Cryopreservation bags and method of use thereof for closed system, high capacity cell-banking

Patent metadata

Field	Value
Publication number	US-9879217-B2
Application number	US-201514826720-A
Country	US
Kind code	B2
Filing date	Aug 14, 2015
Priority date	Aug 14, 2014
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The disclosure provides a cell freezing and storage bag assembly and a method for using the assembly in banking eukaryotic cells for later seed train expansion. The bag is constructed principally of fluorinated ethylene propylene (FEP) fabric, and is designed to be filled such that the cell suspension has a very thin cross-section. The bag design includes at least an inlet conduit and an outlet or inoculation conduit, which can be sterilely welded to the source of the eukaryotic cells. The use of at least two sterile-weldable conduits allows for “closed system” filling of the bags, which significantly reduces the risk of contamination relative to other cell-banking methods. The bag also include a sleeve, which can be thermo-welded to form an enclosure, which protects the inlet and outlet conduits against contamination and mechanical damage during freezing, storage and subsequent thawing. In the method, once each bag is filled, its corresponding inlet conduit is sealed, and both the inlet and outlet conduits are enclosed within the bag's sleeve. This closed system method obviates the need for sterile environments (e.g. laminar flow unit).

First claim

What is claimed is:

1. A high capacity, closed, cryopreservation cell banking system, comprising:
a. a user interface for controlling and monitoring the dispensing of liquid biological materials into a plurality of high capacity, liquid nitrogen temperature resistant cryopreservation bags;
b. a plurality of cryopreservation bags, each bag comprising:
i. a first fluorinated ethylene propylene (FEP) enclosure;
ii. at least two sterile-weldable fluid conduits and adapted for use in a high capacity biological material, closed cryopreservation banking system;
iii. a first rigid FEP inlet conduit, in fluid connection with the first FEP enclosure, and connectable to the first of the two sterile-weldable fluid conduits, wherein said first conduit is a first flexible PVC tube;
iv. a second rigid FEP outlet conduit, in fluid connection with the first FEP enclosure, and connectable to the second of the two sterile-weldable fluid conduits, wherein said second conduit is a second flexible PVC tube; and
v. a FEP pouch, which is initially open at one end, and which is contiguous with the first FEP enclosure, but thermosealed therefrom, and encircling both the rigid inlet conduit and the rigid outlet conduit, such that the pouch is neither in fluid communication with the first enclosure, nor the inlet nor outlet ports; and wherein the pouch extends sufficiently far enough such that when the first enclosure has been filled with the biological material, the PVC tubes may be withdrawn into the pouch, which is then thermosealed to form a second enclosure, which now contains both PVC tubes;

c. a plurality of fluid connections, adapted to fill the plurality of bags;
d. a thermo-sealing means, for sealing the sterile-weldable fluid conduits and bags; and
e. a plurality of cassettes for containing, freezing, storing & thawing the bags.

2. The system of claim 1 , wherein the second enclosure of the bag contains a sleeve or other means for receiving and containing a label, which may be used to recite information about the bag's contents.

3. The system of claim 1 , wherein the rigid FEP inlet conduit is connected to the inlet flexible PVC tubing via a rigid plastic barb-type taper connector, and wherein the rigid FEP outlet conduit is connected to the outlet flexible tubing via a rigid plastic barb-type connector, each connector tapered on both sides such that they can fit tightly inside their respective conduits and PVC tubing.

4. The system of claim 2 , wherein the conduits further comprise retaining rings.

5. The system of claim 2 , wherein the second enclosure protects the PVC tubes against damage and contamination that can occur during freezing, storing or thawing of the bag.

6. The system of claim 1 , wherein the bag has been filled with the biological material and wherein the PVC tubes have been withdrawn into the thermosealed second enclosure.

7. The system of claim 6 , wherein the second enclosure further contains a label.

8. The system of claim 1 , wherein each cryopreservation bag further comprises:
a. a porous filter, affixed to and in fluid communication with the bag's second PVC tube, which tube is fluidly connected to the bag's rigid FEB outlet conduit, said filter allowing for gas sterilization of the bag; and
b. an orifice, which is suitable for receiving a hangar for hanging the bag;
wherein the first and second PVC tubes both remain sterilely fixed to the rigid inlet and outlet conduits, respectively, when the bag is being filled, frozen, stored or thawed.

9. A closed system process for banking large volumes of cells using the system of claim 1 , comprising the following steps:
a. connecting the high capacity bag(s) to the high capacity closed banking system;
b. bringing a source of cells into fluid connection with the system;
c. allowing the cells to come into fluid contact with the bags and to fill each bag to a desired capacity;
d. aseptically thermo-sealing each of the inlet PVC tubes;
e. withdrawing both the inlet tubing and the outlet tubing into each pouch;
f. thermo-sealing each pouch to enclose and protect the PVC tubes and the labeling;
g. placing the bags into the cassettes; and
h. freezing the bags within their respective cassettes thereby banking the large volumes of cells.

10. The process of claim 9 , further comprising the following steps:
a. exposing each sealed bag to a water bath containing water at a suitable thawing temperature;
b. cutting the thermo-sealed enclosure to allow access to the outlet or inoculation conduit;
c. sterilely welding the inoculation conduit to tubing, which is in conditional fluid communication with a target bag, vessel or bioreactor;
d. draining each bag into the target bag, vessel or bioreactor; thereby thawing and dispensing the banked cells.

Assignees

• Merial Inc
• Merial Inc

Inventors

• Coupier Hervé François

Classifications

• B01L2300/1894

  Cooling means; Cryo cooling · CPC title

• C12M37/02

  Filters · CPC title

• C12M37/00

  Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination (C12M23/38 takes precedence; filtration in general and filters per se B01D24/00-B01D41/00; autoclaves B01J3/04; treatment of microorganisms with electrical or wave energy C12N13/00) · CPC title

• C12M37/04

  Seals · CPC title

• B01L2300/0681

  Filter · CPC title