METHODS FOR TISSUE DECELLULARIZATION

Abstract

The present invention provides A method of producing a decellularised extracellular matrix (ECM) scaffold of at least a portion of a lobular organ with no common artery, the method comprising: a) closing afferent blood vessels to substantially seal a target lobular organ or portion thereof with no common and/or major artery within a non-human donor or a dead/brain dead human donor; b) optionally: (i) cleaning coagulum and/or blood from at least a portion of the closed afferent blood vessels; and/or (ii) perfusing the organ or portion thereof to confirm closure of the afferent blood vessels; c) removing the sealed organ or portion thereof from the donor; and d) perfusing the sealed organ or portion thereof with detergent and enzymatic solutions to obtain the decellularised ECM scaffold. Methods for producing an artificial organ, and artificial organs produced by the methods are also provided.

Claims

1. A method of producing a decellularised extracellular matrix (ECM) scaffold of at least a portion of a lobular organ with no common artery, the method comprising: a) closing afferent blood vessels to substantially seal a target lobular organ, or a portion thereof, with no common and/or major artery within a non-human donor or a dead/brain dead human donor; b) optionally: (i) cleaning coagulum and/or blood from at least a portion of the closed afferent blood vessels; and/or (ii) perfusing the target lobular organ or the portion thereof to confirm closure of the afferent blood vessels to form a sealed organ; c) removing the sealed organ or a portion thereof from the non-human donor or a dead/brain dead human donor; and d) perfusing the sealed organ or the portion thereof with a detergent and enzymatic solutions to obtain the decellularised ECM scaffold.

2. The method according to claim 1, wherein the detergent is sodium deoxycholate (SDC), sodium dodecyl sulphate (SDS), Triton X-100, or a combination thereof.

3. The method according to claim 1, wherein the SDC concentration is about 1% to about 10% (w/v), or the SDS concentration is about 0.05% to about 1% (w/v), or the Triton X-100 concentration is about 0.05% to about 5% (w/v).

4. The method according to claim 1, wherein the sealed organ or the portion thereof is perfused with the detergent for about 1 to about 4 hours.

5. The method according to claim 1, wherein the sealed organ or the portion thereof is perfused with the detergent at a rate from about 0.2 ml/min to about 1 ml/min.

6. The method according to claim 1, wherein the sealed organ or the portion thereof is perfused for a single cycle.

7. The method according to claim 1, wherein the sealed organ or the portion thereof is freely floating in solution during perfusion.

8. The method according to claim 1, wherein the lobular organ, or the portion thereof with no common artery is a) a thymus or a lobe thereof, b) thyroid gland or a lobe thereof, c) parathyroid gland or d) salivary gland.

9. The method according to claim 8, wherein the lobular organ is the thymus, and wherein the afferent blood vessels closed to seal the thymus comprise a right or left common carotid artery, a right subclavian artery, a right internal mammary artery, a right costocervical trunk, a right aortic arch, a left aortic arch, a left costocervical trunk, a left internal mammary artery and a left subclavian artery, a left internal mammary artery, a left costocervical trunk and a left aortic arch in the non-human donor or the dead/brain dead human donor.

10. The method according to claim 9, wherein the right common carotid artery is closed and the thymus is perfused through the left common carotid artery, or wherein the left common carotid artery is closed and the thymus is perfused through the right common carotid artery.

11. The method according to claim 8, wherein the blood vessels closed to seal the thyroid comprise the right or left common carotid artery after junction with a superior thyroid artery.

12. The method according to claim 11, wherein each thyroid lobe is perfused through the right or left common carotid artery.

13. A decellularised ECM scaffold obtained by the method of claim 1.

14. A method for producing an artificial organ, the method comprising: repopulating a decellularised ECM scaffold of claim 13 with stromal cells, to form a repopulated scaffold, culturing the repopulated scaffold in vitro for about 4 to about 7 days; and optionally seeding the repopulated scaffold with donor cells.

15. The method according to claim 14, wherein the stromal cells are a combination of epithelial and mesenchymal cells, and wherein the decellularised ECM scaffold is repopulated with the epithelial and mesenchymal cells at a ratio of about 2:1 to about 5:1.

16. An artificial organ comprising the decellularised ECM scaffold of claim 13.

17. (canceled)

18. A method for treating a subject with DiGeorge syndrome, comprising transplanting the artificial organ of claim 16 into the subject, wherein the artificial organ is a thymus, thereby treating the DiGeorge syndrome in the subject.

19. An in vitro method for testing a compound for its ability to elicit a pharmacological, immunological or toxicological response, the method comprising contacting the artificial organ of claim 16 with the compound, thereby testing the compound for its ability to elicit the pharmacological, immunological or toxicological response.

20. A method of organ transplantation, the method comprising surgically implanting the artificial organ of claim 16 into a patient.

21. A method of treating a disease, the method comprising surgically implanting the artificial organ according to claim 16 into a patient in need thereof, thereby treating the disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The invention will now be described in detail, by way of example only, with reference to the figures.

[0041] FIG. 1 shows a schematic representation of a surgical procedure for ligating specific artery branches that allow the formation of an artificial perfusion system around the thymus. Knots 1-9 are formed in the afferent blood vessels (knot 6 on the left aortic arch should not be tight at this stage), then the catheter is inserted into the left common carotid. The left internal jugular vein is incised; followed by incision of the left aortic arch below knot 6; then the right internal jugular vein is cut. Buffer solution, such as PBS, is flushed slowly through the catheter to wash the aortic arch and remove coagulum, then knot 6 on the left aortic arch is closed tightly. The cannulated/catheterised thymus is then removed by cutting the closed arteries near knots 1-9, then the associated veins are cut starting from the subclavian vein.

[0042] FIG. 2 shows rat thymi before (A, B) and after (C, D) decellularisation through perfusion of detergent and enzymatic solutions (DET) at both gross macroscopy (B, D) and by histology (Haematoxylin and Eosin, H&E; A, C).

[0043] FIG. 3 shows the histology of decellularised thymus scaffold of the present invention: Haematoxylin and Eosin (H&E) (A), Picrosirius Red (collagen, B) and Masson's Trichrome (ECM, C) staining demonstrate the absence of cell nuclei and preservation of capsular and reticular ECM of the thymus.

[0044] FIG. 4 shows gross microscopy of a rat thymus scaffold before (panel A) and after (panel B) decellularisation by the method of the present invention. H&E staining of the thymus at low magnification (4×, panel C) and at higher magnification (panel D, scale bar=100 um), demonstrates preservation of parenchymal lobular and vascular structures.

[0045] FIG. 5 shows the appearance of a thymus scaffold obtained by the methods of the present invention before (A), soon after (B) and following 4 days of culture (C) after injection of stromal cells. Extra thymic tissue (darker areas in the images) allows connection to the cannula.

[0046] FIG. 6 shows histology (H&E) of the repopulated scaffold cultivated for 4 days (panel A). Immunohistochemistry (IHC) images demonstrate healthy stromal cells, which are negative for Caspase3 and most are positive for progenitor/stem cell marker p63 (panel B). Only few cells (yellow *) are Caspase3-positive indicating apoptosis (panel C). Repopulating stromal cells express Cytokeratin 5 (CK5), CK8 and alpha6 integrin (CD49f) (panel E). Moreover, most epithelial cells (EpCAM-positive) are proliferating (Ki67-positive) (panel D), thus indicating that the stromal cells are repopulating the scaffold and maintain a progenitor phenotype within the 3D structure (Scale bars=50 um).

[0047] FIG. 7 shows gross macroscopy (panel A, scale bar=2 mm) and histology (H&E) of repopulated scaffolds engrafted in NSG mice and matured in vivo for 8 weeks (B), 11 weeks (C), and 21 weeks (D), respectively. H&E staining demonstrates the presence of small round cells of hematopoietic origin interacting with the organised stromal cells. Notably there is a progressive stromal cell maturation as demonstrated by the presence of Hassall's bodies within the scaffold at 11 weeks' time-point. Scale bars=50 μm.

[0048] FIG. 8 shows immunohistochemistry of repopulated scaffolds grafted in NSG mice matured in vivo, demonstrating maturation of the human stroma (EpCAM and ECadherin positive cells, red) expressing functional markers such as HLA-DR (green) (A). Anti-Human Vimentin (green) recognises organised human mesenchymal cells (B), while anti-human CD3 (green) demonstrates the presence of maturing thymocytes in contact with human thymus stromal cells (C). Detection of AIRE-1 positive cells (3,3′-Diaminobenzidine, DAB staining) in the grafted thymus scaffolds demonstrates functional maturation of thymus cells, which is important in their role to eliminate self-reactive T cells (D).

[0049] FIG. 9 shows that cells within the repopulated decellularised ECM scaffold differentiate into the correct lymphoid lineages. A. FACS analysis of dissociated human engineered thymus scaffold matured in vivo shows that the vast majority (80%) of CD45+ cells developed from CD34+ progenitors are CD3+, lymphoid lineage; B. Single positive CD4 and CD8 cells express mature markers such as CD3+ and TCRab+.

[0050] FIG. 10 shows that single and double CD4+ and CD8+ thymocytes are created at proportions equivalent to normal tissue. A. FACS analysis of dissociated fresh human thymus showing single and double positive CD4+ and CD8+ thymocytes. B. FACS analysis of thymocytes developed in vitro from TN progenitors within the engineered thymus scaffold (rat), 6 days' time point; this demonstrates that engineered human thymus supports T cell development ex vivo.

[0051] FIG. 11 shows gross microscopy of a pig thymus before (left panel) and after (right panel) perfusion decellularisation (2 cycles of 4 hours perfusion at 4% SDC at 0.6-0.8 ml/min).

[0052] FIG. 12 shows histological analysis of the pig thymus after perfusion-decellularisation demonstrating preservation of ECM and vascular structures: H&E left panel; Masson Trichrome (MT); Picrosirius Red (PS) demonstrate preservation of ECM proteins and vasculature structures.

[0053] FIG. 13 shows gross microscopy of a human thymus before (left panel) and after (right panel) perfusion decellularisation (2 cycles of 4 hours perfusion at 4% SDC at 0.6-0.8 ml/min).

[0054] FIG. 14 shows H&E staining of the human thymus scaffold after perfusion decellularisation demonstrating removal of cell components and preservation of large and small vascular structures.

EXAMPLES

Example 1

[0055] Rat thymi were sealed by ligating the right common carotid artery, the right subclavian artery, the right internal mammary artery, the right costocervical trunk, the right aortic arch, the left aortic arch, the left costocervical trunk, the left internal mammary artery and the left subclavian artery (see FIG. 1) and were then extracted from subjects. The sealed thymi were decellularised in parallel using the following detergent-enzymatic perfusion treatment (DET), which was perfused by cannulation of the left common carotid artery while freely floating in solution:

a. perfusion with MilliQH2O for 96 hrs at 0.2 mL/min, 4° C.;
b. perfusion with 4% SDC for 1.5 h at 0.2 mL/min, RT;
c. perfusion with MilliQH2O for 24 hrs at 0.2 mL/min, RT;
d. perfusion with 0.1 mg/ml DNase (warm 37° C.) at 0.2 mL/min for 30 min; and
e. perfusion with PBS at 0.2 mL/min for 1 hr, RT.

[0056] The scaffolds were then processed for sterilization (gamma irradiation, 1782Gy).

[0057] The parallel decellularisation of the thymi illustrates the efficiency and reproducibility of the method of the present invention. As shown in FIG. 2 the decellularisation results in transparent thymic tissue, whilst maintaining the capsule. As shown in FIGS. 2C and 3A, histological analysis of the decellularised thymi by H&E staining demonstrates the absence of any cell nuclei, DNA or cellular debris. FIGS. 3B and C demonstrate preservation of capsular and reticular ECM of the thymus structure. Thymic ECM was detected as a homogenous reticulum and staining intensity reflects the abundance of ECM that is higher in the capsule of the organ (red in FIG. 3B and blue in FIG. 3C).

Example 2

[0058] A rat thymus was extracted and decellularised according to the protocol outlined in Example 1. Rat weight was 173 g.

[0059] Gross microscopy of the decellularised thymus is shown in FIG. 4 using H&E staining before (4A) and after (4B) decellularisation. Low magnification (FIG. 4C) and an area of higher magnification (FIG. 4D) demonstrates preservation of parenchymal lobular as well as of vascular structures all over the organ tissue. No DNA or cellular debris was detected. This demonstrates whole organ (bi-lobular) decellularisation through a single cannula at histological level.

Example 3

[0060] A thymus scaffold was obtained according to the protocol outline in Example 1. The thymus was then repopulated with a 5:1 (respectively) combination of Thymic Epithelial Cells (TEC) and Thymic Mesenchymal Cells (TMC) delivered simultaneously by direct needle injection, following ex vivo expansion from a tissue thymus donor. The cells were injected with a needle syringe (1 injection per lobe, 40p1 each, 2M TEC: 0.4M TMC per injection) in the thymic lobes shown as transparent tissue in FIG. 5A-C. Extra thymic tissue (darker areas in FIG. 5A-C) allowed connected to the cannula that was maintained during culture.

[0061] Repopulated scaffolds were maintained under in vitro conditions to allow for cell migration over the reticular ECM of the scaffold and initial differentiation (e.g. cell polarization, up/downregulation of adhesion molecules) over a period of 4 days. For in vitro culture the scaffolds were connected to a cannula and placed in a petri dish submerged in cFAD (epithelial) medium, which consists of DMEM and Ham's F12 medium (v/v 3:1), supplemented with 10% foetal bovine serum (FBS), insulin (5 μg/mL), 3,3,5-triiodo-L-thyronine (T3) (2×10.sup.−9 M), hydrocortisone (0.4 μg/mL), cholera toxin (1×10.sup.−10 M), and 1% penicillin/streptomycin.

[0062] FIG. 5 shows the appearance of the scaffold before (FIG. 5A), soon after (FIG. 5B) and following 4 days of culture after injection of the TEC and TMC cells (FIG. 5C). Histological analysis of the scaffold by H&E staining after 4 days of culture is shown in FIG. 6A, which reveals that cells adhere to and along the scaffold ECM. Only a small area of the scaffold is not yet repopulated, but demonstrates the presence of an intact matrix within the scaffold (black *). Immunohistochemistry (IHC) images demonstrate that healthy stromal cells were negative for Caspase3 and most were positive for progenitor/stem cell marker p63 (FIG. 6B). Only a small number of cells (yellow *) were Caspase3-positive indicating apoptosis (FIG. 6C). Repopulating stromal cells expressed Cytokeratin 5 (CK5), CK8 and alpha6 integrin (CD49f) (FIG. 6E). Moreover, more epithelial cells (EpCAM-positive) were proliferating (Ki67-positive) (FIG. 6D), thus indicating that the stromal cells were repopulating the scaffold and maintaining a progenitor phenotype within the 3D structure.

Example 4

[0063] Repopulated rat thymic scaffolds were prepared according to the protocol outlined in Example 3 above. After 4 days of in vitro culture with the stromal cells the repopulated scaffolds were engrafted subcutaneously into NSG mice for 8 and 11 weeks.

[0064] Histological analysis (H&E) of the two thymus scaffolds matured in vivo for 8 weeks (FIG. 7B), 11 weeks (FIG. 7C) and 21 weeks (FIG. 7D) demonstrated the presence of small round cells of hematopoietic origin interacting with the organized thymus stromal cells. Notably, there was a progressive stromal cell maturation as demonstrated by the presence of Hassall's bodies within the scaffold at 11 weeks' time-point (FIG. 7C) and increase of thymocyte density (FIG. 7D).

Example 5

[0065] Repopulated rat thymic scaffolds were prepared according to the protocol outlined in Example 3 above and were repopulated with human stromal cells identified by EpCAM and ECadherin positive cells. After 4 days of in vitro culture with the stromal cells the repopulated scaffolds were engrafted subcutaneously into NSG mice for 11 weeks.

[0066] Immunohistochemistry analysis demonstrated the presence of repopulating human stroma identified by EpCAM and ECadherin positive cells. Functional maturation of stromal cells was demonstrated by the up-regulation of MHC Class II surface receptor (HLA-DR-positive cells) (FIG. 8A). Presence of mesenchymal cells was detected by anti-human vimentin immunostaining (FIG. 8B), while anti-human CD3 immunostaining demonstrated the presence of maturing thymocytes within the same area (FIG. 8C). Detection of autoimmune response element 1 (AIRE-1) positive cells in the grafted thymus scaffolds demonstrates functional maturation of thymus cells, which is important in their role to eliminate self-reactive T cells and thereby induce tolerance (FIG. 8D). This demonstrates the capacity of human cultivated stromal cells to survive long term in vivo and organize functional structures similar to the human native thymus (i.e. Hassall's body and expression of HLA-DR).

Example 6

[0067] A portion of the thymus scaffolds repopulated and transplanted in vivo into NSG mice, were dissociated and analysed by FACS analysis that demonstrated high survival of human cells (63.9%, middle panel FIG. 9A); the vast majority of live cells were CD45+(92.9%, right panel FIG. 9A). Importantly, 80% of the CD45+ cells were also CD3+(FIG. 9B, left panel) thus demonstrating that the thymus scaffold is an inductive microenvironment for the lymphoid lineage; the presence of single positive CD4+ and CD8+ cells that express TCRab demonstrate that mature functional T cells have been produced within the scaffold in vivo (FIG. 9B, middle and right panels).

Example 7

[0068] A thymus scaffold was repopulated in vitro with stromal cells, and maintained in culture for 7 days before being injected with immature triple negative thymocytes (TN). The thymocytes were analysed by FACS 7 days after injection into the thymus scaffold where they matured towards both single and double positive CD4+ and CD8+ cells (FIG. 10B). Importantly the ratio of single positive CD4/CD8 in the scaffold is similar to the native thymus (FIG. 10A,B).

[0069] The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 639429.

REFERENCES

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