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Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

<p>The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.</p><p>Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.</p><p>Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.</p><p>In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.</p><p>Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.</p>

<p>The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium.</p><p>Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.</p>

The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells. Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation. Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics. In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity. Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well. / <p>QC 20101129</p>

The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium. Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex. / QC 20101125

There has been an increase in the carbon dioxide (CO2) emissions in the last 3 decades. A large share of these emissions is produced by the transport sector. In 2010 alone, global transport accounted for 7 GtCO2 eq and approximately 23% of total energy-related CO2 emissions. In order for the decarbonisation of the transport sector, one of the most important strategies is to reduce the use of fossil fuels. Fossil fuel consumption can be reduced by rolling out more battery electric vehicles (BEVs) on public roads. This is one of the methods by which the concept of electromobility is promoted. In order to increase the share of EVs, many countries have implemented different policies that promote the electrification of the transport sector. With respect to freight transport, electric commercial vans are one of the feasible choices.  This Master thesis involves a quantitative study which focus on the “total cost of ownership” (TCO) of light commercial vehicles (LCVs). Two diesel vans currently used in Curitiba, Brazil were selected - the Sprinter van by Mercedes-Benz and the Master van by Renault. In addition, their electric counterparts were also chosen; in conjunction, a sensitivity analysis with respect to fuel prices and annual distance driven was conducted. The results showed that the TCO of the electric LCVs is around 1.6 to 1.7 times higher than their diesel versions. As far as the two van model types were concerned, the Mercedes-Benz Sprinter had a higher TCO than the Renault Master over the chosen vehicle lifetime for both the diesel and electric versions, with the difference around 7.5% for the diesel versions and approximately 13% for the electric versions.  Based on the results of the TCO study, possible economic policies and fiscal instruments were recommended with regards to light commercial freight transport for Curitiba. / Det har skett en ökning av koldioxidutsläppen (CO2) under de senaste 3 decennierna. En stor del av dessa utsläpp produceras av transportsektorn. Bara 2010 svarade, global transport för 7 GtCO2 ekvivalenter och cirka 23% av de totala energirelaterade koldioxidutsläppen. För att avkolning av transportsektorn är en av de viktigaste strategierna att minska användningen av fossila bränslen. Fossil bränsleförbrukning kan minskas genom att rulla ut fler elektriska fordon (EF) på allmänna vägar när det gäller transportsektorn i allmänhet. Detta är en av metoderna som begreppet elektromobilitet främjas. För att öka andelen elbilar har många länder genomfört olika policyer som främjar elektrifiering av transportsektorn. När det gäller godstransport, är elektriska kommersiella lastbilar och skåpbilar två av de möjliga valen.  Detta examensarbete involverar en kvantitativ studie som fokuserar på “totala ägandekostnaderna” (TÄK) för lätta nyttofordon. Två dieselbilar som för närvarande används i Curitiba, Brasilien valdes - Sprinter-skåpbilen från Mercedes-Benz och Master-skåpbilen av Renault. Dessutom valdes deras elektriska motsvarigheter; i samband med detta genomfördes en känslighetsanalys avseende bränslepriser och årlig körd distans. Resultaten visade att T för elektriska LCV är cirka 1.6 till 1.7 gånger högre än deras dieselversioner. När det gäller de två typerna av skåpbilar hade Mercedes-Benz Sprinter en högre TCO än Renault Master under den valda fordonstiden för både diesel - och elektriska versioner, med skillnaden cirka 7.5% för dieselversionerna och cirka 13% för de elektriska versionerna.  Baserat på resultaten av TCO-studien rekommenderades möjlig ekonomisk politik och finanspolitiska instrument när det gäller lätt kommersiell godstransport för Curitiba.