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Oct 15, 2013 (Vol. 33, No. 18)

Growing and Preparing Adult Stem Cells

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    Closed hoods with integrated incubation chambers are a key design feature in the Xvivio system. According to BioSpherix, incubators open only into aseptic space, and contamination risk is reduced. The hood is controlled for various cell parameters, the same as with the incubator. When cells are removed from the incubator, they experience no disruption in temperature, CO2, pH, O2, etc. Technicians work in soft, flexible, clear plastic-gloved windows, isolated from cells.

    Diverse factors account for the interest in (and success of) the adult stem cell field in recent years.

    These factors include breakthroughs in reprogramming, ethical and regulatory issues more tractable than those encountered with embryonic stem cells, and (with a wink to The Graduate) … plastics. Diverse factors will likely contribute to the field’s future as well. At the “Adult Stem Cell Therapy and Regenerative Medicine Conference” factors such as manipulation, transduction, visualization, and homing were considered, as was cell culture.

    How the cells are nurtured can be as important as how they are deployed. When it comes to preparing cells for possible (re)introduction into animals, every precaution needs to be taken—from the obvious (keeping them sterile and cross-contamination-free), to the subtle (housing and manipulating them in conditions that mimic their inherent physiology). Mimetic culture can support viability as well as promote the desired differentiation state.

    It’s not enough to keep cells in a 37°C, 5% CO2 incubator, passage them in a laminar flow hood, and then view them on a benchtop microscope. According to Kevin Murray, director of sales and marketing for BioSpherix, cells should be maintained in uninterruptable conditions: “Whatever conditions [you use for] culturing your cells…you should also use for processing them. For instance, incubators are set up to mimic body temperature and control for CO2, which is basically how they manage pH in the media. Another aspect we advocate is oxygen control, because cells in the body don’t see room air.”

    Researchers generally take great care to match the body’s physiological conditions in their incubators, but the one thing that hardly anybody controls for is oxygen. “And oxygen is tied to the expression of a multitude of genes, to cell signaling—many of the things that researchers ultimately are looking at, whether they know it or not,” Murray said.

    While the cells may not venture out of the incubator for very long, many of these genes may be very oxygen-labile—taking hours to upregulate but literally minutes to downregulate. BioSpherix’ Xvivo system is a set of equipment modules—from hypoxic incubators to containment hoods to processing chambers that can house microscopes, centrifuges, and a variety of other equipment, including automation and robotics, all in a controlled environment. The barrier/isolator modules are designed to fit together in a range of configurations to maintain a constant environment in which cultures can be manipulated without being exposed to the room’s atmosphere.

    Only a very small percentage of the millions or billions of cells carefully grown and expanded ex vivo and introduced into an animal will survive to engraft and replicate, Murray said. “One of the main reasons they die is that they get very used to room air—about 21% O2—and when you inject them into the body of an animal they’re probably in an environment that’s less than 5% O2.” Mountain climbers need to acclimate to the atmosphere of Mount Everest, Murray pointed out, and something similar is likely true for injected cells.

  • Mimetic Cultureware

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    The surfaces of polystyrene microcarriers may be treated to enhance attachment of cells, such as mesenchymal stem cells, to increase cell yield and viability. For example, Corning Microcarriers may be coated with the company’s Synthe­max substrate, a synthetic product designed for human pluripotent cell culture and neuronal cell culture.

    Growing and preparing cells poses a variety of requirements. For adherent cells, such as mesenchymal stem cells, a key requirement is something that not only supports their ability to attach and spread out, but also allows them to maintain their functionality. Much of this requirement used to be met by biologically based supplements to the culture medium such as serum. As researchers moved toward “leaner” media, serum was often replaced with surface coatings such as fibronectin.

    “Our customers that are expanding stem cells for clinical and preclinical use would rather not use the animal- or human-derived material,” explained Paula Flaherty, technology manager, life sciences at Corning. These can be a source of pathogens, and they sometimes require additional screening to demonstrate that they are pathogen-free. In addition, there can be batch-to-batch variability both in the raw material and in the coating protocol. Accordingly, there is growing demand for defined, animal-free culture environments, including synthetic extracellular matrices (ECMs).

    Flaherty discussed advances in developing integrated solutions—including media, surfaces, and vessel design—to replicate the functionality of naturally derived proteins. For example, the Corning® PureCoat™ ECM Mimetic cultureware is coated with covalently attached, chemically synthesized collagen I or fibronectin peptides. The company also offers Synthemax®, a synthetic vitronectin peptide-based product specifically designed for human pluripotent cell culture and neuronal cell culture, which is sold as coated plates and polystyrene microcarrier beads, and is available lyophilized to allow users to coat their own culture vessels and glass slides.

    Mesenchymal stem cells from various sources including bone marrow and adipose grown on ECM Mimetic or Synthemax in serum-free media showed equivalent population doubling, attachment, tri-lineage differentiation potential, karyotype, and functionality to those grown in standard tissue cultureware in serum-based medium.

    That equivalence can also be achieved for stem cells using a combination of Corning’s CellBIND charge-based surfaces and Cellgro serum-free, xeno-free, defined medium, Flaherty said.

    Flaherty also emphasized that the ability to scale-up production is crucial to meeting the needs of cell therapeutics. Many of the synthetic (and charged) surfaces are available from 24-well plates up through a variety of sizes of single- and multi-layered T flasks. In addition, using Synthemax microcarriers in scalable spinner flasks “combines the best of both worlds of adherent culture but in a suspension mode so you can get much higher volumes of cells, at much greater density,” Flaherty said.

  • Lentiviral Reprogramming

    Cells used for therapy generally aren’t just taken from a donor, grown, and given to a patient. Typically they’re genetically manipulated in some way to give them their desired properties, whether that means replacing a defective gene, conveying pathogen resistance, or making the cells able to withstand otherwise toxic treatments. Lentiviral technology, based on HIV, is the most efficient system known for reprogramming cell function, said Boro Dropulic, Ph.D., founder and CSO of Lentigen.

    Dr. Dopulic recalled that in 2003, when he was still CSO of VIRxSYS, he led the team that first used lentiviral vectors in human trials. He presented a short overview of the field as it has progressed from that groundbreaking work through more recent, promising data coming out of current lentiviral clinical trials.

    The VIRxSYS trial utilized a replication-deficient virus with an antisense HIV payload to target HIV-1. “To test HIV vectors in the very first instance it was appropriate to test in an HIV patient, because we really needed to know the outcome of the interactions between an HIV vector and a wild-type virus. Because sooner or later, even if you’re treating patients with cancer or another disease, [you’ll encounter] a patient infected with HIV,” Dr. Dropulic explained. “So it was important to establish the safety in a best case/worst case scenario.” That Phase I trial of five enrolled patients demonstrates the safety of the lentiviral vector.

    Others have now followed in their tracks, Dr. Dropulic said, including “the first lentiviral vector trial getting clinical success.” This trial, run by Drs. Patrick Aubourg and Nathalie Cartier in France, showed that when stem cells transduced with a lentiviral vector expressing the ABCD1 gene were introduced into patients with X-linked adrenoleukodystrophy, the patients were able to derive benefits equivalent to hematopoietic cell transplantation, the standard of care. Another project, for β-thalassemia, was so successful that the formerly anemic recipients had to be periodically phlebotomized.

    Among other projects, Lentigen is currently involved in a Phase I trial for glioblastoma. The standard of care for this type of brain cancer is surgical resection followed by the chemotherapy agent temozolomide.

    “The problem with temozolomide is that with multiple rounds of treatment the drug is toxic to the hematopoietic stem cell compartment, so we’re genetically modifying these cells with the MGMT gene in order to make them resistant to the effects of temozolomide,” Dr. Dropulic explained. “We’ve seen in our clinical trial … that when you transduce these cells with this MGMT gene you actually can increase the dosing for these patients from an average of one and one-half cycles to five cycles.”



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