ABSTRACT
The aim of cellular agriculture is to use cell-culturing technologies to produce alternatives to agricultural products. Cultured meat is an example of a cellular agriculture product, made by using tissue engineering methods. This study aims to improve the understanding of the potential environmental impacts of cultured meat production by comparing between different bioprocess design scenarios. This was done by carrying out a life cycle assessment (LCA) for a bioprocess system using hollow fiber bioreactors, and utilizing bench-scale experimental data for C2C12 cell proliferation, differentiation and media metabolism. Scenario and sensitivity analyses were used to test the impact of changes in the system design, data sources, and LCA methods on the results to support process design decision making. We compared alternative scenarios to a baseline of C2C12 cells cultured in hollow fiber bioreactors using media consisting of DMEM with serum, for a 16-day proliferation stage and 7-day differentiation stage. The baseline LCA used the average UK electricity mix as the energy source, and heat treatment for wastewater sterilization. The greatest reduction in environmental impacts were achieved with the scenarios using CHO cell metabolism instead of C2C12 cell metabolisim (64-67 % reduction); achieving 128 % cell biomass increase during differentiation instead of no increase (42-56 % reduction); using wind electricity instead of average UK electricity (6-39 % reduction); and adjusting the amino acid use based on experimental data (16-27 % reduction). The use of chemical wastewater treatment instead of heat treatment increased all environmental impacts, except energy demand, by 1-16 %. This study provides valuable insights for the cultured meat field to understand the effects of different process design scenarios on environmental impacts, and therefore provides a framework for deciding where to focus development efforts for improving the environmental performance of the production system.
Subject(s)
Bioreactors , Wastewater , Amino Acids , Animals , Life Cycle Stages , MeatABSTRACT
Organoids are three-dimensional multicellular tissue constructs. When cultured in vitro, they recapitulate the structure, heterogeneity, and function of their in vivo counterparts. As awareness of the multiple uses of organoids has grown, e.g. in drug discovery and personalised medicine, demand has increased for low-cost and efficient methods of producing them in a reproducible manner and at scale. Here we focus on a bioreactor technology for organoid production, which exploits fluid flow to enhance mass transport to and from the organoids. To ensure large numbers of organoids can be grown within the bioreactor in a reproducible manner, nutrient delivery to, and waste product removal from, the organoids must be carefully controlled. We develop a continuum mathematical model to investigate how mass transport within the bioreactor depends on the inlet flow rate and cell seeding density, focusing on the transport of two key metabolites: glucose and lactate. We exploit the thin geometry of the bioreactor to systematically simplify our model. This significantly reduces the computational cost of generating model solutions, and provides insight into the dominant mass transport mechanisms. We test the validity of the reduced models by comparison with simulations of the full model. We then exploit our reduced mathematical model to determine, for a given inlet flow rate and cell seeding density, the evolution of the spatial metabolite distributions throughout the bioreactor. To assess the bioreactor transport characteristics, we introduce metrics quantifying glucose conversion (the ratio between the total amounts of consumed and supplied glucose), the maximum lactate concentration, the proportion of the bioreactor with intolerable lactate concentrations, and the time when intolerable lactate concentrations are first experienced within the bioreactor. We determine the dependence of these metrics on organoid-line characteristics such as proliferation rate and rate of glucose consumption per cell. Finally, for a given organoid line, we determine how the distribution of metabolites and the associated metrics depend on the inlet flow rate. Insights from this study can be used to inform bioreactor operating conditions, ultimately improving the quality and number of bioreactor-expanded organoids.
ABSTRACT
Scaffold materials suitable for the scale-up and subsequent commercialization of tissue engineered products should ideally be cost effective and accessible. For the in vitro culture of certain adherent cells, synthetic fabrication techniques are often employed to produce micro- or nano-patterned substrates to influence cell attachment, morphology, and alignment via the mechanism of contact guidance. Here we present a natural scaffold, in the form of decellularized amenity grass, which retains its natural striated topography and supports the attachment, proliferation, alignment and differentiation of murine C2C12 myoblasts, without the need for additional functionalization. This presents an inexpensive, sustainable scaffold material and structure for tissue engineering applications capable of influencing cell alignment, a desired property for the culture of skeletal muscle and other anisotropic tissues.
Subject(s)
Muscle, Skeletal/growth & development , Poaceae , Tissue Engineering/methods , Tissue Scaffolds , Animals , Cell Adhesion , Cell Line , Cell Proliferation , Mice , Muscle Fibers, Skeletal , Myoblasts/physiology , NanostructuresABSTRACT
Herein we describe the manufacture and characterisation of biocompatible, porous polystyrene membranes, suitable for cell culture. Though widely used in traditional cell culture, polystyrene has not been used as a hollow fibre membrane due to its hydrophobicity and non-porous structure. Here, we use microcrystalline sodium chloride (4.7⯱â¯1.3⯵m) to control the porosity of polystyrene membranes and oxygen plasma surface treatment to reduce hydrophobicity. Increased porogen concentration correlates to increased surface pore density, macrovoid formation, gas permeability and mean pore size, but a decrease in mechanical strength. For tissue engineering applications, membranes spun from casting solutions containing 40% (w/w) sodium chloride represent a compromise between strength and permeability, having surface pore density of 208.2⯱â¯29.7 pores/mm2, mean surface pore size of 2.3⯱â¯0.7⯵m, and Young's modulus of 115.0⯱â¯8.2â¯MPa. We demonstrate the biocompatibility of the material with an exciting cell line-media combination: transdifferentiation of the AR42J-B13 pancreatic cell line to hepatocyte-like cells. Treatment of AR42J-B13 with dexamethasone/oncostatin-M over 14 days induces transdifferentiation towards a hepatic phenotype. There was a distinct loss of the pancreatic phenotype, shown through loss of expression of the pancreatic marker amylase, and gain of the hepatic phenotype, shown through induction of expression of the hepatic markers transferrin, carbamoylphosphate synthetase and glutamine synthetase. The combination of this membrane fabrication method and demonstration of biocompatibility of the transdifferentiated hepatocytes provides a novel, superior, alternative design for in vitro liver models and bioartificial liver devices.
ABSTRACT
Here a zirconium amine tris(phenolate) is used as the initiator for the production of polylactide for biomedical applications, as a replacement for a tin initiator (usually tin octanoate). The ring opening polymerization (ROP) was carried out in the melt at 130°C. The zirconium-catalyzed PLA (PLA-Zr) required 30min, resulting in a polydispersity index (PDI) of 1.17, compared to 1h and PDI=1.77 for tin-catalyzed PLA (PLA-Sn). PLA-Zr and PLA-Sn supported osteosarcoma cell (MG63) culture to the same extent (cell number, morphology, extracellular matrix production and osteogenic function) until day 14 when the PLA-Zr showed increased cell number, overall extracellular matrix production and osteogenic function. To conclude, the reduction in reaction time, controllable microstructure and biologically benign nature of the zirconium amine tris(phenolate) initiator shows that it is a more effective initiator for ROP of polylactide for biomedical applications.
Subject(s)
Zirconium/chemistry , Amines , Dioxanes , Polyesters , PolymerizationABSTRACT
This study presents experimental data of a fluidized-bed bioreactor for the cultivation of encapsulated pancreatic beta-cells. The fluidization quality for the bioreactor was evaluated at different flow rate using bed-expansion parameters. Homogeneous distribution of microcapsules was achieved at a flow rate of 2000 µL/min. This enabled efficient contact between the encapsulated cells and medium, which contributed to high cell viability. Microcapsule breakage was <4% on day 7 and confirmed the stability of encapsulated systems under fluidized culture. Importantly, endocrine beta-cells cultured in the bioreactor were shown to be dramatically more responsive to changes in glucose concentration compared to static culture (P < 0.001). On the basis of these results, cultivation of encapsulated cells in a fluidized bioreactor, especially for pancreatic beta-cells that are limited in supply, is a promising approach to address the lack of a safe method for storage and handling of cells between laboratories and clinical sites prior to transplantation.
ABSTRACT
Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.
Subject(s)
Bioreactors , Cell Culture Techniques/methods , Tissue Culture Techniques/methods , Carcinoma, Hepatocellular/pathology , Hep G2 Cells , Humans , Liver Neoplasms/pathologyABSTRACT
The scale-up of tissue engineering cell culture must ensure that conditions are maintained while also being cost effective. Here we analyse the stability of hepatocyte growth factor (HGF) to investigate whether concentrations change under dynamic conditions, and compare commercial recombinant human HGF as an additive in 'standard medium', to HGF secreted by the osteosarcoma cell line MG63 as a 'preconditioned medium'. After 3 h under flow conditions, HGF in the standard medium degraded to 40% of its original concentration but HGF in the preconditioned medium remained at 100%. The concentration of secreted HGF was 10 times greater than the working concentration of commercially-available HGF. Thus HGF within this medium has increased stability; MG63-derived HGF should therefore be investigated as a cost-effective alternative to current lyophilised powders for use in in vitro models. Furthermore, we recommend that those intending to use HGF (or other growth factors) should consider similar stability testing before embarking on experiments with media flow.
Subject(s)
Biotechnology/methods , Culture Media/chemistry , Hepatocyte Growth Factor/chemistry , Cell Culture Techniques/methods , Drug Stability , HumansABSTRACT
The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to 0:05 Pa are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges 2.75 x 10(-5) m(2) s(-1) to 3 x 10(-5) m(2) s(-1) (equivalent to 2.07 ml min(-1) to 2.26 ml min(-1)) and 1.077 x 10(5) Pa to 1.083 x 10(5) Pa (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells.
Subject(s)
Myocytes, Cardiac/physiology , Animals , Bioreactors , Cell Aggregation , Cell Culture Techniques , Cells, Cultured , Models, Biological , Oxygen/physiology , Rats , Stress, PhysiologicalABSTRACT
Hollow fibre membrane bioreactors (HFB) provide a novel approach towards tissue engineering applications in the field of regenerative medicine. For adherent cell types, HFBs offer an in vivo-like microenvironment as each fibre replicates a blood capillary and the mass transfer rate across the wall is independent from the shear stresses experienced by the cell. HFB also possesses the highest surface area to volume ratio of all bioreactor configurations. In theory, these factors enable a high quantity of the desired cellular product with less population variation, and favourable operating costs. Experimental analyses of different cell types and bioreactor designs show encouraging steps towards producing a clinically relevant device. This review discusses the basic HFB design for cell expansion and in vitro models; compares data produced on commercially available systems and addresses the operational differences between theory and practice. HFBs are showing some potential for mammalian cell culture but further work is needed to fully understand the complexities of cell culture in HFBs and how best to achieve the high theoretical cell yields.
Subject(s)
Bioreactors , Tissue Engineering/methods , Animals , Humans , Mammals , Membranes , Regenerative Medicine/methodsABSTRACT
The focus of much scientific and medical research is directed towards understanding the disease process and defining therapeutic intervention strategies. The scientific basis of drug safety is very complex and currently remains poorly understood, despite the fact that adverse drug reactions (ADRs) are a major health concern and a serious impediment to development of new medicines. Toxicity issues account for â¼21% drug attrition during drug development and safety testing strategies require considerable animal use. Mechanistic relationships between drug plasma levels and molecular/cellular events that culminate in whole organ toxicity underpins development of novel safety assessment strategies. Current in vitro test systems are poorly predictive of toxicity of chemicals entering the systemic circulation, particularly to the liver. Such systems fall short because of (1) the physiological gap between cells currently used and human hepatocytes existing in their native state, (2) the lack of physiological integration with other cells/systems within organs, required to amplify the initial toxicological lesion into overt toxicity, (3) the inability to assess how low level cell damage induced by chemicals may develop into overt organ toxicity in a minority of patients, (4) lack of consideration of systemic effects. Reproduction of centrilobular and periportal hepatocyte phenotypes in in vitro culture is crucial for sensitive detection of cellular stress. Hepatocyte metabolism/phenotype is dependent on cell position along the liver lobule, with corresponding differences in exposure to substrate, oxygen and hormone gradients. Application of bioartificial liver (BAL) technology can encompass in vitro predictive toxicity testing with enhanced sensitivity and improved mechanistic understanding. Combining this technology with mechanistic mathematical models describing intracellular metabolism, fluid-flow, substrate, hormone and nutrient distribution provides the opportunity to design the BAL specifically to mimic the in vivo scenario. Such mathematical models enable theoretical hypothesis testing, will inform the design of in vitro experiments, and will enable both refinement and reduction of in vivo animal trials. In this way, development of novel mathematical modelling tools will help to focus and direct in vitro and in vivo research, and can be used as a framework for other areas of drug safety science.
ABSTRACT
Bioartificial livers have yet to gain clinical acceptance. In a previous study, a theoretical model was utilized to create operating region charts that graphically illustrated viable bioartificial liver configurations. On this basis a rationale for the choice of operating and design parameters for the device was created. The concept is extended here to include aspects of liver zonation for further design optimization. In vivo, liver cells display heterogeneity with respect to metabolic activity according to their position in the liver lobule. It is thought that oxygen tension is a primary modulator of this heterogeneity and on this assumption a theoretical model to describe the metabolic zonation within an in vitro bioartificial liver device has been adopted. The distribution of the metabolic zones under varying design and operating parameters is examined. In addition, plasma flow rates are calculated that give rise to an equal distribution of the metabolic zones. The results show that when a clinically relevant number of cells are contained in the BAL (10 billion), it is possible to constrain each of the three metabolic zones to approximately one-third of the cell volume. This is the case for a number of different bioreactor designs. These considerations allow bioartificial liver design to be optimized.
Subject(s)
Hepatocytes/metabolism , Liver, Artificial , Liver/metabolism , Models, Theoretical , Equipment Design , Oxygen/metabolismABSTRACT
One-step surfactant-free, water-droplet templating has been developed as a fabrication method for a poly(lactide-co-glycolide) (PLGA) film that can be used as a model to investigate the relationship between solvent, monomer ratio, polymer concentration and humidity on its structure. The resulting material is a honeycomb-structured film. Formation of this structure was highly sensitive to solvent, monomer ratio, polymer concentration and humidity. Surfactant-free, water-droplet templating thus allows investigation of fabrication parameters and that PLGA monomer ratio selection is important for scaffold structure but not for MG63 cell attachment and proliferation.
Subject(s)
Polyglactin 910/chemistry , Surface-Active Agents/analysis , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Cell Line , Humans , Humidity , SolventsABSTRACT
Bioartificial livers (BALs) are a potentially effective countermeasure against liver failure, particularly in cases of acute or fulminant liver failure. It is hoped these devices can sustain a patient's liver function until recovery or transplant. However, no large-scale clinical trial has yet proven that BALs are particularly effective and evidently design issues remain to be addressed. One aspect of BAL design that must be considered is the mass transfer of adequate oxygen to the hepatocytes within the device. We present here a mathematical modeling approach to oxygen mass transport in a BAL. A mathematical model based upon Krogh cylinders is outlined to describe a diffusion-limited hollow fiber bioreactor. In addition, operating constraints are defined on the system--cells should not experience hypoxia and the cell population should be of adequate size. By combining modeling results with these operating constraints and presenting the results graphically, "operating region" charts can be constructed for the hollow fiber BAL (HF-BAL). The effects of varying various operating parameters on the BAL are then established. It is found that smaller radii and short, thin walled fibers are generally advantageous while cell populations in excess of 10 billion could be supported in the BAL with a plasma flow rate of 200 mL/min. For fibers of intermediate length and lumen radius, the minimum number of fibers required to produce a viable design ranges approximately from 7,000-10,000. In theory, this may be enough to support patients with failing livers.
Subject(s)
Liver, Artificial , Models, Theoretical , Bioreactors , Hepatocytes/metabolism , Humans , Oxygen/metabolismABSTRACT
The interactions of post-culture treatments reagents used for fixing, lysing and cell quantification on poly(lactide-co-glycolide) (PLGA) flat sheet membrane scaffolds are presented. Lysing with Alkaline buffer solution/Triton X-100/MilliQ water (ATM) and fixing with 10% Neutral Buffered Formalin (10% NBF) had no affect on membrane structure while fixing with 95% ethanol caused smoothing of the surface, shrinkage and a reduction in surface area of 55, 48 and 33, for 100:0, 75:25 and 50:50 (PLA:PGA), respectively. PicoGreen assay was selected for cell (560pZIPv.neo) quantification since the background noise would not affect readings for cell numbers over 3,000 cells/cm(2), while the background reading was too high for MTT and Methylene Blue (MB). MB at 0.5% (w/v) was, however, deemed suitable for visualising cell morphology on the membranes. Furthermore ATM buffer was suitable for the PicoGreen assay, which allows the same samples to be used for quantification of alkaline phosphatase activity.
Subject(s)
Biocompatible Materials/chemistry , Cell Culture Techniques/methods , Lactic Acid/chemistry , Membranes, Artificial , Osteoblasts/cytology , Osteoblasts/physiology , Polyglycolic Acid/chemistry , Tissue Engineering/methods , Cell Line , Cell Proliferation , Materials Testing , Polylactic Acid-Polyglycolic Acid CopolymerABSTRACT
We develop a simple mathematical model for forced flow of culture medium through a porous scaffold in a tissue-engineering bioreactor. Porous-walled hollow fibres penetrate the scaffold and act as additional sources of culture medium. The model, based on Darcy's law, is used to examine the nutrient and shear-stress distributions throughout the scaffold. We consider several configurations of fibres and inlet and outlet pipes. Compared with a numerical solution of the full Navier-Stokes equations within the complex scaffold geometry, the modelling approach is cheap, and does not require knowledge of the detailed microstructure of the particular scaffold being used. The potential of this approach is demonstrated through quantification of the effect the additional flow from the fibres has on the nutrient and shear-stress distribution.
Subject(s)
Bioreactors , Models, Biological , Tissue Engineering/methods , Culture Media , Humans , Perfusion , Porosity , Rheology , Stress, Mechanical , Tissue Engineering/instrumentationABSTRACT
Providing a scaffold that can supply nutrients on a large scale (several cubic centimeters) is the key to successfully regenerating vascularized tissue: biodegradable membranes are a promising new scaffold suited to this purpose. Poly(lactic-co-glycolic-acid) (PLGA) flat sheet membranes of different lactide:glycolide ratios, prepared by phase inversion using 1-methyl-2-pyrrolidinone (NMP) as the solvent and water as the nonsolvent, were compared by assessing attachment, proliferation and osteogenic function of human bone derived cells (HBDC). Three different lactide:glycolide ratios, 50:50, 75:25, and 100:0, were compared to tissue culture polystyrene (TCPS). For attachment, 50:50 and 75:25 had similar numbers to TCPS but 100:0 had significantly fewer cells than TCPS. 50:50 and 75:25 had significantly lower HBDC numbers after 7 days but 100:0 had similar numbers compared to TCPS. For proliferation the cell number on the membranes were similar to each other. After 3 weeks, osteoblastic function of the HBDC, shown by mineralization and alkaline phosphatase activity, was present but was significantly lower compared to the TCPS control but similar when the membranes were compared. PLGA membranes fabricated from a range of ratios support HBDC culture so the optimum scaffold composition can be selected based on other factors, such as degradation rate.
Subject(s)
Biocompatible Materials/chemistry , Bone and Bones/cytology , Lactic Acid/chemistry , Polyglycolic Acid/chemistry , Tissue Engineering , Tissue Scaffolds/chemistry , Adult , Cell Adhesion , Cell Proliferation , Cells, Cultured , Female , Humans , Membranes, Artificial , Polylactic Acid-Polyglycolic Acid Copolymer , Polystyrenes/chemistryABSTRACT
Strategies to expand human bone marrow stromal cells (HBMSC) for bone tissue engineering are a key to revolutionising the processes involved in three-dimensional skeletal tissue reconstruction. To facilitate this process we believe the use of biodegradable porous poly(DL-lactide-co-glycolide) (PDL LGA) hollow fibres as a scaffold used in combination with HBMSC to initiate natural bone repair and regeneration offers a potential solution. In this study, the biocompatibility of 75:25 PDL LGA fibres with HBMSC and the capacity of a PDL LGA fibre-associated HBMSC-monolayer to establish an osteogenic phenotype in vivo was examined. A high proportion of HBMSC survived when expanded on PDL LGA fibres for 6 days, with only 10% of the propidium iodide (pI)-labelled population represented in the sub-G1 DNA peak on analysis by flow cytometry. Tracking carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labelled HBMSC by flow cytometry indicated that HBMSC attachment to the P(DL)LGA fibres does not interfere with their rate of proliferation. Furthermore, in response to osteogenic stimuli, HBMSC expanded on PDL LGA fibres can differentiate, as expected, along the osteogenic lineage with associated alkaline phosphatase activity. Following implantation into SCID mice, osteogenic-conditioned PDL LGA fibre-HBMSC graft resulted in type I collagen deposition and associated bone mineralisation and osteoid formation, as evidenced by immunohistochemistry and histology. These studies provide evidence that porous PDL LGA hollow fibre-HBMSC graft is an innovative biomaterial that offers new approaches to mesenchymal cell expansion, which could be utilised as a scaffold for skeletal tissue generation.
Subject(s)
Bone Marrow Cells/cytology , Bone and Bones/cytology , Bone and Bones/physiology , Lactic Acid , Polyglycolic Acid , Polymers , Tissue Engineering , Biocompatible Materials , Bone and Bones/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Humans , Polylactic Acid-Polyglycolic Acid Copolymer , Stromal Cells/cytology , Tissue ScaffoldsABSTRACT
Mass transfer limitations of scaffolds are currently hindering the development of 3-dimensional, clinically viable, tissue engineered constructs. We have developed a poly(lactide-co-glycolide) (PLGA) hollow fibre membrane scaffold that will provide support for cell culture, allow psuedovascularisation in vitro and provide channels for angiogenesis in vivo. We produced P(DL)LGA flat sheet membranes using 1, 4-dioxane and 1-methyl-2-pyrrolidinone (NMP) as solvents and water as the nonsolvent, and hollow fibre membranes, using NMP and water, by dry/wet- and wet-spinning. The resulting fibres had an outer diameter of 700 micro m and an inner diameter of 250 micro m with 0.2-1.0 micro m pores on the culture surface. It was shown that varying the air gap and temperature when spinning changed the morphology of the fibres. The introduction of a 50 mm air gap caused a dense skin of 5 micro m thick to form, compared to a skin of 0.5 micro m thick without an air gap. Spinning at 40 degrees C produced fibres with a more open central section in the wall that contained more, larger macrovoids compared to fibres spun at 20 degrees C. Culture of the immortalised osteogenic cell line 560pZIPv.neo (pZIP) was carried out on the P(DL)LGA flat sheets in static culture and in a P(DL)LGA hollow fibre bioreactor under counter-current flow conditions. Attachment and proliferation was statistically similar to tissue culture polystyrene on the flat sheets and was also successful in the hollow fibre bioreactor. The P(DL)LGA hollow fibres are a promising scaffold to address the size limitations currently seen in tissue engineered constructs.
Subject(s)
Cell Culture Techniques/methods , Glycolates/chemistry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Membranes, Artificial , Tissue Engineering/methods , Biocompatible Materials/chemistry , Cell Adhesion , Cell Differentiation , Cell Line , Cell Proliferation , Humans , Lactic Acid , Materials Testing , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid CopolymerABSTRACT
While methods for the production of scaffolds with the appropriate mechanical properties and architecture for tissue engineering are attracting much attention, the effects of subsequent sterilization processes on the scaffold properties have often been overlooked. This study sought to determine the effects of sterilization with ethanol, peracetic acid, ultraviolet irradiation, and antibiotic solution on the structure of 50:50 (mol:mol) 65:35, and 85:15 poly(D,L-lactic-co-glycolic acid [PLGA]) flat-sheet and hollow-fiber scaffolds. All methods resulted in scaffold sterilization, but scanning electron microscopy revealed deformations to the scaffold surface for all treatments. The extent of surface damage increased with treatment duration. This was further investigated by measurement of pore sizes, water flux, breaking strain, and Young's modulus. External pore size and water flux was found to be increased by all treatments in the following order: ethanol (largest), antibiotics, ultraviolet light, and peracetic acid. Pore sizes were 0.25 to 0.17 microm and water flux ranged from 0.01 kg x m(-2) x s(-1) to 3.34 kg x m(-2) x s(-1). For all samples, the Young's modulus was 1.0 to 31.1 MPa and breaking strain was 1.2 to 2.4 MPa. The results of this study suggest that antibiotic treatment shows the most potential to sterilize PLGA hollow fibers for tissue engineering.