Paul J. Price

Viable cultured neurons in ambient carbon dioxide and hibernation storage for a month.

Neurobasal is a bicarbonate-buffered medium optimized for the growth of embryonic rat hippocampal neurons at pH 7.3 in 5% CO2. Neurons die within hours in this or in other 26 mM bicarbonate buffers when transferred to ambient CO2 (0.2%). Death is associated with a rapid rise in medium pH to 8.1. A new CO2-independent modification of Neurobasal (Hibernate E), when supplemented with B27, can maintain neuron viability for at least 2 days in ambient CO2. This same medium can also be used to store viable brain tissue for up to a month with refrigeration. These advances should facilitate studies of neuron physiology outside the incubator as well as storing and transporting neuronal tissue.

Engineering mammalian cells for solid-state sensor applications.

A fundamental advance in the development and application of cell- and tissue-based biosensors would be the ability to achieve air-dry stabilization of mammalian (especially human) cells with subsequent recovery following rehydration. The would allow for the preparation of sensors with extended shelf lives, only requiring the addition of water for activation. By understanding and subsequently employing the tactics used by desiccation-tolerant extremophiles, it may be possible to design stabilized mammalian cell-based biosensors. The approaches required to realize this goal are discussed and illustrated with several examples.

Use of Liquid Medium Concentrates to Enhance Biological Productivity.

Liquid medium concentrates (LMC) were initially developed in response to industrial customer demand for improved efficiency and productivity of mammalian cell bioreactors. The resultant technology, which is undergoing its second generation of improvement, exploits biochemical properties intrinsic to the constituent nutrients to improve solubility, stability, and biological performance. Customer-perceived benefits, relative to liquid media produced by conventional technologies, include both enhanced biological productivity and improved manufacturing efficiency. The key concept which drives superior performance is that in concentrate technology all nutrient components of a complex biochemical formulation are fully pre-solubilized as a minimal number (generally three) of 50X LMC sub-groupings precedent to complete admixture. Under conventional procedures, critical nutrient potencies may be reduced relative to theoretical values due to poor solubilization and partial removal by precipitation and filtration. Use of LMC intermediates for the formulation of liquid media produces superior initial correlation with theoretical nutrient potency and may result in superior performance stability. Delivery of the full complement of nutrients to the biomass may be limiting to culture survival, attainment of maximal cell density, optimization of the specific cellular productivity, and prolongation of the bioreactor production cycle. The advantages of LMC become increasingly apparent with serum reduction or elimination, augmented cell densities, enhanced medium residence time and increasing development of balanced nutrient formulations. Complex nutrient media, formulated as LMC, are stable for up to one year in many cases, are compatible with various hulk containers, and exhibit superior biochemical potency and biological performance relative to conventionally-prepared culture media.

Continuous, Automated Reconstitution of Liquid Media Concentrates

Two years of customer experience with liquid media concentrates (LMC) have confirmed the superior consistency, quality and stability of IX liquid media produced from concentrated intermediates. However, facilitated processes and reduced cost for media manufacture have been the primary motivations for conversion by large volume industrial users from powdered medium to LMC. Recent technical advances have increased the breadth of formulations, enhanced lipid solubility and vitamin stability, increased fold concentration, and qualified alternative concentrate buffering approaches. Most large-scale users of LMC have reconstituted medium in formulation tanks similar to their current method for producing liquid medium from powder, experiencing a 50–75% reduction in overall time required for high volume medium manufacture. Some users have consistently reconstituted medium from LMC in closed vessels containing sterile diluent water. We now report development of an automated admix device for continuous apportionate reconstitution of LMC. The unit is designed for compatibility with a clean room environment, with size (6 ft2 of floor space) and weight (less than 400 lbs.) parameters to minimize facility costs. A prototype unit is capable of on-line preparation of liquid medium at continuously adjustable delivery rates up to 200 liters per hour with a mixing precision tolerance of less than ±1%. Its modular design permits regulated delivery of up to five LMC nutrient feeds. Second generation mixing devices based upon this prototype design are targeted for 1994 field evaluation to expand this capability to flow rates of 1500 liters per hour incorporating an unlimited number of independently-regulated LMC feed streams driven by a process logic controller and software interface to facilitate initial validation and batch documentation. Utilizing appropriate LMC intermediates, the mixing device can be applied to large volume preparation of nutrient medium for bioreactor feeding or of buffered salt solutions used for cell/tissue washing or chromatographic purification. Nutrient media reconstituted using this mixing device may be sterile-filtered and delivered either directly to a large surge tank or manifolded to multiple storage vessels. Technical advances in LMC sub-grouping and stabilization combined with the automated reconstitution capacity of the apportionate mixing device provides the potential for continuous preparation of a homogeneous 250,000 liter batch of liquid medium or buffered salt solution from bulk concentrates.

Improved bioreactor productivity and manufacturing efficiency using liquid medium concentrates

Synopsis Liquid media concentrates (LMC), designed for a broad range of classical and novel culture formulations, have exhibited superior performance in large-scale animal cell bioreactors relative to conventional media delivery options. Various options for concentrate reconstitution reduce the complexity and cost of high volume media preparation. Collaborative field studies conducted over the past two years confirm that use of LMC can improve bioreactor productivity and manufacturing efficiency in a broad range of animal cell culture environments.

Serum-Free Media for the Growth and Maintenance of the Differentiated State of Keratinocytes, Hepatocytes, and Neural Cells

Culture media are generally supplemented with animal sera to promote cellular proliferation. However, serum contains components which alter the phenotypic expression of the proliferating cells and decrease its usefulness as a model system. Serum addition may produce undesirable artifacts, resulting from: (1)serum lot variability; (2)cellular differentiation and/or senescence; and (3)alteration of the effective dose of the presumptive toxicant. To avoid such artifacts, we have developed several serum-free media (SFM) which both support cell growth and maintain normal phenotypic expression of keratinocytes, hepatocytes, and neural cells.

Sustained Inducibility of Cytochrome P450 Activity in Rat Hepatocytes Cultured in a Serum-Free Medium

Liver microsomal oxygenases are multicomponent enzyme systems which metabolize a wide variety of xenobiotics. A major component of the system is a group of enzymes collectively known as cytochrome P450 (CP450). A major limitation in the use of rodent hepatocyte cultures in toxicity testing and pharmacokinetic studies has been the rapid loss of phase 1 reactions catalyzed by the CP450-dependent mono-oxygenases. Using a sandwich matrix and a serum-free medium developed by GIBCO, total rat CP450 could be maintained for at least 9 days at 75–80% day “0” levels. Metabolic studies of the microsomal fraction of primary adult rat hepatocytes, measured by the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin and of 3,4-benzo-[a]-pyrene to 3-hydroxybenzo-[a]-pyrene, demonstrated maintenance of activity over the same 9 days comparable to the “0” time controls.