Production of poxviruses with adherent or non adherent avian cell lines
A pox virus and cell line technology, applied in the direction of viruses, embryonic cells, animal cells, etc., can solve problems such as high cost
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Embodiment 1
[0107] Example 1: Generation and establishment of attached cells
[0108] Open the eggs, separating the yolk from the white during the opening process. Embryos were removed from the yolk either directly or by means of a Pasteur pipette, or by means of a small absorbent filter paper (Whatmann 3M paper), which had been pre-cut in the form of a perforated ring with a punch. The diameter of the hole is about 5mm. The ringlets were sterilized in an oven using dry heat for about 30 minutes. The small paper ring is placed on the surface of the yolk and centered on the embryo so that the paper ring surrounds the embryo. The latter was then cut with small scissors, and the whole was taken out and placed in a Petri dish filled with PBS or saline. Excess yolk and placenta are therefore washed out of the medium carrying the embryos out of the ring, so no excess vitellin is collected with a Pasteur pipette.
[0109] In both cases, embryos were placed in tubes containing physiological...
Embodiment 2
[0120] Example 2: Passage of cells
[0121] One of the characteristics of stem cells, especially somatic and embryonic stem cells, is their ability to proliferate in vitro for relatively long periods of time. For propagating and passaging cells, change and replace the medium with fresh medium several hours before passaging. figure 1 The curves shown in illustrate the characteristics of cell growth and establishment.
Embodiment 3
[0122] Example 3: Doubling time and mean differentiation time
[0123] 3.1 Starting with the cells established in culture and the cells shown in the previous examples, the average time to division was calculated. For all independent isolates obtained, the rate of proliferation increased slightly during serial passaging, thus resulting in a change in the mean division time during cell establishment. During the attachment phase, cells were initially seeded on inactivated feeder layers and grown at 1 to 2 × 10 6 Cells per 100mm Petri dish (55cmm 2 Petri dishes) were regularly passaged at a constant initial seeding density. Table 2 illustrates the doubling time (d) and mean division time (MDT in hours) as a function of culture time for 3 established cell types. The average doubling time was observed to decrease during the establishment process.
[0124] Table 2:
[0125] cells / day 50 100 150 200 250 300 350 400 450 500 550 ...
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