M. Adam

Compressed collagen gel: a novel scaffold for human bladder cells

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo‐tissue level. This controlled, cell‐independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell‐seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation. Copyright

Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials

Synthetic biomaterials play an important role in regenerative medicine. To be effective they must support cell attachment and proliferation in addition to being non-toxic and non-immunogenic. We used a suspension-adapted Chinese hamster ovary-derived cell line expressing green fluorescent protein (GFP) to assess cell attachment and growth on synthetic biomaterials by direct measurement of GFP-specific fluorescence. To simplify operations, all cell cultivation steps were performed in orbitally-shaken, disposable containers. Comparative studies between this GFP assay and previously established cell quantification assays demonstrated that this novel approach is suitable for rapid screening of a large number of samples. Furthermore the utility of our assay system was confirmed by evaluation of cell growth on three polyvinylidene fluoride polymer scaffolds that differed in pore diameter and drawing conditions. The data presented here prove the general utility of GFP-expressing cell lines and orbital shaking technology for the screening of biomaterials for tissue engineering applications.

Lentiviral Vectors for Rapid and Efficient Recombinant CHO Cell Line Generation

Recombinant cell line generation by classical transfection techniques followed by genetic selection is a time-consuming process often leading to an unpredictable outcome as transgene integration is a rare, random event. In contrast, lentiviral vectors deliver transgenes into transcriptionally active regions of the target chromatin, favoring stable protein expression. Here we report proof-of-principle experiments for successful generation of recombinant stable cell lines without the use of chemical selection by lentiviral vector-based transduction of CHO cells. All steps from infection to the recovery of clonal cell lines were performed in serum-free suspension cultures. Green fluorescent protein (GFP) was used both as a model protein for expression and a tool for selection. Pools of transduced cells were periodically re-sorted by FACS and analyzed by flow cytometry for up to 12 weeks. 14 days after lentiviral infection clonal cell lines were isolated by single-cell sorting. The GFP-specific fluorescence remained stable over 3 months. Our results demonstrated the feasibility of a rapid recombinant cell line generation in the absence of chemical selective agents using lentiviral vectors.

GFP-Expressing Bladder Fibroblasts for Applications in Tissue Engineering

The availability of green fluorescent protein (GFP)-expressing primary cells is of great interest for the monitoring of cell growth on and within three-dimensional (3D) scaffolds for tissue engineering applications. We used piggyBac (PB)-transposon-mediated nucleofection for gene delivery to generate primary bladder fibroblasts that stably express the GFP gene. Alternatively, cells were transduced with a lentivirus vector carrying the GFP gene. Homogenous GFP-positive cell populations were obtained by cell sorting (lentivirus transduction) or by puromycin selection (PB-mediated gene delivery). Both methods resulted in stable GFP-positive cell pools. However, instability of GFP expression was observed in some pools originating from nucleofection. The successful generation of GFP-expressing human bladder fibroblasts allowed us to monitor cell growth on poly(lactic acid-ɛ-caprolactone) scaffolds using GFP-specific fluorescence as a surrogate marker for cell number.

Towards the Use of CHO Produced Recombinant Extracellular Matrix Proteins as Bioactive Elements in a 3-D Scaffold for Tissue Engineering

The goal of tissue engineering is to regenerate functional tissue to replace injured or diseased tissue using a combination of patient’s cells and a physical structure that supports cell attachment and proliferation (scaffold). The goal of this project is to create a new bioactive scaffold which mimics the natural extracellular matrix (ECM). The new scaffold will consist of a synthetic polymer coated with a layer of recombinant ECM proteins produced by CHO cells thus conferring cell recognition signals to the polymer. In a first step, we have established a powerful system to screen a variety of polymers for their ability to allow cell attachment and to promote cell growth using a stable GFP-expressing CHO cell line. This system allows rapid comparisons of polymer chemistries and physical parameters (porosity, weaving structure, etc.) and allows choosing the best performing polymers by simple fluorescence measurement. Subsequently, we cloned genes encoding two main ECM proteins (collagen I and tropoelastin) into mammalian expression vectors. We showed that recombinant elastin and collagen are both expressed and secreted by CHO DG44 cells. In a next step, CHO cells genetically modified to overproduce recombinant ECM proteins will be seeded and grown on synthetic polymers. It is expected that the secreted recombinant ECM proteins will adhere to the scaffold. After removal of the genetically modified CHO cells, the new bioactive scaffold can be seeded with patient’s cells to regenerate tissue.

GFP expressing cell line as screening tool for biomaterials

Introduction: For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. The applicability of poly(trimethylene carbonate) (PTMC), random copolymers of TMC and e-caprolactone poly (TMC-CL), and networks based on these polymers as scaffolding materials was investigated. Methods: TMC-based (co)polymers were synthesized by ringopening polymerization. Tubular structures were prepared by dipping glass mandrels in polymer solutions containing dispersed, sieved sugar particles, followed by g-irradiation and cross-linking, and leaching. For mechanical- and biocompatibility tests, films of different thicknesses were prepared by compression molding, solvent casting, and spin-coating. Results and Discussion: PTMC and poly(TMC-CL) are flexible materials, with E-modulus values below 10 MPa and elongations at break higher than 500%. After g-irradiation in vacuo at 25– 100 kGy, networks with gel contents up to 73 wt% were obtained. The networks showed excellent creep resistance under static and dynamic loading conditions. Good cell attachment and proliferation behavior of mesenchymal stem cells, endothelial cells, and smooth muscle cells on polymer films and networks was observed. In lipase solutions, the films degraded substantially within one month by surface erosion. Porous tubular structures, with pore sizes in the range of 80 – 130 mm and a porosity of approximately 85%, could readily be prepared. A pulsatile bioreactor that allows mechanical stimulation of smooth muscle cells and endothelial cells seeded in the porous structures is being constructed. Conclusions: TMC-based (co)polymers and networks are flexible, elastic, biocompatible, and biodegradable. Porous tubular scaffolds based on these materials have much potential in tissue engineering of small diameter blood vessels.Our bodies are constantly exposed to different sorts of mechanical forces, from muscle tension to wound healing. Connective tissue adapts its extracellular matrix (ECM) to changes in mechanical load and the influence of mechanical stimulation on fibroblasts has been studied for a long time [1, 2]. When exposed to forces, fibroblasts are known to respond with expression and remodeling of ECM proteins, in particular collagen type I [3]. In this study the effect of dynamic culture conditions on human dermal fibroblasts was evaluated in terms of deposition and remodeling of ECM, with the aim of producing an ECM based scaffold. The fibroblasts were grown on compliant polymer supports either in a bioreactor with a pulsating flow or under static conditions. By applying dynamic culture conditions, the collagen deposition on the polymer supports increased fivefold. Scanning electron microscopy showed that polymer fibers were well integrated with cells and ECM and alignment along the polymer fibers was observed. Scaffold design should aim at creating structures that can help guiding the cells to form new, functional tissue. The presented system may present a new way of producing designed extracellular matrix based scaffolds for tissue engineering.Synthesis and surface activation of synthetic biodegradable polymers as support for cell produced ECMWe have previously demonstrated that porous poly-(epsiloncalprolactone) films with regularly spaced, controlled pore sizes provide adhesion and support for cultured dermal fibroblasts. We have determined the effects of applying various sized porous films (n¼3 for each treatment) on 4mm punch biopsy wounded mice to assess wounding response. Films with pores ranging in size from 3–20 microns, elicited a mild lymphocytic and foreign body perifollicular immune response, regardless of pore size but this treatment failed to significantly shorten wound healing time or increase the rate of wound closure. By 21 days after wounding the grafted porous films had become fully incorporated into or completely biodegraded in the wounded tissue. Finally, we assessed the proof of principle that live cultured fibroblasts can be delivered using porous films and sustained in model SCID mouse wounds. Human fibroblasts (30,000 cells) were subconfluently cultured on 5 micron porous films. These cell/film combinations were then transplanted onto wounded mice but failed to significantly affect wound healing. However, these transplanted fibroblast cells were readily detected using anti-human HLA antibodies in wounded SCID mice skin 21 days after treatment, when the wounds had completely healed. Taken together, these data demonstrate for the first time the feasibility of using porous films to deliver living human cells into skin wounds as part of our aim to use cell therapy to improve the wound healing response.The aim of this work is to develop an artificial artery for use in bypass surgery. The hybrid artery consists of a porous tubular scaffold made from a polyurethane elastomer. The surface of the polymer is then modified with recombinant proteins in order to encourage the growth of organised layers of vascular cells.