In Vivo Vascularization Chamber for the Implantation of Embryonic Kidneys.

A major obstacle to the implantation of ex vivo engineered tissues is the incorporation of functional vascular supply to support the growth of new tissue and to minimize ischemic injury. Existing prevascularization systems, such as arteriovenous (AV) loop-based systems, require microsurgery, limiting their use to larger animals. We aimed to develop an implantable device that can be prevascularized to enable vascularization of tissues in small rodents, and test its application on the vascularization of embryonic kidneys. Implanting the chamber between the abdominal aorta and the inferior vena cava, we detected endothelial cells and vascular networks after 48 h of implantation. Loading the chamber with collagen I (C), Matrigel (M), or Matrigel + vascular endothelial growth factor) (MV) had a strong influence on vascularization speed: Chambers loaded with C took 7 days to vascularize, 4 days for chambers with M, and 2 days for chambers with MV. Implantation of E12.5 mouse embryonic kidneys into prevascularized chambers (C, MV) was followed with significant growth and ureteric branching over 22 days. In contrast, the growth of kidneys in non-prevascularized chambers was stunted. We concluded that our prevascularized chamber is a valuable tool for vascularizing implanted tissues and tissue-engineered constructs. Further optimization will be necessary to control the directional growth of vascular endothelial cells within the chamber and the vascularization grade. Impact Statement Vascularization of engineered tissue, or organoids, constructs is a major hurdle in tissue engineering. Failure of vascularization is associated with prolonged ischemia time and potential tissue damage due to hypoxic effects. The method presented, demonstrates the use of a novel chamber that allows rapid vascularization of native and engineered tissues. We hope that this technology helps to stimulate research in the field of tissue vascularization and enables researchers to generate larger engineered vascularized tissues.

Effect of Hypoxia on Branching Characteristics and Cell Subpopulations during Kidney Organ Culture.

During early developmental stages, embryonic kidneys are not fully vascularized and are potentially exposed to hypoxic conditions, which is known to influence cell proliferation and survival, ureteric bud branching, and vascularization of the developing kidney. To optimize the culture conditions of in vitro cultured kidneys and gain further insight into the effect of hypoxia on kidney development, we exposed mouse embryonic kidneys isolated at E11.5, E12.5, and E13.5 to hypoxic and normal culture conditions and compared ureteric bud branching patterns, the growth of the progenitor subpopulation hoxb7+, and the expression patterns of progenitor and differentiation markers. Branching patterns were quantified using whole organ confocal imaging and gradient-vector-based analysis. In our model, hypoxia causes an earlier expression of UB tip cell markers, and a delay in stalk cell marker gene expression. The metanephric mesenchyme (MM) exhibited a later expression of differentiation marker FGF8, marking a delay in nephron formation. Hypoxia further delayed the expression of stroma cell progenitor markers, a delay in cortical differentiation markers, as well as an earlier expression of medullary and ureteral differentiation markers. We conclude that standard conditions do not apply universally and that tissue engineering strategies need to optimize suitable culture conditions for each application. We also conclude that adapting culture conditions to specific aspects of organ development in tissue engineering can help to improve individual stages of tissue generation.

Bioprinting Scaffolds for Vascular Tissues and Tissue Vascularization.

In recent years, tissue engineering has achieved significant advancements towards the repair of damaged tissues. Until this day, the vascularization of engineered tissues remains a challenge to the development of large-scale artificial tissue. Recent breakthroughs in biomaterials and three-dimensional (3D) printing have made it possible to manipulate two or more biomaterials with complementary mechanical and/or biological properties to create hybrid scaffolds that imitate natural tissues. Hydrogels have become essential biomaterials due to their tissue-like physical properties and their ability to include living cells and/or biological molecules. Furthermore, 3D printing, such as dispensing-based bioprinting, has progressed to the point where it can now be utilized to construct hybrid scaffolds with intricate structures. Current bioprinting approaches are still challenged by the need for the necessary biomimetic nano-resolution in combination with bioactive spatiotemporal signals. Moreover, the intricacies of multi-material bioprinting and hydrogel synthesis also pose a challenge to the construction of hybrid scaffolds. This manuscript presents a brief review of scaffold bioprinting to create vascularized tissues, covering the key features of vascular systems, scaffold-based bioprinting methods, and the materials and cell sources used. We will also present examples and discuss current limitations and potential future directions of the technology.

Comprehensive analysis of chromatin signature and transcriptome uncovers functional lncRNAs expressed in nephron progenitor cells.

Emerging evidence from recent studies has unraveled the roles of long noncoding RNAs (lncRNAs) in the function of various tissues. However, little is known about the roles of lncRNAs in kidney development. In our present study, we aimed to identify functional lncRNAs in one of the three lineages of kidney progenitor cells, i.e., metanephric mesenchymal (MM) cells. We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA-seq and ChIP-seq. We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells. Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2, a key regulatory gene of MM cells. We further identified three transcript variants of Gm29418 by Rapid Amplification of cDNA Ends (RACE), and confirmed that the transcription-start-sites (TSSs) of these variants were consistent with the result of Cap Analysis Gene Expression (CAGE). In support of the enhancer-like function of Gm29418 on Six2 expression, we found that knock-down of Gm29418 by two independent anti-sense locked nucleic acid (LNA) phosphorothioate gapmers suppressed Six2 mRNA expression levels in MM cells. We also found that over-expression of Gm29418 led to an increase in Six2 mRNA expression levels in a mouse MM cell line. In conclusion, we identified a lncRNA, Gm29418, in nephron progenitor cells that has an enhancer-like function on a key regulatory gene, Six2.

Effect of fluid shear stress on in vitro cultured ureteric bud cells.

Most kidney cells are continuously exposed to fluid shear stress (FSS) from either blood flow or urine flow. Recent studies suggest that changes in FSS could contribute to the function and injury of these kidney cells. However, it is unclear whether FSS influences kidney development when urinary flow starts in the embryonic kidneys. In this study, we evaluated the influence of FSS on in vitro cultured ureteric bud (UB) cells by using a pumpless microfluidic device, which offers the convenience of conducting parallel cell culture experiments while also eliminating the need for cumbersome electronic driven equipment and intricate techniques. We first validated the function of the device by both mathematical model and experimental measurements. UB cells dissected from E15.5 mouse embryonic kidneys were cultured in the pumpless microfluidic device and subjected to FSS in the range of 0.4-0.6 dyn mm-2 for 48 h (dynamic). Control UB cells were similarly cultured in the device and maintained under a no-flow condition (static). We found from our present study that the exposure to FSS for up to 48 h led to an increase in mRNA expression levels of UB tip cell marker genes (Wnt11, Ret, Etv4) with a decrease in stalk cell marker genes (Wnt7b, Tacstd2). In further support of the enrichment of UB tip cell population in response to FSS, we also found that exposure to FSS led to a remarkable reduction in the binding of lectin Dolichos Biflorus Agglutinin. In conclusion, results of our present study show that exposure to FSS led to an enrichment in UB tip cell populations, which could contribute to the development and function of the embryonic kidney when urine flow starts at around embryonic age E15.5 in mouse. Since UB tip cells are known to be the proliferative progenitor cells that contribute to the branching morphogenesis of the collecting system in the kidney, our finding could imply an important link between the FSS from the initiation of urine flow and the development and function of the kidney.

An optimal serum-free defined condition for in vitro culture of kidney organoids.

Kidney organoid is an emerging topic of importance for research in kidney development and regeneration. Conventional culture systems for kidney organoids reported thus far use culture media containing serum, which may compromise our understanding and the potential clinical applicability of the organoid system. In our present study, we tested two serum-free culture conditions and compared their suitability for the maintenance and growth of kidney organoids in culture. One of the serum-free culture conditions was the combination of keratinocytes serum free medium (KSFM) with knockout serum replacement (KSR) (KSFM + KSR), and the other was the combination of knockout DMEM/F12 (KD/F12) and KSR (KD/F12 + KSR). With cell aggregates derived from E12.5 mouse embryonic kidneys, we found that KD/F12 + KSR was superior to KSFM + KSR in promoting the growth of the aggregate with expansion of Six2+ nephron progenitor cells (NPC) and elaborated ureteric branching morphogenesis. With KD/F12 + KSR, we found that lower concentrations of KSR at 5-10% were superior to a higher concentration (20%) in promoting the growth of aggregates without affecting the expression levels of NPC marker genes. We also found that NPC in aggregates retained their differentiation potential to develop nephron tubules through mesenchyme-to-epithelial transition (MET), after being maintained in culture under these conditions for up to 7 days. In conclusion, we have identified a defined serum-free culture condition suitable for the maintenance and growth of kidney organoids that retain the differentiation potential to develop nephron structures. This defined serum-free culture condition may serve as a useful platform for further investigation of kidney organoids in vitro.

Microfluidics for Antibiotic Susceptibility and Toxicity Testing.

The recent emergence of antimicrobial resistance has become a major concern for worldwide policy makers as very few new antibiotics have been developed in the last twenty-five years. To prevent the death of millions of people worldwide, there is an urgent need for a cheap, fast and accurate set of tools and techniques that can help to discover and develop new antimicrobial drugs. In the past decade, microfluidic platforms have emerged as potential systems for conducting pharmacological studies. Recent studies have demonstrated that microfluidic platforms can perform rapid antibiotic susceptibility tests to evaluate antimicrobial drugs' efficacy. In addition, the development of cell-on-a-chip and organ-on-a-chip platforms have enabled the early drug testing, providing more accurate insights into conventional cell cultures on the drug pharmacokinetics and toxicity, at the early and cheaper stage of drug development, i.e., prior to animal and human testing. In this review, we focus on the recent developments of microfluidic platforms for rapid antibiotics susceptibility testing, investigating bacterial persistence and non-growing but metabolically active (NGMA) bacteria, evaluating antibiotic effectiveness on biofilms and combinatorial effect of antibiotics, as well as microfluidic platforms that can be used for in vitro antibiotic toxicity testing.

Determination of antibiotic EC50 using a zero-flow microfluidic chip based growth phenotype assay.

Current existing assay systems for evaluating antimicrobial activity suffer from several limitations including excess reagent consumption and inaccurate concentration gradient preparation. Recently, microfluidic systems have been developed to provide miniaturized platforms for antimicrobial susceptibility assays. However, some of current microfluidic based assays require continuous flows of reagents or elaborate preparation steps during concentration preparation. In this study, we introduce a novel microfluidic chip based growth phenotype assay that automatically generates a logarithmic concentration gradient and allows observing the growth of pathogenic bacteria under different concentrations of antibiotics in nanoliter batch culture reactors. We chose pathogen bacterium Pseudomonas aeruginosa as a model strain and evaluated the inhibitory effects of gentamicin and ciprofloxacin. We determined the EC50 values and confirmed the validity of the present system by comparing the EC50 values obtained through conventional test tube method. We demonstrated that the EC50 values acquired from present assay are comparable to those obtained from conventional test tube cultures. The potential application of present assay system for investigating combinatorial effects of antibiotics on multidrug resistant pathogenic bacteria is discussed and it can be further used for systematic evaluation of antifungal or antiviral agents.

New tools and new biology: recent miniaturized systems for molecular and cellular biology.

Recent advances in applied physics and chemistry have led to the development of novel microfluidic systems. Microfluidic systems allow minute amounts of reagents to be processed using µm-scale channels and offer several advantages over conventional analytical devices for use in biological sciences: faster, more accurate and more reproducible analytical performance, reduced cell and reagent consumption, portability, and integration of functional components in a single chip. In this review, we introduce how microfluidics has been applied to biological sciences. We first present an overview of the fabrication of microfluidic systems and describe the distinct technologies available for biological research. We then present examples of microsystems used in biological sciences, focusing on applications in molecular and cellular biology.

Cell-based dose responses from open-well microchambers.

Cell-based assays play a critical role in discovery of new drugs and facilitating research in cancer, immunology, and stem cells. Conventionally, they are performed in Petri dishes, tubes, or well plates, using milliliters of reagents and thousands of cells to obtain one data point. Here, we are introducing a new platform to realize cell-based assay capable of increased throughput and greater sensitivity with a limited number of cells. We integrated an array of open-well microchambers into a gradient generation system. Consequently, cell-based dose responses were examined with a single device. We measured IC50 values of three cytotoxic chemicals, Triton X-100, H2O2, and cadmium chloride, as model compounds. The present system is highly suitable for the discovery of new drugs and studying the effect of chemicals on cell viability or mortality with limited samples and cells.

Direct oxygen supply with polydimethylsiloxane (PDMS) membranes induces a spontaneous organization of thick heterogeneous liver tissues from rat fetal liver cells in vitro.

Oxygen is a vital nutrient for growth and maturation of in vitro cells (e.g., adult hepatocytes). We previously demonstrated that direct oxygenation through a polydimethylsiloxane (PDMS) membrane increases the oxygen supply to cell cultures and improves hepatocyte functions. In this study, we removed limits on oxygen supply to fetal rat liver cells through the use of direct oxygenation through a PDMS membrane to investigate in vitro growth and maturation. We chose fetal liver cells because they are considered a feasible source of liver progenitor cells for regenerative medicine therapy due to their highly efficient maturation and proliferation. Cells from 17-day-old pregnant rats were cultured under 5% and 21% oxygen atmospheres. Some cells were first cultured under 5% oxygen, and then switched to a 21% oxygen atmosphere. When oxygen supply was enhanced by a PDMS membrane, the rat fetal liver cells organized into a complex tissue composed of an epithelium of hepatocytes above a mesenchyme-like tissue. The thickness of this supportive tissue was directly correlated to oxygen concentration and was thicker under 5% oxygen. When cultures were switched from 5% to 21% oxygen, lumen-containing structures were formed in the thick mesenchymal-like tissue and the albumin secretion rate increased. In addition, cells adapted their glycolytic activity to the oxygen concentrations. This system promoted the formation of a functional and organized thick tissue suitable for use in regenerative medicine.

Gas-permeable membranes and co-culture with fibroblasts enable high-density hepatocyte culture as multilayered liver tissues.

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three-dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell-cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate-based format, enabling high density hepatocyte culture as a stable 3D-multilayer. Multilayered co-cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen-conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co-cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate-based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co-cultures were maintained for at least 1 week; the so-cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate-based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening.

Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12.

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.

RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44.

We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase--and protein kinase C-dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1).

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.