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1.
Adv Biol (Weinh) ; 5(4): e2000136, 2021 04.
Article in English | MEDLINE | ID: mdl-33852182

ABSTRACT

Neural differentiation is studied using a simultaneous application of 3D scaffold culture and hydrodynamic and electrical stimuli in purpose-designed recirculation bioreactors operated with continuous fluid flow. Pheochromocytoma (PC12) cells are seeded into nonwoven microfibrous viscose-rayon scaffolds functionalized with poly-l-lysine and laminin. Compared with the results from static control cultures with and without electrical stimulation and bioreactor cultures with the fluid flow without electrical stimulation, expression levels of the differentiation markers ß3-tubulin, shootin1, and ephrin type-A receptor 2 are greatest when cells are cultured in bioreactors with fluid flow combined with in-situ electrical stimulus. Immunocytochemical assessment of neurite development and morphology within the scaffolds confirm the beneficial effects of exposing the cells to concurrent hydrodynamic and electrical treatments. Under the conditions tested, electrical stimulation by itself produces more pronounced levels of cell differentiation than fluid flow alone; however, significant additional improvements in differentiation are achieved by combining these treatments. Fluid flow and electrical stimuli exert independent and noninteractive effects on cellular differentiation, suggesting that interference between the mechanisms of differentiation enhancement by these two treatments is minimal during their simultaneous application. This work demonstrates the beneficial effects of combining several different potent physical environmental stimuli in cell culture systems to promote neurogenesis.


Subject(s)
Bioreactors , Tissue Scaffolds , Cell Differentiation , Electric Stimulation , Neurogenesis
2.
Integr Biol (Camb) ; 13(3): 73-85, 2021 03 17.
Article in English | MEDLINE | ID: mdl-33704437

ABSTRACT

Mixed populations of cardiosphere-derived stem and progenitor cells containing proliferative and cardiomyogenically committed cells were obtained from adult rat hearts. The cells were cultured in either static 2D monolayers or dynamic 3D scaffold systems with fluid flow. Cardiomyocyte lineage commitment in terms of GATA4 and Nkx2.5 expression was significantly enhanced in the dynamic 3D cultures compared with static 2D conditions. Treatment of the cells with 5-azacytidine (5-aza) produced different responses in the two culture systems, as activity of this chemical epigenetic conditioning agent depended on the cell attachment and hydrodynamic conditions provided during culture. Cell growth was unaffected by 5-aza in the static 2D cultures but was significantly reduced under dynamic 3D conditions relative to untreated controls. Myogenic differentiation measured as Mef2c expression was markedly upregulated by 5-aza in the dynamic 3D cultures but downregulated in the static 2D cultures. The ability of the physical environment to modulate the cellular cardiomyogenic response to 5-aza underscores the interactivity of biochemical and physical stimuli applied for cell differentiation. Accordingly, observations about the efficacy of 5-aza as a cardiomyocyte induction agent may not be applicable across different culture systems. Overall, use of dynamic 3D rather than static 2D culture was more beneficial for cardio-specific myogenesis than 5-aza treatment, which generated a more ambiguous differentiation response.


Subject(s)
Myocytes, Cardiac , Stem Cells , Animals , Cell Differentiation , Cells, Cultured , Epigenesis, Genetic , Rats
3.
Biofabrication ; 12(1): 015013, 2019 12 02.
Article in English | MEDLINE | ID: mdl-31600744

ABSTRACT

The ability to spatially organise the microenvironment of tissue scaffolds unlocks the potential of many scaffold-based tissue engineering applications. An example application is to aid the regeneration process of peripheral nerve injuries. Herein, we present a promising approach for three-dimensional (3D) micropatterning of nerve cells in tissue scaffolds for peripheral nerve repair. In particular, we demonstrate the 3D micropatterning of PC12 cells in a gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogel using ultrasound standing waves (USWs). PC12 cells were first aligned in 3D along nodal planes by the USWs in Gtn-HPA hydrogel precursor solution. The precursor was then crosslinked using horseradish peroxidase (HRP) and diluted hydrogen peroxide (H2O2), thus immobilising the aligned cells within 90-120 s. This micropatterning process is cost effective and can be replicated easily without the need for complex and expensive specialised equipment. USW-aligned PC12 cells showed no adverse effect in terms of viability or ability to proliferate. To our best knowledge, this is the first report on the effect of USW alignment on neural cell differentiation. Differentiated and USW-aligned PC12 cells showed directional uniformity after 20 d, making this technique a promising alternative approach to guide the nerve regeneration process.


Subject(s)
Hydrogels/chemistry , Neurons/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Cell Proliferation , Hydrogels/radiation effects , Neurons/chemistry , PC12 Cells , Rats , Tissue Engineering/instrumentation , Ultrasonics
4.
Integr Biol (Camb) ; 11(6): 264-279, 2019 06 01.
Article in English | MEDLINE | ID: mdl-31322680

ABSTRACT

The effect of exogenous electrical stimulation on cell viability, attachment, growth, and neurogenesis was examined using PC12 cells in microfibrous viscose-rayon scaffolds immersed in culture medium. The scaffolds were applied either in their nonconductive state or after coating the fibres with 200 nm of gold to give a scaffold sheet resistivity of (13 ± 1.3) Ω square-1. The cells were treated for 12 days using direct current electrical stimulation of 2 h per day. No cytotoxic effects were observed when up to 500 mV (8.3 mV mm-1) was applied to the scaffolds without gold, or when up to 100 mV (1.7 mV mm-1) was applied to the scaffolds with gold. Compared with unstimulated cells, whereas electrical stimulation significantly enhanced cell growth and attachment in the nonconductive scaffolds without gold, similar effects were not found for the conductive scaffolds with gold. Neural differentiation in the presence of nerve growth factor was improved by electrical stimulation in both scaffolds; however, neurite development and the expression of key differentiation markers were greater in the nonconductive scaffolds without gold than in the scaffolds with gold. Application of the same current to scaffolds with and without gold led to much higher levels of neurogenesis in the scaffolds without gold. This work demonstrates that substantial benefits in terms of cell growth and neural differentiation can be obtained using electric fields exerted across nonconductive microfibrous scaffolds, and that this approach to electrical stimulation can be more effective than when the stimulus is applied to cells on conductive scaffolds.


Subject(s)
Cell Proliferation , Electric Stimulation , Neurogenesis , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials , Cell Cycle , Cell Differentiation , Cell Survival , Cellulose/chemistry , Electric Conductivity , Gold , Ions , Neurites/metabolism , Neurons/cytology , PC12 Cells , Rats , Time Factors
5.
Biotechnol Bioeng ; 114(9): 2142-2148, 2017 09.
Article in English | MEDLINE | ID: mdl-28475237

ABSTRACT

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit+ and Mef2c+ cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc.


Subject(s)
Organ Culture Techniques/instrumentation , Spheroids, Cellular/cytology , Stem Cells/cytology , Tissue Scaffolds , Animals , Cell Differentiation/physiology , Cell Proliferation/physiology , Cell Survival/physiology , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Organ Culture Techniques/methods , Spheroids, Cellular/physiology , Stem Cells/physiology , Zebrafish/anatomy & histology , Zebrafish/physiology
6.
J Biotechnol ; 223: 13-23, 2016 Apr 10.
Article in English | MEDLINE | ID: mdl-26880539

ABSTRACT

This study investigated fission yeast (Schizosaccharomyces pombe) and hairy roots of tomato (Solanum lycopersicum) as in vitro production vehicles for biological synthesis of CdS quantum dots. Cd added during the mid-growth phase of the cultures was detoxified within the biomass into inorganic sulphide-containing complexes with the quantum confinement properties of semiconductor nanocrystals. Significant differences were found between the two host systems in terms of nanoparticle production kinetics, yield and quality. The much slower growth rate of hairy roots compared with yeast is a disadvantage for commercial scaled-up production. Nanoparticle extraction from the biomass was less effective for the roots: 19% of the Cd present in the hairy roots was recovered after extraction compared with 34% for the yeast. The overall yield of CdS quantum dots was also lower for the roots: relative to the amount of Cd taken up into the biomass, 8.5% was recovered in yeast gel filtration fractions exhibiting quantum dot properties whereas the result for hairy roots was only 0.99%. Yeast-produced CdS crystallites were somewhat smaller with diameters of approximately 2-6 nm compared with those of 4-10nm obtained from the roots. The average ratio of inorganic sulphide to Cd for the purified and size-fractionated particles was 0.44 for the yeast and 1.6 for the hairy roots. Despite the limitations associated with hairy roots in terms of culture kinetics and product yield, this system produced CdS nanoparticles with enhanced photostability and 3.7-13-fold higher fluorescence quantum efficiency compared with those generated by yeast. This work demonstrates that the choice of cellular host can have a significant effect on nanoparticle functional properties as well as on the bioprocessing aspects of biological quantum dot synthesis.


Subject(s)
Quantum Dots/metabolism , Schizosaccharomyces/growth & development , Solanum lycopersicum/growth & development , Bioreactors , Cadmium/metabolism , Kinetics , Solanum lycopersicum/metabolism , Particle Size , Plant Roots/metabolism , Quantum Dots/chemistry , Schizosaccharomyces/metabolism , Semiconductors
7.
Methods Mol Biol ; 1340: 3-21, 2015.
Article in English | MEDLINE | ID: mdl-26445827

ABSTRACT

Many technologies that underpin tissue engineering as a research field were developed with the aim of producing functional human cartilage in vitro. Much of our practical experience with three-dimensional cultures, tissue bioreactors, scaffold materials, stem cells, and differentiation protocols was gained using cartilage as a model system. Despite these advances, however, generation of engineered cartilage matrix with the composition, structure, and mechanical properties of mature articular cartilage has not yet been achieved. Currently, the major obstacles to synthesis of clinically useful cartilage constructs are our inability to control differentiation to the extent needed, and the failure of engineered and host tissues to integrate after construct implantation. The aim of this chapter is to distil from the large available body of literature the seminal approaches and experimental techniques developed for cartilage tissue engineering and to identify those specific areas requiring further research effort.


Subject(s)
Cartilage, Articular , Chondrocytes , Regeneration , Regenerative Medicine/methods , Tissue Engineering/methods , Animals , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Cartilage, Articular/physiopathology , Cartilage, Articular/surgery , Cell Culture Techniques , Cell Differentiation , Chondrocytes/metabolism , Chondrocytes/pathology , Chondrocytes/transplantation , Chondrogenesis , Graft Survival , Humans , Stem Cell Transplantation , Stem Cells/metabolism , Stem Cells/pathology , Tissue Culture Techniques , Tissue Scaffolds
8.
Methods Mol Biol ; 1340: 25-40, 2015.
Article in English | MEDLINE | ID: mdl-26445828

ABSTRACT

As the only cell type found in healthy adult cartilage, chondrocytes are the obvious and most direct starting point for cartilage tissue engineering. Human adult, juvenile, neonatal, and fetal chondrocytes have all been demonstrated to produce cartilage matrix components in vitro for production of engineered tissues. In this chapter, procedures are outlined for isolation of chondrocytes from human fetal and adult cartilage. Methods for expansion and cryopreservation of the cells and characterization of gene expression using quantitative polymerase chain reaction (Q-PCR) analysis are also described.


Subject(s)
Adult Stem Cells/physiology , Cell Culture Techniques , Cell Differentiation , Chondrocytes/physiology , Fetal Stem Cells/physiology , Regenerative Medicine/methods , Tissue Engineering/methods , Cell Proliferation , Cell Separation , Cells, Cultured , Chondrogenesis , Cryopreservation , Gene Expression Regulation , Humans , Phenotype , Real-Time Polymerase Chain Reaction
9.
Methods Mol Biol ; 1340: 53-64, 2015.
Article in English | MEDLINE | ID: mdl-26445830

ABSTRACT

Human adult mesenchymal stem cells are present in fat tissue, which can be obtained using surgical procedures such as liposuction. The multilineage capacity of mesenchymal stem cells makes them very valuable for cell-based medical therapies. In this chapter, we describe how to isolate mesenchymal stem cells from human adult fat tissue, propagate the cells in culture, and cryopreserve the cells for tissue engineering applications. Flow cytometry methods are also described for identification and characterization of adipose-derived stem cells and for cell sorting.


Subject(s)
Adipose Tissue/cytology , Adult Stem Cells/physiology , Chondrocytes/physiology , Mesenchymal Stem Cells/physiology , Regenerative Medicine/methods , Tissue Engineering/methods , Adult Stem Cells/metabolism , Adult Stem Cells/transplantation , Biomarkers/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Lineage , Cell Separation , Cells, Cultured , Chondrocytes/metabolism , Chondrocytes/transplantation , Chondrogenesis , Cryopreservation , Flow Cytometry , Humans , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Phenotype , Tissue Scaffolds
10.
Methods Mol Biol ; 1340: 221-33, 2015.
Article in English | MEDLINE | ID: mdl-26445842

ABSTRACT

Mechanical forces, including hydrodynamic shear, hydrostatic pressure, compression, tension, and friction, can have stimulatory effects on cartilage synthesis in tissue engineering systems. Bioreactors capable of exerting forces on cells and tissue constructs within a controlled culture environment are needed to provide appropriate mechanical stimuli. In this chapter, we describe the construction, assembly, and operation of a mechanobioreactor providing simultaneous dynamic shear and compressive loading on developing cartilage tissues to mimic the rolling and squeezing action of articular joints. The device is suitable for studying the effects of mechanical treatment on stem cells and chondrocytes seeded into three-dimensional scaffolds.


Subject(s)
Bioreactors , Cartilage/cytology , Chondrocytes/physiology , Chondrogenesis , Mechanotransduction, Cellular , Regenerative Medicine/instrumentation , Tissue Engineering/instrumentation , Animals , Biomechanical Phenomena , Cartilage/metabolism , Cartilage/transplantation , Cell Culture Techniques , Cells, Cultured , Cellular Microenvironment , Chondrocytes/metabolism , Chondrocytes/transplantation , Equipment Design , Humans , Physical Stimulation , Regeneration , Regenerative Medicine/methods , Stress, Mechanical , Tissue Engineering/methods , Tissue Scaffolds
12.
Adv Healthc Mater ; 3(10): 1655-70, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24711346

ABSTRACT

Compared with preformed anisotropic matrices, an anisotropic matrix that allows users to alter its properties and structure in situ after synthesis offers the important advantage of being able to mimic dynamic in vivo microenvironments, such as in tissues undergoing morphogenesis or in wounds undergoing tissue repair. In this study, porous gradients are generated in situ in a hydrogel comprising enzymatically crosslinked gelatin hydroxyphenylpropionic acid (GTN-HPA) conjugate and carboxylmethyl cellulose tyramine (CMC-TYR) conjugate. The GTN-HPA component acts as the backbone of the hydrogel, while CMC-TYR acts as a biocompatible sacrificial polymer. The hydrogel is then used to immobilize HT1080 human fibrosarcoma cells in a microfluidic chamber. After diffusion of a biocompatible cellulase enzyme through the hydrogel in a spatially controlled manner, selective digestion of the CMC component of the hydrogel by the cellulase gives rise to a porosity gradient in situ instead of requiring its formation during hydrogel synthesis as with other methods. The influence of this in situ tunable porosity gradient on the chemotactic response of cancer cells is subsequently studied both in the absence and presence of chemoattractant. This platform illustrates the potential of hydrogel-based microfluidics to mimic the 3D in vivo microenvironment for tissue engineering and diagnostic applications.


Subject(s)
Cell Culture Techniques/instrumentation , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Microfluidic Analytical Techniques/instrumentation , Tissue Scaffolds/chemistry , Cell Culture Techniques/methods , Cell Line, Tumor , Cell Movement , Cell Survival , Gelatin/chemistry , Humans , Microfluidic Analytical Techniques/methods , Porosity , Propionates/chemistry
13.
Adv Healthc Mater ; 3(5): 725-36, 2014 May.
Article in English | MEDLINE | ID: mdl-24151286

ABSTRACT

Since rates of tissue growth vary significantly between tissue types, and also between individuals due to differences in age, dietary intake, and lifestyle-related factors, engineering a scaffold system that is appropriate for personalized tissue engineering remains a significant challenge. In this study, a gelatin-hydroxyphenylpropionic acid/carboxylmethylcellulose-tyramine (Gtn-HPA/CMC-Tyr) porous hydrogel system that allows the pore structure of scaffolds to be altered in vivo after implantation is developed. Cross-linking of Gtn-HPA/CMC-Tyr hydrogels via horseradish peroxidase oxidative coupling is examined both in vitro and in vivo. Post-implantation, further alteration of the hydrogel structure is achieved by injecting cellulase enzyme to digest the CMC component of the scaffold; this treatment yields a structure with larger pores and higher porosity than hydrogels without cellulase injection. Using this approach, the pore sizes of scaffolds are altered in vivo from 32-87 µm to 74-181 µm in a user-controled manner. The hydrogel is biocompatible to COS-7 cells and has mechanical properties similar to those of soft tissues. The new hydrogel system developed in this work provides clinicians with the ability to tailor the structure of scaffolds post-implantation depending on the growth rate of a tissue or an individual's recovery rate, and could thus be ideal for personalized tissue engineering.


Subject(s)
Biocompatible Materials/chemistry , Hydrogel, Polyethylene Glycol Dimethacrylate/administration & dosage , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials/toxicity , COS Cells , Carboxymethylcellulose Sodium , Cell Proliferation/drug effects , Cell Survival/drug effects , Cellulase , Chlorocebus aethiops , Female , Gelatin , Hydrogel, Polyethylene Glycol Dimethacrylate/toxicity , Phenylpropionates , Porosity , Rats , Rheology , Tyramine
14.
Adv Biochem Eng Biotechnol ; 134: 135-53, 2013.
Article in English | MEDLINE | ID: mdl-23463360

ABSTRACT

: Hairy roots are a convenient experimental tool for investigating the interactions between plant cells and metal ions. Hairy roots of species capable of hyperaccumulating Cd and Ni have been applied to investigate heavy metal tolerance in plants; hairy roots of nonhyperaccumulator species have also been employed in metal uptake studies. Furnace treatment of hairy root biomass containing high concentrations of Ni has been used to generate Ni-rich bio-ore suitable for metal recovery in phytomining applications. Hairy roots also have potential for biological synthesis of quantum dot nanocrystals. As plant cells intrinsically provide the confined spaces needed to limit the size of nanocrystals, hairy roots cultured in bioreactors under controlled conditions are a promising vehicle for the manufacture of peptide-capped semiconductor quantum dots.


Subject(s)
Metals, Heavy/metabolism , Nanoparticles/metabolism , Plant Cells/metabolism , Plant Roots/metabolism , Bioreactors , Cadmium/metabolism , Cell Culture Techniques/methods
15.
Biotechnol Bioeng ; 110(1): 318-26, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22811278

ABSTRACT

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water-soluble metabolites through their high water-content matrix. The ability to image three-dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell-biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB-tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well.


Subject(s)
Biocompatible Materials/chemistry , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Tissue Scaffolds/chemistry , Animals , COS Cells , Cell Adhesion/physiology , Chlorocebus aethiops , Electrons , Focal Adhesions/chemistry , Hydrogel, Polyethylene Glycol Dimethacrylate/chemical synthesis , Microscopy, Confocal , Polymerization , Porosity , Tissue Engineering/instrumentation , Tissue Engineering/methods , Tomography
16.
Biotechnol Prog ; 29(1): 176-85, 2013.
Article in English | MEDLINE | ID: mdl-23125060

ABSTRACT

Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose-derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual-chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1-fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual-chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem-cell source.


Subject(s)
Adipose Tissue/cytology , Osteogenesis , Stem Cells/cytology , Tissue Engineering , Bioreactors , Cell Culture Techniques , Cell Differentiation , Cells, Cultured , Humans , Polyglycolic Acid/chemistry , Tissue Scaffolds/chemistry
17.
Curr Med Chem ; 20(8): 1047-55, 2013.
Article in English | MEDLINE | ID: mdl-23210789

ABSTRACT

Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used. As in whole plants, proteolysis in cultured plant cells can lead to significant degradation of foreign proteins after synthesis; however, substantial progress has been made to counter the destructive effects of proteases in plant systems. Although protein secretion into the culture medium is advantageous for product recovery and purification, measures are often required to minimise extracellular protease activity and product losses due to irreversible surface adsorption. Disposable plastic bioreactors, which are being used increasingly in mammalian cell bioprocessing, are also being adopted for plant cell culture to allow rapid scale-up and generation of saleable product. This review examines a range of technical and regulatory issues affecting the choice of industrial production platform for foreign proteins, and assesses progress in the development of in vitro plant systems for biopharmaceutical production.


Subject(s)
Biological Products/chemistry , Bioreactors , Plant Cells/chemistry , Proteins/chemistry , Cell Culture Techniques , Protein Engineering/trends
18.
Biomaterials ; 33(16): 4078-88, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22391264

ABSTRACT

Development of cartilage lesions in osteoarthritis and following traumatic injury has important consequences on the weight bearing and articulation of joints, has severe impact on the quality of life of affected individuals and is of significant socioeconomic impact. Hyaline cartilage is a highly specialised tissue with a limited ability to self repair. Development of three-dimensional scaffolds which maintain the correct chondrocyte phenotype during expansion of cells in vitro and their application in regenerative strategies for cartilage repair is therefore a major research objective of many laboratories. This study examined the matrix components elaborated by cultured foetal cartilage rudiment cells, a mixture of chondroblasts/chondroprogenitor cells and committed chondrocytes, in monolayer, cell pellet cultures and in the synthetic scaffolds sodium alginate and polyglycolic acid (PGA). The ability of fibroblast growth factor (FGF)-2 and FGF-18 to promote chondrogenesis in pellet cultures was also examined. While the scaffolds did not completely replicate the matrix organisation evident in native cartilage, type II collagen and aggrecan were nevertheless prominent matrix components. FGF-2 and FGF-18 further promoted the production of cartilage-specific matrix components in pellet culture as FGF-18 stimulated the production of type X collagen and perlecan and may be indicative of a more terminally differentiated phenotype induced in the rudiment cells with this growth factor.


Subject(s)
Cartilage/metabolism , Extracellular Matrix/metabolism , Aggrecans/metabolism , Alginates/chemistry , Cartilage/cytology , Cartilage/embryology , Cells, Cultured , Chondrogenesis/drug effects , Collagen Type II/metabolism , Culture Media , Fetus/cytology , Fetus/metabolism , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factors/pharmacology , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Immunohistochemistry , Polyglycolic Acid/chemistry , Stem Cells/metabolism
19.
Trends Biotechnol ; 30(3): 166-76, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22071143

ABSTRACT

Joint injury and disease are painful and debilitating conditions affecting a substantial proportion of the population. The idea that damaged cartilage in articulating joints might be replaced seamlessly with tissue-engineered cartilage is of obvious commercial interest because the market for such treatments is large. Recently, a wealth of new information about the complex biology of chondrogenesis and cartilage has emerged from stem cell research, including increasing evidence of the role of physical stimuli in directing differentiation. The challenge for the next generation of tissue engineers is to identify the key elements in this new body of knowledge that can be applied to overcome current limitations affecting cartilage synthesis in vitro. Here we review the status of cartilage tissue engineering and examine the contribution of stem cell research to technology development for cartilage production.


Subject(s)
Cartilage/growth & development , Chondrogenesis , Tissue Engineering/methods , Humans , Stem Cell Research
20.
Biotechnol Bioeng ; 109(4): 1060-73, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22095592

ABSTRACT

The effect of dynamic mechanical shear and compression on the synthesis of human tissue-engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA-alginate scaffolds were precultured in shaking T-flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak-to-peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T-flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3- and 10-fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4-fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue-engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II.


Subject(s)
Batch Cell Culture Techniques/instrumentation , Biomechanical Phenomena , Bioreactors , Chondrocytes/cytology , Growth Plate/cytology , Joints/physiology , Stress, Mechanical , Tissue Engineering/instrumentation , Alginates , Cells, Cultured/cytology , Cells, Cultured/metabolism , Chondrocytes/metabolism , Collagen Type II/biosynthesis , Compressive Strength , Equipment Design , Glucuronic Acid , Glycosaminoglycans/biosynthesis , Growth Plate/embryology , Hexuronic Acids , Humans , Perfusion , Polyglycolic Acid , Pressure , Shear Strength , Tissue Scaffolds , Weight-Bearing
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