Abstract
An apparatus and method for preparing and culturing cells comprises a gas permeable chamber having at least an inlet and an outlet for introducing culture medium and cells. A cell growth substrate placed at the end(s) of the gas permeable chamber for cells anchored or embedded. The present invention discloses a simple but efficient cell culture method by slowly rotating the cell culture apparatus horizontally combining with a partially filled culture media in the gas permeable container in order to be able to expose the carriers and submerge the carriers intermittently, to achieve an optimal cell culture environment through the efficient oxygen transfer, CO2 balance, with sufficient nutrient supply, without vigorous agitation to achieve oxygen transfer. The horizontally rotation movement with the present cell growth apparatus efficient the nutrition, carbon dioxide and oxygen transfer during cultured process for production of cellular protein products, viruses or harvesting the cells.
Claims
1. A cell-cultivation apparatus, comprising: a gas permeable chamber with at least one open end to accommodate culture medium; a sealing member detachably mounted at the at least one open end; and a growth substrate disposed in the gas permeable chamber.
2. The cell-cultivation apparatus according to claim 1, wherein the sealing member comprises a cap and a rigid neck or a rigid tube, and the rigid neck or the rigid tube is detachably mounted at the at least one open end to engage with the cap.
3. The cell-cultivation apparatus according to claim 1, wherein the growth substrate is porous matrices in a disc-shape, in a sheet shape or a strip-shape.
4. The cell-cultivation apparatus according to claim 1, wherein the gas permeable chamber is with two the open ends.
5. The cell-cultivation apparatus according to claim 4, wherein two the sealing members detachably and respectively mounted at two the open ends, and one of two the sealing members includes a cap or a substrate holder disposed to fix the growth substrate.
6. The cell-cultivation apparatus according to claim 1, wherein the gas permeable chamber further comprises a close end.
7. The cell-cultivation apparatus according to claim 6, further comprising a basket disposed at the close end to fix the growth substrate.
8. The cell-cultivation apparatus according to claim 6, wherein the gas permeable chamber further comprises a fixing portion at the close end for fixing the growth substrate.
9. The cell-cultivation apparatus according to claim 6, further comprising an opening connected with a tube at the close end, wherein the tube is closed or opened by a fitting.
10. A device for rotating the cell-cultivation apparatus according to claim 1, comprising: a motor; a holder disposed to hold the cell-cultivation apparatus; and a shaft connected between the motor and the holder to be driven by the motor to rotate the cell-cultivation apparatus.
11. The device according to claim 10, further comprising a timing controller connected to the motor to control a rotation process of the cell-cultivation apparatus.
12. A method for culturing cell with the cell-cultivation apparatus according to claim 1, comprising: partially filling the culture medium into the gas permeable chamber to cover the growth substrate; rotating the gas permeable chamber upside-down to expose the growth substrate from the culture medium; and rotating the gas permeable chamber upside-down to submerge the growth substrate in the culture medium.
13. The method according to claim 12, further comprising maintaining the gas permeable chamber to be still with an interval time between the rotating steps.
14. The method according to claim 13, wherein the interval time is less than 60 minutes.
15. The method according to claim 12, wherein a rotation speed is greater than 0 and smaller than or equal to 2 rpm.
16. The method according to claim 12, wherein a rotation speed is smaller than 10 rpm.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1A and FIG. 1B show side views of a cell-cultivation apparatus of the first embodiment of present invention with pre-assemble and post-assemble.
[0031] FIG. 2A and FIG. 2B show side views of a cell-cultivation apparatus of the second embodiment of present invention with pre-assemble and post-assemble.
[0032] FIG. 3A and FIG. 3B show side views of a cell-cultivation apparatus of the third embodiment of present invention with pre-assemble and post-assemble.
[0033] FIG. 4A and FIG. 4B show side views of a cell-cultivation apparatus of the fourth embodiment of present invention with pre-assemble and post-assemble.
[0034] FIG. 5A and FIG. 5B show side views of a cell-cultivation apparatus of the fifth embodiment of present invention with pre-assemble and post-assemble.
[0035] FIG. 6A and FIG. 6B show side views of a cell-cultivation apparatus of the sixth embodiment of present invention with pre-assemble and post-assemble.
[0036] FIG. 7A and FIG. 7B show side views of a cell-cultivation apparatus of the seventh embodiment of present invention with pre-assemble and post-assemble.
[0037] FIG. 8 shows a side view of a cell-cultivation apparatus of the eighth embodiment of present invention.
[0038] FIG. 9A and FIG. 9B are schematically stereoscopic diagrams showing a device of the first and the second embodiments of present invention for driving the cell-cultivation apparatus.
[0039] FIG. 10A, FIG. 10B and FIG. 10C are schematically stereoscopic diagrams showing how the device rotates the cell-cultivation apparatus.
[0040] FIG. 11 is schematically stereoscopic diagram showing the device of the third embodiment of present invention for driving the cell-cultivation apparatus.
[0041] FIG. 12A, FIG. 12B and FIG. 12C are schematically side-view diagrams illustrating a cell-cultivation apparatus of gas permeability during rotating according to the present invention.
[0042] FIG. 13 shows a side view of a cell-cultivation apparatus of the nineth embodiment of present invention.
[0043] FIG. 14A and FIG. 14B show side views of a cell-cultivation apparatus of the tenth embodiment of present invention with pre-assemble and post-assemble.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] In according with the present invention, there is provided an apparatus and a method for culturing cells. The embodiments of the present invention can be used to culture different variety cells, such as eukaryotic and prokaryotic cells, particularly animal cells and/or mammalian cells. More particularly, anchorage-dependent animal cells.
[0045] The cell-cultivation apparatus of present invention includes a gas permeable chamber with at least one open end, a sealing member is detachably mounted at the at least one open end, and a growth substrate is disposed in the gas permeable chamber. The sealing element is a cap or a substrate holder, and the growth substrate is porous matrices in a disc-shape or a strip-shape. Below describing detailed parts of the cell-cultivation apparatus in different embodiments.
[0046] The apparatus for preparing and culturing cells shown in FIG. 1A, FIG. 1B show side views of a cell-cultivation apparatus of the first embodiment of the present invention with pre-assemble (FIG. 1A) and post-assemble (FIG. 1B) for ease of understanding. The cell-cultivation apparatus includes a gas permeable chamber 101 which is preferably a bottle shape chamber with a first open end 111 and a second open end 112. A sealing member may include a cap 102 detachably mounted at the first end 111. A growth substrate holder 104 and the growth substrate 105 are detachably mounted at the second end 112 of the gas permeable chamber 101. The growth substrate 105 is a porous sheet or disc that is fixed by a wedge ring 106 molded in the growth substrate holder 104. The growth substrate holder 104 is open at top for culture medium and gas transfer and exchange during cell culture. The gas permeable chamber 101 is preferably made by silicone rubber which can be semi-rigid under certain thickness from 0.5 mm to 2 mm, more preferably, from 0.5 to 1 mm, and it can be stand still without requiring extra supporting means. The gas permeable chamber 101 is closed by the cap 102. In order to secure the cap 102 to be air-tight with the gas permeable chamber 101, a rigid neck or a rigid tube 103 with external threads 23 is detachably mounted on the gas permeable chamber 101 in order to engage with the internal threads 22 of the cap 102. Silicone rubber has proven to be good materials for the gas exchange membrane. A silicone rubber can be manufactured economically in any desired shape by injection molding. Silicone rubber is available commercially in many thicknesses, shapes, and specific gas permeability. It has high tear resistance and good chemical resistance to the medium ordinarily used in cell culturing, and it is therefore also especially easy to handle. It is understood that the threads for engagement may be directly made on the open end of the gas permeable chamber 101 in case that silicone rubber is rigid enough for processing.
[0047] Referring to FIG. 1A and FIG. 1B again, the gas permeable chamber 101 may accommodate the culture medium 12, and the culture medium 12 in liquid phase may be enclosed within the gas permeable chamber 101 of the cell-cultivation apparatus 11 after post assembling. The culture medium 12 does not full in the gas permeable chamber 101 to leave a little space for gas. The gas may be in and out of the gas permeable chamber 101 due to gas permeability of the cell-cultivation apparatus 11.
[0048] The apparatus for preparing and culturing cells shown in FIG. 2A and FIG. 2B show side views of a cell-cultivation apparatus of the second embodiment of the present invention with pre-assemble (FIG. 2A) and post-assemble (FIG. 2B) for ease of understanding. Compared to the first embodiment, in addition to one part with the thread 23 for engaging with the cap 22, the rigid tube 103 provides a narrow part to plug into the first end 111 of the gas permeable chamber 101 and a wide part to lean on the gas permeable chamber 101. Furthermore, the gas permeable chamber 101 is preferably made by silicone rubber with thickness from 0.5 to 2 mm, more preferably, from 0.5 to 1 mm, which can be semi-rigid under certain thickness and can be stand still without requiring extra supporting means.
[0049] The apparatus for preparing and culturing cells shown in FIG. 3A, FIG. 3B shows a side view of a cell-cultivation apparatus of the third embodiment of the present invention with pre-assemble (FIG. 3A) and post-assemble (FIG. 3B) for ease of understanding. The cell-cultivating apparatus includes a gas permeable chamber 101 with a first open end 111 and a second open end 112, wherein a cap 102 with threads is detachably mounted in the first end, and another cap 102′ with threads 22′ and a growth substrate 105 are detachably mounted at the second end of the gas permeable chamber 101. The growth substrate 105 is a porous sheet or disc that is fixed by a wedge ring 106 molded in another cap 102′. In order to secure the caps 102, 102′ to be air tight, rigid necks 103, 103′ with external threads 23, 23′ are detachably mounted on the gas permeable chamber 101 both on top and bottom open ends in order to engage and secure with internal threads 22, 22′ of the caps 102, 102′.
[0050] The apparatus for preparing and culturing cells shown in FIG. 4A, 4B and FIG. 5A, FIG. 5B show side views of a cell-cultivation apparatus of the fourth and fifth embodiments of the present invention with pre-assemble (FIG. 4A, FIG. 5A) and post-assemble (FIG. 4B, FIG. 5B) for ease of understanding. The cell-cultivation apparatus 11 includes a gas permeable chamber 101 with a first open end and a second open end, wherein a cap 102 is detachably mounted at the first end, and a growth substrate holder 104 and the growth substrate 105 is detachably mounted at the second end of the gas permeable chamber 101. The growth substrate 105 is a plurality of discs or strips of porous matrices, such as BioNOC II carriers (CESCO Bioengineering Co., Ltd., Taiwan), that are disposed in the growth substrate holder 104. The growth substrate holder 104 is a basket that has top openings 107 for transfer and exchange of culture medium and air during operation. The gas permeable chamber 101 is closed by a rigid cap 102 with threads. In order to secure the cap 102 to be air tight, a rigid neck or a rigid tube 103 with external threads is detachably mounted on the gas permeable chamber 101 in order to engage and secure with the cap 102.
[0051] The apparatus for preparing and culturing cells in FIG. 6A, FIG. 6B and FIG. 7A, FIG. 7B show side views of a cell-cultivation apparatus of the sixth and seventh embodiments of the present invention with pre-assemble (FIG. 6A, FIG. 7A) and post-assemble (FIG. 6B, FIG. 7B) for ease of understanding. The cell-cultivation apparatus includes a gas permeable chamber 101 with an open end 111 and a close end 113, wherein a cap 102 is detachably mounted at the open end 111, and a growth substrate holder 104 and a growth substrate 105 is detachably mounted at the close end 113 of the gas permeable chamber 101. The growth substrate 105 is a plurality of discs or strips of porous matrices, such as BioNOC II carriers (CESCO Bioengineering Co., Ltd., Taiwan), that are disposed in the growth substrate holder 104. The growth substrate holder 104 is a basket that has top openings 107 for transfer and exchange of culture media and air during operation. The gas permeable chamber 101 is closed by a rigid cap 102. In order to secure the cap 102 to be air tight, a rigid neck or a rigid tube 103 with external threads is detachably mounted on the gas permeable chamber 101 in order to engage and secure with the cap 102. It's optional to use other designs to secure the cap such as flanges.
[0052] Accordingly, the gas permeable chamber of silicon rubber is beneficial for users to see through during cell cultivation. That is, the growth substrate and culture medium in the gas permeable chamber are visible for user to observe them from outside of the gas permeable chamber. It is also noted that the growth substrate may be deposited at a suitable position within the gas permeable chamber where it may be repeatedly submerged in or exposed out of the culture medium in the gas permeable chamber.
[0053] The apparatus for preparing and culturing cells shown in FIG. 8 shows a side view of a cell-cultivation apparatus of the eighth embodiment of the present invention, especially for close operation. The cell-cultivation apparatus includes a gas permeable chamber 101 with a close end 113 and an open end 111, wherein a plurality of openings with tubes 108, 108′ are detachably mounted at the close end 113 of the gas permeable chamber 101, and these openings with tubes 108, 108′ are close by fittings 109, 109′ such as luer fittings. A growth substrate holder 104 and a growth substrate 105 is detachably mounted at the open end 111 of the gas permeable chamber 101. The growth substrate 105 is a plurality of discs or strips of porous matrices, such as BioNOC II carriers (CESCO Bioengineering Co., Ltd., Taiwan), that are disposed in the growth substrate holder 104. The growth substrate 105 could also be a sheet of porous matrices. The growth substrate holder 104 is a basket that has top openings 107 for transfer and exchange of culture media and air during operation.
[0054] FIG. 9A and FIG. 9B show devices for rotating the cell cultivation apparatus for cell growth according to the present invention. The devices 202, 202′ respectively shown in FIG. 9A and FIG. 9B include at least one clamp or holder or platform 203 disposed to hold the cell-cultivation apparatus, and the clamp or the holder or the platform 203 is connected to a motor 201 through a shaft 204 and bearings 205, preferably two-way step motor. Shown from FIG. 10A, FIG. 10B to FIG. 10C, the motor of the device 202 may rotate slowly clockwise or counterclockwise until reach 180 degree of angle, and then stops and holds the cell cultivation apparatus 11 with an interval time. A timing controller (not shown) is used to control the interval time. Then the motor of the device 202 rotates again counterclockwise or clockwise slowly until reach 180 degree of angle, and then stops again and holds the cell cultivation apparatus with the interval time. The degree of angle is not necessary to reach 180 degree but to expose and submerge the growth substrate is the target that determines the minimum degree of angle for rotation.
[0055] The apparatus for preparing and culturing cells in FIG. 11 shows the cell cultivation apparatus of the present invention where the device 302 includes a plurality of clamps or holders 203 is disposed to hold the cell-cultivation apparatus, the clamps or the holders 203 connected to a motor 201 through a shaft 204 and bearings 205, 205′, preferably two-way step motor. The motor 201 rotates slowly clockwise or counterclockwise until reach 180 degree of angle, and then stops and holds the cell cultivation apparatus with an interval time. A timing controller (not shown) is used to control the interval time. Then the motor 201 rotates again counterclockwise or clockwise slowly until reach 180 degree of angle, and then stops again and holds the cell cultivation apparatus with the interval time. The degree of angle is not necessary to reach 180 degree but to expose and submerge the growth substrate 105 (referring to FIG. 1A, and so on) is the target that determines the minimum degree of angle for rotation. The rotation speed is smaller than 10 rpm, or greater than 0 and smaller than or equal to 2 rpm, or preferably, greater than 0 and smaller than or equal to 1 rpm. The interval time at upright and bottom down position is about or less than 60 minutes, preferably, about 10 minutes. Accordingly, the device 302 executes a rotation process for cell cultivation apparatus, and the rotation process includes clockwise or counterclockwise rotating the cell cultivation apparatus and maintain the cell cultivation apparatus to be still with the interval time between two rotating steps.
[0056] The apparatus for preparing and culturing cells shown in FIG. 12A, FIG. 12B and FIG. 12C shows the mechanism of a cell-cultivation apparatus of the present invention, as an example from all designs, where in the culture medium 12 is partially filled in the gas permeable chamber 101, the growth substrate 105 is at the bottom of the gas permeable chamber 101 at the beginning (for example) and is submerged in the culture medium 12. The amount of the filled culture medium 12 is judged by the capability to expose the cell growth substrate 105 completely when rotating the gas permeable chamber 101 and the growth substrate 105 is at top position.
[0057] After mounting the cell cultivating apparatus on the driving device, as disclosed in FIG. 10A and FIG. 12A, and start rotation, the gas permeable chamber 101 is then rotated clockwise or counterclockwise where in part of the gas permeable chamber 101 is exposed to gaseous phase and part of the gas permeable chamber 101 is submerged to culture medium 12 of liquid phase. The gas permeable chamber 101 keeps rotating clockwise or counterclockwise continuously (middle state shown in FIG. 12B) until the growth substrate 105 reach at the top position (shown in FIG. 12C) where the growth substrate 105 in the higher end of the gas permeable chamber 101 is thus emerge from the culture medium and exposed to the gaseous environment. Then rotating the gas permeable chamber 101 upside-down to submerge the substrate in the medium. The partial filled of culture medium 12 in the gas permeable chamber 101 can be used to eliminate the boundary effect of oxygen and carbon dioxide transfer by skimpily remove the liquid with gaseous head space by rotating a partial liquid filled gas permeable chamber 101. The partial filled of culture medium in the gas permeable chamber 101 can also be used to expose the growth substrate 105 to the gaseous phase and enhance gas transfer rate for both oxygen and carbon dioxide. Through this special design and mechanism, the oxygen and carbon dioxide transfer rate can be further accelerated, and a result of high-density cell culture could be reached, without requiring vigorous agitation and/or bubble sparging.
[0058] Shown in FIG. 13, in the ninth example, the gas permeable chamber 101 may provide a narrow portion 33 for fitting of the clamp of the device 202 (shown in FIG. 9A) for driving. An additional member 34 may be mounted onto the narrow portion 33 for supporting. The member 34 may be transparent without blocking visibility. There may be some marks 35 on the member 34 for liquid level aligning. Optionally, the marks 35 for liquid level aligning may be directly attached onto the suitable position of wall of the gas permeable chamber 101.
[0059] FIG. 14A and FIG. 14B show side views of a cell-cultivation apparatus of the tenth embodiment of present invention with pre-assemble and post-assemble. In addition to the narrow portion 33 in the nineth example, the gas permeable chamber 101 further includes a fixing portion 37 at the close end of the gas permeable chamber 101. Moreover, the marks 35 are directed formed on the wall of the gas permeable chamber 101 and the wall of the gas permeable chamber 101 is transparent enough for a user to see the level of culture medium in the gas permeable chamber 101. A sheet of the growth substrate 105 may be put into the gas permeable chamber 101 and mounted by the fixing portion 37. And the bottom close end of the gas permeable chamber 101 may be transparent enough for a user to see the sheet of the growth substrate 105.
[0060] The present invention is directed to a reliable, simple, inexpensive, disposable, sterile and efficient method for culturing cells and/or tissues and harvesting cellular products produced by cells cultured thereof. More specifically, the present invention provides a novel method for efficiently culturing any cells whether eukaryotic, prokaryotic, mammalian or animal wherein both oxygen and nutrients needed to ensure cell growth are readily available without causing damage to cells. Furthermore, the method of the present invention prevents or greatly reduces the metabolite waste accumulation, avoid introducing shear forces on growing cultures, and protect cells from direct exposure to gas, air bubbles and gases. Further still, the instant invention provides a method for an easier and more convenient means for producing and harvesting secreted cellular products such as proteins, antibodies from cell or tissue cultures.
[0061] The VERO cells, a common cell line for virus expression, were grown according to the instant invention. The first step was to open the cap on the gas permeable vessel (60 ml total volume) made by silicone rubber, where around 20 pieces of BioNOC II (occupy around 1.5 ml) are packed and fixed at the bottom end of the gas permeable vessel, then 1×10.sup.7 VERO cells were introduced with 50 ml culture medium into the vessel, the cap was closed and make sure it was air tight, make sure the carriers can be exposed to gaseous headspace after reversing the vessel, mounted the gas permeable vessel on the clamp set on the device, set up rotation rate as 0.3 rpm, top interval time is 10 seconds, and bottom interval time is 10 seconds, and started the culture. The device rotated the gas permeable vessel clockwise with 0.3 rpm it until reach 180 degree of angle until the gas permeable vessel was upside down and the carriers were exposed, then will hold 10 seconds, then the device rotated the gas permeable vessel counterclockwise with 0.3 rpm until reached 180 degree of angle until the gas permeable vessel stood upright and the carriers were submerged in the culture medium, another 10 seconds was hold at the position before starting another cycle. After 3 days culture, the cell density reached 3.891×10.sup.7 by estimating the cell number with sampled carriers. After 6 days culture, the cell density reaches 7.91×10.sup.7, and the carriers were filled with cells when observed under microscope.
[0062] The embodiments described above are merely illustrative of the technical spirit and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and practice the present invention. The scope of the patent, that is, the equivalent changes or modifications made by the spirit of the present invention, should still be included in the scope of the patent of the present invention.
