Transwell for cell culture, method for manufacturing using synthetic bioink, and in vitro tissue model

Abstract

The invention relates to an advanced manufactured transwell (AM-transwell). The AM-transwell comprises a lower chamber, an upper chamber, a lower chamber, an upper chamber and a lower chamber, an upper chamber; a membrane disposed between the lower chamber and the upper chamber; and one or more struts. The one or more struts form at least a portion of the lower chamber. One or more of the lower chamber, the upper chamber, the membrane, and the one or more struts are printed using synthetic bio-ink. Methods for making and using the AM-transwell are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 392,724, filed on July 27, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present application relates to 3D printed structures. background

[0003] Traditionally, in vitro assessment of epithelial and endothelial barrier function has been achieved using commercially available plastic transwells (e.g., Corning) containing porous membranes made of polyester (PET) or polycarbonate (PC). These membranes are "stiff" and do not allow changes in their mechanical properties. Overview

[0004] The systems and methods disclosed herein include advanced manufactured (AM) transwells 3D printed using synthetic bioink. The 3D printed AM-transwells contain printable membranes with adjustable thickness, porosity, and mechanical properties. The 3D printed AM-transwells can be surface modified to allow epithelial and endothelial cell barrier formation, followed by barrier function assessment.

[0005] In an embodiment, an advanced fabricated transwell (AM-transwell), the AM-transwell comprises: 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 an embodiment, one or more pillars form at least a portion of the lower chamber.

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

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

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

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

[0011] In an embodiment, one or more of the lower chamber, the upper chamber, the membrane, and the one or more pillars are printed using synthetic bio-ink.

[0012] In an embodiment, the synthetic bio-ink comprises one or more of a degradable peptide ink and a triacrylate peptide ink.

[0013] In an embodiment, the synthetic bio-ink comprises one or more of the following: 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; 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 any range or value therebetween; and UV386A (386nm visible dye) in an amount of about 0.1% to about 0.5%, and any range or value therebetween.

[0014] In an embodiment, the synthetic biological ink further comprises: a balance of water.

[0015] In an embodiment, the synthetic bio-ink further comprises: a buffer solution comprising a 0.1 M HEPES aqueous solution and 1X PBS, with a pH of 7.2.

[0016] In an embodiment, the synthetic bio-ink further comprises: a monocysteine ​​peptide in an amount of 0.5 mM to 20 mM, and any range or value therebetween; and a dicysteine ​​peptide in an amount of 0.5 mM to 20 mM, and any range or value therebetween.

[0017] In embodiments, the bi-cysteine ​​peptide is matrix metalloproteinase (MMP) degradable.

[0018] In an embodiment, the monocysteine ​​peptide comprises one or more of RGDS, PHSRNKRGDS, IKVAV, AG73, GFOGER, Bm-Binder, and Fn-Binder.

[0019] In an embodiment, a method for preparing an advanced manufactured transwell (AM-transwell) includes: a) printing one or more of a lower chamber, an upper chamber, and a 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.

[0020] In an embodiment, step b) prints at least 3 assembled AM-transwells and any ranges or values ​​therein. In an embodiment, step b) prints at least 20 assembled AM-transwells. In an embodiment, step b) prints at least 50 assembled AM-transwells.

[0021] In an 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 an embodiment, the 3D printing technology is digital light projection (DLP) printing technology.

[0022] In embodiments, the method further comprises: c) optionally storing the assembled AM-transwell at 4°C until needed.

[0023] In embodiments, the method further comprises: c) transferring each of the AM-transwells to 1X DPBS Ca+ / Mn+ in a tube; d) pouring out the 1X DPBS Ca+ / Mn+ from the tube and washing each AM-transwell twice more in DPBS Ca+ / Mn+ for about 5 minutes to form a first washed AM-transwell; and 3) transferring the first washed AM-transwell to a sterile tube. In embodiments, the method further comprises: c) transferring each of the AM-transwells to about 35 mL DPBS Ca+ / Mn+ in an about 50 mL tube; d) pouring out the DPBS Ca+ / Mn+ from the tube and washing each AM-transwell twice more in about 35 mL DPBS Ca+ / Mn+ for about 5 minutes to form a first washed AM-transwell; and e) transferring the first washed AM-transwell to a sterile 50 mL tube. In embodiments, the volume of DPBS Ca+ / Mn+ and the size of the tube in steps c) and d) depends on the number of AM-transwells to be transferred. The AM-transwells only need to be enclosed in some buffered deionized water solution.

[0024] In embodiments, the method further comprises: f) incubating the first washed AM-transwell in 1X DPBS Ca- / Mn- supplemented with antibiotic / antimycotic (anti-anti) overnight to form a sterilized AM-transwell.

[0025] In embodiments, the method further comprises: g) pouring off the 1X DPBS Ca- / Mn-anti-anti and washing the incubated AM-transwell in about 35 mL of 1X PBS / 0.1M HEPES anti-anti buffer solution for about 4 hours to form a second washed AM-transwell; and h) pouring off the 1X PBS / 0.1M HEPES anti-anti buffer solution from the tube and incubating the second washed AM-transwell in about 35 mL of cell culture medium in the tube for about 2 days or over a weekend. In embodiments, the method further comprises: g) pouring off the 1X DPBS Ca- / Mn-anti-anti and washing the incubated AM-transwell in about 35 mL 1XPBS / 0.1M HEPES anti-anti buffer solution for about 4 hours to form a second washed AM-transwell; and h) pouring off the 1X PBS / 0.1M HEPES anti-anti buffer solution from the tube and incubating the second washed AM-transwell in about 35 mL cell culture medium in an about 50 mL tube for about 2 days or over a weekend.

[0026] In an embodiment, a method of using an advanced fabrication transwell (AM-transwell) comprises: a) seeding cells on at least one side of a membrane of an AM-transwell as described herein for in vitro cell studies.

[0027] In an embodiment, a method of using an advanced fabrication transwell (AM-transwell) comprises: a) seeding cells on both sides of a membrane of an AM-transwell as described herein for in vitro cell studies.

[0028] Those skilled in the art will appreciate that this summary is illustrative only and is not intended to be limiting in any way. Other aspects, inventive features and advantages of the devices and / or processes described herein (limited only by the claims) will become apparent in the detailed description set forth herein and in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The details of one or more embodiments 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 description, drawings, and claims.

[0030] Figure 1 shows a commercially available plastic transwell (prior art);

[0031] Figure 2A shows an upper perspective view of an AM-transwell according to an embodiment of the present invention;

[0032] Figure 2B Shows Figure 2A The bottom perspective view of the AM-transwell;

[0033] Figure 3A shows a top view of an AM-transwell according to an embodiment of the present invention;

[0034] Figure 3B Shows the Figure 3A Side view of the AM-transwell;

[0035] Figure 4 A photograph depicting an AM-transwell 3D printed using synthetic bio-ink and a digital light projection (DLP) printer;

[0036] Figure 5 Depicts a photograph of a membrane 3D printed using synthetic bio-ink and a digital light projection (DLP) printer;

[0037] Figure 6 A photograph showing an exemplary 3D printed AM-transwell with a cylindrical shape using synthetic bio-ink and an LS80 printer;

[0038] Figure 7 A photograph depicting an exemplary 3D printed AM-transwell having a cubic shape using synthetic bio-ink and a BLF20 printer;

[0039] Figure 8 A photograph depicting an exemplary 3D-printed AM-transwell with a cylindrical shape using synthetic bio-ink and a BLF20 printer.

[0040] Fig. 9 Schematic diagram showing the experimental setup of the AM-transwell permeability assay;

[0041] Fig.10 Schematic diagram showing the experimental setup of the serial dilution AM-transwell permeability assay;

[0042] Fig.11 A standard curve graph of concentration (μg / mL) vs relative fluorescence units for the AM-transwell (bioinks AG71 to AG76) permeability assay is shown;

[0043] Fig.12 A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bioinks AG71 to AG76) permeability assay;

[0044] Fig.13A A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bio-ink AG71) permeability assay;

[0045] Fig. 13B A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bio-ink AG72) permeability assay;

[0046] Fig. 13C A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bio-ink AG73) permeability assay;

[0047] Fig.13D A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bio-ink AG74) permeability assay;

[0048] Fig.13E A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bio-ink AG75) permeability assay;

[0049] Fig.13F A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (bio-ink AG76) permeability assay;

[0050] Fig.14 A graph showing concentration (μg / mL) vs relative fluorescence units for AM-transwell (bioinks AG77 to AG80) permeability assays is shown;

[0051] Fig.15 A graph showing the time of permeability assays of AM-transwells (bioinks AG77 to AG80) vs. the percentage of FITC-dextran in the lower chamber compartment;

[0052] Fig.16A A graph showing the time of AM-transwell (bioink AG77) permeability assay vs. the percentage of FITC-dextran in the lower chamber compartment;

[0053] Fig. 16B A graph showing the time of AM-transwell (bioink AG78) permeability assay vs. the percentage of FITC-dextran in the lower chamber compartment;

[0054] Fig. 16CA graph showing time vs. percentage of FITC-dextran in the lower chamber compartment of the AM-transwell (AG79) permeability assay;

[0055] Fig.16D A graph showing time vs. percentage of FITC-dextran in the lower chamber compartment of an AM-transwell (AG80) permeability assay; and

[0056] Fig.17 Shown is a graph of compartments versus the percentage of medium recovered from the AM-transwells after three days. Detailed Description

[0057] The following detailed description of various embodiments of the present invention refers to the accompanying drawings, which illustrate specific embodiments in which the present invention can be practiced. Although the exemplary embodiments of the present invention have been described in detail, it should be understood that various other modifications will be apparent to those skilled in the art without departing from the spirit and scope of the present invention, and these modifications can be easily made. Therefore, the scope of the appended claims is not limited to the examples and descriptions described herein, but the claims should be interpreted as covering all features of the patentable novelty in the present invention, including all features that are considered to be equivalent to those skilled in the art of the present invention. Therefore, the scope of the present invention is limited only by the appended claims and the full scope of equivalents to which these claims are entitled. Commercial Transwell

[0058] FIG. 1 shows a commercially available plastic transwell 100 (prior art). 3D printed Transwell

[0059] Figure 2A shows an upper perspective view of an AM-transwell 200 according to an embodiment of the present invention; and Figure 2B Shows Figure 2A A perspective view of the AM-transwell 200 from below.

[0060] Figure 3A shows a top view of an AM-transwell 300 according to an embodiment of the present invention; and Figure 3B Shows the Figure 3A Side view of the AM-transwell 300.

[0061] like Figure 2A-2B and Figure 3A-3BAs shown, the AM-transwell 200 , 300 has a lower chamber 210 , 310 , an upper chamber 220 , 320 , a membrane 230 , 330 disposed between the lower chamber 210 , 310 and the upper chamber 220 , 320 , and one or more pillars 240 , 340 .

[0062] In an embodiment, one or more pillars 240 , 340 form at least a portion of the lower chamber 210 , 310 .

[0063] In an embodiment, the AM-transwell is a cylinder, a cube, a cuboid, a truncated cone, a truncated pyramid, a truncated sphere, or a combination thereof. In an embodiment, the AM-transwell is a cube. For example, see Figure 7 In an embodiment, the AM-transwell 200, 300 is a cylinder. For example, see Figure 2A-2B and Figure 3A-3B .

[0064] In an embodiment, the height 202, 302 of the AM-transwell 200, 300 is about 10 mm to about 16 mm, and any range or value therebetween, and the thickness 204, 304 thereof is about 1 mm to about 3 mm, and any range or value therebetween. In an embodiment, the height 202, 302 of the AM-transwell 200, 300 is about 13 mm, and the thickness 204, 304 thereof is about 2 mm.

[0065] In an embodiment, the thickness 202 , 302 of the AM-transwell 200 , 300 is dependent on the interface 308 of the mini-lobe 306 .

[0066] In an embodiment, the height 212, 312 of the lower chamber 210, 310 is about 2 mm to about 4 mm, and any range or value therebetween, and the height 222, 322 of the upper chamber 220, 320 is about 8 mm to about 12 mm, and any range or value therebetween. In an embodiment, the height 212, 312 of the lower chamber 210, 310 is about 3 mm, and the height 222, 322 of the upper chamber 220, 320 is about 10 mm.

[0067] In an embodiment, the thickness 332 of the film 230, 330 is about 1 mm to about 3 mm, and any range or value therebetween. In an embodiment, the thickness 332 of the film 230, 330 is about 2 mm.

[0068] In an embodiment, the inner pores of the upper chamber 220, 320 support the growth of lung epithelial cells (SAEC, small airway epithelial cells) inside.

[0069] In an embodiment, the length 242, 342 of one or more struts 240, 340 is about 3.7 mm to about 5.7 mm, and any range or value therebetween. In an embodiment, the length 242, 342 of one or more struts 240, 340 is about 4.72 mm.

[0070] In an embodiment, the height 244, 344 of one or more struts 240, 340 is about 2 mm to about 4 mm, and any range or value therebetween. In an embodiment, the height 244, 344 of one or more struts 240, 340 is about 3 mm.

[0071] In an embodiment, the width 246 of one or more struts 240, 340 is about 0.6 mm to about 2.6 mm, and any range or value therebetween. In an embodiment, the width 246 of one or more struts 240, 340 is about 1.64 mm.

[0072] In an embodiment, one or more struts 240, 340 support the growth of pulmonary endothelial cells (PAEC, pulmonary airway endothelial cells) on the bottom.

[0073] In an embodiment, one or more of the lower chamber, the upper chamber, the membrane, and the one or more pillars are printed using synthetic bio-ink.

[0074] In an embodiment, the synthetic bio-ink comprises one or more of a degradable ink and a triacrylate peptide ink.

[0075] In an embodiment, the synthetic bio-ink comprises one or more of: 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 any range or value therebetween; and UV386A in an amount of about 0.1% to about 0.5%, and any range or value therebetween.

[0076] In an embodiment, the synthetic bio-ink further comprises: water, in an amount as the balance.

[0077] In an embodiment, the synthetic bio-ink further comprises: a buffer solution comprising a 0.1 M HEPES aqueous solution and 1X PBS, with a pH of 7.2.

[0078] In an embodiment, the synthetic bio-ink further comprises: a monocysteine ​​peptide in an amount of 0.5 mM to 20 mM, and any range or value therebetween; and a dicysteine ​​peptide in an amount of 0.5 mM to 20 mM, and any range or value therebetween.

[0079] In embodiments, the bicysteine ​​peptide is MMP-degradable.

[0080] In an embodiment, the monocysteine ​​peptide comprises one or more of RGDS, PHSRNKRGDS, IKVAV, GFOGER, and an ECM-binding agent (BM-binding agent, FN-binding agent).

[0081] Figure 4 depicts a photograph of an AM-transwell 3D printed using synthetic bio-ink and a digital light projection (DLP) printer; and Figure 5 Depicted is a photograph of a membrane 3D printed using synthetic bioink and a digital light projection (DLP) printer. Exemplary bio-ink formulations and printing settings for the LS80 printer

[0082] Figure 6 A photograph of an exemplary 3D-printed AM-transwell with a cylindrical shape using synthetic bio-ink and an LS80 printer is shown. Example 1 (AG71) Registration Actual concentration unit PEGDA700 PEGDA700 2.85-3.15 ％ PEGDA6000 40% PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X MMP MMP – Cross-linker 9.5-10.5 mM PHSRNK-RGDS PHSRNKRGDS 1.9-2.1 mM HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 1.425-1.575 ％ UV386 UV386A 0.114-0.126 ％ water margin Example 2 (AG72) Registration Actual concentration unit PEGTAC PEGTAC 2.85-3.15 ％ PEGDA6000 40% PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 1.425-1.575 ％ UV386 UV386A 0.171-0.189 ％ water margin Example 3 (AG73) Example 4 (AG74) Registration Actual concentration unit PEGDA700 PEGDA700 3.325-3.675 ％ PEGDA6000 40% PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 1.425-1.575 ％ UV386 UV386A 0.171-0.189 ％ water margin Example 5 (AG75) Registration Actual concentration unit PEGDA700 PEGDA700 3.325-3.675 ％ PEGDA6000 40% PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X MMP MMP – Cross-linker 9.5-10.5 mM PHSRNK-RGDS PHSRNKRGDS 1.9-2.1 mM HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 1.425-1.575 ％ UV386 UV386A 0.171-0.189 ％ water margin Example 6 (AG76) Print settings for LD80 printer Region 1 value unit Layer thickness 20 μm Irradiance 13 <![CDATA[mW / Cm 2 ]]> Curing time 1000 ms PumpUpTime 1500 ms Pump down time (PumpDownTime) 1500 ms PumpUpDelay 500 ms Pump Down Delay (PumpDownDelay) 1500 ms Pump Speed 300 ms Grassroots Irradiance 13 <![CDATA[mW / Cm 2 ]]> Curing time 2000 ms Pump working time 1000 ms Pump downtime 1000 ms Pump operation delay 1000 ms Pump stop delay 1000 ms Pump speed 1000 ms

[0083] Figure 6A photograph of an exemplary 3D-printed AM-transwell with a cylindrical shape using synthetic bio-ink and an LS80 printer is shown.

[0084] AM-transwells were reproducible using all synthetic bioink formulations and printing settings of the LS80 printer. Exemplary bio-ink formulations and printing settings for the BLF20 printer

[0085] Figure 7 depicts a photograph of an exemplary 3D printed AM-transwell having a cubic shape using synthetic bio-ink and a BLF20 printer; and Figure 8 A photograph depicting an exemplary 3D-printed AM-transwell with a cylindrical shape using synthetic bio-ink and a BLF20 printer. Example 7 (AG77) Registration Actual concentration unit PEGTAC PEGTAC 2.85-3.15 ％ PEGDA6000 40% PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 1.425-1.575 ％ UV386 UV386A 0.152-0.168 ％ water margin Example 8 (AG78) Example 9 (AG79) Registration Actual concentration unit PEGDA700 PEGDA700 3.325-3.675 ％ PEGDA6000 40PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 01.425-1.575 ％ UV386 UV386A 0.152-0.168 ％ water margin Example 10 (AG80) Registration Actual concentration unit PEGDA700 PEGDA700 3.325-3.675 ％ PEGDA6000 40PEGDA6000 4.75-5.25 ％ HEPES 1M HEPES pH 7.3 0.095-0.105 M 10XPBS DPBS10XCa,Mg- / - 0.95-1.05 X MMP MMP – Cross-linker 9.5-10.5 mM PHSRNK-RGDS PHSRNKGDS 1.9-2.1 mM HPA Hydroxypropyl acrylate (HPA) 9.5-10.5 ％ NAP NAP 1.425-1.575 ％ UV386 UV386A 0.152-0.168 ％ water margin Print Settings for BLF20 Printer (Cubic Transwell Design)

[0086] Figure 7 A photograph depicting an exemplary 3D-printed AM-transwell with a cube shape using synthetic bio-ink and a BLF20 printer. Print settings for BLF20 printer (round (cylindrical) design) Region 1 value unit Irradiance 13 <![CDATA[mW / Cm 2 ]]> Curing time 4000 ms StepSpeed 150 ms PumpEverynthLayer 1 ms Pump Distance 300 ms Pump working time 1500 ms Pump operation time delay 500 ms Pump downtime 1500 ms Pump downtime delay 1500 ms Build startup speed (SpeedtoBuildStart) 3335 ms Grassroots Irradiance 13 <![CDATA[mW / Cm 2 ]]> Curing time 8000 ms Step speed 200 ms Pumping every n layers 1 ms Pumping distance 1000 ms Pump working time 1000 ms Pump time delay 1000 ms Pump downtime 1000 ms Pump downtime delay 1000 ms Build startup speed 3335 ms

[0087] Figure 8 A photograph depicting an exemplary 3D-printed AM-transwell with a cylindrical shape using synthetic bio-ink and a BLF20 printer.

[0088] AM-transwells were reproducible using all synthetic bioink compositions and printing settings in the BLF19 / 20 and FS20 printers. Permeability of AM-Transwell AM-Transwell Printing

[0089] Before loading the bio-ink, clean the printer vat. After cleaning, add 20 mL of bio-ink to the printer vat. Load the printer setup into the digital light projection (DLP) printer and print. After printing, offload the 3D printed AM-transwell into 1X PBS Ca+ / Mn+. Clean the printer vat and platform. AM-Transwell Sterilization

[0090] Unload the 3D printed AM-transwell into 1X PBS Ca+ / Mn+. Wash the scaffold three times in 1X PBS Ca+ / Mn+. Dispose of the 1X PBS Ca+ / Mn+ waste in the acylate waste container. Incubate the scaffold and 3D printed AM-transwell in 0.1M HEPES / 1X PBS supplemented with antibiotic / antimycotic (anti-anti) overnight. Permeability assay

[0091] In a biosafety cabinet, pour out the sterilization buffer. After pouring out, transfer the 3D printed AM-transwell to a twelve-well plate. Add 1.2 mL of PAEC medium to the apical compartment (upper chamber). Gently remove all bubbles from under the 3D printed AM-transwell.

[0092] Prepare the FITC-dextran solution in a biosafety cabinet with little to no light. Calculation of FITC-dextran solution

[0093] Final experimental concentration = 400 μg / mL

[0094] Stock solution concentration = 10 mg / mL

[0095] Total volume = 15 mL

[0096] (400 μg / mL)(15 mL) / (10,000 μg / mL)=0.6 mL=600 μL FTIC-dextran 10K

[0097] 300 μL of FITC-dextran solution was added to the apical compartment (upper chamber) of the 3D printed AM-transwell so that permeability into the basal compartment (lower chamber) could be measured over time. 600 μL of FITC-dextran solution was added to a commercially available plastic transwell (e.g., Corning) as a control.

[0098] Fig. 9A schematic diagram of the experimental setup for the AM-transwell permeability assay is shown.

[0099] At 1, 2 and 4 hours, 100 μL aliquots (n=3) from the basolateral chamber (lower compartment) were measured for fluorescence at 490 / 520 nm on a plate reader.

[0100] The basolateral compartment (lower chamber) was replenished with an amount of FITC-dextran medium equal to the aliquot removed for fluorescence measurement. Standard curve generation

[0101] Fig.10 A schematic diagram of the serial dilution experimental setup for AM-transwell permeability assay using known concentrations of FITC-dextran is shown.

[0102] For the standard curve, take 250 μL of 400 μg / mL FITC-dextran solution into 1.750 mL PAEC medium. Pipette up and down to mix thoroughly. Transfer 1 mL of the dilution to an Eppendorf tube containing 1 mL PAEC medium and mix thoroughly. This will dilute the original concentration by half. Continue diluting a total of eight times. Measure the fluorescence of a 100 μL aliquot at 490 / 520 nm. Generate a standard curve using known concentrations. Determine the best fit line. Use the best fit equation to quantify the FITC-dextran in the experimental samples. In the best fit equation, x is the concentration (μg / mL) and Y is the relative fluorescence. Calculation of initial dilution for standard curve

[0103] Initial concentration = 400 μg / mL

[0104] Final concentration = 50 μg / mL

[0105] (50μg / mL)(2mL) / (400μg / mL)=0.25mL

[0106] Diluent = 2000-250 = 1750 μL

[0107] Fig.11 Shown is a standard curve graph of concentration (μg / mL) vs relative fluorescence units for the AM-transwell (AG71 to AG76) permeability assay.

[0108] Fig.12 Shown is a graph of time vs FITC-dextran concentration (μg / mL) for AM-transwell (AG71 to AG76) permeability assays.

[0109] Changes in FITC-dextran concentration over time in 3D-printed AM-transwells compared to commercially available plastic transwells (e.g., Corning). A slight decreasing trend in FITC-dextran concentration was observed for AG73 to AG76, while a slight increasing trend in FITC-dextran concentration was observed for AG71 and AG72.

[0110] Fig.13A A graph showing time vs FITC-dextran concentration (μg / mL) for AM-transwell (AG71) permeability assay; Fig. 13B A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (AG72) permeability assay; Fig. 13C A graph showing time vs FITC-dextran concentration (μg / mL) for AM-transwell (AG73) permeability assay; Fig.13D A graph showing time vs FITC-dextran concentration (μg / mL) of AM-transwell (AG74) permeability assay; Fig.13E A graph showing time vs FITC-dextran concentration (μg / mL) for AM-transwell (AG75) permeability assay; Fig.13F Shown is a graph of time vs FITC-dextran concentration (μg / mL) for AM-transwell (AG76) permeability assay. Permeability assay

[0111] A standard curve was created by preparing 1 / 2 serial dilutions starting at 50 μg / mL and measuring their fluorescence at 490 / 520 nm.

[0112] In a twelve-well plate, fill each well with 1.2 mL of PAEC medium. Carefully transfer the 3D printed AM-transwell into the twelve-well plate. Gently remove all bubbles from underneath the 3D printed AM-transwell.

[0113] Once the bubbles were removed, 175 μL of 400 μg / mL FITC-dextran solution was added to the apical compartment (upper chamber) of the 3D printed AM-transwell.

[0114] Cover the twelve-well plate with foil and incubate at 37 °C.

[0115] Fig. 9 A schematic diagram of the experimental setup for the AM-transwell permeability assay is shown.

[0116] At 1, 2, 4 and 21 hours, 100 μL aliquots (n=3) from the basolateral chamber (lower compartment) were measured for fluorescence at 490 / 520 nm on a plate reader.

[0117] The basolateral compartment (lower chamber) was replenished with an amount of FITC-dextran medium equal to the removed aliquot.

[0118] Commercially available plastic transwells (eg, Corning) were used as controls. Standard curve generation

[0119] Fig.10 A schematic diagram of the serial dilution experimental setup for the AM-transwell permeability assay is shown.

[0120] For serial dilutions, take 250 μL of 400 μg / mL FITC-dextran solution to 1.750 mL of endothelial (PAEC) culture medium. Pipette up and down to mix thoroughly. Transfer 1 mL of this first dilution to a new Eppendorf tube containing 1 mL of PAEC culture medium and mix thoroughly. Continue serial dilutions for a total of eight times. Transfer 100 μL of each dilution (three replicates) to a 96-well plate and measure fluorescence at 490 / 520 nm. Generate a standard curve using known concentrations. Determine the best fit line. Use the best fit equation to quantify the FITC-dextran in the experimental sample. In the best fit equation, x is the concentration (μg / mL) and Y is the relative fluorescence. Calculation of FITC-dextran stock solution

[0121] Stock solution = 10 mg / mL

[0122] Final assay concentration = 400 μg / mL

[0123] Final volume = 4 mL

[0124] Stock solution volume = (400 μg / mL)(4 mL) / 10,000 μg / mL) = 0.16 mL = 160 μL Calculation of initial dilution for standard curve

[0125] Initial concentration = 400 μg / mL

[0126] Final concentration = 50 μg / mL

[0127] (50μg / mL)(2mL) / (400μg / mL)=0.25mL

[0128] Diluent = 2000 μL-250 μL = 1750 μL 21-hour analysis

[0129] After the final 21-hour fluorescence measurement, remove the FITC-dextran medium from the top chamber (upper chamber) of the 3D printed AM-transwell. Assuming a concentration of 400 μg / mL, dilute the solution at a ratio of 1:16, with a theoretical concentration of 25 μg / μL. Take 100 aliquots of 100 μL and measure the fluorescence at 490 / 520 nm on a plate reader.

[0130] Fig.14 Shown is a standard curve graph of concentration (μg / mL) vs relative fluorescence units for the AM-transwell (AG77 to AG80) permeability assay.

[0131] Fig.15 Shown is a graph of time vs. percentage of FITC-dextran in the basolateral compartment for AM-transwell (AG77 to AG80) permeability assays.

[0132] The concentration of FITC-dextran in 3D-printed AM-transwells compared to commercially available plastic transwells (e.g., Corning) changes over time. Data are presented as the average of the mass percentage of FITC-dextran in the basal compartment (lower chamber) at time T relative to the total FITC-dextran in the system.

[0133] Fig.16A A graph showing time vs. percentage of FITC-dextran in the basal compartment of the AM-transwell (AG77) permeability assay; Fig. 16B A graph showing time vs. percentage of FITC-dextran in the basal compartment of the AM-transwell (AG78) permeability assay; Fig. 16C shows a graph of time vs. percentage of FITC-dextran in the basal compartment of an AM-transwell (AG79) permeability assay; and Fig.16D Shown is a graph of time vs. percentage of FITC-dextran in the basolateral compartment of the AM-transwell (AG80) permeability assay.

[0134] The permeability of each replicate, each fluorescence measurement was measured in triplicate and compared to the average of the formulation and the average of a commercially available plastic transwell (e.g., Corning) as a control. Data are presented as the average of the mass percentage of FITC-dextran in the basal compartment (lower chamber) at time T to the total FITC-dextran in the system. The culture medium was recovered after three days. method

[0135] The changes in liquid volume in the apical compartment (upper chamber) and basal compartment (lower chamber) of the 3D-printed AM-transwell (AG71) after a period of time were measured. First, the 3D-printed AM-transwell was stored in PBS in a 50 mL tube. Pour out the PBS and transfer the 3D-printed AM-transwell to a sterile 50 mL tube. 35 mL of HEPES / PBS anti-anti was added to the 50 mL tube with the 3D-printed AM-transwell and sterilized overnight. After sterilization, the sterilization buffer was poured out and the 3D-printed AM-transwell was incubated in the culture medium for at least 4 hours. After the culture medium incubation, the 3D-printed AM-transwell was transferred to a twelve-well plate. 1.2 mL of culture medium was added to the basal compartment of the 3D-printed AM-transwell. Gently remove all bubbles from under the 3D-printed AM-transwell. After removing the bubbles, 0.5 mL of culture medium was carefully added to the basal compartment of the 3D AM-transwell. 175 μL of culture medium was carefully added dropwise to the apical compartment, avoiding overflow. The 3D printed AM-transwells were incubated in an incubator at 37 °C for three days. After the third day, the plates were removed from the incubator. For each 3D printed AM-transwell, all culture medium was recovered from the basal compartment into a 5 mL tube. Similarly, all culture medium was carefully recovered from the apical compartment into a 0.5 mL Eppendorf tube. Then, the total volume of culture medium recovered from each compartment was measured and recorded. Initial conditions formula AG71 unit Number of AM-Transwells 3 Each Initial apical volume 175 μL Initial base volume 1.7 mL Top recycling serial number Final apical volume unit Recycling percentage unit N1 60 μL 34.3 ％ N2 70 μL 40.0 ％ N3 76 μL 43.4 ％ Substrate recycling serial number Final base volume unit Recycling percentage unit N1 1.68 mL 98.8 ％ N2 1.78 mL 104.7 ％ N3 1.65 mL 97.1 ％ Total Recycling serial number Final total volume unit Recycling percentage unit N1 1.74 mL 92.8 ％ N2 1.85 mL 98.7 ％ N3 1.73 mL 92.1 ％

[0136] Fig.17 Shown is a graph of compartments versus the percentage of medium recovered from the AM-transwells after three days.

[0137] The volume of the apical compartment (upper chamber) can be increased to accommodate more culture medium.

[0138] More medium can be added to the outside of the AM-transwell to keep the hydrogel more hydrated and retain some medium in the apical compartment.

[0139] The cells can be cultured with daily changes of medium to prevent the apical compartment from becoming too dry to support a cell monolayer, as indicated by >50% medium remaining in the apical compartment after three days. Method for preparing AM-Transwell

[0140] The method for preparing an AM-transwell comprises: a) printing one or more of the lower chamber, the upper chamber and the membrane of the AM-transwell using 3D printing technology; b) assembling and / or printing the AM-transwell as described herein to form an assembled AM-transwell.

[0141] In an embodiment, step b) prints at least 3 assembled AM-transwells. In an embodiment, step b) prints at least 20 assembled AM-transwells. In an embodiment, step b) prints at least 50 assembled AM-transwells.

[0142] In embodiments, the method further comprises: c) optionally storing the assembled AM-transwell at 4°C until needed.

[0143] In an embodiment, the method further comprises: c) transferring each of the AM-transwells to DPBS+ / + in a tube; d) pouring out the DPBS+ / + from the tube and washing each AM-transwell twice more in DPBS+ / + for about 5 minutes to form a first washed AM-transwell; and e) transferring the first washed AM-transwell to a sterile 50 mL tube.

[0144] In embodiments, the method further comprises: f) incubating the first washed AM-transwell in DPBS- / - 1X anti-anti- overnight to form an incubated AM-transwell.

[0145] In embodiments, the method further comprises: g) pouring off the DPBS- / -1X anti-anti and washing the incubated AM-transwell in about 35 PBS / HEPES 1X anti-anti for about 4 hours to form a second washed AM-transwell; and h) pouring off the PBS / HEPES 1X anti-anti from the tube and incubating the second washed AM-transwell in about 35 mL of cell culture medium in an about 50 mL tube for about 2 days or over a weekend.

[0146] In an embodiment, 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. In an embodiment, the 3D printing technology is digital light printing (DLP) printing technology. Methods of using AM-Transwell

[0147] The method of using the AM-transwell comprises: a) seeding cells on at least one side of the membrane of the AM-transwell described herein. Methods of using AM-Transwell

[0148] The method of using the AM-transwell comprises: a) seeding cells on both sides of the membrane of the AM-transwell described herein for in vitro cell studies.

[0149] The embodiments and examples described herein are intended to best explain the present invention and its practical application, so that those skilled in the art can prepare and utilize the present invention. However, those skilled in the art will recognize that the foregoing description and examples are provided for illustration and example purposes only. The description is not intended to be exhaustive or to limit the present invention to the precise form disclosed. Many modifications and variations may be made according to the above teachings without departing from the spirit and scope of the following claims. The present invention is particularly intended to be as broad as the following claims and their equivalents.

[0150] Any reference to an embodiment or element or behavior of a system and method referred to herein in the singular may include an embodiment containing a plurality of such elements, and any reference to any embodiment or element or behavior in the plural may include an embodiment containing only a single element. References in the singular or plural are not intended to limit the currently disclosed systems or methods, their components, behaviors, or elements to a single or plural configuration. References to any behavior or element based on any information, behavior, or element may include an embodiment in which the behavior or element is based at least in part on any information, behavior, or element.

[0151] As used herein, the terms "approximately," "about," "substantially," and similar terms are intended to have a broad meaning consistent with the common and recognized usage by those of ordinary skill in the art to which the subject matter of the present disclosure belongs. Those skilled in the art who read the present disclosure should understand that these terms are intended to allow the description of certain features described and claimed without limiting the scope of these features to the precise numerical ranges provided. Therefore, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or changes to the subject matter described and claimed are considered to fall within the scope of the present disclosure as described in the appended claims.

[0152] It should be noted that, as used herein, the term "exemplary" and variations thereof, used to describe various embodiments, are intended to indicate that these embodiments are possible examples, representatives, or illustrations of possible embodiments (and these terms are not intended to imply that these embodiments are necessarily the preeminent or best examples).

[0153] As used herein, the term "coupled" and its variants mean that two components are directly or indirectly connected to each other. Such connection can be stable (e.g., permanent or fixed) or removable (e.g., removable or releasable). Such connection can be achieved by coupling the two components directly to each other, or by coupling the two components to each other using a separate intermediate component and any additional intermediate components that are coupled to each other, or by coupling the two components to each other using an intermediate component that is integrally formed with one of the two components as a single entity. If "coupled" or its variants are modified by other terms (e.g., directly coupled), the general definition of "coupled" provided above will be modified by the ordinary language meaning of the other terms (e.g., "directly coupled" means that the two components are connected without any separate intermediate components), resulting in a narrower definition than the general definition of "coupled" provided above. Such coupling can be mechanical, electrical or fluid.

[0154] Any embodiment disclosed herein may be combined with any other embodiment, and references to "an embodiment," "some embodiments," "alternative embodiments," "various embodiments," "one embodiment," etc. are not necessarily mutually exclusive and are intended to indicate that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment. As used herein, these terms do not necessarily all refer to the same embodiment. Any embodiment may be combined with any other embodiment, inclusively or exclusively, in any manner consistent with the aspects and embodiments disclosed herein.

[0155] The reference of "or" may be interpreted as an inclusive meaning, that is, any term described using "or" may represent a single, more than one, or all of the terms. The reference of at least one of the terms listed with a conjunction may be interpreted as an inclusive "or" to indicate a single, more than one, or all of the terms. For example, the reference of "at least one of 'A' and 'B'" may include only 'A', only 'B', and both 'A' and 'B'. Elements other than 'A' and 'B' may also be included.

[0156] References to element positions herein (e.g., "top," "bottom," "above," "below") are only used to describe the orientation of the various elements in the drawings. It should be noted that the orientation of the various elements may be different according to other exemplary embodiments, and the present disclosure is intended to encompass such variations.

[0157] Although the figures and descriptions may illustrate a particular order of method steps, the order of these steps may be different from that depicted and described, unless otherwise specified above. In addition, unless otherwise specified above, two or more steps may be performed simultaneously or partially simultaneously. Such variations may depend on, for example, the selected software and hardware systems and the designer's choice. All of these variations are within the scope of the present disclosure. Similarly, the software implementation of the method may be implemented by standard programming techniques, using rule-based logic and other logic to implement various connection steps, processing steps, comparison steps, and decision steps.

[0158] Where technical features in the drawings, detailed description or any claims are followed by reference signs, these reference signs are introduced to improve the intelligibility of the drawings, detailed description and claims. Therefore, the presence or absence of these reference signs will not have any limiting effect on the scope of any claim element.

[0159] The systems and methods described herein may be implemented in other specific forms without departing from their characteristics. The foregoing embodiments are intended to illustrate rather than limit the systems and methods. Therefore, the scope of the systems and methods described herein is indicated by the appended claims rather than the foregoing description, and changes within the meaning and scope of the equivalents of the claims are also included therein.

Claims

1. An advanced manufactured transwell (AM-transwell), the AM-transwell comprising: a) Lower room; b) Upper chamber; c) a membrane disposed between the lower chamber and the upper chamber; and d) one or more pillars, wherein the one or more pillars form at least a portion of the lower chamber, wherein one or more of the lower chamber, the upper chamber, the membrane, and the one or more pillars are printed using synthetic bio-ink. 2 . The AM-transwell according to claim 1 , wherein the AM-transwell is a cylinder, a cube, a cuboid, a truncated cone, a truncated pyramid, a truncated sphere, or a combination thereof. The AM-transwell according to claim 1 , wherein the AM-transwell is a cylinder. 4 . The AM-transwell according to claim 1 , wherein the AM-transwell has a height of about 10 mm to about 16 mm and a thickness of about 1 mm to about 3 mm. The AM-transwell according to claim 1 , wherein the AM-transwell has a height of about 13 mm and a thickness of about 2 mm. The AM-transwell according to claim 1 , wherein the height of the lower chamber is about 2 mm to about 4 mm, and the height of the upper chamber is about 8 mm to about 12 mm. 7 . The AM-transwell according to claim 1 , wherein the height of the lower chamber is about 3 mm, and the height of the upper chamber is about 10 mm. The AM-transwell according to claim 1 , wherein the thickness of the membrane is about 1 mm to about 3 mm.

9. The AM-transwell according to claim 1, wherein the thickness of the membrane is about 2 mm. 10 . The AM-transwell according to claim 1 , wherein the synthetic bio-ink comprises one or more of a degradable peptide ink and a triacrylate peptide ink.

11. The AM-transwell according to claim 1, wherein the synthetic bio-ink comprises one or more of the following: 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%; PEGTAC in an amount of about 1% to about 5%; PEO in an amount from about 0.1% to about 5%; NAP in an amount of about 1% to about 3%; LAP in an amount from about 1% to about 3%; and UV386A in an amount of about 0.1% to about 0.5%.

12. The AM-transwell according to claim 13, wherein the synthetic bio-ink further comprises: Water, the amount of which serves as the balance.

13. The AM-transwell according to claim 13, wherein the synthetic bio-ink further comprises: The buffer solution includes 0.1 M HEPES aqueous solution and 1X PBS, pH 7.

2.

14. The AM-transwell according to claim 13, wherein the synthetic bio-ink further comprises: a monocysteine ​​peptide in an amount of 0.5 mM to 20 mM; and The amount of the di-cysteine ​​peptide is 0.5 mM to 20 mM. 15 . The AM-transwell according to claim 16 , wherein the single cysteine ​​peptide comprises one or more of RGDS, PHSRNKRGDS, IKVAV, Bm-binder, and FN-binder. The AM-transwell according to claim 16 , wherein the bi-cysteine ​​peptide is degradable by MMP.

17. A method for preparing an advanced manufactured transwell (AM-transwell), the method comprising: a) printing one or more of the lower chamber, the upper chamber and the membrane of the AM-transwell using 3D printing technology; and b) assembling and / or printing the AM-transwell of claim 1 to form an assembled AM-transwell.

18. 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.

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

20. The method according to claim 19, further comprising: c) Store the assembled AM-transwell at 4°C until needed.

21. The method of claim 19, further comprising: c) transfer each of the AM-transwells into DPBS+ / + in a tube; d) pouring off the DPBS+ / + from the tube and washing each AM-transwell twice more in DPBS+ / + for about 5 minutes to form a first washed AM-transwell; and e) Transfer the first washed AM-transwell to a sterile 50 mL tube.

22. The method according to claim 23, further comprising: f) The first washed AM-transwell was incubated overnight in DPBS- / - supplemented with antibiotic / antimycotic (anti-anti) to form a sterilized AM-transwell.

23. The method according to claim 24, further comprising: g) pouring off the DPBS- / -anti-anti and washing the incubated AM-transwell in about 35 mL PBS / 0.1 M HEPES anti-anti buffer solution for about 4 hours to form a second washed AM-transwell; and h) Pour off the PBS / 0.1 M HEPES anti-anti buffer solution from the tube and incubate the second washed AM-transwell in about 35 mL of cell culture medium in a about 50 mL tube for about 2 days or over a weekend.

24. The method of claim 19, wherein step b) prints at least 3 assembled AM-transwells.

25. The method of claim 19, wherein step b) prints at least 20 assembled AM-transwells.

26. The method of claim 19, wherein step b) prints at least 50 assembled AM-transwells.

27. A method of using an advanced manufactured transwell (AM-transwell), the method comprising: a) seeding cells on at least one side of the membrane of the AM-transwell of claim 1.

28. A method of using an advanced manufactured transwell (AM-transwell), the method comprising: a) Seeding cells on both sides of the membrane of the AM-transwell of claim 1 for in vitro cell research.

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