Passaging and harvesting formulation and method for human pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-9885019-B2
Application number	US-201214117687-A
Country	US
Kind code	B2
Filing date	May 17, 2012
Priority date	May 17, 2011
Publication date	Feb 6, 2018
Grant date	Feb 6, 2018

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 karyo-type after passaged with the formulations for extended period of time.

First claim

What is claimed is:

1. A formulation for harvesting and subsequent passaging of human pluripotent stem cells or human mesenchymal stem cells comprising:
sodium citrate, wherein said sodium citrate is at a concentration of 1-15 mMol/Liter;
a salt, said salt comprising KCL at a concentration of 203-316 mMol/Liter and
a phosphate-buffered saline solution, wherein the phosphate-buffered saline solution is Ca2+/Mg2+-free Dulbecco's phosphate buffered saline (DPBS);
wherein said formulation has an osmolarity of 418-570 mOsmol/Liter; and
wherein said formulation is a non-enzymatic cell detachment solution.

2. The formulation of claim 1 , wherein the human pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

3. The formulation of claim 1 , wherein the salt further comprises a salt selected from the group consisting of NaCl, Na 2 HPO 3 , NaH 2 PO 3 , K 2 HPO 3 , KH 2 PO 3 , and NaHCO 3 .

4. The formulation of claim 1 , wherein the formulation is pH buffered with bicarbonate, phosphate, ethanolamine, triethanolamine, or trometamol.

5. The formulation of claim 1 , wherein the formulation has a pH between 7.2-7.8.

6. A method for harvesting and subsequent passaging of human pluripotent stem cells (hPSCs)cultured in vitro comprising:
incubating the hPSCs in a formulation of claim 1 in cell culture plates or vessels for about 2-20 minutes, wherein said hPSCs detach from the cell culture plates or vessels in clusters having cell viability between about 85-100%, and wherein the average cluster size is about 10-1000 μm.

7. The method of claim 6 , 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.

8. The method of claim 6 , wherein the average cluster size is about 40-500 μm.

9. The method of claim 6 , 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.

10. A method for harvesting and subsequent passaging of human pluripotent stem cells (hPSCs) comprising:
plating the hPSCs in medium,
aspirating spent medium,
washing with DPBS,
adding the formulation of claim 1 ,
removing the formulation of claim 1 ,
incubating,
adding culture media, and
collecting detached clusters.

11. The method of claim 6 , wherein the formulation of claim 1 is not removed.

Assignees

• Nie Ying
• Rowley Jonathan Allen
• Fellner Thomas
• Walsh Patrick
• Lonza Walkersville Inc

Inventors

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

Classifications

• 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

• C12N2500/14

  Calcium; Ca chelators; Calcitonin · CPC title

• C12N5/0662 Primary

  Stem cells · CPC title

• 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