Three-dimensional cell culture system and method

Abstract

A high throughput three dimensional cell culture system and related methods are disclosed herein. The cell culture system is configured to provide relatively uniform cell distribution across the surface of the well to improve imaging analysis.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 309,950, filed on February 14, 2022, which is incorporated herein by reference. Background Art

[0003] Three-dimensional (3D) cell culture is a promising technology for the development and identification of therapeutic treatments. Relative to two-dimensional cell culture systems, the benefits of three-dimensional cell culture can be to more closely simulate biological functions. High-quality cell sample preparation is very important for various clinical and research applications. 3-D cell culture can closely represent in vivo properties. High-throughput 3D cell culture methods and systems that can effectively and reproducibly culture cells at an appropriate cost are needed. Summary of the invention

[0004] Disclosed herein is a method for producing 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, thereby forming a concave meniscus within the well; (b) crosslinking the first acrylate and the first thiolated polymer of the first mixture to form a first cured hydrogel layer, wherein the first cured hydrogel layer comprises 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, wherein the second cured hydrogel layer comprises an upper surface that is less concave than the concave upper surface of the first cured hydrogel layer. The upper surface of the second cured layer can be substantially planar.

[0005] Crosslinking the first acrylate and the first thiolated polymer to form the first cured hydrogel layer may include exposing the first mixture to ultraviolet light while the first mixture is disposed (positioned) in the hole and before the second mixture has been delivered into the hole. Crosslinking the second acrylate and the second thiolated polymer to form the second cured hydrogel layer may include exposing the second mixture to ultraviolet light while both the first cured hydrogel layer and the second mixture are disposed in the hole. The time period for exposing the first mixture to ultraviolet light may be longer than the time period for exposing the second mixture to ultraviolet light. The second mixture may be exposed to ultraviolet light for less than 12 seconds, and the first mixture may be exposed to ultraviolet light for more than 12 seconds. In some embodiments, the second mixture may be exposed to ultraviolet light for less than 15 seconds, and the first mixture may be exposed to ultraviolet light for more than 15 seconds. In some embodiments, the second mixture and the first mixture are exposed to ultraviolet light for 5 seconds to 20 seconds (e.g., 10 seconds to 20 seconds), respectively. The wavelength of the ultraviolet light may be 320nm and 390nm. The intensity of the ultraviolet light may be 250mW / cm 2 .

[0006] The second mixture may further include a plurality of cells. The plurality of cells may be unaggregated cells. The plurality of cells may have been stained before (c). The stained plurality of cells may be stained with one or more of the following: (i) a dye specific to the nucleus, (ii) a dye specific to the cell membrane, (iii) a dye specific to living cells, and (iv) a dye specific to dead cells. The plurality of cells may be stained with one or more of the following: lipophilic carbocyanine dyes, bisbenzimides, xanthene dyes or propidium iodide. Before the second mixture is delivered to the hole, the plurality of cells may be labeled with a fluorescent tag.

[0007] The second mixture may optionally or further include laminin, so that the laminin is non-covalently distributed in the second solidified hydrogel. In some embodiments, the second mixture does not contain 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.

[0008] The method may further include adding a cell culture medium to the three-dimensional hydrogel system. The cell culture medium may include one or more of the following: sodium phosphate, potassium chloride, sodium bicarbonate, ferric nitrate, calcium chloride, or potassium chloride. The kinematic viscosity of the first mixture when delivered into the well may be between 0.5 square millimeters per second and 10 square millimeters per second. The kinematic viscosity of the second mixture when delivered into the well may be between 0.5 square millimeters per second and 10 square millimeters per second.

[0009] One or both of the first acrylate and the second acrylate can be multi-arm PEG acrylate. One or both of the first acrylate and the second acrylate can be PEG diacrylate. The number average molecular weight of the PEG diacrylate of the first mixture can be greater than three thousand Daltons. In some embodiments, the number average molecular weight of the PEG diacrylate of the first mixture can be between three thousand Daltons and four thousand Daltons. In some embodiments, the number average molecular weight of the PEG diacrylate of the first mixture can be between about 7 thousand Daltons and 15 thousand Daltons. The number average molecular weight of the PEG diacrylate of the second mixture can be greater than three thousand Daltons. In some embodiments, the number average molecular weight of the PEG diacrylate of the second mixture can be between three thousand Daltons and four thousand Daltons. In some embodiments, the number average molecular weight of the PEG diacrylate of the second mixture can be between 7 thousand Daltons and 15 thousand Daltons.

[0010] One or both of the first thiolated polymer and the second thiolated polymer can be thiolated gelatin. The degree of thiolation of the thiolated gelatin can be between 20% and 30%. In some embodiments, the weight ratio of PEG diacrylate to thiolated gelatin in the second mixture is between 1:4 and 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 and 8:1 (e.g., between 2:1 and 8:1). The second mixture can include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and the weight ratio of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone to thiolated gelatin is between 1:20 and 1:100, such as 1:50.

[0011] The second mixture may include laminin, and the weight ratio of thiolated gelatin to laminin is between 1:1 and 8:1, such as 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 no greater than 1.0 mg / mL. The concentration of laminin in the second mixture may be between 0.2 mg / mL and 1.0 mg / mL or between 0.2 mg / mL and 0.6 mg / mL.

[0012] The hole can be a hole in a hole array of a multi-well plate. The hole array can include at least 96 holes. The hole array can include at least 384 holes. The hole array can include at least 1536 holes. The delivery of the first mixture and the second mixture can be completed on 384 holes in less than one hour. The delivery of the first mixture can be completed on 384 holes in less than 20 minutes. The delivery of the second mixture can be completed on 384 holes in less than 20 minutes. The delivery of the first mixture and the second mixture can be completed on 1536 holes in less than one hour. The delivery of the first mixture can be completed on 1536 holes in less than 30 minutes. The delivery of the second mixture can be completed on 1536 holes in less than 30 minutes.

[0013] The delivery of the first mixture may include delivery by a robotic system. The delivery of the second mixture may include delivery by a robotic system. Delivering the first mixture may 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 may be greater than the second amount. Delivering the second mixture may 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 may be greater than the second amount.

[0014] The second mixture may include cells. The cells may be substantially uniformly distributed throughout the second solidified hydrogel layer. Disclosed herein is a hydrogel formed by a method described herein.

[0015] Incorporated by Reference

[0016] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Various features of the present 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 which sets forth illustrative embodiments in which the principles of the invention are utilized, and in the accompanying drawings:

[0018] Figure 1A A monolayer hydrogel is shown with cells concentrated at the pore walls.

[0019] Figure 1B The bilayer hydrogel shows that the cells are evenly dispersed throughout the entire area of ​​the wells.

[0020] Figure 2A is a graph showing the growth of HCT-116 cells in a gel made of Gelin-S and Ac-PEG-Ac.

[0021] Figure 2B is a graph showing the activity comparison between Gelin-S based gel and RGD based gel.

[0022] Figure 2C is a graph showing the activity comparison between Gelin-S based gel, RGD based gel and pH balanced RGD gel.

[0023] Figure 3A and 3B is a cross-sectional view of a hole 100, 200 containing a hydrogel component. Specifically, Figure 3A A well 100 is depicted that has been filled with two hydrogel layers, wherein the upper surface 111 of the first layer 110 has a greater concavity than the upper surface 121 of the second layer 120 that has been deposited on the first layer 110 . Figure 3B Describes a filled Figure 3A The hydrogel in FIG. 1 has a pore 200 of the same total volume of hydrogel, but the pore 200 is formed by depositing only a monolayer 210. The concavity of the upper surface 211 of this monolayer 210 is greater than that of Figure 3A The upper surface 111 of the second layer 110 of the double-layer structure shown is more concave.

[0024] Figure 4 shows the deposition of a single hydrogel layer ( Figure 4A ) and double-layer hydrogel deposition ( Figure 4B ) were compared with the cell counting analysis.

[0025] Figure 5 An example of a computer system configured to implement the methods provided herein is shown. DETAILED DESCRIPTION

[0026] Many hydrogel-forming mixtures are too viscous and spontaneously crosslink into a gel form at room temperature within a short time scale (e.g., minutes). This presents challenges for automation, which typically relies on liquid handling robots that have difficulty handling viscous liquids and materials with inconsistent properties. A hydrogel manufacturing method is needed in which the hydrogel-forming mixture remains in a relatively low viscosity form so that the hydrogel can be dispensed into a high-throughput compatible well array prior to crosslinking while maintaining a relatively uniform dispersion of cells within the hydrogel system or a portion thereof.

[0027] Disclosed herein are systems and methods for high-throughput three-dimensional cell culture. The mixture forming the hydrogel can naturally wick to the side of the hole, thereby concentrating the hydrogel (and the cells in the hydrogel) on the outer wall (e.g., periphery) of the hole. The methods and systems described herein are configured to explain the tendency of the mixture forming the hydrogel to concentrate the hydrogel (and the cells therein) on or near the outer wall of the hole by using a double-layer deposition process, which allows cells to grow robustly in the center of the hole (and along the periphery of the hole). Therefore, the cells of the present disclosure can be more evenly dispersed in the entire area of ​​the hole. The manufacturing method may include curing the first layer of hydrogel in the hole to produce a concave upper surface. The curing process may involve a photoinitiator. Curing may include light exposure (e.g., exposure to UV light). The method may include depositing a second layer of hydrogel including cells on top of the first layer and curing the second layer of hydrogel. The second layer may include one or more proteins (e.g., laminin) configured to interact with cells (such as mammalian cells). The first layer and the second layer may have the same refractive index after curing. The porosity of the hydrogel can be configured to be large enough to allow the diffusion of nutrients and dyes, but small enough to retain cells, such as immune cells.

[0028] Thiolated polymer

[0029] The hydrogel (of the hydrogel layer) may include and / or be formed from a thiolated polymer. The thiolated polymer may be gelatin or polyethylene glycol (PEG). The number average molecular weight of the thiolated polymer may be 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, 10 kDa, 10.5 kDa, 11 kDa, 11.5 kDa, 12 kDa, 12.5 kDa, 13 kDa, 13.5 kDa, 14 kDa, 14.5 kDa, 15 kDa, 15.5 kDa, 16 kDa, 16.5 kDa, 17 kDa, 17.5 kDa, 18 kDa a, 18.5kDa, 19kDa, 19.5kDa, 20kDa, 20.5kDa, 21kDa, 21.5kDa, 22kDa, 22.5kDa, 23kDa, 23.5kDa, 24kDa, 24.5kDa, 25kDa, 25.5kDa, 26kDa, 26.5kD a, 27kDa, 27.5kDa, 28kDa, 28.5kDa, 29kDa, 29.5kDa, 30kDa, 30.5kDa, 31kDa, 31.5kDa, 32kDa, 32.5kDa, 33kDa, 33.5kDa, 34kDa, 34.5kDa or 35kDa.The number average weight of the thiolated polymer may be less than 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, 19kDa, 18.5kDa, 18kDa, 17.5kDa, 17kDa, 16.5kDa, 16kDa, 15.5kDa, 15kDa, 14.5kDa, 14kDa, 13.5kDa, 13kDa, 12.5kDa, 12kDa, 11.5kDa, 11kDa, 10 .5kDa, 10kDa, 9.5kDa, 9kDa, 8.5kDa, 8kDa, 7.5kDa, 7kDa, 6.5kDa, 6kDa, 5.5kDa, 5kDa, 4.5kDa, 4kDa, 3.5kDa, 3kDa, 2.5kDa, 2kDa, 1.5kDa or 1kDa.The number average molecular weight of the thiolated polymer may be from 1 kDa to 2 kDa, from 1.5 kDa to 2.5 kDa, from 2 kDa to 3 kDa, from 2.5 kDa to 3.5 kDa, from 3 kDa to 4 kDa, from 3.5 kDa to 4.5 kDa, from 4 kDa to 5 kDa, from 4.5 kDa to 5.5 kDa, from 5 kDa to 6 kDa, from 5.5 kDa to 6.5 kDa, from 7 kDa to 8 kDa, from 7.5 kDa to 8.5 kDa, from 9 kDa to 10 kDa, from 9.5 kDa to 10.5 kDa, from 10 kDa to 11 kDa, from 10.5 kDa to 11.5 kDa, from 11 kDa to 12 kDa, from 13 kDa to 14 kDa, from 15 kDa to 16 kDa, from 17 kDa to 18 kDa, from 19 kDa to 20 kDa, from 21 kDa to 22 kDa, from 23 kDa to 24 kDa, from 25 kDa to 26 kDa, from 27 kDa to 28 kDa, from 29 kDa to 30 kDa, from 31 kDa to 32 kDa, from 33 kDa to 34 kDa, from 35 kDa to 36 kDa, from 37 kDa to 38 kDa, from 39 kDa to 40 kDa, from 39 kDa to 41 kDa, from 3 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 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 28 kDa, 29 kDa to 30 kDa, 31 kDa to 32 kDa, 33 kDa to 34 kDa 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, and 34 kDa to 35 kDa.

[0030] The degree of thiolation of the thiomer can be greater than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. The degree of thiolation of the thiomer can be less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. The degree of thiolation of the thiomer can be from 5% to 15%, from 10% to 20%, from 15% to 25%, from 20% to 30%, from 30% to 40%, or from 35% to 45%.

[0031] As described herein, the degree of thiolation can refer to the percentage of free (e.g., unreacted) thiols (e.g., "thiol groups," -SH groups) out of the total number of thiol groups on a molecule. The free thiols can be measured using any technique known to those skilled in the art, such as Ellman's assay. The Ellman's assay uses 5.5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) to react with the free thiols to produce a mixed disulfide 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 thiols in solution.

[0032] The number average molecular weight of the thiolated PEG can be 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 number average molecular weight of the thiolated PEG can be 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 number average molecular weight of the thiolated PEG can be from 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.

[0033] The number average molecular weight of the thiolated gelatin can be 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, 10 kDa, 10.5 kDa, 11 kDa, 11.5 kDa, 12 kDa, 12.5 kDa, 13 kDa, 13.5 kDa, 14 kDa, 14.5 kDa, 15 kDa, 15.5 kDa, 16 kDa, 16.5 kDa, 17 kDa, 17.5 kDa, 18 kDa a, 18.5kDa, 19kDa, 19.5kDa, 20kDa, 20.5kDa, 21kDa, 21.5kDa, 22kDa, 22.5kDa, 23kDa, 23.5kDa, 24kDa, 24.5kDa, 25kDa, 25.5kDa, 26kDa, 26.5kD a, 27kDa, 27.5kDa, 28kDa, 28.5kDa, 29kDa, 29.5kDa, 30kDa, 30.5kDa, 31kDa, 31.5kDa, 32kDa, 32.5kDa, 33kDa, 33.5kDa, 34kDa, 34.5kDa or 35kDa. The number average weight of the thiolated gelatin may be less than 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, 1 9kDa, 18.5kDa, 18kDa, 17.5kDa, 17kDa, 16.5kDa, 16kDa, 15.5kDa, 15kDa, 14.5kDa, 14kDa, 13.5kDa, 13kDa, 12.5kDa, 12kDa, 11.5kDa, 11kDa, 10 .5kDa, 10kDa, 9.5kDa, 9kDa, 8.5kDa, 8kDa, 7.5kDa, 7kDa, 6.5kDa, 6kDa, 5.5kDa, 5kDa, 4.5kDa, 4kDa, 3.5kDa, 3kDa, 2.5kDa, 2kDa, 1.5kDa or 1kDa.The number average molecular weight of the thiolated gelatin can be from 1 kDa to 2 kDa, from 1.5 kDa to 2.5 kDa, from 2 kDa to 3 kDa, from 2.5 kDa to 3.5 kDa, from 3 kDa to 4 kDa, from 3.5 kDa to 4.5 kDa, from 4 kDa to 5 kDa, from 4.5 kDa to 5.5 kDa, from 5 kDa to 6 kDa, from 5.5 kDa to 6.5 kDa, from 7 kDa to 8 kDa, from 7.5 kDa to 8.5 kDa, from 9 kDa to 10 kDa, from 9.5 kDa to 10.5 kDa, from 10 kDa to 11 kDa, from 10.5 kDa to 11.5 kDa, from 11 kDa to 12 kDa, from 13 kDa to 14 kDa, from 15 kDa to 16 kDa, from 17 kDa to 18 kDa, from 19 kDa to 20 kDa, from 21 kDa to 22 kDa, from 23 kDa to 24 kDa, from 25 kDa to 26 kDa, from 27 kDa to 28 kDa, from 29 kDa to 30 kDa, from 31 kDa to 32 kDa, from 33 kDa to 34 kDa, from 35 kDa to 36 kDa, from 37 kDa to 38 kDa, from 39 kDa to 40 kDa 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 a, 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 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, and 34 kDa to 35 kDa.

[0034] Acrylate

[0035] The hydrogel (of the hydrogel layer) may include and / or be formed by acrylates. Acrylates may include polyethylene glycol (PEG) with one or more acrylate groups. In certain embodiments, PEG acrylates may be multi-arm PEG acrylates. In certain embodiments, acrylates may be PEG diacrylates. The number average molecular weight of PEG acrylates (e.g., PEG diacrylates) may be 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 number average weight of PEG can be less than 10kDa, 9.5kDa, 9kDa, 8.5kDa, 8kDa, 7.5kDa, 7kDa, 6.5kDa, 6kDa, 5.5kDa, 5kDa, 4.5kDa, 4kDa, 3.5kDa, 3kDa, 2.5kDa, 2kDa, 1.5kDa or 1kDa. The number average molecular weight of PEG acrylate can be 1kDa to 2kDa, 1.5kDa to 2.5kDa, 2kDa to 3kDa, 2.5kDa to 3.5kDa, 3kDa to 4kDa, 3.5kDa to 4.5kDa, 4kDa to 5kDa, 4.5kDa to 5.5kDa, 5kDa to 6kDa, 5.5kDa to 6.5kDa, 7kDa to 8kDa, 7.5kDa to 8.5kDa or 9kDa to 10kDa.

[0036] Photoinitiator

[0037] Acrylates and thiolated polymers can be crosslinked using a photoinitiator. The photoinitiator can include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959).

[0038] The photoinitiator can be a cationic or free radical photoinitiator. Free radical photoinitiators are compatible with water and act on molecules containing acrylate or styrene groups. The photoinitiator can include Irgacure 2959, 184 or 651.

[0039] Hydrogel curing conditions

[0040] The first or second hydrogel layer can be cured using light (e.g., UV light) having an average wavelength 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 300 nm-320 nm, 310 nm-330 nm, 320 nm-340 nm, 330 nm-350 nm, 340 nm-360 nm, 350 nm-370 nm, 360 nm-380 nm, 370 nm-390 nm, or 380 nm-400 nm.

[0041] The first hydrogel layer may solidify for more than 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, or 30 seconds. The first hydrogel layer may solidify for less than 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, or 10 seconds. The first hydrogel layer may solidify for 5 seconds to 15 seconds, 10 seconds to 20 seconds, 15 seconds to 25 seconds, 20 seconds to 30 seconds, 25 seconds to 35 seconds, 30 seconds to 40 seconds, 35 seconds to 45 seconds, or 40 seconds to 50 seconds.

[0042] The second hydrogel layer can cure for more than 1 second, 3 seconds, 5 seconds, 8 seconds, or 10 seconds. The second hydrogel layer can cure for less than 15 seconds, 10 seconds, 8 seconds, 5 seconds, or 3 seconds. The second hydrogel layer can cure for 1 second to 5 seconds, 3 seconds to 8 seconds, 5 seconds to 10 seconds, or 8 seconds to 15 seconds.

[0043] Culture medium

[0044] The cell culture comprising the hydrogel system may include a culture medium. The culture medium may include one or more of the following: sodium phosphate, potassium chloride, sodium bicarbonate, ferric nitrate, calcium chloride, or potassium chloride. The culture medium may include any suitable culture medium known in the art. Suitable culture media may include, for example, Dulbecco's Phosphate Buffered Saline, Earle's Balanced Salts, Hanks' Balanced Salts, Tyrode's Salts, Alsever's Solution, Gey's Balanced Salt Solution, Kreb's-Henseleit Buffer Modified, Kreb's-Ringer Bicarbonate Buffer, Puck's Saline, Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Eagle's Medium / Nutrient F-12 Ham, Nutrient Mixture F-10 Ham (Ham's F-10), Medium 199, Minimum Essential Medium (Minimum Essential Medium) and so forth. Medium Eagle), RPMI-1640 medium, Ames' Medium, BGJb medium (Fitton-Jackson Modification), Crick's Medium, CMRL-1066 medium, Fischer's Medium, Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15 medium (Leibovitz), McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth Medium, Williams' Medium E, or a combination thereof. The medium may be modified or supplemented. The culture medium may further include albumin, selenium, transferrin, fetoprotein, sugars, amino acids, vitamins, growth factors, cytokines, hormones, antibiotics, lipids, lipid carriers, cyclodextrins, or combinations thereof.

[0045] Cell Type

[0046] The cell can be cultured, grown or maintained in a culture system comprising a hydrogel layer of a hydrogel system, the hydrogel system comprising a plurality of (e.g., two) hydrogel layers. The cell can be a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a mammalian cell or a plant cell. In some embodiments, the cell is derived from a tumor. The cell can be derived from a tissue. The cell can be a hepatocyte, a gastrointestinal cell, a lymphoid cell, a pancreatic cell, a kidney cell, a lung cell, a tracheal cell, a vascular cell, a skeletal muscle cell, a heart cell, a skin cell, a smooth muscle cell, a connective tissue cell, a corneal cell, a urogenital cell, a mammary cell, a germ cell, an endothelial cell, an epithelial cell, a fibroblast, a neural cell, a Schwann cell, a fat cell, an osteocyte, a bone marrow cell, a chondrocyte, a pericyte, a mesothelial cell, a stromal cell, a stem cell, a progenitor cell, a lymphocyte, a blood cell, an endoderm-derived cell, an ectoderm-derived cell or a mesoderm-derived cell. The cells may include T cells, B cells, monocytes, macrophages, dendritic cells, eosinophils, basophils, mast cells or natural killer cells. For example, the cells may be obtained by resection, biopsy, fine needle aspiration, brushing or swab.

[0047] Cell labeling

[0048] Cells can be labeled with stains or markers. Cells can be labeled with dyes specific to the nucleus, dyes specific to the cell membrane, dyes specific to live cells, dyes specific to dead cells, or fluorescent labels. Fluorescent labels can be attached to antibodies or derivatives thereof. Cells can be labeled with one or more of the following: lipophilic carbocyanine dyes, bisbenzimides, xanthene dyes, or propidium iodide. Cells can be labeled with, for example, Hoeschst33342, CellBrite NIR 680, Calcein-AM, or live or dead NucFix Red. In some embodiments, cells are stained, labeled, or marked before being introduced into the hydrogel system. In other embodiments, cells are stained after being introduced into the hydrogel system.

[0049] Extracellular matrix components

[0050] One or more hydrogel layers can include one or more components of extracellular matrix or its derivatives. For example, the second hydrogel layer (for example, the layer delivered on the top of the first layer) can include one or more components of extracellular matrix or its derivatives. The component can include a protein produced by cells, and the protein keeps the tissue together, provides tensile strength or promotes cell signaling. The extracellular matrix components can include collagen, fibronectin, laminin, hyaluronate, elastin or proteoglycan. The extracellular matrix components can be purified from human or animal sources, or produced by recombinant methods known in the art.

[0051] Hydrogel

[0052] The hydrogel may include one or more (or all) of the following: (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.

[0053] Before solidification, the kinematic viscosity of the mixture of thiomer and acrylate can be between 0.5 square millimeter / second and 10 square millimeter / second. Before solidification, the kinematic viscosity of the first and / or second mixture of thiomer and acrylate can be between 0.5 square millimeter / second and 10 square millimeter / second. The kinematic viscosity of the mixture can be greater than 0.1 square millimeter / second, 0.5 square millimeter / second, 1.0 square millimeter / second, 1.5 square millimeter / second, 2.0 square millimeter / second, 2.5 square millimeter / second, 3.0 square millimeter / second, 3.5 square millimeter / second, 4.0 square millimeter / second, 4.5 square millimeter / second, 5.0 square millimeter / second, 5.5 square millimeter / second, 6.0 square millimeter / second, 6.5 square millimeter / second, 7.0 square millimeter / second, 7.5 square millimeter / second, 8.0 square millimeter / second, 8.5 square millimeter / second, 9.0 square millimeter / second or 9.5 square millimeter / second. The kinematic viscosity of the mixture may be less than 10.0 mm2 / sec, 9.5 mm2 / sec, 9.0 mm2 / sec, 8.5 mm2 / sec, 8.0 mm2 / sec, 7.5 mm2 / sec, 7.0 mm2 / sec, 6.5 mm2 / sec, 6.0 mm2 / sec, 5.5 mm2 / sec, 5.0 mm2 / sec, 4.5 mm2 / sec, 4.0 mm2 / sec, 3.5 mm2 / sec, 3.0 mm2 / sec, 2.5 mm2 / sec, 2.0 mm2 / sec, 1.5 mm2 / sec, 1.0 mm2 / sec or 0.5 mm2 / sec. The kinematic viscosity of the mixture can be 0.5 square mm / s to 1.5 square mm / s, 1.0 square mm / s to 2.0 square mm / s, 1.5 square mm / s to 2.5 square mm / s, 2.0 square mm / s to 3.0 square mm / s, 2.5 square mm / s to 3.5 square mm / s, 3.0 square mm / s to 4.0 square mm / s, 3.5 square mm / s to 4.5 square mm / s, 4.0 square mm / s to 5.0 square mm / s, 4.5 square mm / s to 5.5 square mm / s. mm / s, 5.0 square mm / s to 6.0 square mm / s, 5.5 square mm / s to 6.5 square mm / s, 6.0 square mm / s to 7.0 square mm / s, 6.5 square mm / s to 7.5 square mm / s, 7.0 square mm / s to 8.0 square mm / s, 7.5 square mm / s to 8.5 square mm / s, 8.0 square mm / s to 9.0 square mm / s, 8.5 square mm / s to 9.5 square mm / s or 9.0 square mm / s to 10.0 square mm / s.

[0054] The weight ratio of acrylate to thiolated polymer in the hydrogel or hydrogel system can be greater than 1:1 or 1:2. The weight ratio of acrylate to thiolated polymer in the first layer or the second layer can be less than 1:2 or 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 between 1:4 and 2:1.

[0055] The weight ratio of the thiolated polymer to the 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 between 1:20 and 1:100. The weight ratio of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone to thiolated gelatin can be 1:50.

[0056] The weight ratio of the thiolated polymer to the extracellular matrix component can be 3: 1, 4: 1 or 5: 1. The weight ratio of the thiolated gelatin to the 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.

[0057] Array

[0058] The hydrogel or hydrogel system can be placed in the wells of the array (e.g., the wells of a porous plate). The array can include a plurality of wells. The wells can be wells in a porous plate. The well array 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.

[0059] use

[0060] The three-dimensional cell culture systems and methods described herein can be used to identify therapies for the treatment of diseases, or to identify which tissues may be sensitive to known therapies. The three-dimensional cell culture systems and methods described herein can be used to identify reagents for studying cell interactions and reactions in three-dimensional cultures. The three-dimensional cell culture systems described herein can be used to generate cell aggregates that can simulate the response of in vivo tissues to stimuli.

[0061] In certain aspects, the methods and systems disclosed herein can be used to determine the course of treatment for a subject. For example, such methods and systems may involve screening compounds to identify compounds that provide patient-specific or tissue-specific responses. The subject providing the tissue or cell sample may be healthy. The subject may be positive for a disease. The disease may be an infection. The infection may be viral, bacterial, or fungal. The subject may suffer from an autoimmune disease. The subject may be positive for a tissue-specific disease. The methods and systems may involve screening that can be used to identify tissue-specific responses within a particular population of individuals.

[0062] The subject may be positive for a disease. The disease may be liver disease, heart disease, kidney disease, pancreatic disease, colon disease, skin disease, brain disease, thymus disease, bone disease or lung disease. 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. 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.

[0063] Reagent test kit

[0064] The kit may include one or more containers containing one or more of the components provided in the disclosure and instructions for use. Specifically, such kits may include one or more compositions described herein, as well as instructions describing the intended application and proper use and / or disposal of these compositions. The kit may contain components in appropriate concentrations or quantities for conducting various experiments.

[0065] Computer Systems

[0066] The present disclosure provides computer systems for implementing the methods provided herein. Figure 5An example of a computer system 1001 is shown. The computer system 1001 includes a central processing unit (CPU, also referred to herein as a "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 a memory or memory location 1010 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1015 (e.g., a hard disk), a communication interface 1020 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage area, and / or an electronic display adapter. The memory 1010, the storage unit 1015, the interface 1020, and the peripheral devices 1025 communicate with the CPU 05 via a communication bus (solid line), such as a motherboard. The storage unit 1015 may be a data storage unit (or data repository) for storing data. The computer system 1001 may be operably coupled to a computer network ("network") 1030 by means of the communication interface 1020. The network 1030 may be the Internet, the Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 1030 is a telecommunications network and / or a data network in some cases. The network 1030 may include one or more computer servers that may implement distributed computing such as cloud computing. In some cases, the network 1030 may implement a peer-to-peer network with the aid of the computer system 1001, which may enable coupling of a device to the computer system 1001 to act as a client or server.

[0067] The CPU 1005 may execute a series 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 program or otherwise configure the CPU 1005 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1005 may include fetching, decoding, executing, and writing back.

[0068] CPU 1005 may be part of a circuit, such as an integrated circuit. One or more other components of system 1001 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0069] 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 may in some cases include one or more additional data storage units, which are located external to the computer system 1001, such as located on a remote server that communicates with the computer system 1001 via an intranet or the Internet.

[0070] Computer system 1001 can communicate with one or more remote computer systems via network 1030. For example, computer system 1001 can communicate with a remote computer system of a user (e.g., a remote cloud server). Examples of remote computer systems include personal computers (e.g., portable PCs), tablets or tablet-type PCs (e.g., Galaxy Tab), phones, smartphones (e.g. iPhone, Android-supported devices, ) or a personal digital assistant. A user can access the computer system 1001 through the network 1030.

[0071] Methods as described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 1001 (e.g., memory 1010 or electronic storage unit 1015). The machine executable or machine readable code may be provided in the form of software. During 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 cases, the electronic storage unit 1015 may be excluded and the machine executable instructions may be stored on the memory 1010.

[0072] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during runtime. The code may be supplied in a programming language that may be selected to enable execution of the code in a pre-compiled or as-compiled manner.

[0073] Various aspects of the system and method provided herein (such as computer system 1001) can be embodied in programming. Various aspects of technology can be regarded as "products" or "articles" generally in the form of machine (or processor) executable code and / or associated data, which are executed on a type of machine-readable medium or implemented in a type of machine-readable medium. Machine executable code can be stored in an electronic storage unit, such as a memory (e.g., a read-only memory, a random access memory, a flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of computers, processors, etc. or their associated modules, such as various semiconductor memories, tape drives, hard disk drives, etc. that can provide non-temporary storage for software programming at any time. All or part of the software can sometimes communicate through the Internet or various other telecommunication networks. Such communication, for example, can realize loading software from one computer or processor into another computer or processor, such as loading from a management server or a host computer into a computer platform of an application server. Thus, another type of medium that can carry software elements includes light waves, electrical waves, and electromagnetic waves, such as those used across physical interfaces between local devices over wired and optical landline networks and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered as mediums that carry software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0074] Thus, machine-readable media such as computer executable code may take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, etc., such as may be used to implement, for example, a database shown in the accompanying drawings, etc. 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 fiber, including the wires that comprise a bus within a computer system. Carrier transmission media may take the form of an electrical or electromagnetic signal, or an acoustic or light wave, 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 diskette, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, a punched card tape, any other physical storage medium having a pattern of holes, a RAM, a ROM, a PROM and an EPROM, a FLASH-EPROM, any other memory chip or memory cartridge, a carrier wave that transmits data or instructions, a cable or link that transmits such a carrier wave, or any other medium from which a computer can read programming code and / or data. A variety of these forms of computer readable media may be involved in conveying one or more sequences of one or more instructions to a processor for execution.

[0075] The computer system 1001 may include or communicate with an electronic display 1035, the electronic display including a user interface (UI) 1040 for providing, for example, an electronic output of an identified gene fusion. 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 a robotic arm for performing, for example, one or more mixing, deposition, or imaging steps. Examples of robotic arms include, but are not limited to, multi-pipette arms, cameras, and palletizers for moving multiwell plates.

[0076] The method and system 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 .

[0077] Examples

[0078] Example 1: Gel Production

[0079] Pre-cured mixtures used to form hydrogels can often naturally wick to the sides of the wells, forming a concave meniscus within the wells. In pre-cured mixtures containing cells, cells may concentrate at the periphery of the wells due to wicking, leaving the center portion of the wells with relatively low cell density. This is less than ideal for imaging and promoting cell-cell interactions.

[0080] A two-stage plate inoculation method is disclosed herein, in which a cell-free mixture is added to a well, and then cross-linked in the well to form a first hydrogel layer, leaving a concave cavity (or a "basket" of space) in the center of the well. Next, a second mixture containing cells is added to the well on top of the first hydrogel layer. After solidification, the second mixture forms a second solidified hydrogel layer, the concavity of the upper surface of the second solidified hydrogel layer being smaller than the concavity of the upper surface of the first hydrogel layer (e.g., a substantially planar upper surface). Cells are distributed throughout the second layer, resulting in cells being relatively evenly distributed across the xy plane of the well. Images can be taken using z-stack imaging, in which multiple (e.g., 10) images of the xy axis can be taken at a specified distance (e.g., 100 microns) on the z-axis (top to bottom of the well), and then projected to create a single image.

[0081] The mixture for forming the first hydrogel layer and the second hydrogel layer was prepared according to the following table:

[0082] Table 1 - Hydrogel components

[0083]

[0084] Add 125 mg of AC-PEG-AC (e.g., Formula I) to a 1.5 mL microcentrifuge tube. Add 0.2% irgacure solution to the tube containing AC-PEG-AC and mix. Resuspend 50 mg of Gelin-S bottle in 500 μL of 0.1% irgacure solution. Combine the AC-PEG-AC solution and Gelin-S mixture and immediately plate into a 384-well source plate. Immediately after the first layer is plated, use an intensity of 250 mW / cm 2 The film was cured with UV light (320nm-390nm) for 15 seconds.

[0085]

[0086] Cells were stained as described in Example 2 and centrifuged at 300 xg for 5 minutes to produce a cell pellet. The supernatant was removed from the cell pellet.

[0087] To form the second cured hydrogel layer, the AC-PEG-AC solution, laminin solution, and Gelin-S mixture were mixed with the stained cell pellet as shown in Table 1. The stained cells were resuspended in the gel mixture to form a seeded gel mixture. The seeded gel mixture was plated on top of the first layer and immediately heated to 250 mW / cm 2The mixture was cured under UV light (320nm-390nm) for 10 seconds. When the mixture of the first layer and the mixture of the second layer were added to the hole, the kinematic viscosity of each mixture was about 9.65 square millimeters / second. Figure 1B As seen (DAPI channel only), a second layer of stained cells resulted in an even distribution of cells across the well, as compared to a single layer of seeded gel in the same well ( Figure 1A ; DAPI channel only). Images were acquired with a confocal microscope using a 4× air objective, using the DAPI channel corresponding to excitation of the fluorophore Hoechst. Images can include a pseudo-complex of 4 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 100 μm apart in the Z axis and projected using maximum projection to create a single image (e.g., Z-stack imaging). Cells in each well were counted and the distribution was quantified as Figure 4A and 4B It can be seen that Figure 4B shows the distribution in the bilayer hydrogel formed in this example, and Figure 4A Shown with Figure 4B 3 shows the distribution of cells in a similar monolayer hydrogel with the same volume of hydrogel in the well, which was formed by the components of the second layer of hydrogel described in Table 1. The maximum projection image of all wells in each condition (352 images per condition) was cropped into 5 boxes of equal size, corresponding to the upper left corner, lower left corner, lower right corner, upper right corner and center of the image. Cells were counted for each cropped image using a custom algorithm, and the counts for all wells were used to create a box plot. As can be seen in Figure 3, the monolayer 210 hydrogel in well 200 Figure 3B Compared with the double-layer hydrogel in hole 100 Figure 3A Example cross section of. Figure 3A and 3B The holes 100 and 200 have the same volume of hydrogel, but the concavity of the upper surface 211 of the monolayer 210 is greater than Figure 3A The upper surface 121 of the second layer of the illustrated bi-layer-like structure is more concave. In some embodiments, the upper surface 121 of the second layer is substantially planar.

[0088] Example 2: Cell Staining with Hoechst Nuclear Stain and CellBrite Cytoplasmic Membrane Stain

[0089] From the last passage, the HCT-116 cells (colorectal cancer cell line (ATCC CCL-247)) of T75 flasks were grown in DMEM supplemented with 10% FBS and 1×Primocin at 37°C for 4 days. The cells were released from the bottom of the flask using 0.25% trypsin-EDTA by washing the cells twice with 2mL trypsin-EDTA, and then adding 2mL trypsin-EDTA, and the flask was returned to the incubator at 37°C for 7 minutes. The cells were then washed to one corner of the flask using 4mL fresh warm culture medium. The cells and culture medium were then transferred to a 15mL conical tube. The conical tube was centrifuged at 300×g for 5 minutes to precipitate the cells. The cells were then counted using an automated cell counter. The cells were then stained with Hoechst 3342 (Abcam ab228551; for identification of cell nuclei) and Cell Brite NIR 680 (Biotium 20070; for identification of cell membrane and cytoplasm) by resuspending the cells in PBS and staining solution (1 uL / mL CellBrite NIR) in a 15 mL conical tube.

[0090] 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 as used in Example 1 above.

[0091] Example 3: Cell Viability

[0092] Due to the synthetic nature of the PEG component, PEG-based hydrogels have advantages due to their relatively low batch-to-batch variability. However, hydrogels based entirely on PEG components are acidic, which may adversely affect the viability of 3D cell cultures in such hydrogels. In addition, adjusting the pH of the PEG-only hydrogel to a neutral range can reduce its rigidity and density to the extent that such hydrogels can no longer retain cells therein. Some specific gel formulations described herein utilize 3.4kDa PEG-diacrylate, which maintains the consistency of the above-mentioned synthetic components (e.g., minimal batch-to-batch variability). The pore size produced by 3.4kDa PEGDA is large enough to allow nutrients and dyes to diffuse, but small enough to retain cells, including small immune cells that may be required for immunotherapy screening.

[0093] The viability of a tumor cell line (HCT-116) was evaluated at 3 and 5 days of growth in Gelin-S and Ac-PEG-Ac based gels.

[0094] 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.

[0095] HCT-116 cells were resuspended in the gel at a concentration of 5M cells / mL. 5 μL of gel + cell mixture per well was added to 48 wells of two 384-well microplates (24 wells per plate). The gel was cross-linked by exposure to UV light for 15 seconds. 50 μL of Dulbecco's Modified Essential Media (DMEM) supplemented with 10% FBS was added to each well. The microplate was then placed at 37°C with 5% CO 2 Incubator. 3 days after cell inoculation, remove 1 microplate from the incubator and perform a luminescence-based viability assay (CellTiter Glo 3D, Promega) according to the manufacturer's instructions. Use Varioskan plate reader (ThermoScientific) to read luminescence. The luminescence value is subtracted from the blank, and the average luminescence and standard error of the average value for all 24 samples are calculated. The average luminescence after 3 days is 3,198,142.9, with a SEM of 229,246.8. After 5 days of cultivation, remove the second microplate from the incubator and use the same luminescence assay to assess viability. After 5 days, the average luminescence is 4,754,192.3, with a SEM of 913,546.9. This is an increase of 48.7% over the values ​​observed on the 3rd day. Figure 2A The growth of HCT-116 cells in Gelin-S and Ac-PEG-Ac based cells is shown. These data show that cells in Gelin-S and Ac-PEG-Ac based gels are viable at 3 days in culture and continue to grow after 5 days in culture.

[0096] Example 4: Cell viability: comparison with PEG+RGD hydrogel

[0097] Gelin-S + Ac-PEG-Ac ("gelatin") hydrogels were compared with Ac-PEG-Ac + GRGDSPC ("RGD") hydrogels.

[0098] 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.

[0099] 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.

[0100] HCT-116 cells are resuspended in each gel type with a concentration of 5M cells / mL. For each gel type, 5 μL gel+cell mixtures are added to 24 wells of a 384-well microplate per well. The gel is cross-linked by exposure to UV light for 15 seconds. 50 μL of Dulbecco's modified minimal medium (DMEM) supplemented with 10% FBS is added to each well. The microplate is then placed in a 5% CO2 incubator at 37°C. 72 hours after cell inoculation, the microplate is removed from the incubator, and a luminescent viability assay (CellTiter Glo 3D, Promega) is performed according to the manufacturer's instructions. Luminescence is read using Varioskan plate reader (Thermo Fisher Scientific). For each gel type, blank is subtracted from the luminescent value, and the average luminescence of all 24 samples and the standard error of the mean value are calculated. The average luminescence of the RGD-based gel samples was -3,1647.3 with a SEM of 4,128.5, while the average of the Gelin samples was 3,198,142.9 with a SEM of 229,246.8. Figure 2B A comparison between Gelin-S based gels and RGD based gels is shown. These data indicate that cells in the RGD condition were not viable after 3 days in culture, whereas cells in the Gelin condition were still viable.

[0101] Example 5: Cell viability: comparison with PEG+RGD hydrogel and pH-balanced PEG+RGD hydrogel

[0102] As observed in Example 4, cells cannot survive in RGD gels. It was observed that the typical pH of RGD gels is in the range of 4-5, and it is assumed that low pH may have a negative impact on cell viability. In order to test whether RGD gels can be improved by adjusting the pH value to within the physiological range of 7.4, 1M NaOH was added dropwise to the RGD gel formulation (as 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 with Gelin-S+Ac-PEG-Ac ("Gelin") hydrogels and Ac-PEG-Ac+GRGDSPC ("RGD") hydrogels without pH balance. Gelin and RGD-based gels were prepared as above. The pH of Gelin gel was measured to be 7.3 without any modification. HCT-116 cells were resuspended in the gel at a concentration of 5M cells / mL. 5 μ L gel+cell mixture per well is added to 72 wells of 3 384-well microplates (24 wells per plate). Gel is cross-linked by being exposed to UV light for 15 seconds. 50 μ L Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% FBS is added to each well. The microplate is then placed in 5% CO2 incubator at 37°C. 3 days after cell inoculation, the microplate is taken out from the incubator, and the viability assay (CellTiter Glo3D, Promega) based on luminescence is carried out according to the manufacturer's instructions. Varioskan plate reader (Thermo Fisher Scientific) is used to read luminescence. The luminescence value is subtracted from blank, and the average luminescence and standard error of the mean value of all 24 samples are calculated. For the RGD condition, the average luminescence after 3 days was -31647.26 with a SEM of 4128.54; for the balanced RGD condition, the average luminescence was 3206783.46 with a SEM of 232717.981, and for the Gelin condition, the average luminescence was 3198142.86 with a SEM of 229246.80. 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 the Gelin gel, it was observed that the neutral pH RGD gels could not maintain their structure after several days of culture. Figure 2C A comparison of the viability between Gelin-S based gels, RGD based gels and pH balanced RGD gels is shown. By day 5, the pH balanced RGD gels had completely dissolved, allowing cells to pass through the gels and adhere to the bottom of the plate. Additionally, balancing the pH of these gels requires additional steps and may be difficult to automate. This suggests that Gelin gels have the desired combination of durable structural integrity and neutral pH.

[0103] Example 6: Hydrogel plate inoculation

[0104] Due to the small size of the wells and the polarity of the hydrogel liquids in question, the hydrogel liquid tends to adhere or wick to one or both corners of the wells rather than even covering the well surface. Many of the steps taken, including the double layer, are designed to avoid this situation as this can cause significant problems when collecting and analyzing image-based data. The gel components of the first layer were mixed together and equal volumes of gel were added to all 8 wells in column 1 of a standard U-bottom 96-well microplate. Flat-bottom 384-well microplates were inoculated using an Opentrons liquid handling robot running on a custom program and configured with a 2nd generation P208 channel pipette as follows: First, the robot simultaneously aspirated 20 microliters (uL) of gel from the 96-well plate and moved to the 384-well plate so that the tip was positioned 0.2 mm above the bottom of wells A1, C1, E1, G1, I1, K1, M1, and O1. Each channel then dispenses 20 μL to evenly coat the bottom of the well with gel, waits 0.5 seconds, and then aspirates 15 μL, leaving a 5 μL layer of gel. The robot then returns to column 1 of the 96-well source plate, aspirates 5 μL (so the tip now contains 20 μL), and moves to the B1, D1, F1, H1, J1, L1, N1, and P1 wells of the 384-well plate. The robot then dispenses 20 μL and removes 15 μL as before, so now all wells in column 1 of the 384-well plate contain a 5 μL layer of gel. This process is repeated for the remaining 23 columns of the 384-well plate. The total time for this procedure is approximately 20 minutes. The plate is then exposed to UV light for 15 seconds to crosslink the gel. Next, the components of the second layer of gel are mixed together with cells from the patient sample and added to each well in column 2 of the 96-well source plate in equal volumes. The above seeding process was then repeated for the second layer of gel, so that at the end of the process, each well in the 384-well plate would get a second layer of 5 μL of gel containing patient cells. Again, this process took about 20 minutes. The plate was then exposed to UV light for 10 seconds. From start to finish, the entire procedure lasted about 1 hour.

[0105] Example 7: Hydrogel plate inoculation

[0106] A 384-well microplate was placed on the plate carrier of a MultiFlo FX automated liquid dispenser equipped with a 5 μL 8-channel peristaltic pump cassette. A 50 milliliter (mL) conical tube filled with 40 mL of deionized water was mounted near the dispenser, and the end of the cassette tubing was placed in the water and the tubing dead volume was filled with the water to be dispensed. Using a custom program, 15 microliters (μL) of water was dispensed into each well of the 384-well plate. The dispensing process lasted approximately 30 seconds. The water was then purged from the tubing. Next, the liquid in the wells was manually pumped out, leaving only a water residue covering the bottom of the wells. The gel components of the first layer of hydrogel were then mixed together in a 50 mL conical tube. The tube was then mounted near the MultiFlo, and the end of the cassette tubing was placed in the gel and primed. Using a custom program, 5 μL of gel was dispensed into each well of the 384-well plate, a process that took approximately 15 seconds. The plate was then crosslinked under UV light for 15 seconds. Next, the components of the second layer of gel were mixed together with cells from the patient sample and added to a second 50mL conical tube. As with the first layer, 5μL was dispensed into each well of the microplate. The gel was then exposed to UV light for 10 seconds to crosslink. This process lasted about 20 minutes from start to finish.

[0107] Example 8: Hydrogels and Hydrogel Plating in the Absence of Laminin

[0108] The hydrogel was prepared essentially as described in Example 1, except the following components as shown in Table 2 were used.

[0109]

[0110] Add 100 mg AC-PEG-AC (e.g., Formula I) to a 1.5 mL microcentrifuge tube. Add 0.1% irgacure solution to the tube containing AC-PEG-AC and mix. Resuspend 50 mg Gelin-S bottle in 2.5 mL 0.1% irgacure solution. Combine the AC-PEG-AC solution and Gelin-S mixture and immediately plate into a 384-well source plate. Immediately after the first layer is plated, use an intensity of 250 mW / cm 2 The film was cured with UV light (320nm-390nm) for 15 seconds.

[0111] Cells were stained as described in Example 2 and centrifuged at 300 xg for 5 minutes to produce a cell pellet. The supernatant was removed from the cell pellet.

[0112] To form the second cured hydrogel layer, the AC-PEG-AC solution and the Gelin-S mixture were mixed with the stained cell pellet as shown in Table 2. The stained cells were resuspended in the gel mixture to form a seeded gel mixture. The seeded gel mixture was plated on top of the first layer and immediately heated with an intensity of 250 mW / cm 2 The film was cured with UV light (320nm-390nm) for 15 seconds.

[0113] Although 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. Without departing from the present invention, those skilled in the art will now expect many variations, changes and substitutions. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used to practice the present invention. It is contemplated that the following claims define the scope of the present invention and thus cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for producing 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 pores, thereby forming a concave meniscus within the pores; (b) crosslinking the first acrylate and the first thiolated polymer of the first mixture to form a first cured hydrogel layer, wherein the first cured hydrogel layer comprises a concave upper surface; (c) delivering a second mixture comprising a second acrylate and a second thiolated polymer into the wells 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, wherein the second cured hydrogel layer includes 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 upper surface of the second cured layer is substantially planar.

3. The method of claim 1 or claim 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 within the well and before the second mixture has been delivered into the well.

4. The method of any of the preceding claims, wherein cross-linking 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 wells. 5 . The method of claim 4 , wherein the first mixture is exposed to ultraviolet light for a period of time that is longer than the period of time that the second mixture is exposed to ultraviolet light.

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 greater than 12 seconds.

7. The method of any one of the preceding claims, wherein the first mixture and the second mixture are exposed to ultraviolet light for 10 seconds to 20 seconds.

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

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

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

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

12. The method of any one of claims 10 to 11, wherein the plurality of cells have been stained prior to (c).

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

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

15. The method of claim 10 or claim 11, wherein the plurality of cells have been labeled with a fluorescent tag prior to (c).

16. The method according to any one of the preceding claims, wherein the second mixture does not comprise laminin.

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

18. The method of any one of the preceding claims, 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 preceding claim, 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 of any one of the preceding claims, further comprising adding cell culture medium to the three-dimensional hydrogel system.

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

24. The method of any one of the preceding claims, wherein the kinematic viscosity of the first mixture when delivered into the well is between 0.5 mm2 / sec and 10 mm2 / sec.

25. The method of any one of the preceding claims, wherein the kinematic viscosity of the second mixture when delivered into the well is between 0.5 mm2 / s and 10 mm2 / s.

26. The method of any one of the preceding claims, wherein one or both of the first acrylate and the second acrylate are multi-arm PEG acrylates.

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

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

29. The method of claim 28, wherein the PEG diacrylate of the first mixture has a number average molecular weight of between three and four thousand Daltons.

30. The method of claim 27, wherein the PEG diacrylate of the second mixture has a number average molecular weight greater than three thousand Daltons.

31. The method of claim 30, wherein the PEG diacrylate of the second mixture has a number average molecular weight of between three and four thousand Daltons.

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

33. The method of claim 32, wherein the degree of thiolation of the thiolated gelatin is between 20% and 30%.

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

2.

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

1.

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

50.

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

1.

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. The method of any one of claims 27 to 38, wherein the concentration of laminin in the second mixture is no greater than 1.0 mg / mL.

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

41. A method according to any of the preceding claims, wherein the well is a well in an array of wells of a multi-well plate.

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

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

44. The method of claim 41, wherein the array of holes comprises at least 1536 holes.

45. The method of claim 41, wherein steps (a) through (d) are completed on 384 wells in less than one hour.

46. ​​The method of claim 41, wherein steps (a) to (b) are completed on 384 wells in less than 20 minutes.

47. The method of claim 41, wherein steps (c) to (d) are completed on 384 wells in less than 20 minutes.

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

49. The method of any one of claims 1 to 48, wherein delivering the first mixture comprises (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. The method of claim 49, wherein the first amount is greater than the second amount.

51. The method of any preceding claim, wherein (b) comprises exposing the wells to UV light.

52. The method of any of the preceding claims, wherein delivering the second mixture comprises delivering via a robotic system.

53. The method of any of the preceding claims, wherein delivering the second mixture comprises (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. The method of claim 53, wherein the first amount is greater than the second amount.

55. The method of any preceding claim, wherein (d) comprises exposing the wells to UV light.

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

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

58. A hydrogel formed by the method of any preceding claim.

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