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Bioreactors

Bioreactors are controlled, closed systems used for the creation of cultures. They have many uses, ranging from the production of beer to more refined purposes such as creating large batches of stem cells of uniformly high quality. Microfluidic devices are another recent development similar to microbioreactors. Microfluidic devices and microbioreactors allow tight control and management of throughputs for better understanding of niches and more efficient drug screening. They also reveal paracrine/autocrine factors for enhanced control of differentiation. Both allow manipulation of the microenvironment through adjustment of cytokine gradients and flow rate.

Bioreactors have long been used in large-scale animal cell production, and converting them to stem cell generation is a matter of process engineering. Bioreactor engineering enables better modification and management of niches for maximum expansion, differentiation, and application of new cell-based medicines. Critical parameters include oxygen tension; 3-D scaffolds; physical forces such as mechanical, electrical, hydrodynamic force or strain; and flow shear. Different process outcomes and tissue-specific cell types require different bioreactor designs. What works for beer does not necessarily work for stem cells, regardless of type.

Stem cells are of two main types: pluripotent stem cells and adult stem cells. The pluripotent stem cells include both embryonic and induced pluripotent stem cells. Embryonic stem cells are pluripotent and can develop into all types of cells. This reprogramming of somatic cells is what causes them to become pluripotent. Adult stem cells, such as hematopoietic, neural, and mesenchymal stem cells, have a more limited differentiation potential and can only generate a subset of cells.

Stem cells hold great potential for biomedical applications across a wide spectrum, among them regenerative medicine, drug discovery, and cell therapy. Unfortunately, the current static tissue culture vessels, the Petri dish, can produce only small numbers of cells. Stem cell use is exceeding generation capacity of static vessels with cell counts reaching 10¹⁰–10^12. Bioreactors need to be scalable and capable of quickly shifting the number and volume of cells they grow in order to provide the large numbers of stem cells that researchers need.

Stem cells require a regulated environment to grow properly. Because cells may be cultured either as free cells or aggregates fixated to solid substrates (such as the wall of the container) or in suspension, different bioreactor configurations are desirable. Bioreactors provide the necessary niche factors for proper growth into the appropriate cell configuration, regulating parameters such as oxygen, synthetic and decellularized extracellular matrix, paracrine/autocrine signaling, and physical forces such as mechanical and electrical forces and flow shear. The appropriate bioreactor offers precise control and recreation of niche factors by means of modulating or regulation of operation parameters. In this environment, the stem cells can expand and differentiate.

Stirred tank bioreactors produce free cells in suspension. This system is easy to scale up, provides a homogeneous culture environment, and is suitable for aggregate culture. However, it is subject to high shear stress. Another bioreactor type useful for cells or clusters in suspension is the rotary cell culture system (RCCS). The RCCS provides good mass transfer, controlled oxygenation, and low shear stress. However, the limited volume generated by the RCCS is relatively small and potentially inadequate for stem cell research. The wave bioreactor is suitable for hematopoietic stem cell culture and offers low shear stress, easy scalability, and gentle mixing, but it can be quite expensive.

For adherent cells, the microcarrier-based bioreactor may be either the stirred tank or the 4-D rotary cell culture system manufactured by Synthecon. The rotating wall bioreactor is a National Aeronautics and Space Administration (NASA) development to simulate the microgravity of space for cell growth. The bioreactor and inner cartridge rotate at the same angular rate, producing microgravity and low fluid shear. Cells grow as they would in vivo, and the RCCS allows mass transfer and efficient oxygenation through a permeable membrane. Rotating wall bioreactors were first used for bone marrow stem cell research and then expanded into other types of stem cells. However, rotating wall bioreactors are hard to scale up.

Adherent cell bioreactors regulate cell growth and differentiation, allow high-density cell culture, serve as delivery systems, and are easy to scale up. Cell harvest is difficult, however. Another option is a bioreactor with immobilized cells on a fixed or fluid 3-D scaffold. Tissue scaffolds (synthetic extracellular matrices) may be constructed of natural or synthetic materials such as Matrigel, alginate, collagen, hyaluronic acid, polyethylene terephthalate, and self-assembling peptide gels. Surface topology and tensile strength are among the factors that can alter cell adhesion, proliferation, and differentiation. The type of material used in constructing the scaffold depends on tissue. For instance, human embryonic stem cell cultures and soft tissues have worked with hydrogels with tunable properties while the engineering of bone and heart muscle requires a stronger and more porous material such as mineralized silk. Pore size affects the type of engineered bone tissue. The microencapsulation-based stirred tank and RCCS protect from shear stress and offer a 3-D environment, allow spatial organization, and regulate proliferation and tissue formation, but cells need to be released from the hydrogels and harvest is difficult.

The hollow-fiber membrane reactor provides low shear stress in a close approximation to the natural cell environment but is difficult to scale up and lacks a homogeneous environment because of the oxygen gradient and nutrients.

Ideally, bioreactors would offer high production capacity and high quality. Using sensors or probes, researchers can control and maintain hydrodynamic and physiological factors that affect proliferation and differentiation, including regulation of pH, nutrients, dissolved oxygen, extracellular matrix, and shear force. Perfusion and agitation reactors can improve control of oxygen supply and transfer of mass. Perfusion also reduces accumulation of toxic waste such as ammonia or carbon dioxide and, because it allows long-term high-density culture growth, reduces material costs, labor, and risk of contamination.

Stirred tank bioreactors have been studied for stem cell expansion and differentiation over the past several years because they are simple, scalable, dependable large-scale mammalian cell cultures. Stirring provides a homogenous environment for better cell growth and better mass transfer. On the negative, stirring might change the binding of the receptor and ligand, altering physiology and metabolism. Stir tank bioreactors allow easy monitoring of oxygen and pH. Stirred tank reactors have been used for adult stem cells, with a nine-fold higher expansion compared to static cultures. Stirred tank reactors have also had success with mouse cells.

The wave bioreactor uses disposable plastic bags inflated through ports that also allow air circulation. Rocking the bag in a wavelike motion allows good mixing and mass transfer and enhances oxygen transfer while holding the cells in suspension with low shear damage and bubble formation. Using sensors and feedback systems allow bioreactors to control oxygen tension more precisely. The system is safe in clinical environments and has been used in an industrial setting for stem cells and other mammalian cells, although the system is too expensive for most stem cell applications.

Transitioning to bioreactors may pose challenges. In the past few years, EMD Millipore has abandoned the Petri dish for significantly larger-scale bioreactors in anticipation of rapid future growth in the demand for stem cells. Already, stem cell therapy is utilized in the treatment of leukemia. One constraint is the availability of vast quantities of stem cells of suitable quality. In 2010, EMD Millipore began its stem cell initiative and provides reactors that comply with all the regulatory standards government demands. The EMD Millipore production system involves 3- to 50-liter bioreactors. Volume production comes with its own complications, including parameters different from those of the small-scale Petri dish production. New parameters include, but are not limited to, cell count in the initial culture, type of media to be used, temperature, air flow, and stir rate. The ability to differentiate and characteristic cell surface markers are often used to measure quality of a stem cell, but specific attributes of what makes a stem cell “good” and available to function from the beginning to the end of the process is lacking. Continuous monitoring of the bioreactor is necessary in order to check that the cells are retaining their ability to differentiate. One advantage of the bioreactor over the Petri dish is regular sampling is an option. This quality control allows immediate remediation if a flaw develops.

The bioreactor environment is somewhat delicate as well. Cells grow on tiny spheres that allow more surfaces for them to multiply on. However, the spheres make manipulation more of a challenge. In addition, there is no possibility of using an aggressive removal method since that process would destroy the cells and potentially damage the internal configuration of the bioreactor.

A major manufacturer of bioreactors is the Swiss firm Bioengineering. For 40 years, it has been producing commercial and customized, large and small bioreactors and fermentors. Its inventory reflects the variety available for potential buyers of bioreactors. Bioengineering offers a model designated RALF, a bench-top bioreactor in 2-, 3.7-, 5-, and 6.7-liter sizes. It comes in basic and advanced models, takes little space and power, and has the capability of doing cell and microbial culture because of the free configuration of gas lines. It also has process management software that includes process automation, recipe features, management of access, and an audit trail. The turn key system is available from stock in two weeks or less. Other models are larger and more sophisticated, including the P, which comes in sizes ranging from 100 to 1,000 liters. It is a production-scale bioreactor and is marketed as highly flexible, with open frame construction for ease of maintenance and installation and with a modular control system that promotes fast process change. It is also customizable and reprogrammable.

Celltrion Inc. in Korea has the largest cell cultivation plant in the world, a 300,000-liter facility that includes four production trains with five 16,000-liter bioreactors each, six other production trains with five 19,000-liter bioreactors, and 100-plus tanks for buffering, harvesting, and other functions. With storage and quality control facilities, the site positions the company founded in 2002 to become a major producer of cells.

Bioreactors can be small enough to fit on a desk or large enough to require a tank farm. They have many types of configurations. They can be expensive, and growing stem cells at the appropriate scale and quality can be a challenge. However, they represent a niche that cannot be filled by the Petri dish.

John H. Barnhill

Independent Scholar

See Also: Stem Cell Aging; Stem Cell Companies: Overview; Tissue Engineering (Scaffold).

Читать книгу The SAGE Encyclopedia of Stem Cell Research - Группа авторов - Страница 120

Bioreactors

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