Interaction of the EGFR signaling pathway with porcine cumulus oocyte complexes and oviduct cells in a coculture system

It has become increasingly recognized that coculture has a beneficial effect on the in vitro maturation (IVM) of oocytes and embryo development in many species. However, these effects of coculture on IVM have been documented only for their positive conditioning roles without any evidence on the precise mechanisms underlying the action of coculture systems on the development of cumulus oocyte complexes (COCs). It has been suggested that the epidermal growth factor receptor (EGFR) signaling pathway is important for development of COCs, mediated by several epidermal growth factor (EGF)-like proteins with downstream mitogen-activated protein kinase 1/3 signaling. Therefore, we hypothesized that canine oviduct cells (OCs) in a coculture system, which shows improvement of oocyte quality in several species, are associated with EGFR signaling by exposure to progesterone (P4; imitating its production before ovulation and its continuous increase while oocytes reside in the oviduct to complete maturation in dogs). We designed three experimental groups: control, OCs coculture exposed to P4, and OCs coculture without exposure to P4. The result showed that the OCs coculture exposed to P4 strongly expressed EGF-like proteins and significantly improved COCs and subsequent embryo development. Furthermore, the expression of EGFR-related genes in cumulus cells and GDF9 and BMP15 in oocytes was upregulated in the P4-treated group. This study provides the first evidence that OCs exposed to P4 can induce strong expression of EGF-like proteins, and OCs effectively mediate improved porcine COCs development and subsequent embryo development by altering EGFR signaling related mRNA expression.     

Osteochondral tissue coculture: An in vitro and in silico approach

Osteochondral tissue engineering aims to regenerate functional tissue-mimicking physiological properties of injured cartilage and its subchondral bone. Given the distinct structural and biochemical difference between bone and cartilage, bilayered scaffolds, and bioreactors are commonly employed. We present an osteochondral culture system which cocultured ATDC5 and MC3T3-E1 cells on an additive manufactured bilayered scaffold in a dual-chamber perfusion bioreactor. Also, finite element models (FEM) based on the microcomputed tomography image of the manufactured scaffold as well as on the computer-aided design (CAD) were constructed; the microenvironment inside the two FEM was studied and compared. In vitro results showed that the coculture system supported osteochondral tissue growth in terms of cell viability, proliferation, distribution, and attachment. In silico results showed that the CAD and the actual manufactured scaffold had significant differences in the flow velocity, differentiation media mixing in the bioreactor and fluid-induced shear stress experienced by the cells. This system was shown to have the desired microenvironment for osteochondral tissue engineering and it can potentially be used as an inexpensive tool for testing newly developed pharmaceutical products for osteochondral defects.     

Thermodynamics of hydrogen metabolism in methanogenic cocultures degrading ethanol or lactate

Pure cultures of Desulfovibrio vulgaris or Pelobacter acetylenicus do not grow with lactate or ethanol, respectively, under obligately proton- reducing conditions. However, a small part of these substrates was oxidized and molecular hydrogen was produced up to 4.2 and 3.2 kPa, respectively. During growth in syntrophic methanogenic cocultures with Methanospirillum hungatei as partner, maximum hydrogen partial pressures were significantly lower (0.7 to 2.5 kPa) than in the corresponding pure cultures. Calculation of Gibbs free energies for the prevailing culture conditions showed that H₂ partial pressures were kept in a range at which both, H₂-producing and H₂-consuming reactions, were thermodynamically permissive in pure as well as in syntrophic mixed cultures.     

The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

The manufacturing of viable and functional β‐cell spheroids is required for diabetes cell therapy and drug testing. Mesenchymal stromal/stem cells (MSCs) are known to improve β‐cell viability and functionality. We therefore investigated the aggregation behavior of three different β‐cell lines (rat insulinoma‐1 cell line [INS‐1], mouse insulinoma‐6 cell line [MIN6], and a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells [1.1B4]), two MSC types, and mixtures of β‐cells and MSCs under different conditions. We screened several static systems to produce uniform β‐cell and MSC spheroids, finding cell‐repellent plates the most suitable. The three different β‐cell lines differed in their aggregation behavior, spheroid size, and growth in the same static environment. We found no major differences in spheroid formation between primary MSCs and an immortalized MSC line, although both differed with regard to the aggregation behavior of the β‐cell lines. All spheroids showed a reduced viability due to mass transfer limitations under static conditions. We therefore investigated three dynamic systems (shaking multi‐well plates, spinner flasks, and shaking flasks). In shaking flasks, there were no β‐cell‐line‐dependent differences in aggregation behavior, resulting in uniform and highly viable spheroids. We found that the aggregation behavior of the β‐cell lines changed in a static coculture with MSCs. The β‐cell/MSC coculture conditions must be refined to avoid a rapid segregation into distinct populations under dynamic conditions.     

Inflammatory molecules produced by meniscus and synovium in early and end-stage osteoarthritis: a coculture study

The aim of this study was to identify the molecules and pathways involved in the cross-talk between meniscus and synovium that may play a critical role in osteoarthritis (OA) pathophysiology. Samples of synovium and meniscus were collected from patients with early and end-stage OA and cultured alone or cocultured. Cytokines, chemokines, metalloproteases, and their inhibitors were evaluated at the gene and protein levels. The extracellular matrix (ECM) changes were also investigated. In early OA cultures, higher levels of interleukin-6 (IL-6) and IL-8 messenger RNA were expressed by synovium and meniscus in coculture compared with meniscus cultured alone. RANTES release was significantly increased when the two tissues were cocultured compared with meniscus cultured alone. Increased levels of matrix metalloproteinase-3 (MMP-3) and MMP-10 proteins, as well as increased release of glycosaminoglycans and aggrecan CS846 epitope, were observed when synovium was cocultured with meniscus. In end-stage OA cultures, increased levels of IL-8 and monocyte chemoattractant protein-1 (MCP-1) proteins were released in cocultures compared with cultures of meniscus alone. Chemokine (C-C motif) ligand 21 (CCL21) protein release was higher in meniscus cultured alone and in coculture compared with synovium cultured alone. Increased levels of MMP-3 and 10 proteins were observed when tissues were cocultured compared with meniscus cultured alone. Aggrecan CS846 epitope release was increased in cocultures compared with cultures of either tissue cultured alone. Our study showed the production of inflammatory molecules by synovium and meniscus which could trigger inflammatory signals in early OA patients, and induce ECM loss in the progressive and final stages of OA pathology.     

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone–butanol–ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful “coculture” for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.     

Syntrophomonas erecta subsp. sporosyntropha subsp. nov., a spore-forming bacterium that degrades short chain fatty acids in co-culture with methanogens

Two obligate anaerobic bacterial strains (5-3-Z(T) and Y4-1) were isolated from river sediment and rice field mud, respectively. They degraded straight-chain fatty acids with 4-8 carbon atoms in syntrophic association with methanogens, however, neither tested branch-chain fatty acids nor could benzoate be degraded. The strains formed spores when cocultured with methanogens on butyrate, or when grew on butyrate plus dimethyl sulfoxide (DMSO) in pure culture. The cells were slightly curved rods with Gram-negative cell wall structure, and contained small amount of poly beta-hydroxyalkanoate. The strains could not degrade butyrate alone, nor could use fumarate, sulfate, thiosulfate, sulfur or nitrate as electron acceptors except DMSO for butyrate degradation. The generation time of strain 5-3-Z(T) was about 12h when growing on crotonate at 37 degrees C. The growth of the new strains occurred in the range of pH 5.5-8.4, and of temperature 20-48 degrees C, and at NaCl concentration of 0-700 mM. The G+C content of the genomic DNA of strain 5-3-Z(T) was 40.6mol%. Phylogenetic analysis based on 16S rRNA gene similarity showed the two strains to be a member of species Syntrophomonas erecta (98.4-98.9% sequence similarity), however they differed from the existing strains in both phenotypic and genetic characteristics. Therefore, a new subspecies of S. erecta, S. erecta subsp. sporosyntropha was proposed. The type strain was 5-3-Z(T) (=CGMCC1.5032(T)=JCM13344(T)).     

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.