Cell/tissue culturing device, system and method
A device, system and method for axenically culturing and harvesting cells and/or tissues, including bioreactors and fermentors. The device is preferably disposable but nevertheless may be used continuously for a plurality of consecutive culturing/harvesting cycles prior to disposal of same. This invention also relates to batteries of such devices which may be used for large-scale production of cells and tissues. According to preferred embodiments of the present invention, the present invention is adapted for use with plant cell culture.
1 . A method for culturing cells and/or tissue capable of expressing a recombinant protein in at least one disposable device comprising:
providing said at least one device for axenically culturing and harvesting cells and/or tissue in at least one cycle which comprises a sterilisable disposable container which comprises a reusable harvester which comprises a flow controller for harvesting of at least a desired portion of culture medium containing cells and/or tissue when desired, and wherein said device can be continuously used for at least one further consecutive culturing/harvesting cycle, wherein a remainder of said medium containing cells and/or tissue, remaining from a previously harvested cycle may, serve as inoculant for a next culture and harvest cycle; and;
providing axenic inoculant via said harvester;
providing sterile said culture medium and/or, sterile additives; and
allowing said cells and/or tissue to grow in said medium to a desired yield.
2 . The method of claim 1 , wherein said cells and/or tissue are plant cells and/or tissue.
3 . The method of claim 2 , wherein said cells and/or tissue are plant root cells and/or tissue.
4 . The method of claim 1 , wherein said at least one disposable device comprises a battery of said devices.
5 . A method for producing a recombinant protein in cells and/or tissue cultured in at least one disposable device, the method comprising:
(a) genetically modifying a cell and/or tissue to express a desired recombinant protein;
(b) providing said at least one device for axenically culturing and harvesting cells and/or tissue in at least one cycle which comprises a sterilisable disposable container which comprises a reusable harvester which comprises a flow controller for harvesting of at least a desired portion of culture medium containing cells and/or tissue when desired, and wherein said device can be continuously used for at least one further consecutive culturing/harvesting cycle, wherein a remainder of said medium containing cells and/or tissue, remaining from a previously harvested cycle may, serve as inoculant for a next culture and harvest cycle; and
(c) providing axenic inoculant comprising said genetically modified cells and/or tissue via said harvester;
(d) providing sterile said culture medium and/or, sterile additives;
(e) allowing said cells and/or tissue to grow in said medium to a desired yield; and
(f) harvesting said cells expressing said recombinant protein from said cells or medium.
6 . The method of claim 5 , wherein said cells and/or tissues are plant cells or tissues.
7 . The method of claim 6 wherein said plant cells are plant root cells and/or tissue.
8 . The method of claim 5 , wherein said genetically modified cell is transformed with an exogenous polynucleotide which comprises a polynucleotide sequence encoding said desired recombinant protein and at least one polynucleotide sequence selected from the group comprising a plant promoter sequence, a plant signal peptide, a targeting signal, and/or a plant terminator sequence.
9 . The method of claim 5 , wherein said genetically modified cell is a transformed carrot cell.
10 . The method of claim 5 , wherein said recombinant protein is a Human lysosomal protein.
11 . The method of claim 5 , wherein said recombinant protein is a Human glucocerebrosidase.
12 . The method of claim 5 , wherein said cell is genetically modified to express a recombinant protein as set forth in SEQ ID NO: 8.
13 . The method of claim 5 , wherein said cell is transformed with an exogenous polynucleotide comprising SEQ ID 7.
14 . The method of claim 8 , wherein said plant promoter sequence is as set forth in SEQ ID NO: 9.
15 . The method of claim 8 , wherein said targeting signal is as set forth in SEQ ID NO: 11.
16 . The method of claim 8 , wherein said plant signal peptide is as set forth in SEQ ID NO: 10.
17 . The method of claim 8 , wherein said plant terminator sequence is as set forth in SEQ ID NO: 12.
18 . The method of claim 8 , wherein said exogenous polynucleotide is as set forth in SEQ ID NO: 13.
19 . The method of claim 5 , wherein said container comprises an internal fillable volume of 50 or 100 liters or more.
20 . The method of claim 5 , wherein said device further comprises an air inlet for introducing sterile gas in the form of bubbles into said culture medium through a first inlet opening and wherein said air inlet is connectable to a suitable sterile gas supply.
21 . The method of claim 20 , wherein said sterile air is supplied at a rate of greater than 1 liter per minute.
22 . The method of claim 20 , further comprising providing additional oxygen to said air inlet.
23 . The method of claim 5 , wherein said culture medium comprises at least 40 grams per liter sugar.
24 . A culture of cells genetically modified to express a recombinant protein, the culture comprising:
cells expressing a recombinant protein cultured in at least one device for culturing and harvesting cells and/or tissue in at least one cycle which comprises a sterilisable disposable container which comprises a reusable harvester which comprises a flow controller for harvesting of at least a desired portion of culture medium containing cells and/or tissue when desired, and wherein said device can be continuously used for at least one further consecutive culturing/harvesting cycle, wherein a remainder of said medium containing cells and/or tissue, remaining from a previously harvested cycle may, serve as inoculant for a next culture and harvest cycle.
25 . The culture of claim 24 , wherein said cells are plant cells.
26 . The culture of claim 25 wherein said plant cells are root plant cells.