Three-dimensional cell culture system and method

Abstract

Disclosed herein is a high-throughput three-dimensional cell culture system and related methods configured to distribute cells relatively evenly across the surface of a well for improved image analysis.

Description

[Background technology]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 309,950, filed February 14, 2022, which is incorporated herein by reference.

[0002] Three-dimensional (3D) cell culture is a promising technology for application in the development and identification of therapeutic treatments. 3D cell culture can have the advantage of more closely mimicking biological functions compared to 2D cell culture systems. High-quality cell sample preparation is important for a variety of clinical and research applications. 3-D cell culture can closely represent in vivo characteristics. There is a need for high-throughput 3D cell culture methods and systems that can effectively and reproducibly culture cells at an affordable cost. Summary of the Invention

[0003] Disclosed herein is a method of fabricating a three-dimensional hydrogel system in a well, the method comprising: (a) delivering a first mixture comprising a first acrylate and a first thiolated polymer into the well to form a concave meniscus in the well; (b) crosslinking the first acrylate and the first thiolated polymer of the first mixture to form a first cured hydrogel layer, the first cured hydrogel layer comprising a concave upper surface; (c) delivering a second mixture comprising a second acrylate and a second thiolated polymer into the well on top of the first cured hydrogel layer; and (d) crosslinking the second acrylate and the second thiolated polymer of the second mixture to form a second cured hydrogel layer, the second cured hydrogel layer comprising a top surface on top of the first cured hydrogel layer that is less concave than the concave upper surface of the first cured hydrogel layer. The top surface of the second cured layer can be substantially planar.

[0004] The step of crosslinking the first acrylate and the first thiolated polymer to form a first cured hydrogel layer can include exposing the first mixture to ultraviolet light while the first mixture is disposed (located) in the well and before the second mixture is delivered to the well. The step of crosslinking the second acrylate and the second thiolated polymer to form a second cured hydrogel layer can include exposing the second mixture to ultraviolet light while both the first cured hydrogel layer and the second mixture are disposed in the well. The first mixture can be exposed to ultraviolet light for a time longer than the time the second mixture is exposed to ultraviolet light. The second mixture can be exposed to ultraviolet light for less than 12 seconds and the first mixture is exposed to ultraviolet light for more than 12 seconds. In some embodiments, the second mixture can be exposed to ultraviolet light for less than 15 seconds and the first mixture is exposed to ultraviolet light for more than 15 seconds. In some embodiments, the second mixture and the first mixture are each exposed to ultraviolet light for between 5 seconds and 20 seconds (e.g., between 10 seconds and 20 seconds). The ultraviolet light can have a wavelength of between 320 nm and 390 nm. The ultraviolet light can have an intensity of 250 mW / cm 2 The strength of the sintered body may be 100%.

[0005] The second mixture can further include a plurality of cells. The plurality of cells can be non-aggregated cells. The plurality of cells can be stained prior to (c). The stained plurality of cells can be stained with one or more of: (i) a dye specific to cell nuclei, (ii) a dye specific to cell membranes, (iii) a dye specific to live cells, and (iv) a dye specific to dead cells. The plurality of cells can be stained with one or more of a lipophilic carbocyanine dye, a bisbenzimide, a xanthene dye, or propidium iodide. The plurality of cells can be marked with a fluorescent tag prior to delivering the second mixture to the well.

[0006] The second mixture may optionally or further include laminin such that the laminin is non-covalently distributed within the second cured hydrogel. In some embodiments, the second mixture does not include laminin. The first mixture may further include a photoinitiator. The photoinitiator may include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. The second mixture may further include a photoinitiator. The photoinitiator may include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0007] The method can further include adding cell culture medium to the three-dimensional hydrogel system. The cell culture medium can include one or more of sodium phosphate, potassium chloride, sodium bicarbonate, ferric nitrate, calcium chloride, or potassium chloride. The dynamic viscosity of the first mixture is 0.5 mm when delivered to the well. 2 / s~10mm 2 The kinematic viscosity of the second mixture when delivered to the well may be between 0.5 mm / s. 2 / s~10mm 2 / s.

[0008] One or both of the first acrylate and the second acrylate can be a multi-arm PEG acrylate. One or both of the first acrylate and the second acrylate can be a PEG diacrylate. The PEG diacrylate of the first mixture can have a number average molecular weight greater than 3 kilodaltons. In some embodiments, the PEG diacrylate of the first mixture can have a number average molecular weight of 3-4 kilodaltons. In some embodiments, the PEG diacrylate of the first mixture can have a number average molecular weight of about 7-15 kilodaltons. The PEG diacrylate of the second mixture can have a number average molecular weight greater than 3 kilodaltons. In some embodiments, the PEG diacrylate of the second mixture can have a number average molecular weight of 3-4 kilodaltons. In some embodiments, the PEG diacrylate of the second mixture can have a number average molecular weight of 7-15 kilodaltons.

[0009] One or both of the first thiolated polymer and the second thiolated polymer can be thiolated gelatin. The thiolated gelatin can have a degree of thiolation of 20% to 30%. In some embodiments, the weight ratio of PEG diacrylate to thiolated gelatin in the second mixture is between 1:4 to 2:1, such as 1:2. In some embodiments, the weight ratio of PEG diacrylate to thiolated gelatin in the second mixture is between 1:1 to 8:1, such as 2:1 to 8:1. The second mixture can include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in a weight ratio of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone to thiolated gelatin of 1:20 to 1:100, such as 1:50.

[0010] The second mixture may contain laminin in a weight ratio of thiolated gelatin to laminin of 1:1 to 8:1, for example 4:1. The concentration of laminin in the second mixture may be at least 0.1 mg / mL. The concentration of laminin in the second mixture may be 1.0 mg / mL or less. The concentration of laminin in the second mixture may be between 0.2 and 1.0 mg / mL, or between 0.2 and 0.6 mg / mL.

[0011] The well can be a well in an array of wells of a multi-well plate. The array of wells can include at least 96 wells. The array of wells can include at least 384 wells. The array of wells can include at least 1536 wells. The delivery of the first and second mixtures can be completed in less than 1 hour across 384 wells. The delivery of the first mixture can be completed in less than 20 minutes across 384 wells. The delivery of the second mixture can be completed in less than 20 minutes across 384 wells. The delivery of the first and second mixtures can be completed in less than 1 hour across 1536 wells. The delivery of the first mixture can be completed in less than 30 minutes across 1536 wells. The delivery of the second mixture can be completed in less than 30 minutes across 1536 wells.

[0012] The delivery of the first mixture can include delivering via a robotic system. The delivery of the second mixture can include delivering via a robotic system. The delivery of the first mixture can include dispensing a first amount of the first mixture into the well and aspirating a second amount of the first mixture from the well. The first amount can be greater than the second amount. The delivery of the second mixture can include dispensing a first amount of the second mixture into the well and aspirating a second amount of the second mixture from the well. The first amount can be greater than the second amount.

[0013] The second mixture can include cells. The cells can be substantially uniformly distributed throughout the second cured hydrogel layer. Disclosed herein are hydrogels formed by the methods described herein.

[0014] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0015] The various features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0016] [Figure 1A] A monolayer of hydrogel is shown with cells concentrated on the walls of the well. [Figure 1B] Shown is a hydrogel bilayer with cells evenly distributed across the area of ​​the well. [Figure 2A] FIG. 1 is a graph showing the proliferation of HCT-116 cells in gels made from Gelin-S and Ac-PEG-Ac. [Figure 2B] 1 is a graph showing a comparison of viability between Gelin-S and RGD-based gels. [Figure 2C] 1 is a graph showing a comparison of viability between Gelin-S based gels, RGD based gels and pH balanced RGD gels. [Figure 3A] FIG. 1 is a cross-sectional view of a well 100, 200 containing a hydrogel component. Specifically, the figure shows a well 100 filled with two hydrogel layers, a first layer 110 having an upper surface 111 that is more concave than an upper surface 121 of a second layer 120 deposited on the first layer 110. [Figure 3B]FIG. 1 is a cross-sectional view of a well 100, 200 containing a hydrogel component. Specifically, this figure shows well 200 filled with the same total volume of hydrogel as the previous figure. However, well 200 was formed by depositing only a monolayer 210. The top surface 211 of this monolayer 210 is more concave than the top surface 111 of the second layer 110 of a similar two-layer structure shown in the previous figure. [Figure 4A] 1 shows a cell number analysis of a monolayer hydrogel layer deposition. [Figure 4B] 1 shows a cell count analysis of two hydrogel layer depositions. [Diagram 5] 1 illustrates an example of a computer system configured to implement the methods provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Many hydrogel-forming mixtures are excessively viscous and spontaneously crosslink into a gel form over short time scales (e.g., minutes) at room temperature. This creates challenges for automation, which often relies on liquid-handling robots that have problems handling viscous liquids and materials with inconsistent properties. There is a need for a hydrogel fabrication method in which the hydrogel-forming mixture remains in a relatively low-viscosity form prior to crosslinking to dispense the hydrogel into an array of wells that is compatible with high throughput while maintaining a relatively uniform distribution of cells within the hydrogel system or portions thereof.

[0018] Disclosed herein are systems and methods for high throughput three-dimensional cell culture. The hydrogel-forming mixture naturally wicks up at the sides of the well, allowing the hydrogel (and cells within the hydrogel) to concentrate on the outer walls (e.g., perimeter) of the well. The methods and systems described herein are configured to cause the hydrogel-forming mixture to tend to concentrate the hydrogel (and cells therein) on or near the outer walls of the well by using a two-layer deposition process that allows for strong growth of cells in the center of the well (and along the perimeter of the well). Thus, the cells of the disclosure may be more evenly distributed throughout the area of ​​the well. The method of fabrication may include curing a first layer of hydrogel in the well to form a concave top surface. The curing process may include a photoinitiator. The curing may include light exposure (e.g., exposure to UV light). The method may include depositing and curing a second layer of hydrogel containing cells on top of the first layer. The second layer may include one or more proteins (e.g., laminin) configured to interact with cells, such as mammalian cells. The first and second layers can have the same refractive index after curing. The porosity of the hydrogel can be configured to be large enough to allow diffusion of nutrients and dyes, yet small enough to retain cells, such as immune cells.

[0019] Thiolated Polymers The hydrogel (of the hydrogel layer) can include and / or be formed from a thiolated polymer, which can be gelatin or polyethylene glycol (PEG). Thiolated polymers are 1kDa, 1.5kDa, 2kDa, 2.5kDa, 3kDa, 3.5kDa, 4kDa, 4.5kDa, 5kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 7.5kDa, 8kDa, 8.5kDa, 9kDa, 9.5kDa, 10kDa , 10.5kDa, 11kDa, 11.5kDa, 12kDa, 12.5kDa, 13kDa, 13.5kDa, 14kDa, 14.5kDa, 15kDa, 15.5kDa, 16kDa, 16.5kDa, 17kDa, 17.5kDa, 18kDa, 18.5kDa, 19k Da, 19.5 kDa, 20 kDa, 20.5 kDa, 21 kDa, 21.5 kDa, 22 kDa, 22.5 kDa, 23 kDa, 23.5 kDa, 24 kDa, 24.5 kDa, 25 kDa, 25.5 kDa, 26 kDa, 26.5 kDa, 27 kDa, 27.5 kDa, 28 kDa, 28.5 kDa, 29 kDa, 29.5 kDa, 30 kDa, 30.5 kDa, 31 kDa, 31.5 kDa, 32 kDa, 32.5 kDa, 33 kDa, 33.5 kDa, 34 kDa, 34.5 kDa or 35 kDa.The thiolated polymers are 35 kDa, 34.5 kDa, 34 kDa, 33.5 kDa, 33 kDa, 32.5 kDa, 32 kDa, 31.5 kDa, 31 kDa, 30.5 kDa, 30 kDa, 29.5 kDa, 29 kDa, 28.5 kDa, 28 kDa, 27.5 kDa, 27 kDa, 26.5 kDa, 26 kDa, 25.5 kDa, 25 kDa, 24.5 kDa, 24 kDa, 23.5 kDa, 23 kDa, 22.5 kDa, 22 kDa, 21.5 kDa, 21 kDa, 20.5 kDa, 20 kDa, 19.5 kDa, 19 kDa, 18.5 kDa In some embodiments, the molecular weight of the polymer may be less than 1 kDa, 18 kDa, 17.5 kDa, 17 kDa, 16.5 kDa, 16 kDa, 15.5 kDa, 15 kDa, 14.5 kDa, 14 kDa, 13.5 kDa, 13 kDa, 12.5 kDa, 12 kDa, 11.5 kDa, 11 kDa, 10.5 kDa, 10 kDa, 9.5 kDa, 9 kDa, 8.5 kDa, 8 kDa, 7.5 kDa, 7 kDa, 6.5 kDa, 6 kDa, 5.5 kDa, 5 kDa, 4.5 kDa, 4 kDa, 3.5 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1.5 kDa or 1 kDa.The thiolated polymer can have a number average molecular weight of 1 kDa to 2 kDa, 1.5 kDa to 2.5 kDa, 2 kDa to 3 kDa, 2.5 kDa to 3.5 kDa, 3 kDa to 4 kDa, 3.5 kDa to 4.5 kDa, 4 kDa to 5 kDa, 4.5 kDa to 5.5 kDa, 5 kDa to 6 kDa, 5.5 kDa to 6.5 kDa, 7 kDa to 8 kDa, 7.5 kDa to 8.5 kDa, 9 kDa to 10 kDa, 9.5 kDa to 10.5 kDa, 10 kDa to 11 kDa, 10.5 kDa to 11.5 kDa, 11 kDa to 12 kDa, 11.5 kDa to 12.5 kDa, 12 kDa to 13 kDa, 12.5 kDa to 13.5 kDa, 13 kDa to 14 kDa, 13.5 kDa to 14.5 kDa, 14 kDa to 15 kDa, 14.5 kDa to 15.5 kDa, 15 kDa to 16 kDa, 15.5 kDa to 16.5 kDa, 16 kDa to 17 kDa, 16.5 kDa to 17.5 kDa, 17 kDa to 18 kDa, 17.5 kDa to 18.5 kDa, 18 kDa to 19 kDa, 18.5 kDa to 19.5 kDa, 19 kDa to 20 kDa, 19.5 kDa to 20.5 kDa, 20 kDa to 21 kDa, 20.5 kDa to 21.5 kDa, 21 kDa to 22 kDa, 21.5 kDa to 22.5 kDa, 22 kDa to 23 kDa, 22.5 kDa to 23.5 kDa, 23 kDa to 24 kDa, 23.5 kDa to 24.5 kDa, 24 kDa to 25 kDa, 24.5 kDa to 25.5 kDa, 25 kDa to 26 kDa, 25.5 kDa to 26.5 kDa, 26 kDa to 27 kDa, 26.5 kDa to 27.5 kDa, 27 kDa to 28 kDa, 27.5 kDa to 28.5 kDa, 28 kDa to 29 kDa, 28.5 kDa to 29.5 kDa, 29 kDa to 30 kDa, 29.5 kDa to 30.5 kDa, 30 kDa to 31 kDa, 30.5 kDa to 31.5 kDa, 31 kDa to 32 kDa, 31.5 kDa to 32.5 kDa, 32 kDa to 33 kDa, 32.5 kDa to 33.5 kDa, 33 kDa to 34 kDa, 33.5 kDa to 34.5 kDa, 34 kDa to 35 kDa.

[0020] The thiolated polymer can have a degree of thiolation of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50%. The thiolated polymer can have a degree of thiolation of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5%. The thiolated polymer can have a degree of thiolation of 5%-15%, 10%-20%, 15%-25%, 20%-30%, 30%-40%, or 35%-45%.

[0021] The degree of thiolation as described herein can refer to the percentage of free (e.g., unreacted) sulfhydryl groups (e.g., "thiol groups", -SH groups) out of the total number of sulfhydryl groups on a molecule. Free sulfhydryl groups can be measured using any technique known to those of skill in the art, such as the Ellman assay. In the Ellman assay, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) is used to react with free sulfhydryl groups to produce a mixture of disulfides and 2-nitro-5-thiobenzoic acid (TNB). TNB has a high molar extinction coefficient in the visible range and can therefore be directly related to the total number of free sulfhydryl groups in solution.

[0022] The thiolated PEG can have a number average molecular weight of greater than 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, 7.5 kDa, 8 kDa, 8.5 kDa, 9 kDa, 9.5 kDa or 10 kDa. The thiolated PEG can have a number average molecular weight of less than 10 kDa, 9.5 kDa, 9 kDa, 8.5 kDa, 8 kDa, 7.5 kDa, 7 kDa, 6.5 kDa, 6 kDa, 5.5 kDa, 5 kDa, 4.5 kDa, 4 kDa, 3.5 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1.5 kDa or 1 kDa. The thiolated PEG can have a number average molecular weight of 1 kDa to 2 kDa, 1.5 kDa to 2.5 kDa, 2 kDa to 3 kDa, 2.5 kDa to 3.5 kDa, 3 kDa to 4 kDa, 3.5 kDa to 4.5 kDa, 4 kDa to 5 kDa, 4.5 kDa to 5.5 kDa, 5 kDa to 6 kDa, 5.5 kDa to 6.5 kDa, 7 kDa to 8 kDa, 7.5 kDa to 8.5 kDa, or 9 kDa to 10 kDa.

[0023] Thiolated gelatin is available in the following sizes: 1kDa, 1.5kDa, 2kDa, 2.5kDa, 3kDa, 3.5kDa, 4kDa, 4.5kDa, 5kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 7.5kDa, 8kDa, 8.5kDa, 9kDa, 9.5kDa, 10kDa , 10.5kDa, 11kDa, 11.5kDa, 12kDa, 12.5kDa, 13kDa, 13.5kDa, 14kDa, 14.5kDa, 15kDa, 15.5kDa, 16kDa, 16.5kDa, 17kDa, 17.5kDa, 18kDa, 18.5kDa, 19k Da, 19.5 kDa, 20 kDa, 20.5 kDa, 21 kDa, 21.5 kDa, 22 kDa, 22.5 kDa, 23 kDa, 23.5 kDa, 24 kDa, 24.5 kDa, 25 kDa, 25.5 kDa, 26 kDa, 26.5 kDa, 27 kDa, 27.5 kDa, 28 kDa, 28.5 kDa, 29 kDa, 29.5 kDa, 30 kDa, 30.5 kDa, 31 kDa, 31.5 kDa, 32 kDa, 32.5 kDa, 33 kDa, 33.5 kDa, 34 kDa, 34.5 kDa or 35 kDa. Thiolated gelatin is 35kDa, 34.5kDa, 34kDa, 33.5kDa, 33kDa, 32.5kDa, 32kDa, 31.5kDa, 31kDa, 30.5kDa, 30kDa, 29.5kDa, 29kDa, 28.5kDa, 28kDa, 27.5kDa, 27kDa, 26.5kDa, 26kDa, 25.5kDa, 25kDa, 24.5kDa, 24kDa, 23.5kDa, 23kDa, 22.5kDa, 22kDa, 21.5kDa, 21kDa, 20.5kDa, 20kDa, 19.5kDa, 19kDa, 18.5kDa a, 18 kDa, 17.5 kDa, 17 kDa, 16.5 kDa, 16 kDa, 15.5 kDa, 15 kDa, 14.5 kDa, 14 kDa, 13.5 kDa, 13 kDa, 12.5 kDa, 12 kDa, 11.5 kDa, 11 kDa, 10.5 kDa, 10 kDa, 9.5 kDa, 9 kDa, 8.5 kDa, 8 kDa, 7.5 kDa, 7 kDa, 6.5 kDa, 6 kDa, 5.5 kDa, 5 kDa, 4.5 kDa, 4 kDa, 3.5 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1.5 kDa or less than 1 kDa.Thiolated gelatin is available in the following sizes: 1kDa~2kDa, 1.5kDa~2.5kDa, 2kDa~3kDa, 2.5kDa~3.5kDa, 3kDa~4kDa, 3.5kDa~4.5kDa, 4kDa~5kDa, 4.5kDa~5.5kDa, 5kDa~6kDa, 5.5kDa~6.5kDa, 7kDa~8kDa, 7.5kDa~8.5kDa, 9kDa~10kDa, 9.5kDa~10.5kDa, 10kDa~11kDa, 10.5kDa~11.5kDa, 11kDa~12kDa , 11.5kDa~12.5kDa, 12kDa~13kDa, 12.5kDa~13.5kDa, 13kDa~14kDa, 13.5kDa~14.5kDa, 14kDa~15kDa, 14.5kDa~15.5kDa, 15kDa~16k Da, 15.5kDa~16.5kDa, 16kDa~17kDa, 16.5kDa~17.5kDa, 17kDa~18kDa, 17.5kDa~18.5kDa, 18kDa~19kDa, 18.5kDa~19.5kDa, 19kDa~20 kDa, 19.5kDa~20.5kDa, 20kDa~21kDa, 20.5kDa~21.5kDa, 21kDa~22kDa, 21.5kDa~22.5kDa, 22kDa~23kDa, 22.5kDa~23.5kDa, 23kDa~ 24kDa, 23.5kDa~24.5kDa, 24kDa~25kDa, 24.5kDa~25.5kDa, 25kDa~26kDa, 25.5kDa~26.5kDa, 26kDa~27kDa, 26.5kDa~27.5kDa, 27kD The number average molecular weight may be from 1 to 28 kDa, 27.5 kDa to 28.5 kDa, 28 kDa to 29 kDa, 28.5 kDa to 29.5 kDa, 29 kDa to 30 kDa, 29.5 kDa to 30.5 kDa, 30 kDa to 31 kDa, 30.5 kDa to 31.5 kDa, 31 kDa to 32 kDa, 31.5 kDa to 32.5 kDa, 32 kDa to 33 kDa, 32.5 kDa to 33.5 kDa, 33 kDa to 34 kDa, 33.5 kDa to 34.5 kDa, or 34 kDa to 35 kDa.

[0024] Acrylates The hydrogel (of the hydrogel layer) can include and / or be formed from an acrylate. The acrylate can include polyethylene glycol (PEG) having one or more acrylate groups. In some embodiments, the PEG acrylate can be a multi-arm PEG acrylate. In some embodiments, the acrylate can be a PEG diacrylate. The PEG acrylate (e.g., PEG diacrylate) can have a number average molecular weight of greater than 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, 7.5 kDa, 8 kDa, 8.5 kDa, 9 kDa, 9.5 kDa, or 10 kDa. The PEG can have a number average molecular weight of less than 10 kDa, 9.5 kDa, 9 kDa, 8.5 kDa, 8 kDa, 7.5 kDa, 7 kDa, 6.5 kDa, 6 kDa, 5.5 kDa, 5 kDa, 4.5 kDa, 4 kDa, 3.5 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1.5 kDa or 1 kDa. The PEG acrylate can have a number average molecular weight of 1 kDa to 2 kDa, 1.5 kDa to 2.5 kDa, 2 kDa to 3 kDa, 2.5 kDa to 3.5 kDa, 3 kDa to 4 kDa, 3.5 kDa to 4.5 kDa, 4 kDa to 5 kDa, 4.5 kDa to 5.5 kDa, 5 kDa to 6 kDa, 5.5 kDa to 6.5 kDa, 7 kDa to 8 kDa, 7.5 kDa to 8.5 kDa, or 9 kDa to 10 kDa.

[0025] Photoinitiators The acrylate and thiolated polymers can be crosslinked using a photoinitiator, which can include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959).

[0026] The photoinitiator can be a cationic photoinitiator or a radical photoinitiator. Radical photoinitiators are compatible with water and act on molecules containing acrylate or styrene groups. Photoinitiators can include Irgacure 2959, 184, or 651.

[0027] Hydrogel curing conditions The first or second hydrogel layer can be cured using light (e.g., UV light) having an average wavelength of less than 400 nm, 390 nm, 380 nm, 370 nm, 360 nm, 350 nm, 340 nm, 330 nm, 320 nm, or 310 nm. The first or second hydrogel layer can be cured using light having an average wavelength of at least 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, or 400 nm. The first or second hydrogel layer can be cured using light having an average wavelength of 300nm to 320nm, 310nm to 330nm, 320nm to 340nm, 330nm to 350nm, 340nm to 360nm, 350nm to 370nm, 360nm to 380nm, 370nm to 390nm, or 380nm to 400nm.

[0028] The first hydrogel layer can be cured for more than 5, 10, 15, 20, 25 or 30 seconds. The first hydrogel layer can be cured for less than 60, 50, 40, 30, 20 or 10 seconds. The first hydrogel layer can be cured for between 5 and 15 seconds, 10 and 20 seconds, 15 and 25 seconds, 20 and 30 seconds, 25 and 35 seconds, 30 and 40 seconds, 35 and 45 seconds or 40 and 50 seconds.

[0029] The second hydrogel layer can be cured for more than 1 second, 3 seconds, 5 seconds, 8 seconds, or 10 seconds. The second hydrogel layer can be cured for less than 15 seconds, 10 seconds, 8 seconds, 5 seconds, or 3 seconds. The second hydrogel layer can be cured for between 1 second and 5 seconds, 3 seconds and 8 seconds, 5 seconds and 10 seconds, or 8 seconds and 15 seconds.

[0030] Culture medium The cell culture comprising the hydrogel system can include a medium. The medium can include one or more of sodium phosphate, potassium chloride, sodium bicarbonate, ferric nitrate, calcium chloride, or potassium chloride. The medium can include any suitable medium known in the art. Suitable media can include, for example, Dulbecco's phosphate buffered saline, Earle's balanced salts, Hanks' balanced salts, Tyrode's salts, Alsever's solution, Gay's balanced salt solution, modified Krebs-Henseleit buffer, Krebs-Ringer bicarbonate buffer, Pack's saline, Dulbecco's modified Eagle's medium, Dulbecco's modified Eagle's medium / nutrient F-12 Ham's, nutrient mixture F-10 Ham's (Ham's F-10), Medium 199, Eagle's minimum essential medium, RPMI-1640 medium, Ames' medium, BGJb medium (Fitton-Jackson modified), Crick's medium, CMRL-1066 medium, Fisher's medium, Glasgow minimum essential medium (GMEM), Iscove's modified Dulbecco's medium (IMDM), L-15 medium (Leibovitz), McCoy's modified 5A medium, NCTC medium, Swim's S-77 medium, Weymouth's medium, William's medium E, or combinations thereof. Media can be changed or supplemented. The medium can further include albumin, selenium, transferrin, fetuin, sugars, amino acids, vitamins, growth factors, cytokines, hormones, antibiotics, lipids, lipid carriers, cyclodextrin, or combinations thereof.

[0031] Cell type The cells can be cultured, grown or maintained in a culture system comprising a hydrogel layer of a hydrogel system comprising multiple (e.g., two) hydrogel layers. The cells can be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian or plant cells. In some embodiments, the cells are derived from a tumor. The cells can be derived from a tissue. The cells can be hepatic cells, gastrointestinal cells, lymphoid cells, pancreatic cells, kidney cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells, connective tissue cells, corneal cells, urogenital cells, breast cells, germ cells, endothelial cells, epithelial cells, fibroblasts, neural cells, Schwann cells, adipocytes, bone cells, bone marrow cells, chondrocytes, pericytes, mesothelial cells, stromal cells, stem cells, progenitor cells, lymphatic cells, blood cells, endoderm-derived cells, ectoderm-derived cells, or mesoderm-derived cells. The cells can include T cells, B cells, monocytes, macrophages, dendritic cells, eosinophils, basophils, mast cells, or natural killer cells. The cells can be obtained, for example, by excision, biopsy, fine needle aspiration, brushing, or swab.

[0032] Cell labeling The cells can be labeled with a stain or marker. The cells can be labeled with a dye specific to the cell nucleus, a dye specific to the cell membrane, a dye specific to live cells, a dye specific to dead cells, or a fluorescent tag. The fluorescent tag can be attached to an antibody or a derivative thereof. The cells can be labeled with one or more of a lipophilic carbocyanine dye, a bisbenzimide, a xanthene dye, or propidium iodide. The cells can be labeled with, for example, Hoechst 33342, CellBrite NIR 680, Calcein-AM, or Live-or-Dye NucFix Red. In some embodiments, the cells are stained, labeled, or marked before being introduced into the hydrogel system. In other embodiments, the cells are stained after being introduced into the hydrogel system.

[0033] Extracellular matrix components One or more hydrogel layers can include one or more components of the extracellular matrix or its derivatives. For example, a second hydrogel layer (e.g., a layer delivered on top of the first layer) can include one or more components of the extracellular matrix or its derivatives. These components can include proteins produced by cells that hold tissue together, provide tensile strength, or promote cell signaling. Extracellular matrix components can include collagen, fibronectin, laminin, hyaluronate, elastin, or proteoglycans. Extracellular matrix components can be purified from human or animal sources or produced by recombinant methods known in the art.

[0034] Hydrogels The hydrogel may include one or more (or all) of: (1) PEG diacrylate; (2) one or more thiolated polymers (e.g., thiolated gelatin or thiolated PEG); (3) water; (4) laminin; and (5) Irgacure. The hydrogel of the hydrogel system may include at least two layers. The weight ratio of acrylate to thiolated polymer in each layer may be the same. The acrylate may be PEG diacrylate. The thiolated polymer may be thiolated PEG or thiolated gelatin.

[0035] Before curing, the kinetic viscosity of the mixture of thiolated polymer and acrylate was 0.5 mm 2 / s~10mm 2 Before curing, the kinematic viscosity of the first and / or second mixture of thiolated polymer and acrylate may be between 0.5 mm / s. 2 / s~10mm 2 The kinematic viscosity of the mixture may be between 0.1 mm / s. 2 / s, 0.5mm 2 / s, 1.0mm 2 / s, 1.5mm 2 / s, 2.0mm 2 / s, 2.5mm 2 / s, 3.0mm 2 / s, 3.5mm 2 / s, 4.0mm2 / s, 4.5mm 2 / s, 5.0mm 2 / s, 5.5mm 2 / s, 6.0mm 2 / s, 6.5mm 2 / s, 7.0mm 2 / s, 7.5mm 2 / s, 8.0mm 2 / s, 8.5mm 2 / s, 9.0mm 2 / s or 9.5mm 2 The kinematic viscosity of the mixture can exceed 10.0 mm / s. 2 / s, 9.5mm 2 / s, 9.0mm 2 / s, 8.5mm 2 / s, 8.0mm 2 / s, 7.5mm 2 / s, 7.0mm 2 / s, 6.5mm 2 / s, 6.0mm 2 / s, 5.5mm 2 / s, 5.0mm 2 / s, 4.5mm 2 / s, 4.0mm 2 / s, 3.5mm 2 / s, 3.0mm 2 / s, 2.5mm 2 / s, 2.0mm 2 / s, 1.5mm 2 / s, 1.0mm 2 / s, or 0.5 mm 2 The kinematic viscosity of the mixture may be less than 0.5 mm / s. 2 / s~1.5mm 2 / s, 1.0mm 2 / s~2.0mm 2 / s, 1.5mm 2 / s~2.5mm 2 / s, 2.0mm 2 / s~3.0mm 2 / s, 2.5mm 2 / s~3.5mm 2 / s, 3.0mm 2 / s~4.0mm 2 / s, 3.5mm 2 / s~4.5mm 2 / s, 4.0mm 2 / s~5.0mm 2 / s, 4.5mm 2 / s~5.5mm 2 / s, 5.0mm 2 / s~6.0mm 2 / s, 5.5mm 2 / s~6.5mm 2 / s, 6.0mm 2 / s~7.0mm 2 / s, 6.5mm 2 / s~7.5mm 2 / s, 7.0mm 2 / s~8.0mm 2 / s, 7.5mm 2 / s~8.5mm 2 / s, 8.0mm 2 / s~9.0mm 2 / s, 8.5mm 2 / s~9.5mm 2 / s, or 9.0 mm 2 / s~10.0mm 2 / s.

[0036] The weight ratio of acrylate to thiolated polymer in the hydrogel or hydrogel system can be 1:1 or greater than 1:2. The weight ratio of acrylate to thiolated polymer in the first layer or second layer can be 1:2 or less than 1:1. The weight ratio of PEG diacrylate to thiolated gelatin can be 1:2. The weight ratio of PEG diacrylate to thiolated gelatin can be 1:4 to 2:1.

[0037] The weight ratio of thiolated polymer to photoinitiator in the hydrogel can be 40:1, 45:1, 50:1, 55:1 or 60:1. The weight ratio of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone to thiolated gelatin can be 1:20 to 1:100. The weight ratio of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone to thiolated gelatin can be 1:50.

[0038] The weight ratio of thiolated polymer to extracellular matrix component can be 3:1, 4:1 or 5:1. The weight ratio of thiolated gelatin to laminin can be 4:1. The concentration of the extracellular matrix component can be less than 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL or 0.1 mg / mL. The concentration of the extracellular matrix component can be at least 0.1 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, 0.35 mg / mL, 0.40 mg / mL or 0.5 mg / mL.

[0039] array The hydrogel or hydrogel system can be disposed in a well of an array (e.g., a well of a multiwell plate). The array can include a plurality of wells. The wells can be wells of a multiwell plate. The array of wells can include at least 5, 10, 20, 50, 100, 150, 200, 300, 400, 500, 1000, 2000, 3000, 4000, or 5000 wells. The wells can include at least 96 wells, 384 wells, or 1536 wells.

[0040] use The three-dimensional cell culture systems and methods described herein can be used to identify therapies for disease treatment or to identify which tissues are likely to be sensitive to known therapies. The three-dimensional cell culture systems and methods described herein can be used to identify reagents for use in studying cellular interactions and responses in three-dimensional culture. The three-dimensional cell culture systems described herein can be used to create cellular aggregates that can mimic in vivo tissue responses to stimuli.

[0041] In certain aspects, the methods and systems disclosed herein may be useful for determining the course of treatment for a subject. For example, such methods and systems may include screening compounds to identify compounds that result in a patient-specific or tissue-specific response. The subject providing the tissue or cell sample may be healthy. The subject may be positive for a disease. The disease may be an infectious disease. The infectious disease may be viral, bacterial, or fungal. The subject may have an autoimmune disease. The subject may be positive for a tissue-specific disease. The methods and systems may include screens that can be used to identify tissue-specific responses within a particular population of individuals.

[0042] The subject may be positive for a disease. The disease may be a disease of the liver, heart, kidney, pancreas, colon, skin, brain, thymus, bone, or lung. The disease may be cancer. The disease may be bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer. The cancer may be, for example, melanoma, ovarian cancer, hypopharyngeal cancer, kidney cancer, lung cancer, prostate cancer, breast cancer, pancreatic cancer, hepatocellular carcinoma, or head and neck cancer. The disease may be a bacterial infection, a viral infection, or a fungal infection.

[0043] kit The kits may include one or more containers housing one or more components provided in the present disclosure and instructions for use. Specifically, such kits may include one or more compositions described herein, along with instructions describing the intended use and proper use and / or placement of these compositions. The kits may contain components in appropriate concentrations or amounts for carrying out various experiments.

[0044] Computer Systems The present disclosure provides a computer system for implementing the methods provided herein. Figure 5 shows an example of a computer system 1001. The computer system 1001 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 1005, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1001 also includes memory or memory locations 1010 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1015 (e.g., hard disk), a communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1010, the storage unit 1015, the interface 1020, and the peripheral devices 1025 are in communication with the CPU 1005 via a communication bus (solid lines), such as a motherboard. The storage unit 1015 may be a data storage unit (or data repository) for storing data. Computer system 1001 may be operatively coupled to a computer network ("network") 1030 using communication interface 1020. Network 1030 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. Network 1030 may be a telecommunications and / or data network in some cases. Network 1030 may include one or more computer servers that may enable distributed computing, such as cloud computing. Network 1030 may be able to implement a peer-to-peer network in some cases using computer system 1001, thereby enabling devices coupled to computer system 1001 to operate as clients or servers.

[0045] The CPU 1005 may execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. The instructions may be directed to the CPU 1005, which may then be programmed or configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 1005 may include fetch, decode, execute, and writeback.

[0046] The CPU 1005 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1001 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0047] The storage unit 1015 can store files such as drivers, libraries, and saved programs. The storage unit 1015 can store user data such as user preferences and user programs. The computer system 1001 can optionally include one or more additional data storage units external to the computer system 1001, such as located on a remote server in communication with the computer system 1001 via an intranet or the Internet.

[0048] The computer system 1001 can communicate with one or more remote computer systems via the network 1030. For example, the computer system 1001 can communicate with a user's remote computer system (a remote cloud server). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a phone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®, or a personal digital assistant). A user can access the computer system 1001 via the network 1030.

[0049] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored on electronic storage locations of the computer system 1001, such as, for example, on the memory 1010 or electronic storage unit 1015. The machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by the processor 1005. In some cases, the code may be retrieved from the storage unit 1015 and stored on the memory 1010 for immediate access by the processor 1005. In some circumstances, the electronic storage unit 1015 may be eliminated and the machine executable instructions are stored in the memory 1010.

[0050] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or it may be compiled during run-time. The code may be provided in a programming language that may be selected to enable the code to be executed in a pre-compiled or as-compiled manner.

[0051] Aspects of the systems and methods provided herein, such as the computer system 1001, can be embodied in programming. Various aspects of the technology can be thought of as an "article of manufacture" or "product" typically in the form of machine (or processor) executable code and / or associated data, carried or embodied on some type of machine-readable medium. The machine executable code can be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, processor, or the like, such as various semiconductor memories, tape drives, disk drives, etc., that can provide non-transitory storage for software programming at any time, or associated modules thereof. All or a portion of the software can sometimes be communicated over the Internet or various other telecommunications networks. Such communication can, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, another type of medium that may carry software elements includes optical, electrical and electromagnetic waves, such as those used across physical interfaces between local devices, over wired and optical networks, and in various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered software-bearing media. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0052] Thus, a machine-readable medium such as a computer executable code may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of any computer(s) such as may be used to implement the databases shown in the figures. Volatile storage media include dynamic memory such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire and optical fibers, including the wires that comprise a bus within a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transports data or instructions, a cable or link that transports such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0053] The computer system 1001 may include or communicate with an electronic display 1035 that includes, for example, a user interface (UI) 1040 for providing an electronic output of the identified gene fusions. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces. The computer system 1001 may include or communicate with, for example, a robotic arm for performing one or more mixing, depositing, or imaging steps. Examples of robotic arms include, but are not limited to, a multi-pipette arm, a camera, and a palletizer for moving multi-well plates.

[0054] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 1005. EXAMPLES

[0055] Example 1 - Preparation of gel The pre-curing mixture for forming a hydrogel can often naturally wick up at the sides of the well, forming a concave meniscus within the well. In the pre-curing mixture containing cells, the cells can wick to concentrate at the periphery of the well, leaving the center of the well with a relatively low cell density. This is less desirable for imaging and promoting cell-cell interactions.

[0056] Disclosed herein is a two-step plating method in which an acellular mixture is added to the well and then crosslinked within the well to form a first hydrogel layer, leaving a concave cavity (or "basket" space) in the center of the well. A second mixture containing cells is then added to the well on top of the first hydrogel layer. After hardening, the second mixture forms a second hardened hydrogel layer having a top surface that is less concave (e.g., a top surface that is substantially planar) than the top surface of the first hydrogel layer. The cells are distributed throughout the second layer, resulting in a relatively uniform distribution of cells throughout the xy plane of the well. Images can be acquired using z-stack imaging, in which multiple (e.g., 10) images in the xy axis are taken at specified distances (e.g., 100 microns) apart in the z axis (from the top to the bottom of the well) and then projected to create a single image.

[0057] The mixtures forming the first and second hydrogel layers were formulated according to the following table. [Table 1]

[0058] 125 mg of AC-PEG-AC (e.g., Formula I) was added to a 1.5 mL microcentrifuge tube. 0.2% Irgacure solution was added to the tube containing AC-PEG-AC and mixed. 50 mg of a Gelin-S bottle was resuspended in 500 μL of 0.1% Irgacure solution. The AC-PEG-AC solution and Gelin-S mixture were combined and immediately plated into a 384-well source plate. Immediately after plating the first layer, an intensity of 250 mW / cm was applied. 2 The resin was cured with UV light (320nm to 390nm) for 15 seconds. [ka]

[0059] The cells were stained as described in Example 2 and centrifuged at 300×g for 5 minutes to produce a cell pellet. The supernatant was removed from the cell pellet.

[0060] To form the second cured hydrogel layer, the AC-PEG-AC solution, laminin solution, and Gelin-S mixture were combined with the pellet of stained cells as shown in Table 1. The stained cells were resuspended in the gel mixture to create the seeded gel mixture. The seeded gel mixture was plated on top of the first layer and immediately illuminated with an intensity of 250 mW / cm. 2 The mixture was cured with UV light (320 nm to 390 nm) for 10 seconds. The kinematic viscosity of the first layer mixture and the second layer mixture when added to the well was approximately 9.65 mm. 2 / s. As seen in Figure 1B (DAPI channel only), the second layer of stained cells resulted in a more uniform distribution of cells throughout the well compared to a single layer of seeded gel in the same well (Figure 1A, DAPI channel only). Images were acquired on a confocal microscope using a 4x air objective, using the DAPI channel corresponding to excitation of the fluorophore Hoechst. Images can contain a pseudocomposite of four channels: DAPI, Cy5, FITC, and Cy3, corresponding to excitation of the fluorophores Hoechst, CellTracker Deep Red, Calcein AM, and NucFix red, respectively. For each channel, 10 images were taken at 100 micron intervals in the z-axis and projected using maximum projection to create a single image (e.g., z-stack imaging). As seen in Figures 4A and 4B, cells were counted and distribution quantified throughout each well, with Figure 4B showing the distribution within a bilayer hydrogel formed in this example, while Figure 4A shows the distribution of cells in a similar monolayer hydrogel of the same volume as the hydrogel of Figure 4B, formed from ingredients from the second layer of the hydrogel shown in Table 1. Maximum projection images from all wells of each condition (352 per condition) were cropped into five equally sized boxes corresponding to the upper left corner, lower left corner, lower right corner, upper right corner, and center of the image. Cell counts for each cropped image were performed using a custom algorithm, and counts for all wells were used to create box plots. An exemplary cross-section of the monolayer 210 hydrogel of Figure 3B in well 200 compared to the bilayer hydrogel of Figure 3A in well 100 is seen in Figure 3. The wells 100, 200 in Figures 3A and 3B have the same volume of hydrogel, but the top surface 211 of the monolayer 210 is recessed relative to the top surface 121 of the second layer of a similar two-layer structure shown in Figure 3A. In some embodiments, the top surface 121 of the second layer is substantially planar.

[0061] Example 2 - Cell staining with Hoechst nuclear stain and CellBrite cell membrane dye A T75 flask of HCT-116 cells (colon cancer cell line (ATCC CCL-247)) was grown in DMEM supplemented with 10% FBS and 1× Primocin for 4 days at 37° C. since the last passage. The cells were detached from the bottom of the flask using 0.25% trypsin-EDTA by washing the cells twice with 2 mL trypsin-EDTA, followed by adding 2 mL trypsin-EDTA and returning the flask to the 37° C. incubator for 7 minutes. The cells were then washed down to the corners of the flask using 4 mL of fresh warm medium. The cells and medium were then transferred to a 15 mL conical tube. The conical tube was centrifuged at 300×g for 5 minutes to pellet the cells. The cells were then counted using an automated cell counter. The cells were then stained with Hoechst 3342 (Abcam ab228551 to identify cell nuclei;) and CellBrite NIR680 (Biotium 20070 to identify cell membranes and cytosol) by resuspending the cells in PBS and staining solution (1 μL / mL CellBriTe NIR) in a 15 mL conical tube.

[0062] The cells were incubated with the stain for 30-60 minutes at 37° C. The cells were then pelleted by centrifugation at 300×g for 5 minutes and the staining mixture was removed to produce a pellet of stained cells for use in Example 1 above.

[0063] Example 3 - Cell viability Due to the synthetic nature of the PEG component, PEG-based hydrogels are advantageous because they have relatively little lot-to-lot variability. However, hydrogels based entirely on PEG components are acidic, which can adversely affect the viability of 3D cell cultures in such hydrogels. Furthermore, adjusting the pH of PEG-only hydrogels to the neutral range can reduce the stiffness and density to the point where such hydrogels are unable to retain cells inside. Some specific gel formulations described herein utilize 3.4 kDa PEG-diacrylate, which retains the consistency (e.g., minimal lot-to-lot variability) of synthetic components as described above. The pore sizes generated by 3.4 kDa PEGDA are large enough to allow diffusion of nutrients and dyes, yet small enough to retain cells, including small immune cells that may be necessary for screening immunotherapies.

[0064] The viability of a tumor cell line (HCT-116) was assessed at 3 and 5 days of growth in Gelin-S and Ac-PEG-Ac based gels.

[0065] Gelin gel was prepared as follows: 2.5% w / v, 3.4 kDa Ac-PEG-Ac, 0.1% w / v Gelin-S, 0.25 mg / mL laminin, and 0.1% w / v Irgacure 2959.

[0066] HCT-116 cells were resuspended in the gel at a concentration of 5 M cells / mL. 5 μL of the gel+cell mixture was added per well to 48 wells of two 384-well microplates (24 wells per plate). The gel was crosslinked by exposure to UV light for 15 seconds. 50 μL of Dulbecco's modified essential medium (DMEM) supplemented with 10% FBS was added to each well. The microplate was then placed in an incubator at 37°C and 5% CO2. Three days after cell seeding, one microplate was removed from the incubator and a luminescence-based viability assay (CellTiter Glo 3D, Promega) was performed according to the manufacturer's specifications. Luminescence was read using a Varioskan plate reader (Thermo Scientific). The blank was subtracted from the luminescence values, and the mean luminescence and standard error of the mean were calculated across all 24 samples. The mean luminescence after 3 days was 3,198,142.9 with an SEM of 229,246.8. After 5 days of culture, the second microplate was removed from the incubator and viability was assessed using the same luminescence assay. After 5 days, the mean luminescence was 4,754,192.3 with an SEM of 913,546.9. This represents a 48.7% increase compared to the value observed on day 3. Figure 2A shows the proliferation of HCT-116 cells in Gelin-S and Ac-PEG-Ac based gels. These data suggest that cells in Gelin-S and Ac-PEG-Ac based gels are viable on day 3 of culture and continue to proliferate for 5 days of culture.

[0067] Example 4 - Cell viability: Comparison with PEG+RGD hydrogel Gelin-S+Ac-PEG-Ac ("Gelin") hydrogel was compared to Ac-PEG-Ac+GRGDSPC ("RGD") hydrogel.

[0068] Gelin gel was prepared as follows: 2.5% w / v, 3.4 kDa Ac-PEG-Ac, 0.1% w / v Gelin-S, 0.25 mg / mL laminin, and 0.1% w / v Irgacure 2959.

[0069] RGD gels were prepared as follows: 10% w / v 10 kDa Ac-PEG-Ac, 0.05% w / v Irgacure 2959, 2 mM GRGDSPC peptide, and 0.25 mg / mL laminin.

[0070] HCT-116 cells were resuspended in each gel type at a concentration of 5 M cells / mL. For each gel type, 5 μL of gel+cell mixture was added per well to 24 wells of a 384-well microplate. Gels were crosslinked by exposure to UV light for 15 seconds. 50 μL of Dulbecco's modified essential medium (DMEM) supplemented with 10% FBS was added to each well. The microplate was then placed in an incubator at 37°C and 5% CO2. 72 hours after cell seeding, the microplate was removed from the incubator and a luminescence-based viability assay (CellTiter Glo 3D, Promega) was performed according to the manufacturer's specifications. Luminescence was read using a Varioskan plate reader (Thermo Scientific). Blanks were subtracted from the luminescence values, and the mean luminescence and standard error of the mean were calculated across all 24 samples for each gel type. The mean luminescence of the RGD-based gel samples was -3,1647.3 with an SEM of 4,128.5, whereas the mean of the Gelin samples was 3,198,142.9 with an SEM of 229,246.8. Figure 2B shows a comparison of the Gelin-S and RGD-based gels. These data suggest that cells in the RGD condition became nonviable after 3 days in culture, whereas cells in the Gelin condition remained viable.

[0071] Example 5 - Cell viability: Comparison of PEG+RGD and pH-balanced PEG+RGD hydrogels As observed in Example 4, cells do not survive in RGD gels. The typical pH of RGD gels was observed to be in the range of 4-5, and it was hypothesized that low pH may have a negative effect on cell viability. To test whether RGD gels could be improved by adjusting the pH to the physiological range of 7.4, 1 M NaOH was added dropwise to the RGD gel formulation (above) and the pH was measured after each addition. The total amount of NaOH added was negligible relative to the total volume of the gel. These pH balanced RGD-based hydrogels ("Balanced RGD") were compared to non-pH balanced Gelin-S+Ac-PEG-Ac ("Gelin") hydrogels and Ac-PEG-Ac+GRGDSPC ("RGD") hydrogels. Gelin and RGD-based gels were prepared as described above. The pH of the Gelin gels was measured to be 7.3 without any modification. HCT-116 cells were resuspended in the gels at a concentration of 5 M cells / mL. 5 μL of gel+cell mixture was added per well to 72 wells (24 wells per plate) of three 384-well microplates. Gels were crosslinked by exposure to UV light for 15 seconds. 50 μL of Dulbecco's modified essential medium (DMEM) supplemented with 10% FBS was added to each well. The microplates were then placed in an incubator at 37°C and 5% CO2. Three days after cell seeding, the microplates were removed from the incubator and a luminescence-based viability assay (CellTiter Glo 3D, Promega) was performed according to the manufacturer's specifications. Luminescence was read using a Varioskan plate reader (Thermo Scientific). Blanks were subtracted from luminescence values ​​and the mean luminescence and standard error of the mean were calculated across all 24 samples. After 3 days, the mean luminescence was -31647.26 with an SEM of 4128.54 in the RGD condition, 3206783.46 with an SEM of 232717.981 in the balanced RGD condition, and 3198142.86 with an SEM of 229246.80 in the Gelin condition. These data suggest that a neutral pH within the physiological range is important for maintaining cell viability.Both pH-balanced RGD gels and Gelin gels meet this requirement. However, unlike Gelin gels, it was observed that RGD gels with neutral pH do not maintain their structure after several days in culture. Figure 2C shows a comparison of viability between Gelin-S-based gels, RGD-based gels, and pH-balanced RGD gels. By day 5, the pH-balanced RGD gels were completely dissolved, allowing the cells to drop through the gel and attach to the bottom of the plate. Furthermore, pH balancing of these gels requires an additional step and can be difficult to automate. This demonstrates that Gelin gels have the desired combination of sustained structural integrity and neutral pH.

[0072] Example 6 - Plating of Hydrogels Due to the small size of the wells and the polarity of the hydrogel fluid in question, the hydrogel fluid tends to stick to or wick up into one or two corners of the well, rather than covering the surface of the well. Many of the steps performed involving the bilayer are designed to avoid this scenario, as it creates major problems in collecting and analyzing image-based data. The gel components of the first layer were mixed together and added in equal volumes to all eight wells in column 1 of a standard U-bottom 96-well microplate. An Opentrons liquid handling robot, running a custom program and configured with a second generation P208 channel pipette, was used to seed a flat-bottom 384-well microplate as follows: First, the robot simultaneously aspirated 20 microliters (μL) of gel from the 96-well plate and moved it to the 384-well plate such that the tip was positioned 0.2 mm above the bottom of wells A1, C1, E1, G1, I1, K1, M1, and O1. Next, 20 μL was dispensed into each channel to evenly coat the bottom of the well with gel, and then 0.5 seconds was waited before aspirating 15 μL, leaving a 5 μL layer of gel. The robot was then moved back to column 1 of the 96-well source plate, aspirated 5 μL (the tip now contained 20 μL), and moved to wells B1, D1, F1, H1, J1, L1, N1, and P1 of the 384-well plate. The robot then dispensed 20 μL as before and removed 15 μL, so that all wells in column 1 of the 384-well plate now contained a 5 μL layer of gel. This process was repeated for the remaining 23 columns of the 384-well plate. The total time for this procedure was approximately 20 minutes. The plate was then exposed to UV light for 15 seconds to crosslink the gel. The components of the second layer of gel were then mixed with cells from the patient samples and added in equal volumes to each well of column 2 of the 96-well source plate. The above seeding process was then repeated for the second layer of gel, such that at the end of the process each well of the 384-well plate received 5 μL of a second layer of gel containing patient cells. Again, this process took approximately 20 minutes. The plate was then exposed to 10 seconds of UV light. From start to finish, the entire procedure lasted approximately 1 hour.

[0073] Example 7 - Plating of Hydrogels A 384-well microplate was placed on the plate carrier of a MultiFlo FX automated liquid dispenser equipped with a 5-μL eight-channel peristaltic pump cassette. A 50 milliliter (mL) conical tube filled with 40 mL of deionized water was attached near the dispenser and primed by placing the end of the cassette tube in the water to fill the tube's dead volume with the water to be dispensed. A custom program was used to dispense 15 microliters (μL) of water into each well of the 384-well plate. The dispensing process lasted approximately 30 seconds. The water was then purged from the tube. The liquid in the wells was then manually brushed off, leaving only a residue of water covering the bottom of the well. Next, the gel components of the first layer of the hydrogel were mixed together in a 50-mL conical tube. The tube was then attached near the MultiFlo and the end of the cassette tube was placed in the gel and primed. A custom program was used to dispense 5 μL of gel into each well of the 384-well plate, the process took approximately 15 seconds. The plate was then cross-linked under UV light for 15 seconds. The components of the second layer of gel were then mixed with cells from the patient sample and added to a second 50 mL conical tube. As with the first layer, 5 μL was dispensed into each well of the microwell plate. The gel was then exposed to UV light for 10 seconds to cross-link. This process lasted approximately 20 minutes from start to finish.

[0074] Example 8 - Laminin-free hydrogels and plating of hydrogels Hydrogels were made essentially as described in Example 1, except that the following ingredients were used as shown in Table 2: [Table 2]

[0075] 100 mg of AC-PEG-AC (e.g., Formula I) was added to a 1.5 mL microcentrifuge tube. 0.1% Irgacure solution was added to the tube containing AC-PEG-AC and mixed. 50 mg of a bottle of Gelin-S was resuspended in 2.5 mL of 0.1% Irgacure solution. The AC-PEG-AC solution and Gelin-s mixture were combined and immediately plated into a 384-well source plate. Immediately after plating the first layer, an intensity of 250 mW / cm was applied. 2 The resin was cured with UV light (320nm to 390nm) for 15 seconds.

[0076] The cells were stained as described in Example 2 and centrifuged at 300×g for 5 minutes to produce a cell pellet. The supernatant was removed from the cell pellet.

[0077] To form the second cured hydrogel layer, the AC-PEG-AC solution and Gelin-S mixture were combined with the pellet of stained cells as shown in Table 2. The stained cells were resuspended in the gel mixture to create the seeded gel mixture. The seeded gel mixture was plated on top of the first layer and immediately illuminated at 250 mW / cm. 2 The coating was cured for 15 seconds under UV light (320 nm to 390 nm) of 1000 nm intensity.

[0078] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in carrying out the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for producing a three-dimensional hydrogel system in a well, the method comprising: (a) forming a concave meniscus in a well by delivering a first mixture into the well, the first mixture comprising a first acrylate and a first thiolated polymer; (b) crosslinking the first acrylate and the first thiolated polymer of the first mixture to form a first cured hydrogel layer comprising a concave upper surface; (c) delivering a second mixture comprising a second acrylate and a second thiolated polymer into the well on top of the first cured hydrogel layer; (d) crosslinking the second acrylate and the second thiolated polymer of the second mixture to form a second cured hydrogel layer on top of the first cured hydrogel layer, the second cured hydrogel layer including an upper surface that is less concave than the concave upper surface of the first cured hydrogel layer.

2. The method of claim 1 , wherein the top surface of the second curable layer is substantially planar.

3. 3. The method of claim 1 or 2, wherein crosslinking the first acrylate and the first thiolated polymer to form the first cured hydrogel layer comprises exposing the first mixture to ultraviolet light while the first mixture is disposed in the well and before the second mixture is delivered to the well.

4. 4. The method of claim 1, wherein crosslinking the second acrylate and the second thiolated polymer to form the second cured hydrogel layer comprises exposing the second mixture to ultraviolet light while both the first cured hydrogel layer and the second mixture are disposed within the well.

5. 5. The method of claim 4, wherein the first mixture is exposed to ultraviolet light for a longer period of time than the second mixture is exposed to ultraviolet light.

6. 6. The method of claim 5, wherein the second mixture is exposed to ultraviolet light for less than 12 seconds and the first mixture is exposed to ultraviolet light for more than 12 seconds.

7. The method of any of claims 1 to 6, wherein the first mixture and the second mixture are exposed to UV light for between 10 seconds and 20 seconds.

8. The method according to any one of claims 4 to 7, wherein the ultraviolet light has an average wavelength of from 320 nm to 390 nm.

9. The ultraviolet light is 200 to 300 mW / cm ２ The method according to any one of claims 4 to 8, wherein the strength of the

10. The method of any one of claims 1 to 9, wherein the second mixture further comprises a plurality of cells.

11. The method of any one of claims 1 to 10, wherein the plurality of cells are non-aggregated cells.

12. 12. The method of claim 10 or 11, wherein the plurality of cells is stained prior to (c).

13. 13. The method of claim 12, wherein the stained plurality of cells are stained with one or more of: (i) a dye specific to cell nuclei, (ii) a dye specific to cell membranes, (iii) a dye specific to live cells, and (iv) a dye specific to dead cells.

14. 14. The method of claim 13, wherein the stained plurality of cells are stained with one or more of a lipophilic carbocyanine dye, a bisbenzimide, a xanthene dye, or propidium iodide.

15. 12. The method of claim 10 or 11, wherein the plurality of cells are marked with a fluorescent tag prior to (c).

16. The method of any one of claims 1 to 15, wherein the second mixture does not contain laminin.

17. 16. The method of any one of claims 1 to 15, wherein the second mixture further comprises laminin, such that laminin is non-covalently distributed within the second cured hydrogel.

18. The method of any one of claims 1 to 17, wherein the first mixture further comprises a photoinitiator.

19. The method of claim 18, wherein the photoinitiator comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

20. The method of any one of claims 1 to 19, wherein the second mixture further comprises a photoinitiator.

21. The method of claim 20, wherein the photoinitiator comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

22. The method according to any one of claims 1 to 21, further comprising the step of adding a cell culture medium to the three-dimensional hydrogel system.

23. 23. The method of claim 22, wherein the cell culture medium comprises one or more of sodium phosphate, potassium chloride, sodium bicarbonate, ferric nitrate, calcium chloride, or potassium chloride.

24. The kinematic viscosity of the first mixture when delivered to the well is less than 0.5 mm ２ / s ~ 10mm ２ The method according to any one of claims 1 to 23, wherein

25. The kinematic viscosity of the second mixture when delivered to the well is less than 0.5 mm ２ / s ~ 10mm ２ The method according to any one of claims 1 to 24, wherein:

26. The method of any one of claims 1 to 25, wherein one or both of the first acrylate and the second acrylate is a multi-arm PEG acrylate.

27. The method of any one of claims 1 to 26, wherein one or both of the first acrylate and the second acrylate is a PEG diacrylate.

28. 28. The method of claim 27, wherein the PEG diacrylate of the first mixture has a number average molecular weight greater than 3 kilodaltons.

29. 29. The method of claim 28, wherein the PEG diacrylate of the first mixture has a number average molecular weight of 3 to 4 kilodaltons.

30. 28. The method of claim 27, wherein the PEG diacrylate of the second mixture has a number average molecular weight greater than 3 kilodaltons.

31. 31. The method of claim 30, wherein the PEG diacrylate of the second mixture has a number average molecular weight of 3 to 4 kilodaltons.

32. The method of any one of claims 1 to 31, wherein one or both of the first thiolated polymer and the second thiolated polymer is a thiolated gelatin.

33. 33. The method of claim 32, wherein the thiolated gelatin has a degree of thiolation of 20% to 30%.

34. The method according to any one of claims 27 to 33, wherein the weight ratio of PEG diacrylate to thiolated gelatin in the second mixture is from 1:4 to 2:1, such as 1:

2.

35. The method according to any one of claims 27 to 33, wherein the weight ratio of PEG diacrylate to thiolated gelatin in the second mixture is from 1:1 to 8:1, such as from 2:1 to 8:

1.

36. 36. The method of any one of claims 27 to 35, wherein the second mixture comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in a weight ratio of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone to thiolated gelatin of 1:20 to 1:100, such as 1:

50.

37. 37. The method of any one of claims 27 to 36, wherein the second mixture comprises laminin in a weight ratio of thiolated gelatin to laminin of 1:1 to 8:1, such as 4:

1.

38. 38. The method of any one of claims 27 to 37, wherein the concentration of laminin in the second mixture is at least 0.1 mg / mL.

39. 39. The method of any one of claims 27 to 38, wherein the concentration of laminin in the second mixture is 1.0 mg / mL or less.

40. 40. The method of any one of claims 38 to 39, wherein the concentration of laminin in the second mixture is 0.2 to 1.0 mg / mL or 0.2 to 0.6 mg / mL.

41. The method of any one of claims 1 to 40, wherein the well is one well of an array of wells on a multi-well plate.

42. 42. The method of claim 41, wherein the array of wells comprises at least 96 wells.

43. 42. The method of claim 41 , wherein the array of wells comprises at least 384 wells.

44. 42. The method of claim 41 , wherein the array of wells comprises at least 1,536 wells.

45. 42. The method of claim 41, wherein steps (a) through (d) are completed within one hour across 384 wells.

46. 42. The method of claim 41, wherein steps (a) through (b) are completed in less than 20 minutes across 384 wells.

47. 42. The method of claim 41, wherein steps (c) through (d) are completed within 20 minutes across 384 wells.

48. 48. The method of any one of claims 1 to 47, wherein delivering the first mixture comprises delivering via a robotic system.

49. 49. The method of any one of claims 1 to 48, wherein the step of delivering the first mixture comprises the steps of: (i) dispensing a first amount of the first mixture into the well; and (ii) aspirating a second amount of the first mixture from the well.

50. 50. The method of claim 49, wherein the first amount is greater than the second amount.

51. The method of any one of claims 1 to 50, wherein (b) comprises exposing the wells to UV light.

52. 52. The method of any one of claims 1 to 51, wherein delivering the second mixture comprises delivering via a robotic system.

53. 53. The method of any one of claims 1-52, wherein the step of delivering the second mixture comprises the steps of: (i) dispensing a first amount of the second mixture into the well; and (ii) aspirating a second amount of the second mixture from the well.

54. 54. The method of claim 53, wherein the first amount is greater than the second amount.

55. 55. The method of any one of claims 1-54, wherein (d) comprises exposing the wells to UV light.

56. 56. The method of any one of claims 1 to 55, wherein the second mixture comprises cells.

57. 57. The method of claim 56, wherein the cells are substantially uniformly distributed throughout the second cured hydrogel layer.

58. 58. A hydrogel formed by the method of any one of claims 1 to 57.

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