January 1, 2009 (Vol. 29, No. 1)

New Methodologies and Technologies Abound in Both Quality and Quantity

Stem cell therapy holds great promise for various diseases. In fact, in what many are hailing as a medical milestone, European researchers have successfully transplanted a trachea built from a patient’s own stem cells to replace a failing airway damaged by tuberculosis, according to a recent article in The Lancet.

The team stripped a donor trachea of all its cells and used the remaining collagen as a scaffold that was seeded with the patient’s stem cells. Four days after surgery, the engineered windpipe was almost indistinguishable from normal bronchi; four months post-surgery, the patient shows no signs of rejection and is enjoying a normal quality of life.

This success may help future stem cell applications overcome some of the current challenges, which speakers at the “Stem Cell Summit” to be held next month, talked to GEN about in advance of the meeting. The scientists interviewed for this article were especially concerned about regulatory issues—is it a device, biologic, or both; financial matters—it’s an early field and federal funding for embryonic stem cell research has been put on hold; quality and quantity assurance of stem cells to enhance patient outcomes; and understanding their mechanism of action.

Despite the challenges, however, a recent BBC Research market report (July 2008) estimates the U.S. market for stem cell technology is expected to increase from $112 million in 2007 to $423 million in 2012.

Since the 1970s, Stuart Williams, Ph.D, CSO at Tissue Genesis, has been experimenting with isolating cells from fat to test their potential use in building blood vessels. The resulting mix of adult stem cells has regenerative qualities. “I started using these cells in lab experiments and learned how to isolate them. I realized this type of therapy needed to be automated in a totally closed system.”

This conclusion led to the development of the company’s TG1 1000 cell isolation system. The compact desktop unit provides isolated cells in about an hour, according to the company, and accepts fat tissue from the same device used for tissue harvesting.

Dr. Williams explains that the most critical component of the process is the company’s enzyme mixture to digest tissue and keep the cells viable. Initial efforts involved using the cells to reline artificial blood vessels. His group took Gortex-like tubing commonly used in vascular procedures and lined the inner surface with the patient’s own cells derived from fat, which re-created the natural blood vessel lining rapidly after the implant.

“We only need about 30 grams of fat for vascular grafts. Each gram provides about a million therapeutic cells and is consistent from patient to patient,” explains Dr. Williams. The stromal cells take cues from the extracellular matrix proteins they are surrounded with. So if they are removed from fat and placed in a new environment, like the heart, they will take their cues from heart tissue. “You can put them in a 2-D or 3-D environment and the matrix will have a dramatic effect on their phenotype and function.”

The company has set up a consortium that explores these cells for orthopedic (spinal fusions), cardiac (congestive heart failure, myocardial infarction), and inflammation (currently for veterinarian use only) applications.

Dr. Williams says that they are now working with the FDA to gain approval of the device for specific therapeutic applications. “The device itself is just a complex centrifuge digestion system. What you use the cells for will dictate within FDA regulations whether it will go through an IDE process or an IND process and whether it will be registered as a biologic or a combination product.”

Using Harvest Technologies’ BMAC system, bone marrow aspirate concentrate (BMAC) can be prepared at a patient’s bedside from a small volume of bone marrow that is percutaneously aspirated from the patient’s hip.

Cord Blood

Isolating cells without touching their surface is a way to avoid creating molecular activity or reactions. This is a key concept behind BioE ’s cord blood closed-bag processing system, PrepaCyte®-CB. “We wanted to optimize negative selection—get rid of unwanted cells—to obtain a highly purified population or group of populations, without touching the cells,” says Michael Haider, president and CEO.

The process is biological rather than mechanical. “We use mother nature and various materials depending on the cell population we’re trying to remove.” Only a small amount of biologic is required, and simple gravity is used for cell isolation. Since the dynamic range of the biologic is broad, exact mixtures are not required, Haider adds.

Approximately 98.5% of all red blood cells are removed in about 30 minutes, leaving nucleated and stem cells. Total nucleated cell count is the industry-standard measurement for cord blood samples and directly correlates to patient outcomes in transplants. Few cord blood samples make the cut—only about 25% are suitable for banking; 75% are thrown out due to low volume or cell count.

Currently, PrepaCyte-CB is a class II device, which allows sales to institutions regulated by an IND. Dr. Haider says the company’s main focus over the next six months is to obtain FDA 510(k) approval, which will open up global markets and allow sales to private cord blood banks.

“We believe this is setting a new pathway and enablement where the biologic is an enclosed system at the time of manufacture and all the work occurs by the biological fluid. The FDA is moving toward tighter regulations. The industry needs to continue to improve on the quality of the material that’s transplanted into the patient —this is usually a life or death issue,” Dr. Haider adds.

Services for Community

Progenitor Cell Therapy has expanded its services to include consulting, product development, cGMP-compliant contract cell manufacturing, and creating clinical trial products. The company recently received AABB accreditation in cord blood activities—processing, storage, and distribution.

It has worked with a wide range of cell products other than stem cells including T cells, skin products, mesenchymal stem cells, neural products for CNS disorders, and protein products for cancer. Robert Preti, Ph.D., company president and CSO, reports that one of the greatest challenges of bringing stem cells into the clinic is “converting great science into technical feasibility as an industry.”

Additional challenges for this industry are many, he adds. Regulatory framework is less problematic than a few years ago because the FDA has been working with industry so companies now understand the basic framework around cell therapies. Clinical challenges are still complex—how do the cells work, what are they supposed to do once infused/transplanted, and will the cells be economically feasible?

Financing, especially now, is a huge concern. “There’s a certain amount of investor wariness that has taken place. It’s more difficult now to get funding to advance these therapies and the lack of federal funding for embryonic stem cell research has put a hold on that field,” states Dr. Preti. Manufacturing challenges arise from characterization of the product. “If you can figure out mechanism of action—how to characterize a cell product that typically is not a homogenous group of cells?—how do you define it? and what is the potency measurement?” He says that safety is not an issue with adult stem cells, but the biggest problem is identity of the product and potency; how do they work? and do they work?

“Since we don’t have the ability to fully characterize the cell product itself, we are forced to view the process as the product, and when we do that, we have to define the process very carefully,” Dr. Preti states. This points to other challenges like logistics—getting the product delivered to the applications site and setting up facilities. “When you are dealing with stem cells, you are talking about cures versus palliative treatment. That’s a lofty goal. And well worth the effort.”

Improving Cell Harvesting

Stem cells are traditionally isolated from bone marrow by a chemical process called Ficoll density gradient separation, which precipitates the cells out and takes anywhere from 8–24 hours.

Harvest Technologies has developed a system, BMAC™, that provides at least twice the number of total nucleated cells over the Ficoll process, says Gary Tureski, president. “Our technology provides a simple point-of-care procedure for concentrating these cells in about 15 minutes.”

This process also eliminates culturing cells. “You have challenges when you culture because you don’t know if the culturing—which is accelerating cell division at an unnatural rate—is causing a problem or the culture medium is causing problems,” adds Kevin Benoit, vp and GM of strategic development.

The FDA has granted IDE approval for a 48-patient feasibility study in the U.S. using BMAC to treat patients with end-stage critical limb ischemia. Patients will be injected with their own stem cells, processed by the company’s system, into the affected limb, in an attempt to prevent amputation.

BMAC is also being used in Europe in several cardiac procedures. It is infused into coronary arteries, post-myocardial infarction, to repair a portion of heart damage, injected into heart muscle as adjunctive treatment with bypass surgery to improve heart function, and used in combination with laser cardiac procedures.

The company has submitted an investigation device exemption for a double-blind placebo cardiac safety study—to inject BMAC cells into the myocardium during bypass surgery in patients with severely compromised heart function. It is expecting a favorable response by the FDA to start the study by the end of 2008, according to Tureski.

Synthetic Scaffold for 3-D Cell Culture

A synthetic peptide hydrogel discovered by Shuguang Zhang at MIT in 1992, now serves as a 3-D extracellular matrix cell culture. 3DM holds the exclusive license to the technology and has partnered with BD Biosciences to commercialize the BD Puramatrix™ research product. The firm is also developing the material for clinical applications in regenerative medicine.

Puramatrix is a peptide (16 amino acid residues) that self-assembles to form a nano-fiber structure or hydrogel in aqueous solution. The strength of the scaffold can be adjusted for specific cell types, with the ability to deliver and release therapeutic proteins and stem cells in a localized area.

Since it is fully synthetic and devoid of animal-derived materials, there is no risk of infection. It is also highly reproducible, yet also customizable—other cells and factors can be added to it. The nano-fiber structure resembles natural collagen; enabling cell proliferation equal to that within collagen.

“As opposed to growing cells on just plastic, cells in a Puramatrix culture typically behave more like they would in normal tissue,” says Lisa Spirio, Ph.D., CTO and cofounder.

She adds that it works well with stem cells, and the company has in vitro and in vivo data showing that Puramatrix supports maintenance of stem cell phenotypes, as well as differentiating stem cells into various cells of interest. The company is actively collaborating with various orthopedic, cardiac, and drug companies.

Wound healing and dental bone clinical trials are expected to begin in early 2009 (first set of trials will not include stem cells). “It’s likely that these applications will require separate FDA approval,” Dr. Spirio says.

Adult mouse neural stem cell embedded in 3DM’s peptide scaffold product, Puramatrix, a synthetic hydrogel peptide that serves as a 3-D extracellular matrix, and supports the maintenance of stem cell phenotypes

Cell-Based Product Manufacturing

Some of the challenges companies face with stem cells, according to Alan Smith, Ph.D., CEO and president of Cognate Bioservices, include having adequate facilities, appropriate cleanrooms, and processes that can be scaled-up and manufactured under GMP.

“We also find people looking for help with regard to developing appropriate quality control release testing and information on what the FDA is looking for in this area. I think a lot of people, especially small companies, don’t have a comprehensive understanding of good manufacturing practice and what that entails.” Dr. Smith adds that his company helps identify suitable reagents that are compatible with GMP and helps with receipt and testing of raw materials.

In addition, it provides stem cell services, including development of cell-based therapeutics using bone marrow cells for tissue repair and regeneration. He explains that there are differences in processing stem cells based on their source. Bone-marrow-derived stem cells are typically in single cells in suspension versus adipose stem cells, which are tissue-based.

“Working out those to optimize yields in manufacturing is often time consuming and critical because the cost of goods for the product is dependent on the number of stem cells one can derive from the starting material.”

Additional challenges in regards to stem cells include reliability and robustness of the process to yield consistent outcomes. To address this, the company breaks the operation down into segments: procurement, shipping/logistics, manufacturing, scale-up, and quality control, said Dr. Smith.

“We try to build in fail-safes to each of those aspects in addition to the quality control of the product that’s in tandem to the manufacturing process.” He adds that the firm has developed some techniques that allow for certain aspects of the stem cell process to occur in a closed-system fashion. The company is currently expanding its stem cell capabilities, including additional types of stem cells such as cord blood.

Although the promise of stem cell therapy started with the first successful bone marrow transplants in 1968, it is just beginning to reach the clinic. Thanks to companies’ success in developing new technologies and methods of isolating, growing, and producing adult stems cells, their quality and quantity is increasing. There still remain many hurdles—such as regulatory and financial—before they can be used to treat patients. Yet, many companies remain optimistic that working together with the FDA will create a standard of quality that will help move the field from the bench to the bedside.

Tissue Genesis’ TG1 1000 cell isolation system

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