Methods of measuring cell purity for making quality control determinations and related compositions

• US 10,392,598 B2
• Filed: 06/17/2016
• Issued: 08/27/2019
• Est. Priority Date: 06/19/2015
• Status: Active Grant

• Current Assignee: Emory University
• Original Assignee: Emory University
• Inventors: Xu, Chunhui, Nie, Shuming, Han, Jingjia
• Primary Examiner(s): Nur, Abdullahi
• Application Number:
  US15/738,109
• Publication Number:
  US 20180171287A1
• Time in Patent Office: 1,166 Days
• CPC Class Codes: C08G 65/334 containing sulfur
• CPC Class Codes: C12N 5/0081 Purging biological preparations of unwanted cells
• CPC Class Codes: G01J 3/44 Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

This disclosure relates to methods of detecting and quantifying low concentrations of cells in a sample with targeted nanoparticles having Raman reporters using surface-enhanced Raman scattering for detection. In certain embodiments, the sample is a group of cells derived from stem cells that have been differentiated into specific cell types and one is detecting residual stem cells or other progeny in order to quantify the purity of the sample. In certain embodiments, the targeted nanoparticles contain two or more Raman reporters for the purpose of multiplexing. In certain embodiments, purity measurements are used to make quality control determinations.

Claims

1. A method of detecting human pluripotent stem cells in a sample comprising:

mixing a sample comprising cells suspected of containing human pluripotent stem cells with nanoparticles, wherein the nanoparticles comprise;

a) a gold or silver core;

b) a Raman reporter molecule next to the surface of the gold or silver core;

c) a layer of thiol polyethylene glycol encapsulating the Raman reporter molecule; and

d) a specific binding agent capable of selectively binding with a target cell surface marker of human pluripotent stem cells;

wherein the mixing is done under conditions such that the specific binding agent binds target cell surface marker providing nanoparticles bound to human pluripotent stem cells;

manipulating the sample under conditions such that nanoparticles not bound to human pluripotent stem cells are separated from the sample providing a purified sample;

exposing the purified sample to electromagnetic radiation providing scattering associated with the Raman reporter molecule;

detecting an intensity of scattering associated with the Raman reporter molecule being exposed to the electromagnetic radiation indicating that the human pluripotent stem cells are in the sample;

quantifying the number of human pluripotent stem cells in the sample based on the intensity of scattering;

and wherein the number of human pluripotent stem cells in the sample is quantified to be less than 0.01 percent of the total cells in the sample.

2. The method of claim 1, wherein the nanoparticles comprise

a first set of nanoparticles comprising a first specific binding agent and a first Raman reporter, wherein the first specific binding agent is a SSEA-5 antibody and

a second set of nanoparticles comprising a second specific binding agent and a second Raman reporter, wherein the second specific binding agent is a TRA1-60 antibody;

wherein the first Raman reporter and the second Raman reporter are not the same molecule and are configured such that exposing the purified sample to electromagnetic radiation result in a first scattering intensity associated with the first Raman reporter and a second scattering intensity associated with the second Raman reporter, wherein the first scattering intensity and the second scattering intensity are at different and distinct wavelengths.

3. The method of claim 2, further comprising detecting the first and second scattering intensity.

4. The method of claim 3, further comprising quantifying the number of target cells in the sample based on the first and second scattering intensity.

5. The method of claim 1, wherein quantifying the number of target cells provides a numerical measurement, wherein the numerical measurement above a threshold value when compared to the threshold value indicates the sample is not acceptable for use in a medical application or procedure or further processing or manipulations, and wherein the numerical measurement at or below a threshold value when compared to the threshold value indicates the sample is acceptable for use in a medical application or procedure or further processing or manipulations.

6. A composition comprising a nanoparticle, wherein the nanoparticles comprise:

a) a gold or silver core;

b) a Raman reporter molecule about the surface of the gold or silver core;

c) a layer of thiol polyethylene glycol encapsulating the Raman reporter molecule; and

d) a specific binding agent capable of selectively binding with a target cell surface marker,

wherein the specific binding agent is a pentameric TRA1-60 immunoglobulin M antibody, and

wherein the immunoglobulin density is between 50 and 150 antibodies per particle.

7. The composition of claim 6, wherein the thiol polyethylene glycol has an average molecular weight of between 15 and 25 kDa.

8. A composition comprising a nanoparticle, wherein the nanoparticles comprise:

a) a gold or silver core;

b) a Raman reporter molecule about the surface of the gold or silver core;

c) a layer of thiol polyethylene glycol encapsulating the Raman reporter molecule; and

d) a specific binding agent capable of selectively binding with a target cell surface marker,

wherein the specific binding agent is a monomeric SSEA-5 immunoglobulin G antibody, and

wherein the immunoglobulin density is between 15 and 30 antibodies per particle.

9. The composition of claim 8, wherein the thiol polyethylene glycol encapsulating the Raman reporter has an average molecular weight between 2 and 10 kDa.

10. The method of claim 8, wherein the binding agent is a pentameric TRA1-60 immunoglobulin M (IgM) antibody and the immunoglobulin density is between 50 and 150 antibodies per particle.

11. The method of claim 10 wherein, the thiol polyethylene glycol has an average molecular weight of between 15 and 25 kDa.

12. The method of claim 8, wherein the binding agent is a monomeric SSEA-5 immunoglobulin G antibody and the immunoglobulin density is between 15 and 30 antibodies per particle.

13. The method of claim 12, wherein the thiol polyethylene glycol encapsulating the Raman reporter has an average molecular weight between 2 and 10 kDa.