CN211972297U - Cell separation device for bioreactor - Google Patents
Cell separation device for bioreactor Download PDFInfo
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- CN211972297U CN211972297U CN202020183859.1U CN202020183859U CN211972297U CN 211972297 U CN211972297 U CN 211972297U CN 202020183859 U CN202020183859 U CN 202020183859U CN 211972297 U CN211972297 U CN 211972297U
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Abstract
The present disclosure discloses a cell separation device for a bioreactor, comprising: a cell separation assembly disposed within the tank and submerged below a liquid level of the mixture and comprising a filtration membrane configured to filter the mixture and direct separated liquid into a liquid gutter, the liquid gutter comprising a radially outer side wall and a radially inner side wall, an interior cavity of the liquid gutter being in fluid communication with the tank only through the radially inner side wall or one or more openings on the radially outer side wall; and a tangential flow drive assembly comprising one or more paddles located radially inward of the cell separation assembly and rotatable about the axis of rotation to drive the mixture into a tangential circulatory flow along both the inside and outside surfaces of the cell separation assembly. The cell separation device achieves low shear force and does not easily clog the filter membrane.
Description
Technical Field
The present disclosure generally relates to the field of bioreactors. More specifically, the present disclosure relates to a cell separation device for a bioreactor.
Background
In animal cell expansion, an increasing number of processes involve separation of cells from culture medium or other liquids, including perfusion culture, liquid change or cell washing, etc. Conventional separation equipment includes an ATF system, a TFF system, and the like. These systems utilize diaphragm pumps or centrifugal pumps as the motive force to pump the mixture of cells and culture medium into the hollow fiber column and utilize the fiber pores of the hollow fiber column for separation of cells from the culture medium. These systems employ diaphragm pumps or centrifugal pumps as the motive force for pumping the cell culture medium mixture, which suffer from the disadvantage of excessive shear forces. Animal cells (especially human T cells or stem cells) are very sensitive to shearing force, and too high shearing force can cause tumor formation of stem cells or loss of cell markers, thereby seriously affecting the use safety of cell therapy products.
Another separation device utilizes a filtration system that floats above the level of the mixture to separate the cells from the culture medium. The mixture flow direction of the separation device is consistent with the filtration direction of the filter membrane, and the filter membrane is easily blocked during the use process. The filter membrane blockage often means the failure of the whole process, which not only brings huge economic loss to enterprises, but also delays the precious biological samples and treatment time of patients.
SUMMERY OF THE UTILITY MODEL
It is an object of the present disclosure to provide a cell separation device that overcomes at least one of the deficiencies of the prior art.
The subject technology of the present disclosure is illustrated in accordance with aspects described below. For convenience, various examples of aspects of the subject technology are described as clauses (1, 2, 3, etc.) of the reference numerals. These terms are provided as examples and do not limit the subject technology of the present disclosure.
1. A cell separation device for a bioreactor comprising a tank for containing a mixture of cells and a liquid, wherein the cell separation device comprises:
a cell separation assembly disposed within the tank and submerged below a liquid level of the mixture, the cell separation assembly comprising a filter membrane configured to filter the mixture and direct separated liquid into a liquid gutter, the liquid gutter comprising a radially outer sidewall and a radially inner sidewall, an internal cavity of the liquid gutter being in fluid communication with the tank only through the one or more openings in the radially inner sidewall and through the filter membrane, or only through the one or more openings in the radially outer sidewall and through the filter membrane; and
a tangential flow drive assembly comprising one or more paddles located radially inward of the cell separation assembly and rotatable about an axis of rotation to drive tangential flow of the mixture along the filter membrane.
2. The cell separation device of clause 1, wherein the cell separation device further comprises a liquid collection assembly disposed outside the tank and comprising a collection container fluidly connected to the liquid draft trough.
3. The cell separation device of clause 2, wherein the liquid collection assembly comprises a collection tube fluidly connecting the collection container to the liquid draft trough.
4. The cell separation device according to clause 3, wherein a pump is provided on the collection tube and configured to pump the separated liquid from the liquid guiding gutter to the collection container.
5. The cell separation device of clause 4, wherein the pump comprises a peristaltic pump.
6. The cell separation apparatus according to clause 3, wherein a pressure sensor is provided in the collection tube to monitor a liquid pressure in the collection tube.
7. The cell separation device of any one of clauses 1-6, wherein the cell separation assembly further comprises a support sheet positioned between the liquid draft channel and the filter membrane, the support sheet configured to support the filter membrane.
8. The cell separation device according to clause 7, wherein the filter membrane is fixed to the support sheet by heat fusion, adhesion, or laser welding.
9. The cell separation device according to clause 7, wherein the support sheet is attached to the liquid guiding gutter by heat fusion, male-female fitting, or laser welding.
10. The cell separation apparatus according to clause 3, wherein the liquid guide groove is provided with a guide tube connected to the collection tube.
11. The cell separation device according to clause 10, wherein the flow guide tube extends vertically upward from the top wall of the liquid guide channel.
12. The cell separation device according to any one of clauses 1-6, wherein the liquid draft channel is provided with a support column configured to support the cell separation assembly above and spaced apart from the bottom of the tank.
13. The cell separation device according to clause 12, wherein the support column is configured to be connected to a radially inner side wall of the liquid guiding gutter by radiating arms radially therefrom.
14. The cell separation device according to clause 12, wherein the support column is configured to extend downward from a bottom wall of the liquid guiding gutter.
15. The cell separation device according to any one of clauses 1 to 6, wherein the filter membrane is made of PTFE, PP, PC, nylon, PES, or a sintered porous material.
16. The cell separation device according to any one of clauses 1-6, wherein the pore size of the filtration membrane is about 5 μm, about 10 μm, about 20 μm, about 50 μm, about 100 μm, or about 200 μm.
17. The cell separation apparatus according to clause 7, wherein the support sheet is made of a perforated soft material.
18. The cell separation apparatus of clause 17, wherein the soft material comprises a soft gel material.
19. The cell separation device according to clause 17, wherein the shape of the hole is substantially circular, substantially square, or substantially hexagonal.
20. The cell separation device according to clause 7, wherein the support sheet is made of a fibrous porous material.
21. The cell separation device according to clause 7, wherein the support sheet has a thickness of between 50 μm and 300 μm.
22. The cell separation device according to any one of clauses 1 to 6, wherein the tangential flow drive assembly further comprises a motor for driving the rotation of the paddle.
23. The cell separation device according to any one of clauses 1 to 6, wherein the paddle is provided with a diameter slightly smaller than an inner diameter of the cell separation assembly to provide a tangential flow effect in a narrow space.
24. The cell separation device according to any one of clauses 1 to 6, wherein the diameter of the paddle is configured to be 30 to 45% of the inner diameter of the can.
25. The cell separation device according to any one of clauses 1 to 6, wherein a distance between a radially outermost point of the paddle and a radially inner surface of the filter membrane is between 1mm and 5 mm.
26. The cell separation device of any one of clauses 1-6, wherein the paddle is in the form of a fully axial flow paddle.
27. The cell separation device according to any one of clauses 1 to 6, wherein the paddle is in the form of a single layer or multiple layers like an ear paddle, a sea paddle, or a ribbon paddle.
Additional features and advantages of the disclosed subject technology will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosed subject technology. The advantages of the subject technology of the present disclosure will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology of the present disclosure as claimed.
Drawings
Various aspects of the disclosure will be better understood upon reading the following detailed description in conjunction with the drawings in which:
FIG. 1 is a schematic view of a cell separation device according to an embodiment of the present disclosure;
FIG. 2 is a partial cross-sectional view of a cell separation assembly of the cell separation device shown in FIG. 1;
FIG. 3 is a cross-sectional view of a cell separation assembly of the cell separation device shown in FIG. 1; and
FIGS. 4 and 5 are schematic views of a support sheet of the cell separation device shown in FIG. 1.
Detailed Description
The present disclosure will now be described with reference to the accompanying drawings, which illustrate several embodiments of the disclosure. It should be understood, however, that the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, the embodiments described below are intended to provide a more complete disclosure of the present disclosure, and to fully convey the scope of the disclosure to those skilled in the art. It is also to be understood that the embodiments disclosed herein can be combined in various ways to provide further additional embodiments.
It should be understood that like reference numerals refer to like elements throughout the several views. In the drawings, the size of some of the features may be varied for clarity.
It is to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting of the disclosure. All terms (including technical and scientific terms) used in the specification have the meaning commonly understood by one of ordinary skill in the art unless otherwise defined. Well-known functions or constructions may not be described in detail for brevity and/or clarity.
As used in this specification, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. The terms "comprising," "including," and "containing" when used in this specification specify the presence of stated features, but do not preclude the presence or addition of one or more other features. The term "and/or" as used in this specification includes any and all combinations of one or more of the associated listed items. The terms "between X and Y" and "between about X and Y" as used in the specification should be construed to include X and Y. The term "between about X and Y" as used herein means "between about X and about Y" and the term "from about X to Y" as used herein means "from about X to about Y".
In the description, when an element is referred to as being "on," "attached" to, "connected" to, "coupled" to, or "contacting" another element, etc., another element may be directly on, attached to, connected to, coupled to, or contacting the other element, or intervening elements may be present. In contrast, when an element is referred to as being "directly on," "directly attached to," directly connected to, "directly coupled to," or "directly contacting" another element, there are no intervening elements present. In the description, one feature is disposed "adjacent" another feature, and may mean that one feature has a portion overlapping with or above or below an adjacent feature.
In the specification, spatial relations such as "upper", "lower", "left", "right", "front", "rear", "high", "low", and the like may explain the relation of one feature to another feature in the drawings. It will be understood that the spatial relationship terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, features originally described as "below" other features may be described as "above" other features when the device in the figures is inverted. The device may also be otherwise oriented (rotated 90 degrees or at other orientations) and the relative spatial relationships may be interpreted accordingly.
FIG. 1 shows a cell separation device 1 according to an embodiment of the present disclosure. The cell separation apparatus 1 is used for a small-sized disposable bioreactor. The disposable bioreactor comprises a tank 2 to contain a mixture of cells and culture medium, or a mixture of cells and other liquids. As shown, the cell separation device 1 includes a tangential flow drive assembly 3, a cell separation assembly 4, and a liquid collection assembly 5. The tangential flow drive assembly 3 and the cell separation assembly 4 can be placed in the bioreactor within the tank 2, while the liquid collection assembly 5 can be placed in the bioreactor outside the tank 2. The tangential flow drive assembly 3 is used to circulate a mixture of cells and media (or other liquid) tangentially along the cell separation assembly 4, the cell separation assembly 4 is used to separate the cells from the media (or other liquid) during the tangential circulation of the mixture, and the liquid collection assembly 5 is used to collect the media or other liquid separated from the cells.
The cell separation assembly 4 is placed within the tank 2 and submerged below the level of the mixture of cells and medium (or other liquid). The cell separation module 4 is in the form of a generally hollow cylinder and comprises a filter membrane 41, a support sheet 42 and a liquid guide channel 43 which are adjacent to one another, as shown in figures 2 and 3. The filter membrane 41 is used for filtering a mixture of cells and culture medium (or other liquid), and the culture medium (or other liquid) flows to the liquid guiding groove 43 through the filter membrane 41 while blocking the cells from passing through the filter membrane 41. The support sheet 42 serves to support the filter membrane 41 so that the filter membrane 41 is kept flat. The liquid guiding groove 43 is used for accommodating the culture medium (or other liquid) passing through the filtering membrane 41 and guiding the culture medium to flow out of the tank 2.
As shown in fig. 3, liquid guiding channel 43 is in the form of a hollow cylinder and includes a radially outer side wall 44 and a radially inner side wall 47, and a top wall 45 and a bottom wall 46 extending between radially outer side wall 44 and radially inner side wall 47. The radially outer side wall 44, the radially inner side wall 47, the top wall 45 and the bottom wall 46 enclose the inner cavity of the liquid guiding channel 43 and are in fluid communication with the tank 2 only through the one or more openings 49 in the radially inner side wall 47 and through the filter membrane 41.
The liquid guide groove 43 is also provided with a guide pipe 48 for guiding the contained culture medium (or other liquid) out of the liquid guide groove 43. The draft tube 48 may extend vertically upward from the top wall 45 of the liquid draft channel 43, for example, to above the level of the mixture of cells and medium (or other liquid).
As shown in FIG. 1, the liquid guiding gutter 43 is further provided with a support column 49 to support the liquid guiding gutter 43 and the whole cell separation module 4 above the bottom of the tank 2 and spaced from the bottom of the tank 2 so that the mixture of cells and culture medium (or other liquid) can circulate through the gap below the cell separation module 4. Support posts 49 may extend downwardly from bottom wall 46 of liquid channel 43. In other embodiments, the support posts 49 may be located within the hollow cylinder of the liquid guiding gutter 43 and connected to the radially inner side wall 47 of the liquid guiding gutter 43 by radiating arms radially therefrom. In some embodiments, the height of support columns 49 is designed so that the gap between cell separation assembly 4 and canister 2 is as small as possible to avoid turbulence that affects cell growth.
The filter membrane 41 may be made of PCTE, PETE, PTFE, PP, PC, nylon, PES, or sintered porous material. The filter membrane 41 is subjected to hydrophilic and positive or negative charge treatment, so that it is not easily adsorbed by cells to clog the filter membrane. The filter membrane 41 may have various pore sizes (including about 5 μm, about 10 μm, about 20 μm, about 50 μm, about 100 μm, about 200 μm, etc.) depending on the biological process (e.g., stem cell, tumor cell, CHO cell, or microcarrier process, etc.).
The filter membrane 41 is fixed to the support sheet 42 by heat fusion, adhesion, laser welding, etc., and the support sheet 42 is connected to the radially inner side wall 47 of the liquid guiding groove 43 by heat fusion, male-female fit, laser welding, etc.
In some embodiments, radially outer side wall 44, radially inner side wall 47, top wall 45 and bottom wall 46 of liquid channel 43 enclose the internal cavity of liquid channel 43 and the internal cavity is in fluid communication with tank 2 only through the one or more openings in radially outer side wall 44 (and not radially inner side wall 47) and through filter membrane 41. The filter membrane 41 is fixed to the support sheet 42 by heat fusion, bonding, laser welding, etc., and the support sheet 42 is connected to the radially outer side wall 44 of the liquid guiding groove 43 by heat fusion, male-female fitting, laser welding, etc.
The tangential flow drive assembly 3 comprises one or more blades 31 and a motor 32 for driving the blades 31 in rotation. The paddle 31 is placed inside the tank 2 and is immersed in the mixture of cells and culture medium (or other liquid). The paddle 31 may be located radially inward of the cell separation assembly 4 and rotated about the axis of rotation 33 to drive the mixture of cells and media (or other liquid) to circulate tangentially (i.e., vertically) along the inner and outer surfaces of the cell separation assembly 4 (e.g., including the filter membrane 41, the radially outer sidewall 44 and the radially inner sidewall 47 of the liquid guiding channel 43, etc.). The rotation of the paddle 31 does not generate excessive shear forces and therefore does not cause damage to the animal cells. The body of the motor 32 is placed outside the can body 2, and its driving shaft is connected directly to the rotating shaft 33 of the paddle 31 or connected to the rotating shaft 33 of the paddle 31 through a rotational speed adjusting mechanism such as a gear box to drive the rotating shaft 33 and the paddle 31 to rotate. The motor 32 may be connected to a controller (not shown).
The output power of the motor 32 is one of the critical design parameters. The greater the output power of the motor 32, the greater the tangential flow velocity of the mixture and the longer the service life of the filter membrane 41. Especially in the case of high-density cell culture, an increase in the tangential flow velocity can reduce the clogging velocity of the filter membrane 41.
The distance between the paddle 31 and the filter membrane 41 is also one of the critical design parameters. The closer the distance, the significantly higher the rate at which the liquid in the mixture passes through the filter membrane 41, but also the local shear force of the liquid, which has an effect on the physiological properties of the stem cells. The diameter of the paddle 31 is set slightly smaller than the inner diameter of the filter membrane 41 (e.g., the distance between the radially outermost point of the paddle 31 and the radially inner surface of the filter membrane 41 is between about 1mm and about 5 mm) to provide a tangential flow effect in a narrow space. In some embodiments, the diameter of the paddles 31 may be designed to be about 30% -45% of the inner diameter of the can body 2. The blades 31 may take the form of fully axial flow blades, such as single or multi-layer, ear-like blades, marine blades, and ribbon blades, among others.
The liquid collection assembly 5 is placed outside the tank 2. The liquid collection assembly 5 comprises a collection container 51 and a collection tube 52. The collection pipe 52 is connected at one end to the collection vessel 51 and at the other end to the guide pipe 48 of the liquid guide tank 43 to guide the culture medium (or other liquid) of the liquid guide tank 43 to the collection vessel 51. In some embodiments, the collection tube 52 may be flexible, such that the collection container 51 may be placed at any suitable location outside the canister 2. A pump 53 (e.g., peristaltic pump, etc.) may be disposed on collection tube 52 and connected to the controller to assist in drawing and pumping the media (or other liquid) to collection vessel 51. The controller can control the pump speed of the pump 53 to vary to adjust the drainage rate based on process requirements, the pump speed and feed weight of other feed pumps, or the signals from some sensors (e.g., a live cell sensor).
In some embodiments, a pressure sensor 54 connected to a controller may be provided on collection tube 52 to monitor the liquid pressure within collection tube 52. When the pressure sensor 54 detects that the cells clog the filter membrane 41 and the pressure in the collection pipe 52 rises, the controller stops the operation of the pump 53 and sends an alarm signal to the operator.
The operation of the cell separation apparatus 1 will be described below. Firstly, the bioreactor is placed in a super clean bench to carry out operations such as culture medium addition, inoculation and the like, and then the bioreactor is placed on a control bench and connected with a corresponding sensor. The controller sends an activation signal to the motor 32, and the motor 32 drives the paddle 31 to rotate about the rotation axis 33, thereby driving the mixture of cells and culture medium (or other liquid) to circulate tangentially (i.e., vertically) along the inner and outer surfaces of the cell separation assembly 4 (e.g., including the filter membrane 41, the radially outer sidewall 44, the radially inner sidewall 47 of the liquid guiding gutter 43, etc.). The controller activates the pump 53 when a fluid change, wash, or cell or microcarrier separation is required. The suction created by pump 53 causes the culture medium (or other liquid) to separate from the cells at filter membrane 41 and into liquid diversion channel 43 and through collection tube 52 into collection container 51.
After a certain volume of waste medium is drained from the tank 2, the pump 53 may be operated in reverse, so that the liquid in the collection pipe 52 can flow in reverse and back flush against the filter membrane 41 to prevent cells from clogging the filter membrane 41.
The cell separation apparatus 1 according to the embodiment of the present disclosure drives the mixture to flow using the paddle 31, so that a low shear force can be achieved without affecting the cells.
In addition, the cell separation device 1 according to the embodiment of the present disclosure performs filtration using a tangential flow of the mixture along the filtration membrane, and is not likely to clog the filtration membrane, thereby being usable for a long time.
Although exemplary embodiments of the present disclosure have been described, it will be understood by those skilled in the art that various changes and modifications can be made to the exemplary embodiments of the present disclosure without substantially departing from the spirit and scope of the present disclosure. Accordingly, all changes and modifications are intended to be included within the scope of the present disclosure as defined in the appended claims. The disclosure is defined by the following claims, with equivalents of the claims to be included therein.
Claims (27)
1. A cell separation device for use in a bioreactor including a tank for containing a mixture of cells and a liquid, the cell separation device comprising:
a cell separation assembly disposed within the tank and submerged below a liquid level of the mixture, the cell separation assembly comprising a filter membrane configured to filter the mixture and direct separated liquid into a liquid gutter, the liquid gutter comprising a radially outer sidewall and a radially inner sidewall, an internal cavity of the liquid gutter being in fluid communication with the tank only through the one or more openings in the radially inner sidewall and through the filter membrane, or only through the one or more openings in the radially outer sidewall and through the filter membrane; and
a tangential flow drive assembly comprising one or more paddles located radially inward of the cell separation assembly and rotatable about an axis of rotation to drive tangential flow of the mixture along the filter membrane.
2. The cell separation device of claim 1, further comprising a liquid collection assembly disposed outside the tank and comprising a collection container fluidly connected to the liquid diversion trench.
3. The cell separation device of claim 2, wherein the liquid collection assembly comprises a collection tube fluidly connecting the collection container to the liquid flow-directing trough.
4. The cell separation device of claim 3, wherein a pump is disposed on the collection tube and configured to pump separated liquid from the liquid guiding gutter to the collection container.
5. The cell separation device of claim 4, wherein the pump comprises a peristaltic pump.
6. The cell separation apparatus according to claim 3, wherein a pressure sensor is provided in the collection tube to monitor a pressure of the liquid in the collection tube.
7. The cell separation device of any one of claims 1-6, wherein the cell separation assembly further comprises a support sheet positioned between the liquid flow-directing groove and the filter membrane, the support sheet configured to support the filter membrane.
8. The cell separation device according to claim 7, wherein the filter membrane is fixed to the support sheet by heat fusion, adhesion, or laser welding.
9. The cell separation device according to claim 7, wherein the support sheet is attached to the liquid guiding groove by heat fusion, male-female fitting, or laser welding.
10. The cell separation apparatus according to claim 3, wherein the liquid guide channel is provided with a guide tube connected to the collection tube.
11. The cell separation device of claim 10, wherein the draft tube extends vertically upward from the top wall of the liquid flow guide.
12. The cell separation device according to any one of claims 1 to 6, wherein the liquid draft channel is provided with a support column configured to support the cell separation assembly above and spaced apart from the bottom of the tank.
13. The cell separation device of claim 12, wherein the support column is configured to be connected to a radially inner side wall of the liquid draft channel by radiating arms radially therefrom.
14. The cell separation device of claim 12, wherein the support posts are configured to extend downwardly from a bottom wall of the liquid draft channel.
15. The cell separation device according to any one of claims 1 to 6, wherein the filter membrane is made of PTFE, PP, PC, nylon, PES or sintered porous material.
16. The cell separation device according to any one of claims 1 to 6, wherein the pore size of the filtration membrane is about 5 μm, about 10 μm, about 20 μm, about 50 μm, about 100 μm, or about 200 μm.
17. The cell separation apparatus according to claim 7, wherein the support sheet is made of a perforated flexible material.
18. The cell separation apparatus of claim 17, wherein the soft material comprises a soft gel material.
19. The cell separation device of claim 17, wherein the shape of the aperture is substantially circular, substantially square, or substantially hexagonal.
20. The cell separation device according to claim 7, wherein the support sheet is made of a fibrous porous material.
21. The cell separation device according to claim 7, wherein the thickness of the support sheet is between 50 μm and 300 μm.
22. The cell separation device of any one of claims 1-6, wherein the tangential flow drive assembly further comprises a motor for driving rotation of the paddle.
23. The cell separation device according to any one of claims 1 to 6, wherein the diameter of the paddle is set slightly smaller than the inner diameter of the cell separation assembly to provide a flow effect of a tangential flow in a narrow space.
24. The cell separation device according to any one of claims 1 to 6, wherein the diameter of the paddle is configured to be 30% to 45% of the inner diameter of the can.
25. The cell separation device according to any one of claims 1 to 6, wherein a distance between a radially outermost point of the paddle and a radially inner surface of the filter membrane is between 1mm and 5 mm.
26. A cell separation device according to any one of claims 1 to 6, wherein the paddle is in the form of a fully axial flow paddle.
27. The cell separation device according to any one of claims 1 to 6, wherein the paddle is in the form of a single or multiple layer like an ear paddle, sea paddle or ribbon paddle.
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| WO2021164509A1 (en) * | 2020-02-19 | 2021-08-26 | 上海艾众生物科技有限公司 | Cell separation apparatus for bioreactor |
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| WO2021164509A1 (en) * | 2020-02-19 | 2021-08-26 | 上海艾众生物科技有限公司 | Cell separation apparatus for bioreactor |
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Effective date of registration: 20220630 Address after: Room 211, building 21, 1158 Jiuting Central Road, Jiuting Town, Songjiang District, Shanghai, 201615 Patentee after: Alite Biotechnology (Shanghai) Co.,Ltd. Address before: 5 / F, building 13, No. 68, Zhongchuang Road, Songjiang District, Shanghai, 201613 Patentee before: Shanghai Aizhong Biotechnology Co.,Ltd. |
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