Cell suspension culture device equipped with a fluid splitter

Abstract

The present invention relates to a cell suspension culture device (1) comprising a culture chamber (2), a configured fluid inlet (11), and a fluid outlet (14). The device (1) further comprises a fluid divider (21) disposed within the culture chamber (2) and positioned between the fluid inlet (11) and the fluid outlet (14) with respect to the fluid supply direction. The fluid divider (21) has at least one fluid inlet (22) that is in fluid communication with the fluid inlet (1), and further has a plurality of fluid inlet / outlet (25) that are in fluid communication with the fluid inlet (22). The fluid divider (21) is configured to introduce a plurality of fluid flows into the culture chamber (2) via the plurality of fluid inlet / outlet (25). In one embodiment, the device (1) includes at least one scaffold holder (50).

Description

Technical Field

[0001] The present invention relates to a cell suspension culture device equipped with a fluid diverter. The culture device can be configured to enable both suspension culture and adherent culture.

[0002] The present invention also relates to a method of operating a cell suspension culture device, the use of the device, and a process for assembling the culture device.

Background Art

[0003] Cell culture devices, i.e., microgravity generating devices, are known in which cells grow in suspension from a moving fluid in a flow chamber or a culture chamber.

[0004] Such devices range in size from a few milliliters to several tens of liters and are used in the laboratory to conduct tests on cells grown in suspension, and in an industrial environment for large-scale production of cells for scientific or industrial use, and further in the vaccine manufacturing process.

[0005] In known devices, suspension is generated either actively or passively. In "active" devices, suspension is generated by rotating components driven by dedicated actuators. On the other hand, in "passive" devices, such rotating components do not exist.

[0006] Furthermore, all of these devices, whether "active" or "passive", do not ensure proper diffusion of oxygen and nutrients in the area of the motion field where cells are suspended. As a result, the rate of cell growth and production is low, and furthermore, the rate can only be evaluated empirically.

[0007] In international patent application 2020095143(A1) by the same applicant, an attempt was made to improve passive devices for handling flow into a culture chamber. This improvement includes providing a valve mechanism driven by a mechanical or magnetic operating device.

[0008] The applicant noted that while the device described in International Publication No. 2020095143(A1) offers significant advantages over the prior art, there is room for improvement in several respects. In particular, the device has room for improvement in handling cells and / or particles in suspension, as cells tend to accumulate and adhere to the bottom of the culture chamber. [Overview of the project]

[0009] Therefore, the main objective of the present invention is to overcome the aforementioned drawbacks in relation to the prior art.

[0010] The objective of the present invention is to improve the handling of cells and / or particles in cell suspension culture devices.

[0011] More specifically, the object of the present invention is to improve the handling of suspended cells and / or particles by optimizing the movement field generated within the culture chamber.

[0012] Therefore, an object of the present invention is to provide a cell suspension culture device that enables optimal cell growth within the culture chamber through a movement field generated within the culture chamber.

[0013] These and other objectives are achieved by fluid dividers, cell suspension culture devices equipped with fluid dividers, and methods for operating cell culture devices, according to the following description, the appended claims, and the embodiments described later.

[0014] Aspects of the present invention constitute an integral part of the technical content of this patent document and are described herein. These aspects may be used during the term of this patent to limit the claims and / or to define additional claims.

[0015] The applicant acknowledges that the above-mentioned objective can be achieved by providing a fluid divider that can be placed inside the culture chamber of a cell suspension culture device, particularly at the bottom of the culture chamber.

[0016] This fluid diverter alters the streamlines within the chamber so that at least one toroidal field and / or one rotational field are generated.

[0017] Computational and experimental studies have confirmed and verified that such a fluid splitter, when placed within the culture chamber of a cell suspension culture device, enables the following: - To reduce cell adhesion to the walls of culture devices. - To make it easier to control the shear stress experienced by cells and to allow it to be arbitrarily changed depending on the type of cell being cultured. - To improve the homogenization of nutrients and oxygen, and to avoid the formation of concentration gradients within the culture volume.

[0018] The following are numbered embodiments of the present invention. Embodiment 1

[0019] A fluid divider, -At least one fluid inlet, -Equipped with at least one fluid inlet / outlet section, or multiple fluid inlet / outlet sections, and in fluid communication with at least one fluid inlet, The fluid splitter is configured and intended to work in conjunction with cell culture devices, and this device is - Culture chamber and, - At least one fluid inlet configured to introduce fluid into the culture chamber, -Includes at least one fluid outlet configured to discharge fluid from the culture chamber, Preferably, in a coupled configuration where the fluid diverter is coupled to a cell culture device, the following: · conveying fluid from at least one fluid inlet to the culture chamber, · a fluid diverter configured to introduce at least one fluid stream or a plurality of fluid streams into the culture chamber via a fluid introduction outlet or a plurality of fluid introduction outlets. Aspect 2

[0020] The aspect according to aspect 1, wherein in a coupled configuration where the fluid diverter is coupled to a cell culture device, the fluid diverter is configured to generate turbulent flow in the fluid. Aspect 3

[0021] The aspect according to aspect 1 or 2, wherein the fluid diverter is designed and intended for use within the culture chamber. Aspect 4

[0022] The aspect according to aspect 1 or 2 or 3, wherein the fluid diverter is intended to be used in cooperation with a cell suspension culture device. Aspect 5

[0023] Use of a fluid diverter according to any of the preceding aspects for introducing a plurality of fluid streams into a culture chamber of a cell culture device, preferably a cell suspension culture device. Aspect 6

[0024] The use according to aspect 5, including generating at least one toroidal fluid motion and / or one rotational fluid motion within the culture chamber. Aspect 7

[0025] A cell culture device, preferably a cell suspension culture device, wherein the cell culture device comprises: - a culture chamber, - at least one fluid inlet configured to be able to introduce fluid directed towards the culture chamber, - at least one fluid outlet configured to be able to discharge fluid from the culture chamber, - a fluid diverter at least partially disposed within the chamber and disposed between at least one fluid inlet and at least one fluid outlet with respect to the direction of fluid travel, A fluid divider is, -At least one fluid inlet and at least one fluid inlet communicating with the fluid, - A fluid divider comprising at least one fluid inlet and at least one fluid inlet / outlet section or a plurality of fluid inlet / outlets communicating with each other, Preferably, the fluid divider is • Fluid is transported into the culture chamber from at least a portion of the fluid inlet. A device configured to introduce one or more fluid flows into a culture chamber through a fluid inlet / outlet compartment or a plurality of fluid inlet / outlet compartments. (Aspect 8)

[0026] The embodiment described above, wherein the fluid divider is a single-piece structure and is preferably manufactured by 3D printing. Embodiment 9

[0027] An embodiment of any of the above, wherein the fluid inlet and outlet are defined on the side and / or bottom of the fluid divider. Embodiment 10

[0028] The embodiment according to any one of the preceding descriptions, wherein each of the multiple fluid inlet / outlet outlets is oriented laterally to the walls of the culture chamber, particularly laterally to the side walls and / or bottom walls of the culture chamber. Embodiment 11

[0029] The embodiment according to any one of the preceding descriptions, wherein each of the multiple fluid inlet and outlet outlets has an axis defining the direction of the fluid outlet, and each axis is lateral to the wall of the culture chamber, particularly lateral to the bottom wall and / or side wall of the culture chamber. Embodiment 12

[0030] The fluid diverter comprises at least one stem having a fluid supply conduit, and further comprises a body in fluid communication with the fluid supply conduit, wherein the fluid supply conduit is in fluid communication with at least one fluid inlet, and in particular in direct fluid communication (i.e., without an intermediate member), and is configured to transport the fluid flow to the body, according to any one of the above embodiments. Embodiment 13

[0031] The embodiment described above, wherein the fluid supply conduit is an internal conduit, that is, it is cut into the body of the stem of the fluid divider. Embodiment 14

[0032] The embodiment described above, wherein the main body has a bottom portion that defines a recess formed in an annular shape around the stem. Embodiment 15

[0033] An embodiment according to any of the above, wherein the recess at the bottom of the stem corresponds to a protrusion on the bottom wall of the chamber, particularly a protrusion defined by a convex edge. Embodiment 16

[0034] An embodiment of any of the above, wherein at least one fluid supply conduit is located in the central part of the fluid divider. Embodiment 17

[0035] The embodiment described above, wherein the main body is deployed within the culture chamber. Embodiment 18

[0036] The embodiment described above, wherein the main body has a larger volume than the stem. Embodiment 19

[0037] The embodiment described in any of the preceding descriptions, wherein the main body is tapered from bottom to top. Embodiment 20

[0038] The embodiment described above, wherein the body of the fluid divider includes a peripheral portion, and a plurality of fluid inlet openings are defined in the peripheral portion. Embodiment 21

[0039] The embodiment described above, wherein the peripheral edge is the periphery of the fluid divider body. Embodiment 22

[0040] An embodiment as described above, wherein the outlets of multiple fluid inlet and outlet outlets are angularly offset from one another. Embodiment 23

[0041] The embodiment described in any one of the preceding descriptions, wherein the outlets of multiple fluid inlet and outlet ports are angularly offset from each other with respect to the circumferential direction. Embodiment 24

[0042] The embodiment described above, wherein the outlets of multiple fluid inlet and outlet ports are angularly offset from each other by the same angle, and the offset angle is defined between consecutive fluid inlet and outlet ports. Embodiment 25

[0043] An embodiment of any one of the above, wherein the fluid divider has an annular conduit positioned between at least one fluid inlet and a plurality of fluid inlet and outlet ports, and the outlets of the plurality of fluid inlet and outlet ports are angularly offset along the annular conduit. Embodiment 26

[0044] An embodiment as described above, wherein the outlets of multiple fluid inlet and outlet terminals are arranged radially. Embodiment 27

[0045] An embodiment as described above, wherein the supply conduit extends to the lower part of the fluid divider and the main body extends to the upper part of the fluid divider. Embodiment 28

[0046] An embodiment of any one of the above, wherein the fluid supply conduit is configured to define at least one fluid inlet and to transport fluid to a plurality of fluid inlet and outlet ports. Embodiment 29

[0047] The fluid divider has the following dual function, as described in any of the above descriptions. - A fluid is delivered from at least one fluid inlet to the culture chamber, and this fluid is divided, in particular by dividing it equally (i.e., into flows having corresponding or similar flow rates), thereby creating multiple fluid flows, which are then supplied to the culture chamber through multiple fluid inlet and outlet ports. - To allow fluid from the culture chamber to flow out of the culture chamber. Embodiment 30

[0048] The fluid diverter is a component of a fluid processing interface configured to cooperate with the culture chamber to allow fluid to flow into and out of the culture chamber, as described in any of the preceding embodiments. Embodiment 31

[0049] -The fluid divider further includes at least one fluid outlet for draining fluid from the culture chamber to the outside of the chamber, -Optionally, the fluid outlet is in communication with at least one fluid outlet section, as described in any of the preceding embodiments. Embodiment 32

[0050] An embodiment of any one of the foregoing, wherein the fluid splitter is configured to transport fluid from the culture chamber through a fluid outlet to at least one fluid outlet and discharge fluid from the device. Embodiment 33

[0051] An embodiment of any of the above, wherein the fluid divider is located at least partially in the lower part of the chamber. Embodiment 34

[0052] An embodiment of any of the above, wherein at least one fluid outlet is located at the bottom of the cell suspension culture device. Embodiment 35

[0053] An embodiment of any one of the foregoing, wherein the fluid divider includes a fluid outlet conduit connecting at least one fluid outlet and at least one fluid outlet section. Embodiment 36

[0054] -A fluid outflow conduit runs between a first end and a second end, with the first end defined as the top, for example, the apex, of the fluid divider. -An embodiment of any of the above, wherein at least one fluid outlet is defined at the second end. Embodiment 37

[0055] An embodiment according to any of the above, wherein the first end is configured to guide fluid from the culture chamber into the outflow conduit. Embodiment 38

[0056] The embodiment described above, wherein a fluid outflow conduit is formed inside at least a portion of the main body. Embodiment 39

[0057] An embodiment of any of the above, wherein the fluid shunt is engaged with the lower part of the device. Embodiment 40

[0058] An embodiment of any of the above, wherein the device has a lower part defined below the bottom wall of the culture chamber. Embodiment 41

[0059] An embodiment of any of the above, wherein the stem of the fluid shunt engages with the housing of the device defined at the bottom of the device. Embodiment 42

[0060] An embodiment of any one of the foregoing, wherein the fluid splitter includes a fluid outlet conduit extending between a first end and a second end, the first end being defined at the top, e.g., the apex, of the fluid splitter, and at least one fluid outlet being defined at the second end. Embodiment 43

[0061] An embodiment of any one of the foregoing, wherein the fluid divider includes at least one fluid distribution conduit defined within the body and positioned between at least one fluid supply conduit and one or more outlets of a plurality of fluid inlet and outlet conduits. Embodiment 44

[0062] The embodiment described above, wherein the fluid distribution conduit is an internal conduit, i.e., formed within the body of the fluid divider. Embodiment 45

[0063] An embodiment of any one of the foregoing, wherein at least one fluid distribution conduit is lateral to at least one fluid supply conduit, extends at least partially in a curved manner, and is in fluid communication with one or more fluid inlets and outlets of a plurality of fluid inlets and outlets. Embodiment 46

[0064] An embodiment according to any one of the preceding descriptions, wherein at least one fluid distribution conduit has an annular or annular shape. Embodiment 47

[0065] An embodiment of any of the above, wherein the fluid distribution conduit is deployed in an at least partially annular manner. Embodiment 48

[0066] An embodiment of any one of the foregoing, wherein the fluid splitter includes at least one connecting conduit located between at least one fluid distribution conduit and at least one fluid supply conduit, the connecting conduit being configured to transport fluid from at least one fluid supply conduit to at least one fluid distribution conduit. Embodiment 49

[0067] The embodiment described above, wherein the connecting conduit has a curved outer shape. Embodiment 50

[0068] An embodiment of any of the above, wherein at least one fluid supply conduit is located in the central part of the fluid divider. Embodiment 51

[0069] An embodiment of any one of the foregoing, wherein the fluid divider includes a single fluid distribution conduit defined within the body and positioned between at least one fluid supply conduit and a plurality of fluid inlet and outlet conduits. Embodiment 52

[0070] An embodiment of any one of the foregoing, wherein a single fluid distribution conduit is configured to distribute fluid to all outlets of a plurality of fluid inlets and outlets. Embodiment 53

[0071] An embodiment of any of the above, wherein the fluid divider includes a single fluid inlet that is in fluid communication with a single fluid distribution conduit. Embodiment 54

[0072] An embodiment according to any of the above, wherein a single fluid distribution conduit has an annular shape. Embodiment 55

[0073] An embodiment according to any of the above, wherein a single fluid distribution conduit has an annular outer shape that forms an open ring. Embodiment 56

[0074] - Fluid divider, • Multiple fluid inlets, It includes multiple fluid distribution conduits, each of which is in fluid communication with a corresponding fluid inlet. -An embodiment of any of the above, wherein, in a plurality of fluid inlet and outlet locations, a plurality of fluid inlet and outlet compartments or groups are identified, and each fluid inlet and outlet compartment or group includes at least one fluid inlet and outlet and is in fluid communication with a corresponding fluid distribution conduit. Embodiment 57

[0075] The embodiment described in any one of the preceding descriptions, wherein multiple fluid inlets and outlets are divided into multiple fluid inlet compartments or groups. Embodiment 58

[0076] The embodiment described in any one of the preceding descriptions, wherein each fluid inlet / outlet group includes multiple fluid inlets / outlets. Embodiment 59

[0077] The embodiment according to any one of the above, wherein each fluid distribution conduit is in fluid communication with a fluid inlet / outlet group including multiple fluid inlets and outlets. Embodiment 60

[0078] The embodiment described above, wherein each fluid inlet includes the same number of fluid inlet and outlet sections. Embodiment 61

[0079] The embodiment described above, wherein each fluid distribution conduit is supplied independently of the other fluid distribution conduits. Embodiment 62

[0080] An embodiment of any of the foregoing, wherein the device is configured to independently supply fluid distribution conduits, for example, intermittently and / or sequentially. Embodiment 63

[0081] An embodiment of any of the foregoing, wherein the device is configured to supply fluid to one or more fluid distribution conduits over a specific period of time. Embodiment 64

[0082] An embodiment according to any one of the preceding descriptions, wherein at least one fluid supply conduit is located in the center of the fluid splitter, and the fluid splitter includes a plurality of fluid distribution conduits that branch off from the fluid supply conduit and reach the periphery of the fluid splitter body. Embodiment 65

[0083] The embodiment described above, wherein each fluid distribution conduit opens to a corresponding fluid inlet or outlet. Embodiment 66

[0084] An embodiment of any of the above, wherein the fluid diverter includes a single fluid supply conduit. Embodiment 67

[0085] The embodiment described in any one of the preceding descriptions, wherein the conduits of multiple fluid distribution conduits branch radially from a single fluid supply conduit. Embodiment 68

[0086] An embodiment according to any of the above, wherein each fluid distribution conduit unfolds in at least a partially curved manner. Embodiment 69

[0087] The embodiment described above, wherein each fluid distribution conduit extends radially from at least one fluid supply conduit. Embodiment 70

[0088] An embodiment of any one of the above, wherein the fluid diverter has a single fluid supply conduit configured to supply fluid to a plurality of fluid supply conduits. Embodiment 71

[0089] An embodiment of any of the above, wherein each fluid distribution conduit unfolds in at least a partial spiral shape. Embodiment 72

[0090] The embodiment described above, wherein the device includes an additional chamber or a spare chamber, and a fluid shunt is housed at least partially in the additional chamber or spare chamber. Embodiment 73

[0091] An embodiment of any of the above, wherein an additional chamber or spare chamber is located upstream of the culture chamber. Embodiment 74

[0092] The embodiment described above, wherein the fluid supply conduit is housed in the additional chamber or auxiliary chamber. Embodiment 75

[0093] - The culture chamber includes a bottom wall having a bottom opening and a convex edge extending around the bottom opening. - The auxiliary chamber is separated from above by its bottom opening, With respect to the direction of fluid flow, it is positioned between at least a portion of the fluid inlet of the device and the internal volume of the chamber. -A fluid divider that, when fluid flows out of the auxiliary chamber, forms a fluid passage together with the convex edge of the bottom wall of the chamber, as described in any of the above descriptions. Embodiment 76

[0094] An embodiment of any of the above, wherein the bottom wall of the chamber includes a recess around the convex edge. Embodiment 77

[0095] An embodiment as described above, wherein the convex edge is positioned between the concave and bottom opening. Embodiment 78

[0096] The embodiment described above, wherein the convex edge has a convex portion, and the fluid divider includes a recess in one of its bottom walls corresponding to the convex portion of the convex edge. Embodiment 79

[0097] An embodiment as described above, wherein the fluid passage is defined between the bottom wall of the fluid divider and the bottom wall of the culture chamber. Embodiment 80

[0098] - The convex edge extends circumferentially around the bottom opening, -The recess extends circumferentially around the convex edge, -An embodiment described in any of the above, in which the fluid passage opening is arranged in an annular shape. Embodiment 81

[0099] - The device includes a second fluid inlet configured to communicate fluidly with a pre-chamber and supply fluid directed to the pre-chamber, -The pre-chamber is located downstream of the second fluid inlet and upstream of the internal volume of the culture chamber with respect to the direction of fluid flow, as described in any of the preceding embodiments. Embodiment 82

[0100] - The device includes multiple fluid inlets, -A fluid divider comprising a plurality of fluid distribution conduits, each conduit associated with a corresponding fluid inlet, and receiving fluid coming from that corresponding fluid inlet, as described in any of the preceding embodiments. Embodiment 83

[0101] - The device, • First fluid inlet section, • Second fluid inlet section, • Includes a third fluid inlet, - Fluid divider, A first fluid distribution conduit associated with a first fluid inlet and positioned between at least one fluid supply conduit and one or more of a plurality of fluid inlet and outlet conduits, A second fluid distribution conduit associated with a second fluid inlet and in fluid communication with one or more of the multiple fluid inlet and outlets, The embodiment described above, comprising a third fluid distribution conduit associated with a third fluid inlet and in fluid communication with one or more outlets of a plurality of fluid inlet and outlets. Embodiment 84

[0102] An embodiment according to embodiment 82 or embodiment 83, wherein the fluid divider does not have a fluid outlet conduit. Embodiment 85

[0103] The device includes at least two fluid supply lines for supplying fluid to the culture chamber, i.e., -The first fluid supply line has a fluid divider as a fluid receiving mechanism into the chamber. -The second fluid supply line is configured to receive an additional fluid flow into the culture chamber, as described in any one of the preceding embodiments. Embodiment 86

[0104] An embodiment of any of the above, wherein the second supply line supplies fluid into the culture chamber downstream of the reserve chamber. Embodiment 87

[0105] An embodiment of any of the above, wherein the fluid splitter is configured to change the flow line of the fluid received into the culture chamber. Embodiment 88

[0106] The embodiment described above, wherein the fluid divider is primarily made of solid material. Embodiment 89

[0107] An embodiment of any of the above, wherein the fluid divider has one or more hollow sections configured to allow a fluid to pass through the fluid divider. Embodiment 90

[0108] An embodiment as described above, wherein the fluid divider is primarily made of solid material, and its cavity is configured solely for the passage of fluid. Embodiment 91

[0109] The fluid divider is made of a biocompatible and sterilizable material, as described in any of the preceding embodiments. Embodiment 92

[0110] An embodiment of any of the foregoing, wherein the fluid divider and / or chamber is produced by 3D printing. Embodiment 93

[0111] The embodiment described above, wherein the fluid splitter is configured to divide the flow flowing into the cell culture device into multiple fluid flows supplied into the culture chamber. Embodiment 94

[0112] The embodiment described above, wherein the fluid diverter is a generator of toroidal fluid motion, and more particularly a generator of turbulent toroidal fluid motion. Embodiment 95

[0113] The embodiment described above, wherein the fluid divider is a fluid distributor. Embodiment 96

[0114] The embodiment described above, in which the fluid divider is a separate component from the culture chamber. Embodiment 97

[0115] An embodiment according to any of the above, wherein the culture chamber has side walls and a lower section, and the side walls and the lower section are integral. Embodiment 98

[0116] An embodiment described in any of the preceding descriptions, wherein the side walls of the chamber are integral. Embodiment 99

[0117] An embodiment described above in which the fluid divider has an approximately "mushroom shape". Embodiment 100

[0118] The fluid diverter comprises a stem on which a supply conduit is defined and a body engaged above the stem, wherein the body is tapered from bottom to top, according to any of the above descriptions. Embodiment 101

[0119] The embodiment described above, wherein the body of the fluid divider has an outer shape that is approximately "bell-shaped". Embodiment 102

[0120] An embodiment of any one of the foregoing, wherein the device comprises a closure element, the culture chamber opens upward, and the closure element is configured to fluidly close the culture chamber upward in an assembled state in which it is engaged with the culture chamber. Embodiment 103

[0121] An embodiment of any of the above, wherein the chamber has a substantially axially symmetric external shape. Embodiment 104

[0122] An embodiment of any of the above, wherein the chamber has a substantially cylindrical outer shape. Embodiment 105

[0123] An embodiment of any of the foregoing, wherein the device includes a second fluid inlet at one of its lower parts. Embodiment 106

[0124] An embodiment of any one of the above, wherein the device comprises a pre-chamber located downstream of the second fluid inlet and upstream of the internal space of the culture chamber with respect to the direction of fluid flow, and a fluid splitter forms at least one fluid passage with the bottom of the culture chamber at the fluid outlet of the pre-chamber. Embodiment 107

[0125] The embodiment described above, wherein the second portion of the fluid inlet is connected to the second inlet of the fluid divider. Embodiment 108

[0126] An embodiment of any of the foregoing, wherein the device includes a third fluid inlet at one of its lower parts. Embodiment 109

[0127] The embodiment described above, wherein the third portion of the fluid inlet is connected to the third inlet in the fluid divider. Embodiment 110

[0128] An embodiment of any of the foregoing, wherein the device has an opening in one of its upper parts that allows fluid to be drawn in or discharged from the chamber. Embodiment 111

[0129] An embodiment according to any of the preceding descriptions, wherein the opening is defined by a closing element of the device. Embodiment 112

[0130] An embodiment of any of the above, wherein the fluid shunt is at least partially housed within a housing defined at the bottom of the device. Embodiment 113

[0131] An embodiment of any of the above, wherein the stem of a fluid diverter, on which a fluid supply conduit is defined, is engaged with the housing. Embodiment 114

[0132] The chamber is elongated, where an elongated chamber means a chamber whose height is greater than its diameter, as described in any of the preceding descriptions. Embodiment 115

[0133] An embodiment of any of the above, wherein the chamber has a diameter and a height, and the height is greater than the diameter. Embodiment 116

[0134] An embodiment of any of the above, wherein the height of the chamber is at least 1.3 times the diameter of the chamber. Embodiment 117

[0135] Use of a cell suspension culture device according to any of the above embodiments and / or appended claims for culturing cells in a suspension state. Embodiment 118

[0136] Use of the cell suspension culture device described in any of the foregoing embodiments and / or appended claims for the testing of suspension culture cells and / or the production of cells for, for example, large-scale scientific or industrial use and / or the production of vaccines. Embodiment 119

[0137] A method for operating a cell culture device, preferably a cell suspension culture device, the embodiment and / or claim described above, comprising the following steps: - Introducing fluid into the device through at least one fluid inlet, -Transporting fluid from at least one fluid inlet to a fluid splitter at least partially located within the culture chamber, downstream of at least one fluid inlet and upstream of at least one fluid outlet, wherein the upstream and downstream arrangements are defined with respect to the direction of fluid flow, - A method comprising introducing multiple fluid flows into a culture chamber through multiple fluid inlet and outlet ports of a fluid divider. Embodiment 120

[0138] An embodiment of any of the above-described method embodiments, wherein the step of introducing multiple fluid flows into a culture chamber includes generating a toroidal fluid motion or field within the culture chamber. Embodiment 121

[0139] An embodiment of any of the above embodiments of the method, wherein the toroidal fluid motion or field within the chamber is at least partially turbulent. Embodiment 122

[0140] An embodiment of any of the above-described embodiments of the method, wherein the toroidal fluid motion or field within the culture chamber includes turbulent and laminar components. Embodiment 123

[0141] An embodiment of any of the above-described method embodiments, wherein the step of introducing multiple fluid flows into a culture chamber includes generating rotational fluid motion or a field within the culture chamber. Embodiment 124

[0142] An embodiment of any of the above-described embodiments of the method, wherein the rotational fluid motion or field within the culture chamber is turbulent. Embodiment 125

[0143] The process of introducing multiple fluid flows into the culture chamber is An embodiment of any one of the above-described method embodiments, comprising generating both toroidal fluid motion and rotational fluid motion within the culture chamber. Embodiment 126

[0144] The method is a cell suspension culture method, as described in any of the above-described embodiments of the method. Embodiment 127

[0145] An embodiment of any of the above-described method embodiments, wherein the fluid is a fluid for cell culture or includes such fluid. Embodiment 128

[0146] An embodiment of any of the above-described embodiments of the method, wherein the fluid includes cells in a suspended state. Embodiment 129

[0147] An embodiment of any one of the methods described above, wherein the step of introducing multiple fluid flows into a culture chamber includes directing the multiple fluid flows laterally against the walls of the culture chamber that divide the culture chamber, such as the bottom wall and / or side walls. Embodiment 130

[0148] An embodiment of the method described above, wherein the step of introducing multiple fluid flows into a culture chamber includes introducing multiple turbulent flows into the culture chamber. Embodiment 131

[0149] An embodiment of any one of the above-described methods, wherein the method includes the step of transporting a fluid from at least one fluid inlet through a fluid divider. Embodiment 132

[0150] An embodiment of the method described above, wherein the method includes the step of introducing an additional fluid flow into the culture chamber. Embodiment 133

[0151] An embodiment of any of the above-described methods, wherein the step of introducing an additional fluid flow into the culture chamber includes introducing the fluid into a defined fluid inlet, the fluid inlet being defined between a fluid splitter and the bottom wall of the culture chamber. Embodiment 134

[0152] An embodiment of any of the above-described methods, wherein the step of introducing a fluid flow into a fluid passage defined between the fluid divider and the bottom wall of the culture chamber includes introducing the fluid into an annular fluid passage defined between the convex edge of the bottom wall of the culture chamber and the bottom wall of the body of the fluid divider. Embodiment 135

[0153] The embodiment described above, wherein the convex edge is positioned between the recess of the bottom wall of the fluid divider and the bottom opening. Embodiment 136

[0154] An embodiment of any of the above-described methods, wherein the method involves introducing multiple fluid flows into a culture chamber in a predetermined order and / or intermittently with respect to one another. Embodiment 137

[0155] An embodiment of any of the above-described methods, wherein the method involves supplying each flow to a culture chamber for a predetermined time. Embodiment 138

[0156] The embodiment described above, wherein the culture device is a bioreactor. Embodiment 139

[0157] The embodiment described above, wherein the culture device is a bioreactor for culturing cells in a suspension state. Embodiment 140

[0158] An embodiment of any of the above, wherein the cell culture device is passive, i.e., does not include any rotating member for generating microgravity or stirring the fluid. Embodiment 141

[0159] An embodiment of any of the above, wherein the fluid divider is a component substantially fixed within the culture chamber. Embodiment 142

[0160] An embodiment of any of the foregoing, wherein the fluid splitter is configured to accelerate and / or energize the flow entering the culture chamber. Embodiment 143

[0161] The embodiment described above, wherein the fluid divider is a turbulence generator. Embodiment 144

[0162] An embodiment of any of the above, wherein the fluid divider is configured to generate turbulence when the fluid is introduced into the culture chamber. Embodiment 145

[0163] The embodiment described in any one of the above-mentioned descriptions, wherein each of the multiple fluid inlet and outlet outlets constitutes a fluid flow restriction section capable of accelerating the fluid flowing out of the fluid divider. Embodiment 146

[0164] An embodiment according to any one of the preceding descriptions, wherein each of the multiple fluid inlet and outlet outlets includes a nozzle. Embodiment 147

[0165] The embodiment according to any one of the above, wherein each of the multiple fluid inlet and outlet outlets has a fluid outlet cross-section smaller than the fluid passage cross-section of the distribution conduit supplied to it. Embodiment 148

[0166] In any of the embodiments described above, the fluid divider conforms to the following equation: k = Q / (n * A * d / 2) During the ceremony: Q is the volumetric flow rate of the fluid, and in particular, the volumetric flow rate at the inlet of the fluid divider (e.g., cm). 3 / s), n is the number of fluid inlets and outlets in the fluid divider. A is the cross-sectional area of ​​the fluid inlet and outlet (for example, cm²). 2 ), • d is the inner diameter of the device's culture chamber (e.g., cm). · TIFF2026510399000002.tif22 has angular frequency dimensions and is expressed in Hz. (Appendix 149)

[0167] The embodiment described above, wherein k is between 10 and 40, and especially between 20 and 30. Embodiment 150

[0168] The embodiment described above, wherein k is between 45 and 75, and in particular between 55 and 65. Embodiment 151

[0169] A cell culture device, preferably a suspension and / or adherent cell culture device, - Culture chamber and, - At least one fluid inlet configured to allow fluid to flow into the culture chamber, - A device comprising at least one fluid outlet configured to allow fluid to flow out of a culture chamber. Embodiment 152

[0170] An embodiment of Embodiment 151, wherein the device comprises a fluid divider at least partially located within a chamber, and is positioned between at least one fluid inlet and at least one fluid outlet with respect to the direction of fluid flow. Embodiment 153

[0171] A fluid divider, -At least one fluid inlet and at least one fluid inlet in fluid communication, - An embodiment according to embodiment 151 or 152, which represents at least one fluid inlet / outlet section or a plurality of fluid inlet / outlets that are in fluid communication with at least one fluid inlet of a fluid divider. Embodiment 154

[0172] A fluid divider, • Fluid is transported to the culture chamber from at least one fluid inlet. The embodiment according to embodiment 153, wherein one or more fluid flows are introduced into the culture chamber through a fluid inlet / outlet compartment or a plurality of fluid inlet / outlet compartments. Embodiment 155

[0173] An embodiment of any one of the foregoing, further comprising at least one retaining portion configured to support one or more scaffolds, preferably configured to support multiple scaffolds. Embodiment 156

[0174] The embodiment according to embodiment 155, wherein the fluid divider includes a positioning element configured to position the holding portion within the culture chamber. Embodiment 157

[0175] The embodiment according to embodiment 156, wherein the positioning element is defined on the top of the fluid shunt and / or includes the support portion of the fluid shunt, e.g., the central support portion. Embodiment 158

[0176] An embodiment of Embodiment 156 or 157, wherein the positioning element is configured to stably and / or uniquely and / or position the retaining portion within the culture chamber in a defined manner or position. Embodiment 159

[0177] The embodiment described above, wherein the fluid divider is incorporated into the culture chamber, particularly in the base and / or bottom wall and / or side wall of the culture chamber. Embodiment 160

[0178] An embodiment according to any one of embodiments 155 to 159, wherein the holding part has a plurality of housings, and each housing is configured to accommodate a corresponding scaffold. Embodiment 161

[0179] An embodiment according to any one of embodiments 155 to 160, wherein the holding portion further comprises an interface portion configured to interface the support with a fluid divider. Embodiment 162

[0180] The embodiment according to embodiment 161, wherein the interface portion is a positioning portion, and this positioning portion is configured to position the scaffolding holding portion to the center, preferably so as to position the scaffolding holding portion relative to the fluid divider. Embodiment 163

[0181] An embodiment according to embodiment 160, 161, or 162, wherein the interface portion or positioning portion is defined in the center of the holding portion, and each housing of the plurality of housings is defined around the interface portion or positioning portion. Embodiment 164

[0182] An embodiment according to any one of embodiments 156 to 163, wherein the positioning element is configured to interface with the interface portion or positioning portion of the holding portion, or in particular to be positioned with respect to it. Embodiment 165

[0183] The embodiment according to embodiment 164, wherein the positioning element and the interface portion or positioning portion have corresponding external shapes. Embodiment 166

[0184] An embodiment according to embodiment 164 or 165, wherein the positioning element and the interface portion or positioning portion have corresponding shapes. Embodiment 167

[0185] An embodiment according to any one of embodiments 156 to 166, wherein the interface portion or positioning portion includes a housing portion, and the positioning element includes a central support portion (preferably defining the upper part of the fluid shunt), and the central support portion is insertable into the housing portion of the holding portion and defines mutual constraints. Embodiment 168

[0186] An embodiment of any of the foregoing, wherein the device further comprises one or more scaffolds, preferably multiple scaffolds. Embodiment 169

[0187] An embodiment of any one of the foregoing, particularly the embodiment of at least embodiment 167 and embodiment 155, wherein one or more scaffolds are supportable or supported by a scaffolding holder. Embodiment 170

[0188] A system comprising multiple culture devices according to the claims of any one of the aforementioned embodiments of a device and / or an attached device. Embodiment 171

[0189] The embodiment according to embodiment 170, wherein the cell culture devices are arranged in parallel with each other. Embodiment 172

[0190] Use of a cell culture device according to any of the above-mentioned embodiments and / or attached device claims for tissue engineering, or the system according to embodiment 170 or 171. Embodiment 173

[0191] The following steps: - A step of providing side walls and a base to form a culture chamber for a cell culture device, preferably a step of the cell culture device conforming to any of the embodiments of the device and / or claims of the attached device described above, - A step of providing a fluid divider, wherein the fluid divider is optionally incorporated into a base, and therefore this step is part of the step of providing a base. - A step of providing at least one retaining part configured to support one or more scaffolding, - A cell culture device assembly process comprising the step of engaging at least one scaffolding holder with a fluid diverter. Embodiment 174

[0192] The embodiment described in embodiment 173, - The step of providing side walls and a base to form a culture chamber is, • The process involves providing the side wall and base separately from each other, and the fluid divider having an integral structure with the base. The process includes engaging the side wall and the base with each other, preferably including positioning the fluid divider within the culture chamber, -Optionally, the step of engaging at least one scaffolding holder with the fluid divider is performed before or after the step of positioning the fluid divider within the culture chamber. Embodiment 175

[0193] The embodiment according to embodiment 173 or 174, wherein the step of engaging at least one scaffolding support with a fluid diverter involves connecting the central support of the fluid diverter to the housing of the scaffolding support. Embodiment 176

[0194] A process for removing at least one scaffolding support, - A step of providing a device according to any of the above embodiments and / or any of the attached claims, further, • At least one scaffolding support unit, The process involves providing one or more scaffolds engaged with at least one scaffolding holder, - A step of removing an obstruction element 5 that at least partially separates the culture chamber 2, preferably by detaching it from the side wall 4, A process comprising the step of removing at least one scaffold from the scaffolding support. Embodiment 177

[0195] An embodiment of Embodiment 176, wherein the step of removing at least one scaffolding includes moving (in particular by moving) at least one scaffolding stopper from a position that restrains at least one scaffolding to a position that makes at least one scaffolding detachable from the scaffolding holder. Embodiment 178

[0196] An embodiment of any one of the foregoing, wherein the culture device comprises one or more fluid supply conduits, each fluid supply conduit being in the form of a continuous channel and / or having a substantially constant diameter or no significant or abrupt change in diameter. Embodiment 179

[0197] The embodiment described above, wherein the device is reusable or disposable. Embodiment 180

[0198] An embodiment according to any one of embodiments 155 to 179, wherein the fluid inlet and outlet are configured to introduce fluid to the location of or near the scaffolding support, particularly below the support.

[0199] Terms and Definitions In the following detailed description, please note that corresponding parts / components / elements are indicated by the same reference number. Drawings may depict the subject matter of the invention without scale, and therefore parts / components / elements shown in the accompanying drawings and related to the subject matter of the invention may be represented only schematically.

[0200] In this specification, terms such as “up,” “upper side,” “upper,” “down,” “lower side,” “side,” “inside,” “inside,” “outside,” “horizontal,” “horizontally,” “vertical,” “vertically,” “front,” “front,” “rear,” “rear,” “right,” and “left,” or similar terms and variations thereof, mean at least one spatial direction that the object of the invention may take under the conditions of use (see, for example, one or more of the accompanying drawings).

[0201] Unless otherwise specified, the terms “state” and “configuration” may be used interchangeably within the context of this specification. The phrase “at least one” may be used interchangeably with “each.” Expressions such as “each” do not necessarily imply that there are multiple elements they refer to. For example, “each element” may refer to a single element or multiple elements, depending on the context in which it is used and the embodiment being referenced.

[0202] Unless otherwise specified, terms such as "upstream" and "downstream," or similar or derived terms, refer to the arrangement of parts / components / elements with respect to the direction of fluid flow along the fluid line or circuit in which they are located, or along a particular line or branch of that circuit.

[0203] In the context of this specification, one or more of the following definitions / concepts shall apply, as appropriate, unless otherwise stated and / or the context excludes them.

[0204] - "Cell suspension culture device" means a device configured to enable the culture of cells in a suspension state. The cell suspension culture device may also be configured to enable adherent culture, and for this purpose, it may be provided with, for example, one or more scaffold holders and optionally one or more scaffolds. The cell suspension culture device is preferably a bioreactor.

[0205] - A "culture chamber" refers to a chamber in a cell culture device designed to allow cells to be cultured in a suspension and / or adherent state. The culture chamber defines an internal volume (culture volume) designed to contain fluids and allow cells to actually grow in a suspension and / or adherent state. The culture chamber is typically the primary fluid chamber in a suspension and / or adherent cell culture device. If the suspension and / or adherent cell culture device has multiple chambers (e.g., a main chamber and a secondary chamber), the culture chamber may be the primary fluid chamber, i.e., the chamber with the largest volume in the suspension and / or adherent cell culture device.

[0206] - The "internal volume" of the culture chamber is understood to mean the internal volume of its main fluid-containing section. In other words, in embodiments where the culture chamber has multiple internal volume sections, the internal volume section is understood to refer to the main volume section (therefore, any auxiliary chamber(s) are not included in the internal volume section as understood in this way).

[0207] - A "fluid splitter" means a component that is housed within a culture chamber or incorporated into a part of the culture chamber, preferably the lower or base, and that transports and directs at least one flow flowing into its body, thereby enabling the supply of multiple fluid flows to the culture chamber. Preferably, the fluid splitter also allows for the outflow of fluid from the culture chamber.

[0208] - The term "fluid divider" can be used interchangeably with "fluid distributor" or "turbulence generator."

[0209] - A “scaffold” (sometimes called a “structure”) means one or more artificial structures usable in tissue engineering that are configured to contain and / or support cell cultures and promote their growth until, for example, regeneration of damaged tissue is achieved. To facilitate the above function, one or more scaffolds may have nanometer-sized morphological properties designed to mimic the structure of the tissue to be regenerated. For example, in the case of a bone scaffold, the scaffold mimics the structure of bone.

[0210] The above terms and definitions may be used to interpret the claims as necessary. Where necessary, one or more of these terms and definitions may be included in one or more of the following claims and / or one or more of the aforementioned embodiments, in particular where these claims and / or embodiments use expressions that are subject to one or more terms or definitions.

[0211] To better understand the present invention and to appreciate its advantages, several embodiments are described below, illustrative and not limiting, with reference to the accompanying drawings.

[0212] In the attached diagram, arrows without reference numerals indicate the direction of fluid flow under the operating conditions of the device and fluid shunt, or the direction of assembly or positioning of components. A brief description of the attached diagram is given below. [Brief explanation of the drawing]

[0213] [Figure 1] This diagram shows a fluid divider according to a first embodiment of the present invention, where the outer wall is drawn with dashed lines to more clearly show the defined conduits within the fluid divider, and the conduits are shown with solid lines. [Figure 2] Figure 1 shows a bottom view of the fluid divider. [Figure 3] Figure 3 shows a front cross-section of the cell suspension culture device according to the present invention, with a culture chamber containing the fluid divider shown in Figure 1 located at its lower part. Figure 3 also shows details of the fluid passage that communicates with the culture chamber. [Figure 4]Figure 3 shows a side cross-section of a suspension cell culture device, which contains a culture medium. The toroidal motion generated by the flow passing through the fluid passage is illustrated by the flow passing through the fluid passage defined between the lower surface of the bottom wall of the fluid divider and the upper surface of the bottom wall of the culture chamber, and the flow flowing out from multiple fluid inlet and outlet ports of the fluid divider. [Figure 5] This diagram shows a fluid divider according to a second embodiment of the present invention, where the outer wall is drawn with dashed lines to more clearly show the defined conduits inside the fluid divider, and the conduits are shown with solid lines. [Figure 6] This figure shows a side cross-section of the cell suspension culture device according to the present invention, with a culture chamber containing the fluid divider shown in Figure 5 located at its lower part. [Figure 7] Figure 6 shows a front cross-section of the device. [Figure 8] Figure 6 shows a cross-section of the device realized according to the cutting plan VIII-VIII. It shows the rotational motion field generated by the fluid flowing out of the spirally unfolded fluid distribution conduit. [Figure 9] This diagram shows a fluid divider according to a third embodiment of the present invention, where the outer wall is drawn with dashed lines to more clearly show the defined conduits within the fluid divider, and the conduits are shown with solid lines. [Figure 10] Figure 9 is a top view of the fluid divider shown. [Figure 11] This figure shows a side cross-section of the cell suspension culture device according to the present invention, with a culture chamber containing the fluid divider shown in Figure 9 located at the bottom. [Figure 12] Figure 11 shows a front cross-section of the device. [Figure 13] The cell suspension culture device according to the present invention is shown, comprising a fluid splitter according to the first or second embodiment, connected to an external fluid power circuit equipped with a pump, and allowing fluid to enter and exit the device through the circuit. [Figure 14]This shows a schematic velocity field map (in technical terms, streamline: velocity field) of the front cross-section of one side (right side) of the culture chamber of the cell suspension culture device according to the present invention, indicating that during cell culture, the installation of an elongated culture chamber generates one vortex at the bottom and one at the top of the culture chamber. [Figure 15] Referring again to the front cross-section of one side of the chamber in Figure 14, a color velocity map is shown where the fluid exhibits higher velocity values ​​at the outlet of the flow diverter (left nozzle representing the fluid inlet of the flow diverter) and the inlet of the culture chamber (lower nozzle). [Figure 16] This diagram shows a schematic of a fluid receiving outlet where a fluid shunt influences the side wall of the chamber laterally (diagonally). [Figure 17] An exploded view of a cell culture device according to a further embodiment of the present invention is shown. According to this embodiment of the present invention, the device includes a scaffolding holder that is placed inside the culture chamber in its assembled state (see Figure 23). [Figure 18] Figure 17 shows the lower or base of the device with the fluid shunt incorporated. [Figure 19A-B] The diagram shows two types of scaffolding support units, each configured to support a circular base scaffolding (Figure 19A) and a polygonal base scaffolding (Figure 19B). [Figure 20A-B] Figure 19A shows the step of positioning the scaffolding support (Figure 20A), and Figure 20B shows the step of positioning the scaffolding relative to this support. [Figure 21] This shows the process of positioning each scaffold, with its pre-installed scaffold support section, on top of the fluid diverter. [Figure 22] Figure 17 shows a cross-section of the assembled device configuration, with the scaffolding support and each scaffolding removed (arrows indicate the direction of flow). [Figure 23] Figure 17 shows the assembled device configuration, with the circuit connected to a pump configured to circulate the fluid within the circuit (arrows indicate the direction of flow within the circuit and the culture chamber). [Modes for carrying out the invention]

[0214] Cell suspension culture device The cell suspension culture device according to the present invention is also referred to simply as the "device" or "bioreactor" in this text and is generally shown by numerical reference 1 in the figures. In certain embodiments, as detailed below, the culture device 1 may be configured to enable both suspension culture and adherent culture.

[0215] Cell suspension culture device 1 is passive because it does not contain any rotating parts for generating microgravity or agitating the fluid.

[0216] Device 1 includes a culture chamber 2 that defines an internal volume where cell culture in a suspended state takes place during use.

[0217] The culture chamber 2 has a bottom wall 3 and side walls 4 that divide its internal volume. As shown in the attached Figures 3, 4, 6, 7, 11, 12, 13, 18 and 21-23, the side walls 4 are preferably integrally molded. Providing a culture chamber 2 laterally divided by an integral (integrally molded) side wall 4 is advantageous compared to embodiments that provide side walls of a culture chamber achieved by assembling two or more parts together (such as the device described in International Publication No. 2020095143(A1)). This is because joining means (such as threads) are avoided, and therefore, fluid leakage, pressure, contamination, inaccuracy, or assembly problems that may occur in the assembly of these parts are avoided. Additionally or alternatively, as shown in the attached Figures 3, 4, 6, 7 and 11-13, the bottom wall 3 may also be integral with the side walls 4. In this case as well, the same reasons apply regarding the provision of integral molding and its relative advantages. In another embodiment, as shown in Figures 17, 18, 21, 22, and 23, the bottom wall 3 may be separate from the side walls 4. In such an embodiment, the bottom wall 3 is part of the lower (or base) portion 40 of the culture chamber 2, which is assembled with the side walls 4. The side walls 4 are open at the top.

[0218] Under operating conditions, the upper opening of the culture chamber 2 is closed by a closure element 5. As shown in the attached Figures 3, 4, 6, 7, 11, 12, 13, and 17, the closure element 5 is a separate component from the side wall 4 and is removable from the side wall 4. Thus, the closure element 5 can be mounted on the side wall 4 and close its upper opening. The closure element 5 can be engaged with the side wall 4 by known means, such as a threaded element 6. The closure element 5 provides multiple connection or through-holes 7, or inlets, or nozzles 8, for inserting probes and / or nutrients for sampling, and / or connecting to a tank (storage section), and / or inserting elongated elements (such as tubes or rods) for drawing out fluids.

[0219] Geometrically, as shown in the attached figure, the culture chamber 2 has an axially symmetrical shape and is particularly substantially cylindrical. Preferably, the culture chamber 2 has a substantially constant diameter D. Also, the culture chamber 2 preferably has a height H that is greater than the diameter D. Thus, the culture chamber 2 is elongated, and an elongated culture chamber 2 precisely refers to a culture chamber 2 in which the height H is greater than the diameter D. If the diameter D is not constant, the average diameter can be considered. More specifically, the attached figure shows a culture chamber 2 in which the height H is at least 1.3 times greater than the diameter D. By providing an elongated culture chamber 2, it becomes possible to change the volume of fluid between various applications / uses, for example, it is possible to increase the culture medium when culturing cells for the production of biomolecules. Furthermore, as shown by hydrodynamic simulations and is also clear from Figure 14, the elongated culture chamber 2 allows for the formation of two separate vortex zones, specifically the lower vortex zone 2' and the upper vortex zone 2''. Thanks to this environment, cells remain confined to the lower part (the portion indicated by the lower vortex 2' generated just above the bottom wall 3 of the culture chamber 2), while biomolecules with different characteristics rise to the upper part. This solution makes it possible to incorporate both cells and their products into a single system, avoiding the need to add an external storage unit where biomolecule collection is typically carried out. In some applications, it is understood that a storage unit can be provided where cell products such as antibodies can be placed. The elongated culture chamber 2 does not require the culture chamber to be filled with fluid for operation, as it allows for the provision of an air storage unit at the top sufficient for gas exchange. Essentially, when in use, the device 1 according to the present invention operates in the presence of culture fluid in the culture chamber 2, and further in the presence of gas and / or oxygen within the culture chamber (therefore, the culture chamber does not need to be filled with culture fluid). See Figure 4 for this point. The elongated culture chamber 2 also facilitates the operation of nutrient infusion, oxygen and CO2 control, and pH control, which can be carried out at the top of the device 1, particularly by connections or through-holes 7, or inlets or nozzles 8 defined in the closure element 5.

[0220] Here, the bottom wall 3 of the culture chamber 2 is described in structural detail. The bottom wall 3 has a recess 3a and may have a convex rim 3b and a bottom opening 3c. The convex rim 3b improves cell containment within the culture chamber 2 by slowing the ascent of cells from the recess 3a, thereby reducing the risk of cells being deposited into other cavities below the culture chamber 2. This allows it to operate without a valve (such as the valve of the device in International Publication No. 2020095143(A1)) that regulates the inflow of fluid into the culture chamber. The bottom opening 3c is defined in the center of the bottom wall 3, and the convex rim 3b is located between the recess 3a and the bottom opening 3c. The convex rim is a substantial edge that defines the convex portion 3c relative to the adjacent recess 3a of the bottom wall 3. Thus, the convex rim 3b is raised relative to the adjacent recess 3a, and the direction of fluid flow defined within it is from the convex rim 3b towards the recess 3a. Both the convex rim 3b and the concave rim 3a are shown extending in an annular manner around the bottom opening 3c; in other words, the convex rim 3b and the concave rim 3a have an annular shape that extends circumferentially around the bottom opening 3c. Therefore, by providing the convex rim 3b and the concave rim 3a downstream of the convex rim 3b, particularly directly downstream of the convex rim 3b, it becomes advantageous to generate toroidal fluid motion within the culture chamber 2. This toroidal motion allows for the movement of the fluid (in which the cells are suspended) within the culture chamber 2, which is necessary for cell growth in a suspended state. The bottom opening 3c preferably has a circular shape and is located in the center of the bottom wall 3, i.e., concentric with it.

[0221] Device 1 may further include an additional chamber or pre-chamber 9 defined below it, particularly below the culture chamber 2. This additional chamber or pre-chamber 9 opens into a bottom opening 3c and is defined above it by the bottom opening 3c. In the embodiments of Figures 1 to 8, the additional chamber or pre-chamber 9 functions as a fluid passage chamber located upstream of the culture chamber 2. Essentially, depending on the fluid introduction configuration into the culture chamber 2, the fluid flows into the culture chamber 2 after passing through the pre-chamber 9. The additional chamber or pre-chamber 9 is a substantially cylindrical well defined by a downward recess relative to the bottom wall 3. In the embodiments of Figures 17 to 23, such a pre-chamber 9 is not provided.

[0222] However, in some variations of this embodiment, the presence of a spare chamber is not excluded.

[0223] Device 1 may further comprise a housing 10 configured to house at least a portion of a fluid divider 21, which will be described in detail later in this specification. In the accompanying Figures 3, 4, 6, 7, 11, and 12, the housing includes a hollow section 10 located at the bottom of Device 1, which is configured to restrain a portion of, for example, the stem 29 of the fluid divider 21. The hollow section 10 communicates with an additional chamber or spare chamber 9. In the embodiments of Figures 17 and 18, the lower or base 40 has a fluid divider 21 that is integrally incorporated. In other words, the fluid divider 21 is integral with the lower section 40. Thus, the fluid divider 21 can be formed integrally with the lower section 40. In this way, a robust structure is achieved through integral molding.

[0224] In the embodiments shown in Figures 3, 4, 6, 7, 11, and 12, the device 1, in particular the culture chamber 2, is configured to house a fluid splitter 21. More specifically, the device 1, in particular the culture chamber 2, is configured to house the fluid splitter 21 in the lower part of the device 1 itself. As shown in the attached Figures 3, 4, 6, 7, 11, and 12, the fluid splitter 21 can be engaged in (or near) an additional chamber or spare chamber 9. As shown in the attached Figures 3, 4, 6, 7, 11, and 12, the stem 29 of the fluid splitter 21 is partially housed in a hollow section 10, which forms a housing that communicates with the additional chamber or spare chamber 9.

[0225] Device 1 further comprises at least one fluid inlet 11, 12, 13. The fluid inlets 11, 12, 13 allow fluid toward the culture chamber 2 to flow into Device 1. In the accompanying figures, the fluid inlets 11, 12, 13 are defined at the bottom of the culture chamber 2 and are in fluid communication with at least one fluid inlet 22, 23, 24 of the fluid splitter 21, as will be confirmed in the following more detailed description. Preferably, Device 1 comprises at least first and second fluid inlets 11, 12. The first fluid inlet 11 is in fluid communication with the fluid splitter 21, and the second fluid inlet 12 is in fluid communication with the pre-chamber 9 (see Figures 3, 4, 6, and 7). Alternatively, both the first and second fluid inlets 11, 12 are in fluid communication with the fluid splitter 21 (see Figures 11 and 12). In the embodiments shown in Figures 11 and 12, device 1 further comprises a third fluid inlet 13 (see Figure 11), which is also in fluid communication with the fluid diverter 21. Each of the fluid inlets 11, 12, and 13 is preferably defined at the bottom of device 1. Each of the fluid inlets 11, 12, and 13 has, or is associated with, its own fluid passage channel or conduit or opening, and in particular has its own fluid supply conduits 26, 27, and 28.

[0226] Device 1 further comprises at least one fluid outlet 14. One or more fluid outlets 14 allow fluid originating from the culture chamber 2 to be discharged from Device 1. The fluid outlets 14 may be defined in the lower part and / or upper part of Device 1, particularly in the closing member 5. In the embodiments of Figures 3, 4, 6, and 7, the fluid outlet 14 is defined in the lower part of Device 1 and is in fluid communication with the fluid diverter 21. Optionally, in such embodiments, the closing member 5 may be provided with a through-hole 7 through which fluid can pass, allowing for the insertion of an elongated element for drawing out the fluid. Typically, under the operating conditions of Device 1 according to the embodiments of Figures 3, 4, 6, 7, 22, and 23, the fluid is discharged from the culture chamber 2 via the fluid outlets 14 (discharge of fluid from below). In contrast, in the embodiments of Figures 11 and 12, fluid discharge from the culture chamber 2 is provided solely by a fluid outlet section 14 (see Figure 11) consisting of a through-hole 7 formed on the closing member 5, which allows for the insertion of an elongated element for drawing out the fluid (fluid exits from above). In the latter embodiment, there is no fluid outlet at the bottom of the device 1. The conduit of the device 1 shown in Figure 11 is actually used to direct the fluid into the corresponding inlet 24 of the fluid diverter 21 (in contrast, the conduit of the device 1 in the embodiments of Figures 4 and 6 is used to discharge the fluid from the device 1). Each of the fluid outlet sections 14 has or is associated with its own channel or conduit or fluid passage opening.

[0227] Device 1 further comprises a fluid divider 21, which may be located at or near the bottom 3 of the culture chamber 2, or may be incorporated at least into the lower part 40 of the culture chamber 2.

[0228] fluid diverter The fluid splitter 21 is described herein in particular in relation to the three embodiments shown in the attached Figures 1 to 13. Each embodiment of the fluid splitter 21 corresponds to each embodiment of the device 1 comprising the fluid splitter 21. It is also conceivable that the fluid splitter 21 may be implemented integrated with the culture chamber 2. In use, the fluid splitter 21 is located within the culture chamber 2 and engages with the lower part of the culture chamber 2 and the device 1 (Figures 1 to 13), or is integrated with it (Figures 17, 28, and 21 to 23). When fluid connections are defined below, these refer to the operating conditions of the device 1 and the fluid splitter 21. Before going into detail about each embodiment of the fluid splitter 21, common features between the embodiments are described. Note that the fluid splitter 21 is a static component, i.e., it is not configured to rotate within the culture chamber 2. Therefore, the fluid splitter 21 is a substantially fixed component within the culture chamber 2. Regarding materials, it should be noted that the material for the fluid divider 21 is preferably biocompatible and autoclavable. For example, the fluid divider 21 may be made of a biocompatible and sterilizable resin. The resin used is preferably configured for use in complex and demanding applications and capable of reproducing small parts with a smooth surface finish. The fluid divider 21 (and similarly the culture chamber 2) is preferably made of resin and can be manufactured by 3D printing technology, particularly 3D stereolithography printing technology. The fluid divider 21 may be transparent, so that it can be visually inspected.

[0229] The fluid divider 21 includes at least one fluid inlet 22, 23, 24 that communicates with at least one fluid inlet 11, 12, 13 of device 1 in order to receive fluid from the fluid inlet 11, 12, 13.

[0230] In embodiments where the fluid diverter 21 is integrated with the culture chamber 2, the fluid inlet 22 and the fluid inlet section 11 may coincide, or they may be positioned close to each other but offset from each other. For example, the fluid inlet 22 may be positioned directly downstream of the fluid inlet section 11.

[0231] The fluid splitter 21 further comprises a plurality of fluid inlet / outlet 25 communicating with at least one fluid inlet 22, 23, 24. The fluid inlet / outlet 25 are preferably defined on the sides and / or bottom of the fluid splitter 21. The fluid inlet / outlet 25 are oriented laterally with respect to the bottom wall 3 and / or side wall 4 of the culture chamber 2, so that the fluid flow is directed toward at least one wall 3, 4. This wall 3, 4, by its concave shape, guides the fluid flow out of the fluid splitter 21. In particular, the fluid inlet / outlet 25 may be oriented obliquely, in which case the fluid exiting them collides with the bottom wall 3. The inclination angle of the axis A of the fluid inlet / outlet is inclined with respect to both the horizontal and vertical directions (oblique direction of fluid introduction into the culture chamber), and this angle may vary depending on the application, requirements, and / or the capacity of the suspension cell culture device. As shown in the attached figure, each fluid inlet / outlet may be provided with a corresponding nozzle 25. The fluid inlet and outlet 25 are preferably positioned angularly offset from each other, and in particular, pairs of adjacent fluid inlet and outlet 25 are offset by the same angle α. By angularly offsetting the fluid inlet and outlet, especially by the same angle α, multiple outflows from the fluid splitter 21 can preferably cover a wide angular arc, particularly substantially 360° uniformly (see attached figure), thereby allowing fluid inlet to occur circumferentially around the fluid splitter 21, generating toroidal and / or rotational motion around the fluid splitter 21 within the culture chamber 2. Uniform distribution of all fluid components, including cells, within the culture chamber 2 is a crucial characteristic for promoting nutrient exchange, avoiding cell deposition and the formation of concentration gradients, and thereby increasing cell growth. Furthermore, it is important that cells are uniformly distributed within the culture chamber 2 so that each cell receives an adequate supply of oxygen and nutrients to perform its metabolic functions. The fluid splitter 21 is configured to accelerate and / or energize the inlet flow into the culture chamber 2. This is achieved by providing multiple fluid inlet / outlet ports 25 (nozzles) each having a fluid passage cross-section smaller than the fluid passage cross-section of one or more fluid distribution conduits 36, 37, 38 (described later) to which they are connected.

[0232] The fluid divider 21 is configured to deliver fluid to the culture chamber 2 from at least some of the fluid inlets 11, 12, and 13, and to supply multiple fluid flows to the culture chamber 2 through multiple fluid inlet and outlet ports 25.

[0233] To allow fluid to be introduced, the fluid divider 21 is equipped with at least one fluid supply conduit 26, 27, 28, which is preferably defined at the bottom of the fluid divider 21. At least one supply conduit 26, 27, 28 extends into the central part of the fluid divider 21.

[0234] Structurally, the fluid shunt 21 shown in the attached Figures 1 to 12 comprises a stem 29 and a body 30 connected to the stem 29 and positioned above it. The body 30 is in fluid communication with the stem 29. The fluid shunt 21 is advantageously a single structure, and therefore it is preferable that the stem 29 and the body 30 are integrated. The stem 29 may have a substantially cylindrical and elongated shape. As shown in the attached Figures 1 to 12, the stem 29 may unfold in a linear direction, and in particular, it may unfold in a single linear direction. The fluid supply conduit 26 is defined within the stem 29. Essentially, the fluid supply conduit 26 is an internal conduit, i.e., formed within the body (or casing) of the stem 29 of the fluid shunt 21. This design choice advantageously allows the flow rate of the device 1 to be kept as low as possible. Because the culture chamber 2 is elongated, an increase in height leads to an increase in hydrostatic pressure at the bottom of device 1, which reduces the effectiveness of cell suspension at the bottom of culture chamber 2. In the configuration of device 1 according to the present invention, there is a "point-like" and localized introduction (i.e., introduction through multiple fluid introduction outlets 25 spaced apart from each other), which makes it possible to maintain the same cell suspension effect (the same effect as in the case of a non-elongated culture device), thereby ensuring operation at low and controlled flow rates.

[0235] The stem 29 not only allows for the introduction of fluid into the fluid splitter 21, but also enables the engagement and positioning of the fluid splitter 21 with respect to the device 1 and the culture chamber 2. The body 30 has a larger volume than the stem 29 and, according to the illustrated embodiment, has a tapered shape from bottom to top, thereby minimizing the footprint within the culture chamber 2. The body 30 has a bottom wall 31 and side walls 32, the side walls 32 being perpendicular to the bottom wall 31 and defined by a casing that is substantially rotationally symmetrical. Preferably, the casing of the body 30 has a curved profile, thereby minimizing the impact of the fluid splitter 21 on the environment generated within the culture chamber 2. Preferably, the fluid splitter 21 is a solid component with a cavity provided for the passage of fluid. As shown in the accompanying figures, the body 30 preferably has a substantially "bell-shaped" external form.

[0236] The bottom wall 31 of the main body 30 defines a recess 31a that extends annularly around the stem 29. The recess 31a is defined by the lower surface of the bottom wall 31. The recess 31a corresponds to the protrusion of the convex edge 3b that extends annularly around the bottom opening 3c of the culture chamber 2. The connection between the fluid diverter 21 and the culture chamber 2 is such that a fluid passage opening 33 exists between the recess 31a of the fluid diverter 21 and the protrusion of the convex edge 3b (see details in Figure 3 and the arrows indicating the fluid passage in the region in the cross-sections of Figures 3 and 7). This is particularly applicable to the first and second embodiments, where a preliminary chamber 9 is provided through which the fluid flows via the fluid passage opening 33. Considering the direction of fluid movement, the presence of a further recess 3a extending annularly downstream of the convex edge 3b induces a toroidal motion field, thereby enabling optimal fluid movement within the culture chamber 2.

[0237] As described above, the fluid splitter 21 can be engaged with the culture chamber 2 of the device 1 in the housing 10 and stably positioned. In particular, at least a portion of the stem 29 of the fluid splitter 21 is engaged within the hollow portion defined by the housing 10. Since the housing 10 is concentric with the culture chamber 2, this engagement between the stem 29 of the device 1 and the housing 10 allows the fluid splitter 21 to be centered relative to the culture chamber 2.

[0238] Some similarities between specific embodiments are described below, followed by descriptions of individual embodiments. It should be noted that the development of the three embodiments of the fluid divider 21 described herein with reference to Figures 1-13 and the further embodiment in Figure 18 stems from computational and experimental studies in which cell suspension was verified and guaranteed and cell sedimentation to the bottom wall 3 of the culture chamber 2 was avoided. These effects may also be obtained from other shapes and can be optimized according to specific applications and / or requirements and / or size (see also the section on scalability).

[0239] In the first embodiment (Figures 1-4) and the second embodiment (Figures 5-8), the fluid splitter 21 is configured to allow fluid to be introduced into the culture chamber 2 as well as to be discharged from the culture chamber 2. Therefore, the fluid splitter 21 includes a fluid discharge conduit 34 extending between a first end and a second end. The first end is the upper end and is defined as the top of the fluid splitter 21, for example, the apex, and is configured to guide fluid from the culture chamber 2 into the fluid discharge conduit. To guide the fluid, it may have a substantially funnel-shaped outer form, which may be defined on the outer surface of the apex of the main body 30. The second end is the lower end and has at least one fluid outlet 35 suitable for discharging fluid from the culture chamber 2 to the fluid discharge section 14 of the device 1.

[0240] In all three embodiments, the fluid splitter 21 comprises at least one fluid distribution conduit 36, 37, 38, which is at least partially defined within the body 30 and positioned between the fluid supply conduits 26, 27, 28 and one or more outlets of a plurality of fluid inlet and outlet 25. The fluid supply conduit 26 is common to all three embodiments and is located in the central part of the fluid splitter 21, particularly in its stem 29. The fluid distribution conduits 36, 37, 38 traverse each of the respective fluid supply conduits 26, 27, 28, and at least part of them curve outwards, communicating with one or more fluid inlet and outlet 25. The fluid distribution conduits 36, 37, 38 have an annular shape (first embodiment), an annular shape (third embodiment), or a helical shape (second embodiment).

[0241] The choice to configure one or more fluid supply conduits 26, 27, and 28 as continuous channels having substantially constant diameters, or in any case as continuous channels without significant or abrupt changes in diameter, thereby connecting the outside and inside of the culture chamber 2, is based on the objective of preventing the generation of cell shear stress caused by changes in the diameter of the circuit as the culture fluid passes from the outside to the inside.

[0242] This is an improvement over prior art. In fact, in some bioreactor configurations of prior art, such as International Publication No. 2020095143(A1), this passage is abrupt. This is because the culture medium coming from an external hydrodynamic circuit consisting of tubes is pushed into a base with a duct having a diameter much larger than the diameter of the external tubes. The aforementioned configuration choices implemented in device 1 according to the present invention allow the external and internal circuits ("external" and "internal" are interpreted with respect to the culture chamber 2) to be maintained as uniformly as possible without significant changes in diameter, ensuring uniform pressure throughout the system, thereby reducing the shear stress on the cells. The aforementioned improvement in fluid flow is facilitated by the structure of the fluid diverter 21, which includes one or more fluid supply conduits 26, 27, 28, in particular by the stem 29 from which at least one fluid supply conduit 26, 27, 28 is defined, and the body 30 communicating with at least one fluid supply conduit 26, 27, 28 of the stem 29.

[0243] In the first and third embodiments (Figures 9 to 12), the fluid diverter 21 may include at least one connecting conduit 39 positioned between the fluid distribution conduits 36, 37, 38 and the fluid supply conduits 26, 27, 28, and crossing between them, in order to connect the fluid supply conduits 26, 27, 28 and the respective fluid distribution conduits 36, 37, 38. The connecting conduit 39 is configured to transport fluid from the fluid supply conduits 26, 27, 28 to the fluid distribution conduits 36, 37, 38.

[0244] First embodiment ("donut" shape) The first embodiment shown in Figures 1 to 4 envisions a single fluid distribution conduit 36 ​​that unfolds in an annular shape around the periphery of the body 30 of the fluid divider 21. The single fluid distribution conduit 36 ​​is configured to distribute fluid to all outlets of the multiple fluid inlet and outlet 25. Due to having such an annular unfolding, the fluid distribution conduit 36 ​​has a substantially annular, or "donut-shaped," unfolding. As shown in Figures 1 and 2, the fluid distribution conduit 36 ​​may exhibit an open annular unfolding. This annular unfolding preferably covers an angular arc of at least 270°, and particularly at least 300° (see Figure 2). Furthermore, by unfolding in an annular shape, the fluid distribution conduit 36 ​​demarcates an internal space (inside the ring), thereby allowing the passage of one or more other conduits, such as connecting conduits 39. In fact, as shown in Figure 1, connecting conduits 36 may be arranged in the free internal space, and these connecting conduits 36 have a curved orientation and connect the fluid supply conduit 26 which unfolds vertically with the fluid distribution conduit 36 ​​which is substantially located on a horizontal plane. These orientations are defined with reference to the operating conditions of the fluid divider 21 shown in Figures 3 and 4. However, it is understood that the unfolding and arrangement of the conduits may vary depending on design choices, available volume, and / or other factors.

[0245] The fluid distribution conduit 36 ​​extends near the bottom wall 31 of the main body 30. The fluid inlet and outlet 25 branch off from the fluid distribution conduit 36. In particular, each fluid inlet and outlet 25 is provided with a separate flow path that branches off from a single fluid distribution conduit 36 ​​and is spaced radially apart. As shown in Figure 2, the fluid inlet and outlet 25 are angularly offset by the same angle α (see Figure 2) and are arranged to substantially cover the entire circumference of the main body 30 of the fluid divider 21. To enable toroidal motion around the entire circumference of the fluid divider 21, there may be at least four fluid inlet and outlet 25, and in particular at least six or seven. Figure 2 shows eight fluid inlet and outlet 25. By extending the fluid distribution conduit 36 ​​over an angular arc of at least 270° or at least 300° and providing an appropriate number of fluid inlet and outlet 25 angularly offset along this annular development, it is possible to uniformly supply fluid into the culture chamber 2. Furthermore, since the outlet is directed toward the bottom wall 3 having a recess 3a, it contributes to the toroidal motion generated within the culture chamber 2 (see Figure 2). Fluid introduction from the fluid introduction outlet 25 contributes to the toroidal motion generated by the fluid introduction into the culture chamber 2. This toroidal motion occurs at the fluid passage 33 defined between the fluid splitter 21 and the convex edge 3b of the bottom wall 3 of the culture chamber 2. Essentially, according to the first embodiment, there are two modes of fluid introduction into the culture chamber 2. One is via multiple fluid introduction outlets 25 of the fluid splitter 21, and the other is via fluid flowing in from the second fluid inlet 12, passing through the pre-chamber 9, and being guided to the fluid passage 33. Both modes of fluid introduction generate toroidal motion within the culture chamber 2, and as a result, this toroidal motion is emphasized and enhanced compared to known cell suspension culture devices (particularly those described in WO2020095143A1). More specifically, the toroidal motion originating from the fluid inlet / outlet 25 is turbulent, while the toroidal motion originating from the fluid passage 33 is laminar. The combination of these two types of toroidal motion, turbulent and laminar, is optimal for preventing cells from adhering to the culture chamber 2 and thus optimizing their suspension (embodiments shown in Figures 1-12).

[0246] The fluid diverter 21 according to the first embodiment further includes the aforementioned fluid outlet conduit 34. This fluid outlet conduit 34 branches off from the top of the main body 30, passes through the interior of the main body 30, and emerges laterally from the outer casing 32 of the main body 30. Thus, the fluid outlet 35 is defined outside the main volume of the main body 30 and is located away from the bottom wall 31 of the main body 30. This spacing ensures that the toroidal motion field within the culture chamber 2 does not change.

[0247] As shown in Figures 3 and 4, the device 1 according to the first embodiment has a first fluid inlet 11 and a second fluid inlet 12 at its lower part. The first fluid inlet 11 is in fluid communication with the fluid supply conduit 26 of the fluid divider 21, and the second fluid inlet 12 is in fluid communication with the auxiliary chamber 9. A fluid outlet 14 is also defined at the lower part of the device 1, which is in fluid communication with the fluid outlet conduit 34 of the fluid divider 21.

[0248] Second embodiment ("spiral") The second embodiment shown in Figures 5-8 has multiple fluid distribution conduits 36, 37, and 38 branching from the same fluid supply conduit 26. Essentially, in the second embodiment shown in Figure 5, a connecting conduit 39 between the fluid supply conduit 26 and the fluid distribution conduits 36, 37, and 38 is preferably not provided. However, in some modifications, it is understood that one or more connecting conduits may be provided. Each of the fluid distribution conduits 36, 37, and 38 opens to a corresponding fluid inlet / outlet 25. However, in other embodiments, it is understood that the same fluid distribution conduits 36, 37, and 38 may open to multiple fluid inlet / outlet 25s, and / or that the fluid inlet / outlet 25s are in fluid communication with multiple fluid distribution conduits 36, 37, and 38s.

[0249] Essentially, in the second embodiment, a plurality of fluid distribution conduits 36, 37, 38 branch from the fluid supply conduit 26 "like the spokes of a wheel". Each of the fluid distribution conduits 36, 37, 38 has at least a part with a spiral outer shape (i.e., defines at least a part of a helix). By the spiral deployment of the fluid distribution conduits 36, 37, 38, it becomes possible to generate a rotational flow of fluid around the fluid diverter 21 within the culture chamber 2. In this regard, refer to FIG. 8.

[0250] The fluid distribution conduits 36, 37, 38 are configured to supply fluid at an angle such that the inclination angle θ between the outflow flow and the bottom of the body is 10° - 30°, preferably 12° - 20°, via their respective fluid introduction outlets. The inclination angle θ may preferably be about 15°. The inclination angle θ is defined in the horizontal plane, and this angle is schematically shown in FIG. 8.

[0251] In other embodiments, it is understood that the directions of the fluid distribution conduits 36, 37, 38 may be different from the spiral direction. However, this is limited to the case where their directions are configured to generate such a rotational flow. For this reason, the direction of fluid introduction from the fluid introduction outlet 25 of the fluid diverter 21 needs to have at least one tangential motion component at the periphery of the bottom wall 31 of the body 30 and / or have the aforementioned inclination angle θ.

[0252] The fluid introduction outlets 25 are angularly offset by the same angle α (see FIG. 8) and are arranged to substantially cover the entire circumference of the body 30 of the fluid diverter 21. The second embodiment shown in FIGS. 5 and 8 has three fluid introduction outlets 25, each of which is supplied by a corresponding fluid distribution conduit 36, 37, 38, and these conduits are angularly offset from each other by 120°. However, in other embodiments, it is understood that their number may be other than three and / or they may have different offset angles.

[0253] The fluid diverter 21 according to the second embodiment also includes the aforementioned fluid outflow conduit 34 and fluid outflow outlet 35 (see FIG. 5), which is as described above for the fluid diverter 21 according to the first embodiment. With respect to the fluid inlet portion and the fluid outlet portion, the lower part of the device 1 is configured in the same manner as in the first embodiment and is connected to the fluid diverter 21, that is, it includes the first and second fluid inlet portions 11, 12 and the fluid outlet portion 14.

[0254] Essentially, in the second embodiment, there are also two methods for introducing fluid into the culture chamber 2. One is through the plurality of introduction outlets 25 of the fluid diverter 21, and the other is through the preliminary chamber 9 as described above (this flow generates a toroidal motion). By introducing a plurality of flows from the fluid introduction outlets 25, in the second embodiment, a turbulent rotational motion is generated in the culture chamber 2 (see FIG. 8), while in the first embodiment, it should be noted that a toroidal motion is generated by this inflow. This is due to the different configurations of the fluid diverter 21 (differences in the fluid distribution conduits and the arrangement of the fluid introduction outlets). Therefore, in the culture chamber 2, a gyroscopic motion is generated by the combined effect ("total") of the toroidal motion and the rotational motion. The applicant has confirmed that such a gyroscopic motion is particularly effective and suitable for cell culture in a suspended state. In particular, the turbulent rotational motion suspends the cells and significantly reduces (even substantially eliminates) deposition on the bottom wall 3 of the culture chamber 2. Therefore, the combination of the rotational motion and the toroidal motion is particularly effective. Furthermore, due to the toroidal motion, a laminar flow is created in the culture chamber 2, ensuring a more uniform distribution of the fluid, and thus oxygen and nutrients. It should also be noted that the turbulent rotational motion that rotates the cells around the main axis of the fluid diverter 21 holds the cells within the convection region.

[0255] The third embodiment ("three - split type") The third embodiment shown in Figures 9 to 12 has multiple fluid distribution conduits 36, 37, and 38. The basic concept of fluid distribution in this embodiment is to subdivide the single distribution conduit of the first embodiment into multiple divided sections 36, 37, and 38 (i.e., the first, second, and third distribution conduits below), each having the shape of an annular section. In the specific embodiment shown in detail in Figures 9 and 10, there are three annular sections 36, 37, and 38, and therefore the distribution conduit (understood as the sum of the sections) is substantially "three-part". It is understood that in other embodiments, there may be multiple fluid distribution conduits (multi-part conduits) with a number other than three.

[0256] Returning to the details in Figures 9 and 10, the fluid divider 21 includes the following: -A first fluid distribution conduit 36 ​​is in fluid communication with the first fluid inlet 11 of device 1 via the first fluid supply conduit 26. -A second fluid distribution conduit 37 is in fluid communication with the second fluid inlet 12 of device 1 via the second fluid supply conduit 27. - A third fluid distribution conduit 38 is in fluid communication with the third fluid inlet 13 of device 1 via the third fluid supply conduit 28.

[0257] As shown in Figure 9, a corresponding connecting conduit 39 may be provided between each fluid supply conduit 26, 27, 28 and each fluid distribution conduit 36, 37, 38. In other embodiments, it is understood that the number of fluid distribution conduits 36, 37, 38 and / or fluid supply conduits 26, 27, 28 and / or connecting conduits 39 may be different from three.

[0258] From each of the fluid distribution conduits 36, 37, and 38, flow paths associated with the fluid inlet and outlet 25 branch off and are spaced radially apart. Each of the fluid distribution conduits 36, 37, and 38 opens to multiple fluid inlet and outlet 25, and essentially each fluid distribution conduit 36, 37, and 38 corresponds to a section or group of fluid inlet and outlet 25. A fluid inlet section may contain only one fluid inlet or outlet 25, while a fluid inlet group may contain multiple fluid inlet and outlet 25. In the specific examples in Figures 8 and 9, it is assumed that each of the fluid distribution conduits 36, 37, and 38 opens to a fluid inlet group containing three fluid inlet and outlet 25. In other embodiments, it is understood that the same fluid distribution conduits 36, 37, and 38 may open to groups (or a single fluid inlet or outlet) with a different number of fluid inlet and outlet 25 than three, and / or one fluid inlet or outlet 25 may be in fluid communication with multiple fluid distribution conduits 36, 37, and 38.

[0259] Each fluid distribution conduit 36, 37, and 38 is supplied independently of the other conduits by its corresponding fluid supply conduit 26, 27, and 28. It is advantageous to have multiple fluid distribution conduits 36, 37, and 38, as this allows for intermittent and / or sequential supply of fluid to only one conduit or to multiple conduits simultaneously, depending on the specific application. In particular, an automated system allows for selection of which fluid distribution conduits 36, 37, and 38 (or their respective annular sections) to supply and operate, for how long to supply fluid, and in what order to supply fluid to each other.

[0260] In the third embodiment, the stem 29 of the fluid splitter 21 engages with the additional chamber or spare chamber 9, leaving no space for fluid to pass through as in the first and second embodiments (see Figures 11 and 12). However, it should be noted that a small amount of fluid (leakage fluid) may enter the additional chamber or spare chamber 9 and supply one of the three fluid distribution conduits 36, 37, and 38. More specifically, such leakage fluid may pass through the fluid inlet 23 and supply the fluid distribution conduit 37. Essentially, the stem 29 of the fluid splitter 30 in the third embodiment is preferably wider than the stem of the fluid splitter in the first and second embodiments.

[0261] As shown in Figures 11 and 12, the device 1 is provided with first, second, and third fluid inlets 11, 12, and 13 at the bottom, each in fluid communication with the first, second, and third fluid distribution conduits 36, 37, and 38 via corresponding fluid supply conduits 26, 27, and 28. The third fluid inlet 14 is the part that functioned as a fluid outlet in the first and second embodiments. This is because the fluid divider 21 according to the third embodiment does not have a fluid outflow conduit and a fluid outlet, and therefore cannot drain fluid from the culture chamber 2 through the fluid divider 21. The third fluid supply conduit 28 emerges laterally from the casing of the main body 30, and the third fluid inlet 24 is therefore defined externally to the main volume of the main body 30 and separated from the bottom wall 31 of the main body 30. In other embodiments, the fluid divider 21 may be equipped with a fluid outlet conduit, and the device 1 may be equipped with a corresponding fluid outlet that is in fluid communication with the fluid outlet conduit.

[0262] From the perspective of the motion field generated within the culture chamber 2 by the fluid diverter 21 having a three-part (or multi-part in any case) distribution conduit, it is shown that the motion generated within the culture chamber is toroidal motion, since the distribution conduit is based on the concept obtained by dividing the single distribution conduit in the first embodiment. This toroidal motion is turbulent by analogy with the first embodiment, and the same considerations as above apply. However, in the third embodiment shown in the attached figure, the presence of the stem 29 substantially fills the additional chamber or reserve chamber 9, so no laminar toroidal motion field is generated from it.

[0263] Possibility of integrating a fluid splitter into the culture chamber As predicted above, in further alternative embodiments to the first, second, and third embodiments, the fluid splitter 21 may be incorporated into the culture chamber 2. In particular, as shown in Figures 17, 18, and 21-23, the fluid splitter 21 may be incorporated into the lower or base 40 of the culture chamber 2. In such embodiments, the fluid splitter 21 has a body 30 with a positioning element 41 (in the form of a central support as shown in the accompanying figure), the positioning element 41 preferably defines the upper part of the body 30.

[0264] Flow trends, especially downstream of the fluid inlet and outlet. Upstream of device 1, a closed external hydrodynamic circuit may be provided, in particular, containing a pump 15 (peristaltic pump) configured to move fluid along the circuit under operating conditions (see Figure 13). The schematic diagram in Figure 13 shows device 1 equipped with a fluid diverter 21 according to the first or second embodiment. In fact, a fluid outlet 14 from which fluid flows out is shown (the arrow indicates the direction in which fluid flows out from the nozzle or fitting associated with the fluid outlet 14). In the schematic diagram (not shown in the attached figure), in device 1 equipped with a fluid diverter 21 according to the third embodiment, the part shown as the fluid outlet 14 in Figure 13 becomes a fluid inlet 13 (more specifically, a third fluid inlet), and the arrow indicating the fluid direction indicates the direction in which fluid flows into the nozzle or fitting associated with the fluid inlet 13. In some applications, a storage section may be provided into which cell products such as antibodies are introduced. By providing a storage section, the culture chamber 2 is isolated and protected from the control and monitoring of parameters necessary for cell culture performed in the storage section. This improves oxygen dissolution through appropriate mechanical agitation applied in the storage section, and, most importantly, prevents air bubbles from entering the fluid circulation system and thus impairing cell viability.

[0265] The fluid outflow from the fluid inlet / outlet 25 is inclined with respect to the side wall 4 of the culture chamber 2 (see Figure 8), and by colliding with the wall 4, it creates rotational motion, which is added to the toroidal motion as described below. Figure 16 schematically shows the fluid flow out from the fluid inlet / outlet 25 and colliding with the side wall 4 of the culture chamber 2 at an oblique angle. This fluid flow pattern is applicable to each realized configuration, in particular to the second configuration. The geometric center GC is defined by extending the axis A of the fluid inlet / outlet onto the side wall, with the upstream direction facing the acute angle β and the downstream direction being the opposite, coinciding with the main flow direction. The stagnation point SP is located in the direction of the acute angle β, while the point of maximum pressure tends to move away from the stagnation point depending on the flow gradient. After leaving the fluid inlet / outlet 25, the impinging flow exhibits a turbulent velocity and kinetic energy profile, which changes depending on the set flow rate and the shape of the fluid splitter 21. In the volumetric section near the fluid inlet / outlet 25, the flow is independent of the presence of the side wall 4 of the culture chamber 2, depending on the distance between the side wall 4 and the fluid diverter 21. As the flow moves away from the fluid inlet / outlet, the axial velocity begins to decrease, and the flow tends to expand. The desired motion is created by the flow hitting the wall. In the case of the fluid diverter 21 according to the second embodiment, rotational motion is generated, while in the case of the fluid diverter 21 used according to the first or third embodiment, toroidal motion is generated. Upon reaching the wall, the flow changes direction, is deflected laterally, and generates tangential and normal stresses, which affect the transport of local quantities. Here, the flow is affected by the velocity gradients with respect to both the wall and the surrounding fluid. The created configuration increases turbulence and makes the motion of the main flow more pronounced.

[0266] Similar considerations regarding fluid flow and hydrodynamics within culture chamber 2 also apply to the closed circuit in Figure 23.

[0267] Cell suspension culture device with scaffolding support The cell suspension culture device 1 shown in Figure 17 includes a holding section 50 configured to support multiple scaffolds 51. This holding section 50 is also simply called the "scaffold holding section" 50.

[0268] Device 1, shown in Figure 17, is configured for use in tissue engineering applications. Device 1 is scalable and possesses operational adaptability, enabling its use in applications / productions ranging from small-scale to large-scale.

[0269] The holding unit 50 comprises a plurality of housings 52, each configured to house a corresponding scaffold 51. The holding unit 50 further has an interface unit 53 configured to allow the holding unit 50 to interface with the fluid divider 21. Preferably, the interface surface is a positioning unit 53, configured to position the holding unit 50 relative to the fluid divider 21, and in particular to allow centering. In the embodiments shown in the attached Figures 17-23 (particularly Figures 19A and 19B), the interface surface or positioning unit 53 is defined in the central part of the holding unit 50, and the housings 52 are defined around the interface unit or positioning unit 53, for example, by a wheel spoke-like structure.

[0270] Figures 19A and 19B show two of the many possible configurations for the support section 50. The support section 50 in Figure 19A is configured to support scaffolding 51 with a circular base, such as cylindrical scaffolding 51 (see Figure 20), while the support section in Figure 19B is configured to support scaffolding 51 with a polygonal base, specifically configured to support rectangular scaffolding 51. Figures 19A and 19B show a support section 50 capable of supporting up to six scaffolding 51, which may have the same dimensions or different dimensions. It is understood that the scaffolding support section 50 may be molded into other shapes to support scaffolding 51 of different shapes and dimensions. In fact, the support section 50 may be custom designed and manufactured to support scaffolding 51 of a particular shape. The same device 1 may be equipped with multiple scaffolding support parts 50, and in particular (as shown in Figures 19A and 19B), the device 1 may be equipped with different support parts so that it can be used with different types of scaffolding 51.

[0271] The device 1 equipped with a scaffold-holding unit 50 enables anchorage-dependent (adherent) cell culture by supporting one or more scaffolds 51 that allow cell adhesion. Therefore, the cell culture device 1 according to the embodiment in Figure 17 enables adhesion-dependent cell culture by providing a scaffold-holding unit 50 for each scaffold 51.

[0272] The scaffolding support 50 is engageable with the fluid diverter 21. For this purpose, the fluid diverter 21 includes a positioning element 41 configured to position the support within the culture chamber 2. The positioning element 41 is configured to stably and / or uniquely position the support 50 relative to the fluid diverter 21 within the culture chamber 2. Preferably, the positioning element 41 is defined at the top of the fluid diverter 21 (or defines the top itself). Preferably, the positioning element is defined by a central support 41 extending to the central part of the fluid diverter 21. The positioning element 41 interfaces with and is particularly constrained by an interface surface or positioning part 53 of the support 50. Preferably, the positioning element 41 and the interface surface or positioning part 53 have corresponding external shapes, and may particularly be opposite shapes. Preferably, the positioning part 53 allows the scaffolding support 50 to be centered with respect to the fluid diverter 21 and the culture chamber 2. The interface surface or positioning portion may be in the form of the housing portion 53 of the holding portion 50 (Figures 19A, 19B), and the positioning element may be in the form of the central support portion 41 of the fluid divider 21 (Figure 22), and may be inserted into the housing portion 53 of the holding portion 50 and engage with each other. As shown in Figures 17, 20A, and 20B, the housing portion 53 unfolds in the center of the holding portion 50, preferably in a dome shape, and the lower part may be hollow so that the central support portion 41 is housed in the lower cavity.

[0273] In the embodiments of FIGS. 17 and 18, the lower part 40 of the culture chamber 2 has an integrated fluid diverter 21 (integrated structure with the lower part 40), and thus the body 30 of the fluid diverter 21 (and the stem 29 if present) is integrated with the lower part 40. Additionally or alternatively, although they are shown separately in FIGS. 17 and 21, the lower part 40 may be incorporated into the side wall 4 of the culture chamber 2.

[0274] The applicant has confirmed that embodiments of the fluid diverter 21 that can preferably be assembled at the lower part of the culture chamber 2 are the first embodiment ("donut") and the second embodiment ("spiral"). Furthermore, the applicant has confirmed that these embodiments provide optimal results regarding fluid flow and cell culture. However, alternatively, the shape of the third embodiment of the fluid diverter 21 can also be assembled at the lower part. The attached FIGS. 17-18 and FIGS. 21-23 show the lower part equipped with the assembled fluid diverter 21 according to the first embodiment. Some modifications have been made to this, for example, adapting the upper part as a positioning element for the scaffold holding part (a support that is assembled at the lower part of the culture chamber). The features of the fluid diverter 21 according to one of the three embodiments described above in relation to FIGS. 1-12 are also applicable to the embodiments of FIGS. 17-23. For example, when the fluid diverter 21 is assembled to the base 40 and at the same time shares features with the fluid diverter according to any of the three embodiments described above in relation to FIGS. 1-12, the body 30 and the stem 29 of the fluid diverter 21 can be regarded as integrally formed. Regarding the conduits, fluid inlets and outlets of the fluid diverter 21 and the base 40, the fluid outflow conduit 35 may coincide with the fluid outflow part 14 or may be defined immediately upstream thereof (see FIG. 22). Similarly, the fluid inlet 22 may coincide with the fluid inlet part 11 or may be defined immediately downstream thereof.

[0275] The device 1 may further comprise the following. - Scaffolding stopping element 54. This allows the scaffolding 51 to be positioned relative to the holding part, and in Figures 17, 20, 21 and 23, the scaffolding stopping element consists of a clip 54 with a through cavity (through hole). - Restraint element 55. This is configured to fix the scaffolding to the retaining part and preferably functions in cooperation with the scaffolding stop element. In Figures 17, 20, 21 and 12, the restraint element is threaded (screw-in type). -Optionally, multiple scaffolding structures 51. Note that device 1 may be supplied without scaffolding, as scaffolding can be procured separately.

[0276] In order to restrain the scaffolding 51 to the holding part 50, the holding part 50 may have a restraining part 56. In the embodiment shown in the attached figure, these restraining parts 56 are threaded, and a screw-in type restraining element 55 can be attached to them.

[0277] The process of fixing the restraint elements and scaffolding to the holding part is shown in Figures 20A and 20B. These steps are part of the assembly process of device 1, which will be described later. This section describes the structure of the culture chamber 2 in the embodiment of device 1 shown in Figure 17. This structure is modular. The culture chamber 2 includes the following: - A lower section 40 equipped with a fluid divider 21 (in the attached figure, the fluid divider 21 is incorporated into the lower section 40. However, in other embodiments, the fluid divider 21 may be separated from the lower section). - Side wall 4 (shown separately from the lower part 40 in the figure; however, in other embodiments, the side wall may be integrated with the lower part and / or the fluid divider). - Closing element 5. This element is provided with multiple connection points, or through holes 7, or a fluid inlet or nozzle 8, or a sampling port for cell collection. It also has a threaded support for housing a vent plug.

[0278] Each threaded support portion of the closing element 5 may be fitted with a filter-type threaded vent plug 60 (equipped with a porous membrane), thereby enabling gas exchange (O2, CO2). Furthermore, a connector 61, such as a Luer lock type connector, can be attached to the sampling port (see Figure 23).

[0279] Connectors 61, such as Luer lock type connectors, may be provided at the fluid inlet sections 11, 12, 13 and the fluid outlet section 14, respectively.

[0280] Figure 23 shows a closed circuit in which cell culture medium is continuously delivered from the base 40 (i.e., the bottom wall 3, see the curved arrows in Figures 22 and 23) to the culture chamber 2 using an external peristaltic pump 15 and tubing. This delivery lifts the cells, making it possible to seed them onto the scaffold. For simplification, the scaffold 51 is not shown in Figure 23. However, the scaffold 51 is present under the operating conditions of the device 1 according to the embodiments of Figures 17-23. In Figure 23, as can be seen from the curved arrows, the fluid flows out from below the holding section 50, particularly from the fluid inlet 25 located between the bottom wall 3 and the holding section 50.

[0281] The embodiments shown in Figures 17-23 provide at least the following functions and advantages. - Connect the culture chamber 2 to the pump circuit 15 inside the incubator (where device 1 is located). - Sampling the culture medium during cell culture. - The possibility of seeding cells within the culture volume. - The possibility of using scaffolding of different sizes. - Modularity: Multiple experiments can be performed in parallel using multiple devices 1. In this case, it is preferable that the peristaltic pump 15 is equipped with a multi-channel head.

[0282] system The present invention further relates to a system comprising: a plurality of cell culture devices 1 of the type described above, wherein these cell culture devices 1 are optionally arranged in parallel with one another. The devices 1 of the system can be used to perform experiments, preferably arranged in parallel with one another.

[0283] Scalability of cell suspension culture devices The suspension cell culture device 1 (bioreactor) and its respective culture chamber 1 have large volumes, and in particular, can vary from the order of milliliters to liters, and even to the order of hundreds of liters. In this case, the following can also vary: size, volume, the number of fluid inlet and outlet 25, the number of fluid distribution conduits 36, 37, 38, and other parameters of the fluid dividers 21 configured to operate within these devices 1. Thus, the applicant's objective is to protect the technical solution regardless of the actual dimensions or parameters of the device 1 and / or fluid dividers 21 (e.g., fluid capacity, base chamber diameter, fluid divider parameters, etc.). Essentially, the actual dimensions or parameters of the device 1 and / or fluid dividers 21 may follow one or more "invariant constitutive laws" even as various variables acting within the device 1 change.

[0284] This objective involves parameterization and is based on the need to make Device 1 scalable so that it can be used in a variety of situations, namely, from university research laboratories (using small volumes of less than 100 milliliters) to large pharmaceutical companies (using volumes ranging from 10-20 liters to 500-1000 liters).

[0285] Therefore, the applicant considered the possibility of utilizing the same structure and operating principle in different sizes without the need to redesign the bioreactor 1 for each individual application. More specifically, considering the fluid splitter 21 of the first and third embodiments, the number of fluid inlet and outlet 25 from which the flow that generates additional toroidal motion exits depends on the flow rate set by the pump 15 and the geometry of the culture chamber 2, particularly its diameter D and the cross-section of the fluid inlet and outlet 25 of the fluid splitter 21. Similarly, the same variables may be used for the fluid splitter 21 according to the second embodiment. In this case, their relationship governs the creation of sufficient rotational motion to suspend cells and significantly reduces (and in some cases eliminate) cell deposition against the bottom wall 3 of the culture chamber 2.

[0286] The main parameters considered in the implementation of the model are as follows: • Flow rate set by the pump [cm²] 3 / s](Q) • Number of fluid inlet and outlet in the fluid divider (n) • Cross-sectional area of ​​fluid inlet / outlet [cm²] 2 ](A) • Inner diameter [cm] (d) of the device's culture chamber.

[0287] The four parameters mentioned above are combined according to the following formula.

number

number

[0288] In this way, the optimal configuration for different sizes of the culture chamber 2 can be obtained by changing the above parameters. In this case, for the fluid divider 21, k is maintained in the range of 20 to 30 in the first and third embodiments, and k is maintained in the range of 55 to 65 in the second embodiment.

[0289] It should be noted that the above formula does not include the density and viscosity values ​​of the culture medium. This is because it is assumed that Bioreactor 1 is always used for cell culture or tissue engineering, in which case the culture medium has stable density and viscosity values ​​and can therefore be considered constant in different configurations.

[0290] The versatility and adaptability of device 1 according to the present invention make it possible to easily change the fluid dynamics within the culture chamber 2, in particular, by replacing the fluid shunt 21 (for example, with fluid shunts having different geometries and / or sizes) and / or by changing the flow rate defined by the pump 15, depending on the application and requirements. The fluid shunt 21 was designed using FEM (finite element method) and CFD (computational fluid dynamics) simulations. Figures 14 and 15 show 2D simulations in an axisymmetric configuration, and it should be noted that this does not 100% reflect the actual flow field generated by the fluid shunt 21, as the fluid inlet and outlet 25 are distributed on its sides or bottom. However, these simulations were used to verify the effect of the number, cross-section, and distribution of fluid inlet and outlet 21 of the fluid shunt 21. Subsequently, these aspects were experimentally verified by PIV (particle image velocity measurement) analysis and post-processing analysis using appropriate software. The latter two analyses are also used to determine whether gyroscopic motion ensures cell suspension and prevents cells from settling at the bottom of the culture chamber 21. As can be seen from Figures 14 and 15, the cells are trapped within a bottom vortex (region 2') generated in the culture chamber 2.

[0291] The technical features relating to the function of Device 1 or its parts / components / elements disclosed herein are applicable in the context of corresponding uses of Device 1 or method steps described below, and can therefore be used to identify such uses and methods in the appended claims.

[0292] use The present invention further relates to the use of the aforementioned cell suspension culture device 1. The use of device 1 is intended for cell culture in a suspension state, and can optionally be used for both suspension culture and adherent culture (see embodiments in Figures 17-23).

[0293] Device 1 can be used in the laboratory for testing suspension culture cells, as well as for large-scale production. For example, it can be used to culture cells for scientific or industrial use, or cells used in vaccine production processes.

[0294] This device is used, in particular, in device 1 equipped with a scaffolding support unit 50, for tissue engineering operations or applications.

[0295] During use, device 1, equipped with a scaffolding holder, enables both cell suspension culture (especially in the initial stages of use or operation) and adherent cell culture (when cells adhere to the scaffolding in the later stages) without the need to replace the culture chamber 2. This is a particular advantage.

[0296] How to operate a cell suspension device The present invention also relates to a method for operating the above-described type of cell suspension culture device 1. This method is - A step of introducing fluid into the device 1 through at least one fluid inlet 11, -A process of transporting fluid from at least one fluid inlet 11, 12, 13 downstream of at least one fluid inlet 11, 12, 13 and upstream of the fluid outlet 14 to a fluid divider 21 at least partially located within the culture chamber 2, wherein the upstream and downstream arrangements are defined with respect to the direction of fluid flow. - The process includes introducing multiple fluid flows into the culture chamber 2 through multiple fluid inlet and outlet outlets 25 of the fluid divider 21.

[0297] As described above, this method may include generating turbulent toroidal motion (first and third embodiments) and / or generating turbulent rotational motion (second embodiment) within the culture chamber 2 by introducing fluid through a plurality of fluid inlet and outlet ports 25.

[0298] Furthermore, in particular in the first and second embodiments, this method can result in the generation of additional toroidal (laminar) motion within the culture chamber 2 by introducing fluid at the fluid passage 33 located downstream of the pre-chamber 9.

[0299] The fluid introduction step may include supplying multiple turbulent fluid flows into the culture chamber 2 through multiple fluid inlet / outlet ports 25.

[0300] The method may also, particularly in relation to the third embodiment, include introducing a plurality of fluid flows into the culture chamber 2 in a predetermined order and / or introducing each fluid flow into the culture chamber 2 for a predetermined time.

[0301] Those skilled in the art will understand that, unless otherwise indicated, the specific order of steps described herein is merely illustrative and can be modified within the scope of the disclosures and appended claims. Therefore, unless otherwise indicated or referred to by the same terminology, the steps of this method may be carried out in any convenient or preferred order.

[0302] Process for assembling a cell suspension culture device equipped with a scaffolding support. The assembly of the device equipped with the scaffolding support (see Figure 17) is performed under sterile conditions. If the same device 1 is to be used multiple times, it is necessary to ensure that any liquid present before sterilization is completely removed.

[0303] This process includes the assembly stage of the scaffolding support section 50 and the scaffolding positioning 51.

[0304] -As shown by arrow 1 in Figure 20A, engage the threaded restraint element 55 (hereinafter referred to as "screw" for simplicity) with the restraint portion of the corresponding scaffolding stop element 54 (hereinafter referred to as "clip" for simplicity). Repeat this operation a number of times equal to the number of scaffolding 51 used. - Using a sterilized operating device 70 (e.g., a screwdriver or hex wrench), the screw 55 is secured to the restraint portion 56 of the scaffolding support portion 50 (see Figure 20A, direction of arrow 2). - Using a sterilized instrument (e.g., tweezers), position the scaffold 51 between the clip 54 and the holding part 50 (Figure 20B). - To secure the scaffolding 51, adjust the height of the screw using the operating device 70 so that the clip 54 can secure the scaffolding 51 (Figure 20B). Note that the screw is inserted into the recess of the clip 54. If necessary, rotate the clip 54 with a sterilized instrument.

[0305] It is understood that the assembly process of the scaffolding support unit 50 and the positioning process of the scaffolding 51 described above may be carried out by alternative means, depending on the type of means used to fix the scaffolding 51 to the support unit 50.

[0306] This process also includes the step of assembling the lower part (hereinafter referred to as the "base") 40 of device 1 to obtain the configuration shown in Figure 18. Starting from the exploded view of Figure 17 (see the bottom of the exploded view), this process includes the following: - Insert two O-rings 62 into predetermined grooves on the outer surface of the base 40. - Insert two O-rings 63 into predetermined grooves in the central support column 41 of the fluid divider 21 incorporated into the base 40. - Luer lock type connectors 61 are attached to each fluid inlet 11, 12, 13 (side inlet nozzles) and each fluid outlet 14 (side outlet nozzle) on the base 40 of device 1.

[0307] This process also includes the step of assembling the closing element 5 (hereinafter referred to as the "cap"), which includes the following: - Insert the O-ring 64 into the dedicated groove on the outer surface of the closing element 5. - Screw the Luer lock connector 61 into each threaded port located on the top of the closing element 5 (see Figure 17, then Figure 23, which shows the assembled state with two Luer lock connectors engaged with the cap). - Screw the vent plugs 60 into each threaded retaining part on the closing element 5 (see Figure 17, and further see Figure 23, which shows the assembled state with the two vent plugs engaged with the closing element).

[0308] If cell sampling is required, a tube and valve can be connected to the Luer lock connector 61 to perform cell sampling / culture medium supply.

[0309] This process includes assembling the side wall 4 to the base and assembling the scaffolding support 50 to the base 40, and it should be noted that the order of these steps may be reversed. Furthermore, these steps may vary depending on the properties of the components. For example, if the side wall 4 is integrated with the base 40, the step of assembling the side wall 4 to the base is unnecessary.

[0310] In the structural configuration shown in Figure 17, when the side wall 4 and the base 40 are separated from each other (the side wall is substantially a hollow cylindrical portion), the process of assembling the side wall 4 to the base preferably includes the following: - The surface of the O-ring 62 (base 40) is moistened with water to lubricate it. - Apply pressure until the surfaces of both members are in contact, joining the side walls to the base (note that the base and side walls have similar diameters).

[0311] The process of assembling the scaffolding support to the base (see Figure 21) includes inserting the scaffolding support, to which the scaffolding is fixed as described above, into the base by applying pressure, thereby inserting the central support 41 of the fluid divider 21 into the cavity of the housing portion 53 of the scaffolding support 50 and restraining the support to the fluid divider 21.

[0312] This process may further include connecting the inlet / outlet to the peristaltic pump 15 and introducing the cell suspension / cell medium into the culture chamber 2. The cell medium and cell suspension can be directly introduced into the pre-assembled base 40 and side walls 4. The culture chamber 2 must be filled to at least the height of the outlet nozzle.

[0313] This process involves attaching the closing element 5 to the side wall 4, preferably as follows. - The surface of the O-ring 64 of the closing element 5 is moistened with water to lubricate it, thereby allowing the closing element 5 and the side wall 4 to be easily connected. -The closing element 5 is bonded to the side wall 4 by applying pressure until the surfaces of the two parts come into contact.

[0314] Generally, the order of each step in the device assembly process can be changed as needed.

[0315] At this point, device 1 can be activated. Providing the device includes the following: - Start the peristaltic pump 15 and set the desired flow rate. - Check for air bubbles in the circuit. Due to the presence of air, bubbles may appear in the tubes and culture chamber 2 for the first few seconds. If the device is functioning correctly, the bubbles will disappear quickly. -Stop pump 15. Next, place device 1 inside the incubator. If the peristaltic pump 15 to be used cannot be installed inside the incubator, ensure that the pump's power cable and tubing can easily pass through the door seal of the culture incubator and do not damage the internal environment of the incubator.

[0316] Afterward, the peristaltic pump 15 is activated again. When using anchorage-dependent (adherent) cells, cell adhesion is usually dependent on the test conditions.

[0317] Cell / culture medium sampling can be performed by connecting a sterile syringe or pipette to the sampling port while device 1 remains in the incubator, or by moving device 1 to the laminar flow hood. This can be done while the peristaltic pump 15 is operating. Alternatively, sampling may be performed by moving device 1 from the incubator to the laminar flow hood and removing the closing element 5 of device 1. In this way, the sample can be collected with a pipette or syringe.

[0318] A process for removing at least one scaffold from a cell suspension culture device equipped with a scaffold holder. The present invention further provides a process for removing at least one scaffold 51. This process is performed after deploying or using at least one device 1 equipped with the scaffold holder 50 of the type described above and one or more scaffolds 51. When using anchorage-dependent cells, cell adhesion to the scaffold 51 can be completed within 24 hours after inoculation of the cell culture. At this point, the cellularized scaffold 51 (i.e., the scaffold 51 with cells adhered to its walls) can be removed from the device 1 and subjected to physicochemical or biological characterization. The scaffold holder 50 can support multiple scaffolds 51, thereby enabling statistical and temporal analysis of the cellularized scaffolds. To remove the scaffold 51, the device 1 must be moved from the incubator to the laminar flow hood. The steps for removing at least one scaffold are as follows: -Move device 1 from the incubator to the laminar flow hood. - Preferably, the peristaltic pump 15 is stopped. - Remove the closing element 5. Preferably, this is done by manually separating it from the side wall 4. - Using sterilized instruments such as tweezers, gently rotate the desired scaffolding stopper (clip). - Remove the corresponding scaffolding 51.

[0319] The removal process may then include the following: - Gently push the closing element 5 against the side wall 4 to close the device 1. - Activate the peristaltic pump 15. -Move device 1 from the laminar flow hood to the incubator.

[0320] Further benefits and conclusions The installation of the fluid diverter 21 advantageously allows the flow to be directed only to specific points at the bottom of the bioreactor 1. This is an improvement over prior art, for example, International Publication No. 2020095143(A1), in which more energy and power were required and consumed from the peristaltic pump. This was because the inflow pressure was uniformly distributed across the entire base of the culture chamber. By operating the bioreactor 1 according to the present invention in a "localized" and limited manner, it becomes possible to reduce the fluid flow rate while maintaining the same cell suspension effect, thereby suppressing energy consumption and flow rate to controllable values.

[0321] By installing the fluid divider 21 near the bottom wall 3, or by integrating it with the bottom wall 3, the fluid divider 21 can be "integrated" or integrated into the lower part of the bioreactor 1, making it more compact, easier to assemble, and easier to sterilize.

[0322] Device 1 may be reusable by sterilization or may be disposable. Device 1 is preferably configured to be sterilizable in an autoclave, for example, at about 121°C for about 30 minutes. With respect to materials, the base, closing element 5, scaffold holder 50 and clip 54 may be made of resin, while the side wall 4 may be made of glass (and thus transparent). The screw 55, if provided, may be made of stainless steel. The culture chamber 2 is preferably completely transparent to allow optical inspection.

[0323] The present invention advantageously enables the provision of devices and methods for cell culture in a suspension state, forming an efficient environment for cell growth in a suspension state, and further, as shown in the embodiments in Figures 17-23, enabling this even in an adherent state. In particular, the present invention ensures cell suspension by reducing cell adhesion to the walls of the cell culture device 1, and in the embodiments shown in Figures 17-23, also enables adhesion to the scaffold 51 and avoids cell sedimentation at the bottom. This makes it easier to control the shear stress on the cells, which can be changed as desired depending on the type of cells being cultured, improves the homogenization of nutrients and oxygen, and avoids the formation of concentration gradients within the culture volume.

[0324] It is understood that each element, component, and / or step of the product / method according to the present invention may be replaced by equivalent elements, components, and / or steps (hereinafter referred to as "Equivalents"). Such Equivalents may already exist at the time of the filing date or priority date of this patent document, or at the time of subsequent conception / development.

Claims

1. A cell suspension culture device (1), - Culture chamber (2), - At least one fluid inlet (11, 12, 13) configured to allow fluid toward the culture chamber (2), - At least one fluid outlet (14) configured to discharge fluid from the culture chamber (2), - A fluid divider (21) is provided, at least a portion of which is located within the culture chamber (2), and is positioned between the at least one fluid inlet (11, 12, 13) and the at least one fluid outlet (14) with respect to the direction of fluid flow. The aforementioned fluid divider (21) - At least one fluid inlet (22, 23, 24) that is in fluid communication with the at least one fluid inlet (11, 12, 13), The fluid divider (21) includes a plurality of fluid inlets (25) that are in fluid communication with at least one fluid inlet (22, 23, 24), The aforementioned fluid divider (21) - The fluid from at least one of the fluid inlet sections (11, 12, 13) is transported into the culture chamber (21). - A device configured to introduce multiple fluid flows into a culture chamber (2) through the multiple fluid inlets (25) mentioned above.

2. The device according to claim 1, wherein each of the plurality of fluid inlet / outlet ports (25) is oriented laterally with respect to the walls (3, 4) of the culture chamber (2), and in particular, is oriented laterally with respect to the bottom wall (3) and / or side wall (4) of the culture chamber (2).

3. - The fluid divider (21) A stem (29) equipped with a fluid supply conduit (26), The system includes a main body (30) that is in fluid communication with the fluid supply conduit (26), The fluid supply pipeline (26) is in fluid communication with at least one fluid inlet (11, 12, 13), and in particular is in direct fluid communication with it, and is configured to transport the fluid flow to the main body (30). - The main body (30) is deployed inside the culture chamber (2) and has a larger volume than the stem (29), - The device according to claim 1 or 2, wherein the main body (30) has a peripheral portion, and the plurality of fluid inlet and outlet ports (25) are defined on the peripheral portion.

4. The device according to claim 3, wherein the fluid supply conduit (26) is an internal conduit, and preferably is realized inside the body of the stem (29) of the fluid diverter (21).

5. The device according to claim 3 or 4, wherein the peripheral portion is the circumferential portion of the main body (30), and the outlets of the plurality of fluid inlet and outlet ports (25) are angularly offset from one another.

6. The fluid divider (21) has a dual function: The fluid is transported into the culture chamber (2) from at least one fluid inlet (11, 12, 13), divided, and in particular equally divided, to form multiple fluid flows that are supplied into the culture chamber (2) via the multiple fluid inlet / outlet (25). - A device according to any one of the prior claims, comprising the ability to drain fluid from the culture chamber (2) to the outside of the culture chamber (2).

7. - The fluid divider (21) further comprises at least one fluid outlet (35) configured to allow the fluid from the culture chamber (2) to flow out of the culture chamber (2), - The fluid outlet (35) is in communication with the at least one fluid outlet (14), - The device according to any one of the prior claims, wherein the fluid divider (21) is configured to transport fluid from the culture chamber (2) to the at least one fluid outlet (14) via the fluid outlet (35), thereby discharging fluid from the device (1).

8. - The fluid divider (21) is located at the bottom of the culture chamber (2), and the at least one fluid outlet (14) is located at the bottom of the cell suspension culture device (1), The fluid divider (21) includes a fluid outlet conduit (34) that establishes fluid communication between the at least one fluid outlet (35) and the at least one fluid outlet section (14), - The device according to claim 7, wherein at least a portion of the fluid outlet conduit (34) is formed within the main body.

9. The fluid divider (21) is defined within the main body (30) and comprises at least one fluid distribution conduit (36, 37, 38) positioned between the at least one fluid supply conduit (26, 27, 28) and one or more outlets of the plurality of fluid inlet and outlet (25). The at least one fluid distribution conduit (36, 37, 38) extends laterally with respect to the at least one fluid supply conduit (26, 27, 28), and at least a portion of it is curved. The device according to claim 3, 4, or 5, or at least claim 7 or 8 dependent on claim 3, wherein it is in fluid communication with one or more outlets of the plurality of fluid inlet and outlet outlets (25).

10. - The at least one fluid supply conduit (26, 27, 28) is located in the central part of the fluid divider (21), - The at least one fluid distribution conduit (36, 37, 38) has an annular or partially annular outer shape, The device according to claim 9, wherein the fluid divider (21) includes at least one connecting conduit (39) positioned between the at least one fluid distribution conduit (36, 37, 38) and the at least one fluid supply conduit (26, 27, 28), and the connecting conduit (39) is configured to transport the fluid from the at least one fluid supply conduit (26, 27, 28) to the at least one fluid distribution conduit (36, 37, 38).

11. The fluid divider (21) includes a single fluid distribution conduit (36) having an annular outer shape, the fluid distribution conduit (36) is defined within the main body (30) and is positioned between the at least one fluid supply conduit (26) and the plurality of fluid inlet and outlet ports (25), The single fluid distribution conduit (36) is configured to distribute fluid to all of the outlets of the plurality of fluid inlet and outlet (25), Preferably, the device according to claim 10, wherein the fluid divider (21) includes a single fluid inlet (22) that is in fluid communication with the single fluid distribution conduit (36).

12. - The fluid divider (21) - Multiple fluid inlets (22, 23, 24), - Includes a plurality of fluid distribution conduits (36, 37, 38), each having a partially annular outer shape and being in fluid communication with corresponding fluid inlets (22, 23, 24), - In the plurality of fluid inlet / outlet ports (25), a plurality of fluid inlet compartments or groups may be identified, and each fluid inlet compartment or group includes at least one fluid inlet / outlet port (25) and is in fluid communication with the corresponding fluid distribution conduit (36, 37, 38). Preferably, the device according to claim 10, wherein each fluid distribution conduit (36, 37, 38) is in fluid communication with a fluid introduction group including a plurality of fluid inlet and outlet ports (25).

13. - The at least one fluid supply conduit (26, 27, 28) is located in the central part of the fluid divider (21), - The fluid divider (21) is equipped with a plurality of fluid distribution conduits (36, 37, 38) that branch off from the fluid supply conduits (26, 27, 28) and extend toward the peripheral edge of the main body of the fluid divider (21), - Each fluid distribution conduit (36, 37, 38) is arranged in a spiral shape, at least in part. - Preferably, the device according to claim 9, 10, or 12, wherein the fluid divider (21) includes a single fluid supply conduit (26), and the conduits of the plurality of fluid distribution conduits (36, 37, 38) branch radially from the single fluid supply conduit (26).

14. - The device (1) includes an additional chamber or spare chamber (9) located upstream of the culture chamber (2), - The fluid divider (21) is at least partially housed within the additional chamber or auxiliary chamber (9), - Preferably, the device according to any one of the prior claims, wherein at least one fluid supply conduit (26, 27, 28) is at least partially deployed in the additional chamber or auxiliary chamber (9).

15. - The culture chamber (2) comprises a bottom wall (3) with a bottom opening (3c) and a convex edge (3b) that extends around the bottom opening (3c), - The pre-chamber (9) is partitioned above by the bottom opening (3c) and is positioned between at least one fluid inlet (11, 12, 13) of the device (1) and the internal volume of the culture chamber (2) with respect to the direction of fluid flow. - The fluid divider (21) defines a fluid passage (33) at the fluid outlet of the pre-chamber (9) together with the convex edge (3b) of the bottom wall (3) of the culture chamber (2), - Preferably, the device according to claim 14, wherein the front bottom wall (3) of the culture chamber (2) includes a recess (3a) that extends around the convex edge (3b).

16. The device according to any one of the prior claims, wherein the culture chamber (2) has a diameter (D) and a height (H), and the height (H) is greater than the diameter (D), preferably the height (H) is at least 1.3 times the diameter (D).

17. It further includes at least one holding part (50) configured to support one or more scaffolding (51), preferably a plurality of scaffolding (51), The device according to any one of the prior claims, wherein the fluid divider (21) includes a positioning element (41) configured to position the holding portion (50) within the culture chamber (2).

18. The device according to claim 17, wherein the positioning element (41) is defined on the upper part of the fluid divider (21), and preferably the positioning element (41) includes a support column of the fluid divider (21).

19. The device according to claim 17 or 18, wherein the positioning element (41) is configured to stably and / or uniquely position the holding portion (50).

20. The device according to any one of the prior claims, wherein the fluid divider (21) is incorporated into the culture chamber (2), and in particular into the base (40) and / or bottom wall (3) and / or side wall (4) of the culture chamber (2).

21. Use of the device (1) described in any one of the prior claims for tissue engineering.

22. A method for operating a cell suspension culture device (1) according to any one of prior claims 1 to 20, - Introducing fluid into the device (1) through at least one fluid inlet (11, 12, 13), - A step of transporting fluid from the at least one fluid inlet (11, 12, 13) downstream of the at least one fluid inlet (11, 12, 13) and upstream of the at least one fluid outlet (14) to the fluid divider (21) located inside the culture chamber (2), wherein the upstream and downstream arrangements are defined with respect to the direction of fluid flow, A method comprising the step of introducing multiple fluid flows into the culture chamber (2) through multiple fluid inlet and outlet ports (25) of the fluid divider (21).

23. A method according to claim 22, wherein the step of introducing a plurality of fluid flows into the culture chamber is - To generate toroidal flow motion within the culture chamber (2), and / or A method comprising generating rotational fluid motion within a culture chamber (2).

24. - A step of providing side walls (4) and a base (40) to form a culture chamber (2) of a cell culture device (1), preferably the cell culture device (1) being one of the prior art claims, - A step of providing a fluid divider (21) and at least one holding part (50) configured to support one or more scaffolds (51), wherein optionally the fluid divider (21) is incorporated into the base (40), - An assembly process for a cell culture device (1), comprising the step of engaging at least one scaffolding holding part (50) with the fluid diverter (21).

25. - The step of providing the side walls (4) and base (40) to form the culture chamber (2) is - A step in which the side wall (4) and the base (40) are provided separately from each other, and the fluid divider (21) is an integral structure with the base (40), The process according to claim 24, comprising the step of engaging the side wall (4) and the base (40) with each other, the step of positioning the fluid divider (21) within the culture chamber (2), A process wherein the step of engaging the at least one scaffolding holder (50) with the fluid divider (21) is performed before or after the step of positioning the fluid divider (21) within the culture chamber (2).

Images