Expansion and chondrogenic potential of human articular chondrocytes on macroporous microcarriers

Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro. High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed. Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage.

A brief history of haemotopoietic stem cell Ex vivo expansion

Haematopoiesis is the process by which a hierarchy of mature and progenitor blood cells are formed. These cell populations are all derived from multipotent haematopoietic stem cells (HSC), which reside in the bone marrow ‘niche’ of adult humans. Over the lifetime of a healthy individual, this HSC population replenishes between 1010-1011 blood cells on a daily basis. Dysregulation of this system can lead to a number of haematopoietic diseases, including aplastic anaemias and leukaemias, which result in, or require for disease resolution, bone marrow cell depletion. In 1956, E. Donnall Thomas demonstrated that haematopoiesis could be restored by transplanting bone marrow-derived cells from one man into his identical twin brother, who was suffering from advanced leukaemia. His success drew significant interest in academic research and medicine communities, and 12 years later, the first successful allogeneic transplant was performed. To this day, HSCs remain the most studied and characterised stem cell population. In fact, HSCs are the only stem cell population routinely utilised in the clinic. As such, HSCs function as a model system both for the biological investigation of stem cells, as well as for their clinical application. Herein, we briefly review HSC transplantation, strategies for the ex vivo cultivation of HSCs, recent clinical outcomes, and their impact on the future direction of HSC transplantation therapy.

Fabrication of a corneal-limbal tissue substitute using silk fibroin

Fibroin extracted from silkworm cocoon silk provides an intriguing and potentially important biomaterial for corneal reconstruction. In the present chapter we outline our methods for producing a composite of two fibroin-based materials that supports the co-cultivation of human limbal epithelial (HLE) cells and human limbal stromal (HLS) cells. The resulting tissue substitute consists of a stratified epithelium overlying a three-dimensional arrangement of extracellular matrix components (principally ‘degummed’ fibroin fibers) and mesenchymal stromal cells. This tissue substitute is currently being evaluated as a tool for reconstructing the corneal limbus and corneal epithelium.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Impact of a change in tillage and crop residue management practice on soil chemical and microbiological properties in a cereal-producing red duplex soil in NSW, Australia

The effect of a change of tillage and crop residue management practice on the chemical and micro-biological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0-5 cm of soil, in organic C, total N, electrical conductivity, NH4-N, NO3-N, soil moisture holding capacity, microbial biomass and CO2 respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0-5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.

Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells

The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

Dewatering of microalgal cultures : a major bottleneck to algae-based fuels

Microalgae dewatering is a major obstruction to industrial-scale processing of microalgae for biofuel prodn. The dil. nature of harvested microalgal cultures creates a huge operational cost during dewatering, thereby, rendering algae-based fuels less economically attractive. Currently there is no superior method of dewatering microalgae. A technique that may result in a greater algal biomass may have drawbacks such as a high capital cost or high energy consumption. The choice of which harvesting technique to apply will depend on the species of microalgae and the final product desired. Algal properties such as a large cell size and the capability of the microalgae to autoflocculate can simplify the dewatering process. This article reviews and addresses the various technologies currently used for dewatering microalgal cultures along with a comparative study of the performances of the different technologies.

Microalgal biomass as a fermentation feedstock for bioethanol production

BACKGROUND The increasing cost of fossil fuels as well as the escalating social and industrial awareness of the environmental impacts associated with the use of fossil fuels has created the need for more sustainable fuel options. Bioethanol, produced from renewable biomass such as sugar and starch materials, is believed to be one of these options, and it is currently being harnessed extensively. However, the utilization of sugar and starch materials as feedstocks for bioethanol production creates a major competition with the food market in terms of land for cultivation, and this makes bioethanol from these sources economically less attractive. RESULT This study explores the suitability of microalgae (Chlorococum sp.) as a substrate for bioethanol production via yeast (Saccharomycesbayanus)under different fermentation conditions. Results show a maximum ethanol concentration of 3.83 g L -1 obtained from 10 g L-1 of lipid-extracted microalgae debris. CONCLUSION This productivity level (∼38% w/w), which is in keeping with that of current production systems endorses microalgae as a promising substrate for bioethanol production.

Microalgal growth characteristics and subsequent influence on dewatering efficiency

Dewatering of microalgal culture is a major bottleneck towards the industrial-scale processing of microalgae for bio-diesel production. The dilute nature of harvested microalgal cultures poses a huge operation cost to dewater; thereby rendering microalgae-based fuels less economically attractive. This study explores the influence of microalgal growth phases and intercellular interactions during cultivation on dewatering efficiency of microalgae cultures. Experimental results show that microalgal cultures harvested during a low growth rate phase (LGRP) of 0.03 d-1 allowed a higher rate of settling than those harvested during a high growth rate phase (HGRP) of 0.11 d-1, even though the latter displayed a higher average differential biomass concentration of 0.2 g L-1 d-1. Zeta potential profile during the cultivation process showed a maximum electronegative value of -43.2 ± 0.7 mV during the HGRP which declined to stabilization at -34.5 ± 0.4 mV in the LGRP. The lower settling rate observed for HGRP microalgae is hence attributed to the high stability of the microalgal cells which electrostatically repel each other during this growth phase. Tangential flow filtration of 20 L HGRP culture concentrated 23 times by consuming 0.51 kWh/m3 of supernatant removed whilst 0.38 kWh/m3 was consumed to concentrate 20 L of LGRP by 48 times.

Dewatering of microalgal culture for biodiesel production : exploring polymer flocculation and tangential flow filtration

Background: Conventional biodiesel production relies on trans-esterification of lipids extracted from vegetable crops. However, the use of valuable vegetable food stocks as raw material for biodiesel production makes it an unfeasibly expensive process. Used cooking oil is a finite resource and requires extra downstream processing, which affects the amount of biodiesel that can be produced and the economics of the process. Lipids extracted from microalgae are considered an alternative raw material for biodiesel production. This is primarily due to the fast growth rate of these species in a simple aquaculture environment. However, the dilute nature of microalgae culture puts a huge economic burden on the dewatering process especially on an industrial scale. This current study explores the performance and economic viability of chemical flocculation and tangential flow filtration (TFF) for the dewatering of Tetraselmis suecicamicroalgae culture. Results: Results show that TFF concentrates the microalgae feedstock up to 148 times by consuming 2.06 kWh m-3 of energy while flocculation consumes 14.81 kWhm-3 to concentrate the microalgae up to 357 times. Economic evaluation demonstrates that even though TFF has higher initial capital investment than polymer flocculation, the payback period for TFF at the upper extreme ofmicroalgae revenue is ∼1.5 years while that of flocculation is ∼3 years. Conclusion: These results illustrate that improved dewatering levels can be achieved more economically by employing TFF. The performances of these two techniques are also compared with other dewatering techniques.

Evaluation of oil production from oil palm empty fruit bunch by oleaginous micro-organisms

Oil palm empty fruit bunch (EFB) is a readily available, lignocellulosic biomass that has potential to be utilized as a carbon substrate for microbial oil production. In order to evaluate the production of microbial oil from EFB, a technical study was performed through the cultivation of oleaginous micro-organisms (Rhodotorula mucilaginosa, Aspergillus oryzae, and Mucor plumbeus) on EFB hydrolyzates. EFB hydrolyzates were prepared through dilute acid pre-treatment of the biomass, where the liquid fraction of pre-treatment was detoxified and used as an EFB liquid hydrolyzate (EFBLH). The solid residue was enzymatically hydrolyzed prior to be used as an EFB enzymatic hydrolyzate (EFBEH). The highest oil concentrations were obtained from M. plumbeus (1.9 g/L of oil on EFBLH and 4.7 g/L of oil on EFBEH). In order to evaluate the feasibility of large-scale microbial oil production, a techno-economic study was performed based on the oil yields of M. plumbeus per hectare of plantation, followed by the estimation of the feedstock cost for oil production. Other oil palm biomasses (frond and trunk) were also included in this study, as it could potentially improve the economics of large-scale microbial oil production. Microbial oil from oil palm biomasses was estimated to potentially increase oil production in the palm oil industry up to 25%, at a cheaper feedstock cost. The outcome of this study demonstrates the potential integration of microbial oil production from oil palm biomasses with existing palm oil industry (biodiesel, food and oleochemicals production), that could potentially enhance sustainability and profitability of microbial oil production.

Cultivating teachers’ beliefs, knowledge and skills for leading change in schools

Australian policy initiatives and state curriculum reform efforts affirm a commitment to address student disengagement through the development of inclusive school environments, curriculum, and pedagogy. This paper, drawing on critical social theory, describes three Australian projects that support the cultivation of teachers’ beliefs, knowledge and skills for critical reflection and leading change in schools. The first project reports on the valued ethics that emerged in pre-service teacher reflections about a Service-learning Program at a university in Queensland. The second project reports on a school-based collaborative inquiry approach to professional development with a focus on literacy practices. The final project reports on an initiative in another university in Victoria, to operationalise pedagogical change and curriculum renewal in Victoria, through the Principles of Learning and Teaching (PoLT). These case studies illustrate how critical reflection and development of beliefs, knowledge and skills can be acquired to better meet the needs of schools.

Victoria regia's bequest to modern architecture

This paper will explore the relationship between the giant South American waterlily, the Victoria regia (today named Victoria amazonica), and the 1914 Glashaus exposition building by the German architect, Bruno Taut. Starting with a general botanical introduction of Victoria regia, the paper exposes the first European cultivation of the lily by Joseph Paxton at Chatsworth,England, in 1849. Following this initial cultivation, Paxton subsequently develops a specialist greenhouse for the plant, that later becomes the prototype for all Victoria regia greenhouses. However, from about 1860 as Victoria regia cultivation spreads to continental Europe, a greenhouse that differs from Paxton’s prototype subsequently evolves. An investigation of these later continental European greenhouses, coupled with an exposure of Taut’s own writings concerning Victoria regia, reveals startling similarities to the Glashaus, which ultimately reveals the Glashaus as directly inspired by Victoria regia.

Religious information literacy : using information to learn in church community

What informs members of the church community as they learn? Do the ways people engage with information differ according to the circumstances in which they learn? Informed learning, or the ways in which people use information in the learning experience and the degree to which they are aware of that, has become a focus of contemporary information literacy research. This essay explores the nature of informed learning in the context of the church as a learning community. It is anticipated that insights resulting from this exploration may help church organisations, church leaders and lay people to consider how information can be used to grow faith, develop relationships, manage the church and respond to religious knowledge, which support the pursuit of spiritual wellness and the cultivation of lifelong learning. Information professionals within the church community and the broader information profession are encouraged to foster their awareness of the impact that engagement with information has in the learning experience and in the prioritising of lifelong learning in community contexts.

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63+ progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.