WO2021219075A1

WO2021219075A1 - Cell culture device

Info

Publication number: WO2021219075A1
Application number: PCT/CN2021/090953
Authority: WO
Inventors: Ping-Lun Jiang; Tzu-Sheng Yang; Cheng-Kun Tsai
Original assignee: Medical And Pharmaceutical Industry Technology And Development Center
Priority date: 2020-04-29
Filing date: 2021-04-29
Publication date: 2021-11-04
Also published as: TWI768856B; TW202140769A

Abstract

A cell culture device is provided. The cell culture device includes at least one chamber, a first inlet channel, a first inlet connection channel, and an outlet channel. The first inlet connection channel has a first length and connects a bottom portion of the chamber to the first inlet channel. The first inlet connection channel includes a fluid valve for selectively porting a pressurized medium from the first inlet channel to the chamber. The outlet channel is connected to the chamber. The fluid valve has a second length and a first hydraulic width defining an aspect ratio adapted to taper the pressurized medium along the first inlet connection channel. The fluid valve includes a first recess extending along the second length within the fluid valve to permit the pressurized medium to flow along the second length at a substantially uniform flow velocity.

Description

CELL CULTURE DEVICE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/017, 410, filed April 29, 2020, the entirety of which is incorporated by reference herein.

BACKGROUND Technical Field

The present disclosure relates to a cell culture device, and in particular it relates to a cell culture device having a biomimetic microenvironment.

Description of the Related Art

Cell culture is widely used in biomedical research, tissue engineering, pharmaceutical development, and industrial practices. A two-dimensional (2-D) cell culture technique has been used with in vitro models to study cellular responses to different biophysical or biochemical conditions. Conventional 2-D cell culture techniques rely on adherence to a flat surface, typically a petri dish made of glass or polystyrene, to provide mechanical support for the cells.

However, 2-D cell culture techniques only allow cells to grow in two dimensions. A 2-D cell culture technique cannot simulate the real in vivo microenvironment of cells. It cannot accurately represent how cells grow or how they are affected by different biophysical or biochemical conditions, or how they interact with each other in a living organism.

On the other hand, three-dimensional (3-D) cell culture techniques have been intensively developed in recent years. 3-D cell culture platforms are being developed to better mimic in vivo conditions and are sometimes called spheroid or organoid culture. In addition, 3-D cell culture techniques can provide more explicit observation of cell-cell interactions in realistic biochemical and physiological conditions.

Although currently existing 3-D cell culture techniques have been adequate for their intended purposes, they have not been satisfactory in all respects. The development of a cell culture device that can more stably maintain a biomimetic microenvironment is still one of the goals that the industry currently aims for.

Spatial control is one of many basic principles underlying the operation of microfluidic 3D cell culturing techniques. It could be manipulated to allow for cell patterning and tuning of extracellular microenvironment, to create stratified cultures or co-cultures with gradient formation and medium perfusion.

In conventional arts, the spatial control is usually achieved by a membrane, a matrix, or a scaffold to support surface-attached cell growth, and separation of the culture reactor in multiple compartments. Cells can also be spatially controlled without using a matrix, but with microchambers or droplets, in which suspended cells can settle and cluster to form spheroid. Whichever method is used, it is commonly understood that the importance of the ability to spatially control cells signals a path for combining multiple cell types in a way that more faithfully represents the organization of tissues and organs.

SUMMARY

In accordance with some embodiments of the present disclosure, a cell culture device focusing on improved spatial control of microfluidic environment is provided. The cell culture device includes at least one chamber, a first inlet channel, a first inlet connection channel, and an outlet channel. The first inlet connection channel having a first length that connects a bottom portion of the chamber to the first inlet channel. The first inlet connection channel includes a fluid valve for selectively porting a pressurized medium from the first inlet channel to the chamber. The outlet channel is connected to the chamber. The fluid valve has a second length and a first hydraulic width defining an aspect ratio adapted to taper the pressurized medium along the first inlet connection channel. The fluid valve includes a first recess extending along the second length within the fluid valve to permit the pressurized medium to flow along the second length at a substantially uniform flow velocity.

A detailed description is given in the following embodiments with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure may be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:

FIG. 1A illustrates a schematic diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 1B illustrates an enlarged schematic diagram of region A of FIG. 1A in accordance with some embodiments of the present disclosure;

FIG. 2A illustrates a schematic diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 2B illustrates a schematic side-view diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 3A illustrates a schematic diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 3B illustrates a schematic side-view diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 4A illustrates a schematic diagram of a portion of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 4B illustrates a schematic diagram of a portion of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 5A illustrates an operation diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 5B illustrates an operation diagram of a cell culture device in accordance with some embodiments of the present disclosure;

FIGs. 6A-6C illustrate hydrodynamic simulation results of a cell culture device in accordance with some embodiments of the present disclosure;

FIG. 7 illustrates a diagram of spheroid cell formation in a cell culture device in accordance with some embodiments of the present disclosure;

FIGs. 8A-8D illustrate hydrodynamic simulation results of a cell culture device in accordance with some embodiments of the present disclosure;

FIGs. 9A-9D illustrate hydrodynamic simulation results of a cell culture device in accordance with some embodiments of the present disclosure.

DETAILED DESCRIPTION

The structure of the cell culture device of the present disclosure is described in detail in the following description. In the following detailed description, for purposes of explanation, numerous specific details and embodiments are set forth in order to provide a thorough understanding of the present disclosure. The specific elements and configurations described in the following detailed description are set forth in order to clearly describe the present disclosure. It will be apparent, however, that the exemplary embodiments set forth herein are used merely for the purpose of illustration, and the inventive concept may be embodied in various forms without being limited to those exemplary embodiments. In addition, the drawings of different embodiments may use like and/or corresponding numerals to denote like and/or corresponding elements in order to clearly describe the present disclosure. However, the use of like and/or corresponding numerals in the drawings of different embodiments does not suggest any correlation between different embodiments.

The descriptions of the exemplary embodiments are intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. It should be understood that the drawings are not drawn to scale. In fact, the size of the element may be arbitrarily enlarged or reduced in order to clearly express the features of the present disclosure. In addition, in the embodiments, relative expressions are used. For example, “lower” , “bottom” , “higher” or “top” are used to describe the position of one element relative to another. It should be appreciated that if a device is flipped upside down, an element that is “lower” will become an element that is “higher” .

It should be understood that, although the terms “first” , “second” , “third” etc. may be used herein to describe various elements, components, or portions, these elements, components, or portions should not be limited by these terms. These terms are only used to distinguish one element, component, or portion from another element, component, or portion. Thus, a first element, component, or portion discussed below could be termed a second element, component, or portion without departing from the teachings of the present disclosure.

The terms “about” and “substantially” typically mean +/-10%of the stated value, more typically +/-5%of the stated value, more typically +/-3%of the stated value, more typically +/-2%of the stated value, more typically +/-1%of the stated value and even more typically +/-0.5%of the stated value. The stated value of the present disclosure is an approximate value. When there is no specific description, the stated value includes the meaning of “about” or “substantially” . Furthermore, the phrase “in a range between a first value and a second value” or “in a range from a first value to a second value” indicates that the range includes the first value, the second value, and other values between them.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It should be appreciated that, in each case, the term, which is defined in a commonly used dictionary, should be interpreted as having a meaning that conforms to the relative skills of the present disclosure and the background or the context of the present disclosure, and should not be interpreted in an idealized or overly formal manner unless so defined.

In accordance with some embodiments of the present disclosure, a cell culture device that can culture three-dimensional spheroid cells or organoid cells is provided. The cell culture device uses continuous flowing culture medium to provide a biomimetic microenvironment where spheroid cells or organoid cells can be grown. In accordance with some of the present disclosure, the flow rate of the culture medium can be adjusted or more than one types of culture media can be provided so that the cells can be grown steadily and the functionality of the grown spheroid cell or organoid cell is quite similar to that in real physiological state in vivo.

FIG. 1A illustrates a schematic diagram of a cell culture device 10 in accordance with some embodiments of the present disclosure. It should be understood that in accordance with some embodiments of the present disclosure, additional features may be added to the cell culture device 10 described below. In some other embodiments, some features of the cell culture device 10 may be replaced or omitted.

As shown in FIG. 1A, the cell culture device 10 includes at least one chamber 102, a first inlet channel 202, and an outlet channel 206. The first inlet channel 202 and the outlet channel 206 are connected to the chamber 102. Specifically, in some embodiments, the first inlet channel 202 is located below the chamber 102, and located lower than the outlet channel 206. In addition, the cell culture device 10 includes a first inlet connection channel 204 that connects a bottom portion of the chamber 102 with the first inlet channel 202. The first inlet connection channel 204 is disposed between the chamber 102 and first inlet channel 202. In some embodiments, the cell culture device 10 may further include an outlet connection channel 208 that connects a side surface 102s of the chamber 102 with the outlet channel 206. The detailed positional configuration of the chamber 102, the first inlet channel 202, the first inlet connection channel 204, the outlet channel 206 and the outlet connection channel 208 will be described later.

In addition, the cell culture device 10 may include several cell culture units 100U. The cell culture unit 100U may include one chamber 102, one first inlet connection channel 204, one outlet connection channel 208, and portions of the first inlet channel 202 and the outlet channel 206. However, it should be understood that the number of cell culture units 100U is not limited to what is illustrated in the drawing. In some embodiments, there may be between one and ninety-nine cell culture units 100U. In other words, there may be between one and ninety-nine chambers 102. The number of cell culture units 100U may be adjusted according to need.

FIG. 1B illustrates an enlarged schematic diagram of region A of FIG. 1A in accordance with some embodiments of the present disclosure. As shown in FIG. 1B, the first inlet connection channel 204 connects the first inlet channel 202 to the chamber 102. In some embodiments, the first inlet connection channel 204 is in direct contact with the first inlet channel 202 and the chamber 102. In some embodiments, the medium (indicated by the arrows in the drawing) flows from the first inlet channel 202 through the first inlet connection channel 204 to the chamber 102. That is, the medium may flow in a direction from the bottom to the top of the cell culture device 10 in accordance with some embodiments.

Specifically, as shown in FIG. 1B, the first inlet connection channel 204 includes a fluid valve 204V for selectively porting a pressurized medium from the first inlet channel 202 to the chamber 102. In some embodiments, the first inlet connection channel 204 may further include a main portion 204M connected to the first inlet channel 202. In some embodiments, the main portion 204M is disposed below the fluid valve 204V. In some embodiments, the main portion 204M is disposed between the fluid valve 204V and the first inlet channel 202.

In addition, the first inlet connection channel 204 has a first length L1. The fluid valve 204V has a second length L2 and a first hydraulic width

defining an aspect ratio adapted to taper the pressurized medium along the first inlet connection channel 204. In some embodiments, the first length L1 is greater than second length L2. In some embodiments, the first hydraulic width

may be in a range from 0.4 millimeters (mm) to 1.6 mm, for example, 0.8mm. However, it should be understood that the value of the first hydraulic width

can be adjusted in various embodiments according to the size of the spheroid cell or organoid cell that is to be grown. As shown in FIG. 1B, in accordance with some embodiments, a top surface 204Vt of the fluid valve 204V (or the bottom portion of the chamber 102) may serve as a cell aggregation region CA. In some embodiments, the second length L2 of the fluid valve 204V may be greater than 0.2 millimeters (mm) , and such a range does not affect the flow field. It should be noted that if the second length L2 is too short (e.g., shorter than 0.2 mm) , the difficulty of the manufacturing process will be increased.

In addition, it should be understood that in accordance with the embodiments of the present disclosure, the term “hydraulic width” has substantially the same meaning as the term “hydraulic diameter” , and they can be used interchangeably.

In particular, the fluid valve 204V includes a first recess 204R ₁ extending along the second length L2 within the fluid valve 204V. Since only a portion of the medium can flow through the first recess 204R ₁ to the chamber 102 and another portion of the medium is impeded from flowing to the chamber 102, the medium is pressurized and tapered from the main portion 204M to the fluid valve 204V. With such a configuration, the first recess 204R ₁ permits the pressurized medium to flow along the second length L2 at a substantially uniform flow velocity. Moreover, as shown in FIG. 1B, in accordance with some embodiments, the cell culture device 10 further includes a second recess 204R ₂, and the first recess 204R ₁ and the second recess 204R ₂ are spaced apart by the first hydraulic width

In addition, it should be understood that, the second recess 204R ₂ may have the same configuration and the features as that of the first recess 204R ₁, and thus the following description to the first recess 204R ₁ may also apply to the second recess 204R ₂.

As shown in FIG. 1B, the first inlet channel 202 has a second hydraulic width

and the main portion 204M of the first inlet connection channel 204 has a third hydraulic width

In some embodiments, the ratio of the second hydraulic width

to the third hydraulic width

may be greater than 3: 1. In accordance with some embodiments, a T-junction configuration is formed by the main portion 204M and first inlet channel 202. In particular, the first inlet channel 202 uses a single sheath-flow to build fluid momentum to deliver the medium through the T-junction configuration, where an inertial force is formed to push the medium upward to the fluid valve 204V. In other words, the flowing direction of the medium is altered and stabilized in the main portion 204M.

As described above, the fluid valve 204V includes the first recess 204R ₁ extending along the second length L2. Moreover, the first recess 204R ₁ has a fourth hydraulic width

In some embodiments, the ratio of the third hydraulic width

to the fourth hydraulic width

may be greater than or equal to 10: 1. In some embodiments,

must not be equal to

In some embodiments,

must be greater than

In accordance with some embodiments, cells can be allowed to grow stably, while also the medium can be kept to undergo a constant stimulation so as to control the transfer of medium more stably, enabling less tearing on cell integrity and more interactions between cells in the medium. The fluid valve 204V can provide another sheath-flow to build fluid momentum to deliver the medium. In addition, the interaction between the fluid valve 204V and the main portion 204M and the volumetric exchange bias therebetween lead to the formation of turbulence in cell aggregation and agglomeration. In some embodiments, the medium flows from the first inlet channel 202 through the first inlet connection channel 204 to the chamber 102 to permit turbulence to form in the medium.

In some embodiments, the fourth hydraulic width

of the first recess 204R ₁ may be in a range from 0.05 mm to 0.15 mm. In accordance with some embodiments, the fourth hydraulic width

refers to the average width of the first recess 204R ₁. It should be noted that the range of the fourth hydraulic width

should be well-controlled so that it will not affect the flow field distribution of the medium. If the fourth hydraulic width

is too wide, the discharge will need to be increased accordingly in order to attain for relevant medium concentration as compared to the setting where the fourth hydraulic width

is set within the prescribed range.

Furthermore, in some embodiments, the first recess 204R ₁ has a cross-sectional shape, and the cross-sectional shape may be a meniscus, an arc, a circle, a polygon, a curved shape, or a sector.

In addition, the main portion 204M has a third length L3. In some embodiments, the third length L3 of the main portion 204M is greater than the third hydraulic width

It should be noted that if the third length L3 is smaller than the third hydraulic width

the fluid field may be not uniform and may lead to turbulence.

As shown in FIG. 1B, the chamber 102 is connected to the fluid valve 204V. In some embodiments, the chamber 102 may have a tapered profile, a countersunk profile, or an oval profile. Specifically, an included angle θ may exist between the side surface 102s of the chamber 102 and a side surface 204s of the fluid valve 204V. In some embodiments, the included angle θ may refer to the slope of the chamber 102, and the included angle θ may be in a range from 3 degrees to 30 degrees.

Specifically, in accordance with some embodiments, turbulence may be transiently created at the bottom portion of the chamber 102 due to the profile of the chamber 102. In some embodiments, the turbulence may be enhanced and complemented by adjusting the included angle θ. The medium ported through the fluid valve 204V may be trapped and enriched by the turbulence in the chamber 102 to undergo cell aggregation and agglomeration to form spheroids.

Next, FIG. 2A, illustrates a schematic diagram of a cell culture device 20 in accordance with some other embodiments of the present disclosure. It should be understood that the same or similar components or elements in above and below contexts are represented by the same or similar reference numerals. The materials, manufacturing methods and functions of these components or elements are the same or similar to those described above, and thus will not be repeated herein.

As shown in FIG. 2A, in some embodiments, the cell culture device 20 further includes a second inlet channel 210, and the second inlet channel 210 is connected to the side surface 102s of the chamber 102. In some embodiments, the second inlet channel 210 may be located above the first inlet channel 202. In some embodiments, the first inlet channel 202 and the second inlet channel 210 may be substantially parallel to each other. In some embodiments, the first inlet channel 202 and the second inlet channel 210 may be used to provide the same type or different types of media.

Refer to FIG. 2B, which illustrates a schematic side-view diagram of the cell culture device 20 in accordance with some embodiments of the present disclosure. As shown in FIG. 2B, in some embodiments, the cell culture device 20 further includes a second inlet connection channel 212 connected to the second inlet channel 210 and an outlet connection channel 208 connected to the outlet channel 206. Specifically, in some embodiments, the second inlet connection channel 212 may be connected between the second inlet channel 210 and the chamber 102, and the outlet connection channel 208 may be connected between the outlet channel 206 and the chamber 102.

In some embodiments, the first inlet connection channel 204 may be located lower than the second inlet connection channel 212 and the outlet connection channel 208 may be located higher than the second inlet channel 210. That is, the medium may flow in a direction from the bottom to the top of the chamber 102 in accordance with some embodiments. In some embodiments, the second inlet channel 210 may be located lower than the outlet channel 206. In some embodiments, the first inlet channel 202 may be disposed right below the chamber 102. That is, the medium may flow in a direction from the bottom to the top of the chamber 102 in accordance with some embodiments. In such a configuration, the cells can be grown in an environment with steady flow rate and the functionality of the grown spheroid cell or organoid cell is similar to that in real physiological state in vivo.

Furthermore, as shown in FIG. 2B, in some embodiments, the second inlet channel 210 and the outlet channel 206 may be disposed on opposite sides of the chamber 102. As described above, the chamber 102 may have a tapered profile. In some embodiments, the width W ₁ of the top portion of the chamber 102 may be greater than the width W ₂ of the bottom portion of the chamber 102. In addition, in some embodiments, the chamber 102 may further include a coating layer (not illustrated) on the side surface, e.g., the inner side surface, and the coating layer may be hydrophobic or positively charged.

Moreover, the outlet channel 206 may be configured in a cavity 206c in accordance with some embodiments. In some embodiments, the outlet channel 206 is located at the bottom of the cavity 206c.

Next, refer to FIG. 3A and the FIG. 3B. FIG. 3A illustrates a schematic diagram of the cell culture device 20 in accordance with some embodiments of the present disclosure. FIG. 3B illustrates a schematic side-view diagram of the cell culture device 20 in accordance with some embodiments of the present disclosure. As shown in FIG. 3A and FIG. 3B, in some embodiments, the chamber 102, the first inlet channel 202, the second inlet channel 210 and the outlet channel 206 are configured in a base BS. In some embodiments, the base BS may include a junction portion (e.g., the protruding portions of the first inlet channel 202, the second inlet channel 210 and the outlet channel 206 on the side surface of the base BS) , and the junction portions may connect to another base (e.g., connect to the recessing portions of the first inlet channel 202, the second inlet channel 210 and the outlet channel 206 on another side surface of the base BS) .

In some embodiments, the material of the base BS may include polystyrene (PS) , polymethyl methacrylate (PMMA) , resin, other suitable materials, or a combination thereof. In some embodiments, the base BS having the above elements (the first inlet channel 202, the second inlet channel 210 and the outlet channel 206 and so on) may be formed by a three-dimensional printing process or other suitable processes.

Refer to FIG. 4A, which illustrates a schematic diagram of a portion of a cell culture device 30 in accordance with some other embodiments of the present disclosure. It should be understood that FIG. 4A only illustrates the chamber 102, the first inlet channel 202 and the first inlet connection channel 204 for clarity. In some embodiments, the chamber 102 may have a countersunk profile. Specifically, the bottom portion of the chamber 102 may have a flat area and the side surface 102s may be tilted. In addition, the width W ₁ of the top portion of the chamber 102 may be greater than the width W ₂ of the bottom portion of the chamber 102.

Refer to FIG. 4B, which illustrates a schematic diagram of a portion of a cell culture device 40 in accordance with some other embodiments of the present disclosure. It should be understood that FIG. 4B only illustrates the chamber 102, the first inlet channel 202 and the first inlet connection channel 204 for clarity. In some embodiments, the chamber 102 may have an oval profile. Specifically, the side surface 102s may be curved. In addition, the width W ₁ of the top portion of the chamber 102 may be greater than or equal to the width W ₂ of the bottom portion of the chamber 102.

Refer to FIG. 5A, which illustrates an operation diagram of a cell culture device in accordance with some embodiments of the present disclosure. As shown in FIG. 5A, in some embodiments, the cell culture device further includes at least one medium provider 302, and the medium provider 302 is connected to the first inlet channel 202. In some embodiments, the medium provider 302 may provide the medium with a flow rate from about 1 microliter/hour to about 200 milliliter/hour. It should be noted that the flow rate of the medium should be well-controlled so that a consistent biomimetic microenvironment can be established. Moreover, in some embodiments, the pump pressure of the medium provider 302 may be in a range from about 50 pa to about 12000 pa.

In addition, in some embodiments, the cell culture device further includes a waste liquid collector 304, and the waste liquid collector 304 is connected to the outlet channel 206. The waste liquid collector 304 can transport leftovers from cell aggregation and agglomeration out of the cell culture device.

Specifically, in accordance with some embodiments, the operation process of the cell culture device may include the following stages: the medium provider 302 provides the medium to the first inlet channel 202; a portion of the medium flows into the main portion 204M of the first inlet connection channel 204 while a portion of the medium flows to another cell culture unit 100U; the portion of the medium flowing into the main portion 204M flows to the fluid valve 204V of the first inlet connection channel 204, and then flows to the chamber 102; and the medium then flows from the chamber 102 to the outlet channel 206, and then to the waste liquid collector 304.

Refer to FIG. 5B, which illustrates an operation diagram of a cell culture device in accordance with some other embodiments of the present disclosure. As shown in FIG. 5B, in some embodiments, the cell culture device includes more than one medium providers 302. Specifically, in some embodiments, one medium provider 302 is connected to the first inlet channel 202 and another one medium provider 302 is connected to the second inlet channel 210. However, in some other embodiments, the first inlet channel 202 and second inlet channel 210 may be connected to the same medium provider 302. In some embodiments, the medium providers 302 connected to the first inlet channel 202 and the second inlet channel 210 provide the same type or different types of media. In addition, in some embodiments, the medium providers 302 connected to the first inlet channel 202 and the second inlet channel 210 provide the same type or different types of media to the chamber 102 simultaneously or nonsimultaneously. For example, the medium providers 302 connected to the first inlet channel 202 and the second inlet channel 210 may provide the medium alternately in accordance with some embodiments.

Specifically, in accordance with some embodiments, the operation process of the cell culture device may include the following stages: the medium provider 302 provides the medium to the first inlet channel 202; a portion of the medium flows into the main portion 204M of the first inlet connection channel 204 while a portion of the medium flows to another cell culture unit 100U; the portion of the medium flowing into the main portion 204M flows to the fluid valve 204V of the first inlet connection channel 204, and then flows to the chamber 102; and the medium then flows from the chamber 102 to the outlet channel 206, and then to the waste liquid collector 304. In addition, the operation process of the cell culture device also include the following stages: another medium provider 302 provides the medium to the second inlet channel 210; the medium flows from the second inlet channel 210 through the second inlet connection channel 212 and then to the chamber 102; and the medium then flows from the chamber 102 to the outlet channel 206, and then to the waste liquid collector 304. In this embodiments, the medium provided by different medium providers 302 may be mixed in the chamber 102 so that the cells can be grown in more than one types of media.

Next, refer to FIGs. 6A-6C, which illustrate hydrodynamic simulation results of the cell culture device 10 in accordance with some embodiments of the present disclosure. The hydrodynamic simulation result of FIG. 6A is obtained using a medium flow rate of 2400 microliter/hour and a pump pressure of 50 pa. The hydrodynamic simulation result of FIG. 6B is obtained using a medium flow rate of 24000 microliter/hour and a pump pressure of 4800 pa. The hydrodynamic simulation result of FIG. 6C is obtained using a medium flow rate of 36000 microliter/hour and a pump pressure of 10132 pa.

According to the results of FIGs. 6A-6C, it is known that the cell culture device 10 can provide a flow field with a substantially uniform flow velocity, and a consistent biomimetic microenvironment can be established.

Next, refer to FIG. 7, which illustrates a diagram of spheroid cell formation in a cell culture device in accordance with some embodiments of the present disclosure. As shown in FIG. 7, in accordance with some embodiments, the cells cultured in the cell culture device are aggregated at the bottom of the chamber to form spheroid cells (as indicated by the circle in the drawing) .

Next, refer to FIGs. 8A-8D, which illustrate hydrodynamic simulation results of the cell culture devices in accordance with some embodiments of the present disclosure. FIG. 8A shows the hydrodynamic simulation result of the cell culture device having the first hydraulic width

of 0.4 mm. FIG. 8B shows the hydrodynamic simulation result of the cell culture device having the first hydraulic width

of 0.8 mm. FIG. 8C shows the hydrodynamic simulation result of the cell culture device having the first hydraulic width

of 1.2 mm. FIG. 8D shows the hydrodynamic simulation result of the cell culture device having the first hydraulic width

of 1.6 mm.

According to the results of FIGs. 8A-8D, it is known that although the flow fields are different in the cell culture devices having different first hydraulic widths

the flow rates at the central region of the chamber are still maintained in a low-speed range, for example, in a range from 1 microliter/min to 600 microliter/min.

Next, refer to FIGs. 9A-9D, which illustrate hydrodynamic simulation results of the cell culture devices in accordance with some embodiments of the present disclosure. FIG. 9A shows the hydrodynamic simulation result of the cell culture device where the side surface of the chamber and the side surface of the fluid valve having the included angle θ of 3 degrees. FIG. 9B shows the hydrodynamic simulation result of the cell culture device where the side surface of the chamber and the side surface of the fluid valve having the included angle θ of 16 degrees. FIG. 9C shows the hydrodynamic simulation result of the cell culture device where the side surface of the chamber and the side surface of the fluid valve having the included angle θ of 22 degrees. FIG. 9D shows the hydrodynamic simulation result of the cell culture device where the side surface of the chamber and the side surface of the fluid valve having the included angle θ of 28 degrees.

According to the results of FIGs. 9A-9D, it is known that the flow fields of the central cell aggregation region are mainly controlled by adjusting the amount of fluid flow in the chamber.

To summarize the above, in accordance with some embodiments of the present disclosure, the provided cell culture device can culture three-dimensional spheroid cells or organoid cells. The cell culture device uses continuous flowing culture medium to provide a biomimetic microenvironment where spheroid cells or organoid cells can be grown. In accordance with some of the present disclosure, the flow rate of the culture medium can be adjusted or more than one types of culture media can be provided so that the cells can be grown steadily and the functionality of the grown spheroid cell or organoid cell is quite similar to that in real physiological state in vivo. Therefore, the spheroid cell or organoid cell provided by the cell culture device according to the embodiments of the present disclosure is advantageous to the application of cell therapy and pharmacologic screening test.

Although some embodiments of the present disclosure and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined by the appended claims. For example, it will be readily understood by one of ordinary skill in the art that many of the features, functions, processes, and materials described herein may be varied while remaining within the scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed, that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

A cell culture device, comprising:

at least one chamber;

a first inlet channel;

a first inlet connection channel having a first length, which connects a bottom portion of the at least one chamber to the first inlet channel and comprises a fluid valve for selectively porting a pressurized medium from the first inlet channel to the at least one chamber; and

an outlet channel connected to the at least one chamber;

wherein the fluid valve has a second length and a first hydraulic width defining an aspect ratio adapted to taper the pressurized medium along the first inlet connection channel; and

wherein the fluid valve comprises a first recess extending along the second length within the fluid valve to permit the pressurized medium to flow along the second length at a substantially uniform flow velocity.
The cell culture device as claimed in claim 1, further comprising a second inlet channel connected to a side surface of the at least one chamber.
The cell culture device as claimed in claim 1, wherein the first inlet channel is located lower than the outlet channel.
The cell culture device as claimed in claim 1, wherein the first inlet channel is disposed right below the at least one chamber.
The cell culture device as claimed in claim 1, wherein a width of a top portion of the at least one chamber is greater than a width of the bottom portion of the at least one chamber.
The cell culture device as claimed in claim 1, wherein the at least one chamber has a tapered profile, a countersunk profile, or an oval profile.
The cell culture device as claimed in claim 1, further comprising at least one medium provider connected to the first inlet channel.
The cell culture device as claimed in claim 2, wherein the second inlet channel is located lower than the outlet channel.
The cell culture device as claimed in claim 2, wherein the second inlet channel and the outlet channel are disposed on opposite sides of the at least one chamber.
The cell culture device as claimed in claim 2, wherein the side surface of the at least one chamber is curved.
The cell culture device as claimed in claim 2, further comprising a second inlet connection channel connected to the second inlet channel and an outlet connection channel connected to the outlet channel, wherein the first inlet connection channel is located lower than the second inlet connection channel and the outlet connection channel is located higher than the second inlet channel.
The cell culture device as claimed in claim 2, wherein the at least one chamber further comprises a coating layer on the side surface, and the coating layer is hydrophobic or positively charged.
The cell culture device as claimed in claim 2, wherein the at least one chamber, the first inlet channel, the second inlet channel and the outlet channel are configured in a base, and the base comprises a junction portion that connects to another base.
The cell culture device as claimed in claim 2, further comprising at least one medium provider connected to the first inlet channel and the second inlet channel.
The cell culture device as claimed in claim 14, wherein the first inlet channel and the second inlet channel are each connected to the at least one medium provider, and the medium providers connected to the first inlet channel and the second inlet channel provide the same type or different types of media.
The cell culture device as claimed in claim 15, wherein the medium providers connected to the first inlet channel and the second inlet channel provide the same type or different types of media to the at least one chamber simultaneously or nonsimultaneously.
The cell culture device as claimed in claim 14, wherein the at least one medium provider provides a medium flowing from the first inlet channel through the first inlet connection channel to the at least one chamber to permit turbulence formation in the medium.
The cell culture device as claimed in claim 14, wherein the at least one medium provider provides a medium with a flow rate from about 1 microliter/hour to about 200 milliliter/hour.
The cell culture device as claimed in claim 1, wherein the first recess has a cross-sectional shape, and the cross-sectional shape is a meniscus, an arc, a circle, a polygon, a curved shape, or a sector.
The cell culture device as claimed in claim 1, further comprising a waste liquid collector connected to the outlet channel.
The cell culture device as claimed in claim 1, wherein the at least one chamber comprises a cell aggregation region, and the cell aggregation region is located at a top surface of the fluid valve.
The cell culture device as claimed in claim 1, wherein the first inlet connection channel further comprises a main portion connected to the first inlet channel, wherein the first inlet channel has a second hydraulic width and the main portion has a third hydraulic width, and a ratio of the second hydraulic width to the third hydraulic width is greater than 3: 1.
The cell culture device as claimed in claim 1, wherein the first inlet connection channel further comprises a main portion connected to the first inlet channel, wherein the main portion has a third hydraulic width and the first recess has a fourth hydraulic width, and a ratio of the third hydraulic width to the fourth hydraulic width is greater than or equal to 10: 1.
The cell culture device as claimed in claim 1, wherein the fluid valve further comprises a second recess, wherein the first recess and the second recess are spaced apart by the first hydraulic width.