The production of clinical grade stromal cells is an important part of the NEPHSTROM project. In order to be able to treat the patients in the clinical trial, hundreds of millions of stromal cells are needed. If the therapy is to enter the mainstream, we also need to be able to demonstrate that we will be able to produce cells in sufficient quantities to treat thousands of patients. If clinicians are to confidently prescribe cell therapy treatments, however, the cell therapies must be very pure and consistent.

Cell Isolation

The cells used in NEPHSTROM (and in the majority of stromal cell clinical trials) are extracted from bone marrow. The bone marrow is harvested from the top of the donor’s hip-bone (the ‘iliac crest’). The process is rather unpleasant – as a result, there is a general shortage of bone marrow. Umbilical cord tissue (a waste product, after a baby is born) is a very promising alternative source for stromal cells, which we are enthusiastic to explore, beyond NEPHSTROM.

To isolate the (very small) percentage of stromal cells from within the mass of marrow, we take advantage of the fact that stromal cells have specific protein markers on their surface, that other cells (blood cells) do not. These specific markers can bind to antibodies, which have been coated onto tiny magnetic beads, which then stick to the stromal cells. When a magnetic field is applied, the beads ‘anchor’ the stromal cells in place, while other cells are then removed. Clinical use of magnetic bead technology is quite common, the CD362 antigen was identified by Orbsen (in another FP7 project, PURSTEM), and is patented. The resulting stromal cells are made available as the ‘Orbcel’ product.

Orbcel

Orbsen’s product consists of stromal cells which have been isolated from bone marrow using the CD362 antigen; this delivers a uniquely-well-characterised population of stromal cells, with unprecedented purity.

Research in a series of related projects, some funded by the EU is exploring the use of Orbcel in a range of different diseases, including diabetic complications (REDDSTAR), liver disease (MERLIN) and transplant rejection (VISICORT). Orbcel uses the CD362 antibody to isolate stromal cells from the bone marrow, umbilical cord tissue or fat tissue. This is much more precise than the traditional methods of isolating stromal cells, which rely on the fact that stromal cells stick to the bottom or sides of plastic culture vessels (‘plastic adherence’). Unfortunately, stromal cells are not the only types of cells that are ‘plastic adherent’, and so many ‘stem cell’ treatments are made up of a mix of stem cells and several other cell types. This makes their use as a treatment rather difficult, because the clinician cannot be sure what he is treating his patient with. By contrast, the Orbcel process for isolating stromal cells is approximately 3,000 times more precise than traditional plastic-adherence.

When tested in terms of efficacy against plastic-adherent cells, Orbcel was shown to have at least as strong a therapeutic effect in diabetic complications, in lung disease (acute respiratory distress syndrome – ARDS), rheumatoid arthritis and in inflammatory liver disease (PSC).

Interestingly, Orbcel is also being trialled (in the VISICORT) project, as an immunotherapy – a treatment to reduce the risk of the body rejecting a corneal transplant. If a strong result is achieved here, there is excellent potential for reducing the use of immune-suppressant drugs. This would greatly benefit transplant patients.

Large Scale Cell Production Process

NEPHSTROM uses a new two-step process for cell production (‘cell expansion’). The cells are first isolated, to create a master cell bank. This is then expanded in Terumo BCT’s Quantum® Cell Expansion System bioreactor, to generate the large quantities of cells needed for the clinical trial.

The key challenges of large-scale cell production are to maximise the number of cells produced, while minimising the workload on laboratory staff, and minimising the number of opportunities for contamination or human error. The ideal environment is a closed, automated and sterile machine, which carries out the cell culturing without routine human involvement. NEPHSTROM partner Terumo BCT builds just such machines, and is installing their state of the art Quantum system in each cell production site (LUMC, NUIG, HPG23, and NHSBT).

The Quantum system uses a hollow tube filled with very fine filaments of plastic, into which the ‘seed’ cells from the master cell bank are placed, along with the ‘culture medium’ – the cell-feeding liquid. The hollow tube is placed within the Quantum system, which maintains the medium, monitors the cell production and adjusts the environment to maximise cell output. You can see a video on the cell expansion system here https://youtu.be/TZ3Ki3fxwS0.

Scaling Up

The ability to produce very large quantities of consistent, high-quality cells is essential for the future development of NEPHSTROM, and of regenerative medicine as a whole. Within the project, we are establishing a network of four cell production centres across three countries. Each centre uses the same master cell bank (created using Orbsen CD362) and the same bio-reactor technology (the Terumo BCT Quantum system), and follows standard operating protocols (SOPs).

Cells from the production centres are then shipped to the clinical trial centres, for use in the NEPHSTROM clinical trial

SOPs and Regulatory Authorisation

The NEPHSTROM cell production protocols comply with Good Manufacturing Procedure (GMP) standards; this is essential if the cells are to be acceptable as mainstream therapeutic products. Such products require authorisation by national and international regulators such as the EMA (Europe), FDA (USA), MHRA (UK) and HPRA (Ireland). An essential part of securing this authorisation is demonstration of GMP compliance. The cell isolation and production processes in NEPHSTROM all take place within certified GMP environments.

The Future

In NEPHSTROM we are laying the foundations for future widespread use of stromal cells to treat Diabetic Kidney Disease (DKD) and other diseases. An essential element of that future vision is to be able to produce large numbers of consistent cells. By establishing a network of cell production centres, and by applying rigorously standardised processes, we are demonstrating that this vision of the future is realistic and feasible.