Spelling suggestions: "subject:"microcarrier"" "subject:"microcarriers""
1 |
Developing a standardised manufacturing process for the clinical-scale production of human mesenchymal stem cellsRafiq, Qasim Ali January 2013 (has links)
Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.
|
2 |
Development and optimization of an in vitro process for the production of Oryctes nudivirus in insect cell culturesPushparajan, Charlotte January 2015 (has links)
The coconut rhinoceros beetle, an economically important pest of coconut and oil palms, is effectively managed by application of its natural pathogen, the Oryctes nudivirus (OrNV), which act as a bioinsecticide. While this approach offers an environment-friendly alternative to chemical pesticides, the current method of production in infected larvae suffers from inconsistencies in virus productivity and purity. While the anchorage-dependent DSIR-HA-1179 insect cell line has been identified as a susceptible and permissive host for OrNV and therefore would be suitable for the in vitro mass production of the virus, no attempts have been made toward the mass production of the virus, because of the technological challenges that working with DSIR-HA-1179 cells represent. Thus, the main objective of this research was to develop processes for the in vitro production of OrNV in the DSIR-HA-1179 cell line.
Knowledge of the growth kinetics and metabolic properties of the host cell line in a chosen culture medium, as well as the selection of an appropriate infection strategy, form the basis for the rational development of bioreactor-based virus production processes. However, characterization of these properties in the DSIR-HA-1179 cell line has been virtually precluded, due to its strongly adherent growth characteristics and the lack of a reliable method to accurately dissociate and count cells grown in monolayers. Using TrypLE™ Express enzyme, a technique allowing the precise counting of cells was developed.
The cell line was adapted to grow in four serum-supplemented culture media: TC-100, IPL-41, Sf-900 II and Sf-900 III, which were then individually screened for cell growth and virus production in 25 cm2 attached T-flask cultures. TC-100 supplemented with 10% fetal bovine serum was chosen as a suitable culture medium, based on its capacity for achieving a high cell yield and OrNV production. The cell line metabolism was characterized with respect to nutrient consumption and metabolites production in this culture medium. Glucose, along with glutamine were found to be the nutrients that were consumed faster and to a greater extent, while other amino acids were not consumed to a significant degree. The production of metabolites was characterized by non-production of lactate and ammonia, and production of alanine, as a non-toxic alternative to ammonia.
The influence of cell density (CD) at time of infection (TOI) and multiplicity of infection (MOI) on OrNV production was evaluated in T-flask cultures that were infected at different CDs at the TOI and a range of MOIs. The CD at TOI was found to significantly influence OrNV yields, while MOI influenced the dynamics of infection. The cell density effect was found to exist for the DSIR-HA-1179/OrNV system with the progressive decline in cell-specific yield beginning at low cell densities. It was found that in order to maximize OrNV volumetric yield, a combination of MOI and CD at TOI should be selected that allows to keep the maximum cell density reached by the infected culture within a range between 5.0 and 7.0 x 105 viable cells/ml.
The roller bottle system was evaluated for its potential to scale-up DSIR-HA-1179 cell growth and OrNV production, and culture parameters were optimized for the improvement of cell and virus yields. An inoculum density of 3.3 x 105 cells/ml and culture volume of 60 ml resulted in the highest cell yield of 1.5 x 106 cells/ml, in 490 cm2 roller bottles. It was found that an optimal infection strategy for roller bottle cultures, which represented the most efficient use of viral inoculum, involved infecting cells at a density of 5.0 x 105 cells/ml and at a MOI of 1. The resulting OrNV volumetric yield of 2.5 x108 TCID50/ml, improved significantly the viral yields obtained in attached T-flask cultures infected under similar conditions (6.8 x 107 TCID50/ml).
The microcarrier system was also evaluated for culturing DSIR-HA-1179 cells and producing OrNV in spinner flask bioreactors. Three types of microcarriers (Cytodex-1, Cytodex-3 and Cultispher-G microcarriers) were screened for their ability to support DSIR-HA-1179 growth. Cells attached to Cytodex-1 and 3, but failed to attach to Cultispher-G microcarriers. The final cell density reached in microcarrier culture was dependent on bead type and concentration, and the cell to bead ratio. At an optimal bead concentration of 1 mg/ml and cell to bead ratio of 30, cells grew to a maximum density of 1.7 x 106 cells/ml on Cytodex-1, but only to 1.3 x 106 cells/ml on Cytodex-3 microcarriers. Since it supported higher cell yields, Cytodex-1 was chosen to study the kinetics of OrNV production in this system. Microcarrier cultures infected at a cell density of 5.0 x 105 cells/ml and a MOI of 1, produced OrNV at 1.4 x 108 TCID50/ml, which was higher than the yield obtained in T-flask cultures infected under similar conditions.
A framework of knowledge on the physiology, metabolism and growth kinetics of the DSIR-HA-1179 insect cell line has been developed in this thesis. In addition, the feasibility of using roller bottles and microcarrier systems for the in vitro production of the virus has been ascertained. It is envisaged that these findings will contribute to the future development of a large-scale industrial process for the production of the OrNV biopesticide.
|
3 |
Matrix-Derived Microcarriers for Adipose Tissue EngineeringTURNER, ALLISON EUGENIA BOGART 01 December 2010 (has links)
In vivo, adipose tissue demonstrates only a limited capacity for self-repair, and the long-term treatment of subcutaneous defects remains an unresolved clinical problem. With the goal of regenerating healthy tissues, many tissue-engineering strategies have pointed to the potential of implementing three-dimensional (3-D), cell-seeded scaffolds for soft tissue augmentation and wound healing. In particular, microcarriers have shown promise as both cell expansion substrates and injectable cell-delivery vehicles for these applications. However, limited research has investigated the engineering of tissue-specific microcarriers, designed to closely mimic the native extracellular matrix (ECM) composition. In this work, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via non-cytotoxic protocols. Characterization by microscopy confirmed the efficacy of the fabrication protocols in producing stable beads, as well as the production of a microporous surface topography. The mean bead diameter was 934 ± 51 μm, while the porosity was measured to be 29 ± 4 % using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT-based microcarriers were effectively stabilized with the non-cytotoxic photochemical crosslinking agent rose bengal, with only low levels of protein release measured within a simulated physiological environment. In cell-based studies, the DAT-based microcarriers successfully supported the proliferation and adipogenic differentiation of human adipose-derived stem cells (hASCs) in a dynamic spinner flask system, with a more favorable response observed in terms of adhesion, proliferation, and adipogenesis on the DAT-based microcarriers relative to gelatin control beads. More specifically, dynamically-cultured hASCs on DAT-based microcarriers demonstrated greater lipid loading, as well as higher glycerol-3-phosphate dehydrogenase (GPDH) activity, a key enzyme involved in triacylglycerol biosynthesis, at 7 days and 14 days in culture in an inductive medium. Overall, the results indicated that the DAT-based microcarriers provided a uniquely supportive environment for adipogenesis. Established microcarrier sterility and injectability further support the broad potential of these tissue-specific microcarriers as a novel, adipogenic, clinically-translatable strategy for soft tissue engineering. / Thesis (Master, Chemical Engineering) -- Queen's University, 2010-12-01 14:28:14.628
|
4 |
Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadoresMendonça, Marlei Leandro de 29 April 2013 (has links)
Made available in DSpace on 2016-08-17T18:39:47Z (GMT). No. of bitstreams: 1
5353.pdf: 2805182 bytes, checksum: dbd89699431c4bbf40d6959bb375df30 (MD5)
Previous issue date: 2013-04-29 / Financiadora de Estudos e Projetos / The increasing interest in the employment of multipotent mesenchymal stromal cells (MSCs) to applications in tissue engineering and cell therapy is mainly attributed to their plasticity and regenerative capacity. The MSCs represent a revolution in the treatment of countless diseases and in the understanding of tissue repair mechanisms. Due to their low availability in the tissues (0.00001% to 0.0002%) as well as to the need of high doses for therapeutic applications (approximately 2 x 106 cells/kg patient), and to the inefficiency of traditional in vitro cultures (in monolayer) to meet the existing high demand to these applications, development of new technologies of ex vivo expansion on a large scale became indispensable. The use of spinner flasks-like bioreactors with microcarriers in suspension is an effective alternative system for this expansion. However, bibliographic report of cultures with this system have indicated the formation of large clusters of microcarriers with cells as a possible obstacle to obtain a greater cellular productivity. These clusters hamper the diffusion of nutrients, gases (especially oxigen), and reagents of indirect cellular quantification within them; and they harm the cell recovery at the end of culture. Alginate microspheres have become interesting to reduce the frequency of collisions between microcarriers, due to the fact that the microspheres do not adhere to cells nor to microcarries and can prevent the union of same. The dextran sulfate has been described in the literature as an inhibitor of cell-to-cell contact as well as of the encounter between microcarriers in culture, therefore, it too has become a promising option to avoid the formation of clusters of microcarriers. Thus, the aim of this work was to develop a methodology for the culture of MSCs in spinner flasks with microcarriers Cultispher-S for application in cell therapy and evaluate the influence of alginate microspheres and dextran sulfate on this culture. For this, the lineage hMSCTERT and 3 g/L Cultispher-S were held in 100 mL spinner containing α-MEM culture medium (supplemented with 15% of bovine fetal serum, glucose and glutamine) at 37ºC, with pH control. The results showed that the addition of dextran sulfate (at 0.5% and 1%) and of 100% of alginate microspheres (in relation to the number of Cultspher-S particles) to the culture caused considerable cell death; and the control (without additives) as well as the addition of 50% of alginate microspheres promoted cell growth. The formation of clusters was observed in cultures; however, it was delayed with the addition of 50% of alginate microspheres. This occurrence allowed a cellular expansion factor of 50.4 times (with dilution factor correction) and of 13.1 times (without correction). The recovered cells after expansion kept their immunophenotypic characteristics. / O crescente interesse na utilização de células mesenquimais estromais multipotentes (CMMs) para aplicações na engenharia de tecidos e na terapia celular é atribuído principalmente à sua plasticidade e capacidade regenerativa. As CMMs representam uma revolução no tratamento de inúmeras doenças e no entendimento dos mecanismos de reparo tecidual. Sua baixa disponibilidade nos tecidos (de 0,00001 à 0,0002%) associada à necessidade de elevadas doses para as aplicações terapêuticas (aproximadamente 2 x 106 células/kg paciente) e à ineficiência dos cultivos tradicionais (em monocamada) in vitro para atender à alta demanda existente para essas aplicações, tornaram indispensável o desenvolvimento de novas tecnologias de expansão ex vivo em larga escala. A utilização de biorreatores tipo frascos spinner com microcarregadores em suspensão é um sistema alternativo eficaz para esta expansão. O relato bibliográfico de cultivos com esse sistema, no entanto, tem indicado a formação de grandes aglomerados de microcarregadores com células como um possível obstáculo na obtenção de maior produtividade celular. Tais aglomerados dificultam a difusão de nutrientes, gases (principalmente o oxigênio) e reagentes de quantificação celular indireta em seu interior; e prejudicam a recuperação das células no final do cultivo. Microesferas de alginato tornaram-se interessantes para diminuir a frequência de colisões entre os microcarregadores, pois não se aderem às células nem aos microcarregadores podendo evitar a união dos mesmos. O sulfato de dextrana foi descrito na literatura tanto como inibidor do contato célula-célula, quanto do encontro entre microcarregadores em cultivo, por isso também passou a ser uma opção promissora para evitar a formação de aglomerados de microcarregadores. Assim, os objetivos deste trabalho foram desenvolver uma metodologia de cultivo de CMMs em frasco spinner com microcarregadores Cultispher-S para aplicação em terapia celular e avaliar a influência de microesferas de alginato e do sulfato de dextrana nesse cultivo. Para isso, a linhagem hMSC-TERT e 3 g/L Cultispher-S foram mantidos em spinner de 100 mL contendo meio de cultura α-MEM (suplementado com 15% de soro fetal bovino, glicose e glutamina), a 37ºC, com controle de pH. Os resultados mostraram que a adição de sulfato de dextrana (à 0,5% e 1%) e de 100% de microesferas de alginato (em relação ao número de partículas de Cultispher-S) ao cultivo provocaram morte celular considerável; e que o controle (sem aditivos), assim como a adição de 50% de microesferas de alginato promoveram crescimento celular. A formação de aglomerados foi observada nos cultivos, no entanto, ela foi atrasada com adição de 50% de microesferas de alginato. Esse fato permitiu a obtenção de um fator de expansão de 50,4 vezes (com a correção do fator de diluição) e de 13,1 vezes (sem a correção). As células recuperadas após a expansão mantiveram suas características imunofenotípicas.
|
5 |
Monodisperse Microgels based on Poly(2-Oxazoline)s for Regenerative Cell Replacement TherapyLück, Steffen 16 February 2017 (has links) (PDF)
This work aims towards the development of a modular system for fabrication of monodisperse microgels made of poly(2-oxazoline)s for use in the field of regenerative therapy.
|
6 |
Effects of microcarrier concentration, agitation rate, and serum concentration on the specific growth rate of mouse L cells in batch culturesNorcio, Lawrence P. January 1995 (has links)
No description available.
|
7 |
Cultivation of Hepatitis B virus producing cell line HepG2.2.15 on microcarrier and functional characterization of the Hepatitis B virus polymeraseLupberger, Joachim 11 May 2007 (has links)
Hepatitis B Virus (HBV) Infektionen verursachen entzündliche Erkrankungen der Leber. Insbesondere die frühen Phasen des HBV Lebenszyklus sind noch nicht geklärt, so ist z.B. der Rezeptorkomplex an den HBV bindet unbekannt. Mittlerweile stehen neue Infektionsmodelle zur Verfügung um den HBV Lebenszyklus zu untersuchen. Dies erfordert eine effiziente Zellkultur basierende Methode um große Mengen infektiöser Partikel zu generieren. Ein Ziel der Arbeit war durch Kultivierung auf Mikrocarrier die HBV Produktion der Zelllinie HepG2.2.15 zu steigern. Die Analyse von Protein und HBV Sekretion, Infektiösität und MAP Signalübertragung ergab eine 18x höhere HBV Produktion bei einer reduzierten Sekretion von subviralen Partikeln durch HepG2.2.15 die auf Mikrocarrier kultiviert wurden. Der Anstieg der Virusproduktion korreliert mit einer verstärkten Aktivierung der MAP Kinase ERK-2, die mit HBV Replikation in Verbindung steht. Ein weiterer wenig verstandener Teil des HBV Lebenszyklus ist der Kernimport des HBV Genoms. Spuren der viralen Polymerase finden sich im Zellkern von HBV infizierten Zellen. Ziel der Arbeit war, Motive in der HBV Polymerase zu finden, die in der Lage sind Zelllokalisation zu beeinflussen. Durch Sequenzvergleich wurde eine konservierte zweiseitige Kernlokalisationssequenz im Terminalen Protein der HBV Polymerase identifiziert, die eine Proteinkinase CKII Erkennungsstelle enthält. Inhibition der CKII Aktivität in HBV infizierten primären Hepatozyten sowie die Zerstörung der CKII Erkennungsstelle im Terminalen Protein inhibieren die HBV Replikation. Die Funktionalität der Kernlokalisationssequenz wurde durch Fusion an GFP bestätigt und war Abhängig von CKII Aktivität in der Zelle. Dies wurde in vitro durch Bindung des Adapterproteins Karyopherin-alpha an CKII-phosphoryliertes Terminales Protein bestätigt. Die HBV Polymerase enthält eine konservierte zweiseitige Kernlokalisationssequenz deren Funktionalität durch CKII Phosphorilierung vermittelt wird. / Hepatitis B virus (HBV) infection causes acute and chronic liver inflammation. Especially the early phase of the HBV life cycle is not clearly understood. For example the receptor complex that mediates viral entry is not known. Novel infection models to study the HBV lifecycle are described that demand for a large amount of cell culture generated infectious HBV particles. One aim was to enhance HBV production of the cell line HepG2.2.15 by cultivation on microcarrier substrate. Analysis of protein and viral particle secretion, infectivity, and cellular MAP kinase signaling revealed an up to 18x increased HBV production and a decreased subviral particle secretion by HepG2.2.15 when cultivated on microcarrier. The observed effect was due to an enhanced phospho-activation of MAP kinase ERK-2 that is tightly associated with HBV replication. Another poorly understood part of the HBV lifecycle is the mechanism that delivers the HBV genome into the nucleus. Traces of HBV polymerase can be found in HBV infected cells. The second objective was to identify motifs on the HBV polymerase that determine its subcellular localization. By sequence alignment a conserved bipartite nuclear localization signal was found in the terminal protein of the HBV polymerase encompassing a protein kinase CKII recognition site. Inhibition of CKII kinase in infected primary hepatocytes and destruction of the identified CKII recognition site in the viral polymerase impaired virus production. The functionality of the putative nuclear localization signal was confirmed by fusion to GFP. Moreover, its functionality was depended on CKII activity that was verified by in vitro binding experiments of terminal protein to the import adaptor karyopherin-alpha. This data identified a nuclear localization signal in the HBV polymerase, which functionality is mediated by CKII phosphorylation.
|
8 |
Avaliação da expansão de células estromais mesenquimais em biorreator de fibra ocaSantos, Diogo Peres dos 28 February 2013 (has links)
Made available in DSpace on 2016-06-02T19:56:51Z (GMT). No. of bitstreams: 1
5178.pdf: 2466586 bytes, checksum: be50519a129b12383d84e69ae25b54db (MD5)
Previous issue date: 2013-02-28 / Universidade Federal de Sao Carlos / The use of mesenchymal stromal cells (MSCs) for clinical therapy has been limited by the low amount of cells that can be obtained directly from tissue, making it necessary to develop techniques for in vitro cell number expansion. The current methods of expansion are laborintensive, exhibit unfavorable environments for cell growth, show still modest levels of expansion and low yield in the recovery of these cells. In the search for better alternatives, several types of bioreactors have been assessed, however, with results still discreet. A littlestudied system, which has showed itself very effective in the use with other types of animal cells, is the hollow fiber bioreactor. This bioreactor has relatively homogeneous culture environment, low level of hydrodynamic stress on cells and the process control is made through manipulation external to the culture. Thus, it is proposed in this work the study of the in vitro expansion of MSCs in 15 mL hollow fiber prototype bioreactor designed and built with a configuration specifically conceived for expansion of MSCs for use in therapeutic applications. The inoculum was prepared with MSCs precultured adhered to microcarrier Cultispher-S at concentration of 4 g/L in spinner flask containing 50 mL of α-MEM culture medium with 15% v/v fetal bovine serum. The preculture was performed in CO2 incubator at pH close to 7.3 and temperature of 37°C. For bioreactor expansion cultures, it was used the same culture medium, with addition of 12 g/L of alginate and 4.25-4.50 mM of CaCl2 as gelling agents to immobilize and keep in suspension the microcarriers, in the conditions of pH and temperature used in the preculture. The oxygenation of the culture medium continuously recirculated through the intracapilar space was carried out by air bubbling in an external flask. The oxygenation levels were of 70 to 90% of saturation with air. The experimental results obtained show that the used configuration of hollow fiber bioreactor promoted good conditions for expansion of MSCs without cell aggregation, reaching 15.3-fold expansion and cell recovery levels of 82%. These results also demonstrate the possibility of improving the efficiency of MSCs expansion through the renewal of medium to maintain suitable levels of arginine, nutrient present in limiting amounts, and ammonium, growth inhibitor metabolite. / A utilização de células estromais mesenquimais (MSCs em inglês) para a terapia clínica tem sido limitada pela baixa quantidade de células que podem ser obtidas diretamente do tecido, tornando necessário o desenvolvimento de técnicas de expansão do número de células in vitro. Os métodos atuais de expansão apresentam necessidade de intensa mão de obra, ambientes desfavoráveis para o crescimento celular, níveis de expansão ainda modestos e baixo rendimento na recuperação destas células. Na procura de melhores alternativas, diversos tipos de biorreatores vêm sendo avaliados, porém, com resultados ainda discretos. Um sistema pouco estudado que tem se mostrado muito eficiente no uso com outros tipos de células animais é o biorreator de fibra oca. Este biorreator apresenta ambiente de cultura relativamente homogêneo, baixo nível de forças hidrodinâmicas sobre as células e o controle do processo é feito através de manipulação externa à cultura. Assim, é proposto neste trabalho o estudo da expansão in vitro de MSCs num protótipo de biorreator de fibra oca de 15 mL projetado e construído com uma configuração especialmente concebida para expansão de MSCs a serem utilizadas em aplicações terapêuticas. O inóculo foi preparado com MSCs précultivadas aderidas ao microcarregador Cultispher-S na concentração de 4 g/L em frasco spinner contendo 50 mL de meio de cultura α-MEM com 15% v/v de soro fetal bovino. O précultivo foi realizado em incubadora de CO2 a pH próximo a 7,3 e temperatura de 37°C. Para os cultivos de expansão no biorreator foi utilizado o mesmo meio de cultura, com adição de 12 g/L de alginato e 4,25-4,50 mM de CaCl2 como agentes geleificantes para imobilizar e manter suspensos os microcarregadores, nas condições de pH e temperatura utilizadas no précultivo. A oxigenação do meio de cultura continuamente recirculado pelo espaço intracapilar foi realizada mediante borbulhamento de ar em um frasco externo. Os níveis de oxigenação foram de 70 a 90% da saturação com ar. Os resultados experimentais obtidos mostram que a configuração utilizada propiciou boas condições para a expansão sem agregação celular das MSCs, chegando-se a fatores de expansão estimados de 15,3 vezes e níveis de recuperação de células de 82%. Esses resultados também evidenciam a possibilidade de melhora da eficiência da expansão das MSCs através da renovação do meio de cultivo para a manutenção de níveis adequados de arginina, nutriente presente em quantidades limitantes, e amônia, metabólito inibidor de crescimento.
|
9 |
Developing a process control strategy for the consistent and scalable manufacture of human mesenchymal stem cellsHeathman, Thomas R. J. January 2015 (has links)
Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03 · 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.
|
10 |
Monodisperse Microgels based on Poly(2-Oxazoline)s for Regenerative Cell Replacement TherapyLück, Steffen 23 January 2017 (has links)
This work aims towards the development of a modular system for fabrication of monodisperse microgels made of poly(2-oxazoline)s for use in the field of regenerative therapy.
|
Page generated in 0.164 seconds