Tim Scharnweber

Rapid prototyping of microstructures in polydimethylsiloxane (PDMS) by direct UV-lithography

Microstructuring of polydimethylsiloxane (PDMS) is a key step for many lab-on-a-chip (LOC) applications. In general, the structure is generated by casting the liquid prepolymer against a master. The production of the master in turn calls for special equipment and know how. Furthermore, a given master only allows the reproduction of the defined structure. We report on a simple, cheap and practical method to produce microstructures in already cured PDMS by direct UV-lithography followed by chemical development. Due to the available options during the lithographic process like multiple exposures, the method offers a high design flexibility granting easy access to complex and stepped structures. Furthermore, no master is needed and the use of pre-cured PDMS allows processing at ambient (light) conditions. Features down to approximately 5 µm and a depth of 10 µm can be realised. As a proof of principle, we demonstrate the feasibility of the process by applying the structures to various established soft lithography techniques.

Laser modulated transmembrane convection: Implementation in cancer chemotherapy

Transmembrane diffusion imposes fundamental limits to the uptake of cytostatic drugs executing their function intracellularly. Here, we report that transmembrane convection-a mechanism exploiting the effect of moderately intense 670nm laser light on the density and viscosity of nanoscopic interfacial water layers (IWL) in the cell-forces cancer cells to uptake high doses of cytostatic drugs in a short time. Transmembrane convection is a viable alternative to established uptake forms (i.e., it works complementary to diffusive processes) and breaks the limits imposed by diffusion. We demonstrate the potency of the method in human cervical cancer cells, HeLa, using the anticancer compounds doxorubicin (DOX), methotrexate (MTX) and epigallocatechin gallate (EGCG). The method is applicable to virtually the entire chemotherapeutic arsenal and is expected to help overcome multidrug resistance in cancer cells.

Extraordinary Anticancer Effect of Green Tea and Red Light

OBJECTIVE Increasing observational evidence suggests that epigallocatechin gallate--the major polyphenolic component of green tea--is instrumental in suppressing the growth of cancer cells. Therefore, methods that promise to enhance the suppressive potential of green tea have the highest clinical relevance. BACKGROUND DATA Human cervical cancer cells, HeLa, the first continuous cancer cell line, represent a mainstay model in cancer research. Green tea inhibited their growth, whereas their exposure to moderate levels of laser light resulted in an opposite effect. Both effects are individually documented in the literature. METHODS HeLa cells were supplemented with green tea, irradiated with moderately intense laser light (670 nm) for 1 min, and incubated for 52 h. RESULTS We found an extraordinary inhibition of HeLa cells by a combination of green tea and red light. We achieved an inhibition of 1,460%, compared with non-irradiated samples. CONCLUSION Our result receives clinical relevance from a recent study in which epigallocatechin gallate suppressed the growth of melanoma in vivo.

On the Social Behaviour of Cells

Polystyrene Petri dishes are in use in hundreds of thousands of laboratories world wide. Cell culture experiments performed in them provide fundamental information in a wide range of applications, including but not limited to testing novel biomaterials and pharmaceuticals, and stem cell research. These experiments cost billions of dollars per year. In this study we report on a potential deficiency of polystyrene Petri dishes, possibly caused by an increase in interfacial pH under relevant culture conditions and affecting cell performance. We conclude that cell performance on Petri dishes could be improved by improving the Petri dishes. As a spin-off of our study we postulate the concept that cancer cells and stem cells are social. It is impossible to validate this concept on the basis of the model established in this paper. However, the coherence of our insights may encourage further study and lead to the development of a qualitative improvement of cell culture devices, including Petri dishes and culture flasks, to the identification of potential strategies for chemotherapy and chemoprevention that could suppress progression of metastasis, and to the establishment of improved settings for tissue engineering and stem cell research. An immediate recommendation of our study is to use chemically and biologically inert substrates for important cell culture experiments, for example, nanocrystalline diamond.

Percutaneous left atrial appendage closure with a novel self-modelizing device: A pre-clinical feasibility study

The aim of the study is to evaluate the feasibility and safety of a new left atrial appendage (LAA) occluder. Twelve pigs were included. In 2 pigs the implantation process failed due to pericardial tamponade in 1 pig and device embolization in the other pig. The placement of the devices was controlled via TEE and fluoroscopy. After 6 weeks of implantation the hearts were explanted. The devices were found to be easy to deploy and showed a very good adaptation to the LAA tissue. Eight out of 10 pigs had full closure of the LAA directly after implantation. After six weeks, due to the self-modelizing properties of the device, all pigs had a full closure of the LAA. The macroscopic evaluation of the explanted hearts showed that all devices were securely integrated in LAA tissues. There was one case of mild pericarditis but no macroscopic signs of inflammation on the device surrounding endocardium. The explantation revealed that device loops had penetrated the LAA tissue in three pigs. However, no signs of bleeding, pericardial effusion, or other damage to the LAA wall could be detected and the pigs were in good condition with normal weight gain and no clinical symptoms. The Occlutech® LAA occluder achieved complete closure of the LAA in all pigs, and remained in the LAA, with benign healing and no evidence of new thrombus or damage to surrounding structures. Moreover, the uncompromised survival of all implanted pigs demonstrates the feasibility and safety of the device.

Body Building on Diamonds

Whereas conservative therapies aim to stall the advance of disease, regenerative medicine strives to reverse it. The capacity of most tissues to regenerate derives from stem cells, but there are a number of barriers which have to be circumvented before it will be possible to use stem-cell-based therapies. Such therapies, however, are expected to improve human health enormously, and knowledge gained from studying stem cells in culture and in model organisms is now laying the groundwork for a new era of regenerative medicine. One of the most prominent methods to study stem cell differentiation is to let them to form embryoid bodies. Under favourable conditions any stem cell line will form embryoid bodies. However, the mechanism of the formation of embryoid bodies is not very well understood, and to produce them in the laboratory is in no way trivial — an important technical barrier in stem cell research. Recently, the embryoid body cultivation step has been successfully circumvented for the derivation of osteogenic cultures of embryonic stem cells. Here we report on a simple and reusable system to cultivate embryoid bodies in extremely short times. The method is inspired by the principles that lead to the establishment of the biomimetic triangle.

Selective immobilization of Sonic hedgehog on benzylguanine terminated patterned self-assembled monolayers.

Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O(6)-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.