“Dolly the double” debuts in a cleanroom

“Dolly the double” debuts in a cleanroom

Nicole S. Rohlman


Edinburgh, Scotland — Three controlled environment suites were instrumental in the recent successful breakthrough in cloning of the now-famous sheep, Dolly, through nuclear transfer from a differentiated fetal adult cell. The project, which attracted worldwide attention this year, was a joint effort by PPL Therapeutics and the Roslin Institute.

“Ensuring the absence of bacterial, viral or fungal contamination, which could introduce toxic products into the cell culture line, is critical to the success of the work we have done in nuclear transfer with a differentiated adult cell,” says Ron James, managing director at PPL Therapeutics. “A healthy cell culture line affects passaging (multiplying numbers of cells in the culture) because microbial contamination can inhibit the normal functioning of the cell. If this occurs, nuclear transfer will not be successful.”

The tissue culture suite at PPL`s research facility at Edinburgh is where the breakthrough technology began. In this Class 10,000 controlled environment, specially selected tissue samples are cultured, manipulated and stored until required later in the process. The suite, a 20- by 16-foot room, runs a positive pressure regime for containment purposes.

Microbial resistant materials were of the greatest importance in the design of the tissue culture suite. For that reason, a non-plasticizer PVC-faced, galvanized sheet steel wall and panel system, developed by MSS Clean Technology (Raleigh, NC), was utilized in the construction of the modular suite. The surface finishes of the system do not, by design, support the growth or survival of microbial organisms. The system selected further eliminated contamination from out-gassing, particulate shedding or generation.

The second phase of the nuclear transfer technology moves to the surgery suite at PPL`s Animal Sciences Facility in Orminston, Scotland, approximately 10 miles from the research facility. The facility is located on an old arable Scottish farm complete with green pastures, where modern buildings and controlled environments have replaced old stone barns.

At this point in the process, unfertilized donor eggs are recovered from the super-ovulated sheep in the 21- by 15-foot Class 10,000 controlled environment, running at a minimum of 20 air changes per hour. “Contamination control at both the egg collection and micro-injection stages of the nuclear transfer process are crucial on all levels. Sanitation, material cleanability and durability become the greatest concerns to contamination control in the surgery suite,” James says. “The aggressive cleaning regime required of the surgery suite necessitated material incorporation which could withstand our sanitation needs while being easy to clean.”

The sterilization and cleanability issues in the surgery suite were addressed by the aseptic design of the controlled environment, which included large radius coving on all wall-to-wall and ceiling-to-wall corners and interfaces. Three-way corners are radiused with a three-dimensional corner piece, eliminating the cleaning obstacles associated with 90 degree corners. Flush window and door systems eliminate vertical and horizontal ledges. Panel joints are smoothly sealed with a PVC weld system, providing a homogeneous and monolithic interior resistant to aggressive chemical cleaning regimes.

From the surgery suite, the nuclear transfer process moves to the micro-injection suite. At this stage in the process, the genetic material from the donated egg is extracted using a fine hollow needle, working under a microscope to produce the enucleated egg, which will later be fused with a single cell taken from the cell culture line. The micro-injection controlled environment suite is a 10- by 15-foot Class 10,000 room with a uniflow air-handling system. Here, the same contamination control issues apply as in the tissue culture suite.

The fusion of the single cell with the enucleated egg occurs in the micro-injection suite. The cell culture cabinets are also kept here. They contain the fused embryos needed for the beginning of the embryo development process, which is signified by cell division. The developed embryo is then stored overnight in controlled environment incubators before being surgically implanted in a hormonally prepared ewe, an operation which takes place in the surgery suite.

According to James, the company intends to extend its work to other species — particularly cows and pigs — and to commercialize the technology. This can be done by genetically modifying the cell lines, i.e., introducing human genes into the cells in a precise manner, before using them to make transgenic animals via the nuclear transfer technique.

“Contamination control issues will certainly continue to be crucial with the expansion of our research and the commercialization of our techniques,” James adds.

For more information, contact: PPL Therapeutics, Tel. +44 (0) 131 440 4777 or MSS Clean Technology, Inc. (919) 878-8577.

Nicole S. Rohlman works for MSS Clean Technology, an international cleanroom design/build company specializing in cleanroom consultation, design, manufacture, and installation for the pharmaceutical and biotechnology industries.


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