VASCULARIZED IN VITRO PERFUSION DEVICES, METHODS OF FABRICATING, AND APPLICATIONS THEREOF

• US 20170009194A1
• Filed: 07/06/2016
• Published: 01/12/2017
• Est. Priority Date: 07/06/2015
• Status: Active Grant

• Current Assignee: Advanced Solutions Life Sciences, LLC
• Original Assignee: Advanced Solutions Life Sciences, LLC
• Inventors: Golway, Michael W., Hoying, James B.
• Granted Patent: US 10,392,595 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 29/10 Perfusion
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 5/0691 Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5064 Endothelial cells

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.

Claims

1. A vascularized in vitro perfusion device comprising an adaptable microcirculation, said device comprising:

a supporting structure comprising a gel matrix,

a fabricated network of microfluidic endothelial cell-lined channels, said network comprising an inlet channel, an outlet channel, and at least one cross channel connecting the inlet channel to the outlet channel, said cross channel positioned at least partially within the gel matrix,

an inlet port in fluid communication with the network, and an outlet port in fluid communication with the network, and

a neovasculature, said neovasculature being derived from intact native microvessels incorporated into the gel matrix and subject to maturing conditions,

wherein the network is in vascular communication with the neovasculature to form an adaptable microcirculation.

2. The device according to claim 1, further comprising an enclosure.

3. The device according to claim 2, wherein the enclosure comprises a housing comprising a biocompatible plastic polymer selected from acrylic, styrene and carbonate polymers.

4. The device according to claim 1, wherein the gel matrix is selected from a collagen gel, a fibrin gel, and combinations thereof.

5. The device according to claim 1, wherein the intact native microvessels are derived from adipose tissue.

6. The device according to claim 1, wherein the vascular communication is formed from perfusion-driven sprouting from the network of endothelial cell-lined channels, and inosculation between at least two sprouts and the neovasculature.

7. The device according to claim 2, wherein the at least one inlet port and at least one outlet port are accessed exterior to the enclosure.

8. The device according to claim 1, wherein the inlet channel and the at least one cross channel have a cross-sectional diameter ratio between about 1.1:

1 and about 5;

1.

10. The device according to claim 1, further comprising an external perfusion control system in operational communication with the network through the inlet port.

11. The device according to claim 1, wherein “

adaptable”

comprises an ability to undergo vascular differentiation and/or positive vascular remodeling in response to at least one perfusion-driven hemodynamic force or stimuli.

12. The device according to claim 11, wherein the at least one hemodynamic force is one or more of intravascular pressure and shear stress.

13. The device according to claim 1, further comprising living cells, said cells populating the gel matrix and being derived from one or more tissue types.

14. The device according to claim 13, wherein the cells are all from the same tissue type.

15. The device according to claim 13, wherein the gel matrix comprises at least two sections and each section is populated with cells from a distinct tissue type.

16. The device according to claim 13, wherein the living cells comprise human cells.

17. The device according to claim 13, wherein the living cells are selected from one or more of normal cells, diseased cells, stem cells, endothelial cells, stromal cells, epithelial cells, neuronal cells, connective cells, myocardial cells, hepatocytes, renal cells, tumor cells, liver cells, pancreatic cells, muscle cells, brain cells, kidney cells, and patient-specific cells.

18. The device according to claim 13, wherein “

adaptable”

comprises an ability to undergo vascular remodeling in response to at least one cue provided by the living cells.

19. The device according to claim 18, wherein the at least one cue is provided by the living cells in response to an agent delivered by perfusion.

31. A pharmaceutical agent screening device comprising the vascularized in vitro perfusion device according to claim 13, wherein the living cells comprise target parenchymal cells.

32. The screening device according to claim 31, wherein the target parenchymal cells comprise one or more of kidney cells, liver cells, and cardiac cells.

33. A method for screening putative pharmaceutical agents for target pharmaceutical efficacy using the device of claim 31, the method comprising:

formulating (1) a control perfusion fluid and (2) a test perfusion fluid comprising at least one putative agent;

perfusing the adaptable microcirculation with (1);

perfusing the adaptable microcirculation with (2); and

comparing the results to determine pharmaceutical efficacy.

34. The method according to claim 33, wherein the control perfusion fluid comprises no agents or comprises at least one agent having known pharmaceutical efficacy.

35. The method according to claim 33, wherein the target parenchymal cells comprise cardiac cells and the putative agents are screened for efficacy in modulating cardiac remodeling.

36. A system of vascularized in vitro perfusion devices, the system comprising at least two devices according to claim 13, wherein the devices are assembled to be perfusable in series or in parallel or in some combination thereof.

37. The system according to claim 36, wherein each device comprises a living cell derived from a distinct tissue type.

9. The device according to claim 9, comprising at least two cross channels having different cross-sectional diameters such that a hemodynamic gradient is formed between the cross channels upon perfusion.

20. 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.

21. The method according to claim 20, further comprising:

h) adapting the microcirculation to a desired circulatory profile by modulating perfusion of a perfusion fluid through the microcirculation system.

22. The method according to claim 20, wherein the sacrificial material is a thermo-reversible pluronic hydrogel.

23. The method according to claim 20, wherein the perfusion fluid comprises an angiogenic culturing media.

24. The method according to claim 20, 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.

25. The method according to claim 20, 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.

26. The method according to claim 20, wherein the subjecting step (g) is effectuated via an external perfusion control.

27. The method according to claim 20, 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.

28. The method according to claim 27, wherein the provided shear stress is greater than or equal to 10 dynes/cm².

29. The method according to claim 26, 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.

30. The method according to claim 20, wherein all steps are effectuated in a single workflow.

38. A vascularized in vitro perfusion device comprising an adaptable microcirculation that is stable at physiologically relevant perfusion conditions.