Flexible film closed isolators have been described and used from the late 40s in sterile surgery (6) and from the late 50s in breeding of lab germ-free rodents (7).

A recent application with flexible disposable sterile isolator for the fill & finish of aseptic solution has been described (8).

The set-up here of the cleanroom is a ballroom concept. All items and materials to be introduced in the cleanroom are bar coded or equivalent, sterile and double wrapped before to go through a H2O2 lock chamber (Photo 2) after removing the first wrapping.

The cells coming from the outside could be frozen at minus 70 °C. Their entrance in the cleanroom is done through an Alpha/Beta RTP transfer system (Figure 1) which fits with the Beta entrance door of each individual CT isolator (Figure 2).

The CT isolator is a transparent flexible positive pressure disposable sterile ready to use mini- isolator of approximate150 liters volume with adjacent forearms handling, top view and a 190 RTP Beta door to be connected alternatively to the Alpha door for the entrance of the cells, to the storage isolator and to the service isolator for the loading of the required items allowing the operator to proceed. The ventilation/ filtration system of each of these isolators is composed of an inlet 0,5 micron hydrophobic membrane filter and an outlet HEPA filter. The gas used to inflate the isolator can be either regular compressed air or any combination of compressed gas to increase the development of the cells (with added CO2 for instance). The positive pressure is in the range of 30 to 50 Pa. This mini-isolator includes a “tail” as a sleeve for external thawing.

Before to be used the airtightness of each of the individual isolator is tested according to ISO 10648-2 as its inlet membrane filter is integrity tested by a water intrusion test or equivalent. Each isolator is Gamma sterilized as other types of single use bags with Beta RTP connection (11).

Key point in this configuration is the use of Alpha/Beta RTP systems for various loading and unloading without any risk of cross contamination from viable and non-viable particulates. This type of DPTE® like connection with a rotation is QC/QA by its supplier for the quality of the gaskets and the safety during multiple connections (9-10)

Main point about this very installation is the only use of “closed isolator systems” as described in (4a): “Excluded external contamination from the isolator’s interior by transfer material via aseptic connections to auxiliary equipment rather than using openings to the surrounding environment. Closed systems remain sealed throughout operations” which is a warranty against any cross contamination with the surroundings.

Other important points to consider being in accordance with the User Requirement Specifications (URS) of most of the cell therapy process are:

• Cells transfer
• Thawing
• Centrifugation • Incubation
• Waste

The cells are transferred directly to their isolator from outside of the cleanroom through the RTP system. In the case of frozen cells at minus 70°C the isolator includes a sleeve to be dipped in an external WFI water bath or equivalent during the necessary time to thaw.

Ready to Use sterile RTP containers are used as centrifuge rotors allowing off the shelf refrigerated or not centrifuge to be used without any integration and modification.
Incubation according to the flow of work can be done in an incubation room at 35°C as a part of the cleanroom or in a specific RTP container which have to be placed in an appropriate incubator or in the incubator of the service isolator.