Re-aggregation of stem cell-derived pancreatic beta cells

A β-cell, cell-based technology applied in the field of stem cell-derived pancreatic β-cell reunion, which can solve problems that hinder effective implementation

Pending Publication Date: 2020-06-05
SEMMA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lack of islet donors hinders the effective implementation of this therapy

Method used

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  • Re-aggregation of stem cell-derived pancreatic beta cells
  • Re-aggregation of stem cell-derived pancreatic beta cells
  • Re-aggregation of stem cell-derived pancreatic beta cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0219] Example 1. Method for regrouping cell clusters comprising non-native beta cells.

[0220] This example shows an exemplary method for regrouping cells disclosed from cell clusters comprising stage 6 cells generated using the methods described in U.S. Patent Application Nos. 14 / 684,129 and 14 / 684,101, which Incorporated into this article as a whole. Throughout the examples, the starting clusters are referred to as native clusters. Compared with native clusters, reunited cell clusters contained a higher percentage of cells expressing markers of endogenous mature pancreatic beta cells and exhibited stronger glucose-stimulated insulin secretion both in vitro and in vivo.

[0221] Dissociating native clusters

[0222] Native clusters cultured in 500 mL spinner flasks were removed from the incubator and allowed to settle to the bottom of the flask for 3-5 minutes. The bottle was tilted at a 45° angle and as much liquid as possible inside the bottle was aspirated through t...

Embodiment 2

[0236] Example 2. Method for thawing and culturing cryopreserved reassembled cell clusters

[0237] thaw cells

[0238] The supply of living tissue and cells for autologous and allogeneic transplantation and research is limited in part by how long a tissue or organ can be maintained in a viable state. Increasing the length of time a tissue or organ remains viable may greatly increase the likelihood that a particular tissue will reach a recipient or researcher in a viable state. In some instances of the present disclosure, thawing of cryopreserved cells to obtain functional SCβ cells is performed as described below.

[0239] To thaw and culture cryopreserved SCβ cells, remove the cryopreserved vial and thaw quickly in a 37 °C water bath for 2-3 min. When the vial is thawed and a few ice crystals remain, remove the vial from the water bath and wipe the vial with ethanol before transferring it to the cell culture hood. Prepare a 15 mL conical tube containing at least the sam...

Embodiment 3

[0243] Example 3. Characterization of reassembled cell clusters

[0244] The reconverged cell clusters generated in Example 1 were characterized to determine whether they possessed the functions and characteristics of endogenous islets. The functions and characteristics of the reassembled cell clusters were compared with those of native clusters. The comparison revealed that reassembled cell clusters were more similar to endogenous islets than native clusters.

[0245] Flow Cytometry

[0246] The percentage of cells expressing markers of endogenous mature pancreatic beta cells in the re-clustered and native clusters was measured and compared by flow cytometry.

[0247] Through the TrypLE TM Express incubation to disperse native and re-aggregated clusters into single-cell suspensions. Cells, typically 1-2 million, were washed once in PBS (1 mL) and transferred to 1.7 ml microcentrifuge tubes (Bioscience; 11510). Cells were resuspended in 4% PFA and incubated on ice for ...

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Abstract

The present application discloses cell clusters resembling the function and characteristics of endogenous pancreatic islets, and methods for making and using such cell clusters. The present disclosureprovides an in vitro cell cluster comprising at least one non-native pancreatic beta cell that exhibits an in vitro glucose-stimulated insulin secretion response when exposed to a glucose challenge,wherein the cell cluster is an unsorted cell cluster, and wherein the cell cluster comprises: at least 35% of cells that express NKX6.1 and C-peptide; at least 70% of cells that express chromogranin A; at most about 2% of cells that express SOX2; or at most about 10% of cells that express SOX9. In some cases, when transplanted into a subject, the in vitro cell cluster exhibits an in vivo glucose-stimulated insulin secretion response to a glucose challenge in the subject.

Description

[0001] cross reference [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 535,659, filed July 21, 2017, which is hereby incorporated by reference in its entirety. Background technique [0003] Pancreas or islet transplantation has been used to treat diabetes, such as type 1 diabetes. Islet transplantation does not require major surgery, and the function of the islet graft can be maintained in the recipient for years. However, the lack of islet donors has hindered the effective implementation of this therapy. Artificial pancreases or islets offer an alternative source of transplantable islets. Therefore, there is a need for in vitro reconstitution methods of islets that function and behave similarly to endogenous islets. Contents of the invention [0004] In one aspect, the present disclosure provides an in vitro cell cluster comprising at least one non-native pancreatic beta cell that exhibits an in vitro glucose-stimulated insulin secret...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/39A61K35/545A61K9/50A61P3/10
CPCA61K9/0019A61K9/5036A61K35/39C12N5/0671C12N5/0031C12N5/0676C12N2506/22C12N2509/10C12N2527/00
Inventor 柏叶军乔治·哈尔卜费利西亚·J·帕柳卡
Owner SEMMA THERAPEUTICS INC
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