WO2019070960A1

WO2019070960A1 - Artificial blood barrier

Info

Publication number: WO2019070960A1
Application number: PCT/US2018/054342
Authority: WO
Inventors: Luiz C. SAMPAIO; Doris Anita TAYLOR; Christopher R. BRODA
Original assignee: Sampaio Luiz C; Taylor Doris Anita; Broda Christopher R
Priority date: 2017-10-04
Filing date: 2018-10-04
Publication date: 2019-04-11

Abstract

An artificial blood barrier is made by a process comprising the steps of providing an acellular biomaterial, deforming the acellular biomaterial, and seeding cells on the deformed acellular biomaterial. The acellular biomaterial is optionally provided by decellularizing a biologic tissue or organ. The seeded cells are then cultured in vitro to make the artificial blood barrier.

Description

ARTIFICIAL BLOOD BARRIER

BACKGROUND

[0001] This disclosure relates to methods of making an artificial blood barrier. The artificial blood barrier may be used as a vascular structure, an artificial heart, a blood vessel, or the like, for implementation in a patient (i.e., the host). For example, the engineered blood barrier may be used as an inner lining of an artificial heart or the like.

[0002] Synthetic polymers have low immunogenicity. However, such synthetic polymers, for example, expanded polytetrafluoroethylene or ePTFE, may not be sufficiently compliant for forming a blood barrier suitable for low-pressure dynamic applications.

[0003] Decellularized tissues also have low immunogenicity. Decellularized tissues may be sufficiently compliant. However, such tissues may not provide sufficient attachment for cultured cells and may still exhibit a thrombogenic behavior.

[0004] Therefore, there is a need in the art for methods of engineering a blood barrier that is non- thrombogenic and is sufficiently mechanically compliant.

SUMMARY

[0005] This disclosure describes methods of making an artificial blood barrier.

[0006] The methods may comprise providing an acellular biomaterial (e.g., an extracellular matrix material), such as by decellul arizing a biologic material (e.g., a tissue or an organ). For example, the biologic material may include collagen fibers or collagen gel, and/or may consist of one of heart tissue, urinary bladder tissue, or small intestine tissue, such as porcine small intestinal submucosa.

[0007] The methods may comprise deforming (e.g., applying strain to) the acellular biomaterial. Deforming the acellular biomaterial may comprise, for example, applying a uniaxial or biaxial or circumferential strain and/or applying a strain level of at least 5% on the decellularized tissue.

[0008] The methods may comprise seeding cells on the deformed acellular biomaterial. For example, the cells may comprise endothelial cells or endothelial progenitor cells from umbilical vein or umbilical cord, circulating endothelial progenitor cells from venipuncture, isolation of cells from veni-biopsy, cells from bone marrow, or combinations thereof; and/or the cells may comprise tissue-derived stem cells, amniotic fluid stem cells, amniotic membrane stem cells, embryonic stem cells, or induced pluripotent stem cells. The cells may be seeded at a concentration of at least 500K cells/ 50 μΐ.

[0009] The methods may comprise culturing the cells seeded on the acellular biomaterial in vitro.

[0010] The disclosure also describes artificial blood barrier made by the methods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] For a more detailed description of the embodiments of the disclosure, reference will now be made to the accompanying drawings, wherein:

[0012] Figures 1A-1C are images of histological sections stained with Hematoxylin and Eosin of decellularized submucosa seeded with endothelial cells at varying densities;

[0013] Figures 2A-2C are images of histological sections stained with Masson's trichrome of decellularized submucosa seeded with endothelial cells at varying densities;

[0014] Figures 3A-3C are images of histological sections stained with Hematoxylin and Eosin of decellularized submucosa seeded with endothelial cells at the same concentration, but under varying strain levels;

[0015] Figures 4A-4C are images of histological sections stained with antibodies to the platelet endothelial cell adhesion molecule 1 protein of decellularized submucosa seeded with endothelial cells at the same concentration, but under varying strain levels;

[0016] Figures 5A-5C are images of histological sections stained with antibodies to the vascular endothelial growth factor receptor 2 protein of decellularized submucosa seeded with endothelial cells at the same concentration, but under varying strain levels; and

[0017] Figure 6A-6C are images of histological sections stained with antibodies to the von Willebrand factor protein of decellularized submucosa seeded with endothelial cells at the same concentration, but under varying strain levels. DETAILED DESCRIPTION

[0018] It is to be understood that the following disclosure describes several exemplary embodiments for implementing different features, structures, or functions of the invention. Exemplary embodiments of components, arrangements, and configurations are described below to simplify the disclosure; however, these exemplary embodiments are provided merely as examples and are not intended to limit the scope of the invention. The exemplary embodiments presented below may be combined in any combination of ways, i.e., any element from one exemplary embodiment may be used in any other exemplary embodiment, without departing from the scope of the disclosure.

[0019] All numerical values in this disclosure may be approximate values unless otherwise specifically stated. Accordingly, various embodiments of the disclosure may deviate from the numbers, values, and ranges disclosed herein without departing from the intended scope.

[0020] As one skilled in the art will appreciate, various entities may refer to the same component by different names, and as such, the naming convention for the elements described herein is not intended to limit the scope of the invention, unless otherwise specifically defined herein. Further, the naming convention used herein is not intended to distinguish between components that differ in name but not function.

[0021] This disclosure describes engineered blood barriers that comprise an acellular biomaterial used as a biocompatible scaffolding material on which physiologically relevant human cells are cultured.

[0022] Examples of suitable biocompatible scaffolding materials include decellularized tissues such as heart, urinary bladder, small intestine {e.g., porcine small intestinal submucosa (SIS)), or other tissues comprising collagen fibers, fibrin fibers, and or collagen gel. The biocompatible scaffolding material provides an extracellular matrix that can support cell attachment, cell recruitment, growth, proliferation, differentiation and a variety of other functions. Further, the biocompatible scaffolding material has low immunogenicity. Preferably, the biocompatible scaffolding material is mechanically resilient and has adequate degradative properties suitable for remodeling. [0023] Examples of suitable cells that may be cultured include endothelial cells or endothelial progenitor cells from umbilical vein or umbilical cord, circulating endothelial progenitor cells from venipuncture, isolation of cells from veni-biopsy, cells from bone marrow, amniotic fluid stem cells, amniotic membrane stem cells, embryonic stem cells, induced pluripotent stem cells, tissue- derived (e.g., fat-derived) stem cells, and the like. The cells may also comprise stromal cells or mesenchymal cells that undergo mesenchymal to endothelial transition. The cells may have been withdrawn from the host in which the blood barrier is to be implanted.

[0024] The cells may be seeded onto the biocompatible scaffolding material and cultured in a bioreactor to produce a living barrier.

[0025] The methods of engineering a blood barrier in accordance with the disclosure may comprise the steps of decellularizing a biologic material, for example, a tissue, seeding host cells on the decellularized biologic material under deformation (e.g., having a strain applied to), and growing the cells in vitro.

[0026] An example of a decellularization process comprises immersing the biologic material in hypertonic saline solutions (e.g., 500 mM NaCl solution for 8 hours) and hypotonic saline solutions (e.g., 20 mM NaCl solution for 8 hours), preferably for several cycles (e.g., 2 cycles of immersion in the hypertonic and hypotonic solution), immersing the biologic material in a detergent solution (e.g., 0.1% Sodium Dodecyl Sulfate or SDS for 72 hours), rinsing biologic material in double-distilled water (e.g., for 8 hours), and washing the biologic material in phosphate buffered saline or PBS solution with 0.01% to 0.1% peracetic acid (e.g., for 1 to 24 hours). The process may be performed in an orbital shaker at room temperature.

[0027] An example of a cell seeding process comprises applying a strain (e.g., a uniaxial or biaxial or circumferential strain) to the biocompatible scaffolding material to deform it. Cell seeding may be performed on the biocompatible scaffolding material under strain conditions. Applying strain to the biocompatible scaffolding material and selecting a suitable cell seeding concentration may promote cell coverage over the biocompatible scaffolding material, maximize cell attachment and/or adherence, and ultimately result in a blood barrier that minimizes thrombogenesis. Cell seeding concentration may vary between 200,000 and 1,000,000 cells per 50 microliters, and may preferably be approximately 500,000 cells per 50 microliters. Strain level may vary between 5% and 20%, and is preferably at least 5%. [0028] An example of a cell growth process in vitro may involve culture in a cassette at 37^°C for 4 days.

EXAMPLES

[0029] In some embodiments, a porcine urinary bladder submucosa was decellularized, yielding to 6.13% residual double-stranded DNA, which is below the threshold for a tissue to be considered biocompatible. Further, the Young modulus of the decellularized submucosa was lower than 10 Mega Pascals, making it more compliant than a commonly used polymer such as ePTFE.

[0030] The decellularized porcine urinary bladder submucosa was used as the base scaffold. Uniaxial strain of 0%, 5%, 10%, or 20% may be applied to the decellularized submucosa. The decellularized submucosa may subsequently be seeded with Human Umbilical Vein Endothelial Cells at various seeding densities (200K cells/ 50 μΐ - 1000K cells/ 50 μΐ). The composite consisting of the base scaffold and cells may be cultured in cassettes for 4 days before evaluating endothelial cell coverage and/or thrombogenesis.

Effect of cell seeding concentration

[0031] Figures 1A-1C are images of histological sections stained with Hematoxylin and Eosin of decellularized submucosa seeded with endothelial cells at varying densities: respectively 0 cells/ 50 μΐ (control), 200K cells/ 50 μΐ, and 500K cells/ 50 μΐ. The samples were cultured in cassettes for 4 days. The samples are then exposed to mepacrine labeled human peripheral blood cells for 15 min at 37°C.

[0032] Figures 2A-2C are images of histological sections stained with Masson's trichrome of decellularized submucosa seeded with endothelial cells at varying densities: 2A-2C respectively 0 cells/ 50 μΐ (control), 200K cells/ 50 μΐ, and 500K cells/ 50 μΐ. After culture, the samples are then exposed to mepacrine labeled human peripheral blood cells for 15 min at 37°C.

[0033] The unseeded decellularized submucosa (shown in Figures 1A and 2 A) exhibits platelet aggregation. Increasing the seeding concentration of Endothelial Cells from 200K cells/ 50 μΐ to 500K cells/ 50 μΐ appears to decrease thrombogenicity. Preferred seeding concentration was found at 500K cells/ 50 μΐ.

Effect of scaffolding material strain [0034] Figures 3A-3C are images of histological sections stained with Hematoxylin and Eosin of decellulanzed submucosa seeded with endothelial cells at the same concentration of 1000K cells/ 50 μΐ but under varying strain levels: 3A-3C respectively 0% strain, 5% strain, and 10% strain levels.

[0035] Endothelial Cells are visible for strain levels of 5% or more. Human Umbilical Vein Endothelial Cells seeded on decellulanzed porcine urinary bladder submucosa under 5% strain generated 70-80% cell coverage. In comparison, endothelial cells seeded on decellularized submucosa under 0%, 10%, and 20% strain only generated 10-50%) cell coverage.

[0036] Thus, increasing seeding concentration while applying strain on the base scaffold may promote endothelial cell adherence.

[0037] Figures 4A-4C are images of histological sections stained with antibodies to the platelet endothelial cell adhesion molecule 1 protein (PECAM/CD31 protein) of decellularized submucosa seeded with endothelial cells at the same concentration of 1000K cells/ 50 μΐ but under varying strain levels: 4A-4C respectively 0% strain, 5% strain, and 10% strain levels.

[0038] Figures 5A-5C are images of histological sections stained with antibodies to the vascular endothelial growth factor receptor 2 protein (VEGFR-2 protein) of decellularized submucosa seeded with endothelial cells at the same concentration of 1000K cells/ 50 μΐ but under varying strain levels: 5A-5C respectively 0% strain, 5% strain, and 10% strain levels.

[0039] Figure 6A-6C are images of histological sections stained with antibodies to the von Willebrand factor protein (vWF protein) of decellularized submucosa seeded with endothelial cells at the same concentration of 1000K cells/ 50 μΐ but under varying strain levels: 6A-6C respectively 0% strain, 5% strain, and 10% strain levels.

[0040] Figures 4A-4C, 5A-5C, and 6A-6C show that endothelial cells seeded on decellularized submucosa under strain stained positive for endothelial cell markers, and therefore retained endothelial cell phenotype.

[0041] While the examples described in the Figures show cells seeded on a decellularized tissue, other extracellular matrix materials may be used instead of the decellularized tissue.

[0042] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and description. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the claims to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the claims.

Claims

What is claimed is:

1. A method of making an artificial blood barrier, comprising:

providing an acellular biomaterial;

deforming the acellular biomaterial;

seeding cells on the deformed acellular biomaterial; and

culturing the cells seeded on the acellular biomaterial in vitro.

2. The method of claim 1, wherein providing the acellular biomaterial comprises decellularizing a biologic tissue or organ.

3. The method of claim 2, wherein the biologic tissue or organ includes collagen fibers or collagen gel.

4. The method of claim 2, wherein the biologic tissue or organ consists of one of heart tissue, urinary bladder tissue, or small intestine tissue.

5. The method of claim 4, wherein the biologic tissue or organ consists of porcine small intestinal submucosa.

6. The method of claim 1, wherein the cells comprise endothelial cells or endothelial progenitor cells from umbilical vein or umbilical cord, circulating endothelial progenitor cells from venipuncture, isolation of cells from veni-biopsy, cells from bone marrow, or combinations thereof.

7. The method of claim 1, wherein the cells comprise tissue-derived stem cells, amniotic fluid stem cells, amniotic membrane stem cells, embryonic stem cells, or induced pluripotent stem cells.

8. The method of claim 1, wherein seeding cells on the deformed acellular biomaterial comprises seeding the cells at a concentration of at least 500K cells/ 50 μΐ.

9. The method of any of claims 1-8, wherein deforming the acellular biomaterial comprises applying a strain level of at least 5%.

10. The method of any of claims 1-8, wherein deforming the acellular biomaterial comprises applying a uniaxial or biaxial or circumferential strain.

11. The method of claim 10, wherein deforming the acellular biomaterial further comprises applying a strain level of at least 5%.

12. An artificial blood barrier made by a method comprising:

providing an acellular biomaterial;

deforming the acellular biomaterial;

seeding cells on the deformed acellular biomaterial; and

culturing the cells seeded on the acellular biomaterial in vitro.

13. The artificial blood barrier of claim 12, wherein providing the acellular biomaterial comprises decellularizing a biologic tissue or organ.

14. The artificial blood barrier of claim 13, wherein the biologic tissue or organ includes collagen fibers or collagen gel.

15. The artificial blood barrier of claim 13, wherein the biologic tissue or organ consists of one of heart tissue, urinary bladder tissue, or small intestine tissue.

16. The artificial blood barrier of claim 15, wherein the biologic tissue or organ consists of porcine small intestinal submucosa.

17. The artificial blood barrier of claim 12, wherein the cells comprise endothelial cells or endothelial progenitor cells from umbilical vein or umbilical cord, circulating endothelial progenitor cells from venipuncture, isolation of cells from veni-biopsy, cells from bone marrow, or combinations thereof.

18. The artificial blood barrier of claim 12, wherein the cells comprise tissue-derived stem cells, amniotic fluid stem cells, amniotic membrane stem cells, embryonic stem cells, or induced pluripotent stem cells.

19. The artificial blood barrier of claim 12, wherein seeding cells on the deformed acellular biomaterial comprises seeding the cells at a concentration of at least 500K cells/ 50 μΐ.

20. The artificial blood barrier of any of claims 12-19, wherein deforming the acellular biomaterial comprises applying a strain level of at least 5%.

21. The artificial blood barrier of any of claims 12-19, wherein deforming the acellular biomaterial comprises applying a uniaxial or biaxial or circumferential strain.

22. The artificial blood barrier of claim 21, wherein deforming the acellular biomaterial further comprises applying a strain level of at least 5%.