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Cell systems using spheroids and methods of making and using the same

A technology of spheroids and cells, which is applied in the field of three-dimensional cell culture system, can solve the problem that the dissociation properties of the culture cannot replicate the horizontal environment and indicators

Pending Publication Date: 2020-09-11
TULANE EDUCATIONAL FUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This application was groundbreaking for both peripheral nervous system (PNS) and central nervous system (CNS) applications, but the dissociated nature of the culture failed to replicate the population-level environment and metrics critical to peripheral tissues

Method used

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  • Cell systems using spheroids and methods of making and using the same
  • Cell systems using spheroids and methods of making and using the same
  • Cell systems using spheroids and methods of making and using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0269] Example 1: Human motor neurochip for preclinical neurotoxicity testing

[0270] The aim is to develop organotypic microphysiological models that mimic the morphology of peripheral nerves and support clinically similar physiological measurements. The neural chip design is based on the use of microengineered hydrogel scaffolds ( Figure 1A-1B )made.

[0271] The purpose of this design is to guide and constrain 3D axon growth and cell positioning to simulate nerve fiber bundles ( figure 2 ). Robust nerve growth, bundling, and glial interactions promote morphological and physiological output as a high-content screening assay for neurotoxicity and pharmacological manipulation ( image 3 ).

[0272] The results revealed anatomy resembling native peripheral nerves. This allows for tests of nerve density, fiber type, and myelination as well as studies of axonal growth, cell migration, and glial differentiation ( Figure 4-Figure 7 ).

Embodiment 2

[0273] Example 2: Protocol for Rat Spinal Cord Nanoshuttle Spheroids

[0274] Day 1( Figure 12 ):

[0275] 1. Six spinal cords were isolated from E15 rat pups, making sure to remove all dorsal root ganglia.

[0276] 2. Cut all 6 spinal cords into small pieces with spring scissors.

[0277] 3. Using a 1000 μL pipette, transfer the diced spinal cord and medium to a 1.5 mL microcentrifuge tube.

[0278] 4. Pellet the spinal cord mass by centrifugation at 700 rcf for 2 min 30 sec.

[0279] 5. Remove the supernatant from the microcentrifuge tube, taking care not to disrupt the pellet.

[0280] 6. Add 1 mL of 0.25% Trypsin-EDTA to the microcentrifuge tube, making sure the pellet is broken up and suspended in the trypsin.

[0281] 7. Incubate at 37°C for 15 minutes.

[0282] 8. Quench trypsin-EDTA by adding trypsin + cell suspension to a 15 mL tube containing 1.5 mL of trypsin inhibitor solution.

[0283] 9. Pellet the spinal cord mass by centrifugation at 700 rcf for 5 minut...

Embodiment 3

[0335] Spheroids for directed growth of neurites

[0336] Round bottom / U-shaped bottom plate: Untreated spheroid microplates (96 wells with a distinct "U" shaped round bottom (Corning REF: 4415) were used for one differentiated cell type or a combination of differentiated cell types. The resuspended cells were re-counted. Correspondingly, cells were added to each well at the desired density (from five thousand to one hundred thousand cells) per spheroid using a micropipette. Then, the spheroids The microplate is centrifuged for 5 minutes at a centrifugation speed corresponding to the type of cells in suspension and placed in a 37°C incubator for 24 hours or longer until spheroids form.

[0337] hanging drop plate : The Perfecta3D hanging drop plate from 3D bioMatrix was used for spheroid fabrication. Known amounts of differentiated induced pluripotent cell-derived neurons and glial cells (any number from 5,000 to 100,000) are suspended in a small amount of medium. The ra...

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Abstract

The present disclosure generally relates to a cell culturing system, and specifically to a three-dimensional cell culturing system for neuronal cells that promotes both structural and functional characteristics that mimic those of in vivo peripheral fibers, including cell myelination. Using a dual hydrogel construct and spheroids comprising neuronal cells, the present disclosure provides methods,devices, and systems for in vitro spatially-controlled, three-dimensional models that permit intra- and extra-cellular electrophysiological measurements and recordings. The three-dimensional hydrogelconstructs allow for flexibility in incorporated cell types, geometric fabrication, and electrical manipulation, providing viable systems for culture, perturbation, and testing of biomimetic neural growth with physiologically-relevant results.

Description

[0001] Statement Regarding Federally Funded Research [0002] This invention was made with government support under NIH STTR Grant No. R42-TR001270. The US Government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application is an international application designated the United States of America and filed under 35 U.S.C. §120, which claims priority to U.S. Provisional Application No. 62 / 594,525, filed December 4, 2017, which is hereby incorporated by reference in its entirety. technical field [0005] The present disclosure relates generally to cell culture systems, and in particular to three-dimensional cell culture systems for spheroids that facilitate structural and functional properties that mimic those of nerve fibers in vivo, including cellular myelination and complexation Propagation of the action potential. [0006] Background of the invention [0007] Replicating functional aspects of physiology for benchtop ...

Claims

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Application Information

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IPC IPC(8): A61K35/15A61K35/28A61K35/17A61K35/545A61K35/30G01N33/50
CPCG01N33/5026G01N33/5058C12N2533/54C12N5/0619C12N2533/40C12N2513/00C12N2506/45C12N2502/1335C12N5/0697C12N2502/081C12N2502/086
Inventor M·J·穆尔J·L·科尔里A·D·沙尔玛D·波泽尔J·本恩
Owner TULANE EDUCATIONAL FUND
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