Systems and methods for in vitro and in vivo imaging of cells on a substrate

Patent metadata

Field	Value
Publication number	US-10247719-B2
Application number	US-201615004712-A
Country	US
Kind code	B2
Filing date	Jan 22, 2016
Priority date	Jul 12, 2010
Publication date	Apr 2, 2019
Grant date	Apr 2, 2019

Abstract

Disclosed herein are generally to methods and systems that facilitate imaging of cells on a substrate and more particularly to pre-implantation (in vitro) and post-implantation (in vivo) imaging of cell-seeded substrates implanted in target tissues in the context of stem cell therapy.

First claim

What is claimed is:

1. A system for counting in vivo stem cell-derived retinal pigment epithelium (RPE) cells on a substrate implanted along a curvature of an eye, the system comprising:
a camera configured to take a plurality of two-dimensional images of the substrate on an x-y plane at a plurality of focal depths along a z axis, wherein the plurality of focal depths comprises a maximum focal depth and a minimum focal depth; and
a computer system comprising:
a computer processor configured to execute modules comprising at least:
an object identification module programmed to identify objects from the plurality of two-dimensional images of the substrate that are in focus;
a mapping module programmed to map the identified objects that are in focus from the plurality of two-dimensional images to generate a single image;
an initial cell counting module programmed to determine a first number of stem cell-derived RPE cells on the substrate;
a double cell counting identification module programmed to determine double counting of cells by the computer system by identifying stem cell-derived RPE cells appearing at a same x-y coordinate and appearing at different focal depths along the z axis; and

a final cell counting module programmed to determine a final number of stem cell-derived RPE cells in the substrate based on the first number of stem cell-derived RPE cells on the substrate and accounting for the double counting of cells.

2. The system of claim 1 , wherein the maximum focal depth and minimum focal depth are automatically determined by the computer system or manually determined by a user.

3. The system of claim 1 , wherein the computer processor is further configured to execute a focal depth interval determination module programmed to configure an interval between the plurality of focal depths depending on a pitch of the substrate.

4. The system of claim 1 , wherein the substrate further comprises highly localized fluorophore markers that are configured to be coupled to the stem cell-derived RPE cells and not to native RPE cells and wherein the initial cell counting module is further programmed to identify fluorescence emitted from fluorophores coupled to the stem cell-derived RPE cells.

5. The system of claim 1 , wherein the double cell counting identification module is further programmed to determine whether the stem cell-derived RPE cells appearing at the same x-y coordinate and appearing at different focal depths along the z axis are a single stem cell-derived RPE cell or more than one stem cell-derived RPE cell.

6. The system of claim 1 further comprising:
a light source configured to direct a narrow-band light at the substrate.

7. The system of claim 1 , wherein the computer processor is further configured to execute a cell number approximation module programmed to approximate a number of cells in non-imaged regions of the substrate, and the substrate further comprises markings configured to assist counting a number of stem cell-derived RPE cells.

8. The system of claim 1 , wherein the computer processor is further configured to execute an interdigitation determination module programmed to determine a level of interdigitation between stem cell-derived RPE cells on the substrate and photoreceptors.

9. The system of claim 1 , further comprising:
a first light source configured to direct a first light at the substrate, wherein the first light source has a wavelength within a range that lipofuscin fluoresces but not melanopsin;
a second light source configured to direct a second light at the substrate, wherein the second light source has a wavelength within a range that melanopsin fluoresces but not lipofuscin,
wherein the computer processor is further configured to execute an image generating module programmed to generate a first image of the substrate from fluorescence reemitted after directing the first light source and a second image of the substrate from fluorescence reemitted after directing the second light source,
wherein the initial cell counting module is further programmed to determine a first number of stem cell-derived RPE cells in the first image and a second number of stem cell-derived RPE cells in the second image, and
wherein the final cell counting module is further programmed to compare the first number of stem cell-derived RPE cells to the second number of stem cell-derived RPE cells to determine a degree of pigmentation of stem cell-derived RPE cells on the substrate.

10. The system of claim 9 , wherein a single light source comprises the first light source and the second light source.

11. The system of claim 1 , wherein the computer processor is further configured to execute a spatial separation determination module programmed to determine a degree of spatial separation between the substrate and stem cell-derived RPE cells on the substrate.

12. The system of claim 1 further comprising a photoacoustic imaging device configured to detect a presence of stem cell-derived RPE cells on the substrate.

13. The system of claim 1 further comprising a reflectometer configured to collect indirect structural information of photoreceptors within an eye to assess implantation of stem cell-derived RPE cells, wherein the reflectometer is further configured to monitor rhodopsin pigment in the photoreceptors.

14. The system of claim 1 further comprising an adaptive optics system configured to collect indirect structural information of photoreceptors within an eye with or without a scanning laser ophthalmoscope.

Assignees

• Univ Southern California
• Doheny Eye Inst

Inventors

• Humayun Mark
• Ahuja Ashish
• Le Pere Charles

Classifications

• A61K49/0017

  Fluorescence in vivo · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• G01N33/4833 Primary

  of solid biological material, e.g. tissue samples, cell cultures (tissue in vivo A61B5/00; cell suspensions G01N33/48735) · CPC title

• G06T7/0012

  Biomedical image inspection · CPC title

• A61B3/12

  for looking at the eye fundus, e.g. ophthalmoscopes (A61B3/13 takes precedence) · CPC title