Suspension mode seed train development for adherent cells
The disclosure is directed to a method for seed train expansion of adherent cells comprising culturing cells with a serum-supplemented growth medium in a N-2 vessel; removing the cells from the serum-supplemented medium; inoculating the cells from step into a serum-free growth medium in a N-1 vessel; culturing the cells in the N-1 vessel under suspension conditions; and inoculating a growth medium in a bioreactor with the suspension-cultured cells. In some aspects, the adherent cells are not suspension-adapted. In some aspects, the adherent cells are suspension-adapted. In some aspects, the adherent cells produced by the seed train expansion method are used to produce viral vectors. In some aspects, the viral vectors are AAV vectors.
1 . A method of cell expansion of adherent mammalian cells comprising:
(a) culturing the adherent mammalian cells under adherent conditions with a first growth medium comprising serum in a N-2 container;
(b) removing the adherent mammalian cells from the first growth medium;
(c) inoculating the adherent mammalian cells from step (b) into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a N-1 container;
(d) culturing the adherent mammalian cells in the N-1 container under suspension conditions in the second growth medium;
(e) collecting the adherent mammalian cells or the adherent mammalian cells in the second growth medium from step (d) from the N-1 container;
(f) passaging the adherent mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
(g) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor; and
(h) culturing the adherent mammalian cells in the bioreactor.
2 . The method of claim 1 , wherein the third growth medium comprises a higher serum concentration than the serum concentration in the second growth medium.
3 . The method of claim 1 , wherein the adherent mammalian cells are Hela cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, or COS cells.
4 . The method of claim 1 , further comprising culturing the adherent mammalian cells with the first growth medium in a N-3 container.
5 . The method of claim 4 , further comprising culturing the adherent mammalian cells with the first growth medium in a N-4 container.
6 . The method of claim 5 , further comprising culturing the adherent mammalian cells with the first growth medium in a N-5 container.
7 . The method of claim 1 , wherein the third growth medium in the bioreactor comprises at least one factor which promotes cell adherence.
8 . The method of claim 7 , wherein the at least one factor which promotes cell adherence is serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, or combinations thereof.
9 . The method of claim 8 , wherein the third growth medium in the bioreactor comprises DMEM and 10% FBS.
10 . The method of claim 1 , further comprising contacting the adherent mammalian cells with a first polynucleotide sequence in the bioreactor.
11 . The method of claim 10 , wherein the first polynucleotide sequence is a plasmid.
12 . The method of claim 10 , further comprising contacting the adherent mammalian cells with a second polynucleotide encoding a transgene.
13 . The method of claim 12 , further comprising contacting the adherent mammalian cells with a third polynucleotide that encodes an adenoviral helper gene.
14 . A method of cell expansion of adherent mammalian cells comprising:
(a) culturing the adherent mammalian cells under adherent conditions with a first growth medium comprising serum in a N-3 container;
(b) removing the adherent mammalian cells from the first growth medium;
(c) inoculating the adherent mammalian cells from step (b) into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a N-2 container;
(d) culturing the adherent mammalian cells in the N-2 container under suspension conditions in the second growth medium;
(e) collecting the adherent mammalian cells or the adherent mammalian cells in the second growth medium from step (d) from the N-2 container;
(f) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium into the second growth medium in a N-1 container;
(g) culturing the adherent mammalian cells in the N-1 container under suspension conditions in the second growth medium;
(h) collecting the adherent mammalian cells or the adherent mammalian cells in the second growth medium from step (g) from the N-1 container;
(i) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor; and
(j) culturing the adherent mammalian cells in the bioreactor.
15 . The method of claim 14 , further comprising culturing the adherent mammalian cells with the first growth medium in a N-4 container.
16 . The method of claim 15 , further comprising culturing the adherent mammalian cells with the first growth medium in a N-5 container.
17 . A method of seed-train expansion of adherent mammalian cells comprising:
(a) culturing the adherent mammalian cells under adherent conditions in a first growth medium comprising serum;
(b) removing the adherent mammalian cells from the first growth medium;
(c) suspending the adherent mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium;
(d) culturing the adherent mammalian cells from step (c) under suspension conditions in the second growth medium;
(e) passaging the adherent mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
(f) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor; and
(g) culturing the adherent mammalian cells in the bioreactor.
18 . The method of claim 17 , wherein the adherent mammalian cells of step (a) are passaged at least once under adherent conditions.
19 . The method of claim 17 , wherein the adherent mammalian cells are passaged no more than two times under suspension conditions.
20 . A method of producing a viral particle, comprising:
(a) culturing adherent mammalian cells under adherent conditions in a first growth medium comprising serum;
(b) removing the adherent mammalian cells from the first growth medium;
(c) suspending the adherent mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium;
(d) culturing the adherent mammalian cells from step (c) under suspension conditions in the second growth medium;
(e) passaging the adherent mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
(f) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor;
(g) transfecting the adherent mammalian cells with a polynucleotide sequence encoding a viral capsid protein; and
(h) culturing the adherent mammalian cells in the bioreactor under conditions in which the viral particle is produced.
21 . The method of claim 20 , further comprising isolating the viral particle produced in step (h).
22 . The method of claim 20 , wherein the adherent mammalian cells of step (a) are passaged at least once under adherent conditions.
23 . The method of claim 20 , wherein the adherent mammalian cells of step (e) are passaged at least two, at least three, at least four, or at least five times under suspension conditions.
24 . The method of claim 11 , wherein the plasmid encodes a capsid protein of a recombinant AAV particle.
25 . The method of claim 24 , wherein the adherent mammalian cells are cultured under conditions which produce the recombinant AAV particle.
26 . The method of claim 1 , wherein the adherent mammalian cells of step (f) are passaged at least two times under suspension conditions before being inoculated in the bioreactor.
27 . The method of claim 14 , wherein the adherent mammalian cells of step (g) are passaged at least one time under suspension conditions before being inoculated in the bioreactor.
28 . The method of claim 17 , wherein the adherent mammalian cells of step (e) are passaged at least two times under suspension conditions before being inoculated in the bioreactor.
29 . The method claim 1 , wherein the bioreactor is an adherent bioreactor.
30 . The method of claim 14 , wherein the bioreactor is an adherent bioreactor.
31 . The method of claim 17 , wherein the bioreactor is an adherent bioreactor.
32 . The method of claim 20 , wherein the bioreactor is an adherent bioreactor.