Transcript

1. Extract pig islets

Peter Hosking (Living Cell Technologies)

The production process for the DIABECELL^® product starts, of course, with the removal of the pancreas from the neonatal piglets. Once that’s been accomplished, the pancreas is taken through into our clean room facility, and there we have very sterile conditions, and what we do there is we take the pancreas and we treat it with enzymes. And the enzymes isolate the islets of Langerhans from the exocrine tissue. There’s a number of other steps involved, which are washing, centrifuging, separation, these sorts of things, but essentially what we’re doing in that first stage of the process is separating those islets from the rest of the pancreatic tissue.

2. Islet cell culture

Peter Hosking (Living Cell Technologies)

The next step once we’ve isolated the islets is to culture them for 3 days. We do this in spinner flasks, which are about 1 litre, 1.2 litre size. They sit on a magnetic drive and they agitate. And we have in each spinner flask about 300,000 pancreatic islets, and we culture these in about 300 ml of media. We have to change the media a number of times during the 3 days because the islets require oxygen and glucose and they consume some of the nutrients that are in the media. Then after 3 days, we’re ready to proceed to the next phase, which is the encapsulation process.

3. Islet quality control

Peter Hosking (Living Cell Technologies)

After the islets have been cultured for about 72 hours, we then proceed to the encapsulation stage, and in order to do this, we first have to do quality control tests on the islets themselves. So we’ll initially harvest islets from the spinner flasks, check them for the yield – how many islets have we actually been able to produce, what’s the purity of those islets, what is the viability of those islets and what is the size of those islets? – because the size is very important for the next stage of the process, which is encapsulation.

4. Encapsulate islets to prevent rejection

Peter Hosking (Living Cell Technologies)

To start the encapsulation process, we mix the islets with the alginate solution and we pump them through a needle. Now, if this is the needle, essentially what we’re doing is pumping the solution containing alginate and islets through this needle. At the tip of this needle, we have a jet of air, and as the alginate and islets flow out of the needle, they’re cut off into very, very small drops, and these drops are around about 600 microns in size. They’ll fall from the needle into a container, which has a solution of very concentrated calcium chloride that will immediately form spheres of alginate and encapsulate the islet. That happens because the alginate has negative charges. The calcium and the calcium chloride, of course, have positive charges, and the calcium forms bonds in between the negatively charged alginate, which causes the solidification of the alginate.

So now we have an islet inside a sphere of alginate, the next thing we really have to do is make sure that that capsule has both strength, integrity and porosity so that it can function properly. And what we use for that is a compound called poly-L-ornithine. So we first of all put a coating of poly-L-ornithine onto the alginate, wash it, put a second more dilute coating on, wash it again and then we put a final coat of alginate on. And the final coat of alginate binds to the outer layer of the poly-L-ornithine and forms a protective coat for the capsule.

5. Encapsulated islets quality control

Peter Hosking (Living Cell Technologies)

The next stage is to reculture the islets again because the islets, in order to thrive and prosper, have to be put in media where they’ll have nutrients. They have to be put in an incubator at the right temperature, which is 37 degrees. And so we culture them in what we call T-flasks, which are very traditional cell culture vessels where you have a very thin layer of media on the bottom of a plastic container, and we’ll culture them in these T-flasks for as many days as we need to, before patient implanting.

But we also have a number of other quality control checks that we run. Typically, we’ll do this 2 days to 3 days after we’ve encapsulated the islets. At that stage, we’ll harvest the islets and we’ll run a number of tests on them. Again, we’ll be looking at the purity of the islets, the viability of the islets, and we’re talking about encapsulated islets now. We also measure the insulin production, and that’s the SGS assay, and we have very strict quality control acceptance criteria where each batch must meet our acceptance criteria for insulin response.

6. Transplant into patients

Bob Elliott (Living Cell Technologies)

It’s a simple procedure to put them in, takes about 10 minutes. Under an anaesthetic, a tube’s passed into the belly and the cells just literally run in. There’s no real surgical procedure apart from placing the tube inside the abdomen.

Peter Hosking (Living Cell Technologies)

After the cells are implanted, all the patients that receive them are monitored very closely. Clearly what we’re wanting to see is a reduction in their insulin dosage. We also want to see a reduction in their hypoglycaemic episodes and better control of their blood sugar, and all those things can be monitored by our clinical programme.

Acknowledgement: PRN Films

QUALITY CONTROL TESTS

7. Purity test

Peter Hosking (Living Cell Technologies)

It’s a purity test where we try to determine the amount of insulin-producing cells, and so essentially we use a DTZ stain, which stains those cells sort of a red colour.

8. Viability test

Peter Hosking (Living Cell Technologies)

The viability test tells us that the cells are alive. For viability, we use a stain, it’s called AOPI, and the viable cells stain up in a green colour. If the cells weren’t alive and metabolising, they wouldn’t pick up the stain.

9. Capsule size and strength tests

Peter Hosking (Living Cell Technologies)

So other QC tests that we do are really focused on the capsules themselves rather than the islets, and these are tests where we’re looking at the uniformity of the capsules, the size of the capsules, the quality of the coating. And the capsule obviously is key to our process because it protects the islet from the immune system of the patient. We don’t want to see any irregular shapes, we don’t want to see any large capsules or very small capsules, and we don’t want to see any islets that are partially coming through capsules. All of these things could break capsules and damage them, so we’re looking for very uniform perfect spherical capsules and ideally one islet close to the middle.

10. Sterility test

Peter Hosking (Living Cell Technologies)

Well, one of the other tests done after encapsulation and in fact throughout the process, because it is an aseptic process and done in sterile conditions, clearly the sterility of our final product is of critical importance, so we do monitor that at various stages of the process – certainly after they’ve been harvested, 2 days after the encapsulation, also just before we transport them for a patient, we’ll take a second check.

11. Function test

Peter Hosking (Living Cell Technologies)

The SGS assay is one of our critical quality control parameters. SGS stands for static glucose stimulation, and essentially, the assay is such that it challenges the islet with firstly a low concentration of glucose, then a high concentration of glucose, then a low concentration of glucose again. And so we measure the amount of insulin that is excreted at the low glucose level, then at the high glucose level, and then obviously when there’s no glucose there again, we want the cell to switch off and stop pumping out insulin, and so we also check that that happens. And the SGS assay gives us confidence that the cells that we’re using for patients will in fact produce insulin in the sufficient quantities to cause a good clinical outcome.

12. Pathogen tests

Olga Garkavenko (Living Cell Technologies)

A pathogen is a microorganism that can cause disease in a host. Our main task is to ensure the safety of the tissue that is transplanted from animals to humans and also to follow up our patients to make sure that they did not acquire any silent infection or any unknown pathogens from our pigs. So we have to design a very rigorous programme and search for pathogens that do not cause any disease in animals but can for humans.