Methods and devices for cell-based assays

Abstract

The present invention provides a cellular assay system and its use in cell-based assays.

Description

[0001] Cell-based assays have been widely used in fields such as cell biology, medicine, pharmacology, and toxicology. Cell-based assays are typically performed in multiwell cell culture plates. Depending on the assay, cells can be present as suspensions, two-dimensional monolayers, or three-dimensional organoids / spheroids. To address specialized needs, a number of new systems have been introduced, including microfluidic cell culture devices, three-dimensional membrane-based perfusion bioreactors, and three-dimensional cell culture column systems. While these systems offer advantages, they also suffer from varying degrees of complexity, robustness, flexibility, ease of use, and cost-effectiveness. For example, the flow channels of microfluidic systems are easily clogged by bubbles, hydrogels, and cell clumps. Three-dimensional cell culture column systems are only suitable for preparing multispheroids from proliferating cells because they require the use of hydrogels to immobilize the cells on the column. Non-proliferating cells, such as human hepatocytes, cannot form spheroids in hydrogels. The use of hydrogels also limits the application of these column systems because they can affect, in particular, the diffusion of macromolecules and assay results. Furthermore, immobilizing cells on tiny pillars can be a challenge because the hydrogel needs to solidify in a very short time to avoid evaporation from a small volume.

[0002] In light of the shortcomings of these systems, the present invention discloses a removable needle cell culture system for cell-based assays. This system is not only applicable to 2D monolayer and 3D cell culture but also enables simultaneous testing of different cell types cultured on 2D monolayers, 3D scaffolds, and 3D non-scaffolds. This system provides greater flexibility, ease of handling, and robustness for conducting cell-based assays. Summary of the Invention

[0003] In a first aspect, the present invention relates to a needle suitable for a cell-based assay device, comprising: a needle body and a needle tip, wherein the needle body comprises a needle head, and the needle tip comprises a surface for cell seeding.

[0004] In one embodiment of the needle of the present invention, the needle body comprises at least one tapered section, and the needle body and the needle head together form a T-shape.

[0005] In one embodiment of the needle of the present invention, the needle body and the needle tip are cylindrical.

[0006] In one embodiment of the needle of the present invention, the needle body and the needle tip have a non-circular cross-sectional shape.

[0007] In one embodiment of the needle of the present invention, the needle head is a stepped needle head comprising grooves.

[0008] In one embodiment of the needle of the present invention, the surface is selected from the group consisting of: a flat surface, a flat surface with raised edges at the edges, an inwardly structured surface, a multi-grooved surface, a multi-microporous surface, a multi-micropillar surface, a rough surface, a permeable membrane, a porous segment or a magnetic segment; preferably, the layer includes a layer that promotes cell adhesion, more preferably, a cell adhesion promoting layer selected from a hydrogel.

[0009] In a second aspect, the present invention relates to a needle holder comprising a plurality of openings for receiving the needle of the present invention, wherein the needle holder comprises a T-shaped needle holder formed by an annular portion connected to a cylindrical portion, wherein the diameter of the annular portion is greater than the diameter of the cylindrical portion, and wherein the annular portion comprises the plurality of openings for receiving the needle.

[0010] In one embodiment, the needle holder is made of plastic, preferably polytetrafluoroethylene, polystyrene, polyester and polycarbonate.

[0011] In one embodiment, the needle holder is sized to fit into a well of a multi-well plate.

[0012] In a third aspect, the present invention relates to a device for performing a cell-based assay, comprising:

[0013] A needle holder, wherein the needle holder comprises a plurality of openings for receiving the needles of the present invention and a base having a groove channel.

[0014] In one embodiment of the device for performing a cell-based assay, the needle holder and the base have matching shapes, the opening for receiving the needle being arranged such that the opening is aligned with the recessed channel of the base.

[0015] In one embodiment of the device for performing a cell-based assay, the needle holder further comprises an opening for receiving a cell culture insert, and the base further comprises a reservoir for receiving a liquid, wherein the reservoir is disposed within the base such that the reservoir is aligned with the opening of the needle holder for receiving a cell culture insert.

[0016] In one embodiment of the device for performing a cell-based assay, the device further comprises a pump in fluid connection with the reservoir, the recess channel, and the cell culture insert.

[0017] In one embodiment of the device for performing a cell-based assay, the needle holder comprises:

[0018] multiple openings for receiving needles,

[0019] at least two openings for receiving at least two cell culture inserts,

[0020] at least one opening for receiving a shaft of the pump; and

[0021] The base comprises:

[0022] At least two liquid storage tanks,

[0023] at least one opening for receiving the pump, in particular a pump impeller,

[0024] Entryway,

[0025] Exit channel,

[0026] The inlet channel and the outlet channel of the pump are connected to the reservoir and the groove channel.

[0027] In one embodiment of the device for performing a cell-based assay, the device comprises a needle holder having a plurality of openings for receiving the needles of the present invention, a base comprising a groove channel and a reservoir, wherein the groove channel is sized to receive the needle tips of the needles secured in the openings of the needle holder, wherein the groove channel is connected to the reservoir.

[0028] In one embodiment of the device for performing a cell-based assay, the needle holder and the base are detachable components of the device.

[0029] In one embodiment of the device for performing a cell-based assay, the needle holder and the base are integrated into a single component.

[0030] In one embodiment of the device for performing a cell-based assay, the base comprises more than one recessed channel.

[0031] In one embodiment of the device for performing a cell-based assay, the groove channel has a stepped shape including an upper portion, a step, and a lower portion, wherein a size of the upper portion is larger than a size of the lower portion.

[0032] In one embodiment of the device for performing a cell-based assay, the groove channels are arranged in a straight line or a circle in the base.

[0033] In one embodiment of the device for performing a cell-based assay, the reservoir contains a liquid.

[0034] In one embodiment of the device for performing a cell-based assay, the needle holder, the needle and the base are made of plastic, preferably polystyrene, polyester and polycarbonate.

[0035] In a fourth aspect, the present invention relates to a cell seeding device for seeding cells on the needle surface of a needle as defined herein, the cell seeding device comprising:

[0036] A first portion and a second portion form a plurality of funnel-shaped cavities when assembled, wherein the funnel-shaped cavity includes an upper cavity portion having an opening and a lower cavity portion having an opening, wherein the lower cavity portion of the funnel-shaped cavity is sized to receive the needle tip of the needle.

[0037] In one embodiment of the cell seeding device, the funnel-shaped cavities are arranged in a matrix.

[0038] In one embodiment of the cell seeding device, the matrix is ​​an 8-funnel, 12-funnel, 16-funnel, 24-funnel, or 96-funnel format.

[0039] In one embodiment of the cell seeding device, the lower cavity portion of the funnel-shaped cavity and the inserted needle tip form a liquid seal.

[0040] In one embodiment of the cell seeding device, the cell seeding device is made of plastic, preferably polypropylene, rubber, polystyrene, polyester and polycarbonate.

[0041] In one embodiment of the cell seeding device, the funnel-shaped cavity is formed as a single piece.

[0042] In one embodiment of the cell inoculation device, the cell inoculation device comprises a detachable reducing sleeve in the upper cavity portion, wherein the sleeve comprises a chamber having openings at both ends, and the end of the sleeve adjacent to the lower cavity portion has an opening smaller than the diameter of the needle tip.

[0043] In one embodiment of the cell seeding device, the end of the sleeve adjacent to the lower cavity portion comprises a plurality of micro-openings.

[0044] In one embodiment of the cell seeding device, the sleeve comprises a divider to form a plurality of chambers inside the sleeve, wherein each chamber comprises at least one micro-opening at an end of the sleeve adjacent to the lower cavity portion.

[0045] In one embodiment of the cell seeding device, the upper cavity portion has the same diameter as the lower cavity portion.

[0046] In one embodiment of the cell seeding device, the reducing sleeve is placed into the upper cavity portion of the cell seeding device to allow cells to adhere to the needle tip in a designed shape or pattern.

[0047] In a fifth aspect, the present invention relates to a kit for performing a cell-based assay comprising:

[0048] a plurality of needles as defined herein,

[0049] at least one device according to the invention for performing a cell-based assay,

[0050] at least one cell seeding device of the present invention, and

[0051] Manual for performing cell-based assays and cell seeding.

[0052] In one embodiment of the kit, the kit further comprises reagents for performing a cell-based assay.

[0053] In a sixth aspect, the present invention relates to a method for performing a cell-based assay comprising:

[0054] The needles are prepared by attaching cells to the needle surface and culturing the cells to form a two-dimensional monolayer of cells or a three-dimensional structured cell on the needle surface,

[0055] by filling the reservoir of the base with test medium and assembling the needle holder and the base to form a device for performing a cell-based assay,

[0056] and inserting the cell needle into the opening of the needle holder to fix the needle so that the needle surface is immersed in the test culture medium, and

[0057] The cells are cultured in the test medium for a prescribed time, wherein the test medium flows in the groove channel, and after completion of the culture, substances in the test medium and / or the cells on each of the cell needles are analyzed.

[0058] In one embodiment of the method for performing a cell-based assay of the present invention, the cells are covered by a thin layer of hydrogel on the outermost surface of the needle surface to prevent cell detachment.

[0059] In one embodiment of the method for performing a cell-based assay of the present invention, the cell needle is prepared from a group of target cells, wherein each of the cell needles comprises one type of target cell cultured in a two-dimensional or three-dimensional format.

[0060] In a seventh aspect, the present invention relates to a method for seeding cells on a needle surface of a needle, comprising:

[0061] inserting a needle as defined herein into the opening of the lower cavity portion of the cell seeding device of the present invention to form a liquid seal;

[0062] adding a cell suspension into each opening of the upper cavity portion of the cell seeding device, and culturing the cells for a prescribed time to allow the cells to adhere to the needle surface;

[0063] The device is disassembled and the needles are transferred to a device for performing cell-based assays.

[0064] In one embodiment of the method for seeding cells on the needle surface of a needle, the needle moves along the lower cavity portion to draw liquid into the lower cavity portion.

[0065] In one embodiment of the method for inoculating cells on the needle surface of a needle, the sleeve is used to inoculate a first type of cells on the needle surface, and after the first type of cells attach to the needle surface, the sleeve is removed and a second type of cells are added to the upper cavity portion, and the second type of cells are cultured to form a second cell layer on the cell layer of the first type of cells.

[0066] In one embodiment of the method for seeding cells on a needle surface of a needle, prior to step a), the needle tip comprises a layer of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 shows the removable needle cell culture system. Figure 1A A front view of the movable needle cell culture system is shown. Figure 1B shows a side view of the movable needle cell culture system, Figure 1C Shown is a top view of the removable needle cell culture system. Figure 1D is a cross-sectional view of the base along line AA showing the reservoir and stepped groove channel.

[0068] Figure 2 A needle placed with the tip facing upward.

[0069] Figure 3 A section of the needle is shown towards the needle tip having an inwardly facing surface.

[0070] Figure 4 is a front view (A), a side view (B), a top view (C) of the multi-unit cell inoculation device, a BB line cross-sectional view of the device showing the liquid reservoir formed by the upper part of the funnel (D), a CC line cross-sectional view of the device showing the needle 1 in the narrow tube of the funnel (E), and an FF line cross-sectional view (F) showing the connecting portion of the upper part of the funnel.

[0071] FIG5 shows a front view (A) of a movable needle cell culture system having a circular groove channel, a top view (B) of the system, and a cross-sectional view (C) along line DD showing the base of the needle 6 and the groove channel.

[0072] Figure 6A-C shows some top views of variations of groove channels that can be used in the assay system of the present invention.

[0073] Figure 7 are front and top views of the hollow needle and removable magnetic insert.

[0074] Figure 8 is an example of a needle and funnel-shaped cell seeding strip.

[0075] Figure 9 is an example of a T-shaped insert.

[0076] Figure 10 An example of a T-shaped insert in a 12-well plate.

[0077] Figure 11 is an example of a T-shaped insert with a needle in the hole.

[0078] FIG. 12 shows a reducing sleeve for the upper cavity portion of the funnel-shaped cavity. Figure 12A Shows the front view, Figure 12B Shown is a top view with a single opening, Figure 12C shows a top view with multiple openings, Figure 12D Shows a top view with a sleeve separator, Figure 12E Shown is a top view of the sleeve in the cell seeding device.

[0079] FIG13 is an example of a removable needle cell culture system 1a with a cell culture insert and an integrated centrifugal pump; Figure 13A A perspective view of the cell culture system is shown; Figure 13B A top view of the assembled system with needles and cell culture insert is shown; Figure 13C A bottom view of the assembled system with needles and cell culture insert is shown. DETAILED DESCRIPTION

[0080] Figure 1 depicts an exemplary movable needle cell culture system 1a. The cell culture system includes a separate movable needle 1, a needle holder 2 and a base 3. The needle 1 has a stepped needle head 4a with a groove and a needle tip 6, and the needle surface 6a of the needle tip 6 is used for two-dimensional or three-dimensional cell culture. The needle holder 2 is placed on the base 3, and the needle holder 2 includes an opening 7a for receiving the needle 1 so as to fix the needle 1 in the movable needle cell culture system 1a. The needle holder 2 also serves as a cover for the base 3. The base 3 contains a stepped groove channel 7 and two trapezoidal liquid reservoirs 8. There is a liquid reservoir on each side of the channel, and these liquid reservoirs are connected in an S-shaped arrangement by a narrow groove 9 at the bottom. When in use, the test culture medium (red dot in the figure) is added to one liquid reservoir 8, and the test culture medium will flow into the other liquid reservoir through the narrow groove 9. The needle holder 2 is placed on the base 3. Then, using forceps with, for example, crab tips, the cell needle is inserted into the container opening on the needle holder 2. The forceps secure the needle 1 in the groove. The stepped needle tip 4a is secured by the groove channel, so that the needle tip is immersed in the culture medium in the narrow slot 9. During the incubation period, a seesaw shaker is used to circulate the culture medium back and forth through the narrow slot 9 from one reservoir to the other. The axis of the up-and-down motion is parallel to the channel. Depending on the assay, the results can be read out by analyzing the test culture medium and / or the cells on the cell needle. For example, this system can be used to test the cell needle in a drug metabolism assay using a two-dimensional monolayer or a three-dimensional non-scaffolded hepatocyte format. After incubation, the test compound and its metabolites in the culture medium are analyzed. For cytotoxicity assays, the cell needle can be prepared using a panel of target cells so that they can be tested in the same environment. This allows for more accurate correlation with in vivo results. Cellular toxicity, such as changes in cell morphology or the formation or accumulation of colored or fluorescent substances within the cells, can be monitored using a microscope or spectrophotometer. In some cases, single cells can be transferred to multi-well plates for further assays.

[0081] Figure 2 An exemplary needle 1 is depicted, which includes a flat needle tip 6 and a stepped cylindrical needle head 5. The needle surface 6a of the flat needle tip 6 is used for cell attachment and cell growth. In some cases, the needle surface 6a of the needle 1 may include a slightly raised edge at the edge so that it can retain more culture medium at the needle tip to avoid drying out during assembly of the assay system. Although according to the present invention, needles 1 of various shapes can be used for two-dimensional and three-dimensional cell culture, large circular surfaces are preferred for preparing monolayer cells, narrow rectangular surfaces are preferred for preparing multicellular three-dimensional cells to better diffuse nutrients to the center of the cell cluster, and permeable membrane surfaces are preferred for preparing multi-layered large cell layers to ensure that nutrients reach the deep layers of the cells. In addition to a flat surface, the needle surface 6a can be an inwardly structured surface, for example, a concave surface ( Figure 3A, cone, pyramid or semi-cylinder ( Figure 3 B)), the multi-grooved, multi-microporous, multi-micropillar surface of the needle tip, as well as the roughened surface of the needle tip, can also be used, particularly for three-dimensional cell culture with hydrogels. In another embodiment, a needle 1 for three-dimensional scaffold cell culture includes a porous section toward the needle tip. While there are no particular limitations on the dimensions of the needle 1, the needle surface 6a is preferably sized within the microscope field of view for easy observation. A preferred needle surface size is a circular area with a diameter of 0.1 to 2.5 mm.

[0082] Cells are directly attached to the surface of the needle surface 6a using a cell seeding device 40, in which the needle 1 is placed upward. Therefore, the cells in the cell suspension will settle and adhere to the needle surface 6a of the needle 1 by gravity, and form a single layer or a multi-cellular three-dimensional cell cluster. According to one embodiment of the present invention, the cell seeding device 40 includes two compartments, a first part 41 and a second part 43, both of which contain a semi-funnel-shaped cavity. When the two compartments are assembled together, a funnel-shaped cavity is formed for receiving the needle 1. Each funnel-shaped cavity 44 includes an upper cavity portion 44a having an opening and a lower cavity portion 44b having an opening, wherein the size of the lower cavity portion 44b of the funnel-shaped cavity 44 is set to receive the needle tip 6 of the needle 1. In one embodiment of the present invention, the diameter of the lower cavity portion 44b preferably corresponds to the diameter of the needle tip 6. When the needle 1 is placed in the lower cavity portion 44b, the two compartments are fastened together to seal the gap to prevent leakage of culture medium. In one embodiment of the present invention, a gasket, such as a rubber ring or sheet, may be placed around the contact surface to prevent culture medium leakage. When the cell seeding device 40 is made of an elastic material, such as silicone, the gasket may be omitted. Preferably, particularly for three-dimensional cell culture, the needle tip 6 does not extend into the cavity 44. Therefore, all cells can adhere to the needle surface 6a of the needle tip 6, reducing variability in the cell population between needles. To prevent air bubbles from forming in the lower cavity portion 44b, a common occurrence with small tubes closed at one end, the needle 1 may be moved up and down several times to remove trapped air. In another embodiment, the cell seeding device may be constructed with a single compartment instead of two. A length of tubing can also be used for cell seeding, as a suitably sized tubing and needle can form a syringe-like device, with the tubing serving as the barrel and the needle acting as the piston. Thus, a cell suspension can be drawn into the barrel. If the needle is sufficiently large, the cell suspension can even be added to the tubing using a pipette, with the needle tip positioned below the inner opening of the tubing. For three-dimensional cell culture, the surface of the funnel-shaped cavity is preferably provided with a cell-repellent layer, which can be formed by coating or grafting appropriate chemicals or polymers.

[0083] FIG4 depicts a multifunnel cell seeding device 40 comprising two differently structured cavity compartments. One compartment has a plurality of semicircular channels 42, and the second compartment has a matching number of semifunnel-structured cavities 44 in which the wide upper portions of the funnels are connected ( Figure 4F When assembled, the semicircular channel 42 and the corresponding narrow half-tube 45 of the funnel form a narrow tube for accommodating the needle 1 ( Figure 4E The wide upper portion of the funnel is connected to form a liquid storage tank 46 ( Figure 4D ). Therefore, the cell suspension can be poured into the storage tank 46. Once the cell suspension level rises to the storage tank, the cell suspension can flow freely in the upper part of the funnel. After the multi-funnel rapid cell inoculation device 40 is placed horizontally, the cells will be evenly distributed into the funnel under the action of gravity and settle on the needle surface 6a of the needle tip 6. Each of the needles 1 will receive the same number of cells for growth. The growth status of the cells can be observed from the thin compartment of the semicircular channel. The use of the multi-funnel rapid cell inoculation device 40 not only simplifies the operation, but also reduces the changes caused by multiple pipetting to transfer the cell suspension to each funnel due to the rapid sedimentation of the cells.

[0084] The principle of the multifunnel cell seeding device can also be used to create multiwell plates with cell-repelling surfaces, such as 96-, 384-, or 1536-well plates for preparing spheroids. These plates have square wells with conical or rounded bottoms. Each square well leads to a reservoir formed by a raised edge around the plate. A groove exists between this edge and the outermost well to minimize uneven cell distribution caused by liquid rising around the bank. The upper portions of the walls between the wells and the grooves connect to form an angled roof structure that prevents cells from falling and guides them evenly to adjacent wells. Therefore, when a cell suspension is added to the reservoir in a volume sufficient to cover the well openings, cells can be distributed seamlessly into the wells. Once the cells have settled to the bottom of each well, the culture medium in the reservoir can be removed and subsequently processed as with a conventional multiwell plate. After incubation in an incubator, the cells will form a single spheroid in each well.

[0085] Materials that are non-toxic to cells can be used to manufacture the movable needle cell culture system 1a and the cell inoculation device 40. Plastics such as polystyrene, polyester, polycarbonate, polypropylene and polyoxymethylene are preferred. Due to the various conditions for cell attachment, the needle surface 6a of the needle tip 6 may need to be treated in different ways, for example, gas plasma treatment, polymer grafting and extracellular matrix coating. Polytetrafluoroethylene can be used to manufacture the cell inoculation device 40 and the needle 1 to avoid leakage of culture medium and the generation of bubbles. The narrow hydrophobic gap between the needle 1 and the small diameter tube can act as a selective barrier because it allows air to pass freely but prevents leakage of culture medium. The hydrophobic gap can also be made by coating a hydrophobic material on the contact surface of the needle and the cell inoculation device.

[0086] Using the cell seeding device 40, single cell suspensions, biopsy microtissues, and organoids / spheroids produced by three-dimensional cell culture can be directly seeded onto the needle surface 6a. Once the cells are tightly attached to the needle surface 6a of the needle tip 6, the needle 1 can be transferred to a multi-porous cell culture plate for growth. For cells from, for example, lung cancer cell lines, since the cell needle is placed downward in the multi-porous plate, some surface cells will break away from the cell mass during cell culture. In order to prevent the surface cells from falling off, the attached cells can be covered with a thin layer of hydrogel. Suitable hydrogels are agarose, alginate, and collagen. The thin hydrogel coating can be formed by first immersing the needle tip 6 in a hydrogel solution and then immersing the needle tip 6 in a suitable solution to solidify the hydrogel. For example, agarose can be solidified by a cold medium. Alginate solutions can be solidified with BaCl2 or CaCl2 solutions, and collagen can be solidified with warm alkaline solutions.

[0087] In addition to the thin hydrogel coating, the needle 1 comprising a magnetic segment at the needle tip 6 can also be used to confine cells to the needle surface 6a at the needle tip when cells are treated with paramagnetic nanoparticles to become magnetized cells or cell clusters during cell culture on the needle. Figure 7is an example of a hollow needle 71 with a removable magnetic insert 72, which includes a magnetic block 73 at the needle tip. The head of the needle has a groove 74, which is suitable for accommodating the head 75 of the insert. When the insert is placed in the hollow needle, a magnetic needle is formed, which can attract ferromagnetic material and paramagnetic material at the needle tip 76. The magnetic needle can also be made by embedding a magnetic block in the needle tip, where the magnetic block can be a hard magnetic material or a soft magnetic material. Soft magnetic material is preferred because it can have both magnetized and demagnetized states. When cells are directly magnetized at the needle tip, it is best to introduce a demagnetization washing step in order to remove unbound paramagnetic nanoparticles. In addition, by using magnetized cells, the magnetic needle can directly fix cells from a cell suspension to the needle tip. Therefore, the applications of the movable needle cell culture system can be expanded to non-adherent cell assays, polar cell assays with defined apical-basal polarity using paramagnetic nanoparticles that specifically bind to the apical or basolateral membrane, and assays that allow the direct extraction of target cells from a cell mixture containing several cell types using paramagnetic nanoparticles specific for the target cells. Furthermore, applications can be further expanded to magnetic bead immunoassays, particularly multiplexed assays, in which different targeted beads can be reacted with the sample solution one by one due to the easy and complete transfer of beads by the magnetic needle.

[0088] Cell seeding device 40 can also be used to prepare multi-spheroids using hydrogels that require long incubation periods to solidify. For example, to prepare cells in a low-concentration collagen solution, a small amount of cells suspended in the collagen solution is added to the needle surface 6a of the needle tip 6 in cell seeding device 40, and the cell solution is then covered with a low-density alkaline solution with an appropriate pH. After incubation at 37°C for a certain period of time, the collagen solution slowly forms a hydrogel, which, along with the embedded cells, adheres to the needle surface 6a of the needle tip 6. Each cell in the hydrogel grows to form a spheroid.

[0089] According to one embodiment of the present invention, the movable needle cell culture system 1a uses a stepped groove channel 7 to minimize the volume change of the culture medium in the channel during the up and down movement of the rocker shaker ( Figure 1D In the stepped groove channel, the step 10 is intended to prevent the culture medium from climbing into the upper wide groove 11. However, the capillary force of the narrow groove 9 holds the culture medium within the narrow groove, ensuring that the cells on the needle surface 6a of the needle tip 6 are always in the culture medium. Preferably, the narrow groove has a gap size such that the capillary force of the narrow groove can raise the culture medium to a height higher than the step. Therefore, by introducing the step below the maximum lifting height to prevent the culture medium from climbing further upward, the stepped groove channel can maintain the same volume.

[0090] In another embodiment, the base of the movable needle cell culture system (Figure 5) includes a base having a circular groove channel having a container opening for the cell needle. The container opening is located at the upper part of the channel. When in use, the needles 1 are inserted one by one along the annular groove channel, and the T-shaped needle head 4a is fixed in the container opening so that the cells attached to the needle surface 6a of the needle tip 6 are immersed in the culture medium. Vertical and horizontal orbital motion oscillators can be used to drive the culture medium to flow clockwise or counterclockwise in the groove channel. For assays requiring a high cell number to culture volume ratio, the movable needle cell culture system with a circular channel is preferred. The circular channel can also be made by another method, according to which the channel is formed by placing a T-shaped plug in a matching hole of a multi-well plate (see Figure 9 and Figure 10 ). The top protruding structure of the T-shaped plug-in 102 contains a hole for the cell needle, the size of which is preferably the same as the size of the hole, so that the T-shaped plug-in can be fixed in the middle of the hole and form a circular groove channel. In some cases, when the cells are sensitive to the flow of culture medium, a stepped groove channel may be preferred. In this case, the steps are covered by the culture medium, so that the upper wide groove also serves as a flow channel to reduce the flow rate of the culture medium in the narrow groove, so that the cells on the needle surface 6a of the needle tip 6 are less affected. Several variations of the groove channel are shown in Figure 6. For example, when the determination requires different amounts of cells from several cell types, the gap size of the groove channel can be along the groove channel ( Figure 6A ) are different to suit small size needles 1 and large size needles 1. When using large size needles 1, the design of the holes along the narrow channel ( Figure 6B ) can minimize the total volume. When the bottom of the hole is lower than the bottom of the narrow channel, the hole can also be used for cell culture. Therefore, in combination with the needle 1, the ratio of cells to culture medium can be further increased. The serpentine grooves ( Figure 6C ) can accommodate more cell needles without increasing the length of the base.

[0091] The removable needle assay system can be applied not only to grooved channels but also to tube channels in which the container opening is arranged along a tube. A screw and nut structure is used to secure the needle to the container opening, creating a liquid-tight seal. Circulation of the culture medium in the tube can be driven by a tubular pump. Due to the enclosed space of the tube, the needle 1 does not need to be installed with the needle tip facing downward. Since all directions around the tube can be used to install the cell needle, the removable needle assay system can significantly increase the amount of cells, and it can also adapt to assays requiring a high cell number to volume ratio.

[0092] Figure 8A and Figure 8B A needle 1 and corresponding cell seeding device 40 are depicted.

[0093] Figure 9 An example needle holder 101 according to the present invention is depicted having a plurality of openings 103 for receiving a needle 1. The needle holder 101 comprises a T-shaped insert formed by an annular portion connected to a cylindrical portion 104. The annular portion has a larger diameter than the cylindrical portion 104 and includes a plurality of openings 103 for receiving the needle 1. The needle holder 101 is sized to accommodate the wells of a multi-well plate.

[0094] Figure 10 A multiwell plate 110 is depicted with an inserted needle holder 101. This assembly is a functional system of the present invention for performing a cell-based assay. An exemplary cell-based assay of the present invention using this assembly includes the following steps: filling the wells of the multiwell plate with cell culture medium and adding a different test compound to each well. The needle holder 101 is then placed into the wells of the multiwell plate 110, and a needle 1 with cells seeded on the needle surface 6a of the needle tip is inserted into the opening 103. The cells seeded on the needle surface 6a of the needle tip come into contact with the cell culture medium containing the test compound. The needle surface 6a of the needle tip can be seeded with the same or different types of cells. The multiwell plate 110 is placed in an incubator for a specified period of time, and after the incubation is complete, the cell culture medium and / or the cells seeded on the needle surface 6a of the needle tip are analyzed. Figure 11 Shows Figure 10 Bottom view of the porous assembly shown in .

[0095] Figure 13 shows another embodiment of a device for performing cell-based assays, which incorporates a centrifugal pump 1311 that can be driven by a magnetic mixer. The device for performing cell-based assays comprises a base 1301, a magnetically driven pump, and a needle holder 1307. The base 1301 has a groove channel 1302, two reservoirs 1303 for receiving two cell culture plug-ins 1312, and an opening 1304 for receiving a pump, particularly a centrifugal pump impeller, which are connected by a pump inlet channel 1305 and a pump outlet channel 1306, thereby allowing liquid (e.g., cell culture medium) to circulate through these cavities when assembled. The needle holder 1307 comprises an opening for the cell culture plug-in 1312, an opening 1314 for receiving needle 1, a sampling opening 1308, and a pump shaft bearing opening 1309. Screws 1310 are used to secure the base 1301 to the needle holder 1307.

[0096] Before use, assemble the cell-based assay system including the needle holder 1307 and the base 1301. The assembly of the cell-based assay system includes the following steps:

[0097] 1) Place the base 1301 into the culture dish,

[0098] 2) Place the pump impeller in the pump chamber,

[0099] 3) Place the needle holder 1307 on the base, and

[0100] 4) Tighten the screw 1310 to fix the base 1301 on the needle bracket 1307.

[0101] The assembled cell-based assay system is then filled with cell culture medium, the cell culture insert 1312 and the cell needle are positioned in their respective positions, and the assembled system is covered with a culture dish lid. The system is then placed on the magnetic mixer in the cell culture incubator. By setting the appropriate mixer speed, the impeller rotates at the corresponding speed to drive the culture medium through the cavity at the specified flow rate. The sampling opening 1308 can be used to add compounds and to sample the test medium for analysis. Because the direction of rotation of mixers can vary between different suppliers, a bidirectional impeller, such as a bladeless impeller, is used in the system.

[0102] Cell culture inserts are permeable scaffolds that serve as tools for studying anchorage-dependent and independent cell lines. Cell culture inserts include a membrane on which cells can grow. Preferred cell culture inserts are Cell culture inserts. Cell culture inserts are convenient, sterile, easy-to-use permeable supports for studying both anchorage-dependent and anchorage-independent cell lines. Available commercially from cell culture equipment manufacturers such as Fisher Scientific or Sigma Aldrich.

[0103] In another embodiment, the removable needle cell culture system 1a can also include a container opening 1312a for a cell culture plug-in. The container opening for the cell culture plug-in 1312 is preferably located above the reservoir 1303. The cell culture plug-in 1312 can be used to prepare a cell layer and used as a barrier for chemical compounds. Therefore, more complex determinations can be performed to simultaneously study the adsorption, metabolism, and secretion of chemical compounds. For example, the assembled system comprises an intestinal cell layer plug-in in the reservoir 1303, a renal cell layer in another cell layer plug-in, and the hepatocytes on the pin surface 6a of the pin 1 inoculated in the channel 1302. When a test compound is added to the inner chamber of the intestinal cell layer plug-in, the intestinal cell will absorb the compound and be transported to the channel 1302. The hepatocytes inoculated on the pin surface 6a of the pin 1 metabolize the compound, and then the renal cell excretes the compound into the inner chamber of the intestinal cell layer plug-in. By analyzing the metabolites in the culture medium of the compound and from different compartments, useful data can be obtained.

[0104] Example: Application of cell-based systems in drug metabolism research

[0105] Step 1: The needles were coated with 0.02 mg / mL collagen for three hours and then dried overnight at room temperature.

[0106] Step 2: Use the inoculation device of the present invention to inoculate hepatocytes at a concentration of 0.8-1 million / mL, remove the needle 1 after overnight culture and place the needle in the needle holder 101, as shown in FIG. Figure 9 shown.

[0107] Step 3: Place the needle holder 101 with the cell-coated needles 1 upside down in a 12-well cell culture plate containing 0.8-0.9 mL of cell culture medium and test compound (see Figure 10 ).

[0108] Step 3: Place the 12-well cell culture plate on a rocker shaker and shake at 10 rpm in a cell culture incubator.

[0109] Step 4: Samples were taken at designated time points by transferring 20 μL of culture medium into the wells containing acetonitrile.

[0110] Step 5: Analyze the drug metabolites in the sample using LC-MS / MS.

Claims

1. An apparatus for performing a cell-based assay, the apparatus comprising: a needle holder and a base, the needle holder including a plurality of openings for receiving needles, the base including a grooved channel; The needle comprises a needle body and a needle tip, the needle body comprises a needle head, wherein the needle tip comprises a surface for cell inoculation, wherein the needle holder and the base have matching shapes, and the opening for receiving the needle is arranged so that the opening is aligned with the groove channel of the base, wherein the needle holder further comprises an opening for receiving a cell culture insert, and the base further comprises a reservoir for receiving a liquid, wherein the reservoir is arranged in the base so that the reservoir is aligned with the opening of the needle holder for receiving the cell culture insert, wherein the device further comprises a pump, which is fluidically connected to the reservoir, the groove channel and the cell culture insert.

2. The device for performing a cell-based assay according to claim 1, wherein the needle holder comprises: multiple openings for receiving needles, at least two openings for receiving at least two cell culture inserts, and at least one opening for receiving an impeller shaft of the pump; And the base includes: at least two reservoirs for receiving cell culture medium, at least one opening for receiving said pump, Entryway, Exit channel, The inlet channel and the outlet channel of the pump are connected to the reservoir and the groove channel.

3. A kit for performing a cell-based assay, the kit comprising: a plurality of needles, wherein the needles comprise a needle body and a needle tip, the needle body comprising a needle head, wherein the needle tip comprises a surface for cell seeding, at least one device for performing a cell-based assay as defined in any one of claims 1 to 2, at least one cell seeding device for seeding cells on a surface of a needle, wherein the cell seeding device comprises: a first portion and a second portion that, when assembled, form a plurality of funnel-shaped cavities, wherein the funnel-shaped cavities comprise an upper cavity portion having an opening and a lower cavity portion having an opening, wherein the lower cavity portion of the funnel-shaped cavity is sized to receive the needle tip of the needle, and Manual for performing the described cell-based assays and cell seeding.

4. The kit for performing a cell-based assay according to claim 3, wherein the funnel-shaped cavities are arranged in a matrix.

5. The kit for performing a cell-based assay according to claim 4, wherein the matrix is ​​an 8-funnel, 12-funnel, 16-funnel, 24-funnel, or 96-funnel format. 6 . The kit for performing a cell-based assay according to claim 3 , wherein the lower cavity portion of the funnel-shaped cavity and the inserted needle tip form a liquid seal.

7. A kit for performing a cell-based assay according to claim 3 or 4, wherein the cell seeding device is made of plastic.

8. The kit for performing a cell-based assay according to claim 3, wherein the funnel-shaped cavity is formed as a single piece.

9. A kit for performing a cell-based assay according to claim 3 or 4, wherein the cell seeding device comprises a detachable reducing sleeve located in the upper cavity portion, wherein the reducing sleeve comprises a chamber having openings at both ends, and the end of the reducing sleeve proximal to the lower cavity portion has an opening having a size smaller than the diameter of the needle tip.

10. The kit for performing a cell-based assay according to claim 9, wherein the end of the reducing sleeve proximal to the lower cavity portion comprises a plurality of micro-openings.

11. The kit for performing a cell-based assay according to claim 10, wherein the reducing sleeve comprises dividers to form a plurality of chambers inside the reducing sleeve, wherein each chamber comprises at least one micro-opening at the end of the reducing sleeve proximal to the lower cavity portion.

12. The kit for performing a cell-based assay according to claim 10, wherein the upper cavity portion has the same diameter as the lower cavity portion.

13. The kit for performing a cell-based assay according to claim 10, wherein the reducing sleeve is placed into the upper cavity portion of the cell seeding device to allow cells to attach to the needle tip in a designed shape or pattern.

14. A kit for performing a cell-based assay according to claim 3 or 4, further comprising reagents for performing the cell-based assay.

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