Cell culture, manufacturing method using synthetic bioink, and transwell for in vitro tissue model

Abstract

A high-throughput manufacturing well (AM-well) comprising a lower chamber, an upper chamber, a membrane disposed between the lower chamber and the upper chamber, and one or more legs, wherein the one or more legs form at least a part of the lower chamber. One or more of the lower chamber, the upper chamber, the membrane, and the one or more legs are printed using a synthetic bioink. Also disclosed is a method of fabricating and using the AM-well.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 392,724, filed on July 27, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to 3D - printed structures.

Background Art

[0003] In vitro evaluation of epithelial and endothelial barrier functions has conventionally been performed using commercially available plastic transwells (e.g., Corning) that include porous membranes made from polyester (PET) or polycarbonate (PC). Such membranes are "rigid" and do not permit modification of their mechanical properties.

Summary of the Invention

[0004] The systems and methods of the present disclosure include 3D - printed advanced manufacturing (AM) transwells using synthetic bio - ink. The 3D - printed AM transwells include printable membranes whose thickness, porosity, and mechanical properties are adjustable. The 3D - printed AM - transwells may be surface - modified to enable barrier formation by epithelial and endothelial cells, and then the barrier function may be evaluated.

[0005] In one embodiment, an advanced manufacturing transwell (AM - transwell) includes a) a lower chamber, b) an upper chamber, c) a membrane disposed between the lower chamber and the upper chamber, and d) one or more legs.

[0006] In one embodiment, one or more legs form at least a part of the lower chamber.

[0007] In one embodiment, the AM-transwell is a cylinder, cube, cuboid, frustum of a cone, frustum of a pyramid, truncated sphere, or a combination thereof. In one embodiment, the AM-transwell is a cube. In one embodiment, the AM-transwell is a cylinder.

[0008] In one embodiment, the AM-transwell has a height of about 10 mm to about 16 mm and any range or value therebetween, and a thickness of about 1 mm to 3 mm and any range or value therebetween. In one embodiment, the AM-transwell has a height of about 13 mm and a thickness of about 2 mm.

[0009] In one embodiment, the lower chamber has a height of about 2 mm to about 4 mm and any range or value therebetween, and the upper chamber has a height of about 8 to about 12 mm and any range or value therebetween. In one embodiment, the lower chamber has a height of about 3 mm and the upper chamber has a height of about 10 mm.

[0010] In one embodiment, the membrane has a thickness of about 1 mm to about 3 mm and any range or value therebetween. In one embodiment, the membrane has a thickness of about 2 mm.

[0011] In one embodiment, one or more of the lower chamber, upper chamber, membrane, and one or more of the legs are printed using a synthetic bioink.

[0012] In one embodiment, the synthetic bioink includes one or more of a degradable peptide ink and a triacrylate peptide ink.

[0013] In one embodiment, the synthetic bioink comprises HPA in an amount of about 3% to about 10%, and any range or value therebetween, PEGDA3400 in an amount of about 5% to about 20%, and any range or value therebetween, PEGDA6000 in an amount of about 5% to about 20%, and any range or value therebetween, PEGDA575 in an amount of about 1% to about 20%, and any range or value therebetween, PEGDA700 in an amount of about 1% to about 20%, and any range or value therebetween, PEGTAC in an amount of about 1% to about 5%, and any range or value therebetween, PEO in an amount of about 0.1% to about 5%, and any range or value therebetween, NAP in an amount of about 1% to about 3%, and any range or value therebetween, LAP in an amount of about 1% to about 3%, and one or more of UV386A (visible dye at 386 nm) in an amount of about 0.1% to about 0.5%, and any range or value therebetween.

[0014] In one embodiment, the synthetic bioink further comprises the balance of water.

[0015] In one embodiment, the synthetic bioink further comprises a buffer solution containing 0.1M HEPES aqueous solution and 1×PBS at pH 7.2.

[0016] In one embodiment, the synthetic bioink further comprises monocysteine peptide in an amount of 0.5 mM to 20 mM, and any range or value therebetween, and dicysteine peptide in an amount of 0.5 mM to 20 mM, and any range or value therebetween.

[0017] In one embodiment, the dicysteine peptide is matrix metalloproteinase (MMP) degradable.

[0018] In one embodiment, the monocysteine peptide comprises one or more of RGDS, PHSRNKRGDS, IKVAV, AG73, GFOGER, Bm binder, and FN binder.

[0019] In one embodiment, a method of fabricating an advanced manufacturing well (AM-well) includes a) printing one or more of a lower chamber, an upper chamber, and a membrane of the AM-well using 3D printing technology, and b) assembling and / or printing the AM-well to form an assembled AM-well.

[0020] In one embodiment, step b) prints at least three and any number within the range or value included therein of the assembled AM-wells. In one embodiment, step b) prints at least 20 assembled AM-wells. In one embodiment, step b) prints at least 50 assembled AM-wells.

[0021] In one embodiment, the 3D printing technology is one or more of digital light projection (DLP) printing technology, stereolithography (SLA) printing technology, extrusion 3D printing technology, or selective laser sintering 3D printing technology, or a combination thereof. In one embodiment, the 3D printing technology is digital light projection (DLP) printing technology.

[0022] In one embodiment, the method further includes c) optionally storing the assembled AM-well at 4°C until needed.

[0023] In one embodiment, the method further includes: c) transferring each of the AM-transwells into 1× DPBS Ca+ / Mn+ in a tube; d) decanting the 1× DPBS Ca+ / Mn+ from the tube and washing each AM-transwell an additional two times in DPBS Ca+ / Mn+ for about 5 minutes to form a primary washed AM-transwell; and e) transferring the primary washed AM-transwells into a sterile tube. In one embodiment, the method further includes: c) transferring each of the AM-transwells into about 35 mL of DPBS Ca+ / Mn+ in a about 50 mL tube; d) decanting the DPBS Ca+ / Mn+ from the tube and washing each AM-transwell an additional two times in about 35 mL of DPBS Ca+ / Mn+ for about 5 minutes to form a primary washed AM-transwell; and e) transferring the primary washed AM-transwells into a 50 mL sterile tube. In one embodiment, in steps c) and d), the volume of DPBS Ca+ / Mn+ and the dimensions of the tube depend on the number of AM-transwells being transferred. The AM-transwells may simply be encapsulated in a buffered deionized aqueous solution.

[0024] In one embodiment, the method further includes: f) incubating the primary washed M-transwells overnight in 1× DPBS Ca− / Mn− supplemented with an antibiotic / antimycotic (anti-anti) to form sterilized AM-transwells.

[0025] In one embodiment, the method further includes: g) Decanting 1×DPBS Ca− / Mn− anti−anti and washing the incubated AM−transwell in about 35 mL of 1×PBS / 0.1 M HEPES anti−anti buffer solution for about 4 hours to form a secondarily washed AM−transwell, and h) Decanting the 1×PBS / 0.1 M HEPES anti−anti buffer solution from the tube and incubating the secondarily washed AM−transwell in about 35 mL of cell culture medium in the tube for about 2 days or over the weekend. In one embodiment, the method further includes: g) Decanting 1×DPBS Ca− / Mn− anti−anti and washing the incubated AM−transwell in about 35 mL of 1×PBS / 0.1 M HEPES anti−anti buffer solution for about 4 hours to form a secondarily washed AM−transwell, and h) Decanting the 1×PBS / 0.1 M HEPES anti−anti buffer solution from the tube and incubating the secondarily washed AM−transwell in about 35 mL of cell culture medium in a about 50 mL tube for about 2 days or over the weekend.

[0026] In one embodiment, a method of using a high−throughput manufactured transwell (AM−transwell) includes: a) Seeding cells on at least one side of the membrane of the AM−transwell described herein for in vitro cell studies.

[0027] In one embodiment, a method of using a high−throughput manufactured transwell (AM−transwell) includes: a) Seeding cells on both sides of the membrane of the AM−transwell described herein for in vitro cell studies.

[0028] Those skilled in the art will understand that this summary is illustrative only and is not intended to be limiting in any way. Other aspects, novel features, and advantages of the apparatus and / or processes described herein are defined only by the claims and will be understood in conjunction with the detailed description and the accompanying drawings described herein.

[0029] Details of one or more implementations of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages of the subject matter will become apparent from the specification, the drawings, and the claims.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13-1

Figure 13-2

Figure 13-3

Figure 14

Figure 15

Figure 16-1

Figure 16-2

Figure 17

DETAILED DESCRIPTION OF THE INVENTION

[0031] The detailed description of various embodiments of the present invention refers to the accompanying drawings, which show specific embodiments in which the present invention can be implemented. Although exemplary embodiments of the present invention have been specifically described, it will be understood that various other modifications can be contemplated and easily implemented by those skilled in the art without departing from the spirit and scope of the present invention. Therefore, it is not intended to limit the scope of the appended claims to the examples and descriptions set forth herein. Rather, the claims should be construed to include all novel features of patentability inherent in the present invention, as well as all features that are considered equivalents thereof by those skilled in the art. Accordingly, the scope of the present invention is defined only by the appended claims and the full scope of equivalents recognized in those claims.

[0032] Commercially available Transwell Figure 1 shows a commercially available plastic transwell 100 (prior art).

[0033] 3D-printed Transwell FIG. 2A shows an upper perspective view of the AM-transwell 200 according to an embodiment of the present invention, and FIG. 2B shows a lower perspective view of the AM-transwell 200 of FIG. 2A.

[0034] FIG. 3A shows a top view of the AM-transwell 300 according to an embodiment of the present invention. FIG. 3B shows a side view of the AM-transwell 300 according to FIG. 3A.

[0035] As shown in FIGS. 2A-2B and 3A-3B, the AM-transwells 200, 300 have lower chambers 210, 310, upper chambers 220, 320, membranes 230, 330 disposed between the lower chambers 210, 310 and the upper chambers 220, 320, and one or more legs 240, 340.

[0036] In one embodiment, the one or more legs 240, 340 form at least a part of the lower chambers 210, 310.

[0037] In one embodiment, the AM-transwell is a cylinder, cube, cuboid, frustum of a cone, frustum of a pyramid, truncated sphere, or a combination thereof. In one embodiment, the AM-transwell is a cube. See FIG. 7. In one embodiment, the AM-transwells 200, 300 are cylinders. For example, see FIGS. 2A-2B and 3A-3B.

[0038] In one embodiment, the AM-transwells 200, 300 have a height 202, 302 of about 10 mm to about 16 mm, and any range or value therebetween, and a thickness 204, 304 of about 1 mm to 3 mm, and any range or value therebetween. In one embodiment, the AM-transwells 200, 300 have a height 202, 302 of about 13 mm and a thickness 204, 304 of about 2 mm.

[0039] In one embodiment, the thickness 202, 302 of the AM-transwells 200, 300 depends on the interface 308 of the mini-lobe 306.

[0040] In one embodiment, the lower chambers 210, 310 have a height 212, 312 of about 2 mm to about 4 mm, and any range or value therebetween, and the upper chambers 220, 320 have a height 222, 322 of about 8 - 12 mm, and any range or value therebetween. In one embodiment, the lower chambers 210, 310 have a height 212, 312 of about 3 mm, and the upper chambers 220, 320 have a height 222, 322 of about 10 mm.

[0041] In one embodiment, the membranes 230, 330 have a thickness 332 of about 1 mm to about 3 mm, and any range or value therebetween. In one embodiment, the membranes 230, 330 have a thickness 332 of about 2 mm.

[0042] In one embodiment, the inner walls of the upper chambers 220, 320 support the growth of lung epithelial cells (SAEC, small airway epithelial cells) internally.

[0043] In one embodiment, one or more legs 240, 340 have a length 242, 342 of about 3.7 mm to about 5.7 mm, and any range or value therebetween. In one embodiment, one or more legs 240, 340 have a length 242, 342 of about 4.72 mm.

[0044] In one embodiment, one or more legs 240, 340 have a height 244, 344 of about 2 mm to about 4 mm, and any range or value therebetween. In one embodiment, one or more legs 240, 340 have a height 244, 344 of about 3 mm.

[0045] In one embodiment, one or more legs 240, 340 have a width 246 of about 0.6 mm to about 2.6 mm, and any range or value therebetween. In one embodiment, one or more legs 240, 340 have a width 246 of about 1.64 mm.

[0046] In one embodiment, one or more legs 240, 340 assist in the growth of lung epithelial cells (PAECs, pulmonary airway endothelial cells) at the bottom.

[0047] In one embodiment, one or more of the bottom chamber, the upper chamber, the membrane, and one or more of the one or more legs are printed using a synthetic bioink.

[0048] In one embodiment, the synthetic bioink includes one or more of a degradable ink and a triacrylate peptide ink.

[0049] In one embodiment, the synthetic bioink includes an amount of HPA of about 3% to about 10%, and any range or value therebetween, an amount of PEGDA3400 of about 5% to about 20%, and any range or value therebetween, an amount of PEGDA6000 of about 5% to about 20%, and any range or value therebetween, an amount of PEGDA575 of about 1% to about 20%, and any range or value therebetween, an amount of PEGDA700 of about 1% to about 20%, and any range or value therebetween, an amount of PEGTAC of about 1% to about 5%, and any range or value therebetween, an amount of PEO of about 0.1% to about 5%, and any range or value therebetween, an amount of NAP of about 1% to about 3%, and any range or value therebetween, an amount of LAP of about 1% to about 3%, and any range or value therebetween, and one or more of an amount of UV386A of about 0.1% to about 0.5%, and any range or value therebetween.

[0050] In one embodiment, the synthetic bioink further includes the balance of water.

[0051] In one embodiment, the synthetic bioink further includes a buffer solution containing 0.1M HEPES aqueous solution and 1×PBS at pH 7.2.

[0052] In one embodiment, the synthetic bioink further comprises a monocysteine peptide in an amount of 0.5 mM to about 20 mM and any range or value therebetween, and a dicysteine peptide in the range of 0.5 mM to about 20 mM and any range or value therebetween.

[0053] In one embodiment, the dicysteine peptide is MMP-degradable.

[0054] In one embodiment, the monocysteine peptide comprises one or more of RGDS, PHSRNKRGDS, IKVAV, GFOGER, and an ECM binder (BM binder, FN binder).

[0055] Figure 4 shows a photograph of a 3D printed AM-transwell using the synthetic bioink and a digital light projection (DLP) printer, and Figure 5 shows a photograph of a 3D printed membrane using the synthetic bioink and a digital light projection (DLP) printer.

[0056] Exemplary bioink formulations and print settings for the LS80 printer Figure 6 shows a photograph of an exemplary 3D printed AM-transwell having a cylindrical shape using the synthetic bioink and an LS80 printer.

[0057] [Example 1] (AG71)

[0058]

Table 1

[0059] [Example 2] (AG72)

[0060]

Table 2

[0061] [Example 3] (AG73)

[0062]

Table 3

[0063] [Example 4] (AG74)

[0064]

Table 4

[0065] [Example 5] (AG75)

[0066]

Table 5

[0067] [Example 6] (AG76)

[0068]

Table 6

[0069]

Table 7

[0070] Figure 6 shows a photograph of an exemplary 3D-printed AM-transwell having a cylindrical shape using a synthetic bioink and an LS80 printer.

[0071] The AM-transwells were reproducible using all synthetic bioink formulations and print settings of the LS80 printer.

[0072] Exemplary bioink formulations and print settings for the BLF20 printer Figure 7 shows a photograph of an exemplary 3D printed AM-transwell having a cubic shape using a synthetic bioink and a BLF20 printer, and Figure 8 shows a photograph of an exemplary 3D printed AM-transwell having a cylindrical shape using a synthetic bioink and a BLF20 printer.

[0073] [Example 7] (AG77)

[0074] [Table 8]

[0075] [Example 8] (AG78)

[0076] [Table 9]

[0077] [Example 9] (AG79)

[0078] [Table 10]

[0079] [Example 10] (AG80)

[0080] [Table 11]

[0081] Print settings for the BLF20 printer (cube Transwell design)

[0082] [Table 12]

[0083] Figure 7 shows an exemplary photograph of a 3D printed AM-transwell having a cubic shape using synthetic bioink and a BLF20 printer.

[0084] Print settings for the BLF20 printer (circular (cylindrical) design)

[0085] [Table 13]

[0086] Figure 8 shows an exemplary photograph of a 3D printed AM-transwell having a cylindrical shape using synthetic bioink and a BLF20 printer.

[0087] The AM-transwells were reproducible using all synthetic bioink formations and print settings of the BLF19 / 20 and FS20 printers.

[0088] Permeability of AM-Transwell AM-Transwell printing The printer baths were cleaned before filling with bioink. After cleaning, 20 mL of bioink was added to the printer baths. The printer settings were loaded into a digital light projection (DLP) printer and subsequently printed. After printing, the 3D printed AM-transwells were placed in 1×PBS Ca+ / Mn+. The printer baths and platforms were cleaned.

[0089] Sterilization of AM-Transwell The 3D printed AM-transwells were placed in 1×PBS Ca+ / Mn+. The scaffolds were washed three times in 1×PBS Ca+ / Mn+. The 1×PBS Ca+ / Mn+ waste was discarded into an acrylate waste container. The scaffolds and 3D printed AM-transwells were incubated overnight in 0.1M HEPES / 1×PBS supplemented with an antibiotic / antifungal agent (anti-anti).

[0090] Permeability assay The sterile buffer was decanted inside the biosafety cabinet. After decanting, the 3D-printed AM-transwell was transferred to a 12-well plate. 1.2 mL of PAEC culture medium was added to the apical compartment (upper chamber). All air bubbles were gently removed from beneath the 3D-printed AM-transwell.

[0091] In the biosafety cabinet, prepare the FITC-dextran solution with little or no light exposure.

[0092] Calculation of FITC-dextran solution Final experimental concentration = 400 μg / mL Stock concentration = 10 mg / mL Total volume = 15 mL (400 μg / mL)(15 mL) / (10,000 μg / mL) = 0.6 mL = 600 μL of FTIC-dextran 10K

[0093] 300 μL of the FITC-dextran solution was added to the apical compartment (upper chamber) of the 3D-printed AM-transwell to enable measurement of permeability over time to the basal compartment (lower chamber). 600 μL of the FITC-dextran solution was added to a commercially available plastic transwell (e.g., Corning) as a control.

[0094] Figure 9 shows a schematic diagram of the experimental setup for the permeability assay of the AM-transwell.

[0095] Using a plate reader, measure the fluorescence of 100 μL aliquots (n = 3) from the basal chamber (lower compartment) at 490 / 520 nm at 1 hour, 2 hours, and 4 hours.

[0096] Replenish the basal compartment (lower chamber) with the same volume of FITC-dextran medium as the aliquot removed for fluorescence measurement.

[0097] Creation of standard curve Figure 10 shows a schematic diagram of the experimental setup for the serial dilution established using known concentrations of FITC-dextran for the permeability assay of the AM-transwell.

[0098] For the standard curve, take 250 μL of the 400 μg / mL FITC-dextran solution and add it to 1.750 mL of PAEC medium. Mix well by pipetting up and down. Transfer 1 mL of the dilution to an Eppendorf tube containing 1 mL of PAEC medium and mix well. This dilutes the original concentration by half. Perform a total of 8 serial dilutions. Measure the fluorescence of 100 μL aliquots at 490 / 520 nm. Create a standard curve using the known concentrations. Determine the optimal straight line. Use the optimal equation to quantify the FITC-dextran in the experimental samples. In the optimal equation, x is the concentration (μg / mL) and Y is the relative fluorescence.

[0099] Calculation of initial dilution of standard curve Initial concentration = 400 μg / mL Final concentration = 50 μg / mL (50 μg / mL)(2 mL) / (400 μg / mL) = 0.25 mL Diluent = 2000 - 250 = 1750 μL

[0100] Figure 11 shows a standard curve graph of concentration (μg / mL) versus relative fluorescence units in the permeability assay of the AM-transwells (AG71 to AG76).

[0101] Figure 12 shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwells (AG71 to AG76).

[0102] The concentration of FITC-dextran in the 3D-printed AM-transwell was compared over time with that in a commercially available plastic transwell (e.g., Corning). A slight decreasing trend in the concentration of FITC-dextran was observed from AG73 to AG76, while a slight increasing trend in the concentration of FITC-dextran was observed in AG71 and AG72.

[0103] Figure 13A shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwell (AG71). Figure 13B shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwell (AG72). Figure 13C shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwell (AG73). Figure 13D shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwell (AG74). Figure 13E shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwell (AG75). Figure 13F shows a graph of time versus FITC-dextran concentration (μg / mL) in the permeability assay of the AM-transwell (AG76).

[0104] Permeability assay Start with 50 μg / mL, prepare 1 / 2-step dilutions, and create a standard curve by measuring fluorescence at 490 / 520 nm.

[0105] In a 12-well plate, fill each well to be used with PAEC culture medium. Carefully transfer the 3D-printed AM-transwell to the 12-well plate. Gently remove the air bubbles from under the 3D-printed AM-transwell.

[0106] After the air bubbles are removed, add 175 μL of 400 μg / mL FITC-dextran solution to the top compartment (upper chamber) of the 3D-printed AM-transwell.

[0107] Cover the 12-well plate with foil and incubate at 37 °C.

[0108] Figure 9 shows a schematic diagram of the experimental apparatus for the permeability assay of the AM-transwell.

[0109] Using a plate reader, measure the fluorescence of 100 μL aliquots (n = 3) from the basal chamber (lower compartment) at 490 / 520 nm for 1 hour, 2 hours, 4 hours, and 21 hours.

[0110] Replenish the basal compartment (lower chamber) with the same volume of FITC-dextran medium as the aliquot removed.

[0111] Use a commercially available plastic transwell (e.g., Corning) as a control.

[0112] Standard curve creation Figure 10 shows a schematic diagram of the serial dilution experimental apparatus for the permeability assay of the AM-transwell.

[0113] For serial dilution, take 250 μL of a 400 μg / mL FITC-dextran solution and add it to 1.750 mL of endothelial (PAEC) medium. Mix well by pipetting up and down. Transfer 1 mL of this first dilution to an Eppendorf tube containing 1 mL of PAEC medium and mix well. Perform a total of 8 dilutions. Transfer 100 μL of each dilution (repeated 3 times) to a 96-well plate and measure the fluorescence at 490 / 520 nm. Create a standard curve using known concentrations. Determine the optimal straight line. Use the optimal equation to quantify the FITC-dextran in the experimental samples. In the optimal equation, x is the concentration (μg / mL) and Y is the relative fluorescence.

[0114] Calculation of FITC-dextran stock solution Stock = 10 mg / mL Final assay concentration = 400 μg / mL Final volume = 4 mL Stock volume = (400 μg / mL)(4 mL) / 10,000 μg / mL) = 0.16 mL = 160 μL

[0115] Calculation of initial dilution of standard curve Initial concentration = 400 μg / mL Final concentration = 50 μg / mL (50 μg / mL)(2 mL) / (400 μg / mL) = 0.25 mL Diluent = 2000 μL - 250 μL = 1750 μL

[0116] 21-hour analysis After fluorescence measurement at the end of 21 hours, the FITC-dextran medium is removed from the apical compartment (upper chamber) of the 3D-printed AM-transwell. Assuming a concentration of 400 μg / mL, the solution is diluted 1:16 to a theoretical concentration of 25 μg / mL. A 100 μL aliquot is taken and fluorescence is measured at 490 / 520 nm using a plate reader.

[0117] Figure 14 shows a standard curve graph of concentration (μg / mL) versus relative fluorescence units in the permeability assay of AM-transwells (AG77 to AG80).

[0118] Figure 15 shows a graph of time versus the percentage of FITC-dextran in the basal compartment in the permeability assay of AM-transwells (AG77 to AG80).

[0119] The concentration of FITC-dextran within the 3D-printed AM-transwell was compared over time with a commercially available plastic transwell (e.g., Corning). The data are presented as the mean mass percentage of FITC-dextran in the basal compartment (lower chamber) at time T relative to the total amount of FITC-dextran in the whole system.

[0120] Figure 16A shows a graph of time in the basal compartment and the percentage of FITC-dextran in the permeability assay of AM-transwell (AG77). Figure 16B shows a graph of time in the basal compartment and the percentage of FITC-dextran in the permeability assay of AM-transwell (AG78). Figure 16C shows a graph of time in the basal compartment and the percentage of FITC-dextran in the permeability assay of AM-transwell (AG79). Figure 16D shows a graph of time in the basal compartment and the percentage of FITC-dextran in the permeability assay of AM-transwell (AG80).

[0121] The permeability of each replicate was measured in triplicate and each fluorescence measurement was compared to the average of the composition and a commercially available plastic transwell (e.g., Corning) as a control. The data are presented as the mean of the mass percentage of FITC-dextran in the basal compartment (lower chamber) at time T relative to the total amount of FITC-dextran in the entire system.

[0122] Medium recovery after 3 days Method Changes in the liquid volume in the apical compartment (upper chamber) and the basal compartment (lower chamber) of 3D-printed AM-transwells (AG71) were measured after a certain period of time. The 3D-printed AM-transwells were first stored in PBS in 50 mL tubes. The PBS was decanted, and the 3D-printed AM-transwells were transferred to 50 mL sterile tubes. 35 mL of HEPES / PBS anti-anti was added to the 50 mL tube containing the 3D-printed AM-transwells and sterilized overnight. After sterilization, the sterilizing buffer was decanted, and the 3D-printed AM-transwells were incubated in the medium for at least 4 hours. After incubation in the medium, the 3D-printed AM-transwells were transferred to a 12-well plate. 1.2 mL of medium was added to the basal compartment of the 3D-printed AM-transwells. Bubbles were gently removed from under the 3D-printed AM-transwells. After the bubbles were removed, 0.5 mL of medium was carefully added to the basal compartment of the 3D AM-transwells. 175 μL of medium was added dropwise to the apical compartment while taking care not to overflow. The 3D-printed AM-transwells were incubated in an incubator at 37 °C for 3 days. After 3 days, the plates were removed from the incubator. For each 3D-printed AM-transwell, all the medium was collected from the basal compartment into a 5 mL tube. Similarly, all the medium was carefully collected from the apical compartment into a 0.5 mL Eppendorf tube. Then, the total volume of the medium collected from each compartment was measured and recorded.

[0123] Initial conditions

[0124]

Table 14

[0125] Apical recovery amount

[0126]

Table 15

[0127] Basal recovery amount

[0128]

Table 16

[0129] Total recovery amount

[0130]

Table 17

[0131] Figure 17 is a graph of the ratio of compartments to medium recovered from the AM-Transwell after 3 days.

[0132] The volume of the apical compartment (upper chamber) can be increased to hold more medium.

[0133] More medium can be added outside the AM-Transwell to keep the hydrogel more moisturized and hold the medium in the apical compartment.

[0134] As indicated by more than 50% of the medium remaining in the apical compartment after 3 days, the medium can be changed daily during cell culture to prevent the apical compartment from drying out excessively to maintain the cell monolayer.

[0135] Method for fabricating AM-Transwell The method of making an AM-Transwell includes a) printing one or more of the lower chamber, upper chamber, and membrane of the AM-Transwell using 3D printing technology, and b) assembling and / or printing the AM-Transwell as described herein to form an assembled AM-Transwell.

[0136] In one embodiment, step b) prints at least 3 assembled AM-transwells. In one embodiment, step b) prints at least 20 assembled AM-transwells. In one embodiment, step b) prints at least 50 assembled AM-transwells.

[0137] In one embodiment, the method further comprises c) optionally storing the assembled AM-transwells at 4°C until needed.

[0138] In one embodiment, the method further comprises c) transferring each of the AM-transwells into DPBS+ / + in a tube, d) decanting the DPBS+ / + from the tube and washing each AM-transwell an additional two times in DPBS+ / + for about 5 minutes each to form a primary washed AM-transwell, and e) transferring the primary washed AM-transwells into a 50 mL sterile tube.

[0139] In one embodiment, the method further comprises f) incubating the primary washed AM-transwells overnight in DPBS- / - 1×anti-anti to form incubated AM-transwells.

[0140] In one embodiment, the method further comprises g) decanting the DPBS- / - 1×anti-anti and washing the incubated AM-transwells in about 35 mL of PBS / HEPES 1×anti-anti for about 4 hours to form a secondary washed AM-transwell, and h) decanting the PBS / HEPES 1×anti-anti from the tube and incubating the secondary washed AM-transwells in about 35 mL of cell culture medium in a 50 mL tube for about 2 days or over the weekend.

[0141] In one embodiment, the 3D printing technology is one or more digital light projection printing (DLP), stereolithography (SLA) printing technology, extrusion 3D printing technology, or selective laser sintering (SLS) 3D printing technology. In one embodiment, the 3D printing technology is digital light printing (DLP) printing technology.

[0142] Method for using AM-Transwell The method of using the AM-transwell includes a) seeding cells on at least one side of the membrane of the AM-transwell described herein.

[0143] The method of using the AM-transwell The method of using the AM-transwell includes a) seeding cells on both sides of the membrane of the AM-transwell described herein for in vitro cell studies.

[0144] The embodiments and examples described herein are presented to most appropriately illustrate the invention and its actual implementation forms, so that those skilled in the art can make and use the invention. However, those skilled in the art will understand that the foregoing descriptions and examples are presented for illustrative and exemplary purposes only. The descriptions set forth herein are not intended to be exhaustive, nor are they intended to limit the invention to the specific forms disclosed. Based on the above teachings, many modifications and variations are possible without departing from the spirit and scope of the following claims. The invention is particularly intended to be construed to the maximum extent within the scope of the following claims and their equivalents.

[0145] In this specification, any reference to an implementation, element, or act of a system and method in the singular may include implementations that include a plurality of these elements. Also, in this specification, any reference to an implementation, element, or act in the plural may include implementations that include only a single element. References in the singular or plural are not intended to limit the systems or methods, their components, acts, or elements of the present disclosure to a single or plural configuration. Any reference to an act or element based on any information, act, or element may include implementations in which the act or element is at least partially based on any information, act, or element.

[0146] As used in this specification, the terms "approximately", "about", "substantially", and similar terms are to be construed to have a broad meaning in accordance with the ordinary and accepted usage by those skilled in the art to which the subject matter of the present disclosure pertains. It will be understood by those skilled in the art considering the present disclosure that these terms are intended to enable the description of the specific features described and claimed without limiting them to the exact numerical ranges provided. Accordingly, these terms are to be construed as indicating that even if there are minor or insignificant changes or modifications to the subject matter described and claimed, they are within the scope of the present disclosure as set forth in the appended claims.

[0147] As used in this specification, the term "exemplary" and its variations are used when describing various embodiments and are intended to indicate that such embodiments are examples, representative examples, or an example of possible embodiments (and such terms are not intended to mean that such embodiments are necessarily particularly excellent or outstanding examples).

[0148] As used herein, "coupled" and variations thereof mean that two members are joined directly or indirectly to each other. Such joining may be immutable (e.g., permanent or fixed) or movable (e.g., removable or releasable). Such joining can be achieved when the two members are directly joined to each other, when the two members are joined to each other via another intervening member, or when the two members are joined to each other via an intervening member that is a single unitary body integrally formed with one of the two members. When "coupled" or variations thereof are modified by additional terms (e.g., "directly coupled"), the general definition of "coupled" as described above is modified by the ordinary meaning of the additional term (e.g., "directly coupled" means that two members are joined without passing through another intervening member), resulting in a definition that is more limited than the general definition of "coupled" as described above. Such coupling may be mechanical, electrical, or fluidic.

[0149] Any implementation disclosed herein can be combined with any other implementation, and references to "an implementation", "some implementations", "another implementation", "various implementations", "one implementation", etc. are not necessarily mutually exclusive and are intended to indicate that a particular feature, structure, or characteristic may be included in at least one implementation. These terms as used herein do not necessarily represent the same implementation. Any implementation may be combined inclusively or exclusively with any other implementation in a manner consistent with the aspects and implementations disclosed herein.

[0150] References to "or" may be construed inclusively such that any term described using "or" may indicate any one, more than one, and all of the terms described. References to at least one of a list of terms may be construed as an inclusive "or" indicating any one, more than one, and all of the terms described. For example, the description "at least one of 'A' and 'B'" can include only 'A', only 'B', and both 'A' and 'B'. It can also include elements other than 'A' and 'B'.

[0151] Descriptions in this specification regarding the position of elements (e.g., "above", "below", "upper", "lower") are merely used to describe the orientation of various elements in the drawings. It should be noted that for other exemplary embodiments, the orientations of various elements may be different, and such variations are intended to be included within the scope of this disclosure.

[0152] Although the steps of a method may be shown in a particular order in the drawings and description, the order of these steps may be different from the order shown and described, unless otherwise specified above. Also, unless otherwise specified above, two or more steps may be performed simultaneously or partially simultaneously. Such variations may depend, for example, on the selected software and hardware systems and the choices of the designer. All such variations are within the scope of this disclosure. Similarly, the software implementations of the described methods can be achieved by implementing various connection steps, processing steps, comparison steps, and decision steps using standard programming techniques with rule - based logic and other logics.

[0153] Reference signs attached to technical features in the drawings, detailed description, or claims are provided to facilitate understanding of the drawings, detailed description, and claims. Therefore, the presence or absence of reference signs does not have a limiting effect on the scope of any claim element.

[0154] The systems and methods described in this specification may be implemented in other specific forms without departing from their characteristics. The above-described implementations do not limit the systems and methods described, but are merely exemplary. Therefore, the scope of the systems and methods described in this specification is indicated not by the above description, but by the appended claims, and changes occurring within the meaning and equivalent scope of the claims are to be included therein.

Description of Reference Numerals

[0155] 100 Commercially available plastic well (prior art) 200, 300 AM-well 202, 302 Height of AM-well 204, 304 Thickness of AM-well 210, 310 Lower chamber 212, 312 Height of lower chamber 220, 320 Upper chamber 222, 322 Height of upper chamber 230, 330 Membrane 240, 340 Legs 242, 342 Length of legs 244, 344 Height of legs 246 Width of legs 306 Mini robe 308 Interface 332 Thickness of membrane

Claims

1. A high-throughput manufacturing well (AM-well), comprising: a) a lower chamber, b) an upper chamber, c) a membrane disposed between the lower chamber and the upper chamber, and d) one or more legs forming at least a part of the lower chamber wherein one or more of the lower chamber, the upper chamber, the membrane, and the one or more legs are printed using a synthetic bioink. The AM-well.

2. The AM-well according to claim 1, wherein the AM-well is a cylinder, a cube, a cuboid, a frustum of a cone, a frustum of a pyramid, a truncated sphere, or a combination thereof.

3. The AM-well according to claim 1, wherein the AM-well is a cylinder.

4. The AM-well according to claim 1, having a height of about 10 mm to about 16 mm and a thickness of about 1 mm to 3 mm.

5. The AM-well according to claim 1, having a height of about 13 mm and a thickness of about 2 mm.

6. The AM-well according to claim 1, wherein the lower chamber has a height of about 2 mm to about 4 mm, and the upper chamber has a height of about 8 to about 12 mm.

7. The AM-well according to claim [omitted number in the original], wherein the lower chamber has a height of about 3 mm, and the upper chamber has a height of about 10 mm.

10. The AM-well according to claim 1, wherein the membrane has a thickness of about 1 mm to about 3 mm.

11. The AM-well according to claim 1, wherein the membrane has a thickness of about 2 mm.

12. The AM-well according to claim 1, wherein the synthetic bioink comprises one or more of a degradable peptide ink and a triacrylate peptide ink.

13. The synthetic bioink comprises HPA in an amount of about 3% to about 10%, PEGDA3400 in an amount of about 5% to about 20%, PEGDA6000 in an amount of about 5% to about 20%, PEGDA575 in an amount of about 1% to about 20%, PEGDA700 in an amount of about 1% to about 20%, PEGTA-C in an amount of about 1% to about 5%, PEO in an amount of about 0.1% to about 5%, NAP in an amount of about 1% to about 3%, LAP in an amount of about 1% to about 3%, and UV386A in an amount of about 0.1% to about 0.5% The AM-transwell according to claim 1, comprising one or more of the following.

14. The AM-transwell according to claim 13, wherein the synthetic bioink further comprises a remaining amount of water.

15. The synthetic bioink contains a buffer solution containing 0.1 M HEPES aqueous solution and 1×PBS at pH 7.2 The AM-transwell according to claim 13, further comprising the above.

16. The synthetic bioink contains a monocysteine peptide in an amount of 0.5 mM to 20 mM, and a dicysteine peptide in an amount of 0.5 mM to 20 mM The AM-transwell according to claim 13, further comprising the above.

17. The AM-transwell according to claim 16, wherein the monocysteine peptide comprises one or more of RGDS, PHSRNKRGDS, IKVAV, Bm binder, and FN binder.

18. The AM-transwell according to claim 16, wherein the dicysteine peptide is degradable by MMP.

19. A method for fabricating a high-precision manufacturing transwell (AM-transwell), comprising: a) printing one or more of the lower chamber, upper chamber, and membrane of the AM-transwell using 3D printing technology; and b) assembling and / or printing the AM-transwell according to claim 1 to form an assembled AM-transwell. The method comprising the above.

20. The method according to claim 19, wherein the 3D printing technology is one or more of digital light projection printing (DLP), stereolithography (SLA) printing technology, extrusion 3D printing technology, or selective laser sintering 3D printing technology, or a combination thereof.

21. The method according to claim 19, wherein the 3D printing technology is digital light printing (DLP) printing technology.

22. c) storing the assembled AM-transwell at 4°C until needed. The method according to claim 19, further comprising the above.

23. c) transferring each of the AM-transwells into DPBS++ in a tube; d) decanting the DPBS++ from the tube and washing each of the AM-transwells an additional two times in DPBS++ for about 5 minutes to form a primary washed AM-transwell; and e) transferring the once-washed AM-transwells into 50 mL sterile tubes The method according to claim 19, further comprising this step. **Claim 24** f) further comprising incubating the once-washed AM-transwells overnight in DPBS− / − supplemented with antibiotics / antifungals (anti-anti) to form sterilized AM-transwells, the method according to claim 23 **Claim 25** g) decanting the DPBS− / − anti-anti, and washing the incubated AM-transwells in about 35 mL of PBS / 0.1 M HEPES anti-anti buffer solution for about 4 hours to form twice-washed M-transwells; h) further comprising decanting the PBS / 0.1 M HEPES anti-anti buffer solution from the tube and incubating the twice-washed AM-transwells in about 35 mL of cell culture medium in a about 50 mL tube for about 2 days or over the weekend, the method according to claim 24 **Claim 26** Step b) prints at least 3 assembled AM-transwells, the method according to claim 19 **Claim 27** Step b) prints at least 20 assembled AM-transwells, the method according to claim 19 **Claim 28** Step b) prints at least 50 assembled AM-transwells, the method according to claim 19 **Claim 29** A method of using high-production transwells (AM-transwells), comprising: a) seeding cells on at least one side of the membrane of the AM-transwell according to claim 1 A method comprising this step. **Claim 30** A method of using high-production transwells (AM-transwells), comprising: a) seeding cells on both sides of the membrane of the AM-transwell according to claim 1 for in vitro cell research A method comprising this step.

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