Chapter 8
Culture Methods for Mass Production of Ruminant Endothelial Cells 1
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José L . Moreira , Pedro M . Miranda , Isabel Marcelino , Paula M . Alves , and Manuel J. T. Carrondo * 1
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Instituto de Biologia Experimental e Technologica/Instituto de Technologia Quimica e Biológica, Apartado 12, 2781-901 Oeiras, Portugal Lab. for Engenharia, Bioquímia, Fac, Cièncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte da Caparica, Portugal 2
Mass production of finite cultures of goat jugular endothelial cells is required for the manufacturing in a cost effective manner, of an inactivated vaccine against Heartwater, a cattle disease caused by the bacteria Rickettsia Cowdria ruminantium that is endemic in Sub-Saharan African countries and West Indies. Two different approaches with potential for industrial purpose were tested to produce large amounts of viable caprine jugular endothelial (CJE) cells: static cultures and stirred tanks. Static cultures were easily scaled-up; similar cell growth patterns and final cell concentrations per cm were achieved for a 700 fold increase of the surface area. In these culture systems a critical variable was the inoculum concentration, which should be maintained in the range of 1 to 2 x 10 cells/cm . CJE growth was also possible in stirred tanks, after optimization of the most relevant variables. The optimal conditions for mass production were 6 g/L of Cytodex 3 using an inoculum of 3.3 x 10 cells/cm in vessels operated at 40 rpm (Reynolds number of approximately 1.5 x 10 ). The amount of cells produced in a 250 ml stirred tank was similar to that obtained with a 6320 cm Cell Factory™ reactor. The final decision upon the chosen culture method will depend upon the efficiency of the bacterial infection and vaccine production processes that can be derivedfromthese mass cell growing processes. 2
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© 2004 American Chemical Society
125 Endothelial cells constitute the inner monolayer of blood vessels. Due to their proximity to the flowing blood and tissues, they mediate and regulate a large number of important physiological processes that occur on the wall vessel. Among those are: the formation of the preliminary vascular plexus and the generation of blood vessels during the development of embryos (1,2), blood pressure regulation and vasodilatation (3,4), chronic rejections after surgery (5,6), wound healing (7), immune response (8,9) and the transport of several macromolecules into the brain (10); moreover, these cells are involved in cancer metastasis (11). Endothelial cells are also very relevant for veterinary studies because they can be infected by a bacteria of the Rickettsia family, Cowdria ruminantium, that lives in the vascular system, is taken up by neutrophils and monocytes of wild and domestic ruminants and is the agent of Heartwater (or Cowdriosis), a tickborne disease that is endemic in Sub-Saharan Africa and in the West Indies (12). This disease is one of the most economically important tick-borne diseases of ruminants; thus, an effective vaccination of domestic animals in a cost effective manner is essential for the limitation of its economic consequences in less developed countries. Presently, a vaccine based on the inactivated bacteria is the best candidate for veterinary use (13-15). In vitro, Cowdria ruminantium is a specific parasite of finite cultures of ruminant endothelial cells (12); consequently, a large-scale production of the vaccine requires the mass production of the host cells. This is a particularly difficult issue because these ruminant endothelial cells have a poor ability to proliferate in vitro namely due to their limited number of cell duplications (finite cultures), low specific cell growth rates and maximum cell concentrations, associated with an almost uncontrollable phenotypic variability. Currently there is no method described in the literature for the cultivation of significant amounts of ruminant endothelial cells. Moreover, it is well reported that the culture method used for the growth of several types of endothelial cells is critical, since culture conditions influence their behavior, characteristics and activity. Previous reports have shown that other types of endothelial cells grown under shear stress often present significant changes in cell morphology (16,17), microstructure, size (6,18) and biochemical activities (19), eventually leading to cell differentiation. The most straightforward strategy that completely eliminates the negative effects of shear stress is to culture cells in static conditions. Currently several options are available in the market that allow the scale-up and large-scale cultivation of cells using this methodology, such as roller bottles, Cell Factories™ (Nunc) and CellCube™ (Costar). Nevertheless, given the increased homogeneity, aeration and scalability provided by stirred tanks, these may be the best culture system for large-scale propagation of animal cells. Endothelial cells are anchorage-dependent and require a solid matrix to adhere and grow in stirred conditions. The growth of endothelial cells of human and bovine origin in porous and non-porous microcarriers has been previously described (20-22). From these reports it is clear that the origin of the endothelial cells is critical for the optimization of culture conditions (e.g. microcarrier type and concentration,
126 inoculum concentration and agitation rate) and influences cell growth and maximum cell concentration. The main goal of this work was the optimization of preliminary techniques and the development of new methodologies for mass production of endothelial cells. Static surfaces and stirred tank bioreactors were tested and optimized for the growth of CJE cells. The different culture methods were compared at both technical and economical levels.
Methodologies Caprine jugular endothelium cells (CJE), isolatedfromdifferent goats, were isolated by Eh-. Dominique Martinez (CIRAD/EMVT, Guadeloupe, France). Cells were propagated in T-flasks (Nunc, Roskilde, Denmark) in a humidified atmosphere of 7% C 0 in air at 37°C. Unless stated otherwise, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2 mM of glutamine, 10 % of fetal bovine serum (FBS), penicillin/streptomycin (100 U/mL) and fungizone (1% v/v) was used as culture medium (all final concentrations and from Invitrogen, Glasgow, UK). Routinely, cells were subcultured with a dilution ratio of 1:3 or 1:5 in similar T-flasks whenever 100% confluence was achieved. The cell detachment from the surface of the T-flask was done by trypsinization using a 0.2% (w/v) Trypsin/EDTA solution (from Invitrogen). These cells do not form stable cell lines and start to differentiate after 25 to 30 passages. Cell differentiation can be detected by morphologic changes such as increase in cell volume, decrease in the cell growth rate and maximum concentration. To circumvent this drawback, cells were discarded after passage number 20. 2
Culture Medium Studies Several media were tested to grow CJE cells: DMEM, HAM's F12, DMEM/HAM's F12 (1:1), RPMI 1640, RPMI 1629 and Glasgow Medium (all from Invitrogen), supplemented with 10 and 20% of FBS. In these studies the cells were grown in the conditions referred previously, and then diluted in the new conditions. These experiments were repeated at least 3 times with similar results.
Static Culture Studies Scale-up studies in static cultures were performed using the procedure described above for cell propagation and a defined inoculum concentration
127 (stated in the text). In those studies, various static culture systems were used: 6 well plates, tissue culture flasks (all standard sizes from Nunc), Cell Factory™ (from Nunc), and also roller bottles (from Coming Costar, Badhoevedor, The Netherlands). For roller bottle cultures, a period at low rotation (12 rph) was used during the initial 24 hours (to promote optimal cell attachment) after which the rate was increased to 24 rph. Despite the fact that roller-bottles are not conventional static cultures, the results obtained with this culture system were included in this section for a more comprehensive interpretation of the results. Cell number and viability were evaluated using the trypan blue dye exclusion method (0.4% (w/v) in phosphate buffer saline (PBS)) and a hemacytometer (Brand, Wertheim, Main, Germany) after cell detachment by trypsinization as described above in the cell propagation section (several washing steps using PBS were required for cell detachment in the Cell Factory™).
Stirred tank studies 3
Stirred tank studies were performed in 250 cm spinner flasksfromWheaton Magna-Flex (Techne, New Jersey, USA). The culture medium was DMEM as described above for cell propagation. Several types of microcarriers were used: Cytodex 2, Cytodex 3 and Cytopore 2 from Pharmacia (Upsalla, Sweden), Cultispher G from Percell (Lund, Sweden) and Biosilon from Nunc. The microcarriers were prepared and sterilized as described by the manufacturers. Unless stated otherwise, an inoculum of 10 cells/cm was used in all studies. The inoculation procedure is particularly critical for an efficient cell attachment: cells were left in contact with the microcarriers in half of the bioreactor final volume, for 2 hours, without agitation, followed by 24 hours of smooth agitation (30 to 40 rpm). Immediately after that period the culture medium was adjusted to 250 cm and the agitation rate was set up to the final value. Unless stated otherwise, Cytodex 3 was used at 40 rpm. The bioreactor sampling was done in two steps: in the first step, samples from the supernatant were collected after microcarrier sedimentation. For that purpose the agitation was arrested for a maximum of 3 min. and the number of non-attached cells was evaluated by the trypan blue dye exclusion method. After that, agitation was restarted and a sample containing microcarriers was collected, the microcarriers were allowed to settle and the culture medium was discarded. After two washing steps with PBS, the microcarriers were trypsinized for cell detachment. Cell number was counted and viability was evaluated using the trypan blue dye exclusion method. 5
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Results and Discussion
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The main goal of this work was the development and optimization of culture methods for mass production of finite cultures of caprine endothelial cells (CJE) that, eventually, will be infected with Cowdria ruminantium and used for the production of a vaccine. This final product being intended for a veterinary vaccine, the lowest production cost is a critical factor for the success of the process. Several culture media were tested and their performance for endothelial cell growth evaluated. The results obtained for static cultures (final cell concentration 196 hours after inoculation) are shown in Figure 1. The best culture medium was DMEM, all other alternatives showed a 1520% lower performance. Similar results were obtained after serial propagation in each culture media tested (at least 5 dilutions), the cell viability always being higher than 95%. Other media (M199, MCDB, Iscove's and Iscove's/HAM's F12 1:1) were also tested, leading to much lower cell yields than those presented in Figure 1 (data not shown). Our results indicate that the optimal culture medium depends on the origin of the cells, since they are compared with results previously reported in the literature for human umbilical cord vein endothelial
