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Methods of enhancing development of renal organoids and methods of using the same

An organoid and kidney tissue technology, applied in the field of enhancing the development of kidney organoids and its use, can solve the problems of inability to maintain longitudinal vitality, imperfect glomerular development, and limited physiological translatability.

Pending Publication Date: 2020-04-10
PRESIDENT & FELLOWS OF HARVARD COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in current in vitro organoid systems, glomeruli are underdeveloped and the vasculature is neither perfused nor longitudinally viable, limiting the extent of relevant applications and limiting their translatability to human in vivo physiology

Method used

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  • Methods of enhancing development of renal organoids and methods of using the same
  • Methods of enhancing development of renal organoids and methods of using the same
  • Methods of enhancing development of renal organoids and methods of using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1: Effects of Chemical Additives on Kidney Organoids

[0132] METHODS: To determine the effects of chemical additives on developing organoids, under static and perfused conditions, in basal advanced RPMI medium with VEGF supplement, GMEC medium, PMA supplement, antibiotics or FBS or FCS supplement and other Components treated vesicles (aged 11, 12, 13 and 14 days). The extent and abundance of the vasculature was studied using immunostaining.

[0133] Conclusions: Figure 22 captures most of the tested conditions (ECM, media additives, days / age of organoids, etc.). When cells were treated with antibiotics, VEGF, PMA or GMEC medium, CD31 + Cell population had no measurable effect. A dramatic effect of adding FBS is evident – ​​in this case, the formed vasculature (CD31 + cells) are more abundant. The vasculature was visible in day 11 RV and day 14 RV.

Embodiment 2

[0134] Example 2: Comparing the effect of perfusion on renal vesicles of different ages (Does the age of the RV matter?)

[0135] Methods: Attempts were made to embed developing organoids of different ages (days 9-14) on perfusion chips using a gelatin substrate (2 wt% gelatin, 10 mg / mL fibrin, 0.2% TG). They were perfused with advanced RPMI matrix + 1.5% FBS, FSS at 0.000001 to 10 dyns / cm 2 change between.

[0136] Conclusions: Organoids embedded at day 9 or 10 were too fragile and dissipated or never differentiated under FSS. Figure 22(b) highlights that if day 11 developing organoids are used, they have the potential to form highly differentiated structures with capillary invasion of the glomerular cleft. Results for developing organoids on days 12 and 13 were similar to those on day 11. Significantly reduced levels of vascular invasion of glomerular structures were observed if embedded at day 14.

Embodiment 3

[0137] Example 3: Effect of Perfusion or FSS on Kidney Organoid Development

[0138] Methods: Developing organoids of different ages (day 11-14) were embedded on a perfusion chip using a gelatin substrate (2 wt% gelatin, 10 mg / mL fibrin, 0.2% TG). They were perfused with advanced RPMI matrix + 1.5% FBS, FSS at 0 to 10 dyns / cm 2 change between.

[0139] in conclusion: Image 6 The effect of perfusion on kidney organoid development is shown. Image 6 shows that at higher FSS conditions (0.01dyn / cm 2 or higher), the abundance of the vasculature increased significantly.

[0140] RT-qPCR results further confirmed these results. Specifically, qPCR identified higher levels of vascular markers and precursors, VEGF, FLK1, CD146, and CD31, under high FSS conditions compared to static and low FSS controls.

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Abstract

Described are methods of enhancing development of renal organoids, methods of using the same, and kits.

Description

[0001] related application [0002] This patent document claims the filing date benefit of U.S. Provisional Patent Application No. 62 / 517,536, filed June 9, 2017, which is incorporated herein by reference under 35 U.S.C. § 119(e). [0003] Federally funded research or development [0004] This invention was made with government support under Contract Nos.: Contract Nos. DK007527, U01DK107350, DK039773, and TR002155 awarded by National Institutes of Health (NIH); Contract No. U01DK107350 awarded by NIH (Re)Building a Kidney Consortium; Contract No. N000141612823 awarded by Office Naval Research Vannevar Bush Faculty Fellowship Program; and Contract No. P30 DK079333 awarded by NIH supporting The Washington University KTRC. [0005] Sequence listing citations submitted via EFS-WEB [0006] The application is filed electronically via EFS-Web and includes a Sequence Listing electronically filed in .txt format. This .txt file contains the sequence listing "HU6909SequenceListing" cr...

Claims

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Application Information

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IPC IPC(8): A61L27/44A61L27/38C12N5/071C07K14/47
CPCC07K14/4702C12N15/86C07K2319/41A61K35/545A61P13/12C12N5/0686C12N2506/02C12N2506/45C12N2533/90C12N2521/00C12N2501/165C12N2533/56C12N2533/54C12N2513/00C12N2501/115A61K35/22C12N2500/00C12N2501/119C12N2503/04C12N2533/52G01N33/5082G01N2500/10
Inventor K.A.霍曼N.R.古普塔K.T.科罗尔D.B.科尔斯基M.斯凯拉-斯科特M.T.韦尔里乌斯J.邦温特R.莫里扎恩J.刘易斯
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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