阅读说明：本技术 细胞培养装置及其使用方法 (Cell culture device and method of use thereof ) 是由 塞萨雷·塞加斯 埃尔亚·阿拉夫阿克巴里 安托万·埃斯皮内特 于 2019-11-04 设计创作，主要内容包括：本发明涉及一种细胞培养装置,其包括至少具有至少一个细胞培养基入口和一个细胞培养基出口的流体通道,所述流体通道包括在所述细胞培养基入口和所述细胞培养基出口之间延伸的下壁,所述细胞培养装置还包括至少一个被配置为接收多个细胞的凹槽以及至少一个细胞阱,所述凹槽由所述下壁形成并限定了一个底表面,所述细胞阱被配置为捕获在流体通道中平流运送的细胞然后使所述细胞沉降到所述凹槽的底表面。(The present invention relates to a cell culture device comprising a fluid channel having at least one cell culture medium inlet and one cell culture medium outlet, the fluid channel comprising a lower wall extending between the cell culture medium inlet and the cell culture medium outlet, the cell culture device further comprising at least one recess configured to receive a plurality of cells, the recess being formed by the lower wall and defining a bottom surface, and at least one cell trap configured to capture cells advected in the fluid channel and then to cause the cells to settle to the bottom surface of the recess.)

1. A cell culture device (1) having at least one surface portion (2) defining a planar surface (3), said surface portion being configured to lie on a horizontal support, said cell culture device comprising at least one fluid channel (4) having at least one cell culture medium inlet (5) and one cell culture medium outlet (6), said fluid channel comprising a lower wall (7) extending between said cell culture medium inlet and said cell culture medium outlet,

the cell culture apparatus is characterized by further comprising:

at least one recess (10) configured to receive a plurality of cells (12), the recess (10) being formed by the lower wall (7) and defining a bottom surface (11), and

at least one cell trap (13), the cell trap (13) being configured to capture cells (12) transported advected in a fluid channel and then to sink the cells to a bottom surface (11) of the recess (10),

the cell trap comprises an obstacle (14) having a main direction of extension defining a sedimentation axis, which is parallel to a sedimentation direction of cells within the obstacle, and wherein the sedimentation axis is perpendicular to the surface portion (2).

2. Cell culture apparatus according to claim 1, wherein the lower wall (7) further comprises an upper surface (23) extending on one side of the recess (10), the obstacle (14) extending from the bottom surface (11) beyond the recess (10) of the upper surface of the lower wall (7).

3. Cell culture device according to claim 1 or 2, wherein the fluid channel further comprises an upper wall (20), the obstacle (14) extending at least from the upper wall (20) to the recess (10).

4. Cell culture device according to any of claims 1-3, wherein each obstacle has a hole, in particular a transverse hole, preferably having a transverse slit (15), the width of the hole being between 3 μm and 30 μm, preferably between 5 μm and 20 μm.

5. The cell culture apparatus according to any one of claims 1-4, wherein at least a portion of the fluid channel (4) is configured to receive a liquid flow according to a main flow direction (16) extending between the cell culture medium inlet (5) and the cell culture medium outlet (6), and

wherein the fluid channel (4) comprises an array of cell traps (13), the array of cell traps (13) extending in a direction perpendicular to the main flow direction (16) in a plane parallel to the planar surface (3).

6. Cell culture apparatus according to any of claims 1-5, wherein the fluid channel further comprises a side surface extending between the cell culture medium inlet (5) and the cell culture medium outlet (6), and wherein at least one groove extends from one side surface to the other side surface over the entire width of the fluid channel.

7. Cell culture device according to any of claims 1-6, wherein the lower wall (7) further comprises an upper surface extending at one side of the recess (10), and wherein the fluidic channel further comprises an upper wall (20), wherein a first height of the fluidic channel (4) is defined as the shorter distance between the upper surface (23) of the lower wall (7) and the upper wall (20), a second height of the recess (10) is defined as the shorter distance between the bottom surface (11) of the lower wall and the upper surface (23) of the lower wall, and wherein the second height is 0.15 to 0.85 times the first height, preferably 0.20 to 0.70 times the first height.

8. Cell culture device according to any of claims 1-7, wherein the lower wall (7) further comprises an upper surface extending at one side of the recess (10), wherein a second height of the fluidic channel is defined as the shorter distance between the lower surface of the lower wall and the upper surface of the lower wall, and wherein the second height is between 10 μm and 500 μm, in particular between 15 μm and 300 μm, and preferably between 15 μm and 200 μm.

9. Cell culture apparatus according to any of claims 1-8, comprising at least one harvest inlet (17) and one harvest outlet (18), which are different from the cell culture medium inlet and outlet, at least one groove being in direct fluid connection with harvest inlet (17) and harvest outlet (18).

10. A method of cell culture, the method comprising:

-arranging the cell culture apparatus according to any of claims 1-9 with the surface portion (2) on a horizontal support,

-a seeding step a) wherein a medium comprising cells is injected into the cell culture medium inlet of the at least one fluid channel of the cell culture assembly until at least more than 10% of the cell traps capture at least one cell, preferably more than the majority of the cell traps capture at least one cell.

11. The cell culture method according to claim 10, further comprising, after the seeding step a), an expansion step b) of injecting a medium comprising an active culture agent into the cell culture medium inlet, wherein the medium is injected at a first predetermined flow rate, the method comprising, after the seeding step a), an optional harvesting step e), wherein medium is injected into the cell culture medium inlet and cells are recovered from the cell culture medium outlet at a predetermined second flow rate, which is strictly higher than the first flow rate, the method further comprising, after step a), an optional sampling step f), wherein medium is injected into the cell culture medium inlet and a cell sample is recovered from the cell culture medium outlet at a predetermined fifth flow rate, which is strictly higher than the first flow rate, the injection time and the fifth sampling flow rate being calculated before the seeding step a), so that less than one third of the cells in the recess are recovered.

12. The cell culture method according to claim 10 or 11, further comprising a washing step c) of injecting a washing medium into the cell culture medium inlet after the seeding step a), wherein the medium is optionally injected at a third predetermined flow rate, the method further comprising an optional harvesting step e) after the seeding step a), wherein medium is injected into the cell culture medium inlet at a predetermined second flow rate and cells are recovered from the cell culture medium outlet, the second flow rate being strictly higher than the third flow rate, the method further comprising an optional sampling step f) after step a), wherein medium is injected into the cell culture medium inlet at a predetermined fifth flow rate, the fifth flow rate being strictly higher than the third flow rate, the injection time and the fifth sampling flow rate being calculated before the seeding step a), and cell samples are recovered from the cell culture medium outlet, so that less than one third of the cells in the recess are recovered.

13. The cell culture method according to any one of claims 10-12, further comprising a differentiation step d) of injecting a medium comprising at least a differentiation factor into the cell culture medium inlet after the seeding step a), wherein the medium is optionally injected at a fourth predetermined flow rate, the method further comprising an optional harvesting step e) after the seeding step a), wherein medium is injected into the cell culture medium inlet at a predetermined second flow rate and cells are recovered from the cell culture medium outlet, the second flow rate being strictly higher than the fourth flow rate, the method further comprising an optional sampling step f) after step a), wherein medium is injected into the cell culture medium inlet at a predetermined fifth flow rate and a cell sample is recovered from the cell culture medium outlet, the fifth flow rate being strictly higher than the fourth flow rate, the injection time and the fifth sampling flow rate are calculated before the seeding step a) so that less than one third of the cells in the well are recovered.

14. The cell culture method according to any one of claims 10-13, further comprising a harvesting step e) of injecting a culture medium into the cell culture medium inlet after the seeding step a) and recovering cells from the cell culture medium outlet.

Technical Field

The present invention relates to a cell culture device and a cell culture method using the device, the method comprising seeding, washing, sampling, differentiating, transducing, expanding and/or harvesting cells in the device. The device according to the invention aims to perform the main cell culture steps in the same device, without the need to transfer the cultured cells into a different chamber of the device or to another external chamber outside the device, in order to produce cells in high yield for cell therapy.

Background

Cell therapy is a process of administering functional cells into a patient. The production of cells is a significant challenge for regulatory agencies, manufacturers, health care providers, and patients involved in their applications because it requires the extraction of cells from a patient, the culturing and/or reprogramming of cells, and the introduction of cells into a patient.

In general, cell culture may include at least a seeding step, a processing step (e.g., a transduction step of cells), a proliferation or expansion step, and a harvesting step in a bioreactor. Periodic sampling is necessary for quality control purposes. In conventional methods, cells are typically cultured in cell culture dishes, such as Petri dishes, flasks, bottles, or bags. Generally, multiple culture dishes must be used during the expansion phase to maintain a viable cell density (typically below confluent density) for the cells. Cell culture dishes are not suitable for fine-tuning control of the cellular environment, such as the concentration of nutrients, metabolites, and other agents that drive differentiation and expansion. Therefore, the efficiency and yield of cell culture is limited when using cell culture dishes. Furthermore, handling cells between different culture dishes, as well as handling cell culture medium in and out of the culture dishes, increases the risk of contamination of the cell culture and the cost of the cell culture.

Therefore, bioreactors manufactured using microfluidic technology have been developed to increase the yield of cell culture and/or to increase the control of each step and/or to study at least one of the cell culture steps.

EP 3029135 a1 describes a microfluidic cell culture system comprising a plurality of culture units, each culture unit comprising at least one culture zone, one cell loading inlet and one medium channel. The culture unit is separated from the medium channel by a structure providing a fluid connection between the medium channel and the culture unit and protects the cells from flowing out of the cell culture area. Therefore, cells can be cultured in a tight space, and a culture medium in contact with the cells can be controlled without moving the cells into different bioreactors. Furthermore, aggregates of cultured cells can be maintained in the cell culture zone. However, this system is not suitable for harvesting cultured cells.

WO 2007024701 A3 describes a cell culture microfluidic device suitable for cell culture monitoring. The device comprises a microfluidic channel comprising a plurality of weir-traps (weir-traps). Each weir well is adapted to capture cells. The adhesion and division of each cell can be monitored under dynamic control of the culture medium in the microfluidic channel. However, the device is not suitable for high throughput cell production: the growth of the cell population in each weir-trap is limited to only two cells. Furthermore, the harvesting of the cells is not controllable.

Rousset et al (Rousset, N., Monet, F., & Gervais, T.,2017, Simulation-associated design of microfluidic sample tracks for optimal tracking and culture of non-adhesive single cells, tissues, and spheres scientific reports,7(1),245) describe a device for non-adherent cell culture. The device includes a microfluidic channel comprising a plurality of grooves. Each well is adapted to capture a single cell. When trapped, the cells are protected by the grooves and do not rise in the main stream, from which nutrients or drugs can be supplied to the cells by diffusion. Thus, no structure is required to separate the cells in the recess from the rest of the microfluidic channel, thereby simplifying the manufacture of the cell culture device. However, the device is not suitable for the expansion or production of cells, as each well is suitable for capturing a single cell.

Disclosure of Invention

A cell culture device has been developed that at least partially responds to the above-described problems of the prior art.

The cell culture apparatus having at least one surface portion defining a planar surface, the surface portion configured for lying on a horizontal support, comprising at least one fluid channel having at least one cell culture medium inlet and one cell culture medium outlet, the fluid channel comprising a lower wall extending between the cell culture medium inlet and the cell culture medium outlet, the cell culture apparatus characterized in that it further comprises:

-at least one recess configured to receive a plurality of cells, the recess being formed by the lower wall and defining a bottom surface, and

-at least one cell trap (cell trap) configured to capture a cell flowing in the fluid channel and then to cause the cell to settle to the bottom surface of the recess.

The cell trap comprises an obstacle having a main direction of extension defining a sedimentation axis parallel to a sedimentation direction of cells within the obstacle, and wherein the sedimentation axis is perpendicular to the surface portion.

In other optional aspects of the invention:

-the lower wall further comprises an upper surface extending outside the recess, and the obstacle extends from the bottom surface and outside the recess, beyond the upper surface of the lower wall,

-the fluid channel further comprises an upper wall and the barrier extends at least from the upper wall to the groove,

-the obstacle has a main direction of extension defining a sedimentation axis parallel to a sedimentation direction of the cells within the obstacle, and wherein the sedimentation axis is perpendicular to the surface portion,

-each obstacle has a hole, in particular a transverse hole, preferably a transverse slit, the width of said hole being between 3 μm and 30 μm, and preferably between 5 μm and 20 μm,

-at least a part of the fluidic channel is configured to receive a liquid flow according to a main flow direction extending between the cell culture medium inlet and cell culture medium outlet, and the fluidic channel comprises an array of cell wells extending in a direction perpendicular to the main flow direction in a plane parallel to the planar surface,

-the fluid channel further comprises a side surface extending between the cell culture medium inlet and the cell culture medium outlet, wherein at least one groove extends from one side surface to the other side surface over the entire width of the fluid channel,

-the lower wall further comprises an upper surface extending at one side of the recess, and the fluid channel further comprises an upper wall, a first height of the fluid channel being defined as the shorter distance between the upper surface of the lower wall and the upper wall, a second height of the recess being defined as the shorter distance between the bottom surface of the lower wall and the upper surface of the lower wall, the second height being 0.15 to 0.85 times the first height, preferably 0.20 to 0.70 times the first height.

-the lower wall further comprises an upper surface extending at one side of the recess, and a second height of the fluid channel is defined as the shorter distance between the lower surface of the lower wall and the upper surface of the lower wall, and the second height is between 10 μm and 500 μm, in particular between 15 μm and 300 μm, preferably between 15 μm and 200 μm,

the minimum distance between the two cell wells is between 10 μm and 300 μm, in particular between 20 μm and 150 μm, preferably between 25 μm and 100 μm,

-the cell culture device comprises at least one harvest inlet and one harvest outlet, which are different from the cell culture medium inlet and the cell culture medium outlet, the at least one groove being in direct fluid connection with the harvest inlet and the harvest outlet,

-the cell culture assembly comprises an inlet channel and an outlet channel, the cell culture assembly comprising a plurality of fluid channels, each fluid channel having at least a respective cell culture medium inlet and cell culture medium outlet, the inlet channel being fluidly connected to the cell culture medium inlet of each fluid channel and the outlet channel being fluidly connected to the cell culture medium outlet of each fluid channel.

Another aspect of the invention is a cell culture method comprising arranging the cell culture apparatus with the surface portion on a horizontal support and comprising an inoculation step a) of injecting a medium comprising cells, the medium being injected into the cell culture medium inlet of at least one fluid channel of the cell culture apparatus until at least more than 10% of the cell traps capture at least one cell, and preferably more than the majority of the cell traps capture at least one cell.

In other optional aspects of the invention:

-the method further comprises, after the seeding step a), an expansion step b) of injecting a culture medium comprising an active culture agent at the cell culture medium inlet, wherein the culture medium is injected at a first predetermined flow rate, the method further comprises, after the seeding step a), an optional harvesting step e) of injecting culture medium at the cell culture medium inlet and recovering cells from the cell culture medium outlet at a predetermined second flow rate, which is strictly higher than the first flow rate, the method further comprises, after step a), an optional sampling step f) of injecting culture medium at the cell culture medium inlet at a predetermined fifth flow rate, which is strictly higher than the first flow rate, and recovering a cell sample from the cell culture medium outlet, the injection time and the fifth sampling flow rate being calculated before the seeding step a) so as to recover less than one third of the cells in the well,

-the method further comprises, after the seeding step a), a washing step c) of injecting a washing medium at the cell culture medium inlet, wherein the medium is optionally injected at a third predetermined flow rate, the method further comprises, after the seeding step a), an optional harvesting step e) wherein medium is injected at the cell culture medium inlet and cells are recovered from the cell culture medium outlet at a predetermined second flow rate, the second flow rate being strictly higher than the third flow rate, the method further comprises, after step a), an optional sampling step f) wherein medium is injected at the cell culture medium inlet at a predetermined fifth flow rate, the fifth flow rate being strictly higher than the third flow rate, and a cell sample is recovered from the cell culture medium outlet, the injection time and the fifth sampling flow rate being calculated before the seeding step a) so as to recover less than one third of the cells in the well,

-the method further comprises, after the seeding step a), a differentiation step d) of injecting a medium comprising at least a differentiation factor into the cell culture medium inlet, wherein the medium is optionally injected at a fourth predetermined flow rate, the method further comprises, after the seeding step a), an optional harvesting step e) wherein the medium is injected at the cell culture medium inlet at a predetermined second flow rate and the cells are recovered from the cell culture medium outlet, the second flow rate being strictly higher than the fourth flow rate, the method further comprises, after step a), an optional sampling step f) wherein the medium is injected at the cell culture medium inlet at a predetermined fifth flow rate and the cell sample is recovered from the cell culture medium outlet, the fifth flow rate being strictly higher than the fourth flow rate, the injection time and the fifth sampling flow rate being calculated before the seeding step a), so that less than one third of the cells in the recess are recovered,

-said method further comprises, after the seeding step a), a harvesting step e) of preferably injecting a culture medium into the cell culture medium inlet and recovering at least 80% of the cells from the cell culture medium outlet.

-the cell culture device comprises at least one harvest inlet and one harvest outlet, different from the cell culture medium inlet and the cell culture medium outlet, with at least one recess in direct fluid connection with the harvest inlet and the harvest outlet, the method further comprising after step a) a harvest step e) of injecting medium into the harvest inlet and recovering cells from the harvest outlet,

-the method further comprises a transduction step, after the seeding step a), of injecting a medium comprising a transduction factor, wherein the medium is injected into the cell culture medium inlet at a predetermined flow rate, the predetermined flow rate being 0.5 to 1.5 times the fourth predetermined flow rate.

Definition of

The term "length" of a channel will be used herein to designate the size of the channel in terms of the main flow direction of the fluid through the channel.

The term "height" of a channel is used herein to denote the smallest dimension of the channel in a first direction, which is transverse to the main flow direction.

The term "width" of a channel is used herein to denote the largest dimension of the channel in a second direction, perpendicular to the first direction and perpendicular to the main flow direction.

The term "microchannel" or "microfluidic channel" is used herein to mean a channel comprising at least one inlet and at least one outlet, the height of which is between 100nm and 1 mm. The term "fluid channel" also includes a range of dimensions including, but not limited to, nanometer, micrometer, millimeter, centimeter dimensions.

Drawings

The invention will be described by way of example with reference to the accompanying drawings, in which:

FIG. 1 shows a cell culture device,

figure 2 schematically shows a part of a fluid channel comprising a recess and a cell trap,

FIG. 3 is a top view of the cell well,

figure 4 schematically shows a cross section of the fluid channel along the main flow direction,

figure 5 is a top view of the fluid channel,

FIG. 6 is a top view of a fluid channel comprising a harvesting inlet and a harvesting outlet,

FIG. 7 shows a cell culture process according to a possible embodiment of the invention.

Detailed Description

Conventional structure of cell culture apparatus 1

Referring to fig. 1, a cell culture device 1 has a surface portion 2 defining a planar surface 3 configured to lie flat on a horizontal support. The surface portion 2 is located behind the portion shown of the cell culture apparatus 1 in FIG. 1. The surface portion 2 may be, for example, the surface of a slide glass or a planar surface in a mesh polymer (e.g., PDMS) according to the method of manufacturing the cell culture apparatus 1.

The cell culture apparatus 1 comprises at least a fluid channel 4. The fluid channel 4 is preferably a microfluidic channel. The fluid channel 4 has at least a cell culture medium inlet 5. According to a possible embodiment of the invention shown in fig. 1, the flow channel 4 has eight cell culture medium inlets 5, which are evenly distributed at one end of the flow channel 4. Thus, it is possible to inject a uniform concentration of cells 12 in the fluid channel 4 and/or to inject a liquid culture medium at the same rate over a portion of the fluid channel 4.

The fluid channel 4 comprises at least a cell culture medium outlet 6 (not shown in fig. 1). The cell culture medium outlet is at the other end of the fluid channel 4.

The first direction 8 is defined as being perpendicular to the planar surface 3 and opposite to the direction from the fluid channel 4 towards the planar surface 3. In use, the cell culture device 1 is positioned on a horizontal support and the first direction 8 corresponds to a direction opposite to the direction of gravity.

The fluid channel 4 comprises a lower wall 7 extending between the cell culture medium inlet and the cell culture medium outlet.

Referring to fig. 1 and 2, the fluid channel 4 comprises at least one, and preferably a plurality of wells 10, each well 10 configured to receive a plurality of cells 12. Said recess 10 is formed by the lower wall 7. According to a possible embodiment of the invention shown in fig. 1 and 2, the groove 10 has the shape of a rectangular cuboid and is produced, for example and without limitation, by a two-step photolithographic microfabrication technique. The recess 10 defines at least one bottom surface 11. In case the recess 10 has a circular shape, the bottom surface 11 may define the entire recess 10. The bottom surface 11 is the lowest surface of the recess with respect to the first direction 8.

The cell culture apparatus 1 comprises at least one, preferably a plurality of cell traps 13. Each cell trap 13 is adapted to capture cells 12 advected by the liquid medium flow in the fluid channel 4 and then to cause the captured cells 12 to settle onto the bottom surface 11 of the recess 10. The cell culture zone 9 comprises a recess 10 and at least one cell trap 13.

Cell trap 13

Referring to fig. 2, the lower wall 7 includes an upper surface 23 corresponding to a surface extending on one side of the recess 10. The cell trap 13 is preferably an obstacle 14. The obstacle 14 may be arranged at the boundary between the groove 10 and the rest of the fluid channel 4. Preferably, the cell trap 13 comprises an obstacle 14 extending from the bottom surface 11 beyond the recess 10, beyond the upper surface 23 of the lower wall 7. Preferably, the cell trap 13 comprises obstacles 14 extending at least from the upper wall 20 to the recess 10. The obstacle 14 is preferably a protrusion, bump or bump. The obstacle 14 preferably has a main extension direction defining a sedimentation axis parallel to the sedimentation direction of the cells 12 within said obstacle 14, said sedimentation axis being perpendicular to the surface portion 2. The sedimentation axis may correspond to the first direction 8. Preferably, the obstacles 14 all extend from the bottom surface 11 of the recess 10 to the upper wall 20 of the fluid channel 4 along a part of the entire fluid channel 4 with respect to the axis. Thus, the efficiency of capture of the cell 12 by the obstacle 14 is improved.

When the cells 12 are advected by, for example, a liquid stream flowing in the primary flow direction 16, the cells 12 may encounter the obstruction 14 and be prevented from advecting in the primary flow direction 16. However, the obstacles 14 are configured to cause the cells 12 to settle in the recesses 10 to the bottom surface 11. The dashed arrows in fig. 2 show the capture and sedimentation of cells 12. Therefore, the cells 12 can be efficiently captured without limiting the speed of the injected medium including the cells 12, so that the cells 12 are settled in the well 10. By the combination of the cell trap 13 and the recess 10, the liquid medium containing the cells 12 can be controlled in the fluid channel 4 at a rate that generally ignores sedimentation, while seeding the recess 10 with the cells 12 using sedimentation. The seeding of the cells 12 is simpler and more efficient than in the prior art.

The obstruction 14 may be a weir trap. Thus, the cell culture medium flow line directed at the obstacle 14 does not entirely bypass the obstacle 14. Some of the flow lines pass over the obstacles 14. Thus, if the cells 12 are in a streamline passing through the obstruction 14, the cells 12 may be captured by the obstruction 14. After the cell 12 is captured, the cell 12 partially blocks the open area of the weir trap and the portion of the streamlines passing through the gated trap (barred trap) is reduced, resulting in self-sealing of the trap. The dimensions of the weir trap can therefore be selected so as to control the number of cells 12 captured by the weir trap.

Figures 2 and 3 show a cell trap 13 according to various embodiments of the invention having a transverse slit 15. The width, i.e. the smallest dimension, of the holes or slits 15 is preferably between 3 μm and 30 μm, and preferably between 5 μm and 20 μm. Thus, the cell well 13 may be a weir well in that the width of the aperture may be smaller than the size of the cells 12, while the width of the aperture is large enough to allow streamlines to pass through the aperture.

According to another embodiment of the invention, the obstacle 14 may extend only partially out of the groove 10 from the bottom surface. Thus, the streamline can pass over the obstacle 14 so that the obstacle 14 can capture the cells and settle them.

The obstacle 14, preferably a weir trap, may have a U-shape, cup-shape, crescent-shape, hollowed-out crescent-shape, star-shape, hemispherical shape, any polygonal and/or hollow shape having three or more vertices.

The size of the obstacle 14 may depend on the average diameter D of the cultured cells_cellTo select. Throughout the specification, such parameter D_cellDefined as the average cultured cell diameter, the cells 12 are in suspension for use in the methods of the invention. D_cellMeasurements can be made by various techniques such as impedance cytometry (e.g., without limitation, coulter counter as described in us patent 2656508 or by light microscopy followed by image analysis). Depending on the origin of the cells, D_cellIt may generally be between 3 μm and 150 μm, in particular between 5 μm and 50 μm.

Width W of obstacle 14_tI.e. the size of the obstacle 14 in a second direction 19 transverse to the main flow direction 16 and the first direction 8,preferably greater than D_cellIn particular more than 10 μm, preferably more than 20 μm.

Groove 10

The dimensions of the recess 10 are chosen such that the cells 12 can proliferate in the recess 10. The recess 10 is configured to receive a plurality of cells 12. According to a preferred embodiment of the invention, the area of the grooves protruding on the planar surface 3 is greater than 150 μm²In particular greater than 250000 μm²And preferably greater than 500000 μm²。

Referring to fig. 3, the geometry of the groove 10 may be designed such that cells 12 in the groove 10 (e.g., on the bottom surface 11 of the groove 10) may be protected from advection transport out of the groove 10 at a predetermined flow rate in the fluid channel 4. In other words, the geometry of the groove 10 allows dead water (dead water) to flow in the groove 10 at a predetermined flow rate, while flow occurs in the upper part of the fluid passage 4, i.e. outside the groove 10. The term "dead water" or "void space" is used herein to designate a volume of liquid in which there is a range of flow rates insufficient to pull the cells 12 out of the recess 10, and preferably insufficient to pull the cells 12. Thus, cells 12 in a well 10 can be washed, or any active compound can be brought to the cell 12 by injecting a wash medium and/or a medium containing the active compound at a predetermined flow rate, without having to pull the cell 12 out of the well 10. Thus, in contrast to prior art devices, the manufacture of the cell culture apparatus 1 can be simplified, for example, by avoiding the manufacture of semi-permeable membranes or walls to contain the cells 12 from being dragged by the flow of medium.

The groove preferably extends more than 2D in the second direction 19_cellIn particular more than 15 μm and preferably more than 100 μm. According to a preferred embodiment of the invention in fig. 1, each groove 10 extends over more than half the width W of the fluid channel, preferably the entire width W of the fluid channel. The fluid channel may comprise a side extending between the cell culture medium inlet 5 and the cell culture medium outlet 6. The groove 10 preferably extends from one side to the other over the entire width of the fluid channel 4.

Preferably, the grooves 10 are formed transversely to the main flow direction 16. Therefore, the other grooves 10 are not limitedFor larger flow rates in the fluid channel 4, there is a possibility of stagnant water in the groove 10. Preferably, the length L of the groove 10_dIs smaller than the width W of the groove 10_dMore preferably, the length L of the groove 10_dThan the width W of the groove 10_dFive times lower.

The fluid channel 4 comprises an upper wall 20 with respect to the first direction 8. First height H_cIs defined as the shorter distance between the upper surface 23 of the lower wall 7 and the upper wall 20. The recess 10 preferably has a second height H with respect to the first direction 8_tWhich is defined with respect to the first direction 8 as the shorter distance between the bottom surface 11 of the lower wall 7 and the upper surface 23 of the lower wall 7. In particular, the second height H_tIs the first height H_c0.25 to 0.85 times, and preferably said first height H_c0.40 to 0.70 times. If the second height H of the groove 10_tE.g. to a first height H_cSeveral times lower, then no dead water zones will appear in the recess 10 when liquid medium is injected into the fluid channel 4 to protect the cells in culture. On the other hand, if the second height H of the groove 10_tIs higher than the first height H of the fluid channel 4_cSeveral times larger, the flow of liquid medium in the fluid channel 4 will occur outside the groove 10, making it difficult to create a vortex in the groove 10 to pull the cells 12 out of the groove 10. Height ratio H according to embodiments of the present invention_t/H_cSuch that, depending on the flow rate of the culture medium injected into the fluid passage 4, it is possible to select whether the liquid culture medium in the groove 10 is significantly stabilized or whether a vortex occurs in the groove 10.

Preferably, the second height H of the groove 10_tBetween 10 μm and 1000 μm, in particular between 15 μm and 300 μm, and preferably between 15 μm and 200 μm.

Array of cell wells 13

The fluid channel 4 comprises a plurality of cell traps 13. Preferably, the fluid channel 4 comprises at least one array of cell wells 13, preferably a plurality of cell wells 13. The array of cell traps 13 may be linear, e.g. extending with respect to the second direction 19, and/or two-dimensional, e.g. extending in a plane parallel to the planar surface 3.

The parameters of the array of cell traps 13 and/or the arrangement of the different arrays in the flow channel 4 determine the cell capture rate at each point of the flow channel 4.

The minimum distance between the two cell traps 13, preferably the minimum distance between the two obstacles 14, is a parameter of the cell capture rate during perfusion of the liquid medium containing the cells 12 in the fluid channel 4. The minimum distance between two obstacles 14 is defined as the minimum distance between the wall of one obstacle 14 and the wall of the nearest obstacle 14. According to a preferred embodiment of the invention, the minimum distance between two obstacles 14 is comprised between 10 μm and 300 μm, in particular between 20 μm and 150 μm, preferably between 25 μm and 100 μm. Thus, cell 12 capture during perfusion may be sufficiently efficient to seed the cells 12 in the fluid channel 4 within 10 minutes, preferably within 1 minute, while being sufficiently low to avoid the formation of a cellular blockage at the entrance of the fluid channel 4.

According to a preferred embodiment of the invention, the obstacles 14 are arranged in a regular two-dimensional grid, wherein the grid is inclined with respect to the main flow direction 16. Two successive obstacles 14 define, with respect to the main flow direction 16, an inclination of the grid to the main flow direction 16 which is not zero, preferably between 2 ° and 20 °, in particular between 2 ° and 10 °, more preferably between 2 ° and 5 °.

According to one possible embodiment of the invention, the obstacles may be arranged in the fluid channel 4 such that the surface density of the obstacles 14 protruding in the planar surface 3 varies along the length of the fluid channel. Preferably, the surface density of the obstacles 14 increases relative to the main flow direction 16. Thus, cells 12 may be seeded at a uniform or constant concentration along the fluid channel 4.

Harvesting

Referring to fig. 5 and 6, cells 12, in particular cells 12 cultured in the wells 10 of the cell culture device 1, may be harvested, for example for further therapeutic use.

According to the embodiment of the invention shown in FIG. 5, the cells 12 may be harvested by flushing the well 10 and recovering the cells 12 from the cell culture medium outlet 6 of the fluid channel 4.

According to another embodiment of the invention shown in fig. 6, the fluid channel 4 may comprise at least one harvest inlet 17 and at least one harvest outlet 18, which are different from the cell culture medium inlet 5 and the cell culture medium outlet 6, respectively. The harvest inlet 17 and harvest outlet 18 are arranged such that a controlled flow between the harvest inlet 17 and harvest outlet 18 can advect the cells 12 in the trough 10 in order to harvest them. In contrast to the controlled flow between the cell culture medium inlet 5 and the cell culture medium outlet 6, the controlled flow between the harvest inlet 17 and the harvest outlet 18 is configured such that no dead water is present in the recess 10. According to the embodiment of the invention shown in fig. 6, the harvesting inlet 17 and the harvesting outlet 18 are configured to allow flow with respect to the second direction 19, and the grooves 10 extend in the same direction.

In a preferred embodiment of the invention, each flute 10 is in fluid connection with one harvesting inlet 17 and one harvesting outlet 18. Thus, when controlling the flow between the harvesting inlet 17 and the harvesting outlet 18, the presence of dead water in the trough 10 can be avoided. The recesses 10 may be in fluid connection with one and the same common harvesting inlet 17 and common harvesting outlet 18 and/or be individually connected by different harvesting inlets 17 and harvesting outlets 18.

Parallelization

The cell culture is suitable for cell therapy, i.e. the cell culture device 1 is suitable for harvesting more than 10⁵Individual cells 12, especially more than 10⁶Individual cells 12, preferably more than 10¹⁰And (4) individual cells 12. The fluidic channels 4 may be parallelized to increase the throughput of the cell culture device 1. According to a preferred embodiment of the invention, the cell culture device 1 comprises an inlet channel 21 and an outlet channel 22. The inlet channel 21 is in fluid connection with the cell culture medium inlet 5 and the outlet channel 22 is in fluid connection with the cell culture medium outlet 6. Thus, production of cells 12 can be parallelized. For example, different fluid channels 4 may be superimposed. The different fluid channels 4 may also be arranged in the same plane of the same stack.

Surface treatment

Different types of cells 12, such as adherent cells 12 or non-adherent cells 12, may be cultured in the cell culture apparatus 1. The bottom surface 11 of the recess 10, in particular the surface of the recess 10, and preferably the walls of the fluid channel 4, may be coated with a surface treatment of a type suitable for the cells 12 to be cultured in the cell culture device 1. In general, the surface treatment may include a step of injecting and circulating a liquid medium suitable for the surface treatment in the culture apparatus. The liquid culture medium may be introduced into the fluid channel 4 through the cell culture medium inlet 5 and the cell culture medium outlet 6 or through the harvest inlet 17 and the harvest outlet 18, wherein the surface treatment is passively adsorbed on the culture surface, i.e. on the walls of the recess 10, in particular on the bottom surface 11. This applies in particular to extracellular matrix coatings. Other steps may include plasma polymerization and chemically induced adsorption, such as, but not limited to, polymer grafting.

Cell culture

The cell culture apparatus 1 described above is suitable for culturing cells 12 used for cell therapy, for example.

FIG. 7 shows a cell culture method according to an embodiment of the present invention. The method may comprise the step 61 of arranging the cell culture device 1 such that the surface portion 2 is located on a horizontal support. Thus, the recess 10 is arranged below the rest of the fluid channel 4 with respect to the first direction 8, and the cell trap 13 is adapted to let the cells 12 settle to the bottom surface 11 of the recess. As an alternative, the surface portion 2 may be inclined and the fluid channel 4 also inclined, so that the groove 10 is arranged below the rest of the fluid channel 4 with respect to the first direction 8.

The method of cell culture includes a seeding step 62. At least one cell 12 can be brought to one well 10, in particular to the majority of wells 10, preferably to more than 80% of wells 10, by means of the cell trap 13. The liquid culture medium containing the cells 12 is injected into the cell culture medium inlet until at least up to 10% or more of the cell wells 13 capture at least one cell 12, in particular more than one third of the cell wells 13 capture at least one cell 12, preferably until the majority of the cell wells 13 capture at least one cell 12. Therefore, the cells 12 can be seeded in the cell culture apparatus 1 by injecting the medium containing the cells 12 at a speed independent of the action of gravity on the cells 12. Thus, the cell culture apparatus 1 can be efficiently and uniformly seeded. The number of seeded cells 12 and/or the number of cells 12 captured by the cell trap 13 may be measured during injection by conventional microscopy, for example by recording an image of the fluid channel 4 using an inverted microscope. The captured cells 12 may then be counted by automatic image processing and/or manually. The injection time may be predetermined.

After the seeding step 62, the method may include an amplification step 65 or a propagation step 65. The proper culture environment is critical for the expansion of cells 12: the expanding step 65 comprises injecting a medium comprising at least one active agent into the cell culture medium inlet. The term "active agent" refers herein to any chemical or biological compound that is necessary or beneficial for the expansion of the cells 12. The injection of the culture medium is controlled at a flow rate low enough to avoid dragging the cells 12 out of the well 10 and preferably without inducing turbulence in the well 10. The liquid flow takes place substantially in the rest of the fluid channel 4. Thus, the active agent can be transported to the cells 12 by diffusion from the remainder of the fluid channel 4 to the recess 10 without moving or advecting the cells 12 expanded in the recess 10.

After the seeding step 62, the method may further include a washing step 64. In order to accurately control the chemical or biological treatment of the cells 12, it may be necessary to wash the fluid channel 4. The washing step 64 comprises injecting a washing medium that is free of the compound to be washed. The injection of wash medium is controlled at a flow rate low enough to avoid dragging the cells 12 out of the well 10, and preferably without inducing turbulence in the well 10. The liquid flow takes place substantially in the rest of the fluid channel 4. Thus, the compound to be washed by dilution or can be transported by diffusion from the recess 10 to the rest of the fluid channel 4 without moving or advecting the cells 12 in the recess 10.

After the seeding step 62, the method may further include a differentiation step 63. The term "differentiation" refers herein to the process by which a cell changes from a less specialized cell type to a more specialized cell type. The differentiation step 63 includes injecting a liquid medium containing a differentiating agent. The injection of the differentiating agent-containing medium is controlled at a flow rate low enough to avoid dragging the cells 12 out of the groove 10, and preferably without inducing turbulence in the groove 10. The liquid flow takes place substantially in the rest of the fluid channel 4. Thus, the differentiating agent can be transported to the cell 12 by diffusion from the remainder of the fluid channel 4 to the recess 10 without moving or advecting the cell 12 in the recess 10.

The method may further include a harvesting step 66. The harvesting step 66 may include injecting media into the cell media inlet 5 and recovering cells from the cell media outlet. The harvesting of the cells 2 may be handled by injecting a culture medium at a flow rate high enough to create a vortex in the recess 10 and to draw more than a substantial portion of the cells 12 out of the recess 10 to the rest of the fluid channel 4. The cells 12 can then be advected by the flow of liquid culture medium and transported to the cell culture medium outlet 6 where they can be recovered.

The threshold flow rate between cell culture medium inlet 5 and cell culture medium outlet 6 will be divided as follows:

a mode of operation in which the liquid flow in the recess 10 is apparently locally zero (dead water), or in which the drag force exerted on the cells 12 in the recess 10 is insufficient to lift the cells 12 out of the recess 10, and

another mode of operation, in which the flow of liquid in the groove 10 is associated with a vortex whose drag on the cell 12 is high enough to lift the cell out of the groove 10.

The threshold flow rate may be determined based on the geometry of the fluid channel 4 and the type of cells 12 being cultured, for example by analysis or by computer simulation using a finite element method. One suitable method may be, for example, to determine at what flow rate between cell culture medium inlet 5 and cell culture medium outlet 6, the local drag force on a cell 12 may counteract the force of gravity on the same cell 12. The flow rate of the injection medium in the harvesting step 66, i.e. the second flow rate, is greater than the first predetermined flow rate of the expansion step 65 and/or the washing step 64 and/or the differentiation step 63.

Alternatively, the harvesting step 66 may include injecting media at the harvest inlet 17 and recovering the cells 12 from the harvest outlet 18.

The method may further comprise a sampling step 67. During cell culture, it may be necessary to sample the cultured cells 12 to determine, for example, the activity, potency, viability, selectivity, morphology, and/or, more generally, the different characteristics of the cells 12. The sampling step 67 may comprise injecting the culture medium into the fluid channel 4 at a third flow rate that is greater than the first flow rate. The third flow rate may be selected, for example, between a threshold flow rate and 1.5 times the threshold flow rate. By adjusting the perfusion time in the sampling step 67, less than one third of the cells 12 in the recess 10 are recovered, preferably less than 10% of the cells 12 are recovered. The sampling step 67 may also include sampling the liquid culture medium in the microchannel 4 to further analyze the sterility and composition of the cell culture fluid and the environment.