Passaging and harvesting formulation and method for human pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-10487312-B2
Application number	US-201715843078-A
Country	US
Kind code	B2
Filing date	Dec 15, 2017
Priority date	May 17, 2011
Publication date	Nov 26, 2019
Grant date	Nov 26, 2019

Abstract

Formulations and methods are disclosed for the harvesting and subsequent passaging of human pluripotent stem cells without the use of enzymes and/or scraping to dislodge cells from cell culture vessels. The formulations and methods permit the harvesting of cells as large clusters from the surface of various cell culture vessels including multilayer cell culture vessels. Further, the formulations and methods provide high yields of harvested cells for subsequent passaging and high post-harvest cell viability. Pluripotent stem cells passaged with the formulations according to the methods remain undifferentiated and express typical stem cell markers, while, at the same time, they retain the differentiation capability and are able to differentiate into the cells in all three germ layers and generate teratomas, even after numerous rounds of harvesting and passaging. These hPSCs also maintain normal karyotype after passaged with the formulations for extended period of time.

First claim

What is claimed is:

1. A method for harvesting and subsequent passaging of human pluripotent stem cells “hPSCs”, comprising:
passaging the hPSCs with a preferred non-enzymatic cell detaching solution, at a split ratio of 1:10 to 1:60;
wherein the culture reaches confluence within seven days after split;
wherein said preferred non-enzymatic cell detaching solution is identified as follows:
creating a plurality of non-enzymatic cell detaching solutions, each of said non-enzymatic cell detaching solutions comprising at least one Ca2+ chelator at varying concentrations, and each of said cell detaching solutions having varying osmolarity, wherein said osmolarity is between 273 mOsmol/L to 1014 mOsmol/L;
testing each of said plurality of non-enzymatic cell detaching solutions, said testing comprising: test passaging hPSCs with each of said plurality of non-enzymatic cell detaching solutions, at a split ratio of 1:10 to 1:60 to yield test cells and determining percentage of culture detached at a given treatment time and determining average cluster size of said test cells; and
selecting said preferred non-enzymatic cell detaching solution from said plurality of cell detaching solutions, wherein said selection is based upon desired percentage of culture detached at a given treatment time and desired average cluster size;

wherein said selected preferred non-enzymatic cell detaching solution is used for said passaging step.

2. The method of claim 1 , wherein said passaging occurs in cell culture plates or vessels.

3. The method of claim 1 , wherein the average cluster size is about 40-500 μm.

4. The method of claim 1 , further comprising:
downstream processing of clusters, wherein downstream processing is selected from the group consisting of continuous counter-flow centrifugation technology, formulation, automated vialing and cryopreservation.

5. The method of claim 1 , wherein the hPSCs maintain pluripotency and normal G-banding karyotype at over 50 passages.

6. The method of claim 1 , wherein the split ratio is 1:15 to 1:40.

7. The method of claim 1 , wherein the osmolarity is 250-1050 mOsmol/Liter.

8. The method of claim 1 , wherein the osmolarity is 311-1014 mOsmol/Liter.

9. The method of claim 2 , wherein the cell culture plates or vessels are selected from the group consisting of petri dishes, multi-well cell culture plates, stacked cell culture apparatus, and cell culture factories.

Assignees

• Lonza Walkersville Inc

Inventors

• Nie Ying
• Rowley Jonathan Allen
• Fellner Thomas
• Walsh Patrick

Classifications

• C12N5/0663

  Bone marrow mesenchymal stem cells (BM-MSC) · CPC title

• C12N5/0607

  Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696) · CPC title

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N5/0662 Primary

  Stem cells · CPC title

• C12N2500/14

  Calcium; Ca chelators; Calcitonin · CPC title