Vascularized In Vitro Perfusion Devices, Methods of Fabricating, and Applications Thereof

Abstract

A bona fide adaptable in vitro microcirculation model is provided by integrating a 3-D printed network of endothelial-cell lined perfusion channels, formed via sacrificial casting in a gel matrix, with a native, adaptable microvasculature matured from native microvessels added to the gel matrix. Responsive vascular adaptation exhibited by the in vitro microcirculation is physiologically relevant. Methods for fabricating, devices, models and investigative platforms for pharmaceutical applications, vascular mechanism and microvessel-parenchyma interaction studies, and vascularizing strategies for tissue engineering applications are also disclosed.

What is claimed: 
     
         1 . A method of manufacturing a stable and adaptable in vitro microcirculation system, the method comprising:
 a) casting a network of channels on a polymerized matrix gel with a sacrificial material, said network cast to form at least one perfusion inlet port and inlet channel, and at least one perfusion outlet port and outlet channel, and one or more cross channels, each cross channel being in communication with both an inlet channel and an outlet channel;   b) incorporating an isolate of intact native microvessels into a polymerizable matrix;   c) distributing the polymerizable matrix from step b) over the network of cast channels and polymerizing the matrix to form a continuous polymerized gel matrix comprising both the network of cast channels and the intact native microvessels;   d) incubating the gel matrix under conditions suitable to promote spontaneous growth of a neovasculature from the native microvessel isolate, said incubating optionally taking place before or after step c);   e) flushing the sacrificial material from the cast network to yield a molded network of channels;   f) lining the molded channels with endothelial cells to form a continuous network of endothelial cell-lined channels; and   g) subjecting the network of endothelial cell-lined channels to perfusion with a perfusion fluid sufficient to induce endothelial sprouting from the one or more cross channels and inosculation between at least two sprouts and the neovasculature, thereby forming a stable adaptable microcirculation system.   
     
     
         2 . The method according to  claim 1 , further comprising:
 h) adapting the microcirculation to a desired circulatory profile by modulating perfusion of a perfusion fluid through the microcirculation system.   
     
     
         3 . The method according to  claim 1 , wherein the sacrificial material is a thermo-reversible pluronic hydrogel. 
     
     
         4 . The method according to  claim 1 , wherein the perfusion fluid comprises an angiogenic culturing media. 
     
     
         5 . The method according to  claim 1 , wherein the “casting” step is effectuated by 3-D bioprinting a bio-ink comprising a thermo-reversible hydrogel, and is carried out entirely at ambient temperatures. 
     
     
         6 . The method according to  claim 1 , wherein the “distributing” step is effectuated by 3-D bioprinting a bio-ink comprising an isolate of intact native microvessels suspended in a gelable matrix. 
     
     
         7 . The method according to  claim 1 , wherein the subjecting step (g) is effectuated via an external perfusion control. 
     
     
         8 . The method according to  claim 7 , wherein the polymerized matrix gel of step (a) is contained within a chamber, and subsequent to step (g) a lid is placed over the chamber to form a housing, said housing being configured to permit connection of the inlet and outlet ports to the external perfusion control. 
     
     
         9 . The method according to  claim 1 , wherein the subjecting step (g) comprises defining perfusion hemodynamics to provide a shear stress between the at least one inlet channel and the one or more cross channels sufficient to induce endothelial sprouting. 
     
     
         10 . The method according to  claim 9 , wherein the provided shear stress is greater than or equal to 10 dynes/cm 2 . 
     
     
         11 . The method according to  claim 1 , wherein all steps are effectuated in a single work-flow.