Recent advances in cell therapies and tissue engineering are paving the road to regenerative medicine. The goals of this emerging field are to replace, repair, or regenerate tissues and organs.
The underpinnings for this process are found in the biomanufacturing requirements for stem cells and engineered tissue. Each must deal with a growing body of complex issues that include cost containment and the need for deriving products under GMP. Other challenges include scale-up of manufacturing processes and use of appropriate tissue scaffolds and bioreactors for the creation of 3-D models.
"Broadly speaking," states Thomas B. Okarma, Ph.D., M.D., president, CEO, and director of Geron (www.geron.com), "the cost of goods is among the most critical issues for biomanufacturing cellular therapies. Our ethics advisory board spent six years studying these issues, and their major concern was that the product must be able to be delivered at a low cost.
"For example, when companies use primary adult stem cells, costs can be huge. That is an individualized therapy with different product specs for each isolated cell product. That type of cell is much harder to regulate and more costly to produce."
Geron focuses on human embryonic stem cells (hESCs). "The use of hESCs eliminates donor variability. The hESC technology provides a product-based business model with scalable manufacturing rigor that is unprecedented over the past 30-40 years of cell therapy manufacturing.
"Cell therapy goes way beyond the therapeutic reach of a pill, and we will see cost containment in this field for the first time. hESCs provide the platform to fill all requirements of scalable GLP and GMP manufacturing," points out Dr. Okarma.
The company is developing cell-based therapies for several diseases, based upon differentiated cells, derived from hESCs. "Our company has a GMP master cell bank, a highly characterized collection of hESCs from which to begin manufacturing. We have developed proprietary methods for growing, maintaining, and scale-up of undifferentiated hESCs using feeder cell-free conditions and a chemically defined culture medium," continues Dr. Okarma.
He points out that a key to success for using hESCs is adapting them to culture without feeder cells or serum-containing media. "This eliminates concerns about viral or animal protein contamination. Manufacturing hESCs in this way enables us to control the manufacturing process like any other biological drug."
Some current methods generate embryonic stem cells using a fertilized egg. Stem cells are taken from the inner cell mass of a five-day old blastocyst, destroying the developing embryo.
Advanced Cell Technology (www.advancedcell.com) is pioneering a new method in which stem cells are removed without destroying the embryo. In this technique, a blastomere is removed at the eight-cell stage. The blastomere is cultured with an established embryonic stem cell line and subsequently separated to form new cell lines. The embryonic blastocyst is left intact and can be implanted into the uterus.
The company recently validated their first GMP laboratory in its facility in Worcester, MA. They are focusing on the derivation and differentiation of human embryonic stem cells under GMP conditions.
"There are a number of issues to be dealt with not only in GMP but beyond that, when one enters human safety and efficacy trials," says Robert Lanza, M.D., vp research and scientific development. "The GMPs are similar in some respects to those for any other biotherapeutic. You need appropriate process validation to trace all raw materials and document them from beginning to end. Equipment and standard operating procedures all must be validated.
"But for cell therapies you need to address other considerations as well. Cells must be carefully characterized to show they do not form tumors, that they don’t contain pathogens, and that the passage number is optimal for the specific use. Their growth conditions must allow them to recapitulate normal tissue."
Dr. Lanza emphasizes that it is critical that the cells are carefully characterized before safety and efficacy trials in patients are initiated. "For example, you need to define all your protocols, including your differentiation system, your inclusion/ exclusion criteria, and which passages and cell culture conditions still provide the same phenotype. You need to be able to reproducibly generate a well-defined population of cells, or the FDA may make you repeat your safety and efficacy studies all over again."
Advanced Cell is studying retinal pigment epithelial (RPE) cells and hemangioblast cells. Their stem cell-derived RPEs target age-related diseases of the eye, such as macular degeneration, while hemangioblasts target cardiovascular disease, such as coronary artery disease and congestive heart failure.
Antibodies to Purify Stem Cells
Stem cells also can be harvested from tissue. These are rare cells that both self-renew and produce the functional specialized cells of the organ from which they were derived. Stem Cells (www.stemcells.com) isolates human tissue-derived stem cells via immunopurification techniques, based on the antigenic signature on the cell surface. The company believes this technology has a broad therapeutic approach, similar in importance to traditional pharmaceuticals and genetically engineered biologics.
In this manufacturing process, specific antigens on the stem cell surface serve as tags for their isolation via Mabs. After, initially harvesting cells from human tissue, such as brain, liver, or pancreas, the company uses their library of Mabs to identify, purify, and characterize the human stem cells of interest. A sophisticated cell-sorting step removes inappropriate cells. The purified populations of stem cells then can be expanded, banked, and evaluated using in vivo and in vitro models to demonstrate that they maintain the original stem cell properties.
For example, their scientists transplanted normal human central nervous system stem cells (HuCNS-SC) into the brains of immunodeficient mice and found that the implanted cells differentiated into the three major CNS cell types. Thus, these cells could adapt to the host brain and behave like normal host cells in continuing to divide, migrate, and differentiate.
Like cell therapy, tissue engineering is another fast growing biotech sector. Tissue engineering seeds cells into culture often containing a physical scaffold that helps them grow into complex, sophisticated 3-D models in vitro.
"Bioreactors are key to this process," says Steve Navran, Ph.D., CSO, Synthecon (www.synthecon.com). "Compared to static culture methods, growing cells in bioreactors provides dynamic cell culture conditions that greatly improve growth and survival of cells, because there is a superior transfer of nutrients and gases."
Dr. Navran notes that the type of bioreactor used may impact the success of cell growth. "Chemical and biomedical engineering issues must be considered. You need to preserve the basic principles to maintain the three dimensionality of the cell growth environment and not expose cells to excessive shear forces. Also the type and size of the bioreactor used affects availability of nutrients and oxygen. For example, applications may need more or less cell aggregation.
"Our high aspect ratio bioreactor vessels tend to promote more aggregation, while the more cylindrical perfusion vessels limit large aggregates."
Another issue is scaleup and preservation. "Scaling up is fairly linear in the Rotary Cell Culture System, or RCCS, up to about 500 ml in a batch-fed RCCS bioreactor," says Dr. Navran. "But beyond that, the best way to go is with perfusion. Perfusion RCCS bioreactor systems can go much higher, because feeding and gassing are provided externally."
Going to GMP is always envisioned and must be carefully taken into consideration, according to Dr. Navran. Sometimes it is more cost efficient to outsource. "We have an application approaching human trial stage. We don’t maintain a clean room facility, so we will partner with an outside source. This strategy can be a cost saving measure and a more efficient way to attain cGMP."
Scaffolds for 3-D Cell Structure
Almost 20 years ago, researchers developed porous polymeric scaffolds that enabled scientists to grow tissue in thick layers. Since then, a number of functional tissue equivalents have been grown in the laboratory, including skin, cartilage, tendon, bone, and blood vessels.
Engineered scaffolds are created from anything from loofa sponges to complexes of polymers, such as polyglycolic acid, polylactic acid, or many other mixtures.
"The choice of which type of scaffold to use has an impact on how cells will look and function," says Richard Fry, managing director, Cellon (www.cellon.lu), "as it is clear that form and function are closely linked in biological systems. Cells grown in normal tissue culture flasks look different than cells that grow in vivo.
"If you cultivate cells on two different scaffolds, they may look different. Cells produced on less rigid supports, such as hydrogel scaffolds, are less spatially constricted, particularly when grown in a rotary cell culture system. This allows them to look and potentially function in a similar way to the parental tissue."
Scaffold properties that researchers consider include mechanical strength, degradation time, degradation byproducts, porosity, and fiber orientation.
"There are many types of scaffolds. Some are standard and some need custom mixing, says Fry. "They can come in sheets or tubes. Nonaligned scaffolds are manufactured from spun fibers using a nonwoven textile process. You can further modify the basic scaffold material to enhance cell attachment and growth or modify the physical properties and resorption rate."
Another type of scaffold is an aligned scaffold. These also are fabricated from spun fibers but are available in a variety of custom 3-D shapes and tubular structures. The main advantage of aligned scaffolds is that structures, such as tubing, are then seamless.
In recent years, some attention has focused on multipotential cells that give rise to various specific cell types. BioTissue Technologies (www.biotissue-tec.com) is developing multipotential cells, such as mesenchymal progenitors and periosteal cells. These cells are an attractive tissue engineering approach because they possess the ability to undergo extensive replication and develop into mesenchymal tissue, such as bone and cartilage.
According to the company, challenges in this arena include developing necessary procedures for cell isolation, expansion, and preservation. It is also important to establish methods for phenotypic and functional characterization, as well as characterize how the cells differentiate in vitro.