injection (physiological pH, high NaCl, heparin and fetal bovine serum concentrations). In addition, cell uptake of the PEG-folate-coated lipoplexes was significantly greater in folate receptor α positive breast cancer cells (39% for 25 µg/mL of lipid and 31% for 40 µg/mL) when compared with folate receptor α negative cells (31% for 25 µg/mL of lipid and 23% for 40 µg/mL) and to systems without PEG-folate (≈13% to 16% for all tested conditions), supporting their selectivity towards the receptor. Overall, the results support these systems as appealing vectors for selective delivery of siRNA to cancer cells by folate receptor α-mediated internalization, aiming at future therapeutic applications of interest.

Microfluidic gradient device for studying mesothelial cell migration and the effect of chronic carbon nanotube exposure

Cell migration is one of the crucial steps in many physiological and pathological processes, including cancer development. Our recent studies have shown that carbon nanotubes (CNTs), similarly to asbestos, can induce accelerated cell growth and invasiveness that contribute to their mesothelioma pathogenicity. Malignant mesothelioma is a very aggressive tumor that develops from cells of the mesothelium, and is most commonly caused by exposure to asbestos. CNTs have a similar structure and mode of exposure to asbestos. This has raised a concern regarding the potential carcinogenicity of CNTs, especially in the pleural area which is a key target for asbestos-related diseases. In this paper, a static microfluidic gradient device was applied to study the migration of human pleural mesothelial cells which had been through a long-term exposure (4 months) to subcytotoxic concentration (0.02 µg cm−2) of single-walled CNTs (SWCNTs). Multiple migration signatures of these cells were investigated using the microfluidic gradient device for the first time. During the migration study, we observed that cell morphologies changed from flattened shapes to spindle shapes prior to their migration after their sensing of the chemical gradient. The migration of chronically SWCNT-exposed mesothelial cells was evaluated under different fetal bovine serum (FBS) concentration gradients, and the migration speeds and number of migrating cells were extracted and compared. The results showed that chronically SWCNT-exposed mesothelial cells are more sensitive to the gradient compared to non-SWCNT-exposed cells. The method described here allows simultaneous detection of cell morphology and migration under chemical gradient conditions, and also allows for real-time monitoring of cell motility that resembles in vivo cell migration. This platform would be much needed for supporting the development of more physiologically relevant cell models for better assessment and characterization of the mesothelioma hazard posed by nanomaterials.

Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos.

Proper oocyte maturation is crucial for subsequent embryo development; however, oocyte mitochondrial and lipid-droplet behaviour are still poorly understood. Although excessive lipid accumulation during in vitro production (IVP) of bovine embryos has been linked with impaired cryotolerance, lipid oxidation is essential for adequate energy supply. Fetal bovine serum (FBS) and bovine serum albumin (BSA) are supplements used during IVP, containing high and low lipid content, respectively. This study aimed to understand how these supplements influence oocyte mitochondrial and lipid behaviour during in vitro maturation (IVM) in comparison to in vivo maturation, as well as their influence on development rates and embryo lipid accumulation during IVP. We demonstrate that only in vivo-matured oocytes maintained correlation between lipid content and active mitochondria. IVM media containing FBS increased total lipid content 18-fold and resulted in higher lipid accumulation in oocytes when compared with media with BSA. IVM using a lower FBS concentration combined with BSA resulted in satisfactory maturation and embryo development and also reduced lipid accumulation in blastocysts. In conclusion, IVM causes changes in mitochondrial and lipid dynamics, which may have negative effects on oocyte development rates and embryo lipid accumulation. Moreover, decreasing FBS concentrations during IVM may reduce embryo lipid accumulation without affecting production rates.

Microenvironment-dependent respiration of T-47D cells cultured in alginate biostructures.

The main objective of this paper was to investigate whether the oxygen consumption rate (OCR) of cells entrapped in alginate hydrogels depends on presence of soluble factors present in foetal bovine serum (FBS). Pericellular oxygen concentrations were measured using a photochemical oxygen sensor inserted into bioconstructs made from different formulations of alginate, containing T-47D cells. Cell count was corrected for viability as determined by cell uptake and exclusion of standard live/dead fluorophores, in sections of freshly prepared biostructures. Based on concentration data, OCR of the embedded cells was calculated according to a simple algorithm. OCR was found to vary significantly between the different formulations investigated. Inclusion of high concentrations of FBS in the biostructure matrices elicited significantly higher OCRs, in guluronate-rich gels similar to those previously found in monolayer culture. Guluronate-rich gels also generally permitted highest OCR. Respiration also had a falling tendency with increasing alginate concentration and elastic modulus. Presence of FBS in biostructure matrices elicited higher OCR in T-47D cells. Formulation of biostructures must consider differential diffusion of macromolecular substances.

Modulation of the cancer cell transcriptome by culture media formulations and cell density.

We investigated how varying the composition of cell culture formulations and growing cancer cells at different densities might affect tumor cell genotype. Specifically, we compared gene expression profiles generated by human MDA-MB-231 breast cancer cells cultured in different media [minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), or Roswell Park Memorial Institute (RPMI)-1640 medium] containing different concentrations of fetal bovine serum (FBS) or different sera (equine or bovine) that were grown at different cell densities. More than 2,000 genes were differentially modulated by at least a 2-fold difference when MDA-MB-231 cancer cells were 90% confluent and compared with cultures that were 50% confluent. Altering the concentration of serum produced an even more pronounced effect on MDA-MB-231 cancer cell gene expression in that 2,981 genes were differentially expressed in a comparison between cells cultured in 0.1% FBS and same cell density cultures that were maintained in 10% FBS. A comparison between MDA-MB-231 cancer cells that were 90% confluent in MEM, DMEM, or RPMI-1640 media, all containing 10% FBS, resulted in 8,925 differentially expressed genes. Moreover, one-quarter (25.6%) of genes from our genome-wide expression analysis were expressed at significantly different levels by cells grown in MEM, DMEM, or RPMI-1640 media. Genes associated with epithelial-mesenchymal transition (EMT) were among the genes that were differentially modulated by cells grown in different cell culture formulations and these genes were verified at the protein level. Collectively, these results underscore the importance of accurate reporting and maintenance of uniform culture conditions to ensure reproducible results.

Oxygen tension affects histone remodeling of in vitro–produced embryos in a bovine model

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O2; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.

Low concentrations of cylindrospermopsin induce increases of reactive oxygen species levels, metabolism and proliferation in human hepatoma cells (HepG2)

Human hepatoma cells (HepG2) were exposed to purified cylindrospermopsin (CYN), a potent toxicant for eukaryotic cells produced by several cyanobacteria. CYN was not toxic at concentrations up to 10μgl−1, leading to increased viability and metabolism in cells cultured with 10% fetal bovine serum (FBS). Reduction of FBS concentration to 2% and induction of cytochrome P450 (CYP) isoforms were performed in order to make xenobiotic-metabolizing capacity of HepG2 cells closest to that of ‘normal’ cells. HepG2 cells proliferated less after CYPs-induction, and this induction has lead to similar results of non-induced cells, except for few individual parameters such lipid peroxidation. Foremost, low concentrations of CYN (below or equal 10μgl−1) have induced HepG2 cells proliferation and metabolism increase, which was not expected.

Artocarpin Induces Apoptosis in Human Cutaneous Squamous Cell Carcinoma HSC-1 Cells and Its Cytotoxic Activity Is Dependent on Protein-Nutrient Concentration.

Artocarpin, a natural prenylated flavonoid, has been shown to have various biological properties. However, its effects on human cutaneous squamous cell carcinoma (SCC) have not been previously investigated. We set out to determine whether artocarpin has cytotoxic effects on SCC cells and whether its pharmacological activity is dependent on protein-nutrient concentration. Our results showed that treatment of HSC-1 cells (a human cutaneous SCC cell line) with artocarpin decreased cell viability and induced cell apoptosis by increasing caspase 3/7 activity. These effects were more pronounced at low fetal bovine serum (FBS) concentrations. Artocarpin induced an increase in the level of phospho-p38 and a decrease in the levels of phospho-ERK, phospho-JNK, phospho-Akt, phospho-mTOR, and phospho-S6K. High FBS concentrations in the culture media inhibited and delayed the uptake of artocarpin from the extracellular compartment (culture media) into the intracellular compartment, as determined by high performance liquid chromatography (HPLC) analysis. In conclusion, artocarpin induces apoptosis in HSC-1 cells through modulation of MAPK and Akt/mTOR pathways. Binding of artocarpin to proteins in the FBS may inhibit cellular uptake and reduce the cytotoxic activity of artocarpin on HSC-1 cells. Therefore, artocarpin may have potential use in the future as a form of treatment for cutaneous SCC.

Synthesis of quasi-spherical and square shaped oligoamino-ester graft-from magnetite nanoparticles: Effect of morphology and chemical structure on protein interactions

In this research, oligoamino-ester grafted magnetite nanoparticles (AEMNPs) with dendritic and linear architecture were synthesized and the effect of morphology and chemical structures of these modified MNPs on the secondary structure of adsorbed protein have been investigated. In the first step, the synthesized magnetic nanoparticles were modified with 3-(triethoxysilyl) propylamine (APTES). The sequential reaction of these silica–coated nanoparticles with acryloyl chloride and diethanolamine will produce the first (G1), second (G2) and third (G3) generations of fluffy shaped nanoparticles. Three generations of these nanoparticles with linear structure were obtained in the presence of 2-(diethylamino) ethanol instead of diethanolamine. FT-IR, thermal gravimetric analysis (TGA), dynamic light scattering (DLS) and zeta potential techniques were used to identify the final structure of all dendrimers. Antibiofouling properties of AEMNPs were analyzed in two different concentrations of fetal bovine serum and the effect of various factors such as zeta potential, density and the surface hydroxyl groups were evaluated by DLS, UV-vis and fluorometric methods. Circular dichroism spectra of FBS in the presence of various amounts of nanoparticles provided information about protein secondary structures.

Controlling plasma stimulated media in cancer treatment application

Cold atmospheric plasma (CAP) constitutes a “cocktail” of various reactive species. Accumulating evidence shows the effectiveness of CAP in killing cancer cells and decreasing the tumor size, which provides a solid basis for its potential use in cancer treatment. Currently, CAP is mainly used to directly treat cancer cells and trigger the death of cancer cells via apoptosis or necrosis. By altering the concentration of fetal bovine serum in Dulbecco's modified Eagle's medium and the temperature to store CAP stimulated media, we demonstrated controllable strategies to harness the stimulated media to kill glioblastoma cells in vitro. This study demonstrated the significant role of media in killing cancer cells via the CAP treatment.

Unconventional secretion of synuclein-γ promotes tumor cell invasion.

Synuclein-γ (SNCG) is a chaperone protein and exists mainly in the cytoplasm. SNCG confers chemoresistance, and is a potential unfavorable biomarker for multiple types of cancer. Our previous work demonstrated that SNCG could be detected in the serum of cancer patients and the medium of cultured cancer cells, but the mechanism of SNCG secretion and its biological roles are unknown. Here, we showed that SNCG levels in the culture medium were positively correlated with cancer cell densities and the concentrations of fetal bovine serum added. SNCG secretion was unaffected by brefeldin A, an inhibitor of the classic protein transport pathway, but was antagonized by exosome inhibitor, lysosome inhibitor, ABC transporter inhibitor, and phosphatidylinositide 3-kinase inhibitor, and knockdown of Rab27a. Ultracentrifugation fractionation revealed that intracellular SNCG was present as both free and vesicle-associated forms, but that the extracellular SNCG was mainly free. The results of reciprocal coimmunoprecipitation experiments showed an interaction between SNCG and flotillin-2, a marker of exosomes and lipid rafts. Moreover, we demonstrated that SNCG, both secreted from tumor cells and exogenously added, markedly promoted cancer cell migration and invasion, but had no effect on noncancerous cells. These findings suggest that SNCG is actively secreted by cancer cells via an unconventional secretion pathway and contributes to aggressive phenotypes of cancer cells.

Cytotoxicity of protein corona-graphene oxide nanoribbons on human epithelial cells

Graphene oxide nanoribbons (GONRs) were synthesized using an oxidative unzipping of multi-walled carbon nanotubes. The interactions of the GONRs with various concentrations of fetal bovine serum or human plasma serum indicated that the GONRs were functionalized substantially by the albumin originated from the two different protein sources. Then, concentration-dependent cytotoxicity of the protein-functionalized GONRs on human epithelial cells was studied. Although the GONRs with concentrations ≤50μg/mL did not exhibit significant cytotoxicity on the cells (with the cell viability >85%), the concentration of 100μg/mL exhibited significant cytotoxicity including prevention of cell proliferation and induction of cell apoptosis. These results can provide more in-depth understanding about cytotoxic effects of graphene nanostructures which can be functionalized by the proteins of media.

Optimization of Culture Conditions for Stem Cells Derived from Human Anterior Cruciate Ligament and Bone Marrow

Tissue engineering with stem cells is a fascinating approach for treating anterior cruciate ligament (ACL) injuries. In our previous study, stem cells isolated from the human anterior cruciate ligament were shown to possess extensive proliferation and differentiation capabilities when treated with specific growth factors. However, optimal culture conditions and the usefulness of fetal bovine serum (FBS) as a growth factor in in vitro culture systems are yet to be determined. In this study, we compared the effects of different culture media containing combinations of various concentrations of FBS and the growth factors basic fibroblastic growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) on the proliferation and differentiation of ligament-derived stem cells (LSCs) and bone marrow mesenchymal stem cells (BMSCs). We found that α-MEM plus 10% FBS and bFGF was able to maintain both LSCs and BMSCs in a relatively undifferentiated state but with lower major extracellular matrix (ECM) component gene expression and protein production, which is beneficial for stem cell expansion. However, the differentiation and proliferation potentials of LSCs and BMSCs were increased when cultured in MesenPRO, a commercially available stem cell medium containing 2% FBS. MesenPRO in conjunction with TGF-β1 had the greatest ability to induce the differentiation of BMSCs and LSCs to ligament fibroblasts, which was evidenced by the highest ligamentous ECM gene expression and protein production. These results indicate that culture media and growth factors play a very important role in the success of tissue engineering. With α-MEM plus 10% FBS and bFGF, rapid proliferation of stem cells can be achieved. In this study, MesenPRO was able to promote differentiation of both LSCs and BMSCs to ligament fibroblasts. Differentiation was further increased by TGF-β1. With increasing understanding of the effects of different culture media and growth factors, manipulation of stem cells in the desired direction for ligament tissue engineering can be achieved.

Serum-containing medium effect on isolation rate of dental pulp cells from cryopreserved intact deciduous teeth.

To isolate cells from pulp of intact cryopreserved deciduous teeth. The null hypothesis raised here is to find no difference in the establishment of cell culture after cryopreservation (1) using culture medium supplemented with different concentrations of fetal bovine serum (FBS); and (2) between teeth with different stages of physiological root resorption. Intact deciduous teeth with different root resorption stages were cryopreserved using FBS and Dimethyl Sulfoxide (DMSO) medium (9:1) in a progressive freezing process, by placing the samples in the refrigerator (4 degrees C/60 min) and subsequently transferring them to a -80 degrees C freezer (controlled device -1 degrees C/min/24 hours), and finally into liquid nitrogen (-196 degrees C/30 days). After the thawing process, the cell isolation was performed by enzymatic digestion (type I collagenase). The cells were re-suspended into the culture medium with 10% (G1) or 20% (G2) of FBS. Microscopic analysis was performed after 30 days to visualize the cell attachment. The culture establishment rate was higher in G2 (75%) than G1 (12.5%) (p = 0.041). There was no difference between the different stages of root resorption. It was possible to establish cell cultures from the pulp of intact cryopreserved deciduous teeth. The use of 20% FBS after thawing improved the culture rate.