Serum-free Cell Culture Research Articles

Expression and purification of human Afamin for structure/function analysis

Protein glycosylation plays an important role in protein stability, folding, and secretion but presents a major challenge for protein crystallization. Crystallization of highly glycosylated proteins is often difficult because of conformational and chemical inhomogeneity of the glycan decorations. Therefore, it is almost always necessary to modify the material to obtain a conformational homogenous protein that can crystallize. (1) We have explored several avenues in the case of the human 87 kDa glycoprotein afamin. Afamin (AFM), a member of the albumin gene family, is mainly expressed in the liver and secreted into the bloodstream. (2) Elevated afamin plasma concentrations are associated with major diseases such as metabolic syndrome and cancer;(3) however, pathophysiological functions are largely unknown. Therefore, we are pursuing the crystal structure of various forms of recombinantly expressed human afamin (rhAFM) for structure guided exploration and analysis of its function. Multiple variants of rhAFM are pursued: 1) fully glycosylated wild-type rhAFM (expressed in CHO cell lines); 2) rhAFM complexed with Fab fragments of two anti-AFM mAbs, 3) enzymatic deglycosylation of rhAFM with PGNaseF, 4) partially glycosylated rhAFM (expressed in glycosylation-deficient Lec1-CHO cells); and 5) glycosylation-free rhAFM, obtained from HEK293 cells virally transfected with a cDNA mutant lacking all 5 glycosylation sites by replacement of ASN with ASP. Yields of up to 2 mg/mL glycosilation free mutant rhAFM in HEK273 cells have been comparable to native rhAFM. C-terminal His6-tagged rhAFM was captured by Ni-IMAC from serum-free cell culture supernatants. All variants and Fab-AFM complexes were polished via SEC yielding pure products per SDS-PAGE and immunoblot analysis. Crystals have been obtained from Fab fragments and crystalline spherulites which are useful for microseeding from Fab-AFM complexes.

Nitric oxide synthase activity is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing hormone in bovine granulosa cells

In rabbits and rodents, nitric oxide (NO) is generally considered to be critical for ovulation. In monovulatory species, however, the importance of NO has not been determined, nor is it clear where in the preovulatory cascade NO may act. The objectives of this study were (1) to determine if nitric oxide synthase (NOS) enzymes are regulated by luteinizing hormone (LH) and (2) to determine if and where endogenous NO is critical for expression of genes essential for the ovulatory cascade in bovine granulosa cells in serum-free culture. Time– and dose–response experiments demonstrated that LH had a significant stimulatory effect on endothelial NOS (NOS3) mRNA abundance, but in a prostaglandin-dependent manner. NO production was stimulated by LH before a detectable increase in NOS3 mRNA levels was observed. Pretreatment of cells with the NOS inhibitor L-NAME blocked the effect of LH on the epidermal growth factor (EGF)-like ligands epiregulin and amphiregulin, as well as prostaglandin–endoperoxide synthase-2 mRNA abundance and protein levels. Similarly, EGF treatment increased mRNA encoding epiregulin, amphiregulin, and the early response gene EGR1, and this was inhibited by pretreatment with L-NAME. Interestingly, pretreatment with L-NAME had no effect on either ERK1/2 or AKT activation. Taken together, these results suggest that endogenous NOS activity is critical for the LH-induced ovulatory cascade in granulosa cells of a monotocous species and acts downstream of EGF receptor activation but upstream of the EGF-like ligands.

Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: Development and optimization of a washing step

In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries – 99% – and purifications – protein purity of 81% and a purification factor of 16 out of a maximum of 20 – were achieved using 100mM d-sorbitol in 10mM Tris–HCl as washing buffer and 0.5M d-sorbitol with 150mM NaCl in 10mM Tris–HCl as elution buffer. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.

A severe case of fraudulent blending of fetal bovine serum strengthens the case for serum-free cell and tissue culture applications.

A severe case of fraudulent blending of fetal bovine serum strengthens the case for serum-free cell and tissue culture applications.

Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1–6

Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1–6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6–2.1log10 higher infectivity titers (4.7–6.2log10Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV.

Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular>dialyzed>CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition.

Ectodomain shedding in the downregulation of cellular ACE‐2 by ER stress in lung cells (716.3)

Previous work from this laboratory has demonstrated that angiotensin (ANG) converting enzyme‐2 (ACE‐2) protects against lung fibrosis by limiting the local accumulation of the profibrotic peptide, ANGII by converting ANGII to angiotensin 1‐7 (ANG 1‐7). However, this protective enzyme is down‐regulated in human and experimental lung fibrosis. We currently know of three ways that ACE‐2 is down‐regulated in lung cells: 1) exposure to hyperoxia in fetal lung fibroblasts, 2) treatment with proteasome inhibitors, MG132 or Clasto‐Lactacystin β‐lactone (CLBL) in alveolar epithelial cells (AECs), and 3) expressing the surfactant protein C mutation G100S (which causes lung fibrosis in people). In all of these cases, ACE‐2 was only measured in cell lysates, but not in the cell culture medium to assess “shedding”. We hypothesize that the down‐regulation of cellular ACE‐2 may be mediated by ectodomain shedding by ADAM17/TACE. Western blots of cell lysates revealed a significant decrease in immunoreactive cellular ACE‐2 with CLBL (p= < 0.01) and a potential recovery in ACE‐2 with TAPI‐2, an inhibitor of ADAM17/TACE. Contrary to our hypothesis, a similar effect was also observed in serum‐free cell culture media (p= < 0.05). From this, we can speculate that ADAMS17/TACE may play a partial role in down‐regulating ACE‐2 by degrading it, in addition to other enzymes. With this model, further research can be done to investigate alternative mechanisms that lead to the down‐regulation of cellular ACE‐2 in lung cells.Grant Funding Source: Supported by APS STRIDE Fellowship and PHS HL ‐ 45136

Bat airway epithelial cells: a novel tool for the study of zoonotic viruses.

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.

Enhancement of Human Thyrotropin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

The influence of sodium butyrate (NaBu) on the synthesis of recombinant human thyrotropin (r-hTSH) by CHO cells was investigated for the first time. A volumetric productivity of ~10 μg hTSH/mL was repeatedly obtained, with a 3.3-fold increase over a control culture carried out in the absence of NaBu. Since NaBu can induce CHO cell apoptosis and cell growth arrest, the increase in specific productivity was even higher, i.e., ca. 5-fold. Analysis of the N-glycan composition of r-hTSH obtained with the addition of NaBu to the culture medium showed an approximately 12 % increase in the amount of sialic acid, as well as in total carbohydrate, partly due to the increase in the site occupancy from 2.77 to 2.93 glycans per mole of hTSH. The two hormone preparations were characterized by N-glycan structural analysis, which showed that NaBu increased the bi-antennary structures by ca. 13 % while decreasing the tri-antennary structures by approximately the same amount. The in vivo biological activity and pharmacokinetic behavior (clearance) were found to be similar for the two hormone preparations.

Abstract 2626: Characterization of the invading cells in glioblastoma using a syngeneic intracranial transplant tumor model.

Abstract Local and distant invasion is a hallmark property of glioblastoma (GBM) that prevents curative surgical resection, and may be the key reason for GBM recurrence. Although cancer stem cells (CSCs) have been suggested to cause tumor recurrence based on their resistance to radiotherapy and chemotherapy, it is not clear whether CSCs are also involved in GBM invasiveness. We hypothesize that the invading cells in GBM have stem-like properties. To test our hypothesis, we propose to characterize cells at the invasive front of GBM using a syngeneic intracranial transplant tumor model. Three core regulatory networks are dysregulated in most human GBMs, including the RTK/RAS/PI3K/PTEN, INK/Cyclin D/CDK/RB, and ARF/p53 pathways. We have previously generated a genetically engineered mouse (GEM) model for GBM development via adult-inducible astrocyte-specific perturbation of RB, K-RAS and PTEN. These mice develop astrocytomas that progress to high grade GBM, and faithfully recapitulate the histopathology and molecular characteristics of most human GBMs. We have isolated primary tumor cells from these mice that are maintained in serum-free neural stem cell culture medium and expanded as neurospheres. These primary and low-passage cultures contain a mixed population of cancer stem cells and progenitor cells, and are highly invasive in vitro by matrigel invasion assay. With > 90% penetrance, intracranial injection of 25,000 cells leads to invasive GBM formation and a 32-day median survival in syngeneic host mice. Thus, the model provides an efficient approach for dissecting GBM cell invasion mechanisms in vivo. We will further trace the migration/invasion of GBM cells in the host brain after labeling with lentiviral-expressed mCherry. Host blood vessels are labeled with YFP via Tie2-Cre endothelial expression of a Rosa26-YFP allele. A time course analysis of stem cell marker expression will be used to assess properties of invading cells at the diffusive tumor margin. Laser capture microdissection will be used for invasive rim-cell and non-invasive core-cell isolation, and RNA samples will be compared by unbiased transcriptome studies. Results will provide hypotheses for invasion mechanisms and may identify potential biomarkers for invasive cells. Citation Format: Nailing Zhang, Yurong Song, Sanaz Jansen, Amit Adhikari, Sophie Wang, Lucy Lu, Teresa Sullivan, Terry Van Dyke. Characterization of the invading cells in glioblastoma using a syngeneic intracranial transplant tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2626. doi:10.1158/1538-7445.AM2013-2626

Surface chemistry of gold nanorods: origin of cell membrane damage and cytotoxicity

We investigated how surface chemistry influences the interaction between gold nanorods (AuNRs) and cell membranes and the subsequent cytotoxicity arising from them in a serum-free cell culture system. Our results showed that the AuNRs coated with cetyl trimethylammonium bromide (CTAB) molecules can generate defects in the cell membrane and induce cell death, mainly due to the unique bilayer structure of CTAB molecules on the surface of the rods rather than their charge. Compared to CTAB-capped nanorods, positively charged polyelectrolyte-coated, i.e. poly(diallyldimethyl ammonium chloride) (PDDAC), AuNRs show improved biocompatibility towards cells. Thus, the present results indicate that the nature of surface molecules, especially their packing structures on the surface of AuNRs rather than surface charge, play a more crucial role in determining cytotoxicity. These findings about interfacial interactions could also explain the effects of internalized AuNRs on the structures or functions of organelles. This study will help understanding of the toxic nature of AuNRs and guide rational design of the surface chemistry of AuNRs for good biocompatibility in pharmaceutical therapy.

Histone Deacetylase Inhibitors Promote the Ex Vivo Expansion of Cord Blood CD34+ Cells in Serum Free Cultures Accompanied by the Upregulation of Pluripotency Genes

AbstractAbstract 345Previously, we have reported that various chromatin modifying agents(CMA) can epigenetically alter HSC fate decisions resulting in increased numbers of cord blood(CB)- SCID repopulating cells(SRC) and/or erythroid progenitor cells depending upon the CMA utilized (Blood. 2011,117(17):4632–41). We have hypothesized that treatment of CB CD34+ cells with histone deacetyalse inhibitors(HDACIs) might expand the numbers of CB-SRC and that such cell products could be used as allogeneic stem cell grafts. We developed serum free(SF) and containing(SC) culture conditions and assessed the ability of several HDACIs including valproic acid(VPA), scriptaid(SCR) and CAY10433(C433) to expand CB-SRC Each of these HDACIs were shown to inhibit both class I and II HDACs. The addition of VPA to SF cultures led to a >6,000 fold expansion of CD34+ cells and >25,000-fold expansion of CD34+CD90+ cells as compared to primary cells(PC). By contrast, SC cultures resulted in only a 54-fold expansion of CD34+ cells and a 75-fold expansion of CD34+90+ cells as compared to PC in the presence of VPA.In order to examine the functional characteristics of HDACIs treated CB-CD34+ cells in SF cultures we evaluated aldehyde dehydrogenase activity(ALDH) in these expanded CD34+ cells. A far greater proportion of CD34+ cells were ALDH+ following treatment with VPA in SF cultures as compared to SC cultures (82.2±8.6%, versus 37.0±0.9%). By contrast incubation in the SC cultures induced the generation of greater proportion of ALDH-CD34+ cells as compared to SF cultures (27.0±5.0% versus 3.7±2.0%). These findings indicate that the inclusion of serum to such cultures favors HSC differentiation and eventual HSC depletion while SF conditions favor expansion of more primitive HPC. Purified ALDH+CD34+ cells from SF cultures supplemented with HDACIs contained far greater absolute numbers of BFU-E and CFU-Mix (8.4×107±6.7×107/CB collection) as compared to SC cultures (1.4×107±8.8×106/CB collection; One way ANOVA p=.02). These data indicate that based upon their degree of ALDH activity and functional potential different phenotypically and functionally defined subsets of CD34+ cells are generated in SF as compared to SC cultures.In order to further examine the effects of VPA on the pluripotency of expanded CD34+ cells we examined the expression of Sox2, Oct4 and Nanog in CD34+ cells re-isolated following 7 days of treatment with HDACIs in SF and SC cultures. RNA and protein expression levels of Sox2, Oct4 and Nanog genes were greater in cells generated in SF cultures supplemented with HDACIs than that observed in CD34+ cells cultured in SC. Sox2 and Oct4 bind to the promoter of Nanog and up-regulate Nanog expression. In the SF but not SC cultures VPA treatment resulted in the up-regulation of Nanog indicating the functionality of the Sox2/Oct4 interactions. We also documented using confocal microscopy the elevated expression of the proteins derived from these pluripotency genes both in the cytoplasm and nucleus of CD34+ cells re-isolated after a week of treatment with each of the HDACIs.The functional potential of the re-isolated CD34+ cells following a week of treatment with VPA in SF medium was evaluated by transplanting the expanded cells into NOD/SCID/gamma(c)null mice. SRC capable of multi-lineage hematopoietic differentiation were detected in the marrow of recipients of VPA treated grafts 13-weeks after transplantation (%CD45, 33.5±13.4% with VPA treated grafts versus 13.0±7.7% with grafts generated in the presence of CA). Bone marrow cells from primary recipients receiving HDACI treated CD34+ cells grafts were also capable of generating greater numbers of cells belonging to myeloid and lymphoid lineages(%CD45, 3.3±1.0%) as compared to grafts receiving CA(%CD45, 0.65±0.16%) 15 weeks after their transplantation into secondary recipients.These data indicate that inhibitors of class I and II HDACs under SF culture conditions provide an effective pharmacological approach which can be utilized to promote the ex vivo expansion of functional CB-SRC which are characterized by high ALDH activity and expression of a variety of pluripotency genes. These findings indicate the importance of epigenetic events in determining phenotype and function of dividing SRC in vitro. Disclosures:Hoffman:NYSTEM: Research Funding.

The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34+ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34+ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34+ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.

Sodium butyrate greatly enhances human thyrotropin (hTSH) synthesis by serum-free CHO cell culture, with alterations of the carbohydrate moiety

Sodium butyrate greatly enhances human thyrotropin (hTSH) synthesis by serum-free CHO cell culture, with alterations of the carbohydrate moiety

Conjugated linoleic acids attenuate FSH- and IGF1-stimulated cell proliferation; IGF1, GATA4, and aromatase expression; and estradiol-17β production in buffalo granulosa cells involving PPARγ, PTEN, and PI3K/Akt

Conjugated linoleic acid (CLA) has drawn much interest in last two decades in the area ranging from anticancer activity to obesity. A number of research papers have been published recently with regard to CLA's additional biological functions as reproductive benefits. However, not much is known how this mixture of isomeric compounds mediates its beneficial effects particularly on fertility. In this study, we demonstrated the cross talk between downstream signaling of CLA and important hormone regulators of endocrine system, i.e. FSH and IGF1, on buffalo granulosa cell function (proliferation and steroidogenesis). Experiments were performed in primary serum-free buffalo granulosa cell culture, where cells were incubated with CLA in combination with FSH (25 ng/ml) and IGF1 (50 ng/ml). Results showed that 10 μM CLA inhibits FSH- and IGF1-induced granulosa cell proliferation; aromatase, GATA4, and IGF1 mRNA; and estradiol-17β production. Western blot analysis of total cell lysates revealed that CLA intervenes the IGF1 signaling by decreasing p-Akt. In addition, CLA was found to upregulate peroxisome proliferator-activated receptor-gamma (PPARG) and phosphatase and tensin homolog (PTEN) level in granulosa cells. Further study using PPARG- and PTEN-specific inhibitors supports the potential role of CLA in granulosa cell proliferation and steroidogenesis involving PPARG, PTEN, and PI3K/Akt pathway.

TH2 cytokines increase kallikrein 7 expression and function in patients with atopic dermatitis

Here we report that Th2 cytokines increase kallikrein 7 expression and function in keratinocytes. Our finding describes a link between the Th2 environment and epidermal barrier dysfunction in atopic dermatitis.

The stimulation of proliferation and differentiation of periodontal ligament cells by the ionic products from Ca7Si2P2O16 bioceramics

The ultimate goal of periodontal tissue engineering is to produce predictable regeneration of alveolar bone, root cementum, and periodontal ligament, which are lost as a result of periodontal diseases. To achieve this goal, it is of great importance to develop novel bioactive materials which could stimulate the proliferation, differentiation and osteogenic/cementogenic gene expression of periodontal ligament cells (PDLCs) for periodontal regeneration. In this study, we synthesized novel Ca7Si2P2O16 ceramic powders for the first time by the sol–gel method and investigated the biological performance of PDLCs after exposure to different concentrations of Ca7Si2P2O16 extracts. The original extracts were prepared at 200mgml–1 and further diluted with serum-free cell culture medium to obtain a series of diluted extracts (100, 50, 25, 12.5 and 6.25mgml–1). Proliferation, alkaline phosphatase (ALP) activity, Ca deposition, and osteogenesis/cementogenesis-related gene expression (ALP, Col I, Runx2 and CEMP1) were assayed for PDLCs on days 7 and 14. The results showed that the ionic products from Ca7Si2P2O16 powders significantly stimulated the proliferation, ALP activity, Ca deposition and osteogenesis/cementogenesis-related gene expression of PDLCs. In addition, it was found that Ca7Si2P2O16 powders had excellent apatite-mineralization ability in simulated body fluids. This study demonstrated that Ca7Si2P2O16 powders with such a specific composition possess the ability to stimulate the PDLC proliferation and osteoblast/cemenoblast-like cell differentiation, indicating that they are a promising bioactive material for periodontal tissue regeneration application.

Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging

We report on a novel and straightforward magnetic cell labeling approach that combines three FDA-approved drugs, ferumoxytol (F), heparin (H) and protamine (P) in serum free media to form self-assembling nanocomplexes that effectively label cells for in vivo MRI. We observed that the HPF nanocomplexes were stable in serum free cell culture media. HPF nanocomplexes exhibited a three-fold increase in T2 relaxivity compared to F. Electron Microscopy revealed internalized HPF within endosomes, confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells, and T-cells when compared to controls. In vivo MRI detected 1000 HPF-labeled cells implanted in rat brains. HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

Interaction of Angiogenically Stimulated Intermediate CD163+ Monocytes/Macrophages With Soft Hydrophobic Poly(n‐Butyl Acrylate) Networks With Elastic Moduli Matched to That of Human Arteries

The cell population of peripheral blood monocytes/macrophages (MO) is heterogeneous: The majority of the MO are CD14++ CD16- and named "classical" (= MO1). Furthermore, two other subpopulations were described: CD14++ CD16+ ("intermediate" = MO2) and CD14+ CD16++ ("non-classical" = MO3). It is reported that MO2 possess anti-inflammatory properties and express the MO lineage marker CD163. On a hydrophilic neutrally charged acrylamide-based hydrogel human intermediate (CD14++ CD16+ ), angiogenically stimulated CD163++ monocytes/macrophages (aMO2) maintained a proangiogenic and noninflammatory status for at least 14 days. Here, we explored whether this aMO2 subset adhered to hydrophobic poly(n-butyl acrylate) networks (cPnBA) and also remained in its proangiogenic and noninflammatory status. Because substrate elasticity can impact adherence, morphology, and function of cells, cPnBAs with different Young's modulus (250 and 1100 kPa) were investigated, whereby their elasticity was tailored by variation of the cross-linker content and matched to the elasticity of human arteries. The cPnBAs exhibited similar surface properties (e.g., surface roughness), which were maintained after ethylene oxide sterilization and exposure in serum-free cell culture medium for 18 h at 37°C. aMO2 were seeded on cPnBA samples (1.7 × 10(5) cells/1.33 cm(2) ) in Dulbecco's modified Eagle medium (DMEM high glucose) supplemented with vascular endothelial growth factor 165 (VEGF-A(165) , 10 ng/mL) and fetal calf serum (10 vol%) for 3 and 72 h. On both polymeric samples (n = 3 each), the numbers of adherent cells per unit area were significantly higher (P < 0.01; cPnBA0250: 3 h 13 ± 5 cells/mm(2) , 72 h 234 ± 106 cells/mm(2) ; cPnBA1100: 3 h 14 ± 3 cells/mm(2) , 72 h 198 ± 113 cells/mm(2) ) compared to control cultures (glass, 3 h: 6 ± 3 cells/mm(2) , 72 h: 130 ± 83 cells/mm(2) ) and showed a typically spread morphology. The mRNA expression profile of the aMO2 was not influenced by the substrate elasticity. In the supernatant of aMO2 on cPnBA0250, significantly less VEGF-A(165) product was found than expected based on the mRNA level measured (P < 0.01). Tests with recombinant VEGF-A(165) then demonstrated that significantly more VEGF-A(165) was adhered on cPnBA0250 than on cPnBA1100 (P < 0.01). Seeded on cPnBA, aMO2-unaffected by the elastic moduli of both substrates-seemed to remain in their subset status and secreted VEGF-A(165) without release of proinflammatory cytokines. These in vitro results might indicate that this MO subset can be used as cellular delivery system for proangiogenic and noninflammatory mediators to support the endothelialization of cPnBA.

Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.