People who regularly culture animal cells become so comfortable with standard techniques that novel approaches can seem contrived or even unnatural. However, the typical cycle of seeding cells at very low density in an excess of medium and harvesting (often quite aggressively) just before the point of medium exhaustion is quite an unphysiologic process. Popular culture systems often take cells that originally grew attached to a porous matrix at high densities, with little variability in nutrient and oxygen supply, and adapt them to low-density, styrene-bound or amorphous suspension cultures. Although these methods are well understood and convenient, classical batch-style two-dimensional culture in T-flasks or three-dimensional suspension culture in shake-flasks and bioreactors really aren't physiologically relevant models.

A remarkable number of alternative culture approaches operating with such unusual mechanisms as rocking bags and depth filters have been introduced over recent years. These are often categorized by characteristics such as suspension/adherent or batch/continuous operation (1). However, most share two fundamental features: Cells are subjected to wide swings in nutrient, waste, and pH levels from seeding to harvest, and they are generally growing at highly cyclical but relatively low culture densities.

PRODUCT FOCUS: CELL CULTURE PRODUCTS

PROCESS FOCUS: PRODUCTION, PRODUCT DEVELOPMENT, UPSTREAM PROCESSING

WHO SHOULD READ: MANUFACTURING AND PROCESS ENGINEERS, ANALYTICAL LABORATORY PERSONNEL

KEYWORDS: PERFUSION CULTURE, MEDIA, VACCINES, BIOANALYSIS, CELL THERAPY

LEVEL: INTERMEDIATE

Until recently, the negative consequences of those highly artificial protocols were not well appreciated. But increasing demands of modern drug development, regenerative medicine, and fundamental scientific investigation have inspired the development of alternative approaches. Perfusion culture now exists in a number of (often quite distinct) implementations (2). Hollow-fiber–based technologies in general are used in many applications, from tangential-flow filtration to prokaryotic biofilms in wastewater treatment. Here we consider only their use in the culture of animal cells, referring to this as hollow-fiber perfusion bioreactor (HFPB) technology. Table 1 introduces many of the growing applications of such systems along with the benefits they provide.

Table 1: Features, values, and example applications of hollow-fiber perfusion culture (Click to enlarge)

The Basics of Hollow-Fiber Perfusion

HFPB is a high-density, continuous perfusion culture system. Its hallmark component is a set of thousands of semipermeable hollow fibers in parallel array within a tubular housing or cartridge that's fitted with inlet and outlet ports. The fiber bundles are systematically potted (attached) at each end so that any liquid entering a cartridge will necessarily flow through their interior. Animal cells are generally seeded within a cartridge but outside the hollow fibers in what is referred to as the extracapillary space (ECS) (Figure 1). Culture medium is pumped through the lumen of the hollow fibers, allowing nutrients and metabolic products to diffuse both ways across the fiber walls. Having passed through the cartridge, medium can be either oxygenated and returned to it or collected while fresh medium is introduced (Figure 2).

Figure 1: (Click to enlarge)

Figure 2: (Click to enlarge)

A wide range of materials — e.g., polysulfone and cellulose derivatives — can be used for the hollow fibers. Molecular weight cutoffs begin at 5 kDa and go up to virtually any desired upper limit. The fiber materials can vary in such properties as percent porosity, molecular weight cut-off, and hydrophilicity, and they can be further modified during either manufacturing or their actual application to introduce defined functionalities onto their surfaces. Three fundamental characteristics of an HFPB system are

• extremely high binding culture surface to volume ratios

• immobilization of cells at a very high (biomimetic) density on a porous matrix supporting prolonged culture

• selectable porosity of the fibers for such purposes as concentration of secreted product.

History of HFPB: The first success in hollow-fiber culture demonstrated that such an approach was feasible and suggested some benefits that have since become better understood (3). Shortly thereafter, commercially available systems were presented by such companies as Amicon (now part of Millipore Corporation, www.millipore.com/amicon) and Endotronics (now Biovest International, www.biovest.com), and they demonstrated some utility in the unique potential HFPBs afford. However, those early systems were limited by their less-capable media pumps and fiber materials such as cuprammonium rayon and cellulose acetate, which offered poor gross filtration rates. In the protein production arena, that made them sufficient for hybridoma cultures at limited cell densities, but early units had insufficient nutrient and waste exchange to support the extended culture of such increasingly popular expression lines as CHO and HEK293.

Large-scale cell culture depends on the mass transfer rates of poorly soluble oxygen, which was the Achilles’ heel of earlier attempts at larger-scale hollow-fiber systems. Engineering advances and newer materials are now addressing those limitations: Newer, large-scale hollow-fiber bioreactor systems are being designed with ECS ≤ 1 L (4). In the past few years, system advances and increased demand for the unique features provided have resulted in a resurgence of interest in HFPB.