Method of Reseeding Adherent Cells Grown in a Hollow Fiber Bioreactor System

Patent metadata

Field	Value
Publication number	US-2016304831-A1
Application number	US-201615195601-A
Country	US
Kind code	A1
Filing date	Jun 28, 2016
Priority date	Mar 5, 2008
Publication date	Oct 20, 2016
Grant date	—

Abstract

This invention is directed to methods of directly reseeding harvested adherent cells grown in a hollow fiber bioreactor. Also disclosed is a novel harvest media for use in directly reseeding adherent cells into a hollow fiber bioreactor.

First claim

1 . A method for directly reseeding harvested adherent cells grown in growth media in a hollow fiber bioreactor having an intracapillary space and an extracapillary space wherein the cell growth space is one of the intracapillary space or the extracapillary space, comprising the steps of:
removing the growth media from the cell growth space; washing the cells to remove residual growth media from the cell growth space; loosening the cells from the cell growth space by adding trypsin to the cell growth space; removing the cells and any trypsin from the cell growth space; inactivating the trypsin from the removed cells; and directly reseeding the removed cells and trypsin into the cell growth space of a hollow fiber bioreactor.

2 . The method of claim 1 wherein the step of removing the cells and any trypsin further comprises the step of pushing fluid through the hollow fibers from one of the intracapillary space or the extracapillary space that is not the cell growth space to the cell growth space.

3 . The method of claim 1 wherein the step of inactivating the trypsin further includes the step of adding protein to the removed cells and trypsin.

4 . The method of claim 3 wherein the step of adding protein further includes the step of adding protein to a cell harvest bag before the removed cells and trypsin are added.

5 . The method of claim 1 wherein the step of inactivating the trypsin further includes the step of adding complete media to the removed cells and trypsin.

6 . The method of claim 3 wherein the step of adding protein further includes the step of adding protein directly to the removed cells and trypsin as the cells and trypsin are removed from the cell growth space.

7 . A media for harvesting cells grown in a hollow fiber bioreactor comprising;
base media; protein; 3.9 mM calcium chloride; 9.91 mM anhydrous D-glucose; and 2.24 mM magnesium sulfide.

8 . The medium of claim 7 wherein the base media is alpha-MEM.

9 . A method of making harvest media comprising the steps of;
calculating the amount of ions and protein needed in the media using C h v h +0v circuit =c f v h +c c v c ; and adding the calculated amounts of ions and protein to the media.

Assignees

• Terumo Bct Inc

Inventors

• Antwiler Glen Delbert
• Windmiller David A
• Givens Monique

Classifications

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title

• C12M25/10 Primary

  Hollow fibers or tubes (hollow fiber modules in general B01D63/02) · CPC title

• C12N2500/14

  Calcium; Ca chelators; Calcitonin · CPC title

• C12M25/12

  the culture medium flowing outside the fiber or tube · CPC title

• C12N2500/34

  Sugars · CPC title