Co-culture Strategy Research Articles

Co-culture of a Novel Fermentative Bacterium, Lucifera butyrica gen. nov. sp. nov., With the Sulfur Reducer Desulfurella amilsii for Enhanced Sulfidogenesis.

Biosulfidogenesis can be used to remediate low pH and high metal content waters such as acid mine drainage and recover the present metals. The selection of a cheap electron donor for the process is important for the economic viability. In this work we isolated a novel versatile acidotolerant fermentative bacterium (strain ALET) that is able to use a great variety of substrates including glycerol. Strain ALET is an obligate anaerobe, and cells are motile, rod-shaped, spore-forming, and stain Gram-positive. Growth occurred in a pH range from 3.5 to 7 (optimum 5.5), and temperature range from 25 to 40°C (optimum 37°C). It grows by fermentation of sugars, organic acids and glycerol. It has the ability to use thiosulfate, iron and DMSO as electron acceptors. Its genome is 4.7 Mb with 5122 protein-coding sequences, and a G+C content of 46.9 mol%. Based on 16S rRNA gene sequence analysis, the closest cultured species is Propionispora hippei (91.4% 16S rRNA gene identity) from the Sporomusaceae family (Selenomonadales order, Negativicutes class, Firmicutes phylum). Based on the distinctive physiological and phylogenetic characteristics of strain ALET, a new genus and species Lucifera butyrica gen. nov., sp. nov., is proposed. The type strain is ALET (=JCM 19373T = DSM 27520T). Strain ALET is an incomplete oxidizer and acetate, among other products, accumulates during glycerol conversion. Strain ALET was used to extend the substrate range for sulfur reduction by constructing co-cultures with the acetate oxidizer and sulfur reducer Desulfurella amilsii. The co-culture was tested with glycerol as substrate in batch and chemostat experiments. Acetate formed by fermentation of glycerol by strain ALET resulted in sulfur reduction by D. amilsii. The co-culture strategy offers good perspectives to use a wide range of cost-efficient substrates, including glycerol, to produce sulfide by specialized sulfur reducers. The recovery of heavy metals from metalliferous streams may become economically feasible by this approach.Note: The locus tag for the genes encoded in Lucifera butyrica is LUCI_∗. To avoid repetition of the prefix along the text, the locus tags are represented by the specific identifier.

Enhanced biohydrogen production from dilute acid pretreated sugarcane bagasse by detoxification and fermentation strategy

The pretreated sugarcane bagasse (SCB) was detoxified by laccase, NaBH4 and washing before anaerobic degradation. The detoxified SCB was used for biohydrogen fermentation by MJ1. The biohydrogen productions from the detoxified samples by laccase and NaBH4 were low (below 7 mM). As for detoxification by washing, the biohydrogen production reached 44 mM, nearly 10 times higher than the non-detoxified sample. Co-culture strategy (DSM 1313 and MJ1) was further adopted to enhance the biohydrogen production. The biohydrogen production of 119.7 mM was reached by optimizing the fermentation conditions, which was 2.7 times higher than monoculture and 25 times higher than non-detoxified sample. The biohydrogen productions from detoxified sample indicated that the detoxifications of acid pretreated SCB by laccase and NaBH4 were not suitable for biohydrogen fermentation by MJ1 and washing was an effective detoxification method. Co-culture of DSM 1313 and MJ1 was an efficient fermentation strategy for biohydrogen production from pretreated SCB. The present study provides a new insight into the detoxification research on biohydrogen production.

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.

Co-culture system of hepatocytes and endothelial cells: two in vitro approaches for enhancing liver-specific functions of hepatocytes.

Although hepatocyte transplantation and bioartificial liver support system provide new promising opportunities for those patients waiting for liver transplantation, hepatocytes are easily losing liver-specific functions by using the common in vitro cultured methods. The co-culture strategies with mimicking the in vivo microenvironment would facilitate the maintenance of liver-specific functions of hepatocytes. Considering that hepatocytes and endothelial cells (ECs) account for 80-90% of total cell populations in the liver, hepatocytes and ECs were directly co-cultured with hepatic stellate cells (HSCs) or adipose tissue-derived stem cells (ADSCs) at a ratio of 700:150:3 or 14:3:3 in the present study, and the liver-specific functions were carefully analyzed. Our results showed that the two co-culture systems presented the enhanced liver-specific functions through promoting secretion of urea and ALB and increasing the expressions of ALB, CYP3A4 and HNF4α, and the vessel-like structure in the co-culture system consisted of hepatocytes, ECs and ADSCs. Hence, our results suggested that the directly co-culture of hepatocytes and ECs with HSCs or ADSCs could significantly improve liver-specific functions of hepatocytes, and the co-culture system could further promote angiogenesis of ECs at a later stage. Therefore, this study provides potential interesting in vitro strategies for enhancing liver-specific functions of hepatocytes.

Engineering of artificial microbial consortia of Ralstonia eutropha and Bacillus subtilis for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production from sugarcane sugar without precursor feeding

Ralstonia eutropha is a well-known microbe reported for polyhydroxyalkonate (PHA) production, and unable to utilize sucrose as carbon source. Two strains, Ralstonia eutropha H16 and Ralstonia eutropha 5119 were co-cultured with sucrose hydrolyzing microbes (Bacillus subtilis and Bacillus amyloliquefaciens) for PHA production. Co-culture of B. subtilis:R. eutropha 5119 (BS:RE5) resulted in best PHA production (45% w/w dcw). Optimization of the PHA production process components through response surface resulted in sucrose: NH4Cl:B. subtilis: R. eutropha (3.0:0.17:0.10:0.190). Along with the hydrolysis of sucrose, B. subtilis also ferments sugars into organic acid (propionic acid), which acts as a precursor for HV monomer unit. Microbial consortia of BS:RE5 when cultured in optimized media led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV) with 66% w/w of dcw having 16 mol% HV fraction. This co-culture strategy overcomes the need for metabolic engineering of R. eutropha for sucrose utilization, and addition of precursor for copolymer production.

Simultaneous Saccharification and Fermentation of Corn Husk by Co-Culture Strategy

Lignocellulosic biofuel production mainly carried out by two ways: simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF). In the present study, simultaneous saccharification and fermentation (SSF) was carried out using microwave assisted thermochemically pretreated (0.5 M NaOH for 20 minutes at 120°C in preheated oven) corn husk. Using co-cultures of Saccharomyces cerevisiae and Fusarium oxysporum, SSF process was optimized. Maximum ethanol production (6.24%, v/v) was observed after 24 h of incubation. Further for enhanced ethanol production, effect of different surfactant was carried out on SSF using co-culture strategy. It was found that addition of Tween 60 enhanced the ethanol production upto 6.38% (v/v). Further for addition enhancement of ethanol production, different co-culture strategy was adopted. It was found that maximum ethanol production (6.58% v/v) was obtained when ethanol fermentation was carried out by Fusarium oxysporum followed by Saccharomyces cerevisiae.

Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol

As a promising one-carbon renewable substrate for industrial biotechnology, methanol has attracted much attention. However, engineering of microorganisms for industrial production of pharmaceuticals using a methanol substrate is still in infancy. In this study, the methylotrophic yeast Pichia pastoris was used to produce anti-hypercholesterolemia pharmaceuticals, lovastatin and its precursor monacolin J, from methanol. The biosynthetic pathways for monacolin J and lovastatin were first assembled and optimized in single strains using single copies of the relevant biosynthetic genes, and yields of 60.0mg/L monacolin J and 14.4mg/L lovastatin were obtained using methanol following pH controlled monoculture. To overcome limitations imposed by accumulation of intermediates and metabolic stress in monoculture, approaches using pathway splitting and co-culture were developed. Two pathway splitting strategies for monacolin J, and four for lovastatin were tested at different metabolic nodes. Biosynthesis of monacolin J and lovastatin was improved by 55% and 71%, respectively, when the upstream and downstream modules were separately accommodated in two different fluorescent strains, split at the metabolic node of dihydromonacolin L. However, pathway distribution at monacolin J blocked lovastatin biosynthesis in all designs, mainly due to its limited ability of crossing cellular membranes. Bioreactor fermentations were tested for the optimal co-culture strategies, and yields of 593.9mg/L monacolin J and 250.8mg/L lovastatin were achieved. This study provides an alternative method for production of monacolin J and lovastatin and reveals the potential of a methylotrophic yeast to produce complicated pharmaceuticals from methanol.

Cardiomyocyte coculture on layered fibrous scaffolds assembled from micropatterned electrospun mats

Challenges remain in engineering cardiac tissues with functional and morphological properties similar to those of native myocardium. In the current study, micropatterned fibrous mats are obtained by deposition of electrospun fibers on lithographic collectors to reproduce the anisotropic structure of myocardium, and carbon nanotubes are included in fibers to provide conductivities at the same level of cardiac muscles. The patterned mats are assembled layer-by-layer into patterned scaffolds for coculture of primary cardiomyocytes (CMs) with cardiac fibroblasts (CFs) and endothelial cells (ECs). CMs are organized along the fibers with clear cardiac strips of sarcomeric α-actinin and belt-like connexin-43, showing strong cellular extraction forces and intercellular communications. Compared with square and rectangle patterns, honeycomb (Hc)-patterned scaffolds shows higher ultimate tensile strength and strain to failure. The finite element analysis indicates no apparent stress concentration under stress application in the two orthogonal directions. The Hc-patterned coculture demonstrates significantly higher CM viabilities, deeper penetrations of cells into scaffolds, stronger expression of troponin I, connexin-43 and sarcomeric α-actinin by CMs and more abundant formations of capillary-like networks by ECs than other scaffolds. CMs on Hc-patterned scaffolds display spontaneous beating rates at 101±12times/min after coculture for 5days and remain synchronously beating at 94±8times/min after 15days, which is close to those of adult and neonatal rats. The layered and patterned coculture strategy achieves the spatial arrangement of multiple types of cells and vascularization potential, providing a biomimetic strategy for engineering functional cardiac patches in vitro.

Strategies to Mitigate Variability in Engineering Human Nasal Cartilage

Skin cancer and its associated treitments can have devastating consequences for survivors; this is particularly true when cancer occurs on the nose. Recent work has applied cell-based tissue engineering (TE) strategies to develop nasal cartilage constructs for reconstruction of the nose. In this study, we have generated human nasal cartilage on a clinically approved collagen scaffold to investigate the donor-to-donor variability of TE cartilage and evaluated strategies to mitigate it. We also evaluated the gene expression of the family of fibroblast growth factor receptors (FGFR1-4) and their association with tissue quality. FGFR1 was significantly positively correlated with GAG/DNA; a measure of chondrogenic capacity. We implemented two strategies: hypoxic culture and co-culture with mesenchymal stromal cells (MSCs) to increase tissue quality. Total glycosaminoglycan (GAG) content varied significantly between donors initially, with >10–fold difference between the best and worst donor tissue. Our co-culture strategy was able to increase TE construct quality from poor quality donor tissue while supressing hypertrophy relative to MSCs alone. However, no differences were observed with the use of hypoxic culture. Tissues generated using co-culture with MSCs became vascularized and calcified in vivo, demonstrating a non-stable cartilage phenotype in co-culture and MSCs cartilage constructs.

Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring

BackgroundCellulose-degrading thermophilic anaerobic bacterium as a suitable host for consolidated bioprocessing (CBP) has been proposed as an economically suited platform for the production of second-generation biofuels. To recognize the overall objective of CBP, fermentation using co-culture of different cellulolytic and sugar-fermenting thermophilic anaerobic bacteria has been widely studied as an approach to achieving improved ethanol production. We assessed monoculture and co-culture fermentation of novel thermophilic anaerobic bacterium for ethanol production from real substrates under controlled conditions.ResultsIn this study, Clostridium sp. DBT-IOC-C19, a cellulose-degrading thermophilic anaerobic bacterium, was isolated from the cellulolytic enrichment cultures obtained from a Himalayan hot spring. Strain DBT-IOC-C19 exhibited a broad substrate spectrum and presented single-step conversion of various cellulosic and hemicellulosic substrates to ethanol, acetate, and lactate with ethanol being the major fermentation product. Additionally, the effect of varying cellulose concentrations on the fermentation performance of the strain was studied, indicating a maximum cellulose utilization ability of 10 g L−1 cellulose. Avicel degradation kinetics of the strain DBT-IOC-C19 displayed 94.6% degradation at 5 g L−1 and 82.74% degradation at 10 g L−1 avicel concentration within 96 h of fermentation. In a comparative study with Clostridium thermocellum DSM 1313, the ethanol and total product concentrations were higher by the newly isolated strain on pretreated rice straw at an equivalent substrate loading. Three different co-culture combinations were used on various substrates that presented two-fold yield improvement than the monoculture during batch fermentation.ConclusionsThis study demonstrated the direct fermentation ability of the novel thermophilic anaerobic bacteria on various cellulosic and hemicellulosic substrates into ethanol without the aid of any exogenous enzymes, representing CBP-based fermentation approach. Here, the broad substrate utilization spectrum of isolated cellulolytic thermophilic anaerobic bacterium was shown to be of potential utility. We demonstrated that the co-culture strategy involving novel strains is efficient in improving ethanol production from real substrate.

Upgrading well plates using open microfluidic patterning.

Cellular communication between multiple cell types is a ubiquitous process that is responsible for vital physiological responses observed in vivo (e.g., immune response, organ function). Many in vitro coculture strategies have been developed, both in traditional culture and microscale systems, and have shown the potential to recreate some of the physiological behaviors of organs or groups of cells. A fundamental limitation of current systems is the difficulty of reconciling the additional engineering requirements for creating soluble factor signaling systems (e.g., segregated cell culture) with the use of well-characterized materials and platforms that have demonstrated successful results and biocompatibility in assays. We present a new open-microfluidic platform, the Monorail Device, that is placed in any existing well plate or Petri dish and enables patterning of segregated coculture regions, thereby allowing the direct upgrade of monoculture experiments into multiculture assays. Our platform patterns biocompatible hydrogel walls via microfluidic spontaneous capillary flow (SCF) along a rail insert set inside commercially available cultureware, creating customized pipette-accessible cell culture chambers that require fewer cells than standard macroscale culture. Importantly, the device allows the use of native surfaces without additional modification or treatments, while creating permeable dividers for the diffusion of soluble factors. Additionally, the ease of patterning afforded by our platform makes reconfiguration of the culture region as simple as changing the rail insert. We demonstrate the ability of the device to pattern flows on a variety of cell culture surfaces and create hydrogel walls in complex and precise shapes. We characterize the physical parameters that enable a reproducible SCF-driven flow and highlight specialized design features that increase the ease of use of the device and control of the open microfluidic flow. Further, we present the performance of our platform according to useful coculture criteria, including permeability and integrity of our hydrogel walls and surface-sensitive cell culture. Lastly, we show the potential of this type of platform to create modular multikingdom culture systems that can be used to study soluble factor signaling between mammalian cells, bacteria, and fungi, as well as the potential for adaptation of this technology by researchers across multiple fields.

ERK/Drp1-dependent mitochondrial fission is involved in the MSC-induced drug resistance of T-cell acute lymphoblastic leukemia cells.

The bone marrow microenvironment facilitates the proliferation and survival of leukemia cells, contributing to disease relapse. Bone marrow-derived mesenchymal stem cells (MSCs) are well known to promote cancer chemoresistance via soluble factors and cell adhesion. However, little is known about the effects of MSCs on the mitochondrial dynamics of T-cell acute lymphoblastic leukemia (T-ALL) cells, or how this may influence the chemoresistance of these cells. Here, we tested both indirect (Transwell) and direct coculture strategies, and found that MSCs protected T-ALL cells from chemotherapeutic cell death and cytotoxicity under both culture conditions. In addition, cell viability was higher in the direct contact system compared with the Transwell system. We further showed that exposure of T-ALL cells to MSCs decreased mitochondrial reactive oxygen species (ROS) levels and promoted a pro-glycolytic shift that was characterized by increased glucose uptake and lactate production with concomitant reductions in adenosine triphosphate production and mitochondrial membrane potential. In T-ALL cells cocultured with MSCs, the mitochondrial morphology of T-ALL cells were altered from elongation to fragmentation because of the extracellular signal-regulated kinase activation-mediated phosphorylation of the pro-fission factor, dynamin-related protein 1 (Drp1), at residue S616. Consistent with this, the expression of S616-phosphorylated Drp1 recapitulated the mitochondrial dynamics, mitochondrial ROS levels, metabolic switching and chemoresistance seen in T-ALL cells cocultured with MSCs. These findings suggest that the ability of MSCs to trigger Drp1 activation-induced changes in mitochondrial dynamics is crucial to their ability to protect cells against chemotherapeutic agents.

Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol.

BackgroundResveratrol is a plant natural product with many health-protecting effects which makes it an attractive chemical both for academic studies and industrial purposes. However, the low quantities naturally produced by plants as well as the unsustainable procedures of extraction, purification and concentration have prompted many biotechnological approaches to produce this chemical in large quantities from renewable sources. None of these approaches have considered a microbial coculture strategy to produce this compound. The aim of this study was to prove the functionality of a microbial coculture for the biosynthesis of resveratrol.ResultsIn this work, we have successfully applied a coculture system strategy comprised of two populations of Escherichia coli strains, each with a partial and complementary section of the pathway leading to the biosynthesis of the stilbene resveratrol. The first strain is a pheA knockout mutant previously engineered to excrete p-coumaric acid into the medium through the overexpression of genes encoding a tyrosine ammonia lyase from Rhodothorula glutinis, a feedback resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and a transketolase. The second strain in the coculture was engineered to express the second part of the resveratrol biosynthetic pathway through the introduction of synthetic genes encoding the 4-coumaroyl-CoA ligase from Streptomyces coelicolor A2 and the stilbene synthase either from the peanut Arachis hypogaea or the grapevine Vitis vinifera, the latter synthesized employing a gene harmonization strategy and showing better resveratrol production performance. Batch cultures were performed in mineral medium with glycerol as the sole carbon source, where a final titer of 22.6 mg/L of resveratrol was produced in 30 h.ConclusionsTo our knowledge, this is the first time that a coculture of bacterial strains is used for the biosynthesis of resveratrol from glycerol, having the potential for a greater improvement in the product yield and avoiding the use of precursors such as p-coumaric acid, yeast extract or an expensive inhibitor such as cerulenin.

Improvement of biohydrogen production with novel augmentation strategy using different organic residues

Biohydrogen has been regarded as carbon neutral fuel. Thus it possesses tremendous potential in energy sector. The present study deals with the potentiality of bio-augmented facultative anaerobic bacteria with obligate anaerobe for the improvement of H2 production. The co-culture strategy of Klebsellia pneumoniae and Clostridium acetobutylicum improved hydrogen yield by 37% and 18% respectively as compared to individual organism. COD removal efficiency was also observed higher in case of co-culture. Maximum H2 yield by this augmented system using cane molasses, starchy wastewater and distillery effluent were 9.47, 8.72 and 7.78 mol H2 kg−1 CODreduced respectively. The COD removal efficiency using different organic residues were in the range of 50–70%. Highest H2 production rate of 1125, 642 and 790 mL L−1 h−1 were observed by using cane molasses, starchy wastewater and distillery effluent respectively in CSTR. So, bio-augmented system could be helpful in realizing the goal of “waste to energy” concept.

Study of chondrogenic potential of stem cells in co-culture with chondrons.

Three-dimensional biomimetic scaffolds have widespread applications in biomedical tissue engineering due to similarity of their nanofibrous architecture to native extracellular matrix. Co-culture system has stimulatory effect on chondrogenesis of adult mesenchymal stem cells. This work presents a co-culture strategy using human articular chondrons and adipose-derived stem cells (ASCs) from infrapatellar fat pad (IPFP) for cartilage tissue production. Isolated stem cells were characterized by flowcytometry. Electrospun and polycaprolactone (PCL) scaffolds (900 nm fiber diameter) was obtained from Bon Yakhteh (Tehran-Iran) and human infrapatellar fat pad-derived stem cells (IPFP-ASCs) were seeded on them. IPFP-ASCs on scaffolds were co-cultured with articular chondrons using transwell. After 21 day, chondrogenic differentiation of stem cell was evaluated by determining the genes expression of collagen2, aggrecan and Indian hedgehog using real-time RT-PCR. Genes expression of collagen2, aggrecan by IPFP-ASCs did not alter significantly in comparison with control group. Howevers, expression of Indian hedgehog decreased significantly compared to control group (P< 0.05). These findings indicate that chondrons obtained from osteoarthritic articular cartilage did not stimulate chondrogenic differentiation of IPFP-ASCs in co-culture.

Cell-Based Strategies for Meniscus Tissue Engineering.

Meniscus injuries remain a significant challenge due to the poor healing potential of the inner avascular zone. Following a series of studies and clinical trials, tissue engineering is considered a promising prospect for meniscus repair and regeneration. As one of the key factors in tissue engineering, cells are believed to be highly beneficial in generating bionic meniscus structures to replace injured ones in patients. Therefore, cell-based strategies for meniscus tissue engineering play a fundamental role in meniscal regeneration. According to current studies, the main cell-based strategies for meniscus tissue engineering are single cell type strategies; cell coculture strategies also were applied to meniscus tissue engineering. Likewise, on the one side, the zonal recapitulation strategies based on mimicking meniscal differing cells and internal architectures have received wide attentions. On the other side, cell self-assembling strategies without any scaffolds may be a better way to build a bionic meniscus. In this review, we primarily discuss cell seeds for meniscus tissue engineering and their application strategies. We also discuss recent advances and achievements in meniscus repair experiments that further improve our understanding of meniscus tissue engineering.

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

A novel bioreactor to study the dynamics of co-culture systems

Co-culture strategy has drawn increasing interest in the last a few years to ferment the mixture of glucose and xylose or lignocellulosic hydrolysate. However, existing research has been mostly qualitative which mainly examined the ethanol production performance of the co-culture system such as final ethanol yield and productivity, with little or no attempt made to understand the dynamic interactions between the two microbes. This is partially due to the difficulties associated with monitoring and control of co-culture systems. In this work, we developed a bioreactor and associated protocols and control strategies to facilitate quantitative and systematic study of co-culture systems. In particular, the reported equipment, operation protocols and control strategies can deliver chemostat operation under controlled stable operation conditions by achieving stable oxygen utilization rates at various levels. In addition, the developed membrane-separated co-culture bioreactor enables independent control of the dissolved oxygen (DO) levels in each chamber, and easy tracking of individual biomass of each strain. The new protocol is a dual continuous/pseudo-continuous operation mode, which allows the control of constant biomass by adjusting the operation time of each mode, dilution rate and feed concentration. Experimental results on the co-culture system of Saccharomyces cerevisiae and Scheffersomyces stipitis are provided to demonstrate the capabilities of the developed co-culture bioreactor. With the developed equipment and protocol, simultaneous and complete utilization of both glucose and xylose was achieved around 70h into the experiments we conducted, and was maintained as long as 800h. Such complete utilization can be maintained even longer if desired. In addition, different OUR conditions (ranging 0.0036–0.0045mmolO2/gDCW/h) were tested under controlled chemostat. Under the different operation conditions tested, ethanol yields and conversion rates varied in the range of 0.12–0.44g/g and 0.22–1.95gEtOH/L/h, respectively, which are in line with results reported in the literature.

Coculture with good-quality COCs enhances the maturation and development rates of poor-quality COCs

In pigs, more than half of the recovered cumulus cell–oocyte complexes (COCs) have one or two layers of cumulus cells and are considered morphologically poor. If we could take full advantage of these poor-quality COCs, we could potentially improve the efficiency of in vitro embryo production. During IVM, although some maturation factors are transmitted bidirectionally between the oocyte and the cumulus cells of the same COC, transmission also occurs between different COCs. We hypothesized that morphologically poor COCs fail to undergo complete oocyte maturation because of their insufficient secretion of maturation factors. Here, we investigated whether coculture with morphologically good COCs (having three or more layers of cumulus cells) could improve the maturation and utilization rates of morphologically poor COCs. Our results revealed that the oocyte maturation rate, glutathione level, embryo development capacity, blastocyst quality, and cumulus cell gene expression levels of BCL-2 and proliferating cell nuclear antigen were similar in the coculture and good-quality groups and that these levels were all significantly higher than those in the poor-quality group. Our results strongly suggest that the coculture strategy greatly improved the utilization rate of morphologically poor COCs without reducing their capacity for maturation and subsequent development.

Understanding the effects of mature adipocytes and endothelial cells on fatty acid metabolism and vascular tone in physiological fatty tissue for vascularized adipose tissue engineering.

Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new co-culture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted.