Biotechnology / Bioengineering / Biomaterials / Cell Culture Technology

Research Leader: Pauline Doran

Current Research Projects

Biological production of semiconductor quantum dot nanoparticles using cell cultures

Heavy metals such as cadmium are highly toxic to cells. Some plant and microbial cells detoxify Cd by complexing it with sulphur; under certain conditions CdS nanocrystals are formed. Plants also bind Cd with phytochelatin peptides, which can stabilise the CdS nanocrystal and serve as a complexing agent for further biological functionalisation. An important property of CdS nanocrystals of size 2–20 nanometres is that they behave as semiconductor quantum dots due to quantum confinement effects. Semiconductor nanoparticles have unique electronic and optical properties that depend directly on particle size, and many applications of quantum dots in medical imaging, diagnostics, therapy and microelectronics are being developed. This project involves investigation of plant and microbial cell cultures for biological synthesis of CdS quantum dots, including studying the effect of culture conditions and particle recovery methods on nanocrystal yield and quality.

Calcium oxalate crystals in plant tissue cultures

Calcium oxalate crystals are present in the tissues and organs of many higher plants and are the most prevalent and widely distributed form of biological mineral deposit. Plant-generated crystals have very specific and controlled morphologies that may be different from those of calcium oxalate crystals produced using chemical methods. For example, some plants produce needle-shaped calcium oxalate crystals and this shape isunique to plants. Crystallisation in plant tissues occurs under biochemical and genetic control: different plant species display specific anatomical, morphological and developmental patterns of crystal accumulation. As well as performing important biological functions in plants, oxalate crystals with specific shapes containing transition and heavy metals are also of interest for development of novel biomaterials such as molecular magnets, and for environmental remediation of polluted soils and water. In this project, plant tissue cultures such as hairy roots and suspended cells of several plant species will be investigated for calcium oxalate crystal production. The effect on crystal shape and structure of doping the crystals with metal atoms will be examined to determine whether plant-produced or plant-inspired calcium oxalate crystals display useful properties for industrial applications.

Previous Research Projects

• Bioreactor Culture of Plant Cells and Organs
• Products From Plant Cell and Organ Cultures (including secondary metabolites and foreign proteins)
• Phytoremediation and Phytomining
• Tissue Engineering

Bioreactor Culture of Plant Cells and Organs

In this series of projects, the biochemical engineering aspects of large-scale reactor processes for culture of suspended plant cells and organs such as hairy roots were investigated.

Bioreactor functions such as mixing and oxygen transfer have an important influence on the growth and productivity of plant cells. However, adequate mixing and mass transfer can be difficult to achieve, especially in high-density organ cultures. The tendency of hairy roots to grow in thick, tangled clumps and the presence of prolific root hairs on the tissue surfaces are significant hindrances to liquid–solid oxygen transfer. In this work, several novel bioreactor designs were developed to facilitate oxygen transfer in hairy root cultures.		High-density hairy root culture in a column bioreactor
Adequate mixing can also be difficult to achieve in suspended plant cell cultures. The cells are shear sensitive and the culture broth is usually viscous and non-Newtonian. The ability of plant cells to form macroscopic clumps creates additional intra-particle barriers to oxygen transfer. Theoretical engineering analysis of mixing, mass transfer, shear damage and oxygen requirements in plant cell bioreactors was used to develop new strategies to optimise culture performance.		Suspended plant cell aggregates in an air-driven bioreactor

Wild-type Arabidopsis thaliana hairy roots

Transgenic Arabidopsis thaliana hairy roots without root hairs

Mutant Arabidopsis thaliana hairy roots with short root hairs

Mutant Arabidopsis thaliana hairy roots with excessive root hairs

Products From Plant Cell and Organ Cultures

Synthesis of several different types of product was studied using hairy root, shooty teratoma and suspended plant cell cultures. Much of this previous research focused on plant secondary metabolites, such as solasodine, hyoscyamine, scopolamine, codeine, morphine and podophyllotoxin. As part of this work, systems for cross-species co-culture of plant cells and/or organs were developed for improved synthesis of secondary products.

Western blot of hairy root extracts showing accumulation of complete, assembled IgG1 antibody at 150 kDa, as well as several other antibody fragments.

We have also studied the production of multimeric foreign proteins in plant culture systems. This work includes an analysis of IgG1 antibody synthesis, assembly, secretion and fragmentation in tobacco cell suspensions, hairy roots and shooty teratomas. Plant cell and organ cultures have also been applied for in vitro propagation of plant virus and for production of foreign protein using genetically modified plant-virus-based vectors.

Single-vessel co-culture of hairy roots and shooty teratomas for improved production of scopolamine

Phytoremediation and Phytomining

Plants can be used to remove heavy metals from polluted soils and waterways. ‘Hyperaccumulator’ species are of particular interest for phytoremediation, as they are capable of taking up and storing high concentrations of heavy metals without experiencing toxic effects. The biochemical and physiological mechanisms of hyperaccumulation and the strategies used by hyperaccumulators to tolerate high metal concentrations are not fully understood.

The mechanisms of Cd and Ni hyperaccumulation were studied using hairy roots of two hyperaccumulator species, Thlaspi caerulescens and Alyssum bertolonii. In both cases, hairy roots are capable of hyperaccumulation in the absence of metal translocation to the leaves. This research showed that hyperaccumulator roots possess superior antioxidative defences compared with roots of non-hyperaccumulator plants such as tobacco.

Hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens, in shake-flask culture

Metallic Ni ‘bio-ore’ after furnace treatment of Ni-hyperaccumulator plant biomass

In phytomining, crops of hyperaccumulator plants are used to extract metals from low-grade surface ores or mineralised soils that are too metal-poor for conventional mining. After the crop is harvested and dried, the biomass is treated for commercial metal recovery. The overall outcome is a saleable metal product and land that is more suitable for agriculture than before phytomining operations.

An essential step in phytomining is metal recovery from the plant biomass. Development of new technology in this area is required. Methods for furnace treatment of Ni-hyperaccumulator biomass to produce a Ni-rich ‘bio-ore’ were investigated in collaboration with A/Prof Veena Sahajwalla at the School of Materials Science and Engineering, UNSW.

Tissue Engineering

In this series of projects, human chondrocyte cells, human adult adipose-derived stem cells, biodegradable polymer scaffolds and bioreactor culture were used to produce cartilage tissue in vitro. The aim was to generate cartilage constructs with properties as close as possible to those of human articular cartilage. Tissue-engineered cartilage has many applications in medicine and research. Cartilage constructs produced in the laboratory may be surgically implanted to treat patients with joint injuries or degenerative diseases such as arthritis. Cartilage tissues also have uses in toxicity testing, and for production of cartilage-derived growth factors.

Left: unseeded polymer scaffold Centre and right: Human cartilage constructs produced in a bioreactor