Xenogeneic versus allogeneic serum and macromolecular crowding in human tenocyte cultures.

Allogeneic serum and tissue-specific extracellular matrix have been shown to maintain permanently differentiated cell phenotype in culture. This is of particular importance for human tenocytes, a cell population that readily loses its function during ex vivo culture. With these in mind, herein we extracted human tenocytes using either foetal bovine serum or human serum, cultured them in the absence and presence of carrageenan and Ficoll®, the most widely used macromolecular crowding agents (to induce tissue-specific extracellular matrix deposition), and assessed cellular function, via metabolic activity, viability, proliferation and immunofluorescence for collagen related molecules, non-collagenous molecules and transmembrane molecules. At day 7, longest time point assessed, neither carrageenan nor Ficoll® significantly affected metabolic activity, viability and proliferation in either serum and human serum significantly increased metabolic activity and proliferation. At day 7, in the absence of macromolecular crowding, cells in human serum deposited significantly lower collagen type VI, biglycan, versican and tenomodulin than cells in foetal bovine serum. Interestingly, at day 7, in comparison to the no macromolecular crowding group, carrageenan in foetal bovine serum induced the highest effect, as judged by the highest number of significantly increased molecules (collagen type I, collagen type IV, collagen type V, collagen type VI, transforming growth factor ß1, matrix metalloproteinase 14, lumican, versican, scleraxis and integrin α2ß1). These data, although contradict previous observations where human serum outperformed foetal bovine serum, at the same time, support the use of foetal bovine serum in the development of cell-based medicines.

Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

Trimethylamine increases intestinal fatty acid absorption: in vitro studies in a Caco-2 cell culture system.

Although elevated blood levels of trimethylamine N-oxide (TMAO) have been associated with atherosclerosis development in humans, the role of its gut microbiota-derived precursor, TMA, in this process has not been yet deciphered. Taking this into account, and the fact that increased intestinal fatty acid absorption contributes to atherosclerosis onset and progression, this study aimed to evaluate the effect of TMA on fatty acid absorption in a cell line that mimics human enterocytes. Caco-2 cells were treated with TMA 250 µM for 24 h. Fatty acid absorption was assessed by measuring the apical-to-basolateral transport and the intracellular levels of BODIPY-C12, a fluorescently labelled fatty acid analogue. Gene expression of the main intestinal fatty acid transporters was evaluated by real-time quantitative reverse transcription PCR. Compared to control conditions, TMA increased, in a time-dependent manner and by 20-50 %, the apical-to-basolateral transport and intracellular levels of BODIPY-C12 fatty acid in Caco-2 cells. Fatty acid transport protein 4 (FATP4) and fatty acid translocase (FAT)/CD36 gene expression were not stimulated by TMA, suggesting that TMA-induced increase in fatty acid transport may be mediated by an increase in FAT/CD36 and/or FATP4 activity and/or fatty acid passive transport. This study demonstrated that TMA increases the intestinal absorption of fatty acids. Future studies are necessary to confirm if this may constitute a novel mechanism that partially explains the existing positive association between the consumption of a diet rich in TMA sources (e.g. red meat) and the increased risk of atherosclerotic diseases.

Platelet lysate for expansion or osteogenic differentiation of bone marrow mesenchymal stem cells for 3D tissue constructs.

Background: The use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems. Methods: We evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining. Results: Cell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT. Conclusions: PLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs.

Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation.

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs). Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate. Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs. Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Protein-liposome interactions: the impact of surface charge and fluidisation effect on protein binding.

At the dawn of a new nanotechnological era in the pharmaceutical field, it is very important to examine and understand all the aspects that influence in vivo behaviour of nanoparticles. In this point of view, the interactions between serum proteins and liposomes with incorporated anionic, cationic, and/or PEGylated lipids were investigated to elucidate the role of surface charge and bilayer fluidity in protein corona's formation. 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) liposomes with the presence or absence of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt) (DOTAP), and/or 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DPPE-PEG 5000) lipids were prepared by the thin-film hydration method. The evaluation of their biophysical characteristics was enabled by differential scanning calorimetry and dynamic and electrophoretic light scattering. The physicochemical characteristics of mixed liposomes were compared before and after exposure to foetal bovine serum (FBS) and were correlated to calorimetric data. Our results indicate protein binding to all liposomal formulations. However, it is highlighted the importance of surface charge and fluidisation effect to the extent of protein adsorption. Additionally, considering the extensive use of cationic lipids for innovative delivery platforms, we deem PEGylation a key parameter, because even in a small proportion can reduce protein binding, and thus fast clearance and extreme toxicity without affecting positive charge. This study is a continuation of our previous work about protein-liposome interactions and fraction of stealthiness (Fs) parameter, and hopefully a design road map for drug and gene delivery.

In situ digestion of alcohol-fixed cells for quantitative proteomics.

Currently, the bottom-up approach, in which proteins are digested by enzymes such as trypsin prior to mass spectrometry, is the mainstream approach in mass spectrometer-based proteomics. In this approach, the enzymatic digestion process strongly affects the reproducibility of protein identification and quantification. Here, we quantitatively evaluated the enzymatic digestion of proteins under various conditions by quantitative proteomics using data-independent acquisition and found that proteins precipitated with acetone after solubilization with SDS were fully digestible without re-solubilization. This result implies that organic solvent treatment makes cells amenable to trypsin digestion. Direct trypsin digestion of methanol-fixed cells achieved the same digestion efficiency and quantitative reproducibility as the conventional method. Furthermore, this method was found to be equally applicable to mouse liver samples. The establishment of this method indicates that the sample preparation process in bottom-up proteomics can be simplified while maintaining high digestion efficiency and is expected to become a general method for sample preparation in bottom-up proteomics in the future.

Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment.

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling.

The dark side of foetal bovine serum in extracellular vesicle studies.

Extracellular vesicles (EVs) have been shown to be involved in cell-cell communication and to take part in both physiological and pathological processes. Thanks to their exclusive cargo, which includes proteins, lipids, and nucleic acids from the originating cells, they are gaining interest as potential biomarkers of disease. In recent years, their appealing features have been fascinating researchers from all over the world, thus increasing the number of in vitro studies focused on EV release, content, and biological activities. Cultured cell lines are the most-used source of EVs; however, the EVs released in cell cultures are influenced by the cell culture conditions, such as the use of foetal bovine serum (FBS). FBS is the most common supplement for cell culture media, but it is also a source of contaminants, such as exogenous bovine EVs, RNA, and protein aggregates, that can contaminate the cell-derived EVs and influence their cargo composition. The presence of FBS contaminants in cell-derived EV samples is a well-known issue that limits the clinical applications of EVs, thus increasing the need for standardization. In this review, we will discuss the pros and cons of using FBS in cell cultures as a source of EVs, as well as the protocols used to remove contaminants from FBS.

Allogeneic Serum and Macromolecular Crowding Maintain Native Equine Tenocyte Function in Culture.

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes.

Microencapsulated Sertoli cells sustain myoblast proliferation without affecting the myogenic potential. In vitro data.

Sertoli cells (SeC) isolated from porcine testes have shown direct effects on muscle precursor cells sustaining C2C12 myoblasts proliferation and inhibiting oxidative stress and apoptosis in the early phase of the differentiation process, and stimulating myoblast fusion into myotubes and the expression of markers of myogenic differentiation in the late phase. This suggested that the cocktail of factors secreted by SeC stimulates proliferation in myoblasts without weakening their myogenic potential resulting in the formation of the critical myoblast amount necessary to rebuild the required muscle mass upon a damage. Here, we show that co-culturing C2C12 myoblasts with high doses of SeC microencapsulated in clinical grade alginate-based microcapsules (MC-SeC) for three days in differentiation medium (DM) translates into increased cell numbers and almost absence of myotube formation. However, after removal of MC-SeC, an intense fusion activity into myotubes was observed culminating in a fusion index similar to that of control after additional three days of culture in DM. These data definitely demonstrate that SeC-derived factors preserve the myogenic potential while sustaining cell proliferation in C2C12 myoblasts.

Safety Assessment of Autologous Stem Cell Combination Therapy in Patients With Decompensated Liver Cirrhosis: A Pilot Study.

BACKGROUND: Haematopoietic stem cell (HSC) infusion has demonstrated short-term improvement in liver functions in patients with chronic liver disease. The combination of HSC with mesenchymal stem cells (MSCs), which has an immunomodulatory effect, may augment the effects and enhance the duration of improvements on liver functions. The aim of the present study was to assess the safety of infusing the combination of autologous HSCs and MSCs in decompensated liver cirrhosis. METHODS: In phase I of the study, in vitro assessment was performed to observe the effect of coculturing MSCs with HSCs on their viability and cytokine profiles. Phase II of the study was to assess the safety of combination of stem cell infusions. Bone marrow (50 ml) was aspirated for MSC isolation and expansion using standard protocol. Patients received subcutaneous doses (n = 5) of granulocyte colony-stimulating factor (G-CSF) for stem cell mobilization followed by leukapheresis for harvesting HSCs using CliniMacs. HSCs and MSCs were infused through the hepatic artery under fluoroscopic guidance and were monitored for any adverse effects. RESULTS: In vitro studies revealed 94% viable HSCs in coculture similar to monoculture. HSCs released only interleukin (IL)-8, whereas MSCs secreted IL-8 and IL-6 in monocultures, and both IL-8 and IL-6 were secreted in coculture. G-CSF administration- and bone marrow aspiration-related complications were not observed. Infusion of the cells through the hepatic artery was safe, and no postprocedural complications were noted. CONCLUSION: The combination of autologous HSC and MSC infusion is a safe procedure in patients with decompensated liver cirrhosis, and the outcomes needed to be assessed in larger studies. TRIAL NUMBER: NCT04243681.

Candida albicans biofilm formation and growth optimization for functional studies using response surface methodology.

AIM: Optimization of Candida albicans growth and biofilm formation is essential for understanding the recalcitrance of this pathogen to advance functional analysis on hospital tools and material surfaces. Optimization and quantification of biofilm have always been a challenge using the conventional one variable at a time (OVAT) method. The present study uses central composite design-based response surface methodology for optimization of conditions to induce growth and biofilm formation in Candida albicans on polystyrene microtiter plates. METHODS AND RESULTS: Statistical software package, Stat Soft®, STASTICA version 12.6 was used for data analysis. The variables considered in the design matrix were media pH, temperature, incubation period, shaker speed and inoculum size. A four-pronged quantification approach with XTT assay (cell viability), crystal violet assay (biofilm), calcofluor white assay and wet/dry weight measurements (cell mass) was used to understand different aspects of biofilm formation. Cell viability and cell mass were inversely related; however, biofilm was independent of these two factors. The study also highlighted the fact that foetal bovine serum does not significantly contribute to cell adhesion and in turn in vitro biofilm formation in some of the cultures. CONCLUSIONS: A high-throughput optimization of C. albicans growth and biofilm formation on polystyrene microplate has been developed and validated. SIGNIFICANCE AND IMPACT OF STUDY: This is a first time approach to optimize the interaction of parameters for C. albicans biofilm formation using RSM. Heterogeneity in growth conditions for local strains of C. albicans clinical isolates was observed. This microtiter plate-based method can be used for future screening of therapeutics for the control of C. albicans.

A critical assessment of the estrogenic potency of benzyl salicylate.

Benzyl salicylate (BS) is a natural ingredient of essential oils and a widely used fragrance chemical. A number of in vitro screening studies have evaluated the estrogenic potential of BS with ambiguous results. Lack of dose-response information for the positive control 17ß-estradiol (E2) in most studies makes an assessment of the relative potency and efficacy challenging. Notwithstanding this difficulty, BS has been added as the only fragrance ingredient to the list of the first 14 substances to be screened as potential endocrine disruptors by the European Scientific Committee for Consumer Safety (SCCS) and it is included in the Community rolling action plan (CoRAP) of the European REACH regulation to be assessed for the same property. Here we review all literature evidence and present new data to quantify the in vitro potency and efficacy of BS vs. E2 with full dose response analysis in both an estrogen response element (ERE) depending reporter gene assay and in the MCF7 cell proliferation (E-screen) assay. In both assays, very similar results for BS were found. BS is a partial agonist exhibiting 35-47 % maximal efficacy and it is active only close to the cytotoxic concentration. The extrapolated concentration to achieve 50 % efficacy is 21'000'000 higher as compared to E2 in the reporter gene assay. A ca. 36'000'000 higher concentration of BS as compared to E2 is required to reach equivalent partial cell proliferation stimulation in the MCF7 proliferation assay. This potency is significantly below the agonistic activity of known chemicals which cause estrogenic effects in in vivo assays. Importantly, in this study the weak agonistic activity is for the first time directly related to the activity of E2 in a full quantitative comparison in human cell lines which may help ongoing evaluations of BS by regulatory bodies.

Relationship between serum concentration, functional parameters and cell bioenergetics in IPEC-J2 cell line.

The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract. With the aim to assess if the FBS-dependent functional variations are linked to the bioenergetic aspects, the addition of 5% and 10% FBS in the IPEC-J2 culture medium were tested. Doubling time and TEER measurement indicated that cells cultured at higher FBS dose grow faster and as a more compact monolayer. 10% FBS increases ATP production and mitochondrial oxidative phosphorylation (OxPhos) and does not affect glycolysis. Both at 5% and 10% FBS ATP production mainly comes from OxPhos and FBS concentration does not affect the cell respiration bioenergetic parameters. Noteworthy, IPEC-J2 treated with 5% and 10% FBS have a metabolic potential since both OxPhos and glycolysis increase by > 100% and < 50%, respectively in comparison with baseline metabolism. Moreover, glucose, fatty acids and glutamine constitute the preferred metabolic fuel for mitochondrial respiration at both FBS conditions tested. Accordingly, the cells flexibility to oxidize these substrates shows that IPEC-J2 mitochondria cannot maintain the basal ATP production without oxidizing all the substrates available irrespective of FBS concentration. To sum up, in IPEC-J2 cells OxPhos increases with the FBS-stimulated functional physiological parameters to fulfil ATP requirements.

Feasibility of Human Platelet Lysate as an Alternative to Foetal Bovine Serum for In Vitro Expansion of Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease that affects a lot of people worldwide. Current treatment for OA mainly focuses on halting or slowing down the disease progress and to improve the patient's quality of life and functionality. Autologous chondrocyte implantation (ACI) is a new treatment modality with the potential to promote regeneration of worn cartilage. Traditionally, foetal bovine serum (FBS) is used to expand the chondrocytes. However, the use of FBS is not ideal for the expansion of cells mean for clinical applications as it possesses the risk of animal pathogen transmission and animal protein transfer to host. Human platelet lysate (HPL) appears to be a suitable alternative to FBS as it is rich in biological factors that enhance cell proliferation. Thus far, HPL has been found to be superior in promoting chondrocyte proliferation compared to FBS. However, both HPL and FBS cannot prevent chondrocyte dedifferentiation. Discrepant results have been reported for the maintenance of chondrocyte redifferentiation potential by HPL. These differences are likely due to the diversity in the HPL preparation methods. In the future, more studies on HPL need to be performed to develop a standardized technique which is capable of producing HPL that can maintain the chondrocyte redifferentiation potential reproducibly. This review discusses the in vitro expansion of chondrocytes with FBS and HPL, focusing on its capability to promote the proliferation and maintain the chondrogenic characteristics of chondrocytes.

Human platelet lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review.

BACKGROUND: Foetal bovine serum (FBS), is the most commonly used culture medium additive for in vitro cultures, despite its undefined composition, its potential immunogenicity and possible prion/zoonotic transmission. For these reasons, significant efforts have been targeted at finding a substitute, such as serum free-media or human platelet-lysates (hPL). Our aim is to critically appraise the state-of-art for hPL in the published literature, comparing its impact with FBS. MATERIALS AND METHODS: In June 2019 a systematic search of the entire Web of Science, Medline and PubMed database was performed with the following search terms: (mesenchymal stem cells) AND (fetal bovine serum OR fetal bovine calf) AND (human platelet lysate). Excluded from this search were review articles that were published before 2005, manuscripts in which mesenchymal stem cells (MSCs) were not from human sources, and when the FBS controls were missing. RESULTS: Based on our search algorithm, 56 papers were selected. A review of these papers indicated that hMSCs cultured with hPL showed a spindle-shaped elongated morphology, had higher proliferation indexes, similar cluster of differentiation (CD) markers and no significant variation in differentiation lineage (osteocyte, adipocyte, and chondrocyte) compared to those cultured with FBS. Main sources of primary hMSCs were either fat tissue or bone marrow; in a few studies cells isolated from alternative sources showed no relevant difference in their response. CONCLUSION: Despite the difference in medium choice and a lack of standardization of hPL manufacturing, the majority of publications support that hPL was at least as effective as FBS in promoting adhesion, survival and proliferation of hMSCs. We conclude that hPL should be considered a viable alternative to FBS in hMSCs culture-especially with a view for their clinical use.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

The bone metastasis niche in breast cancer-potential overlap with the haematopoietic stem cell niche in vivo.

BACKGROUND: Bone metastasis is one of the most common complications of advanced breast cancer. During dissemination to bone, breast cancer cells locate in a putative 'metastatic niche', a microenvironment that regulates the colonisation, maintenance of tumour cell dormancy and subsequent tumour growth. The precise location and composition of the bone metastatic niche is not clearly defined. We have used in vivo models of early breast cancer dissemination to provide novel evidence that demonstrates overlap between endosteal, perivascular, HSC and the metastatic niche in bone. METHODS: Estrogen Receptor (ER) +ve and -ve breast cancer cells were labelled with membrane dyes Vybrant-DiD and Vybrant-CM-DiI and injected via different routes in BALBc/nude mice of different ages. Two-photon microscopy was used to detect and quantitate tumour cells and map their location within the bone microenvironment as well as their distance to the nearest bone surface compared to the nearest other tumour cell. To investigate whether the metastatic niche overlapped with the HSC niche, animals were pre-treated with the CXCR4 antagonist AMD3100 to mobilise hematopoietic (HSCs) prior to injection of breast cancer cells. RESULTS: Breast cancer cells displayed a characteristic pattern of homing in the long bones, with the majority of tumour cells seeded in the trabecular regions, regardless of the route of injection, cell-line characteristics (ER status) or animal age. Breast cancer cells located in close proximity to the nearest bone surface and the average distance between individual tumour cells was higher than their distance to bone. Mobilisation of HSCs from the niche to the circulation prior to injection of cell lines resulted in increased numbers of tumour cells disseminated in trabecular regions. CONCLUSION: Our data provide evidence that homing of breast cancer cells is independent of their ER status and that the breast cancer bone metastasis niche is located within the trabecular region of bone, an area rich in osteoblasts and microvessels. The increased number of breast cancer cells homing to bone after mobilisation of HSCs suggests that the HSC and the bone metastasis niche overlap.