Bioprinted kidney tissue

Abstract

The present disclosure relates to bioprinted kidney tissue and methods for making the same. The bioprinted tissue and methods can be used in a variety of applications, such as regenerative medicine.

Description

[Technical field]

[0001] The present disclosure relates to bioprinted kidney tissues and methods for their production. The bioprinted tissues and methods can be used for a variety of applications, such as disease modeling, drug screening, drug testing, kidney replacement, tissue engineering, and regenerative medicine.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to Australian Provisional Patent Application No. 2019 / 903094, filed on 23 August 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] The kidney plays a major role in removing waste products and maintaining fluid volume. The functional unit of the kidney is known as the nephron. Human kidneys contain up to 2 million epithelial nephrons involved in filtering blood, all of which arise from prenatal nephron progenitor cells. Nephron progenitor cells are absent in postnatal human kidneys. Even the absence of this nephron progenitor cell population does not guarantee the capacity for new nephron formation (neonephrogenesis), so subsequent injury, aging, and disease can lead to a reduction in the number of nephrons and resulting chronic kidney disease (CKD). This ultimately leads to end-stage kidney disease (ESKD), which is life-threatening unless treated with some form of renal replacement therapy, such as dialysis (peritoneal dialysis or hemodialysis) or organ transplantation. These are the only available treatment options for ESKD. Both dialysis and kidney transplantation are costly and have significant drawbacks, affecting the quality of life of the patient. Currently, CKD is increasing at 6% per year worldwide, and only one in four patients has access to a donor organ, making sources of replacement kidney tissue a major therapeutic target.

[0004] The directed differentiation of human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS), to different cellular endpoints has made it possible to generate organoid models of various human tissues, such as the kidney. Traditional organoid models, such as those discussed in Takasato et al. (2015) Nature, Vol. 526: 564-568, can produce kidney organoids with complex three-dimensional structures that are multicellular models of the human kidney. These complex multicellular structures contain fully segmented nephrons connected to a collecting duct network surrounded by renal interstitium and endothelial cells. They display gene expression comparable to that of human fetal kidneys in trimester 1 of development.

[0005] Despite this important finding, kidney organoids produced according to the aforementioned method are self-limiting because they do not have a sustained nephron progenitor cell population. The progression of nephron formation in normal human kidney development is sustained from a nephron progenitor cell population. This population has been shown to be present in kidney organoids, but it has also been shown that this population of progenitor cells is lost after generating nephrons, limiting the upper limit of the number of nephrons that can be generated using this approach (Howden et al, EMBO Reports, 2019). A key factor for maximal generation of functional kidney tissue from stem cells is the relative proportion of tissue that is composed of nephrons. A second important factor is the reduction of unwanted non-kidney populations. Thus, there is a need for engineered kidney tissue that has a higher number of nephrons per number of cells used to generate such tissue, and a more uniform distribution of nephrons. A third important factor is the presence of kidney tissue that shows good patterning of the constituent nephrons and evidence of maturation of nephron segments. A fourth critical element for generating transplantable renal replacement tissue is the ability to generate such tissue in a reliable and reproducible manner that is suitable for automation. Summary of the Invention

[0006] The inventors have surprisingly found that in vitro engineered or bioprinted kidney tissues derived from compositions containing stem cell-derived renal progenitor cells can be produced with more or less nephrons resulting per number of cells used to produce the tissue, depending on the spatial parameters applied to bioprint the tissue. Surprisingly, it has been found that it is possible to produce bioprinted tissues with a more uniform distribution of nephrons. The inventors have surprisingly found that by varying the spatial parameters of the bioprinted tissue, more nephrons can be produced from the same amount of starting material (cells), and the resulting tissue has improved properties. Thus, described herein is a bioprinted kidney tissue enriched with mature nephrons, and a method for producing the same.

[0007] According to a first aspect, the present invention provides a bioprinted kidney tissue enriched with nephrons distributed throughout the tissue, in a preferred embodiment, the nephrons are evenly or homogeneously distributed throughout the printed tissue.

[0008] According to a second aspect, the present invention provides a bioprinted kidney tissue comprising a volume of bioink, wherein the bioink comprises a plurality of cells, the bioink is less than about 50 μm in height, and the bioprinted bioink is induced to form the kidney tissue. In another embodiment, after the bioprinted bioink is induced to form the kidney tissue, the height of the bioprinted kidney tissue is about 150 μm or less. In other words, the height of the final bioprinted kidney tissue is about 150 μm or less.

[0009] According to a third aspect, the present invention provides a bioprinted kidney tissue comprising a volume of bioink, wherein the bioink comprises a plurality of cells, the bioink being greater than or equal to 1 mm. 2The cells are bioprinted in layers containing up to 30,000 cells per layer.

[0010] According to a fourth aspect, the present invention provides a method for producing bioprinted kidney tissue comprising bioprinting a volume of bioink onto a surface, the bioink comprising a plurality of cells, the bioink being bioprinted in a layer having a height of about 50 μm or less, and inducing the bioprinted volume of bioink to form a bioprinted kidney tissue.

[0011] According to a fifth aspect, the present invention provides a method for bioprinting a volume of bioink onto a surface, the method comprising the steps of: bioprinting a volume of bioink onto a surface, the volume of bioink being greater than or equal to 1 mm 2 The present invention provides a method for producing bioprinted kidney tissue, the method comprising: bioprinting a plurality of cells into a layer comprising about 30,000 cells or less per layer; and inducing the bioprinted volume of bioink to form the bioprinted kidney tissue.

[0012] According to a sixth aspect, the present invention provides a bioprinted kidney tissue produced according to the method of the fourth or fifth aspect.

[0013] According to a seventh aspect, the present invention provides a bioprinted kidney tissue of any one of the first, second, third or sixth aspects for use in treating renal disease or failure in a subject in need thereof.

[0014] According to an eighth aspect, the present invention provides use of the bioprinted kidney tissue of any one of the first, second, third or sixth aspects in the manufacture of a medicament for the treatment of a kidney disease in a subject in need thereof.

[0015] According to a ninth aspect, the present invention provides a method for the treatment of renal disease or failure in a subject in need thereof comprising administering to the subject bioprinted kidney tissue of any one of the first, second, third or sixth aspects.

[0016] The numbered statements of the present invention are as follows: 1. Bioprinted kidney tissue, enriched for nephrons distributed throughout the tissue. 2. Bioprinted kidney tissue grows to 0.2mm per 10,000 cells printed 2 2. The bioprinted kidney tissue of statement 1, which is a layer of bioprinted tissue comprising a surface area of ​​nephron tissue of . 3. Bioprinted kidney tissue was measured at 1 mm 2 3. The bioprinted kidney tissue of statement 1 or 2, wherein the layer of bioprinted kidney tissue comprises about 30,000 cells or less per layer. 4. The bioprinted kidney tissue of any one of the preceding statements, wherein the bioprinted kidney tissue expresses high levels of any one or more of SULT1E1, SLC30A1, SLC51B, FABP3, HNF4A, CUBN, LRP2, EPCAM and MAFB. 5. The bioprinted kidney tissue of statement 4, wherein the bioprinted kidney tissue comprises nephrons whose proximal and distal tubule segments express maturation markers including HNF4A and SLC12A1. 6. Bioprinted kidney tissue of statement 4 or 5, where the bioprinted kidney tissue expresses the markers HNF4A, CUBN, LRP2, EPCAM and MAFB, respectively. 7. The bioprinted kidney tissue of any one of the preceding statements, wherein the height of the bioprinted kidney tissue is about 50 μm or less at the time of printing. 8. The bioprinted kidney tissue of any one of the preceding statements, wherein the bioprinted kidney tissue has a length of about 1 mm to about 30 mm and a width of about 0.5 mm to about 20 mm. 9. Bioprinted kidney tissue is grown to approximately 5 to 100 nephrons / mm of bioprinted kidney tissue. 2 Including, statement 7 or 8 bioprinted kidney tissue. 10. A bioprinted kidney tissue comprising a volume of bioink comprising a plurality of cells, the bioink being bioprinted in a layer having a height of about 50 μm or less, and the bioprinted bioink being induced to form kidney tissue. 11. The bioprinted kidney tissue of statement 10, wherein the bioink is bioprinted in a layer selected from about 20 μm high to about 40 μm high. 12. Bioprinted kidney tissue from statement 10, where the bioink is bioprinted in a layer approximately 30 μm high. 13. Bioprinted kidney tissue from statement 10, where the bioink is bioprinted in a layer approximately 25 μm high. 14. The bioprinted kidney tissue of any one of statements 10-14, wherein the predetermined volume of bioink comprises about 10,000 cells / μl to about 400,000 cells / μl. 15. The bioprinted kidney tissue of any one of statements 10-14, wherein the plurality of cells comprises partially differentiated cells. 16. The bioprinted kidney tissue of any one of statements 10-15, wherein the plurality of cells comprises renal progenitor cells. 17. The bioprinted kidney tissue of statement 16, wherein the renal progenitor cells include nephron progenitor cells. 18. The bioprinted kidney tissue of statement 16 or 17, wherein the renal progenitor cells comprise ureteral epithelial progenitor cells. 19. The bioprinted kidney tissue of any one of statements 10-15, wherein the plurality of cells comprises intermediate mesoderm cells. 20. The bioprinted kidney tissue of any one of statements 10-15, wherein the plurality of cells comprises metanephric mesenchymal cells. 21. The bioprinted kidney tissue of any one of statements 10 to 15, wherein the plurality of cells comprises renal duct cells. 22. The bioprinted kidney tissue of any one of statements 10-15, wherein the plurality of cells comprises fully differentiated cells. 23. The bioprinted kidney tissue of any one of statements 10-22, wherein the plurality of cells comprises cells derived from a patient. 24. The bioprinted kidney tissue of any one of statements 10-23, wherein the plurality of cells comprises cells from a reporter cell line. 25. The bioprinted kidney tissue of any one of statements 10 to 24, wherein the plurality of cells comprises gene-edited cells. 26. The bioprinted kidney tissue of any one of statements 10-25, wherein the plurality of cells comprises diseased cells, healthy cells, or a combination of diseased cells and healthy cells. 27. Bioprinted kidney tissue grows to 0.2mm per 10,000 cells printed 2 Bioprinted kidney tissue from any one of statements 10-26, including a surface area of ​​nephron tissue of . 28. Bioprinted kidney tissue was measured at 1mm 2 The bioprinted kidney tissue of any one of statements 10-27 containing approximately 30,000 cells or less per well. 29. The bioprinted kidney tissue of any one of statements 10-28, wherein the bioprinted kidney tissue expresses high levels of any one or more of HNF4A, CUBN, LRP2, EPCAM, and MAFB. 30. The bioprinted kidney tissue of statement 29, wherein the bioprinted kidney tissue comprises nephrons, the proximal and distal tubule segments of which express maturation markers, including HNF4A. 31. The bioprinted kidney tissue of statement 29 or 30, wherein the bioprinted kidney tissue expresses the markers HNF4A, CUBN, LRP2, EPCAM and MAFB, respectively. 32. The bioprinted kidney tissue of any one of statements 1-31, wherein the tissue comprises about 5 to about 100 nephrons / 10,000 cells printed. 33. The bioprinted kidney tissue of any one of statements 10-32, wherein the tissue has a uniform distribution of nephrons throughout the bioprinted layer. 34. The bioprinted kidney tissue of any one of statements 10-33, wherein the tissue has a uniform distribution of glomerular structures expressing MAFB throughout the bioprinted layer. 35. The bioprinted kidney tissue of any one of statements 1 to 34, further comprising a biocompatible scaffold. 36. The bioprinted kidney tissue of statement 35, where the bioink is bioprinted onto a biocompatible scaffold. 37. The bioprinted kidney tissue of any one of statements 35 or 36, wherein the biocompatible scaffold is a hydrogel. 38. The bioprinted kidney tissue of any one of statements 35-37, wherein the biocompatible scaffold is biodegradable or bioabsorbable. 39. The bioprinted kidney tissue of any one of statements 10-38, wherein the bioink further comprises one or more biologically active agents. 40. The bioprinted kidney tissue of statement 39, wherein the one or more biologically active agents promote derivation of kidney tissue from the plurality of cells. 41. A method for producing bioprinted kidney tissue, comprising: bioprinting a quantity of bioink onto a surface, the bioink comprising a plurality of cells, the bioink being bioprinted in a layer having a height of about 50 μm or less; and inducing the bioprinted quantity of bioink to form bioprinted kidney tissue. 42. The method of statement 41, wherein in the bioprinting step, the bioink is bioprinted in a layer selected from a height of about 20 μm to a height of about 40 μm. 43. The method of statement 41, wherein in the bioprinting step, the bioink is bioprinted in a layer about 30 μm high. 44. The method of statement 41, wherein in the bioprinting step, the bioink is bioprinted in a layer about 25 μm high. 45. The method of any one of statements 41-44, wherein the predetermined volume of bioink comprises between about 10,000 cells / μl and about 400,000 cells / μl. 46. ​​The method of statement 45, wherein the bioink comprises about 200,000 cells / μl. 47. The method of any one of statements 41-46, wherein the plurality of cells includes partially differentiated cells. 48. The method of any one of statements 41-46, wherein the plurality of cells comprises renal progenitor cells. 49. The bioprinted kidney tissue of statement 48, wherein the renal progenitor cells include nephron progenitor cells. 50. The method of statement 48 or 49, wherein the renal progenitor cells include ureteral epithelial progenitor cells. 51. The method of any one of statements 41-47, wherein the plurality of cells comprises a culture expanded population of intermediate mesodermal cells, preferably stem cell derived intermediate mesodermal cells. 52. The method of any one of statements 41-47, wherein the plurality of cells includes metanephric mesenchymal cells. 53. The method of any one of statements 41-47, wherein the plurality of cells includes renal duct cells. 54. The method of any one of statements 41-47, wherein the plurality of cells includes fully differentiated cells. 55. The method of any one of statements 41-54, wherein the plurality of cells comprises cells derived from a patient. 56. The method of any one of statements 41-55, wherein the plurality of cells includes cells from a reporter cell line. 57. The method of any one of statements 41-56, wherein the plurality of cells comprises gene-edited cells. 58. The method of any one of statements 41-57, wherein the plurality of cells comprises diseased cells, healthy cells, or a combination of diseased and healthy cells. 59. The method of any one of statements 41-58, wherein the bioprinted kidney tissue comprises about 5 to about 100 nephrons / 10,000 cells printed. 60. The method of any one of statements 41-59, wherein the bioprinted kidney tissue has a uniform distribution of nephrons throughout the bioprinted layer. 61. The method of any one of statements 41-60, wherein the bioprinted kidney tissue has a uniform distribution of glomerular structures expressing MAFB throughout the bioprinted layer. 62. The method of any one of statements 41-61, wherein in the bioprinting step, the bioink is bioprinted onto a biocompatible scaffold. 63. The method of statement 62, wherein the biocompatible scaffold is a hydrogel. 64. The method of any one of statements 62 or 63, wherein the biocompatible scaffold is biodegradable or bioabsorbable. 65. The method of any one of statements 41-64, wherein the bioink further comprises one or more biologically active agents. 66. The method of statement 65, wherein said one or more biologically active agents promote derivation of renal tissue from said plurality of cells. 67. The method of any one of statements 41-66, wherein the inducing step includes contacting a predetermined amount of the bioprinted bioink with FGF-9. 68. The method of statement 67, wherein the inducing step includes contacting the predetermined amount of bioprinted bioink with FGF-9 for 5 days. 69. The method of any one of statements 41-68, wherein the plurality of cells are contacted with a cell culture medium containing CHIR before being bioprinted. 70. The method of any one of statements 41-69, wherein the bioprinting step uses an extrusion-based bioprinter. 71. The method of any one of statements 41-70, wherein in the bioprinting step, a dispensing device of the bioprinter is configured to dispense the layer in one or more lines. 72. The method of any one of statements 41-71, wherein in the bioprinting step, a dispensing device of the bioprinter is configured to dispense the layer in one or more lines to form a continuous sheet or patch. 73. Bioprinted kidney tissue produced according to any one of statements 41-72. 74. The bioprinted kidney tissue of any one of statements 1-40, or 73, for use in treating kidney disease or renal failure in a subject in need of such treatment. 75. Use of bioprinted kidney tissue of any one of statements 1 to 40, or 73 in the manufacture of a medicament for the treatment of kidney disease in a subject in need of such treatment. 76. A method for treating renal disease or renal failure in a subject in need thereof, comprising administering to the subject bioprinted kidney tissue of any one of statements 1-40, or 73. 77. The bioprinted kidney tissue of any one of statements 1-40 or 73 for use according to statement 74, the use of statement 75, or the method of statement 76, wherein in said treatment, the bioprinted kidney tissue is implanted under the renal capsule of said subject.

[0017] Any example or embodiment herein is intended to apply mutatis mutandis to any other example or embodiment, unless otherwise stated.

[0018] The present disclosure is intended to be illustrative only and is not limited in scope by the specific examples described herein. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure as described herein.

[0019] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, a reference to a single step, composition of matter, group of steps, or group of compositions of matter shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0020] The present disclosure will now be described with reference to the following non-limiting examples and the accompanying drawings. [Brief description of the drawings]

[0021] [Figure 1]Highly reproducible generation of human pluripotent stem cell-derived kidney organoids by extrusion-based cell bioprinting of day 7 intermediate mesoderm cell paste. A. Bioprinting protocol for differentiating pluripotent stem cells and generating kidney organoids. This figure illustrates the use of bioprinting to replace manual processing and compares the relative cell numbers and organoid generation rates between manual processing (Takasato et al., 2016) and 3D cell paste extrusion bioprinting. B. Brightfield images of micromass cell paste cultures from the day of printing (day 7+0) to day 20 of culture (day 7+20), showing the spontaneous formation of nephrons over time. C. Whole mount immunofluorescence staining of day 7+18 organoids showing evidence of patterned and segmented nephrons including distal tubules (E-CADHERIN, green), proximal tubules (LTL, blue), podocytes (NEPHRIN, white), and occult segment / collecting ducts (GATA3, red). Fusion of channels indicates the relationship of individual nephron segments. D. Whole mount immunofluorescence staining of day 7+18 bioprinted organoids using markers showing the presence of distal tubules / loops of proximal tubule segments (CD13, CUBN, LTL), tubular basement membrane (LAMININ), surrounding interstitium (MEIS1 / 2), Henle TAL (SLC12A1) and endothelium (CD31). E. Brightfield (day 7+7) and whole mount immunofluorescent stained manual and bioprinted kidney organoids generated simultaneously from the same batch of iPSC-derived intermediate mesoderm. Staining shows evidence of patterned and segmented nephrons in both manual and bioprinted organoids (EPCAM, green: epithelium; LTL, blue: proximal tubule; NPHS1, white: glomerulus; GATA3, red: connecting segment / collecting duct). F. Three kidney organoids are bioprinted onto Transwell® inserts. Starting cell numbers are shown. The top row shows the ability to generate organoids when cell numbers are reduced.The bottom row shows the reproducibility of size when bioprinting a specific cell number across multiple wells. G. Six-well Transwell® insert containing nine bioprinted organoids, each containing approximately 96,000 cells. H. Renal organoid differentiation within bioprinted organoids is comparable despite reduced starting cell numbers. Images show H&E stained sections of mature organoids printed as 2x105 or 4x105 cell organoids. [Diagram 2] A. Full histological section of a day 7+18 bioprinted kidney organoid showing clear evidence of nephron development from interconnecting epithelium (arrows). B. Immunostaining of a bioprinted kidney organoid section showing GATA3+ECAD+ connecting segments / collecting ducts with multiple ECAD+GATA3- nephrons attached. C. Immunostaining of a bioprinted kidney organoid section showing an ECAD+ nephron attached to an MAFB+ glomerulus. D. Brightfield histological and immunofluorescence comparison of kidney organoids (5x105 cells per organoid) manually generated using dry cell paste with organoid diameter control vs. dry cell paste with cell number control vs. wet cell paste. [Diagram 3]A. Immunofluorescence observation of organoids from a single starting differentiation used to generate manual organoids (5x105 cells) vs. bioprinted organoids generated from only 4,000 cells. B. Differentiation over time of bioprinted organoids generated using MAFBmTagBFP2 reporter iPSC lines. C. Organoids bioprinted with MAFBmTagBFP2 on the same transwell filter at 4K, 50K, or 100K cells per organoid, showing fluorescent reporter imaging (blue) and differentiation staining (ECAD, green; LTL, blue; GATA3, red; NPHS1, purple). D. Organoids bioprinted with MAFBmTagBFP2 on the same transwell filter at 100K cells per organoid, showing live fluorescent imaging (blue) and differentiation staining (ECAD, green; LTL, blue; GATA3, red; NPHS1, purple). [Figure 4]Application of bioprinted organoids for compound screening in 96-well format. A. Images of all bioprinted organoids in a 96-well transwell format. Bioprinted organoids were generated with a deposition of 1x105 cells per organoid and further cultured for 18 days. B. 96-well plate secured to the print stage in the plate holder immediately prior to deposition. C. Quality control assessment of bioprinted cell number per organoid and cell viability across the 96-well plate. D. Immunofluorescence analysis of response to 10.0uM doxorubicin versus control. Sections of bioprinted organoids were stained with antibodies against MAFB (podocyte marker), cleaved caspase 3 (CC3, apoptosis marker), cytokeratin 8 / 18 (CCK8 / 18, tubule marker), lotus tetranoglobulin lectin (LTL, proximal tubule), and DAPI to label nuclei. In the presence of doxorubicin, there was a podocyte-specific loss of expression of MAFB and induction of apoptosis. E. Gene expression of kidney injury molecule-1 (HAVCR) and apoptotic genes (CASP3, BAX) in response to doxorubicin treatment. F. Gene expression of key podocyte (NPHS1, PODXL) and proximal tubule (CUBN) genes in response to doxorubicin treatment. G. Assessment of cell viability in response to doxorubicin treatment comparing data from bioprinted organoids deposited in 6-well (green) and 96-well (blue) transwell formats. Viability was assessed 72 hours after addition of doxorubicin. H. Application of 96-well bioprinted organoids to screen viability in response to a range of aminoglycoside antibiotics. [Diagram 5]Use of extrusion bioprinting to vary organoid conformation. A. Generation of a series of organoids of increasing length from the same starting cell number (1.1 x 105 cells). This figure is useful to show the relative impact of varying organoid profile / height in bioprinting from ratio 0 (no needle movement during extrusion) to ratio 40 (extrusion with needle moving across the transwell surface). Ratio refers to the ratio of tip movement to extrusion. B. Fluorescent beads were included to measure cell paste spreading across the transwell surface as organoids are produced. The greater the spreading, the fewer beads per surface area. Representative areas from cell paste depositions of ratios 0 and 40 are shown. The white dotted lines indicate the edge of the cell paste. C. Quantification of bead density per unit transwell surface area. Higher ratios result in greater spreading and lower bead density (n=21 organoids total, n=3 per condition except ratio 0 where n=9). D. Tissue height measured at D7+0 immediately after bioprinting (n=27 organoids, from two independent experiments). E. Organoid height measured at days 7+12 for organoids printed in different conformations (n=21 organoids). Red dots in D and E represent the mean values. Note that the scale of the Y-axis is different in D and E. See Figure 6 for details. F. Representative fluorescent imaging of live organoids generated using the MAFBmTGABFP2 reporter strain containing blue fluorescent protein showing the glomerular region of organoids printed in different conformations. G. Quantification of measured mTagBFP2 area and organoid length in bioprinted organoid replicates in different conformations. Each dot represents a single organoid (n=90 organoids in total, see Figure 6). H. Immunofluorescence of representative bioprinted organoids from each conformation showing MAFBmTagBFP2 (glomeruli, blue endogenous fluorescence), epithelium (EPCAM, grey), proximal tubules (LTL, green), and connecting segments / collecting ducts (GATA3, red). [Figure 6-1]Quantification of bead density and MAFBmTagBFP2 reporter signal in organoids in various conformations. A. Representative images of fluorescent bead signal (grayscale) at D7+0 across the entire print pattern, showing all five conformations from left to right: ratio 0 (three replicates), ratio 40, ratio 30, ratio 20, ratio 10. B. Composite images of each conformation at D7+12 showing mTagBFP2 reporter expression (blue) and bead signal (red). Note that the images are placed on a black background. Scale bars are 1 mm in A and B. C. Quantification of total organoid area (see Methods) and mTagBFP2 area of ​​organoid replicates (compare to Figure 7G). D-C and table of organoid numbers and ratios by plate replicates used for quantification in Figure 7G. E. Example of nine organoid replicates produced using ratio 20. Organoids are uniform among the three organoids from separate wells of each plate, and among plates. F. Representative image of sparse labeling with CellTrace Far Red dye to quantitate organoid height at D7+0. XY and orthogonal views are shown. G. Schematic of the scoring method used for quantification. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7]Altering organoid conformation reduces unpatterned tissue and increases nephron number and maturity (see also Figure 9). A. Heatmap comparing scaled log counts per million expression values ​​in bulk RNAseq transcriptional profiles of organoids at ratio 0 (R0), ratio 20 (R20), and ratio 40 (R40). B. Heatmap of scaled log counts per million gene expression values ​​representing the most significantly enriched GO terms in organoids at ratio 40 versus ratio 0. C. Immunofluorescence to validate transcriptional changes showing a decrease in endothelial marker SOX17 and an increase in loop of Henle's loop (TAL) marker SLC12A1 as ratio increases. D. 3D rendering of bioprinted organoids showing distinct morphology in organoids at ratio 0 to ratio 40. Images are rendered to show that the XY plane is tilted at 45 degrees. [Figure 8-1]Single-cell RNAseq comparison of manual organoids, bioprinted R0 "dots", and bioprinted R40 "lines". A. Experimental design. Multiple organoid sets were generated per conformation, and each set was barcoded and combined to form one scRNAseq library per condition. Both bioprinting types were generated from 1.1×105 cells, while manual organoids were generated from 2.3×105 cells. B. Quantification of images confirms increased nephrons in R40 line organoids based on the MAFB reporter region. Black bars represent mean values. R40-Man, p=2.1×10-5; R40-R0, p=2×10-16, based on pairwise t-test with Holm multiple comparison correction. Details of n values ​​per condition, set-level comparisons, and representative images are shown in Figure 9. C. UMAP visualizing transcriptional variation in interstitial lineage cells of scRNA. For details on cluster identities, see Figure 10. D. Proportion of each interstitial cell cluster by replicate and condition. P-values ​​are shown at p<0.2 and represent one-way ANOVA comparing all three conditions. Each point represents a single replicate, and red diamonds represent the mean of n=4. E. UMAP visualizing transcriptional variation in nephron lineage cells in scRNAseq data. Clusters are nephron progenitor-like (3), prepodocyte (4), podocyte (1), pretubule (2), distal tubule (0), and proximal tubule (8). Clusters 5 and 7 represent cycling cells, and cluster 6 was removed to represent doublet cells. For details, see Figure 10. F. Proportion of each nephron cell type per replicate across conditions. P-values ​​are shown at p<0.2 and represent one-way ANOVA comparing all three conditions. For cluster 4, ANOVA followed by Tukey multiple comparison of means gave p = 0.021 at R40 vs. Man. Heatmap showing the filtered number of differentially expressed (DE) genes within each cluster between conformations of clusters of G. nephron lineages.DE testing was performed on the sum of pseudobulk counts of a given cell type, per replicate and condition, to identify statistically significant (adjusted p-value < 0.05) changes, taking into account variation between replicates (n = 4). Gene lists were filtered to remove genes appearing in more than two cell types to focus on specific changes and minimize potential batch effects. H. Violin plot of normalized single-cell expression values ​​of selected genes identified as having statistically significant DE between R40 and manual organoids in the pseudobulk analysis of proximal tubule cells (nephron cluster 8). The violin plot shows the distribution of single-cell expression values ​​as colored shapes, with individual points overlaid as black dots. R40 organoids show increased expression of genes associated with proximal tubule maturation (SLC30A1, SLC51B, FABP3, SULT1E1) and decreased expression of genes associated with early immature tubules (SPP1, JAG1) compared to manual organoids. I. Heatmap showing filtered gene counts differentially expressed within each cluster between stromal lineage cluster conformations. J. Violin plot of normalized single cell expression values ​​of selected genes identified as significantly increased in expression in pseudobulk analysis of stromal cluster 2 cells between R40 and manually-manipulated organoids. K, L. Violin plot of normalized expression values ​​of selected genes identified as significantly increased in expression in pseudobulk analysis of stromal cluster 3 cells in R40 organoids. Genes associated with nephron progenitor identity were significantly increased in K) R40 vs. manually-manipulated organoids (HOXA11, FOXC2) and L) R40 vs. R0 organoids (EYA1, SIX1). [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9]Quantification of large image datasets related to organoids used for single-cell RNAseq. Line organoids are approximately 12 mm long. A. Representative images from three separate wells across replicates and conditions. B. Quantification of MAFB-mTagBFP2 reporter region per set and condition. Data are as in Figure 8B, but now separated by set. C. Quantification of GATA3-mCherry reporter area. Note that the Y-axis scale is different in B and C, as in most cases GATA3 region represents a rather small percentage of the organoid. D. Area of ​​GATA3 as a percentage of the total reporter area measured (MAFB+GATA3), showing a shift of R0 towards a more distal fate. E. Total number of individual organoids used for quantification per set and condition. [Figure 10-1]Analysis of single-cell RNA dataset. A. Variability within the dataset represented as UMAP plot, color-coded by transcription cluster, predicted cell cycle phase, main cell type, and organoid conformation (clockwise from top left). B. Marker genes for main cell types, WT1 and PAX2 (nephron), PDGFRA (stroma), and SOX17 (endothelium). C. Proportion of each cell type in replicate conditions. P-values ​​shown if p<0.2 (one-way ANOVA). D. High-resolution UMAP of nephron cells after retransformation and clustering. Plots are color-coded by transcription cluster, predicted cell cycle phase, and organoid conformation. Cluster IDs are indicated. E. Marker genes identifying each cluster: GATA3 (distal), HNF1B (pretubular), CUBN (proximal), HNF4A (proximal), FOXC2 (prepodocyte), MAFB (prepodocyte / podocyte), PODXL (podocyte), SIX2 (progenitor), EYA1 (progenitor). F. Stromal UMAP (top to bottom) color-coded by transcription cluster, predicted cell cycle phase, and organoid conformation. G. Markers of specific stromal clusters; SIX2, LYPD1, FOXC2, HOXA11 (cluster 3, nephron progenitor-like), WNT5A, LHX9 (cluster 7), ZIC1 and ZIC4 (cluster 10). H. Heatmap of scaled log counts per million pseudo-bulk counts from the scRNAseq set of the top 100 most significantly expressed genes identified in the bulk RNAseq analysis (Figure 7). Each column represents a single cluster from a single replicate (e.g., R40, Nephron, Set1). Hierarchical clustering of a limited set of genes indicates that changes in bulk RNAseq are primarily driven by alterations in nephron and endothelial cells. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11]Generation of kidney tissue patches using 3D extrusion cell bioprinting. A. Illustration of needle tip movement for cell paste extrusion to generate patch organoids containing ~4x105 cells in an area of ​​~4.8mm x 6mm. Lines indicate continuous movement. B. Brightfield imaging of bioprinted kidney tissue patch showing uniform formation of nephron structures, including at the edge and center of the patch. C. Live confocal imaging of MAFBmTAGBFP2 reporter signal throughout the patch organoid at culture D7+12. Scale bar represents 1 mm. D. Confocal immunofluorescence of patched organoids at D7+14 showing a homogenous distribution of nephrons expressing markers of glomerular podocytes (mTagBFP2 [left panel; blue]), proximal tubules (LTL [left panel; green] and HNF4A [right panel; red]), nephron epithelium (EPCAM [left panel; red]), distal tubules / loops of Henle's TAL (SLC12A1 [right panel; green]) and endothelial cells (SOX17 [right panel; grey]). Scale bars represent 100 μm. E. Live confocal imaging of D7+14 patched organoids derived from an HNF4AYFP reporter iPSC line after incubation in TRITC-albumin substrate. Images show uptake of TRITC-albumin (red) into YFP-positive proximal tubules (yellow). The outlined area (whole organoid image) in the top panel is shown at higher magnification in the bottom panel with and without phase contrast overlay. Scale bars represent 100 μm. [Figure 12]MAFBmTagBFP2 reporter expression in organoids correlates with total nephron number. A, B) Examples of low-resolution, high-throughput imaging used to quantify MAFB+ area as a surrogate for nephron volume in organoids. Brightfield and MAFBmTagBFP2 signal were captured for each organoid using a low NA 4x objective lens with a spinning disk system, allowing for fast capture of many samples. Due to the large axial depth of field, these images capture the majority of the signal within each organoid in a single plane. Given the similar thickness (E, F, Figure 5), this planar area is roughly proportional to the total volume of MAFB+ glomeruli and therefore correlates with nephron number. Portions of example images used for quantification of R0 (A) and R40 (B) organoids at D7+12 are shown. R40 organoids are much longer and were captured by stitching images of multiple fields of view. Only a small portion of the organoid is shown. C,D) Samples were fixed and stained at D7+12 with the MAFBmTagBFP2 reporter (blue), the mature podocyte marker NPHS1 (red), and atypical protein kinase C (aPKC, green), a marker of the apical plasma membrane. Each nephron is composed of a round glomerular structure containing a podocyte (e.g., highlighted by white arrows) connected to other tubular segments that are labeled with aPKC but lack NPHS1. Nephrons are found throughout the field of view and are packed together to the point that individual nephrons cannot be easily visually identified. The MAFBmTagBFP2 reporter is specifically expressed in podocytes expressing NPHS1, but is absent in other nephron segments (aPKC+, NPHS1- regions) or other cell types. Images are maximum projections (50 μm span). E,F) In both conditions, nephrons have similar axial morphology in the nephron-containing regions when viewed from orthogonal slice (i.e., along the imaging Z-axis) orientation. Shown are single orthogonal slices rendered from the 3D stack. G,H) Cropped high-resolution fields showing a single glomerulus in each condition confirming co-expression of the MAFBmtagBFP2 reporter and NPHS1 in podocytes. Shown are single confocal slices.All images represent at least n=3 stained samples. [Figure 13] Spatial distribution of interstitial markers by whole-mount immunofluorescence. A-C) Immunofluorescence staining of markers of organoid stromal populations based on scRNA profiling. R0 organoids consist of nephron-containing regions, a central core with few nephrons, and a thin edge of monolayered cells that are not typically observed by brightfield imaging. Organoids of line R40 consist mainly of dense nephron-containing regions and a thin monolayered edge, with no central core. Interstitial population markers (A) MEIS1 / 2 / 3, (B) SIX1, and (C) SOX9 are present in the regions surrounding the nephrons and within the thin monolayered sheet at the edge of each organoid, but are largely absent in the central core of R0 organoids. Representative images of n=3 organoids stained per condition are shown. Images are maximum projections over the entire volume of the organoid. D) UMAP plot representing stromal cells in the scRNA dataset, color-coded to indicate expression of MEIS1, MEIS2, SIX1, and SOX9. These combined markers are present in most cells in the dataset, and the lack of staining in the central core observed in (E) may indicate that this region is less cellular overall. [Figure 14-1]A direct comparison of kidney organoids to human fetal kidneys confirms improved maturation of proximal tubules in the R40 bioprinted line. A) UMAP plot comparing transcriptional identity based on unbiased clustering in Seurat (left), and classification of cells according to similarity to the human fetal kidney (HFK) dataset, as predicted using the scPred method (right). IDs are assigned based on the most similar cell type in the human fetal kidney data. B) Percentage of cells assigned to each cell type ID across replicates. Points indicate values ​​for individual replicates colored by replicate barcode (HTO-1 being set 1). Bars indicate SEM. P-values ​​based on one-way ANOVA indicate a significant difference in the number of cells predicted to be Pre-Pod cells, being most abundant in the R40 dataset. There are more cells predicted to be podocytes and fewer distal and pre-tubular cells in the bioprinted conditions (R40 and R0). However, these variations are not significant. These results support the trends observed in the analysis shown in Figure 5. C) Distribution of maximum similarity scores for each cell classification across conformations plotted by predicted cell type. Most cells show high similarity to predicted fetal kidney cell types. D) Genes identified as significantly increased in R40 compared to manual organoids (SLC51B, FABP3, SULT1E1) are expressed in mature proximal tubule cells in human fetal kidney, confirming these genes' association with mature cell types. A gene significantly lower in R40 versus manual organoids (SPP1) is selectively expressed in less mature cell types, further confirming increased maturity in proximal cells at R40. UMAP shows transcriptional identity in human fetal kidney data. Top left plot is color-coded by differentiating human fetal kidney cell types (renal vesicles and comma-shaped bodies [RV_CSB], blue; proximal early nephron [PEN], red) and mature proximal tubule (PT, green). The bottom left plot shows a "dot plot" style of selected genes, with the size indicating the percentage of HFK cells expressing the gene and the color indicating the normalized expression level.The normalized expression of each gene per cell is shown in individual UMAP plots where expression is color coded. [Figure 14-2] This is a continuation of Figure 14-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0023] As used herein, unless the context otherwise requires, the term "comprising" and variations of terms such as "comprising," "comprises," and "comprised" are not intended to exclude additional additives, components, integers or steps.

[0024] It will be understood that the indefinite articles "a" and "an" are not to be construed as singular indefinite articles or as excluding a plural or plural subject to which the indefinite article refers. For example, "a" cell includes one cell, one or more cells, and a plurality of cells.

[0025] As used herein, "bio-ink" refers to a liquid, semi-solid, or solid composition for use in bioprinting. In some embodiments, the bio-ink includes a cell solution, cell aggregates, cell-containing gel, or multicellular bodies. In some embodiments, the bio-ink further includes a support material. In some embodiments, the bio-ink further includes a non-cellular material that provides certain biomechanical properties that enable bioprinting. In some embodiments, the bio-ink includes an extrusion compound. In some embodiments, the bio-ink further includes an additive to increase the viscosity of the bio-ink and reduce settling of cells prior to bioprinting. Examples of suitable additives include hydrogels and hyaluronic acid.

[0026] As used herein, "bio-printing", "bio-printed", "bio-printing", or "bio-printed" refers to utilizing three-dimensional precise deposition of cells (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, etc.) by methodologies compatible with automated or semi-automated, computer-controlled three-dimensional prototyping devices (e.g., bioprinters). In this case, this refers to bioprinting by extrusion or additive manufacturing, rather than robotic liquid manipulation. Any suitable bioprinter capable of extrusion bioprinting for precise deposition of cell-containing bioinks can be utilized for bioprinting of the present invention. The bioprinter can be, for example, an extrusion bioprinter in which cells are extruded, either as cells alone or as cells suspended in a material that may include a hydrogel, biological matrix, or other compound compatible with cell survival. An example of a suitable bioprinter includes the Novogen Bio-printer® from Organovo, Inc. (San Diego, Calif.). As used herein, bioprinted kidney tissue refers to the kidney organoid prepared through the process of bioprinting, and the terms "bioprinted kidney tissue" and "bioprinted kidney organoid" can be used interchangeably.

[0027] The terms "differentiate," "differentiating," and "differentiated" refer to the transition of a cell from an early or earlier stage in a developmental pathway to a later or more mature stage in a developmental pathway. In this context, it will be understood that "differentiated" does not mean or suggest that a cell has fully differentiated and lost pluripotency or the ability to transition further along that or other developmental pathway. Differentiation may involve cell division.

[0028] As used herein, the term "extrusion bioprinting" refers to the use of three-dimensional precise extrusion of cells (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrations, multicellular aggregates, multicellular bodies, etc.) by an automated or semi-automated, computer-assisted three-dimensional prototyping device (e.g., bioprinter). In extrusion bioprinting, the shape of the cell aggregates, cell number, cell density, and final tissue height (thickness) are controlled by introducing a fine tip motion as the cells are extruded. By scripting the extrusion port motion during the extrusion process, the bioink can be spread over a fixed distance and in a specific configuration in a manner that cannot be controlled or at least accurately reproduced manually. By increasing the amount of tip motion at a given cell extrusion rate, the user can create bioprinted tissues with various cell densities, shapes, and thicknesses as the cells spread over a larger surface area and subsequently aggregate.

[0029] As used herein, the terms "induce," "inducing," "induced," and "induction," with respect to a cell or cells, or a bioink (including a printed bioink), refer to promoting the differentiation, development, or maturation of a cell or cells or a bioink (including a printed bioink). For example, induction can include treating a cell or cells or a bioink (including a printed bioink) for a time and under conditions that change the cell or cells or the bioink (including a printed bioink) from a default genotype and / or phenotype to a different or non-default genotype and / or phenotype. In the context of promoting differentiation, development or maturation of a cell or a plurality of cells or bioinks (including printed bioinks) to form bioprinted kidney tissue, this includes expressing in a cell or a plurality of cells one or more markers associated with kidney tissue, or dividing into progeny cells that are, for example, genotypically distinct (having altered gene expression as measured by genetic analysis such as PCR or microarray) and / or phenotypically distinct (having altered protein form, function and / or expression) that express one or more markers associated with kidney tissue that differ from the identity of the original cell. In one example, "induction" encompasses promoting differentiation, development or maturation of one or more nephron progenitor cells into one or more connecting segments, distal convoluted tubule (DCT) cells, distal convoluted tubule (DST) cells, proximal convoluted tubule (PCT) and proximal convoluted tubule (PST) segments 1, 2 and 3, PCT and PST cells, podocytes, glomerular endothelial cells, nephron epithelium such as ascending and / or descending loops of Henle. In one example, "induction" encompasses causing an increase in expression of one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM, and MAFB. The step of inducing may include contacting the bioprinted bioink with a specific growth factor (e.g., FGF-9) for a time sufficient to form kidney tissue.In some instances, the induction step may also include contacting the bioink with a specific growth factor (e.g., CHIR) for a sufficient period of time before the bioink is bioprinted and further cultured.

[0030] As used herein, unless the context requires otherwise, the term "height" refers to the height of the tissue or the height of the micromass. In one example, the term "height" used with respect to "height of bioprinted kidney tissue" refers to the height of the tissue from the surface on which the tissue is deposited, and refers to the height of the final tissue. In another example, the term "height" is used with respect to "height of a layer of bioprinted bioink" and refers to the height of the cell mass or micromass in the layer. In yet another example, the term "height" is used to specify that "the bioink is bioprinted in a layer of about X μm height", and refers to the cell mass or micromass in the layer being X μm high. If the bioink further includes additives, additional compounds or materials (such as support materials, non-cellular materials, extrusion compounds or additives), the height of the layer of the bioprinted bioink is the height of the cell mass or micromass, and not the height of the bioink itself. The height measured is the height of the layer of the settled cell mass or micromass from the surface on which the tissue is deposited.

[0031] A "progenitor cell" is a cell that can differentiate along one or more developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent, and are capable of at least limited self-renewal.

[0032] As is well known in the art, the stage or state of cell differentiation can be characterized by the expression and / or non-expression of one of a number of markers. In this context, "marker" refers to a nucleic acid or protein that is encoded by the genome of a cell, cell population, cell line, compartment or subset, and whose expression or pattern of expression changes throughout development. The expression of nucleic acid markers can be detected or measured by any technique known in the art, including, but not limited to, amplification of nucleic acid sequences (e.g., polymerase chain reaction) and nucleic acid hybridization (e.g., microarray, northern hybridization, in situ hybridization). The expression of protein markers can be detected or measured by any technique known in the art, including, but not limited to, flow cytometry, immunohistochemistry, immunoblotting, protein array, protein profiling (e.g., two-dimensional gel electrophoresis).

[0033] As used herein, a "nephron progenitor cell" is a progenitor cell derived from metanephric mesenchyme that can differentiate through a transition from primary mesenchyme to epithelium into all nephron segments (except collecting duct), including, but not limited to, connecting segment, distal convoluted tubule (DCT) cells, distal convoluted tubule (DST) cells, proximal tubule and proximal tubule segments 1, 2, 3 (PCT / PST), PCT and PST cells, podocytes, nephron epithelium such as glomerular endothelial cells, ascending and / or descending loops of Henle. Nephron progenitor cells are also capable of self-renewal.

[0034] Non-limiting examples of characteristic or representative markers of metanephric mesenchyme (MM) include, but are not limited to, WT1, SALL1, GDNF and / or HOXD11. Non-limiting examples of characteristic or representative markers of nephron progenitor cells include, but are not limited to, WT1, SIX1, SIX2, CITED1, PAX2, GDNF, SALL1, OSR1 and HOXD11.

[0035] By "ureteral epithelial progenitor cells" is meant epithelial progenitor cells that originate from, are obtained from, or originate from the mesonephric duct or its derived ureteric bud and are capable of developing into kidney tissue and / or structures such as collecting ducts.

[0036] Non-limiting examples of characteristic or representative markers of ureteral epithelial progenitor cells include, but are not limited to, WNT9B, RET, GATA3, CALB1, E-cadherin, and PAX2.

[0037] As described above, nephron progenitor cells and ureteral epithelial progenitor cells are differentiated from intermediate mesoderm (IM) cells in the presence of FGF9 alone or in combination with one or more agents such as retinoic acid (RA) agonists or analogs BMP7, AGN193109 and / or FGF20, preferably a RA antagonist such as heparin.

[0038] "Intermediate mesoderm (IM)" refers to embryonic mesoderm cells that arise from the definitive mesoderm, which originates from the posterior primitive streak and ultimately develops into the urogenital system, including the ureters and other tissues such as the kidneys and gonads. Non-limiting examples of characteristic or representative markers of intermediate mesoderm include PAX2, OSR1 and / or LHX1.

[0039] It will also be understood that the production of IM cells does not mean that the IM cells are a pure or homogenous population of IM cells, absent other cell types (such as definitive mesoderm). Thus, reference to "IM cells" or "population of IM cells" means that the cells or cell population comprises IM cells. Suitably, according to the present invention, IM cells are produced by contacting posterior primitive streak cells with one or more agents that promote differentiation into IM cells, as described in more detail below.

[0040] Preferably, the IM cells are produced by contacting posterior primitive streak cells with one or more agents that promote differentiation of posterior primitive streak cells into IM cells.

[0041] "Primitive streak (PPS)" refers to cells derived from the posterior end of the primitive streak structure formed in the blastula at the early stage of mammalian embryogenesis, or functionally and / or phenotypically corresponding cells. The posterior primitive streak establishes left-right symmetry, determines the site of gastrulation, and initiates germ layer formation. Usually, the posterior primitive streak is a precursor of the mesoderm (i.e., presumptive mesoderm), and the anterior primitive streak is a precursor of the endoderm (i.e., presumptive endoderm). A non-limiting example of a characteristic or representative marker of the posterior primitive streak includes Brachyury (T). A non-limiting example of a characteristic or representative marker of the anterior primitive streak is SOX17. MIXL1 can be expressed in both the posterior and anterior primitive streak.

[0042] It will also be understood that the production of posterior primitive streak cells does not mean that the posterior primitive streak cells are a pure or homogenous population of posterior primitive streak cells, absent other cell types. Thus, reference to "posterior primitive streak cells" or a "population of posterior primitive streak cells" means that the cells or cell population comprise posterior primitive streak cells.

[0043] The terms "human pluripotent stem cells" and "hPSCs" refer to cells that are derived from, obtainable from, or originate from human tissue and exhibit pluripotency. hPSCs can be human embryonic stem cells or human induced pluripotent stem cells.

[0044] Human pluripotent stem cells can be derived from the inner cell mass or can be reprogrammed from a number of fetal or adult somatic cell types using Yamanaka factors. Generation of hPSCs can also be achieved using nuclear transfer of somatic cells.

[0045] The terms "human embryonic stem cell," "hES cell," and "hESC" refer to cells derived from, obtained from, or originating from a human embryo or blastocyst that are self-renewing, pluripotent or totipotent, and capable of producing all of the cell types present in an adult animal. Human embryonic stem cells (hESC) can be isolated, for example, from human in vivo preimplantation embryos, in vitro fertilized embryos, or human blastocysts obtained from one-cell human embryos expanded to the blastocyst stage.

[0046] The terms "induced pluripotent stem cells" and "iPSCs" refer to any type of cell that can be derived, obtained, or derived from a human adult cell that has been reprogrammed into a pluripotent state through expression of exogenous genes, such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4, and c-MYC. hiPSCs exhibit a level of pluripotency comparable to hESCs, but can be derived from autologous patients with or without simultaneous genetic correction prior to differentiation and cell delivery.

[0047] More broadly, the methods disclosed herein can be applied to any pluripotent stem cells derived from any patient, or hPSCs that are subsequently modified using gene editing to generate mutant models, or mutant hPSCs that are corrected using gene editing. Gene editing can be performed via CRISPR, TALEN, or ZF nuclease technology.

[0048] As used herein, "tissue" refers to a collection of cells. In some embodiments, the cells within a tissue are aggregated or fused.

[0049] As used herein, "scaffold" refers to synthetic scaffolds such as polymer scaffolds and porous hydrogels, non-synthetic scaffolds such as preformed extracellular matrix layers, dead cell layers, and decellularized tissues, as well as any other type of preformed scaffold that is integral to the physical structure of the artificial tissue and cannot be removed from the tissue without damaging / destroying the tissue. In further embodiments, decellularized tissue scaffolds include decellularized natural tissues or decellularized cell materials produced by cultured cells in any manner, such as a cell layer in which cells are killed or decellularized, leaving behind the extracellular matrix (ECM) produced during the life period.

[0050] As used herein, an "individual" is a living organism of any mammalian species, including but not limited to humans, primates, apes, monkeys, dogs, cats, mice, rats, rabbits, pigs, horses, etc. The subject can be any mammalian species, living or dead.

[0051] As used herein, "about" or "approximately" means ±10% of the stated value. For example, about 10 includes 9 to 11.

[0052] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as explicitly supporting both meanings or either meaning.

[0053] Bioprinted kidney tissue Disclosed herein is a bioprinted kidney tissue comprising a bioink, the bioink comprising a plurality of cells, the bioink being bioprinted in a layer of about 150 μm or less in height, and the bioprinted bioink being induced to form kidney tissue. In one embodiment, the bioink is bioprinted in a layer selected from about 15 μm to about 150 μm. In one embodiment, the bioink is bioprinted in a layer selected from about 25 μm in height to about 100 μm in height. In a preferred embodiment, the bioink is bioprinted in a layer of about 50 μm or less in height. In one embodiment, the bioink is bioprinted in a layer of about 15 μm in height. In one embodiment, the bioink is bioprinted in a layer of about 20 μm in height. In one embodiment, the bioink is bioprinted in a layer of about 25 μm in height. In one embodiment, the bioink is bioprinted in a layer of about 30 μm in height. In one embodiment, the bioink is bioprinted in a layer of about 35 μm in height. In one embodiment, the bioink is bioprinted in a layer about 40 μm tall. In one embodiment, the bioink is bioprinted in a layer about 50 μm tall. In one embodiment, the bioink is bioprinted in a layer about 60 μm tall. In one embodiment, the bioink is bioprinted in a layer about 70 μm tall. In one embodiment, the bioink is bioprinted in a layer about 80 μm tall. In one embodiment, the bioink is bioprinted in a layer about 90 μm tall. In one embodiment, the bioink is bioprinted in a layer about 100 μm tall.

[0054] In one embodiment, the height of the bioprinted kidney tissue is about 150 μm or less. In other words, the height of the final bioprinted kidney tissue after the bioprinted bioink is induced to form the kidney tissue is about 150 μm or less. In another embodiment, the height of the bioprinted kidney tissue is about 50 μm to about 150 μm. In another embodiment, the height of the bioprinted kidney tissue is about 100 μm to about 150 μm.

[0055] In one embodiment, the bioprinted layer of bioink is 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises about 5,000 to about 100,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 10,000 to about 50,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 5,000 to about 20,000 cells per mm. 2 In one embodiment, the bioprinted layer of the bioink comprises about 10,000 to about 15,000 cells per mm. 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 10,000 cells per mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 15,000 cells per mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2In one embodiment, the bioprinted layer of the bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 70,000 cells per mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 80,000 cells per mm. 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 90,000 cells per mm 2 Each contains approximately 100,000 cells.

[0056] According to a preferred embodiment, the bioprinted kidney tissue is at about 10,000 cells to about 20,000 cells / mm 2 In a further preferred embodiment, the bioprinted kidney tissue comprises a bioprinted layer of the bioink having a height of about 50 μm or less upon printing. 2 In a further preferred embodiment, the bioprinted kidney tissue comprises a bioprinted layer of bioink containing about 20,000 cells / mm and having a height of about 40 μm or less when printed. In a further preferred embodiment, the bioprinted kidney tissue comprises about 14,000 cells / mm 2 and having a height of about 30 μm or less upon printing. In a further preferred embodiment, the bioprinted kidney tissue has a height of about 11,000 cells / mm 2 In a further preferred embodiment, the bioprinted layer of the bioink comprises a thickness of about 1 mm 2 The bioprinted layer of bioink comprises approximately 10,000 cells per well and has a height of approximately 20 μm or less when printed.

[0057] In one embodiment, the bioink comprises from about 10,000 cells / μl to about 400,000 cells / μl. In one embodiment, the bioink comprises from about 10,000 cells / μl to about 100,000 cells / μl. In one embodiment, the bioink comprises from about 100,000 cells / μl to about 400,000 cells / μl. In one embodiment, the bioink comprises from about 50,000 cells / μl to about 200,000 cells / μl. In one embodiment, the bioink comprises about 10,000 cells / μl, about 30,000 cells / μl, about 40,000 cells / μl, about 50,000 cells / μl, about 60,000 cells / μl, about 70,000 cells / μl, about 80,000 cells / μl, about 90,000 cells / μl, about 100,000 cells / μl, about 150,000 cells / μl, about 200,000 cells / μl, about 250,000 cells / μl, about 300,000 cells / μl, or about 400,000 cells / μl. In a preferred embodiment, the bioink comprises about 200,000 cells / μl.

[0058] In some embodiments, the bio-ink comprises partially differentiated cells. In some embodiments, the bio-ink comprises fully differentiated cells.

[0059] In some embodiments, the bioink comprises cells differentiated from human stem cells (HSCs), such as, but not limited to, human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). In some embodiments, the bioink comprises primitive streak cells, such as, but not limited to, posterior primitive streak cells. In some embodiments, the bioink comprises intermediate mesoderm (IM) cells. In some embodiments, the bioink comprises metanephric mesenchyme (MM) cells. In some embodiments, the bioink comprises renal duct cells. In some embodiments, the bioink comprises renal progenitor cells, such as, but not limited to, nephron progenitor cells, ureteral epithelial progenitor cells, or a combination thereof.

[0060] In some embodiments, the cells of the bioink comprise cells derived from a patient. In some embodiments, the cells of the bioink comprise gene-edited cells. In some embodiments, the cells of the bioink comprise cells derived from a patient that are also gene-edited cells. In some embodiments, the cells of the bioink comprise cells from a reporter strain. In some embodiments, the cells of the bioink comprise gene-edited reporter strain cells.

[0061] In some embodiments, the cells of the bio-ink comprise normal healthy cells. In some embodiments, the cells of the bio-ink comprise cells of a kidney disease patient. In some embodiments, the cells of the bio-ink comprise a combination of patient cells and healthy cells.

[0062] In one example, the bioprinted kidney tissue is derived from a culture-expanded population of renal progenitor cells, such as nephron progenitor cells.

[0063] In another example, bioprinted kidney tissue is derived from a culture-expanded population of MM or IM cells, characterized by the method used for culture expansion and / or production.

[0064] In one example, renal progenitor cells are produced by contacting posterior primitive streak cells with one or more agents that promote differentiation of the posterior primitive streak cells into renal progenitor cells, such as IM cells or MM cells. In one example, a method of producing renal progenitor cells includes culturing a population of stem cells in cell culture medium containing a Wnt / β-catenin agonist for about 2-5 days, followed by culturing the cells in cell culture medium containing an FGF, such as FGF9, for about 2-5 days. In one example, a method of producing renal progenitor cells includes culturing a population of stem cells in cell culture medium containing a Wnt / β-catenin agonist for about 2-5 days, followed by culturing the cells in cell culture medium containing an FGF, such as FGF9, for about 3-4 days. In this example, the cells may be cultured for 7 days or more, after which the renal progenitor cells may be dissociated. In this example, the renal progenitor cells are printed around day 10-13. In another example, a method of producing renal progenitor cells includes culturing a population of stem cells in cell culture medium containing a Wnt / β-catenin agonist for about 2-5 days, followed by culturing the cells in cell culture medium containing an FGF, such as FGF9, for about 3-4 days. In another example, renal progenitor cells can be cultured in nephron progenitor cell maintenance medium for about 10-14 days before the renal progenitor cells are printed.

[0065] In another example, bioprinted kidney tissue is derived from a culture-expanded population of bioprinted IM cells according to the methods described herein, characterized by the methods used for culture expansion and / or production.

[0066] Thus, in one example, a method for producing IM cells includes culturing a population of stem cells in cell culture medium containing a Wnt / β-catenin agonist for about 3-5 days, followed by culturing the cells in cell culture medium containing an FGF, such as FGF9, for about 2-5 days. In one example, a method for producing IM cells includes culturing a population of stem cells in cell culture medium containing a Wnt / β-catenin agonist for about 3-5 days, followed by culturing the cells in medium containing an FGF, such as FGF9, for about 3-5 days. In these examples, the cells can be cultured for a total of 7 days, after which the IM cells are dissociated. The term "Wnt / β-catenin agonist" in the context of the present disclosure is used to refer to a molecule that inhibits GSK3 (e.g., GSK3-β) in the context of the canonical Wnt signaling pathway, but is not preferred in other contexts of non-canonical Wnt signaling pathways. Examples of Wnt β-catenin agonists include recombinant WNT3A, CHIR99021 (CHIR), LiCl SB-216763, CAS 853220-52-7, and other commercially available Wnt / β-catenin agonists from vendors such as Santa Cruz Biotechnology and R&D Systems.

[0067] In one example, IM cells are produced by culturing stem cells for 7 days, where days 3-5 include culturing the stem cells in cell culture medium containing a high concentration of CHIR as described above, and the remaining days include culturing the cells in cell culture medium containing FGF at a concentration as described above. For example, IM cells can be produced by culturing stem cells for 7 days, where days 3-5 include culturing the stem cells in cell culture medium containing at least 3 μM CHIR, and the remaining days include culturing the cells in cell culture medium containing at least 100 ng / ml FGF9.

[0068] In another example, IM cells can be produced by culturing stem cells for up to 13 days, after which the IM cells are dissociated. In another example, IM cells can be produced by culturing stem cells for 8 days. In another example, IM cells can be produced by culturing stem cells for 9 days. In another example, IM cells can be produced by culturing stem cells for 10 days. In another example, IM cells can be produced by culturing stem cells for 11 days. In another example, IM cells can be produced by culturing stem cells for 12 days. In another example, IM cells can be produced by culturing stem cells for 13 days. In another example, IM cells can be produced by culturing stem cells for 14 days. In another example, IM cells can be produced by culturing stem cells for 15 days. In another example, IM cells can be produced by culturing stem cells for 10 days or more. In each of these examples, days 3-5 can include culturing the stem cells in cell culture medium containing at least 3 μM CHIR, and the remaining days can include culturing the cells in cell culture medium containing FGF9. For example, days 3-5 may involve culturing the stem cells in cell culture medium containing 3 µM-8 µM CHIR, while the remaining days may involve culturing the cells in cell culture medium containing FGF9.

[0069] In one example, the cells are cultured in cell culture medium containing 3-8 μM of a Wnt / β-catenin agonist before being cultured in cell culture medium containing FGF. In another example, the cells are cultured in cell culture medium containing 4 μM of a Wnt / β-catenin agonist before being cultured in cell culture medium containing FGF. In another example, the cells are cultured in cell culture medium containing 5 μM of a Wnt / β-catenin agonist before being cultured in cell culture medium containing FGF. In another example, the cells are cultured in cell culture medium containing 6 μM of a Wnt / β-catenin agonist before being cultured in cell culture medium containing FGF. In another example, the cells are cultured in cell culture medium containing 7 μM of a Wnt / β-catenin agonist before being cultured in cell culture medium containing FGF. In another example, the cells are cultured in cell culture medium containing 8 μM of a Wnt / β-catenin agonist before being cultured in cell culture medium containing FGF. In these examples, the Wnt / β-catenin agonist can be CHIR. For example, the cells can be cultured in cell culture medium containing 3-8 μM CHIR before being cultured in cell culture medium containing FGF.

[0070] In one example, the IM cell culture medium comprises at least 50 ng / ml of FGF9. In another example, the cell culture medium comprises at least 100 ng / ml of FGF9. In another example, the cell culture medium comprises at least 150 ng / ml of FGF9. In another example, the cell culture medium comprises at least 200 ng / ml of FGF9. In another example, the cell culture medium comprises at least 300 ng / ml of FGF9. In another example, the cell culture medium comprises at least 350 ng / ml of FGF9. In another example, the cell culture medium comprises at least 400 ng / ml of FGF9. In another example, the cell culture medium comprises at least 500 ng / ml of FGF9. In another example, the cell culture medium comprises 50 ng to 400 ng / ml of FGF9. In another example, the cell culture medium comprises 50 ng to 300 ng / ml of FGF9. In another example, the cell culture medium comprises 50 ng to 250 ng / ml of FGF9. In another example, the cell culture medium contains 100 ng to 200 ng / ml of FGF9.

[0071] In another example, the above levels of FGF9 are substituted with FGF2. For example, the IM cell culture medium can contain 50ng-400ng / ml of FGF2. In another example, the cell culture medium can contain 50ng-300ng / ml of FGF2. In another example, the cell culture medium can contain 50ng-250ng / ml of FGF2. In another example, the cell culture medium can contain 100ng / ml-200ng / ml of FGF2.

[0072] In another example, the above levels of FGF9 are substituted with FGF20. For example, the IM cell culture medium can contain 50ng-400ng / ml of FGF20. In another example, the cell culture medium can contain 50ng-300ng / ml of FGF20. In another example, the cell culture medium can contain 50ng-250ng / ml of FGF20. In another example, the cell culture medium can contain 100ng / ml-200ng / ml of FGF20.

[0073] In one example, the IM cell culture medium containing FGF also contains heparin. In one example, the cell culture medium contains 0.5 μg / ml heparin. In another example, the cell culture medium contains 1 μg / ml heparin. In another example, the cell culture medium contains 1.5 μg / ml heparin. In another example, the cell culture medium contains 2 μg / ml heparin. In another example, the cell culture medium contains 0.5 μg / ml to 2 μg / ml heparin. In another example, the cell culture medium contains 0.5 μg / ml to 1.5 μg / ml heparin. In another example, the cell culture medium contains 0.8 μg / ml to 1.2 μg / ml heparin.

[0074] In one example, the bioink is induced to form kidney tissue by contacting the bioink with FGF-9. In another example, the bioink is induced to form kidney tissue by contacting the bioink with FGF-9 for 5 days. In some examples, a plurality of cells may be contacted with cell culture medium containing CHIR for a short period of time, then bioprinted, and further cultured. For example, a plurality of cells may be contacted with cell culture medium containing 3-8 μM CHIR for 1-2 hours, then bioprinted, and further cultured. In another example, a plurality of cells may be contacted with cell culture medium containing 5 μM CHIR for 1 hour, then bioprinted, and further cultured.

[0075] In other examples, the IM or MM cells used to generate the bioprinted kidney tissue can be cultured in media containing different or additional components. Exemplary components and timing for their use in cell culture are discussed below.

[0076] In one example, the cell culture medium can include a Rho kinase inhibitor (ROCKi), such as Y-27632 (StemCell Technologies). In this example, the stem cells are cultured for 24 hours in cell culture medium containing ROCKi, and then cultured for about 3-4 days in cell culture medium containing at least 4 μM CHIR. In this example, the cells can then be cultured for an additional 3-4 days in cell culture medium containing FGF. In one example, the cell culture medium can include 8 μM ROCKi. In another example, the cell culture medium can include 10 μM ROCKi. In another example, the cell culture medium can include 12 μM ROCKi. In another example, the cell culture medium can include 8 μM to 12 μM ROCKi.

[0077] In the above example, after culturing for 24 hours in ROCKi and for about 3-4 days in at least 4 μM CHIR, the cells can be cultured in medium containing FGF9, as well as one or more or all of the Wnt / β-catenin agonists, such as CHIR at a low concentration (e.g., less than 3 μM), heparin at the above concentrations, poly(vinyl alcohol) (PVA) and methylcellulose (MC). In this example, the IM cell culture medium can contain at least 0.05% PVA. In another example, the cell culture medium can contain 0.1% PVA. In another example, the cell culture medium can contain 0.15% PVA. In another example, the cell culture medium can contain 0.1%-0.15% PVA. In one example, the cell culture medium can contain at least 0.05% MC. In another example, the cell culture medium can contain 0.1% MC. In another example, the cell culture medium can contain 0.15% MC. In another example, the cell culture medium can contain 0.1%-0.15% MC.

[0078] In one example, bioprinted kidney tissue is obtained by producing IM cells using the methods described above, isolating the IM cells, preparing a bioink, bioprinting the bioink, and then further culturing the bioink, i.e., the bioprinted cells, in the method of generating bioprinted kidney tissue discussed below. For example, IM cells can be produced using the methods exemplified above, dissociated, and then bioprinted to form kidney tissue. In an example, bioprinting can be performed in culture on a supported filter. For example, IM cells can be produced using the methods exemplified above, dissociated, and then bioprinted to form kidney tissue for a subsequent period (e.g., 12 days) after bioprinting, in a transwell. TM Can be cultured on filters.

[0079] In one example, the plurality of cells can be dissociated using EDTA after being cultured under conditions and for a period of time sufficient to produce target renal cell progenitor cells. In one example, the IM cells can be dissociated using EDTA. In another example, the cells can be dissociated using trypsin or TrypLE or Accutase or collagenase. In one example, the cells are cultured for at least 12 days after bioprinting. In another example, the cells are cultured for at least 13 days after bioprinting. In another example, the cells are cultured for at least 14 days after bioprinting. In another example, the cells are cultured for at least 15 days after bioprinting. In another example, the cells are cultured for at least 20 days after bioprinting. In another example, the cells are cultured for at least 25 days after bioprinting. In another example, the cells are cultured for at least 35 days after bioprinting.

[0080] In one example, the plurality of cells are dissociated after a culture period sufficient to produce target renal cell progenitor cells. In this example, the dissociated cells are bioprinted to produce bioprinted kidney tissue. In one example, the IM cells are dissociated after 7 days in culture (d7) and then bioprinted to produce bioprinted kidney tissue. In one example, the cells are cultured in a cell culture medium that includes FGF. For example, the cells are cultured in a cell culture medium that includes FGF9, FGF2 or FGF20 at the concentrations referenced above after dissociation and / or bioprinting. In one example, the cells are cultured in a cell culture medium that includes 100 ng / ml FGF9 after dissociation and / or bioprinting. In another example, the cells are cultured in a cell culture medium that includes 200 ng / ml FGF9 after dissociation and / or bioprinting. In these examples, the cell culture medium can also include heparin. For example, the cell culture medium can include FGF9 and 1 μg / ml heparin after dissociation and / or bioprinting. In these examples, the cells can be cultured for 4-6 days in cell culture medium containing FGF and heparin after dissociation and / or bioprinting. In one example, the cells can be cultured for 5 days in cell culture medium containing FGF and heparin after dissociation and / or bioprinting.

[0081] In one example, FGF is removed from the cell culture medium 4-6 days after dissociation and / or bioprinting. In another example, FGF is removed from the cell culture medium 5 days after dissociation and / or bioprinting. In one example, no growth factors are provided in the culture medium 5 days after dissociation and / or bioprinting.

[0082] In one example, the cell culture medium used after dissociation and / or bioprinting can also include retinoic acid. In one example, all-trans retinoic acid (atRA) is added to the cell culture medium after dissociation and / or bioprinting. In one example, at least 0.07 μM retinoic acid is added to the cell culture medium. In one example, at least 0.1 μM retinoic acid is added to the cell culture medium. In one example, at least 0.2 μM retinoic acid is added to the cell culture medium. In one example, at least 0.5 μM retinoic acid is added to the cell culture medium.

[0083] In another example, at least 1.5 μM retinoic acid is added to the cell culture medium. In one example, at least 1.8 μM retinoic acid is added to the cell culture medium. In one example, at least 2.0 μM retinoic acid is added to the cell culture medium. In another example, at least 2.5 μM retinoic acid is added to the cell culture medium. In another example, 1.5 μM to 10 μM retinoic acid is added to the cell culture medium. In another example, 1.5 μM to 5 μM retinoic acid is added to the cell culture medium. In another example, 2.0 μM to 8 μM retinoic acid is added to the cell culture medium. In another example, 2.0 μM to 3 μM retinoic acid is added to the cell culture medium.

[0084] In one example, retinoic acid is added to the cell culture medium 4 days after dissociation and / or bioprinting. In another example, retinoic acid is added to the cell culture medium 5 days after dissociation and / or bioprinting. In another example, retinoic acid is added to the cell culture medium 4-6 days after dissociation and / or bioprinting.

[0085] The bioprinted kidney tissue produced according to the methods included in the present disclosure and disclosed herein can be described based on the number of days in culture. The number of days in culture can be divided into two components, including the number of days (X) for the production of IM cells from stem cells and the number of days (Y) for the formation of (bioprinted) IM cells into kidney tissue. In one example, the step that distinguishes the production of IM cells from stem cells from the production of bioprinted kidney tissue from IM cells is the dissociation of IM cells. One way to express the number of days in culture for the production of IM cells from stem cells and the number of days for the formation of bioprinted kidney tissue from IM cells is days (d)X+Y (e.g., d7+12 represents 7 days of production of IM cells from stem cells, followed by dissociation and bioprinting of IM cells, and 12 days of "induction" of kidney tissue formation from IM cells (i.e., Y=days in culture as bioprinted kidney tissue)).

[0086] In one example, the number of days of culture can be 7 days for the production of IM cells from stem cells and 4 to 30 days or more (d7+4 to d7+30, printing date is d7+0) for the formation of kidney tissue from (bioprinted) IM cells. In one example, the bioprinted kidney tissue is kidney tissue from d7+8 to d7+20. In one example, the bioprinted kidney tissue is kidney tissue from d7+10 to d7+15. In one example, the bioprinted kidney tissue is kidney tissue from d7+12. In another example, the bioprinted kidney tissue is kidney tissue from d7+14. In another example, the bioprinted kidney tissue is kidney tissue from d7+15. In another example, the bioprinted kidney tissue is kidney tissue from d7+16. In another example, the bioprinted kidney tissue is kidney tissue from d7+17. In another example, the bioprinted kidney tissue is kidney tissue from d7+18. In another example, the bioprinted kidney tissue is d7+19 kidney tissue. In another example, the bioprinted kidney tissue is d7+20 kidney tissue. In another example, the bioprinted kidney tissue is d7+21 kidney tissue. In another example, the bioprinted kidney tissue is d7+22 kidney tissue. In another example, the bioprinted kidney tissue is d7+23 kidney tissue. In another example, the bioprinted kidney tissue is d7+24 kidney tissue. In another example, the bioprinted kidney tissue is d7+25 kidney tissue.

[0087] In another example, the bioprinted kidney tissue is d7+30 kidney tissue. In another example, the bioprinted kidney tissue is d7+12 to d7+30. In the above referenced examples, the stem cells can be cultured for about 8, 9, 10, 11, 12, 13, or 14 days up to about 28 days (i.e., d8+Y, d9+Y, d10+Y, d11+Y, d12+Y, d13+Y, or d14+Y to about d28+Y).

[0088] According to another embodiment, the bioprinted kidney tissue comprises about 2 to about 100 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 2 to about 50 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 2 to about 45 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 5 to about 30 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 5 to about 20 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 5 to about 10 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue is characterized in terms of % nephrons, % interstitium, and / or % vasculature. "Nephrons" are functional working units of the kidney that play a major role in removing waste products and maintaining fluid volume. They can be identified and counted by those skilled in the art in the bioprinted kidney tissue disclosed herein using various methods. For example, nephrons can be visualized and counted using confocal microscopy and immunofluorescence labeling (e.g., WT1+ glomeruli, MAFB+NPHS1+ podocytes, HNF4A+LTL+ECAD- proximal tubules, SLC12A1+ECAD+ distal tubules, and ECAD+GATA3+ collecting ducts). In this embodiment, the bioprinted kidney tissue can be additionally or alternatively characterized using single-cell RNA sequencing, PCR-based gene expression analysis, or immunohistochemical methods.

[0089] In one embodiment, the bioprinted kidney tissue has a density of 0.2 mm per 10,000 cells printed. 2 In one embodiment, the bioprinted kidney tissue contains a surface area of ​​nephron tissue of 0.2 mm per 10,000 cells printed. 2 ~1.5mm 2In one embodiment, the bioprinted kidney tissue comprises a surface area of ​​0.25 mm per 10,000 cells printed. 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , or 1.5 mm 2 In one embodiment, the bioprinted kidney tissue comprises a surface area of ​​0.2 mm per 10,000 cells printed. 2 In one embodiment, the bioprinted kidney tissue contains a surface area of ​​0.2 mm per 10,000 cells printed. 2 ~1.5mm 2 In one embodiment, the bioprinted kidney tissue contains 0.25 mm of surface area of ​​cells expressing MAFB per 10,000 cells printed. 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , or 1.5 mm 2 of the surface area of ​​cells expressing MAFB.

[0090] In one embodiment, the bioprinted kidney tissue has a uniform distribution of nephrons throughout the bioprinted layer, in contrast to manually aggregated or bioprinted kidney organoids of suboptimal conformation, which are generated as dots or blobs of cells, forming dome-shaped structures over 150 uM high from the transwell, and with unpatterned central regions or cores that lack nephrons, as described in the prior art. This embodiment describes a bioprinted kidney tissue that includes a large number of uniformly distributed nephrons without a core of non-nephron tissue. For example, the bioprinted kidney has a uniform distribution of glomeruli throughout the bioprinted layer, e.g., as labeled by cells expressing MAFB. In another embodiment, the bioprinted kidney tissue expresses one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM, and MAFB throughout the structure. In another embodiment, the bioprinted kidney tissue exhibits increased or elevated expression levels of one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM and MAFB compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526:564-568), i.e., bioprinted kidney organoids generated manually as aggregates or as dots or blobs of cells. In another embodiment, the bioprinted kidney tissue exhibits increased or higher levels of expression of one or more of SLC30A1, SLC51B, FABP3, and SULT1E1 (genes associated with proximal tubule maturation), and / or decreased expression of either or both of SPP1, JAG1 (genes associated with early immature tubules), compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526:564-568), i.e., bioprinted kidney organoids generated manually as aggregates or as dots or blobs of cells.In one embodiment, the bioprinted kidney tissue exhibits low or no expression, or reduced expression of one or more of THY1, DCN, SOX17, FLT1 and PECAM, compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526:564-568), i.e., bioprinted kidney organoids generated manually as aggregates or as dots or blobs of cells. That is, in the above examples, the high and low levels of expression are compared to kidney organoids cultured by the methods described in Takasato et al. (2015) Nature, Vol. 526: 564-568, Takasato et al. (2016) Nat Protocols, 11: 1681-1692, or Takasato et al. (2014) Nat. Cell Biol., 16: 118-127. In this example, high expression is at least 1.5 times higher. In another example, high expression is at least 2 times higher. In another example, high expression is at least 3 times higher. In one example, low expression is at least 1.5 times lower. In another example, low expression is at least 2 times lower. In another example, low expression is at least 3 times lower.

[0091] Expression levels can be measured using techniques such as the polymerase chain reaction with appropriate primers for the marker of interest. For example, total RNA can be extracted from cells, reverse transcribed, and subjected to PCR and analysis.

[0092] The inventors have also surprisingly discovered that the "non-nephron" tissue of the bioprinted kidney tissue shows increased expression or genes associated with nephron precursor identity compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526: 564-568), i.e., bioprinted kidney organoids produced by hand aggregates or as dots or blobs of cells. In another embodiment, the bioprinted kidney tissue shows increased or elevated expression levels of one or more of HOXA11, FOXC2, EYA1, and SIX2 compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526: 564-568), i.e., bioprinted kidney organoids produced by hand aggregates or as dots or blobs of cells.

[0093] In another embodiment, the bioprinted kidney tissue further comprises a biocompatible scaffold. For example, in another embodiment, the bioink is bioprinted onto a biocompatible scaffold. That is, the surface onto which the bioink is printed is a biocompatible scaffold. In one embodiment, the biocompatible scaffold is biodegradable or bioabsorbable. In another embodiment, the biocompatible scaffold is a hydrogel. In another embodiment, the scaffold can be functionalized with one or more agents (e.g., biologically active agents). For example, biologically active agents (cytokines, chemokines, differentiation factors, signaling pathway inhibitors, etc.) can promote, for example, further development or differentiation of cells in the bioink printed thereon.

[0094] In another embodiment, the bioink further comprises one or more biologically active agents. In one example, the one or more biologically active agents promote the derivation of kidney tissue from the plurality of cells. In another embodiment, the bioink further comprises a differentiation medium, a bioprinting medium, or any combination thereof. In some embodiments, the bioprinting medium comprises a hydrogel, such as a modified hydrogel or a functionalized hydrogel, or a mixture of matrix components or extracellular matrix components. In another embodiment, the bioink comprises hyaluronic acid. In one embodiment, the one or more active agents are selected from anti-proliferative agents, immunosuppressants, pro-angiogenic compounds, antibodies or fragments or portions thereof, antibiotics or antibacterial compounds, antigens or epitopes, aptamers, biopolymers, carbohydrates, cell attachment mediators (such as RGD), cytokines, cytotoxic agents, drugs, enzymes, growth factors or recombinant growth factors and fragments and variants thereof, hormone antagonists, hormones, immunological agents, lipids, metals, nanoparticles, nucleic acid analogs, nucleic acids (e.g., DNA, RNA, siRNA, RNAi, and microRNA agents), nucleotides, nutraceutical agents, oligonucleotides, peptide nucleic acids (PNAs), peptides, prodrugs, prophylactic agents, proteins, small molecules, therapeutic agents, or any combination thereof.

[0095] In other embodiments, the bioprinted kidney tissue further comprises a bioink as described herein above disposed adjacent or proximate to another bioprinted bioink, which may optionally include one or more drugs or one or more other cell types as described above. For example, a bioink including a plurality of cells (and optionally one or more drugs) may be bioprinted adjacent or proximate to another bioprinted bioink. For example, a bioink including a plurality of cells (and optionally one or more drugs) may be bioprinted on or next to (including directly on or next to) a line or layer of a bioprinted bioink (optionally including one or more drugs and / or one or more other cell types). For example, a method for producing a bioprinted kidney tissue includes bioprinting a quantity of a first bioink and printing a quantity of a second bioink on a surface, where the first bioink and the second bioink are different. In one example, the first bioink includes a plurality of cells that is different from the plurality of cells in the second bioink. In another example, a first bio-ink may include a plurality of cells and a second bio-ink may not include cells but may include other components, such as, for example, a biologically active agent.

[0096] Method for producing bioprinted kidney tissue In another aspect, the present invention relates to a method for producing a bioprinted kidney tissue. In one embodiment, a method for producing a bioprinted kidney tissue includes bioprinting a volume of bioink onto a surface, the bioink comprising a plurality of cells, the bioink being bioprinted in a layer less than about 150 μm in height, and inducing the bioprinted volume of bioink to form a bioprinted kidney tissue. Preferably, the bioink is bioprinted in a layer less than about 50 μm in height.

[0097] In one embodiment, in the bioprinting step, the bioink comprising the plurality of cells is bioprinted in a layer less than about 150 μm in height. In one embodiment, the bioink is bioprinted in a layer selected from about 15 μm to about 150 μm in height. In one embodiment, the bioink is bioprinted in a layer selected from about 25 μm in height to about 100 μm in height. In a preferred embodiment, the bioink is bioprinted in a layer less than about 50 μm in height. In one embodiment, the bioink is bioprinted in a layer about 15 μm in height. In one embodiment, the bioink is bioprinted in a layer about 20 μm in height. In one embodiment, the bioink is bioprinted in a layer about 25 μm in height. In one embodiment, the bioink is bioprinted in a layer about 30 μm in height. In one embodiment, the bioink is bioprinted in a layer about 35 μm in height. In one embodiment, the bioink is bioprinted in a layer about 40 μm in height. In one embodiment, the bioink is bioprinted in a layer about 50 μm high. In one embodiment, the bioink is bioprinted in a layer about 60 μm high. In one embodiment, the bioink is bioprinted in a layer about 70 μm high. In one embodiment, the bioink is bioprinted in a layer about 80 μm high. In one embodiment, the bioink is bioprinted in a layer about 90 μm high. In one embodiment, the bioink is bioprinted in a layer about 100 μm high.

[0098] In one embodiment, in the bioprinting step, the bioprinted layer of the bioink is greater than or equal to 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises about 5,000 to about 100,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 10,000 to about 50,000 cells per mm. 2In one embodiment, the bioprinted layer of bioink comprises about 5,000 to about 50,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 10,000 to about 40,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 10,000 to about 30,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 10,000 to about 20,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises about 1 mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of the bioink comprises approximately 10,000 cells per mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 15,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 1 mm 2 In one embodiment, the bioprinted layer of bioink comprises approximately 70,000 cells per mm. 2 In one embodiment, the bioprinted layer of bioink comprises approximately 80,000 cells per mm. 2In one embodiment, the bioprinted layer of bioink comprises approximately 90,000 cells per mm. 2 Each contains approximately 100,000 cells.

[0099] According to a preferred embodiment, in the bioprinting step, the bioprinted layer of bioink is 1 mm thick. 2 In a further preferred embodiment, in the bioprinting step, the bioprinted layer of the bioink has a height of about 1 mm to about 20,000 cells per mm. 2 In a further preferred embodiment, the bioprinted layer of bioink comprises about 20,000 cells per mm and has a height of about 40 μm or less. 2 In a further preferred embodiment, in the bioprinting step, the bioprinted layer of bioink has a height of about 1 mm or less. 2 In a further preferred embodiment, in the bioprinting step, the bioprinted layer of bioink has a height of about 1 mm or less. 2 They contain approximately 10,000 cells per well and have a height of approximately 20 μm or less.

[0100] In a preferred embodiment, the bioink is a wet cell paste. In another embodiment, in the bioprinting step, the bioink comprises about 10,000 cells / μl to about 400,000 cells / μl. In one embodiment, the bioink comprises about 10,000 cells / μl to about 100,000 cells / μl. In one embodiment, the bioink comprises about 100,000 cells / μl to about 400,000 cells / μl. In one embodiment, the bioink comprises about 50,000 cells / μl to about 200,000 cells / μl. In one embodiment, the bioink comprises about 10,000 cells / μl, about 30,000 cells / μl, about 40,000 cells / μl, about 50,000 cells / μl, about 60,000 cells / μl, about 70,000 cells / μl, about 80,000 cells / μl, about 90,000 cells / μl, about 100,000 cells / μl, about 150,000 cells / μl, about 200,000 cells / μl, about 250,000 cells / μl, about 300,000 cells / μl, or about 400,000 cells / μl. In a preferred embodiment, the bioink comprises about 200,000 cells / μl.

[0101] In some embodiments, the bio-ink comprises partially differentiated cells. In some embodiments, the bio-ink comprises fully differentiated cells.

[0102] In some embodiments, the bioink comprises cells differentiated from human stem cells (HSCs), such as, but not limited to, human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). In some embodiments, the bioink comprises primitive streak cells, such as, but not limited to, posterior primitive streak cells. In some embodiments, the bioink comprises intermediate mesoderm (IM) cells. In some embodiments, the bioink comprises metanephric mesenchyme (MM) cells. In some embodiments, the bioink comprises renal duct cells. In some embodiments, the bioink comprises renal progenitor cells, such as, but not limited to, nephron progenitor cells, ureteral epithelial progenitor cells, or a combination thereof.

[0103] In some embodiments, the cells of the bioink comprise cells derived from a patient. In some embodiments, the cells of the bioink comprise gene-edited cells. In some embodiments, the cells of the bioink comprise cells derived from a patient that are also gene-edited cells. In some embodiments, the cells of the bioink comprise cells from a reporter strain. In some embodiments, the cells of the bioink comprise gene-edited reporter strain cells.

[0104] By using the methods for producing bioprinted kidney tissue disclosed herein, nephron-enriched bioprinted engineered kidney tissue can be produced. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 2 to about 100 nephrons / 10,000 cells printed. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 2 to about 50 nephrons / 10,000 cells printed. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 5 to about 40 nephrons / 10,000 cells printed. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 5 to about 75 nephrons / 10,000 cells printed. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 5 to about 60 nephrons / 10,000 cells printed. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 5 to about 50 nephrons / 10,000 cells printed. According to another embodiment, the bioprinted kidney tissue prepared according to the methods described and exemplified herein comprises about 5 to about 40 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 5 to about 20 nephrons / 10,000 cells printed. In one embodiment, the bioprinted kidney tissue comprises about 5 to about 10 nephrons / 10,000 cells printed.As detailed herein, nephrons can be identified and counted (e.g., for WT1+ glomeruli, MAFB+NPHS1+ podocytes, HNF4A+LTL+ECAD- proximal tubules, SLC12A1+ECAD+ distal tubules, and ECAD+GATA3+ connecting segments or collecting ducts) in the bioprinted kidney tissue disclosed herein by those skilled in the art using various methods, such as visualization and counting using confocal microscopy and immunofluorescent labeling. In this embodiment, the bioprinted kidney tissue may additionally or alternatively be characterized using single-cell RNA sequencing, PCR-based gene expression analysis, immunofluorescent labeling, or immunohistochemical methods. In one embodiment, the bioprinted kidney tissue is 0.2 mm per 10,000 cells printed. 2 In one embodiment, the bioprinted kidney tissue contains a surface area of ​​nephron tissue of 0.2 mm per 10,000 cells printed. 2 ~1.5mm 2 In one embodiment, the bioprinted kidney tissue comprises a surface area of ​​0.25 mm per 10,000 cells printed. 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , or 1.5 mm 2 In one embodiment, the bioprinted kidney tissue comprises a surface area of ​​0.2 mm per 10,000 cells printed. 2 In one embodiment, the bioprinted kidney tissue contains a surface area of ​​0.2 mm per 10,000 cells printed. 2~1.5mm 2 In one embodiment, the bioprinted kidney tissue contains 0.25 mm of surface area of ​​cells expressing MAFB per 10,000 cells printed. 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , or 1.5 mm 2 of the surface area of ​​cells expressing MAFB.

[0105] According to another embodiment, the bioprinted kidney tissue produced according to the method disclosed herein has a uniform distribution of nephrons throughout the bioprinted layer. That is, in contrast to manually aggregated or bioprinted kidney organoids, which can be generated as dots or blobs of cells as described in the prior art and form dome-shaped structures with interstitial centers lacking nephrons, the bioprinted kidney tissue has a higher number of nephrons and is more uniformly distributed. For example, the bioprinted kidney has a uniform distribution of glomeruli throughout the bioprinted layer, for example, as labeled by cells expressing MAFB. In another embodiment, the bioprinted kidney tissue expresses one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM and MAFB. In another embodiment, the bioprinted kidney tissue shows increased expression levels of one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM and MAFB compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526: 564-568), i.e., bioprinted kidney organoids produced by manually aggregated or as dots or blobs of cells. In one embodiment, the bioprinted kidney tissue shows low or no expression of one or more of THY1, DCN, SOX17, FLT1 and PECAM, or reduced expression of one or more of THY1, DCN, SOX17, FLT1 and PECAM compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526: 564-568), i.e., bioprinted kidney organoids produced by manually aggregated or as dots or blobs of cells. In another embodiment, the bioprinted kidney tissue has nephrons whose proximal and distal tubule segments exhibit maturation markers including HNF4A and SLC12A1, hi another embodiment, the bioprinted kidney tissue exhibits reduced presence of interstitial, fibroblast, and endothelial cells.In another embodiment, the bioprinted kidney tissue exhibits a reduction in off-target populations for nephron cell types.

[0106] In another embodiment, in the bioprinting step, the bioink is bioprinted onto a biocompatible scaffold. That is, the surface onto which the bioink is printed is a biocompatible scaffold. In one embodiment, the biocompatible scaffold is biodegradable or bioabsorbable. In another embodiment, the biocompatible scaffold is a hydrogel. In another embodiment, the scaffold may be functionalized with one or more biologically active agents. For example, biologically active agents (e.g., small molecules, polypeptides including cytokines and chemokines, differentiation factors, signaling pathway inhibitors, etc.) may, for example, promote the viability of cells within the bioink and further development or differentiation of cells within the bioink.

[0107] In another embodiment, the bioink further comprises one or more agents (e.g., biologically active agents). In one example, the one or more biologically active agents promote the derivation of kidney tissue from the plurality of cells. In another embodiment, the bioink further comprises a differentiation medium, a bioprinting medium, or any combination thereof. In some embodiments, the bioprinting medium comprises a hydrogel and / or one or more ECM components. In one embodiment, the bioink comprises hyaluronic acid. In one embodiment, the one or more active agents are selected from anti-proliferative agents, immunosuppressants, pro-angiogenic compounds, antibodies or fragments or portions thereof, antibiotics or antibacterial compounds, antigens or epitopes, aptamers, biopolymers, carbohydrates, cell attachment mediators (such as RGD), cytokines, cytotoxic agents, drugs, enzymes, growth factors or recombinant growth factors and fragments and variants thereof, hormone antagonists, hormones, immunological agents, lipids, metals, nanoparticles, nucleic acid analogs, nucleic acids (e.g., DNA, RNA, siRNA, RNAi, and microRNA agents), nucleotides, nutraceutical agents, oligonucleotides, peptide nucleic acids (PNAs), peptides, prodrugs, prophylactic agents, proteins, small molecules, therapeutic agents, or any combination thereof.

[0108] In other embodiments, the bioprinted kidney tissue further comprises a bioink as described herein above disposed adjacent or proximate to another bioprinted bioink, which may optionally include one or more drugs or one or more other cell types as described above. For example, a bioink including a plurality of cells (and optionally one or more drugs) may be bioprinted adjacent or proximate to another bioprinted bioink. For example, a bioink including a plurality of cells (and optionally one or more drugs) may be bioprinted on or next to (including directly on or next to) a line or layer of a bioprinted bioink (optionally including one or more drugs and / or one or more other cell types). In one embodiment, a method for producing a bioprinted kidney tissue comprises bioprinting a quantity of a first bioink and printing a quantity of a second bioink on a surface, where the first bioink and the second bioink are different. In one example, the first bioink includes a plurality of cells that is different from the plurality of cells in the second bioink. In another example, a first bio-ink may include a plurality of cells and a second bio-ink may not include cells but may include other components, such as, for example, a biologically active agent.

[0109] In one example, inducing the bioprinted bioink to form kidney tissue includes contacting the bioink with FGF-9. In another example, inducing the bioink to form kidney tissue by contacting the bioink with FGF-9 for 5 days. In some examples, the plurality of cells may be contacted briefly with cell culture medium including CHIR, then bioprinted, and further cultured. For example, the plurality of cells may be contacted with cell culture medium including 3-8 μM CHIR for 1-2 hours, then bioprinted, and further cultured. In another example, the plurality of cells may be contacted with cell culture medium including 5 μM CHIR for 1 hour, then bioprinted, and further cultured. In another embodiment, inducing the bioprinted bioink to form kidney tissue includes contacting the bioink with cell culture medium including CHIR after being bioprinted and further cultured for a short period of time. In one embodiment, the method includes culturing the bioprinted bioink in the presence of 5-10 μM CHIR for 1 hour.

[0110] In one embodiment, the plurality of cells comprises a culture expanded population of stem cell derived intermediate mesoderm (IM) cells. The IM cells can be prepared and cultured according to the methods described above in the section entitled "Bioprinted Kidney Tissue." In one embodiment, the inducing step comprises contacting the bioprinted volume of bioink with FGF-9. In another embodiment, the inducing step comprises contacting the bioprinted volume of bioink with FGF-9 for 5 days. In one embodiment, inducing the bioprinted volume of bioink to form bioprinted kidney tissue is performed as described above in the section entitled "Bioprinted Kidney Tissue."

[0111] In extrusion bioprinting, the shape of the cell aggregates, cell number, cell density, and final tissue height (or thickness) can be controlled by fine movements of the tip as the cells are extruded. By scripting the movements of the extrusion port during the extrusion process, the bioink can be spread over a defined distance in a manner that cannot be controlled or at least accurately reproduced by hand. By increasing the amount of tip movement at a given cell extrusion rate (rate), the user can create bioprinted tissues with various cell densities, shapes, and heights (thicknesses) as the cells spread over a larger surface area and subsequently aggregate. According to one embodiment, the bioprinting step uses an extrusion-based bioprinter. In another embodiment, the bioprinting step uses an extrusion-based bioprinter with a 100-500 μl syringe and a needle with an inner diameter of about 100 to about 550 μm.

[0112] In one embodiment, during the bioprinting step, a dispensing device of the bioprinter is configured to dispense the layers in one or more lines, while in another embodiment, during the bioprinting step, a dispensing device of the bioprinter is configured to dispense the layers in one or more lines to form a continuous sheet or patch.

[0113] The extrusion bioprinter used in the methods disclosed herein can be scripted to adjust the extrusion rate of the bioink with the movement of the dispenser. This is called the "ratio". For example, this term refers to the rate at which material is dispensed over a given movement of the tip through which the bioink is extruded. A higher ratio means more tip movement for the same amount of extrusion. Increasing or decreasing the rate of dispensing increases or decreases the area over which a given amount of bioink volume is extruded. Thus, the ratio can be defined as cells / mm tip movement. In one embodiment, a ratio of 40, 30, 20 or 10 corresponds to about 9,000 cells / mm, about 12,000 cells / mm, about 18,000 cells / mm, and about 36,000 cells / mm, where mm is mm of tip movement, preferably the tip is a 25G needle.

[0114] The height of the bioprinted layer of a given amount of bioink at the time of printing decreases as the distribution ratio increases. That is, the height of the bioprinted layer of a given amount of bioink at the time of printing decreases with increasing line length. In a preferred embodiment, the height of the bioprinted layer of bioink is about 50 μm or less. The height of the same bioprinted structure after differentiation (e.g., after the "induction" step in the methods described herein) may vary depending on the number of days of culture. The examples provided herein show tissue structures cultured for an additional 12 days, during which the printed layer of bioink undergoes self-organization of the constituent cells and differentiation into differentiated cell types. In a preferred embodiment, the height of the bioprinted tissue is about 150 μm or less after the culture period.

[0115] In a preferred embodiment, the method of producing bioprinted kidney tissue comprises the steps of: i) bioprinting a volume of bio-ink comprising a plurality of cells onto a surface to produce a layer of said bio-ink, wherein the height of the bio-ink layer is about 50 μm or less and less than 1 mm 2ii) inducing the printed bio-ink to form kidney tissue, the bio-ink containing about 10,000 cells to about 20,000 cells per culture medium, the cells being stem cell-derived IM cells; and ii) inducing the printed bio-ink to form kidney tissue.

[0116] According to another aspect, the present invention provides bioprinted kidney tissue produced according to the methods described herein.

[0117] Tissue engineering of kidney tissue for transplantation To engineer human kidney tissue for the purpose of transplantation into patients with kidney disease and renal failure, it is necessary to increase the number of nephrons formed per engineered structure and per starting cell type, and to create biocompatible structures amenable to transplantation under the kidney capsule. Manually generated organoids or bioprinted dots can be vascularized by recipient animals when transplanted under the kidney capsule. However, problems associated with transplantation of such engineered tissues are "off-target" tissue differentiation and stromal overgrowth. Therefore, better tissues for transplantation are needed.

[0118] As described herein, the inventors have also surprisingly confirmed that the bioprinted kidney tissue disclosed herein has a high nephron content. Without wishing to be bound by a particular theory, the increased number of nephrons formed per structure and per starting cell type may enable the creation of biocompatible structures amenable to implantation under the kidney capsule. These characteristics may indicate that the bioprinted kidney tissue is more suitable for therapeutic applications such as transplantation. For example, the bioprinted kidney tissue may avoid problems of off-target tissue differentiation and interstitial overgrowth. The bioprinted kidney tissue defined herein may represent a better tissue for transplantation.

[0119] According to one aspect, the present invention relates to a bioprinted kidney tissue as disclosed herein or produced according to the methods disclosed herein for use in treating renal disease or failure in a subject in need thereof. Thus, the present invention also relates to the use of a bioprinted kidney tissue as disclosed herein or produced according to the methods disclosed herein for use in transplantation into a patient with renal disease or failure.

[0120] The present invention also relates to a method for treating renal disease or renal failure in a patient, comprising administering to a patient in need thereof the bioprinted kidney tissue disclosed herein or produced according to the method disclosed herein.In one embodiment, the bioprinted kidney tissue is enriched in nephrons throughout the tissue.This is in contrast to the bioprinted kidney organoid, which produces fewer nephrons and distributes them only around the organoid.

[0121] In one embodiment, the bioprinted kidney tissue comprises a bioink, the bioink comprises a plurality of cells, and the bioprinted kidney tissue has a thickness of 0.2 mm per 10,000 cells printed. 2 In one embodiment, the bioprinted kidney tissue contains a surface area of ​​nephron tissue of 0.2 mm per 10,000 cells printed. 2 ~1.5mm 2 In one embodiment, the bioprinted kidney tissue comprises a surface area of ​​0.25 mm per 10,000 cells printed. 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm2 , 1.3mm 2 , 1.4mm 2 , or 1.5 mm 2 In one embodiment, the bioprinted kidney tissue comprises a surface area of ​​0.2 mm per 10,000 cells printed. 2 In one embodiment, the bioprinted kidney tissue contains a surface area of ​​0.2 mm per 10,000 cells printed. 2 ~1.5mm 2 In one embodiment, the bioprinted kidney tissue contains 0.25 mm of surface area of ​​cells expressing MAFB per 10,000 cells printed. 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , or 1.5 mm 2 of the surface area of ​​cells expressing MAFB.

[0122] In one example, the bioprinted kidney tissue has approximately 5 to 100 nephrons per mm of bioprinted kidney tissue. 2 In one example, the bioprinted kidney tissue comprises about 5 to about 75 nephrons per mm of bioprinted kidney tissue. 2 In one example, the bioprinted kidney tissue comprises about 5 to about 50 nephrons per mm of bioprinted kidney tissue. 2 In one example, the bioprinted kidney tissue comprises about 5 to about 20 nephrons per mm of bioprinted kidney tissue. 2In one example, the bioprinted kidney tissue comprises about 20 to about 50 nephrons per mm of bioprinted kidney tissue. 2 Includes.

[0123] In one example, the bioprinted kidney tissue has a uniform distribution of glomeruli throughout the bioprinted layer, for example, as marked by cells expressing MAFB.In another embodiment, the bioprinted kidney tissue expresses one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM and MAFB.In another embodiment, the bioprinted kidney tissue shows increased expression levels of one or more of SLC12A1, CDH1, HNF4A, CUBN, LRP2, EPCAM and MAFB compared to the previously published method (Takasato et al. (2015) Nature, Vol. 526: 564-568), i.e., the bioprinted kidney organoids that are manually aggregated or generated as dots or blobs of cells. In one embodiment, the bioprinted kidney tissue shows low or no expression or reduced expression of one or more of THY1, DCN, SOX17, FLT1 and PECAM compared to previously published methods (Takasato et al. (2015) Nature, Vol. 526: 564-568), i.e., bioprinted kidney organoids generated manually as aggregates or dots or blobs of cells. In another embodiment, the bioprinted kidney tissue has nephrons whose proximal and distal tubule segments show maturation markers including HNF4A and SLC12A1. In another embodiment, the bioprinted kidney tissue shows reduced presence of interstitial, fibroblasts, and endothelial cells.

[0124] Bioprinted kidney tissue can be produced in a variety of sizes suitable for transplantation. In some embodiments, the bioprinted kidney tissue is printed at a height of about 15 μm to about 150 μm. In one embodiment, the bioink is bioprinted in a layer selected from a height of about 25 μm to a height of about 100 μm. In a preferred embodiment, the bioink is bioprinted in a layer of about 50 μm or less. In one embodiment, the bioink is bioprinted in a layer of about 15 μm height. In one embodiment, the bioink is bioprinted in a layer of about 20 μm height. In one embodiment, the bioink is bioprinted in a layer of about 25 μm height. In one embodiment, the bioink is bioprinted in a layer of about 30 μm height. In one embodiment, the bioink is bioprinted in a layer of about 35 μm height. In one embodiment, the bioink is bioprinted in a layer of about 40 μm height. In one embodiment, the bioink is bioprinted in a layer of about 50 μm height. In one embodiment, the bioink is bioprinted in a layer about 60 μm tall. In one embodiment, the bioink is bioprinted in a layer about 70 μm tall. In one embodiment, the bioink is bioprinted in a layer about 80 μm tall. In one embodiment, the bioink is bioprinted in a layer about 90 μm tall. In one embodiment, the bioink is bioprinted in a layer about 100 μm tall. As noted above, the height of the bioprinted tissue may increase slightly after bioprinting, such as during subsequent culture (e.g., during induction of the bioprinted bioink to form kidney tissue) and / or during maintenance. In one embodiment, after a culture period, the bioprinted tissue attains a height not exceeding 150 μm. In one embodiment, after a culture period, the bioprinted tissue attains a height of about 100 μm to 150 μm. In another embodiment, the bioprinted kidney tissue has a length of 1 mm to 30 mm, and a width of 0.5 mm to 20 mm.In another embodiment, the bioprinted kidney tissue has a length of 5 mm to 30 mm, and a width of 0.5 mm to 2 mm. In another embodiment, the bioprinted kidney tissue has a height of up to about 100 μm to 250 μm. In this embodiment, the height (or thickness) is not the height at which the tissue is printed, but the height (or thickness) of the kidney tissue after the bioprinted bio-ink has been induced (e.g., after a culture period).

[0125] In another embodiment, the bioprinted kidney tissue for use in therapy / transplantation further comprises a biocompatible scaffold. For example, in another embodiment, the bioink is bioprinted onto a biocompatible scaffold. That is, the surface onto which the bioink is printed is a biocompatible scaffold. In one embodiment, the biocompatible scaffold is biodegradable or bioabsorbable. In another embodiment, the biocompatible scaffold is a hydrogel. In another embodiment, the scaffold may be functionalized with one or more agents (e.g., biologically active agents). For example, biologically active agents (cytokines, chemokines, differentiation factors, signaling pathway inhibitors, etc.) may, for example, promote further development or differentiation of cells within the bioink printed thereon or promote engraftment and / or survival of the transplanted bioprinted tissue.

[0126] In another embodiment, the bioink or scaffold further comprises one or more biologically active agents that promote the induction of kidney tissue from the plurality of cells. In another embodiment, the bioink or scaffold further comprises a hydrogel, such as a modified or functionalized hydrogel, or a mixture of matrix components or extracellular matrix components. In one embodiment, the one or more active agents are selected from antiproliferative agents, immunosuppressants, proangiogenic compounds, antibodies or fragments or portions thereof, antibiotics or antimicrobial compounds, antigens or epitopes, aptamers, biopolymers, carbohydrates, cell attachment mediators (such as RGD), cytokines, cytotoxic agents, drugs, enzymes, growth factors or recombinant growth factors and fragments and variants thereof, hormone antagonists, hormones, immunological agents, lipids, metals, nanoparticles, nucleic acid analogs, nucleic acids (e.g., DNA, RNA, siRNA, RNAi, and microRNA agents), nucleotides, nutraceutical agents, oligonucleotides, peptide nucleic acids (PNAs), peptides, prodrugs, prophylactic agents, proteins, small molecules, therapeutic agents, or any combination thereof.

[0127] According to the above embodiments, the bioprinted kidney tissue may be used for transplantation into patients, including those with chronic kidney disease, inherited kidney disease, or reduced kidney function after nephroreductive surgery for cancer. In one embodiment, the bioprinted tissue is implanted under the kidney capsule of the recipient. In one embodiment, the bioprinted tissue may be a sheet or a patch.

[0128] Drug Screening According to another aspect, the present invention provides a method of screening a candidate compound for nephrotoxicity or therapeutic effect, the method comprising contacting a bioprinted kidney tissue as described herein with a candidate compound and determining whether the candidate compound is nephrotoxic or therapeutically effective.

[0129] In one embodiment, the method includes contacting the bioprinted kidney tissue with a candidate compound and a nephrotoxic agent and determining whether the candidate compound is therapeutically effective. In one embodiment, determining whether the candidate compound is nephrotoxic or therapeutically effective includes measuring one or more of the expression of one or more genes associated with cell death, expression of one or more genes associated with cell viability, expression of one or more nephron-associated genes, expression of one or more genes associated with glomerular extracellular matrix, expression of one or more genes associated with podocyte, endothelial or mesangial cell types, expression intensity of a reporter gene associated with at least one gene of interest.

[0130] In another embodiment, i) a measured decrease in one or more of the expression of one or more genes associated with cell viability, expression of one or more nephron-associated genes, expression of one or more genes associated with glomerular extracellular matrix, expression of one or more genes associated with podocytes, endothelial cells or mesangial cell types, and the intensity of the reporter genes, and / or ii) a measured increase in the expression of one or more genes associated with cell death indicates nephrotoxicity of the candidate compound.

[0131] In another embodiment, i) an increase in one or more of the expression of one or more genes associated with cell viability, one or more nephron-related genes, one or more genes associated with glomerular extracellular matrix, one or more genes associated with podocytes, endothelial cells or mesangial cell types, and the intensity of the reporter gene, by measuring, and / or ii) a decrease in the expression of one or more genes associated with cell death, by measuring, indicates the therapeutic effect of the candidate compound. In one embodiment, the candidate compound is a small molecule, a polynucleotide, a peptide, a protein, an antibody, an antibody fragment, a serum, a virus, a bacterium, a stem cell, or a combination thereof. In another embodiment, the candidate compound is a serum, such as a serum isolated from a subject with kidney disease.

[0132] In another embodiment, the method may further comprise selecting candidate compounds that are not nephrotoxic and / or are therapeutically effective. EXAMPLES

[0133] Example 1. Human pluripotent stem cells induced to differentiate and produce organoids by manual manipulation. Human pluripotent stem cells were thawed and plated overnight in the presence of 1x RevitaCell (ThermoFisher Scientific Catalog #A2644501) and cultured in GelTrex (ThermoFisher Scientific Catalog #A1413301) or Essential 8 Medium (ThermoFisher Scientific) under standard feeder-free defined conditions with daily medium changes. The day before initiation of differentiation, cells were detached with TrypLE Select (ThermoFisher Scientific Catalog #12563011), counted using trypan exclusion on a Nexcellom Cellometer Brightfield Cell Counter (Nexcelom Biosciences), and plated in GelTrex, Matrigel, or Laminin-521 coated T-25 flasks or 6-well plates in Essential 8 Medium with 1x RevitaCell (ThermoFisher Catalog #A2644501). Intermediate mesoderm induction was performed by culturing iPSCs for 4 days in STEMdiff APEL medium (STEMCELL Technologies catalog no. 5210) or TeSR-E6 medium containing 6-8 μM CHIR99021 (R&D Systems catalog no. 4423 / 10). On day 4, cells were differentiated in STEMdiff APEL medium or TeSR-E6 medium supplemented with 200 ng / mL FGF9 (R&D Systems catalog no. 273-F9-025) and 1 μg / mL heparin (Sigma Aldrich catalog no. H4784-250MG).

[0134] Organoid generation was performed manually after 7 days of differentiation according to Takasato et al. (Nature Protocols 11, 1681-1692. (2016)), and organoids were cultured for an additional 14–18 days before harvesting.

[0135] Example 2. Bioprinting of kidney organoids material and method Stem cells were prepared as described in Example 1. On day 7, cells were dissociated with Trypsin-EDTA (0.25%, Thermo Fisher Cat. No. 25200-072) or TryPLE Select (ThermoFisher Scientific Cat. No. 12563011). The resulting suspension was counted in a Nexcelom Cellometer and viable cells were measured by trypan exclusion. Single cell suspensions of differentiated cells were first counted using a Neubauer hemocytometer (BLAUBRAND Cat. No. BR7-18605) to obtain a cell count, followed by centrifugation at 200-300×g for 3-5 minutes to pellet the cells in 50 mL or 15 mL polypropylene conical tubes. After aspirating the supernatant, the cellular material was either directly transferred into a 100uL gas-tight syringe (Hamilton catalog #7656-01) with a 21-25 gauge removable needle (Hamilton catalog #7804-12) for bioprinting or resuspended in STEMdiff APEL or TESR-E6 medium to the operational cell density prior to bioprinting. All syringes containing cellular bioink were loaded into a NovoGen MMX bioprinter, primed for cellular material flow, and an aliquot of user-prepared bioink was deposited onto the 0.4μm polyester membrane of a 6-well Transwell permeable support (Corning Costar catalog #3450).

[0136] histological staining Kidney tissues were fixed overnight at 4°C in 2% or 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and pre-embedded in HistoGel (Thermo Fisher, Carlsbad, CA), then dehydrated and infiltrated with paraffin using a TissueTek VIP tissue processing system (Sakura Finetek USA, Torrance, CA). Planar or transverse 5 μm sections were obtained using a Leica RM 2135 microtome (Leica Biosystems, Buffalo Grove, IL). Sections were baked, deparaffinized, hydrated, and then stained with SelecTech stains (Leica Biosystems, Richmond, IL; Hematoxylin #3801570, Define #3803590, Blue Buffer #3802915, and Eosin Y 515 #3801615) following standard degenerative staining protocols. Stained slides were serially dehydrated, cleared, and mounted in Permaslip (Alban Scientific Inc, St. Louis, MO #6530B). Images were acquired on a Zeiss Axio Imager A2 with Zeiss Zen software (Zeiss Microscopy, Thornwood, NY).

[0137] Section and whole mount immunofluorescence For paraffin-embedded organoids, antigens were retrieved from deparaffinized sections in citrate buffer, pH 6.0 (Diagnostic BioSystems, Pleasonton, CA #KO35) and blocked with 5% chicken serum diluted in TBS-T (v / v) prior to immunofluorescence. For whole-mount organoids, organoid harvesting, fixation and blocking, as well as immunofluorescence of prepared sections and whole organoids, were performed as described (Vanslambrouck JM, et al. J Am Soc Nephrol 30, 1811-1823 (2019)). Images were acquired as described in Vanslambrouck JM, et al. or using an Andor spinning disk confocal microscope with a Nikon 25x1.05NA silicone immersion objective.

[0138] Diameter measurement The cross-sectional diameter of organoids was assessed over time by image-based analysis using ImageJ (version 1.51). Total images were collected on day 7 after printing at a fixed distance from the plate surface using a 2x objective. Each sample was manually outlined using the ellipse selection tool and used to calculate the area in pixels for each image. Circular area values ​​were converted to diameter (mm) using the following formula:

number

[0139] Extrusion Bioprinting with Dry Paste During extrusion bioprinting optimization, we compared settled wet pastes to dry pastes generated to mimic the loaded cell density used during manual organoid preparation. To achieve this density of dry paste within the extrusion syringe, the prepared syringe was loaded into a proprietary adapter that allowed for centrifugation at 400 x g in a 50 mL polypropylene conical tube. The syringe / adapter assembly was centrifuged for a total of 9 minutes, mimicking the manual protocol.

[0140] result Bioink containing cell paste was bioprinted with a single-point deposition (ratio 0). This single-point deposition (or dot) was used to evaluate whether the starting concentration and conformation during printing influenced the final morphology. After bioprinting, the single-point deposition (ratio 0) tissue formed a dome-shaped structure with similar characteristics to manually generated kidney organoids, and therefore the single-point deposition (ratio 0) may also be referred to as bioprinted organoids.

[0141] By varying the deposition ratio within the custom software interface, different organoid conformations were generated while scaling the organoid length to a constant 1.1 x 10 deposition ratio where each organoid was deposited in a volume of approximately 0.55 μl. 5 Starting with a bioink containing a wet cell paste of a set cell density, the same number of cells were bioprinted and deposition was varied from a line of approximately 3 mm (ratio 10) to a line of approximately 12 mm (ratio 40) of cells. This allowed for evaluation of whether the starting density influenced the final morphology, as detailed below. In each case, we varied the length of the line to ensure that the absolute number of starting cells for each organoid was approximately equal. The organoids in the line were single-point deposited (approximately 10% in total) at the start of the pattern to ensure uniform fluid flow. The organoids in the "dots" were dotted with the same total cell volume to ensure cell number consistency. During deposition, the needle was positioned 300 microns from the transwell surface. In all cases, deposition rates were based on a 25-gauge needle and a 100 μl syringe.

[0142] After bioprinting, bioprinted organoids are cultured in the basolateral compartment of transwell culture plates in either STEMdiff™ APEL or TeSR-E6 medium for 1 h in the presence of 5-10 μM CHIR99021, and then cultured until day 7+5 in STEMdiff™ APEL or TESR-E6 medium supplemented with 200 ng / mL FGF9 and 1 μg / mL heparin (medium for the basolateral compartment only). From day 7+5 to day 7+18, organoids are grown in STEMdiff™ APEL medium or TeSR-E6 medium, without supplementation. Renal organoids can be cultured from day 7+12 to day 7+20 until harvesting. Tissues were maintained under the same conditions as above.

[0143] The resulting bioprinted organoids showed spontaneous formation of nephrons over the subsequent 20 days of culture (Figure 1A,B,E). Immunofluorescence was used to confirm the presence of classically patterned nephrons, revealing the presence of podocytes (NEPHRIN), proximal tubules (LTL, CUBN), distal tubules / loops of Henle's loop (TAL; ECAD, SLC12A1) and connecting / tubular epithelium (GATA3, ECAD) (Figure 1C,D). The presence of additional cellular components was also revealed, including endothelial cells (CD31) and renal interstitium (MEIS1 / 2) (Figure 1D). Histological sections of the bioprinted organoids revealed the presence of adjacent connecting epithelium (ECAD, GATA3) throughout the width of the tissue from which individual nephrons radiate (Figure 2A,B,C). It should be noted that the cell paste represents cells only and does not incorporate any associated ECM or hydrogel matrix. The patterning achieved was compared to results when the cell paste was centrifuged to remove all remaining medium, creating a packed "dry" cell paste. Subsequent culture of organoids derived from the dried paste showed no evidence of nephron formation (Figure 2D).

[0144] To directly compare the cellular complexity of bioprinted versus manually pelleted organoids, both approaches were subjected to the same monolayer differentiation. The resulting kidney organoids were analyzed using brightfield imaging and immunofluorescence, and the results showed that the bioprinted kidney organoids exhibited morphological equivalence to the manually pelleted kidney organoids (Figure 1E).

[0145] Example 3. Bioprinted kidney organoids with high throughput and small organoid size. The automated process of organoid bioprinting applied here allowed the deposition of approximately 1 micromass every 3 seconds, with very high organoid diameter reproducibility (Table 1). 5 While it is possible to manually place micromasses of cells in 24-well transwell plates, bioprinting allowed for precise placement of multiple micromasses on the same filter (3–9 organoids per filter in a 6-well plate) (Figure 1F,G). It was also possible to reduce the number of cells used to generate the initial micromass without losing the histological complexity within the organoid (Figure 1F,H). Thus, the yield and throughput of the kidney organoid generation process could be significantly increased, with only 4 × 10 cells per filter. 3 A pattern of kidney structure was evident in organoids bioprinted from cells (Figure 3A,C). Indeed, the reproducibility of cell paste deposition, as assessed by the printed volume and the resulting average diameter, showed a coefficient of variation of 1%–4% (Table 1). [Table 1]

[0146] The feasibility of using bioprinting cell lines for kidney organoid generation was extensively evaluated using various human induced pluripotent stem cell lines. Control, reporter, and patient-derived iPSC lines successfully generated kidney tissue when bioprinted with this method. For example, the MAFB gene promoter (MAFB mTagBFP2 The use of a specific reporter line in which a blue fluorescent protein was inserted under the control of GFP-GFP enabled assessments such as the visualization of podocyte differentiation in the glomeruli that form at one end of each kidney nephron by fluorescent imaging of live tissue (Figure 3B).

[0147] Example 4. Bioprinted kidney organoids for compound testing in 96-well format. material and method Bioprinted organoids were prepared using the methods outlined in Example 2.

[0148] Assaying bioink viability and concentration Dispensed bioink was sampled before and after printing onto two rows (24 organoids) of a 96-well plate. Printed bioink was dispensed directly into 1.5 mL Eppendorf tubes filled with APEL medium for dilution and counted using a Nexecelom Cellometer (Nexecelom Biosciences) by trypan blue exclusion. Nexecelom results were entered into JMP for visualization and statistical analysis. For analyses comparing only two conditions, t-tests were performed, and for analyses comparing more than two conditions, one-way ANOVA and Tukey comparisons were performed, and bivariate fits were performed with fitted means, linear fit lines, and significant trends were determined with 95% confidence intervals.

[0149] Drug-induced nephrotoxicity test Doxorubicin (Sigma-Aldrich, D1515) stock solutions were prepared in DMSO. Amikacin, tobramycin, gentamicin, neomycin, and streptomycin were all obtained from Sigma Aldrich (St. Louis, MO) and prepared as 25 mg / ml solutions in APEL medium. Dosing for nephrotoxicity testing in 6-well plates was performed by first diluting the DMSO stock of doxorubicin in APEL medium, then further diluting with additional medium to give concentrations ranging from 0.3 to 10 μM. Dosing for nephrotoxicity evaluation in 96-well plates was performed by serial dilutions. For doxorubicin, serial dilutions of the DMSO stock were added to APEL medium to give concentrations ranging from 24 nM to 25 μM. Stock solutions of aminoglycosides were serially diluted in APEL medium to give dosing concentrations ranging from 1.5 μg / mL to 25 mg / mL. Dosing of drugs began after day 21 or 22 of the differentiation protocol. Dosing was performed by applying the total well volume of APEL medium ± test substance to the apical basket of the transwell permeable carrier (4 mL for 6-well plates, 300 μL for 96-well plates). When medium containing the test substance was added to the transwell permeable carrier, the organoids were allowed to completely settle and were exposed to the added compound when the apical and basolateral compartments were equilibrated. Drug-supplemented medium was replaced every other day until the designated harvest time point.

[0150] Assessing organoid viability The viability of kidney organoids after drug treatment was assessed by measuring ATP content with CellTiter-Glo or CellTiter-Glo 3D Viability Assay (Promega, Madison, WI, USA). Briefly, bioprinted organoids in 6-well plates were individually loaded into Precellys tubes (Bertin Technologies, Bretonneux, France) using CellTiter-Glo buffer and dissociated using a Precellys 24 tissue homogenizer (Bertin Technologies, Bretonneux, France). Homogenized organoids were incubated at room temperature for 10 min and then centrifuged at 1000 g for 2 min to separate the buffer from the homogenization beads. Supernatants were transferred to white opaque 96-well plates and luminescence was measured with a microplate reader (BMG Labtech, Germany). The viability results of 6 wells shown are a combination of three independent experiments and are normalized to the respective control ATP levels in each test. To analyze the ATP content of organoids bioprinted in 96-well plates, all media was aspirated and CellTiter-Glo3D reagent was added to the apical chamber of the transwell permeable carrier. The plates were shaken at 400 rpm for 5 min at room temperature and then left to rest for 25 min, after which luminescence was measured in a white opaque 96-well plate in a microplate reader (BMG Labtech, Germany). Viability analysis was shown as a percentage of control by normalizing the ATP content of treated organoids compared to control organoids. Fitting of viability results was performed with GraphPad Prism 7.03 software (La Jolla, CA) using a four-parameter dose-response curve (Equation 1). Y=bottom+(top-bottom) / (1+((X HillSlope ) / (I C 50 HillSlope )))(Formula 1)

[0151] Gene expression analysis by quantitative RT-PCR after drug exposure Total RNA extraction from kidney organoids after drug exposure was performed using the Rneasy Mini kit (Qiagen, Germany) according to the manufacturer's instructions. RNA was quantified spectrophotometrically using a NanoDrop 2000 (Thermo Fisher, Carlsbad, CA). To analyze gene expression, TaqMan Fast One-Step qPCR Master Mix (Applied Biosystems, Foster City, CA), TaqMan probes for genes of interest (ThermoFisher, Carlsbad, CA), and housekeeping gene probes (Applied Biosystems, Foster City, CA) were mixed with RNA in assigned wells. All qPCR reactions were performed and analyzed in a StepOnePlus qRT-PCR system (Applied Biosystems, Foster City, CA). All data were normalized to the housekeeping gene GAPDH before normalizing to control samples.

[0152] result Although the kidney plays a key role in the elimination of xenobiotics, the uptake of various compounds through specific solute channels in the tubules places the kidney at risk of nephrotoxic injury. Preclinical screening of nephrotoxicity using primary renal proximal tubule epithelial cells (RPTECs) often fails to accurately predict organ-specific toxicity due to the rapid dedifferentiation of such cells in 2D culture, which often results in the loss of expression of key transporters and metabolic enzymes. Human kidney organoids may provide a more accurate and predictable tool to model drug responses, which depends in part on the ability to generate large numbers of viable and reproducible patterned organoids with low coefficient of variation (cv). To this end, automated bioprinting was further scaled down (1.0 × 105 starting cells per organoid) and made suitable for the fabrication of individual organoids on 96-well transwell filters (Figure 4A,B). The precision of cell number and cell viability was reproducible across all 96 wells, with overall cell viability ranging from 93 to 99% (Figure 4C). As a proof-of-concept test for the application of this approach to nephrotoxicity testing, the effects of administration of the chemotherapeutic agent doxorubicin, a known podocyte toxin, were first assessed using bioprinted organoids after treatment with 2 µM or 10 µM doxorubicin for 72 h (Figure 4D-F). Immunofluorescence staining of the resulting organoids showed evidence of specific activation of caspase 3 and loss of MAFB staining within podocytes of organoid glomeruli in response to 10 µM doxorubicin (Figure 4D). Quantitative RT-PCR (qRT-PCR) showed upregulation of the kidney injury molecule KIM1 (HAVCR) and Bcl2-associated X protein (BAX), an indicator of apoptosis, at 10 µM (Figure 4E). Doxorubicin also downregulated key podocyte markers NPHS1 and PODXL at 2 µM, while the proximal tubule gene CUBN was only downregulated in response to 10 µM doxorubicin (Figure 4F), suggesting differences in cell type-specific sensitivity due to concentration. To further evaluate dose response, organoids were bioprinted in 6-well or 96-well format, using ATP content as a readout for viability, and treated with doxorubicin from 24 nM to 25 µM.Viability was affected by doxorubicin exposure in a dose-dependent manner, with comparable IC50 values ​​in response to treatment in both 6-well and 96-well formats (6-well IC50: 3.9 ± 1.8 μM, 96-well IC50: 3.1 ± 1.0 μM) (Figure 4G). Aminoglycosides are a class of broad-host-range antibiotics commonly used to treat infections caused by Gram-negative pathogens. Kidney injury due to acute tubular necrosis is a common complication of aminoglycoside therapy resulting from high intracellular accumulation within proximal tubular cells.

[0153] To assess the response of kidney organoids to this class of compounds, organoids were bioprinted in a 96-well format and treated over a wide concentration range with a panel of known nephrotoxic aminoglycosides, including amikacin, tobramycin, gentamicin, neomycin, and streptomycin. Cell viability, as measured by cellular ATP content, decreased in a concentration-dependent manner after 72 h treatment with all aminoglycosides evaluated (Figure 4H).

[0154] Thus, the bioprinted kidney tissue exemplified herein represents a practical approach to drug testing applicable to the evaluation of nephrotoxicity of novel drugs or drug scaffolds with the reproducibility required to support preclinical safety evaluation. Example 5. Bioprinted kidney tissue conformation alters nephron pattern and number.

[0155] Generating bioprinted organoids using the methods disclosed herein not only provides better quality control and increased throughput, but also allows for analysis of the effects of changes in organoid conformation on tissue morphology. During extrusion bioprinting, the scale and conformation of the cellular micromass formed can be controlled by precisely positioning and moving the needle tip in three dimensions as the cells are extruded.

[0156] material and method Bioprinted organoids were prepared using the methods outlined in Example 2.

[0157] Bead-based analysis of cell density and height during printing The cell paste was spiked with 4um Tetraspec beads (Thermo-fisher), 1ul of bead suspension per 50ul of paste. Organoids were imaged within 2-3 hours of bioprinting to capture brightfield and fluorescent bead signal, and imaged again at various time points during organoid culture. Imaging was performed using an Andor Dragonfly pinning disk confocal with a 4x 0.2NA Nikon objective, starting from the transwell surface and capturing z-stacks until no bead signal was detectable. Fiji (Schindelin, J. et al. Nature Methods, 9, 676-682. (2012)) was used to stitch the tiled datasets and generate maximum projections of bead images. A custom Python script was used to count individual beads in each dataset, and the final count data was analyzed in R. The surface area derived from the distribution of beads was used to estimate the height of the organoids at the time of printing as the height they would be if they had a vertical sided geometry, and had the same surface area and volume as the deposited organoids.

[0158] Measuring organoid height at D7+0 Organoid height was assessed by image-based quantification of pre-labeled cells using Fiji (Schindelin, J. et al.). Prior to bioprinting, 10% of the cells were removed and labeled with CellTrace Far Red (ThermoFisher, C34564) according to the manufacturer's instructions. The labeled cells were mixed back with the remaining cells and bioprinted to achieve sparse labeling in the micromass. Two independent sets of organoids were characterized at D7+0 by removing the transwell containing the organoids and placing them flat in a dish (Sarstedt) with a small amount of medium. This allowed imaging at a much shorter working distance while preventing the organoids from drying out. Images were captured using an Andor Dragonfly spinning disk with a Nikon 1.15 NA 40x water immersion objective, capturing images at a voxel size of 0.325 x 0.325 x 0.5 microns. The highest and lowest points of the image stack were measured manually under orthogonal view in Fiji. For each sample, the image was divided equally into three sections (top, middle, bottom) in the XY plane along the Y axis (Figure 6G). Then, in each section, the two highest and two lowest points were recorded over a 300 micron range in the central region of the image (150 microns in both -X and +X directions from the center). That is, for each condition, six highest and six lowest points were collected. The organoid height was calculated as follows: H (mm) = [average (number of slides with the highest 6 points) - average (number of slides with the lowest 6 points)] x voxel depth (mm) Data was compiled in R for analysis and plotting.

[0159] Quantitative imaging of reporter cell lines Bioprinted D7+12 organoids were live imaged for mTagBFP2 intensity using an Apotome.2 fluorescence microscope (Zeiss) via brightfield. For automated imaging, transwells were transferred to glass-bottom 6-well dishes (CellVis) and imaged using an Andor Dragonfly spinning disk confocal with a 4x 0.2NA Nikon objective. Tiled datasets were stitched using Fiji (Schindelin, J. et al. Nature Methods, 9, 676-682. (2012)). Areas of mTagBFP2 signal were segmented and measured using a Python script using the scikit-image library (Van der Walt, S. et al. Peer J, 19, 2e453. (2014)). The total size of each organoid was estimated by calculating the convex hull around each mTagBFP2 region. The length of the organoid was estimated by the length of the major axis of each object. A small number of organoids were excluded from the final analysis based on a ratio of mTagBFP2 positive pixels:total pixels >0.8, indicating errors in the manually verified segmentation.

[0160] Bulk RNAseq transcriptional profiling RNA was extracted from D7+12 organoids using Bioline Isolate II Mini / Micro Kits (Bioline, New South Wales, Australia) according to the manufacturer's instructions. RNA was used to generate libraries for sequencing using an Illumina Novoseq 6000 sequencer. Fastq files were trimmed using Trimmomatic (0.35). Reads were mapped to the human genome (GRCh38) and counts were performed using STAR aligner (2.5.3a) (Dobin, A. et al. Bioinformatics 29, 15-21. (2013)). EdgeR (3.26.5) (Ritchie, ME, et al. Nucleic Acids Research. 43, e47 (2015)) was used for library normalization and differential gene expression testing using quasi-likelihood negative binomial generalized log-linear models.

[0161] result By varying the tip movement speed to keep the cell extrusion rate constant, tissue height (thickness) can be finely controlled as cells spread over a larger surface area and subsequently aggregate (Figure 5A). Tissue conformation was defined in terms of deposition rate, given by the ratio of tip movement along the transwell surface to the volume of deposited cell suspension. The bioprinter was able to print the same total cell number (1.1 × 10 5 We programmed the deposition rate to create organoids containing approximately 1000 cells (ratio 0, no tip movement during extrusion) but varying from a single point deposition (ratio 0, with 12 mm of movement during extrusion) to a line of cells approximately 12 mm long (ratio 40, with 12 mm of movement during extrusion) (Figure 5A,F). Finally, we went from the formation of a classical organoid structure in which micromasses were deposited as "dots" to organoids formed as "lines" of extruded cell paste. With increasing deposition rate, we were able to obtain approximately equal amounts of the same absolute number of starting cells for each organoid (1.1 × 10 5To maintain cell density (cell density), the line length was increased, resulting in a thinner cell mass that spread over a larger surface area. To empirically confirm this, the cell paste was spiked with fluorescent beads, which showed a similar degree of spreading, but were easily imaged and automatically quantified upon printing (Figure 5B, Figure 6). This reduced the number of cells by 1 mm of occupied transwell surface area. 2 This allowed the calculation of the number of beads per cell (Figure 5C). As expected, bead density decreased as cells spread over longer distances, resulting in an approximately three-fold difference between the densest and least dense conditions (Figure 5C). We also used 3D confocal microscopy to measure tissue height in the first 24 hours after bioprinting. By measuring sparsely labeled tissue, we were able to carefully pinpoint the location of the upper and lower cell limits for each organoid, confirming that high deposition rates result in high tissue volumes (Figure 5D, Figure 6F-G).

[0162] Replicate sets of organoids were generated in the measured conformations, allowing for differentiation and patterning over 12 days after bioprinting. The absolute tissue height of each organoid after 12 days of culture was measured and compared to the approximate starting height at the time of cell extrusion (Figure 5D,E). In both conformations, height increased as they grew, but thicker starting organoids remained thicker after culture (Figure 5E). In these experiments, MAFB was used as described above. mTagBFP2 The reporter strain was used to generate cell pastes, allowing efficient imaging across regions of glomerular tissue across replicate live samples (Figure 5F, Figure 6). mTagBFP2Expression of mTagBFP2 coincided with staining for NPHS1 (nephrin) protein, indicating specificity of MAFB-induced blue fluorescence to podocytes within the forming glomerulus (Figure 12). Thus, fluorescent imaging of live organoids allowed quantification of MAFB-positive area as a surrogate for nephron number. An image processing script was applied to calculate the area of ​​each organoid that contained mTagBFP2-positive structures (MAFB-expressing podocytes in the glomerulus) as a measure of nephron number. Organoids with a long, thin starting conformation had a larger total mTagBFP2-positive glomerular area than small, thick organoids, despite being derived from the same number of starting cells (Figure 5G). This trend was consistent across the density gradient and could also be due to a larger volume of total nephron tissue, as all conditions contained glomerular structures. High-resolution imaging of individual glomeruli of glomerular structures identified in each conformation resulted in comparable size regardless of organoid conformation (Figure 12). Thus, thin organoids bioprinted at high deposition rates show increased nephron numbers.

[0163] Similar to glomerular number, changes in organoid conformation appear to affect organoid morphology, with unpatterned interstitial tissue most prominent in the center of ratio 0 organoids (Figure 5H). To further analyze this change in patterning, bulk RNAseq transcriptional profiling was performed to compare ratio 0 "dot" organoids to "line" organoids of two different lengths (ratio 20 and ratio 40). Genes associated with epithelial formation (CDH1, EPCAM), as well as tubular patterning and function (HNF4A, CUBN, LRP2, SLC12A1) were upregulated at ratio 40 (R40), whereas vascular (FLT1, SOX17, PECAM) and interstitial / fibroblast (THY1, DCN) development were upregulated at ratio 0 (R0) (Figure 7A). GO analysis of pathway changes also suggested improvements in membrane trafficking, extracellular 228 organization, and cell-cell adhesion in the R40 lines compared to the bioprinted R0 dots (Figure 7B). Such changes may reflect changes in the relative ratios of cell types or individual levels of gene expression within such cell types. High-resolution imaging of stained organoids at Ratio 0 and Ratio 40, showing the location of glomeruli (endogenous mTagBFP2), proximal tubules (HNF4A), and endothelial cells (SOX17), revealed the presence of a broad rim of tissue containing a vascular network in the dots, which was reduced in the lines (Figure 7D). These conventional micromass dots also exhibited a clear central core without formed nephrons, as evidenced by nonspecific secondary antibody staining (Figure 7D). Conversely, when organoids were bioprinted as lines, nephrons were uniformly present throughout the width of the tissue (Figure 7D).

[0164] Example 6. Comparison of cellular composition and maturation between organoid conformations by single-cell RNAseq While there are clear changes in nephron uniformity when organoid conformation is changed, significant evidence has previously been found of changing patterns between individual organoid differentiation experiments, even when performed with the same cell line. To validate the reproducibility of this change in morphology and to determine whether the relative composition of cell types or maturation of individual constituent cells varies with organoid fabrication mode (manual vs. bioprinting) or conformation (dots vs. lines), we performed extensive transcriptional profiling (single-cell RNA sequencing; scRNAseq) of three organoid conformations (manual organoids, bioprinting ratio 0 deposited "dots" [R0], and bioprinting ratio 40 deposited "lines" [R40]).

[0165] material and method Generation and analysis of single-cell RNA sequencing libraries Four replicate organoid sets were generated, where each replicate was derived from an independent pool of D7 differentiated iPSCs derived from three monolayer culture wells. For each pool, cells were loaded into the bioprinter and a pattern consisting of three R0 "dots" and three R4 "lines" per well was printed across 10-12 wells (2 plates). At the same time, the remainder of the cell pool was used to generate hand-grown organoids. Bioprinted organoids were 1.1 × 10 5 While organoids were generated from cells, the number of manually generated organoids was 2.3 × 10 5 Printed runs were generated from cells because it was technically not possible to manually manipulate the small masses. Replicate sets were processed sequentially on the same day to ensure that cells were always loaded and printed within a short time. Cells were printed approximately 10 minutes after loading, and runs were completed within approximately 20 minutes of loading.

[0166] Organoids were isolated on D7+12 according to previously published methods (Vanslambrouck JM, et al. J Am Soc Nephrol 30, 1811-1823 (2019)). For each of R0 and R40, 9 organoids from 3 wells (3 per condition, 3 per well) were isolated. Manually, 3 organoids per replicate were isolated. Replicates were multiplexed according to the method of Soeckius et al. (Genome Biol. 19, 224. (2018)). Cells were stained with 1 μg of BioLegend TotalSeq-A anti-human hashtag oligo antibody (BioLegend TotalSeq-A0251, 0252, 0253, 0254) for 20 min on ice. Cells were washed 3 times and then pooled in equal ratios for sequencing. For each suspension / condition (manual, R0, R40), a single library consisting of an equal-sized pool of each replicate was generated (sets 1-4). Libraries were produced according to the standard 10x Chromium Next GEM Single Cell 3'Reagent Kits v3.1 protocol, except that "superloading" of the 10x device was performed with approximately 30k cells (Lun, AT, et al. F1000Research 5, 2122. (2016)). Hashtag oligo (HTO) libraries were generated according to BioLegend's protocol. Sequencing was performed using an Illumina Novoseq.

[0167] The 10x mRNA library was demultiplexed using CellRanger (3.1.0) to generate a matrix of UMI counts per cell. The HTO library was demultiplexed using Cite-seq-count (1.4.3) to generate a matrix of HTO counts per cell barcode. All data was loaded into Seurat (3.1.4) to match the HTO library to the mRNA library. Seurat was used to normalize HTO counts and determine a cutoff to assign an HTO ID per cell (cutoff was typically 100–200 counts per cell). Doublets and unassigned cells were excluded, as were cells with mitochondrial content >15% or with fewer than 1000 genes, resulting in sorted datasets with final sizes of 9963 cells for manual sorting, 8912 cells for R0, and 13525 cells for R40. Genes with counts of fewer than 20 cells were excluded. The combined dataset contained a median of 2034 genes per cell and a median UMI count of 5499 per cell.

[0168] Data were normalized using the SCTransform method (Lun, AT, et al. F1000 Research 5, 2122. (2016)) and merged using Seurat to obtain a single data set. Clustering was first performed to identify clusters belonging to the interstitial, nephron, or endothelial compartments. The Clustree package (Wolock SL, et al. Cell Syst, 8: 281-291 (2019)) was used to visualize the clustering and determine a stable clustering resolution. Nephron and interstitial populations were again normalized and clustered with SCTransform to obtain a more fine-resolution view of cellular heterogeneity. At this level of resolution, we were able to identify clusters with high calculated doublet scores, and identities that appeared to combine two known cell types, using Scrublet (0.2.1) (Lindstrom, NO et al. J Amer Soc Nephrol 29, 806-824. (2018)). These were presumed to be indistinguishable doublets consisting of a single HTO identity and were excluded from further analysis. Marker analysis was performed using Seurat FindMarkers function and restricted to positive markers (i.e., increased expression within a cluster) with >0.25 log fold change. Marker lists were exported and cluster identities were determined by comparison with published human single-cell data (Chen, J. et al. Nucleic Acids Research 37, W305-311. (2009)) or by gene ontology analysis using ToppFun (Berg S. et al. Nature Methods, 16, 1226-1232. (2009)).

[0169] Differential expression tests were performed using the sumCountsAcrossCells function in Scater (1.12.2), where counts were summed to generate "pseudo-bulk" counts per cluster, per replicate, generating a matrix of gene counts across the 12 conditions (four replicates per organoid conformation). This matrix of counts was used as input to perform differential expression tests in EdgeR (3.26.5) using a quasi-likelihood negative binomial generalized log-linear model in the glmQLFFit function. For within-cluster differential expression tests, genes that appeared to be differentially expressed in three or more clusters were excluded from further analysis to remove potential batch effects and to focus on genes specific to certain cell types that may be more biologically relevant. Frequent altered genes tended to be mitochondrial and ribosomal genes. Genes were considered differentially expressed if their adjusted p-value was less than 0.05.

[0170] Comparing organoid and human fetal kidney data using cell identity predictions Raw fastq files for the single-cell datasets at weeks 11, 13, 16, and 18 published in Hochane et al. 2018 were downloaded from Gene Expression Omnibus and mapped to the reference genome GRCh38-3.0.0 using the cellranger and Seurat packages (3.1.5). 52was used to perform quality control and analysis. Cells with fewer than 750 features were excluded, raw counts were normalized and scaled using the SCTransform method, and dimensional reduction was performed. Datasets were merged using the fastMNN method provided in the SeuratWrappers package (0.1.0). After initial clustering, subsets identified as nephrons were isolated and analyzed again to identify progenitor, anterior sheath, podocyte, anterior tubule, distal tubule, and proximal tubule cell populations. Podocyte and proximal cell populations were further analyzed to identify the stages of maturation present within these cell lineages. The model used to identify cell types was generated using the scPred package (0.0.0.9) based on the nephron subset of the merged human fetal kidney data as a reference. This resulted in a model that classifies cells into one of the nephron subcategories (progenitor, anterior sheath, podocyte, anterior tubule, distal tubule, proximal tubule). This model was then applied to organoid single-cell datasets to define the constituent cell types.

[0171] result To address variability in experimental outcomes, libraries were generated from four individually barcoded cell pools representing replicates of each condition, allowing for robust assessment of both population and gene expression changes between conditions. Each replicate organoid set was cultured using differentiated iPSCs (MAFBs). mTAGBFP2 GATA3 mCherry ) cells and bioprinted to generate R0 dots and R40 lines, while manual organoids were generated in parallel from the same cells (Figure 8A). Sorted scRNAseq libraries represent over 8000 individual cell transcriptomes per organoid conformation. All generated organoids (n=229 organoids, from 4 replicate sets across 10 plates) contained glomeruli (MAFBs). mTagBFP2 ) and the distal nephron (GATA3 mCherry) Quantification of fluorescence confirmed the presence of previously observed organoid morphology in all conformations, where there was a clear and quantifiable increase in nephron abundance in the bioprinted cell lines, despite the same starting cell numbers (Figure 8B, Figure 9). The bioprinted lines contained many more nephrons compared to manually generated organoids, which were generated from higher starting cell numbers due to the technical limitations mentioned above (manual: 2.3 × 10 5 , R40: 1.1×10 5 , Figure 8B, Figure 9).

[0172] All single-cell datasets were integrated using Seurat (Nature Biotechnology. 36, 411-420. (2018)), which allowed for the broad identification of endothelial, interstitial, and nephron clusters in all organoid conformations (Figure 10A-C).

[0173] To determine the cell types contributing to the differential gene expression seen in bulk profiling ( Figure 7 ), we recreated a "pseudo-bulk" expression profile using a combined transcriptional profile of each major cell type, which confirmed that genes upregulated in bulk RNAseq of the R0 dots were markers of endothelial cells and genes upregulated in the R40 lineage were nephron markers ( Figure S10H ).

[0174] Re-clustering of stromal cells present within all organoid conformations revealed 10 distinct clusters (Figure 8C). There was a trend towards an increase in cluster 7 (expressing WNT5A, LHX9) and a decrease in cluster 10 (expressing ZIC1, ZIC4) in bioprinted organoids, but these differences were not statistically significant (Figure 8D, Figure 10). Overall, all stromal clusters were present in all organoid conformations, with no statistically significant differences in the proportion of each cell type. This was surprising, given that the central region was unpatterned in R0 and manually prepared organoids. However, further analysis of these organoids using immunofluorescence of stromal markers that identify the majority of the data set (MEIS1 / 2 / 3, SIX1 and SOX9) suggested the presence of a region of reduced cellularity in this central region (Figure 13). Thus, the central core appeared to contribute less to the cell clusters.

[0175] High-resolution reclustering of nephron lineage cells in the scRNAseq dataset revealed the presence of all major nephron cell types in all organoid conformations (Figure 8E-F, Figure 10D-E), with clear expression of MAFB in podocytes, HNF4A in proximal tubules, and GATA3 in distal tubule clusters (Figure 10D-E). Bioprinted organoids showed a significant increase in the distribution of early podocytes ("Pre-Pod") compared to manual organoids (mean values ​​of ~5% vs. ~10-15%) (Figure 8F), and a trend toward increased podocyte ("Pod") and distal tubule distribution in manual and R0 organoids, the latter with GATA3 expressing distal tubules in R0 organoids. mCherryThis is supported by an increased percentage of nephrons (Figure 9D). However, all identified cell clusters were present in all organoid conformations (Figure 8F, Figure 10D). We conclude that while patterning is highly similar between all organoid conformations, the total number of nephrons formed is higher in the bioprinted lines.

[0176] Kidney organoids generated as bioprinted lines show improved proximal tubule maturation and increased nephron number. To assess potential differences in maturation, we identified genes within each cell cluster that showed significant differences in expression between conformations. This revealed the greatest differences between the hand-made organoids and the R40 bioprinted line, particularly in the identity of the distal nephron (Figure 8G). There were fewer differences in individual nephron cell types between the R0 and R40 bioprinted organoids, with the greatest number of differentially expressed genes occurring in nephron progenitor cells (Figure 8G). Importantly, there was evidence of improved maturation of the proximal tubule epithelium in the bioprinted R40 line, but not in the bioprinted R0 dots, compared to the hand-made organoids. Genes known to be related to mature tubule function and metabolism, such as key solute channels (SLC30A1, SLC51B, SULT1E1) and fatty acid metabolism-related gene FABP3, were significantly increased in R40 compared to proximal tubule cells of manual organoids (Figure 8H). Conversely, significantly higher expression of markers such as JAG1 and SPP1 in manual organoid cells suggested less maturity (Figure 8H).

[0177] Differential expression analysis within stromal clusters identified the greatest differences between conformations within stromal clusters 0, 1, 2, and 3 (Figure 8I). Clusters 2 and 3, which are most similar to early nephrogenic mesenchyme, showed significant upregulation of kidney development-related genes in the bioprinted R40 line, such as HOXA11, FOXC2, EYA1, and SIX1, as well as developmental signaling-related genes WNT5A and RSPO3 (Figure 8J-L). That is, although this stromal cell type was present in all conformations, in the bioprinted line these cells appear to have a closer identity to early nephron progenitors. This may contribute to the increase in nephrons in the bioprinted line.

[0178] To more clearly compare the maturation of distinct organoid conformations, we used an independent analytical approach to predict the cellular identity of each cell within the organoids based on a direct comparison with human fetal kidney. Using the scPred method, we created a model that predicted cell identity based on transcriptional similarity to a published scRNA training dataset of human fetal kidney (11-18 weeks gestation) (Figure 14A). This model was used to analyze all organoid data again to provide an unbiased prediction of cell types within the organoids. This approach again confirmed a significant increase in prepodocytes within R40 organoids (Figure 14B). Genes shown to be differentially expressed in the R40 proximal tubule cell cluster were selectively expressed within the most mature proximal tubule cells of human fetal kidney (Figure 14D). Thus, it can be concluded that despite experimental variability, the bioprinted line showed improved nephron maturation and increased glomerular number compared to other conformations.

[0179] Example 7. Bioprinted kidney tissue patches with increased nephron numbers The clinical use of stem cell-derived kidney tissue requires the ability to significantly increase the number of nephron structures present in the transplanted tissue. Herein, we surprisingly found that altering the conformation of kidney organoids using extrusion bioprinting allows for maximizing the final number of nephrons from a given starting cell number. This suggests that conformational changes can increase the spread of kidney tissue.

[0180] material and method Proximal tubule functional assay Functional uptake assays were performed on HNF4A at D7+14 cultured on 6-well transwell plates. YFP The study was performed on mouse-derived patch organoids, differentiated and generated as described above. Organoids were incubated overnight (standard 37°C CO2 incubator conditions) with tetramethylrhodamine isothiocyanate-bovine albumin (TRITC-albumin; Sigma-Aldrich) substrate dissolved at 1:500 in TeSR-E6 (STEMCELL Technologies) and added to the basolateral compartment beneath the insert of the transwell. After incubation, organoids were washed three times with Hanks' balanced salt solution (HBSS; Sigma-Aldrich), transferred to glass-bottom 6-well plates, and imaged live on a ZEISS LSM 780 confocal microscope (Carl Zeiss, Oberkochen, Germany).

[0181] result Using the methods described in Examples 2 and 5 and a script ( FIG. 11A ) that generates a series of parallel lines, a bioprinted kidney tissue patch was extruded using the same extrusion parameters as the line with a ratio of 30. The bioprinted kidney tissue patch contained a total of approximately 4×10 5 The resulting kidney tissue patches were visualized by illumination under bright field and detection of endogenous MAFB cells after 12 and 14 days of culture. mTagBFPThe reporter signal and additional kidney markers were analyzed by confocal imaging. These analyses revealed that the epithelial structures and MAFBs were clearly visible throughout the patch. mTagBFP2 The results revealed a uniform distribution of glomeruli expressing IL-1, as well as the absence of central regions lacking nephrons as observed in dot organoids with a ratio of 0 (Figure ​(Figure11B,C). 11B,C). Patch organoids also showed correctly patterned nephrons expressing markers of proximal tubules (LTL and HNF4A) and distal tubules / loops of Henle (SLC12A1), surrounded by interstitial endothelial cells expressing SOX17 (Figure ​(Figure11D). 11D).

[0182] The surrogate renal tissue must contain nephrons with similar functional capabilities to their in vivo counterparts, such as glomerular filtration, and tubular reabsorption / secretion of water and selected solutes. Given the importance of the proximal tubule in solute reabsorption, we developed a proximal tubule-specific iPSC reporter line in which yellow fluorescent protein (YFP) was inserted under the control of the HNF4A promoter (HNF4A YFP Patch organoids were generated from iPS cells. HNF4A YFP The derived bioprinted patches were incubated overnight in a fluorescently labeled protein substrate (TRITC-albumin) with affinity for megalin and cubilin receptors expressed in podocytes of the glomerulus and proximal tubule. Live confocal imaging revealed specific uptake of TRITC-albumin into YFP-positive proximal tubules, confirming functionality of these nephron segments (Figure 11E).

[0183] The relative number of glomeruli per extruded unit cell was shown to increase approximately 2.5- to 5-fold by changing the ratio setting from 0 to 40, so 5 × 10 5 It is expected that a 4.8 × 6 mm patch generated by cell extrusion can contain up to 250–500 nephrons, and thus a 10 × 12 mm patch can generate 1000 nephrons.

[0184] Collectively, these data highlight the potential use of patch organoids to generate functional kidney tissue suitable for a wide range of bioengineering or screening applications.

[0185] Example 8. Comparative Example of Transplantation of Bioprinted Organoids Bioprinted kidney organoids (or single-point deposit (ratio 0) kidney tissue) generated in Example 2 were transplanted into mice. Eight-week-old recipient mice (n=8, non-obese diabetic / severe combined immunodeficiency (NOD / SCID), Charles River Laboratories) were anesthetized with isoflurane and injected with Temgesic (buprenorphine) to relieve pain before surgery. Core temperature was maintained at 37°C. A lateral incision was made to expose the kidney, and a small incision was made in the renal capsule. Bioprinted kidney organoids cultured for 7+18 days were bisected and transplanted under the renal capsule of the left and right kidneys. After 7 and 28 days, the mice were anesthetized and euthanized, and the kidneys were harvested.

[0186] Bioprinted organoids (days 7+18) were fixed with 2% paraformaldehyde (PFA) for 20 min at 4°C. Organoids were permeabilized and blocked with 10% donkey serum in 0.3% TritonX in PBS for 2 h. They were incubated overnight with primary antibodies and detected with secondary antibodies for 2 h at room temperature or overnight at 4°C. Organoids under the mouse kidney capsule were snap frozen in TissueTek or fixed with 2% PFA for 20 min and stored in PBS for whole mount analysis. Frozen kidney sections (5–10 μm thick) were fixed with 2% PFA for 10 min at room temperature and permeabilized with 0.3% TritonX in PBS for 15 min. Bioprinted kidney organoids and mouse kidney structures were detected using the Mouse on Mouse Basic Kit.

[0187] Assessment of immunofluorescence properties of transplanted and non-transplanted organoids can be performed using antibodies such as NPHS1 (AF4269, R&D Systems), WT1 (SC-192, Santa Cruz Biotechnology), CUBILIN (SC20607, Santa Cruz Biotechnology), CD31 (555444, BD Biosciences), ECAD (610181, BD Biosciences), LTL-biotin conjugate (B-1325, Vector Laboratories), or in other examples highlighting organoid-derived tissue or antibodies such as MECA-32 (553849, BD Biosciences) to label mouse-derived cell types, in this example mouse endothelium. Live fluorescent imaging can also be used for bioprinted organoids generated using reporter strains.

[0188] Transplanted organoids can also be examined using paraffin-embedded tissue and stained with various immunochemical stains, such as hematoxylin and eosin, or Periodic Acid Schiff (PAS) stain, and then sectioned for histological examination. Transplanted organoids can also be examined using transmission or scanning electron microscopy.

[0189] The results described herein suggest that bioprinted organoids can be transplanted, remain viable after transplantation, attract vasculature, and show improved maturation.The results of the present invention also suggest that transplantation assays can be used to compare the relative tubule maturation and outcome success between bioprinted organoids generated from different starting cell lines, such as reporter iPSC lines or patient-derived iPSC lines.

Claims

1. layers of kidney tissue in the form of lines; and 0.2 mm per 10,000 cells ２ Nephron tissue has a surface area of 1. A bioprinted kidney tissue comprising:

2. The layer of kidney tissue is 1 mm ２ 2. The bioprinted kidney tissue of claim 1, comprising about 30,000 cells or less per well.

3. 2. The bioprinted kidney tissue of claim 1, wherein the layer of kidney tissue expresses one or more of SULT1E1, SLC30A1, SLC51B, FABP3, HNF4A, CUBN, LRP2, EPCAM and MAFB at higher levels compared to manually aggregated kidney organoids or bioprinted kidney organoids generated as dots or blobs of cells.

4. 4. The bioprinted kidney tissue of claim 3, wherein the kidney tissue comprises nephrons, proximal and distal tubule segments of which express maturation markers including HNF4A and SLC12A1.

5. 5. The bioprinted kidney tissue of claim 4, wherein the kidney tissue expresses one or more of the markers HNF4A, CUBN, LRP2, EPCAM and MAFB.

6. 10. The bioprinted kidney tissue of claim 1, wherein the layer of kidney tissue is about 50 μm or less in height.

7. 10. The bioprinted kidney tissue of claim 1, wherein the layer of kidney tissue has a length of about 1 mm to about 30 mm and a width of about 0.5 mm to about 20 mm.

8. The layer of kidney tissue has a thickness of about 5 to about 100 nephrons per mm ２ 8. The bioprinted kidney tissue of claim 7, comprising:

9. 2. The bioprinted kidney tissue of claim 1, wherein said layer of kidney tissue comprises glomerular structures expressing MAFB uniformly distributed throughout said layer.

10. 1. A method for producing a bioprinted kidney tissue, comprising: bioprinting a quantity of bio-ink onto a surface, the bio-ink comprising a plurality of cells differentiated from human stem cells (HSCs) selected from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), the bio-ink being bioprinted in a layer about 50 μm high or less in the shape of lines; and coaxing the quantity of bio-ink to form a bioprinted kidney tissue.

11. 11. The method of claim 10, wherein the volume of bio-ink comprises about 10,000 cells / μl to about 400,000 cells / μl.

12. 11. The method of claim 10, wherein the bioprinted kidney tissue comprises about 5 to about 100 nephrons / 10,000 cells.

13. The method of claim 10 , wherein the surface is a biocompatible scaffold.

14. 14. The method of claim 13, wherein inducing the predetermined amount of bio-ink comprises contacting the bioprinted kidney tissue with FGF-9.

15. 11. The method of claim 10, wherein the plurality of cells is contacted with CHIR99021 prior to being bioprinted.

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