Cell based assays have been used broadly in the fields of cell biology, medical science, pharmacology, toxicology, and so on. They are usually performed in a multi-well cell culture plate. The cells can be in the form of suspension, 2D monolayer, or 3D organoids/spheroids according to the assay. In order to meet some special needs, many new systems have been introduced. These include microfluidic cell culture device, 3D membrane based perfusion bioreactor, 3D-cell culture pillar system, and so on. Though bringing some advantages, these systems show more or less drawbacks in terms of complexity, robustness, flexibility, easy handling and cost effectiveness. For example the flow channel of the microfluidic system is prone to be blocked by air bubbles, hydrogel and cell clusters. The 3D-cell culture pillar system is only suitable to prepare multi-spheroids from proliferating cells because it has to use hydrogel to immobilize cells to the pillar. Non-proliferating cells like human hepatocytes cannot form spheroids in the hydrogel. The use of hydrogel also limits the applications of this pillar system as the hydrogel can have impacts on, especially, large molecule diffusion and the assay results. Besides, to immobilize the cells to the tiny pillar can be challenge since the hydrogel need to be solidified in a very short time to avoid evaporation of the small volume.
In view of the shortcomings of these systems, the present invention introduces a movable pin cell culture system for cell based assays. The system is not only suitable for 2D monolayer and 3D cell culture, but also is capable of simultaneously testing different type of the cells which are cultured in 2D monolayer, 3D scaffold and 3D non-scaffold. This system is more flexible, easier to handle and robust for carry out the cell based assays.
In a first aspect, the present invention relates to a pin for use in a cell based assay device comprising: a pin body comprising a pin head and a pin tip, wherein the pin tip comprises a surface for cell seeding
In an embodiment of the pin of the present invention, the pin body comprises at least a tapered segment and the pin body forms a T-shape together with the pin head.
In an embodiment of the pin of the present invention, the pin body and pin tip have a cylindrical shape.
In an embodiment of the pin of the present invention, the pin body and pin tip have a non-round cross-cut shape.
In an embodiment of the pin of the present invention, the pin head is a stepwise pin head comprising a groove.
In an embodiment of the pin of the present invention, the surface is selected from the group consisting of a flat surface, a flat surface with a raised rim at the edge, an inward structured surface, a multi-groove surface, a multi micro-well surface, a multi micro-pillar surface, a rough surface, a permeable membrane, a porous segment or a magnetic segment; preferably the layer comprises a layer promoting cell adhesion, more preferably a cell adhesion promoting layer selected from hydrogel.
In a second aspect, the present invention relates to a pin support comprising a plurality of openings for receiving a pin according to the present invention, wherein the pin support comprises a ring-shaped part connected to a cylindrical part to form a T-shaped pin support, wherein the diameter of the ring-shaped part is bigger than the diameter of the cylindrical part, wherein the ring-shaped part comprises the plurality of openings for receiving the pins.
In an embodiment the pin support is made of plastic, preferably polytetrafluoroethylene, polystyrene, polyester and polycarbonate.
In an embodiment the pin support is sized so as to fit in the well of a multi-well plate.
In a third aspect, the present invention relates to a device to perform a cell based assay comprising:
a pin support comprising a plurality of openings for receiving a pin according to the present invention and a base comprising a groove channel
In an embodiment of the device to perform a cell based assay, the pin support and the base have a matched shape and the openings for receiving a pin are arranged such that they are aligned with the groove channel of the base.
In an embodiment of the device to perform a cell based assay, the pin support 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 within the base such that it is aligned with the opening of the pin support for receiving a cell culture insert.
In an embodiment of the device to perform a cell based assay, the device further comprises a pump which is in fluidic connection to the reservoir, to the groove channel and to the cell culture insert.
In an embodiment of the device to perform a cell based assay, the pin support comprises:
a plurality of openings for receiving a pin,
at least two openings to receive at least two cell culture inserts,
at least one opening to receive the shaft of the pump, and
the base comprises:
at least two reservoirs,
at least one opening to receive the pump, in particular a pump impeller,
an inlet channel,
an outlet channel,
wherein the inlet channel and the outlet channel of the pump are connected to the reservoir and to the groove channel
In an embodiment of the device to perform a cell based assay, the device comprises a pin support comprising a plurality of openings for receiving a pin according to the present invention, a base comprising a groove channel and a reservoir, wherein the groove channel is sized so as to receive the pin tips of the pins being secured in the openings of the pin support, wherein the groove channel is connected to the reservoir.
In an embodiment of the device to perform a cell based assay, the pin support and the base are separable parts of the device.
In an embodiment of the device to perform a cell based assay, the pin support and the base are integrated as a single part.
In an embodiment of the device to perform a cell based assay, the base comprises more than one groove channel
In an embodiment of the device to perform a cell based assay, the groove channel has a stepwise shape comprising an upper part, a step and a lower part, wherein the upper part has a bigger dimension than the lower part.
In an embodiment of the device to perform a cell based assay, the groove channel is in a linear or circular arrangement in the base.
In an embodiment of the device to perform a cell based assay, the reservoir contains the liquid.
In an embodiment of the device to perform a cell based assay, the pin support, the pins and the base are made of plastic, preferably polystyrene, polyester and polycarbonate.
In a fourth aspect, the present invention relates to a cell-seeding device for seeding cells on a pin surface of a pin as defined herein, such cell seeding device comprising:
a first part and a second part forming a plurality of funnel shaped cavities when assembled, wherein the funnel shaped cavity comprises an upper cavity part with an opening and a lower cavity part with an opening, wherein the lower cavity part of the funnel shaped cavity is sized so as to receive the tip of the pin.
In an embodiment of the cell seeding device, the funnel shaped cavities are arrayed in a matrix.
In an embodiment of the cell seeding device, the matrix is an 8-, 12-, 16, 24-96-funnel format.
In an embodiment of the cell seeding device, the lower part of the funnel shaped cavity and the inserted tip of the pin form a liquid tight seal.
In an embodiment of the cell seeding device, the cell seeding device is made of plastic, preferably polypropylene, rubber, polystyrene, polyester and polycarbonate.
In an embodiment of the cell seeding device, the funnel shaped cavity is formed as a single piece.
In an embodiment of the cell seeding device, the cell seeding device comprises a removable reduction sleeve in the upper cavity part, wherein the sleeve comprises a chamber having openings at both ends and the end of the sleeve next to the lower cavity part has an opening with a size smaller than the diameter of the pin tip.
In an embodiment of the cell seeding device, the end of the sleeve next to the lower cavity part contains multi micro-openings.
In an embodiment of the cell seeding device, the sleeve contains a separator to form multi-chambers inside the sleeve, wherein each chamber contains at least one micro-opening at the end of the sleeve next to the lower cavity part.
In an embodiment of the cell seeding device, the upper part cavity has the same diameter as the lower cavity part.
In an embodiment of the cell seeding device, the reduction sleeve is placed into the upper part cavity of the cell seeding device to allow cells to attach to the pin tip in a designed shape or pattern.
In a fifth aspect, the present invention relates to a kit for performing a cell based assay comprising:
a plurality of pins as defined herein,
at least one device to perform a cell assay according to the present invention,
at least on cell seeding device according to the present invention and
a manual to perform the cell based assay and the cell seeding.
In an embodiment of the kit, the kit further comprises the reagents to perform the cell based assay.
In a sixth aspect, the present invention relates to method for performing a cell based assay comprising:
preparing pins by attaching cells to the pin surface and culturing the cells to form 2D monolayer cells or 3D structured cell on the pin surface 6a,
filling the reservoir of the base with test medium, assemble the pin support and the base to form the device to perform a cell based assay,
inserting the cell pins into the openings of the pin support to secure the pins so as to immerge the pin surface in the test medium, and
incubating the cells in the test medium for a defined time, wherein the test medium flows in the groove channel, and after completion of the incubation analyzing substances in the test medium and/or the cells on each of the cell pins.
In an embodiment of method for performing a cell based assay of the invention, the cells are covered at the outmost surface of the pin surface by a thin layer of hydrogel to prevent cells from detaching.
In an embodiment of the method for performing a cell based assay of the invention, the cell pins are prepared from a set of target cells, wherein each of the cell pins contains one type of the target cells cultured in 2D or 3D form.
In a seventh aspect, the present invention relates to a method for seeding cells on the pin surface of a pin comprising:
inserting pins as defined herein in the opening of the lower cavity part of a cell seeding device according to the present invention to form a liquid tight seal,
adding cell suspension to each opening of the upper part cavity of the cell seeding device and culturing the cells for a defined time to allow the cells to adhere to the pin surface, and
disassembling the device and transferring the pins to a device to perform a cell based assay as defined herein.
In an embodiment of the method for seeding cells on the pin surface of a pin, the pins are moved along the lower cavity part to draw the liquid into the lower cavity part.
In an embodiment of the method for seeding cells on the pin surface of the pin, the sleeve is used for seeding a first type of cells on the surface of the pin and the sleeve is removed after the first type of cells are attached to the pin surface, and a second type of cells is added to the upper part cavity and the second type of cells are cultured to from a second cell layer on top of the cell layer of the first type of cells.
In an embodiment of the method for seeding cells on the pin surface of a pin, the pin tip contained a layer of cells prior to step a).
The attachment of the cells direct to the surface of the pin surface 6a is performed by using a cell seeding device 40 in which the pins 1 are placed upward. So the cells in the cell suspension will precipitate and attach to the surface 6a of the pin 1 by the gravity and form a monolayer or multicellular 3D cell cluster. According to an embodiment of the present invention, the cell seeding device 40 comprises two compartments, a first part 41 and a second part 43 which both contain half-funnel like cavities. When put together the two compartments 41 and 43 form funnel shaped cavities for receiving pins 1. Each funnel shaped cavity 44 comprises an upper cavity part 44a with an opening and a lower cavity part 44b with an opening, wherein the lower cavity part 44b of the funnel shaped cavity 44 is sized so as to receive the tip 6 of the pin 1. In an embodiment of the invention, the diameter of the lower cavity part 44b preferably corresponds to the diameter of the pin tip 6. When the pins 1 are placed in the lower cavity part 44b, the two compartments 41 and 43 are tightened together to seal the gaps which prevents leakage of culture medium. In an embodiment of the invention, a gasket e.g. rubber ring or rubber sheet, may be placed around the contact surface to prevent leakage of medium. When the cell-seeding device 40 is made of elastic materials e.g. silicone a gasket can be omitted. It is preferred that the pin tip 6 does not protrude into the cavity 44 especially for the 3D cell culture. Thus, all the cells can attach to the surface 6a of the pin tip 6 to reduce the cell number variability among the pins. In order to avoid the formation of air bubbles in the lower cavity part 44b, which happens quite often for a small sized tube with one end closed, the pin 1 can be moved up and down several times to remove the trapped air. In another embodiment, the cell seeding device can also be made as a single compartment instead of two. A segment of tube is also possible to be used for seeding the cell since a suitable sized tube and pin can form a syringe like device in which the tube functions as a barrel and the pin as a piston. Therefore, the cell suspension can be drawn into the barrel. When the size of the pin is large enough, a pipette can even be used to add the cell suspension to the tube where the pin point is set below the inner opening of the tube. For 3D cell culture, the surface of the funnel like cavity is preferable to have a cell repellent layer, which can be formed by coating or grafting appropriate chemicals or polymers.
The principle of multi-funnel cell seeding device can also be applied to make a cell-repellent surface multi-well plate such as e.g. a 96- or 384- or 1536 well plate for the preparation of spheroids, in which the wells of the multi-well plate are square wells with conical or round bottom. All the square wells open to a reservoir that is formed by a raised bank around the plate. There is a groove between the bank and the outmost wells to minimize uneven distribution of the cells caused by liquid rising around the bank. The upper portions of the wall between the wells and the groove are connected to each other forming an angular rooftop structure to prevent cell landing and to equally guide cells to the adjacent wells. Thus the cells can seamlessly be distributed to the wells when the cell suspension is added to the reservoir with a volume enough to cover the openings of the wells. Once the cells precipitate to the bottom of each well, the culture medium in the reservoir can be removed and subsequently it can be handled just like a normal multi-well plate. The cells will form a single spheroid in each well after cultured in an incubator.
Materials non-toxic to cells can be used to make the movable pin cell culture system 1a and the cell seeding device 40. Plastics such as polystyrene, polyester, polycarbonate, polypropylene and polyoxymethylene are preferred. Due to the various conditions of cell attachment, the surface 6a of the pin point 6 may need to be treated by different means such as e.g. gas plasma treatment, polymer grafting, and extracellular matrix coating. Polytetrafluoroethylene may be used to make the cell seeding device 40 as well as the pins 1 to avoid the leakage of the medium and the generation of air bubbles as well. A narrow hydrophobic gap between the pin 1 and the narrow tube can function as a selective barrier because it allows air to pass through freely but prevents the medium from leakage. The hydrophobic gap can also be made by coating of a hydrophobic material on the contact surface of the pin and the cell seeding device.
With the cell seeding device 40, single cell suspension, biopsy micro-tissues and 3D cell culture produced organoids/spheroids can be seeded directly to the pin surface 6a. Once the cells tightly attach to the surface 6a of the pin tip 6, the pins 1 can be transferred to a multi-well cell culture plate for growth. For cells from e.g. lung cancer cell lines, some of the surface cells will break off the cell cluster during the cell culture since the cell pin 1 is placed downward in the multi-well plate. In order to prevent shedding off of surface cells, the attached cells can be covered by a thin layer of hydrogel. Suitable hydrogels are agarose, alginate and collagen. The thin hydrogel coating layer can be formed by first dipping the pin tip 6 into the hydrogel solution and then dipping the pin tip 6 into an appropriate solution to solidify the hydrogel. For example, agarose can be solidified by a cold medium. An alginate solution can be solidified with a BaCl2 or CaCl2 solution, and collagen can be solidified by a warm alkaline solution.
Besides a thin hydrogel coating layer, a pin 1 containing a magnetic segment at the pin tip 6 can also be used to confine the cells to the pin tip surface 6a when the cells are treated with paramagnetic nano-particles to become magnetized cells or cell clusters during the cell culture on the pin.
The cell seeding device 40 can also be used for the preparation of multi-spheroids with hydrogel which requires long time incubation for solidification. For example, to prepare cells in a low concentration of collagen solution, a tiny amount of cells suspended in the collagen solution is added to the surface 6a of the pin tip 6 in the cell seeding device 40 and the cell solution is covered by a lower density alkaline solution with an appropriate pH. After incubation at 37° C. for certain time, the collagen solution will slowly form a hydrogel that sticks on the surface 6a of the pin tip 6 with the embedded cells. Each cell in the hydrogel will grow to form a spheroid.
According to an embodiment of the present invention, the movable pin cell culture system 1a uses a stepwise groove channel 7 to minimize the volume change of the culture medium in the channel during the seesaw movement by a rocker shaker (
In another embodiment, the base of the movable pin cell culture system (
The moveable pin assay system 1a can be applied not only to the groove channel 7, but also to a tube channel where the receptacle openings are arranged along the tube. A screw and nut structure is used to fasten the pins to the receptacle openings for liquid tight seal. The circulation of the medium in the tube can be driven by a tubing pump. Due to the enclosed space of the tube, it is not necessary for the pin 1 to be installed with the pin point downward. Since all directions around the tube can be used to install the cell pins 1, the cell amount can dramatically be increased in the moveable pin assay system 1a and it can fit assays requiring high cell number to volume ratio.
Before use, the cell based assay system 1a comprising the pin support 1307 and the base 1301 are assembled. The assembly of the cell based assay system 1a comprises the following steps:
1) putting the base 1301 into a petri dish,
2) placing the pump impeller 1311 in the pump house 1304,
3) putting the pin support 1307 on the base and
4) fastening the screws 1310 to fasten the base 1301 to the pin support 1307.
Then, the cell culture medium is filled in the assembled cell based assay system, and the cell culture inserts 1312 and the cell pins 1 are placed to their correspondent positions and the assembled system is covered with a petri dish lid. Afterwards place the system on a magnetic mixer in a cell culture incubator. Set a proper rotation speed of the mixer and the impeller 1311 will turn at a speed accordingly so as to drive the culture medium flowing through the cavities with a defined flow rate. The sampling opening 1308 can be used for addition of compounds as well as sampling the test medium for analysis. Since the rotation direction of the mixers may be different from different suppliers a bi-direction impeller, such as bladeless impeller, is used in the system.
Cell culture inserts are permeable supports which serve as tools for the study of anchorage dependent and independent cell lines. Cell culture inserts comprise a membrane on which cells can be grown. A preferred cell culture insert is a Transwell® cell culture insert. Transwell® cell culture inserts are convenient, sterile, easy-to-use permeable support devices for the study of both anchorage-dependent and anchorage-independent cell lines. Transwells® are commercially available from cell culture device manufacturers such as e.g. Fisher Scientific or Sigma Aldrich.
In a further embodiment, the movable pin cell culture system 1a may also comprise receptacle openings for a cell culture insert 1312a. The receptacle openings for the cell culture insert 1312 are preferably located above the reservoir 1303. The cell culture inserts 1312 can be used to prepare cell sheets and function as a barrier for chemical compounds. Therefore, a more complex assay can be performed to simultaneously studying adsorption, metabolism and secretion of a chemical compound. For example, the assembled system 1a contains an intestine cell sheet insert 1312 in one reservoir 1303, a kidney cell sheet in another cell sheet insert 1312 and hepatocyte cells seeded on the surface 6a of the pins 1 in the channel 1302. When a test compound is added to the inner chamber of the intestine cell sheet insert 1312, the intestine cell will absorb the compound and transport to the channel 1302. The hepatocytes seeded on the surface 6a of the pins 1 metabolize the compound and then the kidney cells excrete the compounds into the inner chamber of the kidney cell sheet insert 1312. A useful data can be obtained through the analysis of the compound and metabolites in the medium from the different compartments.
Step 1: Coat the pins 1 with collagen at 0.02 mg/mL for 3 hours and dry the pins at room temperature overnight.
Step 2: Seed the hepatocytes at 0.8-1 million/mL concentration using the seeding device of the present invention, remove the pins after overnight incubation and place the pins in the pin support 101 as shown in
Step 3: Place the pin support 101 with the cell coated pins 1 in an upside down position in a 12 well cell culture plate which contains 0.8-0.9 mL of cell culture medium and a test compound (see
Step 3: Place the 12 well cell culture plate on a rock shaker set to shake at a speed of 10 rpm in cell culture incubator.
Step 4: Sampling at defined time points by transferring 20 μL of the culture medium into a well of a plate which contains acetonitrile.
Step 5: Analyze the samples by using LC-MS/MS for drug metabolites.
Number | Date | Country | Kind |
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20164950.6 | Mar 2020 | EP | regional |
Number | Date | Country | |
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Parent | PCT/EP2021/057179 | Mar 2021 | US |
Child | 17933447 | US |