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Visualization of cellular processes over extended periods of time has been hampered by the cellular requirement for heat, humidity and a physiological pH balanced media. The advances in micro technologies have enabled the production of miniaturized cell culture devices capable of sustaining mammalian cell growth over extended periods of time. In the present work a novel perfusion based micro cell culture chamber with imbedded thermal monitoring and regulation is presented. The chamber sustained the culturing and online monitoring of both cancer cell lines as well as stem cell lines over long periods of time (>2 weeks and >90 hours respectively) after which the chamber became confluent and the experiments were stopped. The culture conditions in the chamber were assessed by means of morphological observations, growths kinetics and whole genome expression profiling on transcriptome DNA microarrays. The results suggested that the culturing conditions in the µCCC were comparable to those in the conventional culture flask, if the intensity of the microscope and ambient light was carefully monitored. Surface modifications of the structural photoresist SU-8 commonly used for realizing micro channels by means of lithography were assessed for their ability to sustain cell culture. The biocompatibility of the surface modifications were assessed by means of morphological observations, growths kinetics and whole genome expression profiling on transcriptome DNA microarrays. The latter method was denoted bio-comparability enabling the distinguishing from biocompatibility. A surface modification was found to result in comparable morphology and growth kinetics to the reference cell culture flask, but showed a significantly different gene expression profile suggesting biocompatibility does not infer bio-comparability. A novel random priming method for initiation of reverse transcription reactions and real time PCRs was developed. The impact of the simple switch of primer was found to increase the yield of cDNA by 100 % corresponding to 80 % of the RNA template was reverse transcribed into cDNA. Furthermore the random priming method increased the amount of detected genes on transcriptome DNA microarrays by 24 % in the Cy-3 channel and 81 % in the Cy-5 channel, suggesting a significant better coverage of the transcriptome.
|Place of Publication||Kgs. Lyngby|
|Publisher||Technical University of Denmark|
|Number of pages||251|
|Publication status||Published - May 2006|