Christine Franke1, Volker Nock1, Maan Alkaisi1
1Electrical and Computer Engineering, University of Canterbury
Cells are able to sense forces and mechanical properties of their microenvironment and can react to those by changing their morphology, protein levels or even applying a cellular force themselves. These cellular reactions can be particularly important in terms of cancer development and metastasis of cancer cells as the tumour microenvironment differs significantly to a healthy tissue.
We developed a 3D polymeric grid to investigate cellular responses to changes in microenvironment properties like stiffness, shape and extracellular matrix proteins. By using optical lithography and inductively coupled plasma etching silicon moulds can be fabricated. The silicon moulds are used for microcontact printing, allowing a precise and effective transfer of protein patterns from a PDMS substrate to a polyacrylamide gel during polymerisation. In combination with the nanoimprint of the 3D structure of the mould, this leads to a hydrogel with defined wells which are protein covered in the lower areas.
Ovarian cancer cells (SKOV3) cultured on these polyacrylamide gels attach solely to the protein (collagen) covered areas of the wells because of the non-adhesive properties of polyacrylamide. Cells trapped into the defined pattern of the wells are able to deform the side walls of the soft hydrogel (1 kPa - 100 kPa). These displacement of the side walls are measurable by analysing series of bright field images of the cell and permit conclusions to be drawn about the lateral cellular forces applied.
This experimental setup allows changing of the stiffness of the microenvironment as well as the size, shape and protein covering of the wells independently within the same system. It enables investigation of which parameter primary influences cellular responses and if and how reactions to one parameter can be overwritten by another one.
Sevgi Onal1, Maan Alkaisi1, Volker Nock1
1Electrical and Computer Engineering, University of Canterbury
Evidence continues to emerge that cancer is not only a disease of genetic mutations, but also of altered mechanobiological profiles of the cells and microenvironment. This mutation-independent element might be a key factor in promoting development and spread of cancer. Biomechanical forces regulate tumor microenvironment by solid stress, matrix mechanics, interstitial pressure and flow. Compressive stress by tumor growth and stromal tissue alters the cell deformation, and recapitulates the biophysical properties of cells to grow, differentiate, spread or invade. Such a solid stress can be introduced externally to change the cell response and to mechanically induce cell lysis by dynamic compression. In this work we report a microfluidic cell-culture platform with an integrated, actively-modulated actuator for the application of compressive forces on cancer cells. Our platform is composed of a control microchannel in a top layer for introducing external force and a polydimethylsiloxane (PDMS) membrane with monolithically integrated actuators. The integrated actuator, herein called micro-piston, was used to apply compression on SKOV-3 ovarian cancer cells in a dynamic and controlled manner by modulating applied gas pressure, localization, shape and size of the micro-piston. We report fabrication of the platform, as well as characterization of the mechanical actuator and cell loading. We further show use of the actuator to perform both, repeated dynamic compression at physiological pressure levels, and end-point mechanical cell lysis, demonstrating suitability for mechanical stimulation to study the role of compressive forces in cancer microenvironments.
Axel Norberg1, Gabriella Lindberg1, Khoon Lim1,Tim Woodfield1
1Department of Orthopaedic Surgery & Musculoskeletal Medicine, University of Otago
Cell based treatments for regeneration of damaged tissues which rely on biomaterials as delivery vehicles still lack the results to justify their cost. One key bottleneck for regenerating tissues of clinically relevant size is oxygen deprivation and many approaches do not consider the profound effect oxygen levels have on cellular functions. In this study, we aim to incorporate hemoglobin(Hb) as a tailorable oxygen deposit within biomaterials through linkage to the growth factor binding protein heparin. We systematically study the effect of Hb-source and heparin concentration on the Hb incorporation efficiency, retention, cytotoxicity and oxygen carrying capacity.
The oxygen binding capacity of commercial Hb(Sigma-Aldrich) and Hb isolated from human whole blood(series of centrifugation/washing steps and lysing of red blood cells via osmotic pressure in MQ-water) was characterized using a spectrophotometer(VarioSkanTM) and an optical oxygen sensor(PreSensTM). Cytotoxicity of materials was evaluated using human chondrocytes in accordance with ISO 10993-5(Alamarblue®). Allylated gelatin(GelAGE) and thiolated heparin(HepSH) were synthesized and cast into disks(h=2mm, Ø=5.5mm) which were photopolymerized (450nm, 3minutes, 30W/cm2, 1/10 mM Ru/SPS, 0-1.4 wt% HepSH, 20wt% GelAGE, 60mM dithiolthreitol) into 3D-constructs followed by compression tests. The constructs were subsequently soaked in 50mg/ml Hb solution for 48 hours and incorporation efficiency was analysed using a spectrophotometer.
Results revealed that commercial Hb rapidly oxidises to met-Hb (96% met-Hb/4% oxy-Hb) while isolated Hb retains its oxygen binding capabilities (7 % met-Hb/93% oxy-Hb) significantly upregulating oxygen also at lower concentrations (10mg/ml). All materials were cytocompatible and could be fabricated into rigid 3D-hydrogels. Results further demonstrated a dose dependent relationship between HepSH concentration, Hb incorporation (0-0.5wt%HepSH=5mg/ml, 1.4wt%HepSH=9mg/ml) and mechanical stiffness (0-0.5 wt%HepSH=40kPa, 1.4wt%HepSH=70kPa). This method of incorporating oxygen carrying Hb into heparin-infused biomaterials provides a novel oxygen regulating system for the development of next generation cell based treatments.
Debolina Sarkar1, Ashley Garrill1, Volker Nock2
1Biological Sciences, University of Canterbury
2Electrical and Computer Engineering, University of Canterbury
The oomycete Phytophthora can cause serious threats to native flora and agriculture and food biosecurity, causing devastating diseases to plants such as Kauri, oak, avocado and soy. In New Zealand, Phytophthora agathidicida and Phytophthora cinnamomi are the causative agent in kauri die-back and is receiving much coverage in the mainstream press as it can kill a mature kauri tree within two years. These organisms can infect their host plants via motile zoospores. They can travel between sites of infection to the healthy trees via water in the soil. The zoospores can detect the electric and the chemical signals released by the plant root surface. They can also discriminate between the growing tips of the roots and the wounded surface on the roots from rest of the root structure, depending on the difference in electrified. This helps them to find the specific target area and initiate the infection.
My research will focus on mimicking the natural environment around the roots of the plant using microfluidic Lab-on-a-Chip (LOC) devices to try to investigate the electrical parameters combined with the chemical, that allow zoospores to infect plant roots. Plant roots can generate electric fields which may attract zoospores. Different LOC devices have been designed that contain anodes and cathodes, that enable the study of the swimming behavior of Phytophthora zoospores in the presence of electric fields. Preliminary data indicates that zoospores show a tendency to aggregate close to the cathode. This device will also allow us to study the signal transmitted from one zoospore the other to intensify the infectious effect. In the future this research may allow us to design devices that modify electric fields around vulnerable plants and attract zoospores away from roots and thus inhibit their infective capability.