Monitoring biological functions of cultured tissues using microdialysis
<p>Continuous monitoring during tissue culture is important for the success of engineered tissue development. It is also challenging due to lack of suitable established monitoring techniques. In this study, microdialysis, a sampling technique for measuring the unbound solute concentrations in...
| Main Authors: | , , |
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| Format: | Thesis |
| Language: | English |
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2007
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| Subjects: |
| _version_ | 1826305859130490880 |
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| author | Li, Z Cui, Z Urban, J |
| author2 | Cui, Z |
| author_facet | Cui, Z Li, Z Cui, Z Urban, J |
| author_sort | Li, Z |
| collection | OXFORD |
| description | <p>Continuous monitoring during tissue culture is important for the success of engineered tissue development. It is also challenging due to lack of suitable established monitoring techniques. In this study, microdialysis, a sampling technique for measuring the unbound solute concentrations in the tissues and organs of the living body, was adopted to monitor functional tissue growth in a bioreactor with explanted bovine caudal intervertebral discs (IVD) as the test tissue.</p> <p>Apart from cell metabolic activities, cell and tissue biological functions were investigated for the development of microdialysis for monitoring purposes. Methodologies of microdialysis with large pore size membrane probes for sampling macromolecular bio-functional markers were established. The effects of pumping methods, including 'push', 'pull' or 'push-and-pull', and the effect of the resulting transmembrane pressure on the fluid balance, and the relative recovery of small molecules and of macromolecules (proteins) were experimentally studied. The validity of the internal reference <em>in-situ</em> calibration was examined in detail. It was concluded that a push-and-pull system was the only effective method to eliminate fluid loss or gain. The relative recovery of small solutes was hardly affected by the applied pumping methods; however the relative recovery of macromolecules was significantly influenced by them. The <em>in situ</em> calibration technique using Phenol Red can provide reliable results for small molecules including glucose and lactic acid. Using lOkDa and 70kDa fluorescent dextrans as the internal standard for <em>in situ</em> calibration of large molecules of similar size, it was found that the pull pump system did not work well but that the push-and-pull pumping method did work well.</p> <p>A novel bioreactor system for <em>in vitro</em> IVD culture with static load and microdialysis monitoring was developed. Explanted IVDs were cultured under three different loads for up to 7 days. A single microdialysis probe with 3000 kDa membrane was inserted into each of the IVDs at a defined location. The <em>in situ</em> calibration technique was proved valid in the experiments and membrane fouling was not significant. The tissue metabolism and extracellular matrix turnover during 7 day culture were continuously monitored to investigate the effect of different loads. Microdialysis proved to be a feasible and efficient method for multi-parameter monitoring of tissue culture.</p> <p>Substantial effort was directed towards the identification of functional macromolecular markers in conjunction with microdialysis sampling. Amongst several proteins sampled, chitinase-3-like protein 1 (CHI3L1), a major soluble protein secreted by cultured IVD cells in alginate beads and by cultured IVD explants was identified following its successful isolation. Then it was established as a suitable functional marker. The effect of physico-chemical and mechanical stimuli (e.g. osmolarity, pH, oxygen tension and mechanical load) on secretion of CHI3L1 by cultured IVD cells and chondrocytes in alginate beads and by cultured IVD explant were investigated. CHI3L1 release was sensitive to physico-chemical stimulation. The production of CHI3L1 was directly correlated with the cell metabolism and this could be readily monitored with microdialysis.</p> |
| first_indexed | 2024-03-07T06:39:14Z |
| format | Thesis |
| id | oxford-uuid:f8b478fa-881e-4299-9ee5-b8ee29f37fe9 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:39:14Z |
| publishDate | 2007 |
| record_format | dspace |
| spelling | oxford-uuid:f8b478fa-881e-4299-9ee5-b8ee29f37fe92022-03-27T12:52:20ZMonitoring biological functions of cultured tissues using microdialysisThesishttp://purl.org/coar/resource_type/c_db06uuid:f8b478fa-881e-4299-9ee5-b8ee29f37fe9Intervertebral diskTissue cultureGrowthTissuesMathematical modelsEnglishPolonsky Theses Digitisation Project2007Li, ZCui, ZUrban, JCui, ZUrban, J<p>Continuous monitoring during tissue culture is important for the success of engineered tissue development. It is also challenging due to lack of suitable established monitoring techniques. In this study, microdialysis, a sampling technique for measuring the unbound solute concentrations in the tissues and organs of the living body, was adopted to monitor functional tissue growth in a bioreactor with explanted bovine caudal intervertebral discs (IVD) as the test tissue.</p> <p>Apart from cell metabolic activities, cell and tissue biological functions were investigated for the development of microdialysis for monitoring purposes. Methodologies of microdialysis with large pore size membrane probes for sampling macromolecular bio-functional markers were established. The effects of pumping methods, including 'push', 'pull' or 'push-and-pull', and the effect of the resulting transmembrane pressure on the fluid balance, and the relative recovery of small molecules and of macromolecules (proteins) were experimentally studied. The validity of the internal reference <em>in-situ</em> calibration was examined in detail. It was concluded that a push-and-pull system was the only effective method to eliminate fluid loss or gain. The relative recovery of small solutes was hardly affected by the applied pumping methods; however the relative recovery of macromolecules was significantly influenced by them. The <em>in situ</em> calibration technique using Phenol Red can provide reliable results for small molecules including glucose and lactic acid. Using lOkDa and 70kDa fluorescent dextrans as the internal standard for <em>in situ</em> calibration of large molecules of similar size, it was found that the pull pump system did not work well but that the push-and-pull pumping method did work well.</p> <p>A novel bioreactor system for <em>in vitro</em> IVD culture with static load and microdialysis monitoring was developed. Explanted IVDs were cultured under three different loads for up to 7 days. A single microdialysis probe with 3000 kDa membrane was inserted into each of the IVDs at a defined location. The <em>in situ</em> calibration technique was proved valid in the experiments and membrane fouling was not significant. The tissue metabolism and extracellular matrix turnover during 7 day culture were continuously monitored to investigate the effect of different loads. Microdialysis proved to be a feasible and efficient method for multi-parameter monitoring of tissue culture.</p> <p>Substantial effort was directed towards the identification of functional macromolecular markers in conjunction with microdialysis sampling. Amongst several proteins sampled, chitinase-3-like protein 1 (CHI3L1), a major soluble protein secreted by cultured IVD cells in alginate beads and by cultured IVD explants was identified following its successful isolation. Then it was established as a suitable functional marker. The effect of physico-chemical and mechanical stimuli (e.g. osmolarity, pH, oxygen tension and mechanical load) on secretion of CHI3L1 by cultured IVD cells and chondrocytes in alginate beads and by cultured IVD explant were investigated. CHI3L1 release was sensitive to physico-chemical stimulation. The production of CHI3L1 was directly correlated with the cell metabolism and this could be readily monitored with microdialysis.</p> |
| spellingShingle | Intervertebral disk Tissue culture Growth Tissues Mathematical models Li, Z Cui, Z Urban, J Monitoring biological functions of cultured tissues using microdialysis |
| title | Monitoring biological functions of cultured tissues using microdialysis |
| title_full | Monitoring biological functions of cultured tissues using microdialysis |
| title_fullStr | Monitoring biological functions of cultured tissues using microdialysis |
| title_full_unstemmed | Monitoring biological functions of cultured tissues using microdialysis |
| title_short | Monitoring biological functions of cultured tissues using microdialysis |
| title_sort | monitoring biological functions of cultured tissues using microdialysis |
| topic | Intervertebral disk Tissue culture Growth Tissues Mathematical models |
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