From Fetal Bovine Serum Research Articles

Comparative Analysis of Serum and Serum-Free Medium Cultured Mesenchymal Stromal Cells for Cartilage Repair.

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.

Proteomics of serum proteins adsorbed onto hydroxyapatite single-crystal particles with an anisotropic structure

Hydroxyapatite (HAp) has a high adsorption affinity for proteins. Here, the adsorption specificity of proteins from fetal bovine serum (FBS) onto three types of HAp particles with different crystal planes was examined via proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fiber-like (f-HAp) and plate-like (p-HAp) HAp single-crystal particles were used as models for a-plane- and c-plane-oriented HAps, respectively, together with randomly oriented HAp particles (HAp-100), as a control. The amount of protein adsorption was as follows: f-HAp > p-HAp > HAp-100. The number of protein-derived spots was as follows: f-HAp > HAp-100 > p-HAp. LC-MS/MS analysis detected the most abundant proteins in the f-HAp sample, followed by p-HAp and HAp-100 before the removal of abundant proteins; after their removal, the highest number of detected proteins was recorded in the p-HAp sample, followed by f-HAp and HAp-100. Approximately 60 % of the proteins adsorbed onto the three HAp particles were extracellular matrix (ECM) proteins, which were identified via gene ontology (GO) analysis as proteins related to blood coagulation, binding, and cellular/metabolic processes. Mimecan, a protein involved in bone formation/differentiation, was among the proteins adsorbed on f-HAp. These results highlight the potential of this technology to control the orientation plane of HAp for highly specific protein adsorption.

Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer.

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flow-through pores of the monolithic skeleton (median pore size; 5.21 μm) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBS-derived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.

Enhancement of protein immobilization on polydimethylsiloxane using a synergistic combination of polydopamine and micropattern surface modification

Understanding protein interactions at biointerfaces is critical for the improved design of biomaterials and medical devices. Polydimethylsiloxane (PDMS) is used for numerous device applications, and surface modifications can enhance protein immobilization and the response to cells. A multifunctional approach combining topographical and biochemical modifications was applied to PDMS by fabricating 10–20 µm scale patterns onto PDMS surfaces and by coating with polydopamine (PDA). The modifications were confirmed by surface characterization and bovine serum albumin (BSA), fibrinogen (Fg), and fetuin-A (Fet-A) were radiolabeled with 125I. The amounts of protein attached to the surface before and after elution with sodium dodecyl sulfate (SDS) were quantified from single and complex multi-protein solutions to determine protein stability and competitive binding. The PDA coatings were the most stable and capable of immobilizing the highest levels of all proteins. Furthermore, combinations of PDA coatings with the smallest micropatterns provided an additional improvement, enhancing the amount immobilized and the stability. The adsorption of BSA and Fg from plasma demonstrated competitive binding and possible orientation changes, respectively. It was determined that Fet-A, a less studied protein, adsorbed from plasma at low levels, but the adsorption from fetal bovine serum (FBS) was significantly greater, providing important quantification data from radiolabeling that is relevant to many cell culture studies. Overall, combining topography and PDA modification has a synergistic effect on improving protein immobilization. These findings provide new insight on the quantities of proteins bound to PDMS and PDA coatings with implications for cell interactions in various biotechnology and medical applications.

Selective profiling of liver-related specific proteins based on sofosbuvir-modified magnetic separation material.

It has great significance in profiling specific proteins throughout for better understanding of complex pathological processes and in-depth pharmacological studies. In this work, an efficient protein profiling strategy was developed based on the specific protein-drug interaction. Sofosbuvir (SOF), as a first-line drug for the treatment of hepatitis C, was modified onto the surface of nanoparticles through stable chemical bonds to fabricate a novel magnetic separation material denoted as Fe3O4@SiO2@PAA@SOF. With sequence coverage as the screening parameter, nine proteins were profiled from fetal bovine serum (FBS) of which eight were liver related. Similarly, the strategy was applied to hepatocellular carcinoma (HCC) patient serum. Eight proteins were profiled and all of them were liver related, demonstrating the superb specificity and selectivity of this strategy for profiling liver-related proteins by virtue of protein-SOF interaction. When serum proteins from HCC patients were compared to those from healthy people, one unique differential protein (D3DQX7) was profiled, which was liver related and was a potential target for ameliorating liver diseases. For further research, this material design concept and protein profiling strategy can be extended to employ other drugs for corresponding studies. Sofosbuvir, as a therapeutic drug for liver diseases, was modified onto the surface of magnetic nanoparticles to fabricate the specific selective separation material (Fe3O4@SiO2@PAA@SOF). Based on protein-SOF interaction, the material was applied to adsorb specific proteins from different serum samples. After MS analysis, specific proteins, most of which were liver related, were successfully profiled from FBS and HCC patient serum, fully demonstrating the superb specificity and selectivity of this protein profiling strategy.

60 High lipid exposure during invitro maturation alters the lipid profile of bovine oocyte and benefits blastocyst development

Lipid metabolism provides a potent source of energy and has an important role in the acquisition of oocyte competence. However, there are conflicting reports about how lipid exposure during invitro maturation (IVM) impacts the gamete and further embryo development. In this study, we performed IVM of oocytes in the presence of lipid-rich culture media and used a broad lipid screening to accurately map the impact on the lipid profile and developmental potential. For that, nonpolar lipids were extracted from fetal bovine serum (FBS) with organic solvents (Bligh-Dyer method) and then used to supplement IVM medium (TCM-199 bicarbonate+10% FBS, hormones, pyruvate and antibiotics). COCs obtained from abattoir ovaries were submitted to IVM (4 biological replicates) in 2 groups: OC (control; IVM medium) and OHL (high lipid; IVM medium supplemented with extra 10% FBS nonpolar lipids). After 24h, we collected mature oocytes and those remaining followed to IVF and then to IVC (synthetic oviductal fluid with amino acids, SOFaa, with 5% FBS) for 7 days at 38.5°C, 20% O2, and 5% CO2 in air in high humidity. Expanded blastocysts were collected (BC and BHL) and blastocyst rates were assessed. Lipid extracts of individual oocytes and embryos (n=10/group) were analysed by multiple reaction monitoring (MRM)-profiling mass spectrometry. A total of 379 lipids from 10 classes were investigated [triacylglycerol (TAG), cholesteryl esters (CE), free fatty acids (FFA), acyl-carnitine, sphingomyelin (SM) and phospholipids derived from choline (PC), ethanolamine (PE), glycerol (PG), serine (PS), and inositol (PI)]. Exploratory data analysis was performed by principal component analysis (PCA; Metaboanalyst 4.0), and fold-change (FC) values were calculated based on the relative intensity of lipid ions (FC > 2 and P<0.05). IVC rates were compared by t-test (α=5%). PCA revealed a clear distinction in the lipid content for both oocytes and blastocysts (control vs. treated). More specifically, there was 2-fold enrichment for total TAG and CE in control groups and a 1.5-fold enrichment for total FFA in the treated groups at the oocyte and the blastocyst stages. Surprisingly, the average blastocyst rate was higher in the group derived from oocytes exposed to a high-lipid environment (41.56±7.73 vs. 22.62±1.67; P=0.003), which led us to investigate specific lipid ions. Groups OHL and BHL had increased contents of structural and signalling phospholipids (PC, SM, PE, and PS) and up to 3 times more oleic and linoleic acids, which have been associated with improved oocyte maturation and blastocyst development. Here, we demonstrate how distinct lipid exposures during IVM can robustly alter the lipid profile of oocytes. But more interestingly, it is clear that these are long-term effects, still observed at the blastocyst stage. More studies are required to verify the metabolic impact of this alternative lipid supplementation; however, these results indicate that high lipid exposure is not necessarily detrimental and, at a certain point, may even counteract lipid accumulation commonly observed during invitro embryo production.

Tracing the In Vivo Fate of Nanoparticles with a “Non-Self” Biological Identity

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.

Existence of Circulating Mitochondria in Human and Animal Peripheral Blood.

Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions. However, we found circulating mitochondria in human and animal blood. Electron microscopy confirmed the presence of mitochondria in adult human blood plasma. Flow cytometry analyses demonstrated that circulating mitochondria from the plasma of human cord blood and adult peripheral blood displayed the immune tolerance-associated membrane molecules such as CD270 and PD-L1 (programmed cell death-ligand 1). Similar data were obtained from fetal bovine serum (FBS) and horse serum of different vendors. Mitochondria remained detectable even after 56 °C heat inactivation. A real-time PCR array revealed purified mitochondria from animal sera expressed several genes that contribute to human T- and B-cell activation. Transwell experiments confirmed the migration capability of mitochondria through their expression of the chemokine receptor CXCR4 in responses to its ligand stromal-derived factor-1α (SDF-1α). Functional analysis established that human plasma mitochondria stimulated the proliferation of anti-CD3/CD28 bead-activated PBMC, up-regulated the percentage of activated CD4+ T and CD8+ T cells, and reduced the production of inflammatory cytokines. These findings suggested that the existence of circulating mitochondria in blood may function as a novel mediator for cell-cell communications and maintenance of homeostasis. Plasma-related products should be cautiously utilized in cell cultures due to the mitochondrial contamination.

Widespread presence of bovine proteins in human cell lines.

HPLC-MS/MS analysis of various human cell lines shows the presence of a major amount of bovine protein contaminants. These likely originate from fetal bovine serum (FBS), typically used in cell cultures. If evaluated against a human protein database, on average 10% of the identified human proteins will be misleading (bovine proteins, but indicated as if they were human). Bovine contaminants therefore may cause major bias in proteomic studies of cell cultures, if not considered explicitly.

Protein Adsorption From Biofluids on Silica Nanoparticles: Corona Analysis as a Function of Particle Diameter and Porosity.

A study on the adsorption of proteins from fetal bovine serum (FBS) on spherical dense and mesoporous silica nanoparticles with a wide range of diameters, from 70 to 900 nm, is presented. Monodisperse populations of particles with a range of diameters were obtained through modifications of the Stöber method. Extensive characterization of the particles was then performed using N2 physisorption, TEM, DLS, and ζ-potential. Following serum exposure, proteomic evaluation in concert with thermogravimetric analysis revealed the associated concentrations of each protein identified in the hard corona. Small particles adsorbed the largest amount of protein, due to their larger external surface area. Proteins with low molecular weights (<50 kDa) constituted the majority of the protein corona, totaling between 60 and 80% of the total mass of adsorbed protein. Here, the higher surface curvature of small particles favors the enrichment of smaller proteins. Porosity does not promote protein adsorption but improves deposition of the low molecular weight protein fraction due to the size-exclusion effect related to pore diameter. These results have important implications for the use of dense and porous silica nanoparticles in biomedical applications.

Separation of one-pot procedure released O-glycans as 1-phenyl-3-methyl-5-pyrazolone derivatives by hydrophilic interaction and reversed-phase liquid chromatography followed by identification using electrospray mass spectrometry and tandem mass spectrometry

Qualitative and quantitative analysis of naturally-occurring complex glycans is essential for glycomics, which focuses on the studies of structure–function correlations of saccharides. We previously reported a one-pot procedure for the non-reductive release from glycoproteins and in situ labeling with 1-phenyl-3-methyl-5-pyrazolone (PMP) of O-glycans (C. Wang et al., Proteomics 11 (2011) 4229). Here we describe an HPLC-based O-glycan analytical strategy that combines a range of techniques including the one-pot procedure, independent separation by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC) and identification by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The complex mixtures of both neutral and sialylated O-glycans as bis-PMP derivatives released from glycoproteins using the one-pot procedure could be well separated by HILIC based on their size or by RP-HPLC depending on the type of linkage and their resulting three-dimensional (3D) structure, and their structure could be characterized by the ESI-MS and MS/MS analysis of the eluted glycan fractions. The validity of the current strategy was confirmed by the analysis of O-glycans released by the one-pot procedure from some standard glycoproteins, including porcine stomach mucin (PSM), bovine submaxillary mucin (BSM) and bovine fetuin. The applicability of the current method to complex biological samples was also demonstrated by the analysis of mucin-type glycans from fetal bovine serum (FBS) and frog egg–jelly coat (FEC). This strategy, a powerful analytical tool that features the combination of different techniques, is useful for the qualitative and quantitative O-glycan analysis of more complex biological samples and has a potential for constructing an O-glycan analysis and structure database.

Atmospheric Pressure Plasma Surface Modification of Poly(D,L‐lactic acid) Increases Fibroblast, Osteoblast and Keratinocyte Adhesion and Proliferation

AbstractAn atmospheric pressure plasma deposition for P(d,L)LA (PLA) film was used to modify polymer surface properties using 1,2‐diaminopropane and acrylic acid as precursors. These two different plasma coatings result in a high density of amino groups (PLA‐NH2) and carboxylic groups (PLA‐COOH) onto PLA surface as demonstrated by Fourier transform infra‐red (FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS). Plasma coatings modified PLA surface wettability and proteins adsorption from fetal bovine serum (FBS), influencing cell adhesion and proliferation of 3T3 mouse fibroblast, MC‐3T3 E1 mouse pre‐osteoblast, and HaCaT cells (human keratinocytes). In particular both coatings increased pre‐osteoblast and keratinocyte adhesion while no effect was observed on fibroblast. Moreover, cell proliferation assessed after 48 h by Tox‐8 assay was significantly higher for osteoblast cells and keratinocyte seeded onto both PLA‐NH2 and PLA‐COOH compared to cells seeded onto normal PLA. On the basis of the obtained data, the atmospheric pressure plasma deposition described might represent an innovative and useful tool for bone and skin tissue engineering. magnified image

Effect of thyroid hormone depletion on cultured murine cerebral cortex astrocytes

Astrocytes play a crucial role in several steps of brain development, such as the proliferation of neural precursors, neuronal migration and differentiation, axonal growth, and synaptogenesis. Astrocyte generation and maturation is dramatically modulated by thyroid hormones (THs). Here, we propose a modified model for studying THs action on astroglial cells, in vitro. We investigated the effect of depletion of THs from fetal bovine serum (FBS) on the expression of the astrocyte maturation markers, GFAP (glial fibrillary acidic protein) and S100β, and the extracellular matrix (ECM) proteins laminin and fibronectin in cultured astrocytes. To accomplish this, murine cortical astrocytes were cultured in medium supplemented with THs-depleted serum, in contrast to the traditional techniques that use normal FBS which contains considerable amounts of THs. Immunostaining revealed that depletion of THs from FBS did not affect astrocyte proliferation, as observed by the number of astrocytes labeled for the proliferation antigen, Ki67. Surprisingly, western blot and RT-PCR assays revealed decreased levels of GFAP and S100β in astrocytes cultured with depleted serum. These events were reversed by addition of THs to the medium. Immunostaining and western blot assays did not reveal any difference in the organization and synthesis of the ECM protein, laminin; whereas the levels of fibronectin were decreased by 50% in THs-depleted serum. The fact that decreased expression of GFAP and fibronectin is associated with hypothyroidism in vivo suggests that our work might provide a useful model to assess in vitro the molecular mechanism underlying astrocytic maturation under conditions of THs deficiency.

Adsorption of Proteins Derived from Fetal Bovine Serum onto Hydroxyapatite Nanocrystals with Quartz Crystal Microbalance Technique

The adsorption of multiple proteins derived from fetal bovine serum (FBS) in phosphate buffer saline (PBS) and alpha minimum essential (aMEM) was in situ analyzed with a quartz crystal microbalance with dissipation technique on gold, titanium and HAp sensors. The adsorption behaviors of FBS proteins were varied depending on the sensors. The DD/Df value of the HAp sensor were clearly different in PBS and aMEM, and others were not changed. The viscoelastic properties of the protein films adsorbed on the HAp sensor in PBS were flexible in comparison with those on the gold and titanium sensors. The D-f plots incidated that the proteins adsorbed on HAp in PBS would lead to competitive adsorption and conformational change and those in aMEM could form a monolayer. The adsorption behavior on the HAp in carbonate buffer saline was found to be similar to that in aMEM. These differential adsorption behaviors on the HAp surface were attributed to the pre-adsorptive ion, such PO43- or CO32- in the solvent.

Human platelet lysate can replace fetal bovine serum for clinical‐scale expansion of functional mesenchymal stromal cells

Human multipotent mesenchymal stromal cells (MSCs) are promising candidates for a growing spectrum of regenerative and immunomodulatory cellular therapies. Translation of auspicious experimental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS). The capacity of human platelet lysate (HPL) to replace FBS for clinical-scale MSC propagation was analyzed. HPL could be efficiently produced from buffy coats. Multiplex analyses allowed a distinct HPL growth factor profile to be delineated. With a previously established two-step clinical-scale procedure, HPL was reproducibly more efficient than FBS in supporting MSC outgrowth. With only 3 x 10(5) primary culture-derived MSCs, a mean of 4.36 x 10(8) HPL-MSCs (range, 3.01 x 10(8)-5.40 x 10(8)) was obtained within a single secondary 11- to 13-day culture step. Although morphologically distinct, HPL-MSCs and FBS-MSCs did not differ significantly in terms of immunophenotype, differentiation potential in vitro, and lack of tumorigenicity in nude mice in vivo. Replacing FBS with HPL prevents bovine prion, viral, and zoonose contamination of the stem cell product. This new efficient FBS-free two-step procedure for clinical-scale MSC propagation may represent a major step toward challenging new stem cell therapies.

Human Mesenchymal Stem Cell Therapy: Platelet Lysate Supports Efficient Preclinical Expansion.

Coinfusion of hematopoietic stem cells with multipotent mesenchymal stromal/stem cells (MSC) to facilitate engraftment and immunomodulatory treatment of graft versus host disease (GvHD) are the two foremost applications of MSC therapy in hematology and oncology. Translation of promising experimental results into clinical applications has been mainly hampered by the strict dependence of MSC propagation from fetal bovine serum (FBS)-derived factors.We analyzed the capacity of different preparations of human platelet lysates (HPL) to replace FBS for clinical scale MSC production with cytomic, proteomic and genomic technology.Using a previously established two-step good manufacturing practice procedure for MSC propagation, we found that HPL can fully replace FBS as a growth factor supplement for clinical scale MSC preparation. HPL was reproducibly more efficient than FBS and supported the outgrowth of >400 million MSC from <0.4 million primary MSC (derived from <2mL bone marrow aspiration within 10 days ex vivo) within only 2 additional weeks of culture. Although morphologically distinct, HPL-MSC and FBS-MSC did not differ regarding their CD13+/29+/73+/90+/105+/146+/34−/45−/133−/HLA-AB+ immunophenotype. Multiplex analyses allowed delineating a distinct cytokine and growth factor profile. Whole transcriptome microarray analyses revealed a differential regulation of >300 genes after only one cycle of human as compared to bovine growth factor stimulation. Allogeneic HPL-MSC infusion in an intent to treat refractory GvHD was feasible and without infusion-related side effects in one patient. This new FBS-free two-step procedure for clinical scale MSC propagation will largely facilitate rational clinical testing of MSC based therapies. Replacing FBS with HPL excludes bovine prion, viral and zoonose contamination of the stem cell product. Based on this protocol a clinical trial for the treatment of steroid-refractory GvHD with HPL-MSC has been initiated.

Transplantation of Autologous Serum-Derived Cultivated Corneal Epithelial Equivalents for the Treatment of Severe Ocular Surface Disease

To evaluate the use of autologous serum (AS)-derived cultivated corneal epithelial transplantation for the treatment of severe ocular surface disease. Retrospective noncomparative case series. Nine eyes from 9 patients with total limbal stem cell deficiency were studied. These consisted of 2 eyes with Stevens-Johnson syndrome, 1 with chemical injury, 1 with ocular cicatricial pemphigoid, 1 with Salzmann corneal dystrophy, 1 with aniridia, 1 with graft-versus-host disease, and 2 with idiopathic ocular surface disease. Autologous serum obtained from patients was used for cultivating corneal epithelial cells on an amniotic membrane substrate. These AS-derived corneal epithelial equivalents were compared with those derived from fetal bovine serum (FBS)-supplemented medium. At the time of surgery, complete removal of the corneal pannus and conjunctiva up to 3 mm from the limbus was performed. Allogeneic (7 cases) and autologous (2 cases) AS-derived cultivated corneal epithelial equivalents were transplanted onto the ocular surface. Postoperative follow-up included serial slit-lamp examinations with fluorescein staining, as well as photographic documentation. Ocular surface reconstruction with corneal epithelialization, graft integrity, visual acuity, and postoperative complications. The corneal epithelial sheets cultivated in AS- and FBS-supplemented media were morphologically similar, and demonstrated the normal expression of tissue-specific keratins and junctional specialization assembly proteins. After transplantation, complete corneal epithelialization was achieved within 2 to 5 days. All eyes demonstrated an improvement in visual acuity by > or =2 lines. During the follow-up period of 14.6+/-4.36 months, the corneal surface of all patients remained stable and transparent, without significant complications. Transplantation of AS-derived cultivated corneal epithelial equivalents was shown to be a feasible method of treating patients with severe ocular surface disease. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Effects of Serum Proteins in Apatite Layer Formed in Culture Medium on Initial Adhesion and Cell Proliferation

In cell culture medium containing serum proteins, a layer with a great role in ostelblast-like cell’s growth formed on a HAp ceramics by a coprecipitation with a deposition of a bone-like apatite and an adsorption of serum proteins. The adsorption of proteins is influential in the apatite deposition and the cell adhesion. A component of serum albumin is forming &lt; 80% of total serum proteins, therefore we focused on the serum albumin. The serum albumin was removed from fetal bovine serum by using an albumin removal kit, and then the albumin free cell culture medium was prepared. The bone-like apatite layer was quickly formed on the HAp ceramics in the albumin free medium, however the high cell adhesion property was not confirmed. As a result of investigation of an initial cell adhesion, the initial cell adhesion property of the bone-like apatite layer was not excellent even though the layer contained the serum proteins. Moreover, the layer without serum proteins showed a meager cell adhesion property in every time of the cultivation. Therefore, it was revealed that the adsorption of the serum proteins only on the surface was not enough to the improvement of the cell growth. And a few days cultivation was required for an appearance of the remarkable effect on the cell growth on the layer formed by a coprecipitation reaction about the bone-like deposition apatite and the serum proteins adsorption.

Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method.

Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.

Establishment of AIDS-related primary-effusion lymphoma (PEL) cell lines that proliferate continuously without serum

The gammaherpesvirus dually-infected (HHV8/EBV) PEL cell line, BC-1, was weaned gradually from fetal bovine serum (FBS) during successive feedings with RPMI 1640 medium containing human transferrin and selenium dioxide as the only additives. A serum-free cell line (sfBC-1) emerged that was 100% major histocompatibility complex (MHC) class II negative, compared with 10% MHC class II-negative cells before serum removal. In contrast, MHC class I expression by sfBC-1 cells slightly exceeded that of BC-1 cells. BC-1 and sfBC-1 cells were indistinguishable in six polymorphic genetic loci, confirming their relatedness and sfBC-1 cells contained HHV8 and EBV. These findings were not attributable to dual infection because the PEL cell line, BCBL-1, which is infected with HHV8 but not EBV, also contained MHC class II positive (45%) and class II negative (55%) cells. Moreover, a serum-free BCBL-1 (sfBCBL-1) cell line was established and the sfBCBL-1 cells were MHC class I up modulated and 100% MHC class II negative. The serum-free cell lines established in this study may be useful for exploring PEL-cell autocrine-growth pathways and for assessing MHC class II-negative PEL cells for tumorigenesis in animal model systems.