Cell operation device and methods for the use thereof

Abstract

This invention discloses a device for preparing, culturing, and collecting cell spheroids. The device comprises multiple independently operable elements, including a first chamber and a second chamber arranged vertically. The first chamber has microchannels and multiple through-holes to form uniform suspended droplets, promoting cell spheroid formation. The second chamber, located below the first chamber, receives the cell spheroids for imaging or subsequent analysis. This invention enables scalable and reproducible three-dimensional cell culture and collection, suitable for applications such as biomedical research and drug screening.

Description

Technical Field

[0001] Overall, this invention discloses an apparatus and method for cell manipulation, specifically, an apparatus and efficient method capable of high-throughput collection of three-dimensional cell spheres while maintaining their structural integrity for subsequent analysis. Prior Technology

[0002] Compared to traditional two-dimensional cell culture, three-dimensional cell spheroid models better reflect the actual physiological state of the human body, and are therefore increasingly widely used in basic research, drug screening, and precision medicine. However, cell spheroid-based detection procedures generally suffer from problems such as being time-consuming, susceptible to contamination, and prone to cell damage, limiting their practicality in large-scale applications. For example, standard procedures such as cell spheroid transfer, centrifugation, and liquid replacement can severely disrupt their structural integrity, leading to cell damage and loss.

[0003] Traditional methods of cell spheroid processing typically rely on manual manipulation using micropipettes. Samples must be transferred to centrifuge tubes for washing, fixation, perforation, and immunostaining before finally being transferred to an imaging analysis platform, such as a glass-bottomed culture dish. This multi-step process is not only time-consuming and cumbersome but also significantly increases the risk of contamination and cell spheroid damage. Furthermore, while cell spheroid generation is often achieved using low-adhesion round-bottomed discs, this method, although effective in cell culture, still faces challenges such as low efficiency and insufficient integration in subsequent processing and imaging analysis.

[0004] Currently, while technologies exist to simplify the culture and manipulation of cell spheroids, such as Insphero's hanging drop culture disc system that can form cell spheroids in a suspended droplet, the subsequent transfer process—adding liquid to cause the droplet to collapse and allow the cell spheroids to settle into the wells below—remains challenging. During immunostaining or washing steps, liquid replacement via micropipette within the wells often involves direct contact with the cell spheroids, further increasing the risk of cell loss or damage. Furthermore, the plastic substrate of these wells reduces optical clarity, making them unsuitable for high-resolution image analysis.

[0005] Another approach, described in recent literature (Kim, H., Roh, H., Kim, H., & Park, JK (2021)), employs a droplet-on-micropillar method to facilitate cell spheroid transfer. This method consists of an aligned droplet array chip (DAC) made of PDMS (polydimethylsiloxane) and a pillar array chip (PAC) to transfer cell spheroids. However, this method is limited by the complexity of culturing cell spheroids in suspended droplets and requires precise alignment of the DAC and PAC during transfer. Furthermore, the large size of the device hinders the acquisition of high-resolution images, thus limiting its effectiveness in subsequent applications.

[0006] Therefore, the present invention provides a novel cell spheroid manipulation device that overcomes the limitations of existing methods and achieves high-efficiency, high-throughput, and gentle manipulation of three-dimensional cell spheroids.

[0007] To date, no system has successfully addressed all the challenges associated with high-throughput, high-integrity cell spheroid manipulation—including gentle handling, precise fluid replacement, minimizing cell loss, and compatibility with high-resolution imaging technologies. Therefore, there is an urgent need for a device and method that enables efficient, scalable, and minimally invasive manipulation of three-dimensional cell spheroids at all stages of bioanalysis.

[0008] The present invention further provides a cell manipulation method that ensures that cell damage and loss are minimized while maintaining the integrity of the cell spheroids, so as to facilitate subsequent analysis. Summary of the Invention

[0009] Therefore, the present invention provides a cell manipulation device comprising multiple independently operable wafer units, each wafer unit including: A first chamber and a second chamber arranged vertically: The first chamber is located above the second chamber; The first chamber includes a microchannel and a plurality of through holes disposed on the bottom surface of the microchannel for promoting the formation of suspended liquid droplets; and The second chamber includes multiple collection orifices for receiving multiple spherical cells from the suspended liquid droplet; The collection hole is located directly below the suspended liquid droplets formed by the plurality of through holes corresponding to the first chamber.

[0010] In some embodiments of the present invention, the first chamber includes a first injection port configured to communicate with the microchannel for receiving a sample solution.

[0011] In some embodiments of the present invention, the first chamber includes a liquid storage tank configured to communicate with the microchannel to generate hydrostatic pressure.

[0012] The apparatus as described in claim 1, wherein the second chamber is disposed on a substrate comprising a transparent glass layer.

[0013] In some embodiments of the present invention, the thickness of the glass layer ranges from about 0.1 mm to 0.17 mm, which can provide structural support and maintain compatibility with image capture.

[0014] In some embodiments of the present invention, the first chamber includes a second injection port, which communicates with the second chamber for injecting culture medium into the second chamber until the culture medium comes into contact with the suspended droplets.

[0015] In some embodiments of the present invention, the wafer unit further includes: a) An inlet provided on the first chamber for introducing culture medium and in fluid communication with the second chamber through an internal channel; and b) An outlet is provided on the first chamber for removing the culture medium and is in fluid communication with the second chamber through an internal channel.

[0016] In some embodiments of the present invention, the first chamber includes a plurality of columns extending vertically toward the second chamber, thereby enhancing the connectivity and structural stability of the device.

[0017] In some embodiments of the present invention, the area of ​​the microchannel ranges from approximately 1×1 mm² to 86×128 mm², and the height ranges from approximately 0.1 mm to 5 mm.

[0018] In some embodiments of the present invention, the diameter of each of the plurality of through holes ranges from about 0.05 mm to 5 mm, and the depth ranges from about 0.1 mm to 5 mm.

[0019] In some embodiments of the present invention, the second chamber includes a plurality of collection holes disposed on a transparent glass layer. Each collection hole is located directly below the droplet formed at the corresponding through-hole.

[0020] In some embodiments of the present invention, the collection hole has a geometric prism shape, which is selected from the following types: rectangular prism, pentagonal prism, heptagonal prism, octagonal prism, triangular prism, square prism, cylindrical prism or trapezoidal prism.

[0021] In some embodiments of the present invention, the area of ​​the collection hole ranges from approximately 0.05 × 0.05 square millimeters to 86 × 128 square millimeters, and the height ranges from approximately 0.05 millimeters to 5 millimeters.

[0022] In some embodiments of the present invention, the first chamber and the second chamber are made of one or more materials selected from the group consisting of: polydimethylsiloxane (PDMS), polycarbonate (PC), polymethyl methacrylate (PMMA), polyolefin copolymer (POC), polystyrene (PS), polypropylene (PP), glass, and hydrocolloids.

[0023] Another object of the present invention is to provide a method for operating the above-mentioned device, comprising: (a) Injecting the cell suspension into the first chamber through the first injection port; (b) Introducing culture medium into a storage tank to generate a hydrostatic pressure difference, thereby forming suspended droplets at multiple through-holes; (c) Inject the culture medium into the second chamber through the second inlet until it comes into contact with the suspended droplets.

[0024] In some embodiments of the present invention, the method further includes: (d) The culture medium is replaced by removing and replenishing it with fresh medium, with a volume ranging from 1 to 3 times the total volume of the microchannel; and (e) Place the assembled device on the microscope stage for high-resolution conjugate focal microscopic observation of collected cell spheres. Simple Explanation of the Diagram

[0025] Figure 1 is a schematic diagram of the device design of the present invention.

[0026] Figure 2 is a schematic diagram of an example of the device of the present invention.

[0027] Figure 3 is an exploded view of the device of the present invention.

[0028] Figure 4 is a side view of the first chamber filled with sample solution.

[0029] Figure 5 is a side view of the second chamber filled with sample solution.

[0030] Figure 6 is a top view of the device of the present invention.

[0031] Figure 7 is a schematic diagram of the design of the second chamber.

[0032] Figure 8 is a schematic diagram of the collection hole design.

[0033] Figure 9 is a schematic diagram of another design for the second chamber.

[0034] Figure 10 is a top view of another design for the second chamber.

[0035] Figure 11 is a side view of the design of the second chamber.

[0036] Figure 12 is a side view of another design for the second chamber.

[0037] Figures 13A to 13H show the cell spheroid formation in the device of the present invention and the comparison results of using an ultra-low attached (ULA) culture dish.

[0038] Figures 14A to 14D show the verification results of the efficiency of cell spheroid collection using the device of the present invention. Implementation

[0039] All technical features disclosed in this specification can be combined in any manner. Alternatives for the same, equivalent, or similar purposes may replace each technical feature disclosed in this specification. Therefore, unless otherwise specified, each disclosed technical feature is merely a general example of a series of equivalent or similar features.

[0040] The terms "preferred" and "preferably" refer to embodiments that may have certain advantages under specific circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are not effective, nor is it intended to exclude other embodiments.

[0041] Example 1: Device Design and Assembly

[0042] Equipment manufacturing

[0043] As shown in Figures 1 and 2, the device disclosed in this invention has several significant advantages, such as the ability to form, culture, and collect three-dimensional cell spheroids in a single integrated system, thus eliminating the need to transfer cell spheroids to different containers during drug screening, staining, or image analysis. This design effectively reduces cell spheroid loss or structural damage caused by manipulation, allowing direct optical observation through a transparent substrate, which is beneficial for subsequent analysis without additional transfer steps. In one embodiment, the device of this invention includes at least one first chamber (10) and one second chamber (20), arranged in a vertically stacked manner and assembled into an integral structure.

[0044] In some embodiments, the first chamber (10) is vertically positioned above the second chamber (20), such that the plurality of through-holes (13) of the first chamber (10) are aligned with the collection hole (21) of the second chamber (20). This vertical arrangement facilitates the downward movement of cell spheroids from the plurality of through-holes (13) to the collection hole (21) by gravity, thereby simplifying the transition process from the cell spheroid formation stage to the analysis stage (as shown in Figures 4 and 5).

[0045] In some embodiments, the first chamber (10) includes an extended microchannel that serves as the primary channel for the flow of cell suspension and culture medium. The bottom area of ​​the microchannel is approximately 3 × 15 mm² to approximately 6 × 25 mm², more preferably approximately 4 × 17 mm² to approximately 6 × 22 mm², and most preferably approximately 5 × 18 mm² to approximately 5 × 20 mm², also encompassing various values ​​and ranges thereof, depending on factors such as the required number of suspended droplets, cell density, and size compatibility. The height of the microchannel can be approximately 0.8 mm to approximately 1.5 mm, more preferably approximately 1.0 mm to approximately 1.3 mm, and most preferably approximately 1.2 mm, also depending on the fluid volume and the required stability of the suspended droplets.

[0046] The bottom of the microchannel in the first chamber (10) is provided with multiple through-holes (13) that allow hydrostatic pressure to form suspended droplets (as shown in Figures 1 and 3). The design of these multiple through-holes (13) helps to control the detachment and stabilization of the suspended droplets, thereby allowing the cell spheres to settle under gravity. In one embodiment, the diameter of the multiple through-holes (13) can be from about 0.5 mm to about 1.5 mm, more preferably from about 0.8 mm to about 1.3 mm, and most preferably from about 1.0 mm to about 1.2 mm. The depth of the multiple through-holes (13) can be from about 0.3 mm to about 1.0 mm, more preferably from about 0.5 mm to about 0.9 mm, and most preferably from about 0.7 mm to about 0.8 mm, and covers all values ​​and ranges therebetween, depending on factors such as liquid viscosity, cell type, and the required volume of suspended droplets.

[0047] The multiple through-holes (13) are further designed to maintain the stability of the suspended droplets, prevent backflow or leakage, and ensure that the cell spheroids can be smoothly collected to the corresponding position in the second chamber (20) by gravity. The multiple through-holes (13) configuration supports high-throughput and reproducible cell spheroid generation.

[0048] The reservoir (12) is connected to the first chamber (10) to supply fresh culture medium (as shown in Figure 1). The reservoir (12) is positioned at a height higher than the first inlet (11) to generate a hydrostatic pressure difference, thereby promoting the flow of the culture medium. In addition, the reservoir (12) has a larger capacity than the first chamber (10), ensuring a continuous and stable supply of culture medium. During operation, hydrostatic pressure drives the liquid containing cell spheroids through multiple through-holes (13) located at the bottom of the microchannel, initiating the formation of suspended droplets. This pressure-driven flow not only stably generates suspended droplets but also continuously supplies nutrients to the developing cell spheroids in the second chamber (20).

[0049] The first chamber (10) is further provided with a second inlet (14), which is in fluid communication with the second chamber (20) through an internal flow path (as shown in Figure 6). This design allows the introduction of culture medium or reagents into the second chamber (20), promoting the removal of cell spheroids from the suspended droplets above, and subsequently collection into the collection port (21) of the second chamber (20). The fluid flow generated through the second inlet (14) effectively transfers the cell spheroids while minimizing mechanical interference.

[0050] In some embodiments, the device is designed to enable stable vertical stacking, precise chamber alignment, and modular disassembly without structural damage, thereby supporting efficient transfer between cell spheroid culture and subsequent imaging or analysis procedures. To achieve this, the device includes a set of modular alignment structures, one example being four copper pillars (300). The copper pillars (300) are spatially arranged to precisely align the first chamber (10) onto the second chamber (20). The copper pillars (300) not only provide structural support for assembly but also allow for easy separation of the two chambers after culture, minimizing operational interference with the cell spheroids.

[0051] The second chamber (20), located directly below the multiple through-holes (13), contains a transparent glass substrate with multiple collection holes (21) (as shown in Figure 2). The collection holes (21) are configured to receive cell spheroids released from the suspended droplets. The transparency of the substrate allows for direct imaging of the cell spheroids, including observation using a conjugate focal or fluorescence microscope.

[0052] The planar area of ​​the collection orifice (21) ranges from approximately 1.0 × 6 mm² to 2.0 × 12 mm², more preferably from approximately 1.2 × 8 mm² to 1.8 × 10 mm², and most preferably from approximately 1.6 × 9 mm²; the depth of the collection orifice (21) is approximately 0.8 mm² to 1.5 mm², more preferably from approximately 1.0 mm² to 1.3 mm², and most preferably from approximately 1.2 mm², including intermediate values ​​and ranges, depending on the size of the target cell spheroids and the fluid exchange requirements. The footprint of the device of the present invention can be approximately 20 × 60 mm² to 30 × 80 mm², more preferably from approximately 22 × 70 mm² to 27 × 75 mm², and most preferably from approximately 25 × 75 mm², a size configuration suitable for a standard microscope stage.

[0053] Cell spheres can be observed directly through a transparent substrate without transferring samples, thus preserving spatial arrangement and reducing sample loss. The collected cell spheres can be directly stained, imaged, or analyzed in the collection well (21), supporting simplified and non-destructive analytical procedures.

[0054] In some embodiments, the thickness of the transparent glass substrate forming the bottom of the second chamber (20) ranges from about 0.1 mm to 0.17 mm. This thickness range is designed to balance optical transparency for high-resolution imaging with sufficient mechanical strength to ensure the structural integrity of the device during operation and cell spheroid transfer.

[0055] The collection wells (21) designed on the transparent glass substrate of the second chamber (20) can adopt a variety of geometric shapes to optimize the fixation and culture conditions of cell spheres. Typical shapes of collection wells (21) include rectangles, pentagons, heptagons, octagons, triangles, squares, cylindrical prisms, trapezoidal prisms, or other polygonal geometries. The diversity of collection well (21) shapes allows the device to be designed according to specific cell types, sphere sizes, or imaging patterns.

[0056] The first chamber (10) and the second chamber (20) of this device can be made of a variety of materials, including but not limited to polydimethylsiloxane (PDMS), polycarbonate (PC), polymethyl methacrylate (PMMA), polyolefin copolymer (POC), polystyrene (PS), polypropylene (PP), glass, and hydrogel materials. The choice of chamber material can be determined based on the required optical properties, biocompatibility, manufacturing process, and mechanical performance requirements.

[0057] In some embodiments, the device is designed such that cells cultured therein form cell spheroids. For example, the surface of the multiple through-holes (13) may be non-adhesive, encouraging cells to aggregate within the multiple through-holes (13) and form spherical structures. The size of the cell spheroids is limited by the geometry of the multiple through-holes (13). In some embodiments, the surface of the multiple through-holes (13) is coated with an ultra-low adhesion material to make the multiple through-holes (13) non-adhesive to cells.

[0058] The device of this invention can culture multiple cell types. In some embodiments, the cell spheroids consist of a single cell type; while in other embodiments, the cell spheroids contain multiple cell types. When culturing multiple cell spheroids, in some cases all spheroids are of the same type, while in other embodiments two or more different types of spheroids can be cultured simultaneously. The cultured cells can be natural cells or genetically modified cells (e.g., cells containing one or more non-natural gene modifications).

[0059] In some embodiments, the cells used are somatic cells; in other embodiments, they are stem cells or precursor cells (e.g., embryonic stem cells, induced pluripotent stem cells), and can be in any differentiation stage (e.g., pluripotency, multipotency, fate determination, immortalization, etc.). Additionally, the cells can be disease cells or disease model cells, such as cells containing one or more cancer cells, or cells that can be induced to a state of excessive proliferation (e.g., transformed cells).

[0060] The cells used may be derived from or derived from any type of tissue or organ, including but not limited to: adrenal glands, bladder, blood vessels, bones, bone marrow, brain, cartilage, cervix, cornea, endometrium, esophagus, gastrointestinal tract, immune system (e.g., T lymphocytes, B lymphocytes, white blood cells, macrophages, and dendritic cells), liver, lungs, lymphatic system, muscles (e.g., cardiac muscle), nervous system, ovaries, pancreas (e.g., islet cells), pituitary gland, prostate, kidneys, salivary glands, skin, tendons, testes, and thyroid gland.

[0061] In some embodiments, the cells used are mammalian cells, such as human, mouse, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.

[0062] Example 2. Device Operation

[0063] As shown in Figure 3, the cell suspension is injected into the microchannel of the first chamber (10) through the first injection port (11). Next, culture medium is introduced into the reservoir (12), and by generating a hydrostatic pressure difference across the multiple through-holes (13), suspended droplets are induced to form in the multiple through-holes (13). The cell spheroids settle under gravity and gradually aggregate to form uniform three-dimensional cell spheroids.

[0064] To collect cell spheroids for imaging observation or subsequent analysis, culture medium is introduced through the second injection port (14) of the device (as shown in Figures 4-5). When the rising liquid comes into contact with the suspended droplet, the cell spheroids detach from the droplet and fall into the corresponding collection well (21). The geometry of the collection well (21) is designed to restrict the position of the cell spheroids, keeping them stable during subsequent liquid changes and staining. This design prevents cell spheroid displacement or mechanical damage during liquid handling and reagent changes.

[0065] After the cell spheres are collected, the device of this invention can be directly mounted on the microscope stage without transferring the cell spheres to other containers, enabling high-resolution conjugate focal microscopy imaging, thereby minimizing sample loss and maintaining the integrity of the spatial structure.

[0066] Example 3: Design type of collection hole

[0067] To further improve the stability of cell spheres during liquid exchange, different collection hole designs are incorporated into the device of this invention to maintain the position of the cell spheres. As shown in Figure 8, four collection hole designs are proposed.

[0068] In some embodiments of the invention, the collection holes are a set of circular holes with a diameter ranging from 0.8 to 1.5 mm and a depth ranging from 0.5 to 1 mm. These collection holes are evenly distributed to physically restrict the position of each cell sphere and ensure its stable fixation.

[0069] In some embodiments of the present invention, the collection holes are hexagonal holes with a side length ranging from 0.5 to 1 mm and a depth ranging from 0.5 to 1 mm. These hexagonal collection holes are arranged in a tightly stacked manner to maximize space utilization and minimize fluid shear stress.

[0070] In some embodiments of the present invention, the collection holes are square holes with a side length ranging from 0.6 to 1.2 mm and a depth ranging from 0.5 to 1 mm. These square collection holes are arranged in a regular grid layout to prevent cell sphere displacement during fluid exchange.

[0071] In some embodiments of the invention, a grid-type collection hole is used, disposed within a groove with a width ranging from 3 to 6 mm and a depth of 1 mm, to provide overall confinement of multiple cell spheres.

[0072] As shown in Figure 7, during operation, the device of the present invention allows for various liquid exchanges without removing the collection pores containing cell spheroids.

[0073] In some embodiments of the present invention, the exchange liquid operates in the form of a closed channel, and the liquid is exchanged directly above the collection orifice through a continuous microfluidic channel, maintaining the integrity of the cell spheres.

[0074] In some embodiments of the present invention, after the first chamber (10) is removed, a top cover (500) is installed to form a sealed flow channel for liquid replacement (see Figure 7).

[0075] In some embodiments of the present invention, after the first chamber (10) is removed, liquid exchange is carried out through an open groove, which facilitates manual liquid aspiration and keeps the sphere fixed in the collection hole without being moved.

[0076] The structural design of the device in this invention allows for more flexible and stable culture medium exchange, ensuring cell viability and spheroid integrity in drug testing or immunostaining. Furthermore, the device can be directly used for high-resolution imaging and analysis without requiring additional operational steps.

[0077] Example 4: Alternative state of the second chamber

[0078] Figure 9-12 shows another embodiment of the second chamber (20), in which the second chamber (20) includes a recessed basin-shaped structure with a certain depth and a specific depth and curvature design to accommodate rising liquid and deposited cell spheres, ensuring that the collected sample position is stable and consistent.

[0079] In some embodiments, the second chamber (20) further includes one or more fluid inlets and outlets configured for controlled addition and removal of liquid culture medium. These inlets and outlets may be located on the side or bottom of the second chamber (20) and are in fluid communication with internal collection pores. Through these inlets and outlets, culture medium or reagents can be introduced or replaced without disturbing the cell spheroids.

[0080] During operation, the first chamber (10) remains assembled above the second chamber (20). Liquid is injected into the second chamber (20) through the second inlet, causing the liquid level in the second chamber (20) to gradually rise. When the liquid surface approaches the plurality of through holes (13), a convex liquid surface is formed. Once the liquid comes into contact with the suspended droplets, the droplets merge with the rising liquid, causing the encapsulated cell spheres to be released. These cell spheres are then collected by gravity sedimentation into the collection hole (21) at the bottom of the second chamber (20).

[0081] This design allows for non-invasive replacement of culture medium and reagents, minimizing hydrodynamic shear forces and preventing accidental movement or loss of cell spheroids during liquid exchange. By maintaining the integrity and positioning of cultured cell spheroids throughout the entire culture medium replacement process, the device supports long-term culture, multiple staining or washing steps, and high-resolution imaging procedures, all performed in a closed and stable environment.

[0082] Example 5: The device of the present invention generates uniform cell spheroids

[0083] Consistent cell spheroids are a key factor in the reproducibility and accuracy of drug testing. Variations in cell spheroid size and shape affect the tumor microenvironment, influenced by non-cytokine factors such as nutrient distribution, pH gradients, and oxygen gradients. Therefore, maintaining cell spheroid consistency is crucial for obtaining reliable and reproducible results in cancer research and drug testing.

[0084] One common method for forming three-dimensional cell spheroids is using ultra-low attachment (ULA) culture discs. In this embodiment, ULA culture discs are used as a control to evaluate the characteristics of the cell spheroids formed by the device of the present invention. As shown in Figure 13a, U87 cancer cells in ULA culture discs formed three-dimensional cancer cell spheroids over time with different initial cell numbers. Figure 13b illustrates the culture results using the device of the present invention.

[0085] Two hours after cell seeding, most cells cultured using the device of this invention (Fig. 13b) formed complete cell spheroid aggregates. However, as shown in Fig. 13a, cells on the ULA culture dish were still in the process of aggregation and had not fully aggregated after two hours. These results indicate that the cell spheroid aggregation efficiency of this device is significantly improved compared to the ULA culture dish. The improved aggregation efficiency may be attributed to the smaller pore size (approximately 1,000 μm) used in the device, resulting in a greater curvature of the suspended droplets. In contrast, the ULA culture dish has a larger pore size (approximately 10,000 μm) and a smaller curvature. The increased curvature of the suspended droplets promotes rapid aggregation of cell spheroids within 2 hours.

[0086] The quantitative results shown in Figure 9C indicate that the diameter of the cell spheroids increased over time, both in the ULA culture dish and in this device, demonstrating continuous cell division and growth. Furthermore, by adjusting the initial cell number, the initial size of the cell spheroids could be controlled, ensuring the consistency and reproducibility of the results. These data demonstrate that cell spheroids, under the optimal culture conditions provided by the device, can maintain their physiological characteristics and continue to expand.

[0087] The average sphericity of the cell spheroids cultured by the device of the present invention reaches 0.75, which is higher than the 0.65 of the cell spheroids in the ULA culture dish. Furthermore, the average surface roughness of the cell spheroids cultured by the device of the present invention is 0.9, while that in the ULA culture dish is 0.85. These results indicate that the device of the present invention can generate cell spheroids of uniform size and shape, making it suitable for drug screening applications.

[0088] Example 6: Verification of Cell Spheroid Collection Efficiency

[0089] To verify the collection efficiency of cell spheres, the cell spheres cultured on the device of this invention were stained with calcium ion indicator Calcium-AM to assess cell viability and perform fluorescence observation. Before contact with the suspended liquid droplet, the cell spheres existed as droplets on the through-hole (13). Subsequently, liquid was introduced through the second inlet, causing the suspended liquid droplet to break and the cell spheres to fall into the collection hole.

[0090] Figures 14A-14D show that six independent cell spheroids were collected intact. Based on the results shown in Figures 14A-14D, the collection efficiency was quantified. Quantitative data indicate that the cell spheroid collection process is simple and efficient, stably collecting most cell spheroids without damaging the cells, and requiring no complex manipulation.

[0091] All the technical features disclosed in this specification can be combined in any way.

[0092] Alternatives for the same, equivalent, or similar purposes may replace each technical feature disclosed in this specification. Therefore, unless otherwise specified, each disclosed technical feature is merely a general example of a series of equivalent or similar features.

[0093] 10: First Chamber 20: Second Chamber 11: First injection port 12: Liquid Storage Tank 13: Multiple through holes 14: Second injection port 21: Collection Hole 300: Copper Pillar

[0094] none

Claims

1. A cell manipulation device, comprising: a plurality of independently operable wafer units, each wafer unit comprising: A first chamber and a second chamber are arranged vertically: the first chamber is located above the second chamber; the first chamber includes a microchannel and a plurality of through-holes disposed on the bottom surface of the microchannel for promoting the formation of suspended droplets; and the second chamber includes a plurality of collection holes for receiving a plurality of cell spheres from the suspended droplets; wherein the collection holes are located directly below the suspended droplets formed corresponding to the plurality of through-holes in the first chamber.

2. The apparatus as claimed in claim 1, wherein the first chamber includes a first injection port configured to communicate with the microchannel for receiving a sample solution.

3. The apparatus of claim 1, wherein the first chamber includes a liquid reservoir configured to communicate with the microchannel for generating hydrostatic pressure.

4. The apparatus as claimed in claim 1, wherein the second chamber is disposed on a substrate comprising a transparent glass layer.

5. The apparatus as claimed in claim 4, wherein the thickness of the glass layer is approximately 0.1 mm to 0.17 mm.

6. The apparatus of claim 1, wherein the first chamber includes a second inlet communicating with the second chamber for injecting culture medium into the second chamber until the culture medium contacts the suspended droplets.

7. The apparatus as claimed in claim 1, wherein the wafer unit further comprises: a) An inlet is provided on the first chamber for introducing culture medium and for fluid communication with the second chamber through an internal channel; and b) an outlet provided on the first chamber for removing culture medium and in fluid communication with the second chamber through an internal channel.

8. The apparatus as claimed in claim 1, wherein the first chamber includes a plurality of columns extending toward the second chamber, the columns being configured to assist in aligning the plurality of through-holes with corresponding collection holes in the second chamber.

9. The device as claimed in claim 1, wherein the bottom surface area of ​​the microchannel ranges from 1×1 mm² to 86×128 mm².

10. The device as claimed in claim 1, wherein the height of the microchannel ranges from 0.1 mm to 5 mm.

11. The apparatus as claimed in claim 1, wherein the diameter of each of the plurality of through holes ranges from 0.05 mm to 5 mm.

12. The apparatus as claimed in claim 1, wherein the depth of each of the plurality of through holes ranges from 0.1 mm to 5 mm.

13. The apparatus as claimed in claim 1, wherein the collection hole is disposed on a transparent glass layer.

14. The apparatus of claim 13, wherein the shape of the collection orifice is selected from one of the following: a rectangular prism, a pentagonal prism, a heptagonal prism, an octagonal prism, a triangular prism, a square prism, a cylinder, or a trapezoidal prism.

15. The apparatus of claim 13, wherein the planar area of ​​the collection orifice ranges from about 0.05 × 0.05 mm² to about 86 × 128 mm².

16. The apparatus of claim 15, wherein the height of the collection orifice ranges from about 0.05 mm to about 5 mm.

17. The apparatus of claim 1, wherein the first chamber and the second chamber are made of one or more of the following materials selected from polydimethylsiloxane (PDMS), polycarbonate (PC), polymethyl methacrylate (PMMA), polyolefin copolymer (POC), polystyrene (PS), polypropylene (PP), glass, and hydrogel.

18. A method of operating the apparatus of claim 1, comprising: a) Injecting the cell suspension into the first chamber through a first injection port disposed in the first chamber and communicating with the microchannel; b) Injecting culture medium into a reservoir in the first chamber to generate a hydrostatic pressure difference, thereby forming suspended droplets at the plurality of through holes; c) Introducing culture medium into the second chamber through a second inlet provided in the first chamber until the culture medium comes into contact with the suspended droplets.

19. The method of claim 18, further comprising: d) The culture medium is replaced by repeatedly removing and adding fresh culture medium, with the culture medium volume ranging from 1 to 3 times the total channel volume.

20. The method of claim 19, further comprising: e) Place the assembled device on the microscope stage for high-resolution conjugate focal microscopy imaging of the cell spheres.

Images