Micropatterning of living cells by laser-guided direct writing: application to fabrication of hepatic-endothelial sinusoid-like structures

Here, we describe a simple protocol for the design and construction of a laser-guided direct writing (LGDW) system able to micropattern the self-assembly of liver sinusoid-like structures with micrometer resolution in vitro. To the best of our knowledge, LGDW is the only technique able to pattern cells "on the fly" with micrometer precision on arbitrary matrices, including soft gels such as Matrigel. By micropatterning endothelial cells on Matrigel, one can control the self-assembly of vascular structures and associated liver tissue. LGDW is therefore uniquely suited for studying the role of tissue architecture and mechanical properties at the single-cell resolution, and for studying the effects of heterotypic cell-cell interactions underlying processes such as liver morphogenesis, differentiation and angiogenesis. The total time required to carry out this protocol is typically 7 h.     

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

The neural tube patterns vessels developmentally using the VEGF signaling pathway

Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues, but the sources and identities of signals that pattern vessels are not well characterized. We hypothesized that the neural tube provides vascular patterning signal(s) that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation. Both surgically placed ectopic neural tubes and ectopic neural tubes engineered genetically were able to recruit a vascular plexus, showing that the neural tube is the source of a vascular patterning signal. In mouse-quail chimeras with the graft separated from the neural tube by a buffer of host cells, graft-derived vascular cells contributed to the PNVP, indicating that the neural tube signal(s) can act at a distance. Murine neural tube vascular endothelial growth factor A (VEGFA) expression was temporally and spatially correlated with PNVP formation, suggesting it is a component of the neural tube signal. A collagen explant model was developed in which presomitic mesoderm explants formed a vascular plexus in the presence of added VEGFA. Co-cultures between presomitic mesoderm and neural tube also supported vascular plexus formation, indicating that the neural tube could replace the requirement for VEGFA. Moreover, a combination of pharmacological and genetic perturbations showed that VEGFA signaling through FLK1 is a required component of the neural tube vascular patterning signal. Thus, the neural tube is the first structure identified as a midline signaling center for embryonic vascular pattern formation in higher vertebrates, and VEGFA is a necessary component of the neural tube vascular patterning signal. These data suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning.     

Electrosensitive polyacrylic acid/fibrin hydrogel facilitates cell seeding and alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.     

Signaling and gene regulatory programs in plant vascular stem cells

A key question about the development of multicellular organisms is how they precisely control the complex pattern formation during their growth. For plants to grow for many years, a tight balance between pluripotent dividing cells and cells undergoing differentiation should be maintained within stem cell populations. In this process, cell-cell communication plays a central role by creating positional information for proper cell type patterning. Cell-type specific gene regulatory networks govern differentiation of cells into particular cell types. In this review, we will provide a comprehensive overview of emerging key signaling and regulatory programs in the stem cell population that direct morphogenesis of plant vascular tissues.     

Programmed cell death and patterning in Drosophila

Selective cell death provides developing tissues with the means to precisely sculpt emerging structures. By imposing patterned cell death across a tissue, boundaries can be created and tightened. As such, programmed cell death is becoming recognized as a major mechanism for patterning of a variety of complex structures. Typically, cell types are initially organized into a fairly loose pattern; selective death then removes cells between pattern elements to create correct structures. In this review, we examine the role of selective cell death across the course of Drosophila development, including the tightening of embryonic segmental boundaries, head maturation, refining adult structures such as the eye and the wing, and the ability of cell death to correct for pattern defects introduced by gene mutation. We also review what is currently known of the relationship between signals at the cell surface that are responsible for tissue patterning and the basal cell death machinery, an issue that remains poorly understood.     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

Improving the integrity of three-dimensional vascular patterns by poly(ethylene glycol) conjugation

Development of functional tissue-engineering constructs may require that multiple cell types be organized in controlled three-dimensional (3-D) microarchitectures with proper nutrient diffusion and vascularization. In the past few years, a variety of microscale techniques have demonstrated the ability to control protein and cell attachment in defined patterns. Nevertheless, maintenance of these patterns over time has been a significant challenge due to nonspecific protein adsorption and cell migration. To this end, we have investigated the effectiveness of poly(ethylene glycol) (PEG) thin films in maintaining the integrity of 3-D cellular patterns, using human umbilical vein endothelial cells (HUVEC) as a model system. These HUVEC constructs were created using extracellular matrix (ECM)-based microfluidic patterning. Our results indicated that PEG-conjugated substrates improve cell pattern integrity as compared to control silicon. The compliance multifactor (a measure of pattern integrity; higher value means lower pattern integrity) was about 3.66 +/- 0.29 on day 5 for PEG-conjugated surfaces, compared with 8.23 +/- 0.42 for control surfaces ECM-based microfluidic patterning coupled with stable PEG-conjugated surfaces may serve as a vital tool for vascularized tissue engineering.     

Laser-guided direct writing for applications in biotechnology.

Laser-induced optical forces can be used to guide and deposit 100 nm - 10 microm-diameter particles onto solid surfaces in a process we call 'laser-guided direct writing'. Nearly any particulate material, including both biological and electronic materials, can be manipulated and deposited on surfaces with micrometer accuracy. Potential applications include three-dimensional cell patterning for tissue engineering, hybrid biological-electronic-device construction, and biochip-array fabrication.     

Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.     

Glucagon-like peptide-1 (GLP-1) inhibits advanced glycation end product (AGE)-induced up-regulation of VCAM-1 mRNA levels in endothelial cells by suppressing AGE receptor (RAGE) expression

Glucagon-like peptide-1 (GLP-1) is one of the incretins, a gut hormone secreted from L cells in the intestine in response to food intake. It has been proposed as a potential therapeutic target for the treatment of patients with type 2 diabetes. However, the direct effects of GLP-1 on vascular injury in diabetes are largely unknown. Since there is a growing body of evidence that advanced glycation end products (AGE) and their receptor RAGE axis plays an important role in vascular complications in diabetes, this study investigated whether and how GLP-1 blocked the deleterious effects of AGE on human umbilical vein endothelial cells (HUVEC). GLP-1 receptor (GLP-1R) was expressed in HUVEC. GLP-1 dose-dependently inhibited RAGE gene expression in HUVEC, which was blocked by small interfering RNAs raised against GLP-1R. An analogue of cyclic AMP also decreased RAGE mRNA level in HUVEC. Further, GLP-1 decreased reactive oxygen species generation and subsequently reduced vascular cell adhesion molecule-1 mRNA levels in AGE-exposed HUVEC. Our present study suggests that GLP-1 directly acts on HUVEC via GLP-1R and it could work as an anti-inflammatory agent against AGE by reducing RAGE expression via activation of cyclic AMP pathways.     

On‐demand three‐dimensional freeform fabrication of multi‐layered hydrogel scaffold with fluidic channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186. © 2009 Wiley Periodicals, Inc.     

Cell deposition system based on laser guidance

We have designed a laser cell deposition system that employs the phenomenon of laser guidance to place single cells at specific points in a variety of in vitro environments. Here, we describe the components of the system: the laser optics, the deposition chamber, the microinjection cell feeding system and our custom system control software application. We discuss the requirements and challenges involved in laser guidance of cells and how our present system overcomes these challenges. We demonstrate that the patterning system is accurate within one micrometer by repeatedly depositing polymer microspheres and measuring their position. We demonstrate its ability to create highly defined living patterns of cells by creating a defined pattern of neurons with neurite extensions displaying normal function. We found that the positional accuracy of our system is smaller than the variations in cell size and pattern disruptions that occur from normal cell movement during substrate adhesion. The laser cell deposition system is a potentially useful tool that can be used to achieve site- and time-specific placement of an individual cell in a cell culture for the systematic investigation of cell-cell and cell-extracellular matrix interactions.     

Genetic regulation of vascular tissue patterning in Arabidopsis

Plants transport water and nutrients through a complex vascular network comprised of interconnected, specialized cell types organized in discrete bundles. To identify genetic determinants of vascular tissue patterning, we conducted a screen for mutants with altered vascular bundle organization in Arabidopsis cotyledons. Mutations in two genes, CVP1 and CVP2 (for cotyledon vascular pattern), specifically disrupt the normal pattern of vascular bundles in cotyledons, mature leaves, and inflorescence stems. The spatial distribution of the procambium, the precursor to mature vascular tissue, is altered in cvp1 and cvp2 embryos, suggesting that CVP1 and CVP2 act at a very early step in vascular patterning. Similarly, in developing stems of cvp1 and leaves of cvp2, the pattern of vascular differentiation is defective, but the maturation of individual vascular cells appears to be normal. There are no discernible alterations in cell morphology in cvp2 mutants. In contrast, cvp1 mutants are defective in directional orientation of the provascular strand, resulting in a failure to establish uniformly aligned vascular cells, and they also show a reduction in vascular cell elongation. Neither cvp1 nor cvp2 mutants displayed altered auxin perception, biosynthesis, or transport, suggesting that auxin metabolism is not generally affected in these mutants.     

PLGA/hydrogel biopapers as a stackable substrate for printing HUVEC networks via BioLP

Two major challenges in tissue engineering are mimicking the native cell-cell arrangements of tissues and maintaining viability of three-dimension (3D) tissues thicker than 300 μm. Cell printing and prevascularization of engineered tissues are promising approaches to meet these challenges. However, the printing technologies used in biofabrication must balance the competing parameters of resolution, speed, and volume, which limit the resolution of thicker 3D structures. We suggest that high-resolution conformal printing techniques can be used to print 2D patterns of vascular cells onto biopaper substrates which can then be stacked to form a thicker tissue construct. Towards this end we created 1 cm × 1 cm × 300 μm biopapers to be used as the transferable, stackable substrate for cell printing. 3.6% w/v poly-lactide-co-glycolide was dissolved in chloroform and poured into molds filled with NaCl crystals. The salt was removed with DI water and the scaffolds were dried and loaded with a Collagen Type I or Matrigel. SEM of the biopapers showed extensive porosity and gel loading throughout. Biological laser printing (BioLP) was used to deposit human umbilical vein endothelial cells (HUVEC) in a simple intersecting pattern to the surface of the biopapers. The cells differentiated and stretched to form networks preserving the printed pattern. In a separate experiment to demonstrate "stackability," individual biopapers were randomly seeded with HUVECs and cultured for 1 day. The mechanically stable and viable biopapers were then stacked and cultured for 4 days. Three-dimensional confocal microscopy showed cell infiltration and survival in the compound multilayer constructs. These results demonstrate the feasibility of stackable "biopapers" as a scaffold to build 3D vascularized tissues with a 2D cell-printing technique.     

Effect of Vascular Formed Endothelial Cell Network on the Invasive Capacity of Melanoma Using the In Vitro 3D Co-Culture Patterning Model

In vitro three dimensional (3D) cancer models were developed to observe the invasive capacity of melanoma cell spheroids co-cultured with the vascular-formed endothelial cell network. An array-like multicellular pattern of mouse melanoma cell line B16F1 was developed by magnetic cell labeling using a pin-holder device for allocation of magnetic force. When the B16F1 patterned together with a vascular network of human umbilical vein epithelial cells (HUVEC), spreading and progression were observed along the HUVEC network. The B16F1 cells over 80 µm distance from HUVEC remain in a compact spheroid shape, while B16F1 in the proximity of HUVEC aggressively changed their morphology and migrated. The mRNA expression levels of IL-6, MDR-1 and MMP-9 in B16F1 increased along with the distance the HUVEC network, and these expressions were increased by 5, 3 and 2-fold in the B16F1 close to HUVEC (within 80 µm distance) as compared to that far from HUVEC (over 80 µm distance). Our results clearly show that malignancy of tumor cells is enhanced in proximity to vascular endothelial cells and leads to intravasation.     

Mono-(2-Ethylhexyl) Phthalate Induces Injury in Human Umbilical Vein Endothelial Cells

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a widespread environmental contaminant and has been proved to have potential adverse effects on the reproductive system, carcinogenicity, liver, kidney and developmental toxicities. However, the effect of MEHP on vascular system remains unclear. The main purpose of this study was to evaluate the cytotoxic effects of MEHP on human umbilical endothelial cells (HUVEC) and its possible molecular mechanism. HUVEC cells were treated with MEHP (0, 6.25, 12.5, 25,50 and 100 µM), and the cellular apoptosis and mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In present study, MEHP induced a dose-dependent cell injury in HUVEC cell via an apoptosis pathway as characterized by increased percentage of sub-G1, activation of caspase-3, -8and -9, and increased ratio of Bax/bcl-2 mRNA and protein expression as well as cytochrome C releasing. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N-Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, could effectively block MEHP-induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicated that MEHP induced apoptosis in HUVEC cells through a reactive oxygen species-mediated mitochondria-dependent pathway.     

Pattern formation during vasculogenesis

Vasculogenesis, the assembly of the first vascular network, is an intriguing developmental process that yields the first functional organ system of the embryo. In addition to being a fundamental part of embryonic development, vasculogenic processes also have medical importance. To explain the organizational principles behind vascular patterning, we must understand how morphogenesis of tissue level structures can be controlled through cell behavior patterns that, in turn, are determined by biochemical signal transduction processes. Mathematical analyses and computer simulations can help conceptualize how to bridge organizational levels and thus help in evaluating hypotheses regarding the formation of vascular networks. Here, we discuss the ideas that have been proposed to explain the formation of the first vascular pattern: cell motility guided by extracellular matrix alignment (contact guidance), chemotaxis guided by paracrine and autocrine morphogens, and sprouting guided by cell–cell contacts. Birth Defects Research (Part C) 96:153–162, 2012. © 2012 Wiley Periodicals, Inc.     

Engineering systems for the generation of patterned co-cultures for controlling cell–cell interactions

Background

Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell–cell interactions with microscale resolution.

Scope of the review

We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell–cell interactions in the resulting biological function of the tissues.

Major conclusions

Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell–cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell–cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell–matrix interactions.

General significance

Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Cell Proliferation and Oxygen Diffusion in a Vascularising Scaffold

The supply of oxygen to proliferating cells within a scaffold is a key factor for the successful building of new tissue in soft tissue engineering applications. A recent in vivo model, where an arteriovenous loop is placed in a scaffold, allows a vascularising network to form within a scaffold, establishing an oxygen source within, rather than external, to the scaffold. A one-dimensional model of oxygen concentration, cell proliferation and cell migration inside such a vascularising scaffold is developed and investigated. In addition, a vascularisation model is presented, which supports a vascularisation front which moves at a constant speed. The effects of vascular growth, homogenous and heterogenous seeding, diffusion of cells and critical hypoxic oxygen concentration are considered. For homogenous seeding, a relationship between the speed of the vascular front and a parameter defining the rate of oxygen diffusion relative to the rate of oxygen consumption determines whether a hypoxic region exists at some time. In particular, an estimate of the length of time that a fixed point in the scaffold will remain under hypoxic conditions is determined. For heterogenous seeding, a Fisher-like travelling wave of cells is established behind the vascular front. These findings provide a fundamental understanding of the important interplay between the parameters and allows for a theoretical assessment of a seeding strategy in a vascularising scaffold.