PRODUCTION OF CELLULAR SPHEROIDS

Abstract

The present invention relates to a microfluidic device (1; 8) for the production of cellular spheroids comprising at least one chamber (2; 9) comprising a fluid inlet (3) for introducing fluid into the chamber (2; 9) and a fluid outlet (4) for removing said fluid from the chamber (2, 9), wherein said at least one chamber (2; 9) comprises a base formed by a substrate (5; 10) comprising at least two recesses (6; 11) to collect a fluid comprising biological cells when the substrate (5, 10) is contacted with the fluid, wherein the size of the at least two recesses (6; 11) decreases from the fluid inlet (3) to the fluid outlet (4) of the at least one chamber (2; 9).

Claims

1. A microfluidic device (1; 8) for the production of cellular spheroids comprising at least one chamber (2; 9) comprising a fluid inlet (3) for introducing fluid into the chamber (2; 9) and a fluid outlet (4) for removing said fluid from the chamber (2, 9), wherein said at least one chamber (2; 9) comprises a base formed by a substrate (5; 10) comprising at least two recesses (6; 11) to collect a fluid comprising biological cells when the substrate (5, 10) is contacted with the fluid, wherein the size of the at least two recesses (6; 11) decreases from the fluid inlet (3) to the fluid outlet (4) of the at least one chamber (2; 9).

2. The microfluidic device (1; 8) according to claim 1, wherein the fluid inlet (3) and the fluid outlet (4) are positioned to direct fluid flowing from the fluid inlet (3) to the fluid outlet (4) through the chamber (2; 9).

3. The microfluidic device (1; 8) according to claim 1, wherein the substrate (5; 10) comprises, consists of or is coated with a biocompatible material.

4. The microfluidic device (1; 8) according to claim 3, wherein the biocompatible material is a silicone or a plastic material or glass.

5. The microfluidic device (1; 8) according to claim 3, wherein the biocompatible material is selected from the group consisting of a polystyrene, cycloolefin-copolymer, polymethylmethacrylate, cyclo-olefin polymer, polydimethylsiloxane, polycarbonate (PC), polypropylene (PP), polyvinylchloride (PVC), perflouropolyether (PFPE), polyurethane, poly(ethyleneterephthalate) (PET), polyester and thiol-enes.

6. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) and/or the substrate (5; 10) is/are coated at least partially with an anti-fouling layer.

7. The microfluidic device (1; 8) according to claim 6, wherein the anti-fouling layer is selected from a material of the group consisting of polyethylene glycol (PEG)-based polymers, preferably PLL-g-PEG or PEGS having different lengths, varying from about 22 to 450 repeating units, and/or corresponding to a molecular weight in the range of 1000 to 20000 Da, polybetaines, such as poly(sulfobetaine) (PSB) or poly (carboxybetaine) (PCB), polyampholytes, fluorinated-polymers, polysaccharides, such as agar or agarose, polyhydroxy-polymers, such as poly(2-hydroxyethyl methacrylate (poly-HEMA) or poly-hydroxypropyl methacrylate (poly-HPMA), poly(ethylene oxide), hydroxypropyl methylcellulose (HPMC), poly(vinyl alcohol) (PVA), poly(2-hydroxyethyl methacrylate) (pHEMA), poly(acrylic acid) (PAA), dextran, hydroxylethylcellulose (HEC), natural biopolymers (Hydrophobines, S-layer protein SbpA), anti-fouling nanointerfaces such as polyelectrolyte multilayers (PEMs), self-assembled monolayers (SAMs), nonionic surfactants such as polyoxyethylene dodecanol, Tween-20, n-dodecyl-D-maltoside (DDM) and pluronics (triblock copolymers PEO-b-poly(propylene oxide)-b-PEO), and silanes such as 3-glycidoxypropyltrimethoxysilane (GPTMS) and 3-chloropropyltrichlorosilane (CPTMS) and (3-aminopropyl)triethoxy silane (APTES).

8. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) have the shape of a hemisphere, a spherical cap, a semi ellipsoid, a cone, a truncated cone, a terraced cone, a pyramid, a truncated pyramid, a terraced pyramid, a torus, or an elliptic paraboloid.

9. The microfluidic device (1; 8) according to claim 8, wherein with the at least two recesses (6; 11) having the shape of a spherical cap the spherical cap has a polar angle () of 30 to 90, preferably 40 to 90, more preferably 50 to 90, more preferably 60 to 90, more preferably 70 to 90, more preferably 80 to 90, more preferably 85 to 90.

10. The microfluidic device (1; 8) according to claim 8, wherein with at least two recesses (6; 11) having the shape of an elliptic paraboloid at least a part of a shell surface (18) of the paraboloid is formed by a parable with the formula y=A*x.sup.B rotated in space, wherein A is between 0.05 and 10, preferably 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and B is 2, 4 or 6.

11. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) have a depth of 50 m to 1 mm, preferably 50 m to 800 m, more preferably 50 m to 500 m.

12. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) have a width or diameter of 100 m to 2 mm, preferably 100 m to 1.5 mm, more preferably 100 m to 1 mm.

13. The microfluidic device (1; 8) according to claim 1, wherein the ratio of depth to width of the at least two recesses (6; 11) is between 1 to 1 and 1 to 3, preferably 1 to 1 to 1 to 2.

14. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) have a different size and shape.

15. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) are spaced apart from each other at a distance from 10 m to 6 mm, preferably 20 m to 6 mm, more preferably 30 m to 6 mm, more preferably 50 m to 6 mm, more preferably 70 m to 6 mm, more preferably 100 m to 6 mm, more preferably 100 m to 5 mm more preferably 100 m to 4 mm.

16. The microfluidic device (1; 8) according to claim 1, wherein the at least two recesses (6; 11) are grouped and the recesses (6; 11) of each group (7a-7e) have the same size and/or the same shape and the recesses (6; 11) of different groups (7a-7e) have different sizes or different sizes and different shapes.

17. The microfluidic device (1; 8) according to claim 16, wherein one group (7a-7e) of recesses (6; 11) is spaced apart to another group of recesses (6; 11) at a distance of 200 m to 10 mm.

18. The microfluidic device (8) according to claim 1, wherein the at least one chamber (9) rejuvenates from the fluid inlet (3) to the fluid outlet (4).

19.-23. (canceled)

24. A method for the production of cellular spheroids comprising the steps of: applying a fluid comprising biological cells into at least one chamber (2; 9) of a device (1; 8) according to claim 1 and thus providing said fluid to the at least two recesses (6; 11); and incubating the device (1; 8) comprising said fluid for at least 2 hours until at least one cellular spheroid is formed in the at least two recesses (6; 11).

25. The method according to claim 24, wherein the device (1; 8) comprising said fluid is incubated for a maximum of six months, preferably for a maximum of three months, more preferably for a maximum of one month, preferably for a maximum of 14 days, preferably for a maximum of 7 days, preferably for a maximum of 96 h, preferably for a maximum of 72 h, preferably for a maximum of 48 h.

26. The method according to claim 24, wherein the device (1; 8) comprising said fluid is incubated for one to seven days.

27. The method according to claim 24, wherein the fluid comprising biological cells is applied into the at least on chamber (2; 9) with a velocity in the range of 4 L/min to 8 L/min.

28. The method according to claim 24, wherein the at least one cellular spheroid formed in at least two recesses (6; 11) is eluted by rinsing the at least one chamber (2; 9) with a discharging fluid at a velocity sufficient to elute the cellular spheroid.

29. The method according to claim 24, wherein with a substrate (5; 10) comprising at least two recesses (6; 11) of different size the cellular spheroids are eluted by successively rinsing the at least one chamber (2; 9) with a discharging fluid with different velocities.

30. The method according to claim 28, wherein the velocity for eluting the spheroid(s) is in the range of 0.5 L/min to 120 L/min, preferably at least 100 L/min for a recess with a width or diameter between 900 m and 1000 m, is at least 30 L/min for a recess with a width or diameter between 500 m and 700 m and/or is at least 20 L/min for a recess with a width or diameter between 150 m and 300 m.

31. The method according to claim 24, wherein the cellular spheroids within the at least two recesses (6, 11) are incubated with at least one antibody (24) binding specifically to the biological cells forming the cellular spheroids.

32. The method according to claim 31, wherein the at least one antibody (24) is labelled with a fluorophore, a peroxidase, preferably a horseradish peroxidase, an alkaline phosphatase or a radioisotope.

Description

[0093] These and further advantageous embodiments of the invention will be explained based on the following description and the accompanying drawings. The person skilled in the art will understand that various embodiments may be combined.

[0094] FIGS. 1 and 2 show in a schematic plan view an embodiment of a microfluidic device according to the invention.

[0095] FIG. 3 shows in a schematic plan view another embodiment of a microfluidic device according to the invention.

[0096] FIG. 4 shows in a schematic plan view an embodiment of a system according to the invention.

[0097] FIG. 5a shows a sectional side view of the embodiment of the microfluidic device according FIG. 1 with cylindrical shaped recesses.

[0098] FIG. 5b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 5a in a plan view.

[0099] FIG. 6a shows a sectional side view of the embodiment of the microfluidic device according to FIG. 1 with disc-shaped recesses.

[0100] FIG. 6b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 6a in a plan view.

[0101] FIG. 7a shows a sectional side view of the embodiment of the microfluidic device according to FIG. 1 with recesses having the shape of spherical caps.

[0102] FIG. 7b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 7a in a plan view.

[0103] FIG. 8a shows a sectional side view of the embodiment of the microfluidic device according to FIG. 1 with recesses having the shape of elliptical paraboloids.

[0104] FIG. 8b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 8a in a plan view.

[0105] FIG. 9a shows a sectional side view of the embodiment of the microfluidic device according to FIG. 1 with hemispherical shaped recesses.

[0106] FIG. 9b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 9a in a plan view and in a sectional side view.

[0107] FIG. 10 shows a graph with the number of spheroids per recess over the diameter of the recess.

[0108] FIG. 11 shows a graph showing the roundness factor of the cellular spheroids over different shaped and sized recesses.

[0109] FIG. 12 shows a graph with the spheroid diameter over different shaped recesses.

[0110] FIG. 13 shows in a schematic plan view an embodiment of a system according to the invention having six microfluidic devices according to FIG. 1 and a gradient generator.

[0111] FIG. 14 shows cellular spheroids in a microfluidic device according to FIG. 9a during incubation with antibodies in a schematic view.

[0112] FIGS. 1 and 2 show in a schematic plan view an embodiment of a microfluidic device 1 according to the invention. The microfluidic device 1 comprises a chamber 2 having a fluid inlet 3 for introducing fluid into the chamber 2 and a fluid outlet 4 for removing said fluid from the chamber 2. Moreover, the chamber 2 comprises a base formed by a substrate 5, which substrate 5 comprises fifteen recesses 6.

[0113] The recesses 6 are grouped in groups 7a to 7e, wherein each group 7a to 7e includes three recesses 6 arranged in a row. The recesses 6 of each group 7a to 7e have the same size and the same shape and the recesses of different groups 7a to 7e have different sizes. For example, the recesses 6 of the first group 7a have a diameter of 200 m, the recesses 6 of the second group 7b have a diameter of 500 m, the recesses 6 of the third group 7c have a diameter of 700 m, the recesses 6 of the fourth group 7d have a diameter of 900 m and the recesses 6 of the fifth group 7e have a diameter of 1000 m.

[0114] In order to grow cellular spheroids fluid comprising biological cells is applied into the chamber 2 through the fluid inlet 3. Consequently, the substrate 5 is contacted with the fluid and the recesses 6 are filled with the fluid. Depending on the required size of the cellular spheroids an incubation time needs to be met before the cellular spheroids can be eluted from the chamber 2. Smaller cell formation form already after two hours.

[0115] In order to elute the cellular spheroids from the recesses a discharging fluid is applied through the fluid inlet 3 to rinse the chamber 2. The discharging fluid is discharged through the fluid outlet 4. The discharging fluid flows through the chamber 2 in the direction 12. Please see FIG. 2. Preferably the discharging fluid is supplied through the fluid inlet 3 at different velocities in order to elute the cellular spheroids according to their size or, respectively in groups according to their size.

[0116] In the example shown in FIG. 2 the discharging fluid is supplied to the fluid inlet 3 with a first low velocity sufficient to elute the smallest circular spheroids from the recesses 6 of the group 7a first. The first velocity is 20 L/min, for example. In a further step the velocity of the discharging fluid is increased to a second velocity. The second velocity is 30 L/min, for example. Due to that, the cellular spheroids formed in recesses 6 of the groups 7b and 7c are eluted. By further increasing the velocity to a third velocity, for example to 100 L/min, the rest of the circular spheroids of the recesses 6 of the groups 7d and 7e are be eluted. Please note, that with a finer graduation of the velocity the cellular spheroids formed in the groups 7b and 7d can also be eluted individually per group. Please not, that the velocities shown in FIG. 2 are only illustrative and can differ from this values depending on the size of the recesses 6. Furthermore, please note that for the elution of the cellular spheroids it is also possible that the discharging fluid is applied through the fluid inlet 3 with a velocity sufficient to elute all cellular spheroids at once from the chamber 2.

[0117] After all cellular spheroids are eluted, the microfluidic device 1 is cleaned with a cleaning fluid and ready for further use.

[0118] Preferably, the fluid comprising the biological cells is applied to the chamber 2 in a continuous manner through the fluid inlet 3 and discharged out of the chamber 2 by the outlet 4 during incubation. This has the advantage that the cells can be continuously supplied with nutrients and that new cells are supplied to the microfluidic device 1.

[0119] FIG. 3 shows in a schematic plan view of another embodiment of a microfluidic device 8 according to the invention. The microfluidic device 8 distinguishes to the microfluidic device 1 according to FIG. 1 in the fact that a chamber 9 of the microfluidic device 2 essentially rejuvenates from the fluid inlet 3 to the fluid outlet 4 and in the fact that the substrate 10 comprises five more groups 7f to 7j of different sized recesses 11, which leads to thirty recesses 11. Due to the rejuvenation the velocity of fluid rinsing through a chamber 9 is changed. Higher velocities result in higher shear forces on the cellular spheroids. Thus, an impact on the cellular spheroids at different shear forces can be monitored in one single channel. For example, with a fluid applied with a volume velocity of 8 L/min at the inlet 3 at the microfluidic device 8 shown in FIG. 3 a shear force at a wider part of the chamber 9 is 0.01 dyn/cm.sup.2 and a shear force near the fluid outlet is 0.07 dyn/cm.sup.2. Accordingly, the shear forces near the fluid outlet are seven times as high as the shear forces at the wider part of the chamber 9.

[0120] Elements of the micro fluidic device 8 being similar to elements of the microfluidic device 1 according to FIG. 1 are indicated with the same reference numerals. Please note that substrate 10 can also comprise a different amount of recesses 11.

[0121] FIG. 4 shows in a schematic plan view an embodiment of a system 13 according to the invention. The system 13 comprises six microfluidic devices 1 according to FIG. 1. The system 13 has one base 14, in which the individual chambers 2 of the microfluidic devices 1 are contained.

[0122] FIG. 5a shows a sectional side view of the embodiment of the microfluidic device 1 according to FIG. 1 with cylindrical shaped recesses 6.

[0123] FIG. 5b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device 1 according to FIG. 5a in a plan view. Due to simplicity reasons FIG. 5b only shows one example of a cell formation per recesses 6 of each group 7a to 7e. To indicate which picture corresponds to which group 7a to 7e, the pictures are indicated with the group reference numerals.

[0124] As can be seen in the pictures of FIG. 5b, there is no spherical cell formation in cylindrical shaped recesses 6. Rather a multitude of cell formations of different form develop in cylindrical shaped recesses 6.

[0125] FIG. 6a shows a sectional side view of the embodiment of the microfluidic device 1 according to FIG. 1 with disc-shaped recesses 6.

[0126] FIG. 6b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 6a in a plan view. Due to simplicity reasons FIG. 6b only shows one example of a cell formation per recesses 6 of each group 7a to 7e. To indicate which picture corresponds to which group 7a to 7e, the pictures are indicated with the group reference numerals.

[0127] As can be seen in the pictures of FIG. 6b, there is no spherical cell formation in disc-shaped recesses 6. Also with disc-shaped recesses 6 a multitude of cell formations of different form develop in the recesses 6.

[0128] FIG. 7a shows a sectional side view of the embodiment of the microfluidic device 1 according to FIG. 1 with recesses 6 having the shape of spherical caps. The spherical caps have different angles . The angle is the angle between a tangent plane 15 in an exemplary point 19 on a surface 17 of the spherical cap and a horizontal plane 16 of the substrate 5, wherein in the point 19 the surface 17 of the spherical cap intersects with the horizontal plane 16 of the substrate. Since the angle corresponds to the polar angle of the spherical cap, a is hereinafter referred to as the polar angle.

[0129] The spherical caps of different groups 7a to 7e have the same depth but vary in their diameter and hence, also in their polar angle . For example, group 7a, which contains the smallest recesses 6, has a polar angle of 90 and group 7e has a polar angle of 30. Please note that spherical caps of different groups 7a to 7e can also have the same diameter with different polar angles or can also have different depths and diameters with the same gradient angels .

[0130] FIG. 7b shows schematically pictures of cell formations formed in the embodiment of the microfluidic device 1 according to FIG. 7a in a plan view. Due to simplicity reasons FIG. 7b only shows one example of a cell formation per recesses 6 of each group 7a to 7e. To indicate which picture corresponds to which group 7a to 7e, the pictures are indicated with the group reference numerals.

[0131] As can be seen in the pictures of FIG. 7b cellular spheroids form in the spherical cap shaped recesses 6.

[0132] FIG. 8a shows a sectional side view of the embodiment of the microfluidic device 1 according to FIG. 1 with recesses 6 having the shape of elliptical paraboloids. A shell surface 18 of the elliptic paraboloids is formed by a parable with the formula y=A*x.sup.B rotated in space. The recesses 6 of different groups 7a to 7e have the same diameter but vary in their depth and hence in their elliptical paraboloid shape. For example, the recesses 6 of group 7a, which are the smallest recesses 6, are formed by a parable with the formula y=0.1*x.sup.2 rotated in space and the recesses 6 of group 7e are formed by a parable with the formula y=2*x.sup.2 rotated in space. Please note that the recesses 6 of different groups 7a to 7e can also have the same depth with a different elliptical paraboloid shapes or can also have different depths and/or diameters with the same elliptical paraboloid shape.

[0133] FIG. 8b schematically shows pictures of cell formations formed in the embodiment of the microfluidic device according to FIG. 8a in a plan view. Due to simplicity reasons FIG. 8b only shows one example of a cell formation per recesses 6 of each group 7a to 7e. To indicate which picture corresponds to which group 7a to 7e, the pictures are indicated with the group reference numerals.

[0134] As can be seen in the pictures of FIG. 8b cellular spheroids form in the elliptical paraboloid shaped recesses 6.

[0135] FIG. 9a shows a sectional side view of the embodiment of the microfluidic device 1 according to FIG. 1 with hemispherical shaped recesses 6.

[0136] FIG. 9b schematically shows pictures of cell formations formed in the embodiment of the microfluidic device 1 according to FIG. 9a in a plan view and in a sectional side view. Due to simplicity reasons FIG. 9b only shows one example of a cell formation per recesses 6 of each group 7a to 7e in the plan view and in the sectional side view. To indicate which recess 6 belongs to which group 7a to 7e, the recesses 6 are indicated with the group reference numerals.

[0137] As can be seen in FIG. 9b, perfectly round cellular spheroids form in hemispheric shaped recesses 6.

[0138] FIG. 10 shows a graph with the number of spheroids per recess over the diameter of the recess.

[0139] FIG. 11 shows a graph showing the roundness factor of the cellular spheroids over different shaped and sized recesses 6, wherein the size 7a to 7e of the recesses 6 is organized according to FIG. 12.

[0140] FIG. 12 shows a graph with the spheroid diameter over different shaped recesses having different size.

[0141] FIG. 13 shows in a schematic plan view an embodiment of a system 20 according to the invention having six microfluidic devices 1 according to FIG. 1 and a gradient generator 22. The gradient generator 22 comprises six fluid outlets 21, wherein each fluid outlet 21 is connected to a fluid inlet 3 of a microfluidic device 1. Furthermore, the gradient generator 22 comprises two fluid inlets 23, via which fluid inlets 23 a substance A and a substance B may be supplied to the gradient generator.

[0142] By providing a substance A and a substance B at the same pressure at the fluid inlets 23 the microfluidic devices 1 are rinsed with different compounds of the substances A and B. As can be seen in FIG. 13, the compounds vary between a compound of 100% A and 0% B and a compound of 0% A and 100% B. With the system 20 it is possible to determine the influence of varying substance concentrations on cellular spheroids.

[0143] Please note, that the gradient generator 22 can also have more fluid inlets 23 and/or more or less fluid outlets 21.

[0144] FIG. 14 shows cellular spheroids in a microfluidic device 1 according to FIG. 9a during incubation with antibodies 24 in a schematic view, wherein FIG. 14 only shows a part of the microfluidic device 1.

Example

Material and Methods

Fabrication of the Microfluidic Devices 1:

[0145] The microfluidic device 1 as depicted in FIGS. 5a to 9a were fabricated by double casting of poly(dimethyl siloxane) (PDMS). The first matrix including concave microwells and channels, are made in poly(methyl methacrylate) (PMMA) using CNC micromilling PDMS is prepared by mixing the prepolymer and curing agent in a weight ratio of 10:1. The mixture is poured onto the PMMA structure placed and cured for 3 h at 75 C. The structure is peeled off from the PMMA matrix subsequently and placed in the oven for 48 h at 100 C. (thermal aging step). As a result, the second matrix with convex patterns was obtained. The thermally aged PDMS master was used for the next replica molding procedure and thus, the PDMS bottom layer of the microsystem was obtained. Holes for tubing (fluid inlet and fluid outlet) were drilled in glass slides and irreversibly bonded to the bottom PDMS layer using an oxygen plasma activation for 1 minute at 90 W. Bonding between the PDMS and glass layer was facilitated by backing the micro fluidic devices 1 for 1 h at 70 C. after alignment.

Cell Culture:

[0146] Hepatocellular carcinoma cells (ATCC, USA) were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% GlutaMax (Life technologies, Thermo Fisher Scientific, USA) and 1% antibiotic/antimycotic solution (Sigma-Aldrich, Austria). The cells were cultivated in 75 cm.sup.2 cell culture flasks under standard cell culture conditions at 37 C. in 5% CO.sub.2 humidified atmosphere as adherent monolayers. At a confluency of 80%, cells were rinsed with Phosphate buffer (PBS, Sigma-Aldrich, Austria) and trypsinized for 10 min at 37 C. for enzymatic detachment. Complemented MEM was added to inactivate trypsin (GE Healthcare, Austria) and cell suspension was centrifuged at 1250 rpm for 5 min and the precipitated pellet was resuspended in fresh medium. Cell were counted and viability was carried out against Trypan Blue reagent by using an automated cell counter (Nano En Tek, Korea) before seeding.

Cell Seeding and Cellular Spheroid Generation:

[0147] After fabrication the chambers 2 of the microfluidic devices 1 as depicted in FIGS. 5a to 9a were perfused at a flow rate of 4 L min.sup.1 with 70% ethanol using a syringe pump (KD Scientific, USA) for 1 hour. After flushing with sterile dH.sub.2O, the chambers 2 were then loaded by an anti-fouling protein solution with a flow rate of 1 L min.sup.1 and incubated over night at room temperature to create a nanobiointerface that prevents cell adhesion and induces controlled cellular spheroid generation in each recess 6. The protein solution is removed by flow injection of cell culture media followed by a cell seeding step. Single cell suspension at a density of 610.sup.6 cells mL.sup.1 was loaded into the chambers 2 using a syringe pump at flow rate of 4 L min.sup.1 for 10 min After one hour of incubation, non-trapped cells were eluted by cell culture medium at a flow rate of 10 L min.sup.1. Cellular spheroid cultivation in the recesses 6 is conducted at constant flow rate of 4 L min.sup.1 at 37 C. and 5% CO.sub.2.

Image Analysis:

[0148] Micrographs of the microfluidic devices 1 as depicted in FIGS. 5a to 9b were analyzed with an image processing and analysis freeware (ImageJ, NIH, USA). Spheroid cultivation was monitored over 3 days with an inverted fluorescence optical microscope (TE2000, Nikon, Japan) equipped with a digital camera (DS-Qi1MC, Nikon, Japan). Spheroid area, perimeter, solidity and roundness were measured by using the BioVoxxel Image Processing and Analysis Toolbox for ImageJ (Biovoxxel, Germany) Data were expressed as meanstandard deviation (SD). All experiments were done independently in triplicates.

Results

[0149] To evaluate the optimal shape of a recess 6 for precise size control of reproducible, uniform-sized, single HepG2 cellular spheroids, variable recess shapes and sizes were tested. In particular, recesses 6 of the shape of a cylinder (100 m and 500 m depth), a spherical cap, an elliptic paraboloid and a hemisphere were tested. After 3 days post-seeding morphology of HepG2 cellular spheroids was evaluated. FIG. 10 shows the impact of the recess shapes on the number of generated HepG2 cellular spheroids in dependence of a recess diameter. The results indicate that hemispherical recesses resulted in the formation of single spheroids within each of the recess independent of recess diameter. In contrast, recesses with different curvatures as well as cylindrical shapes showed a higher probability to form multiple spheroids in each cavity, thus decreasing spheroid production yield and accuracy.

[0150] To assess the influence of recess shape on the quality of spheroid morphology, roundness factor of each micro-tissue was determined for recesses having the shape of a cylinder (100 m and 500 m depth), a spherical cap and a hemisphere. FIG. 11 shows that hemispherical- and the spherical cap-shape induced the formation of reproducible round HegG2 spheroids in each well diameter. Additionally, the elliptical paraboloid shape with a shell surfaces of the paraboloids formed by parables with the formula y=A*x.sup.B with A between 0.14 and 0.3 and B between 2 and 4 resulted in formation of round single HepG2 spheroids comparable to the hemispherical and spherical cap shaped recesses, however, with elliptical paraboloid shaped recesses with a shell surfaces different of that a reduction in roundness was observable. Spheroids with a roundness factor above 0.9 are considered as regular spherical shaped micro-tissues as described in literature (see Zanoni et al (Sci Rep. 2016; (6): 19103).

[0151] In contrast, both of the cylindrical shape resulted in irregular non-circular HepG2 spheroids. To investigate these effects in more detail, spheroid size controllability for recesses with a hemispherical, elliptic paraboloid a spherical cap shape was evaluated. Hemispherical recesses showed the ability to adjust spheroid size in a reproducible manner as shown in FIG. 12. The hemispherical shape led to a linear increase of spheroid size within a single microchannel channel, whereas elliptical paraboloid- and spherical cap shaped recesses resulted in an irregular and less controllable formation of spheroids (no linear increase and correlation).

CONCLUSION

[0152] Here, we show a novel method and device for size control of cellular spheroid generation within a single microchannel based on the influence of recess shape and diameter in a microfluidic device. We showed that cylinder-shaped recesses enable the formation of multiple non-regular cellular spheroids and therefore, these shapes can be considered as unpredictive architecture for precise micro-tissue size control. Further recess shapes as elliptic paraboloid, spherical cap and hemispherical recess were evaluated regarding optimal cell trapping efficiency as well as resulting formation of size-tunable round cellular spheroids. In conclusion, hemispherical recesses lead to the generation of reproducible single cellular spheroid in each individual recess with 100% yield with high controllability in spheroid size and morphology.

[0153] The microfluidic device of the present invention is a rapid evaluation tool for determining the optimal spheroid size for toxicology screenings, stem cell research, tissue engineering and biochemical staining protocols which are the basis for reliable and robust data. In summary, the microfluidic device according to the invention is a facile tool for screening spheroids of different sizes produced by geometric control of micro-recesses, which demonstrates the optimal spheroid size for a broad range of applications.