PharmPro

By Stanley Goldberg, P.E., Director, Glen Mills Incorporated

The culturing of adherent cell clusters and tissue masses has developed from single layer 2-D small quantity systems to high cell densities cultured for long periods of time in a large-scale bioreactor, with cells structured in truly 3-D cell growth configurations. By moving away from violent stirred tank bioreactors with tumbling bead supports to stable scaffolds in rotating bed systems allows for long-term growth and study of fragile systems. Cell densities of 1010 have been attained. The ability to utilize scaffolds of varied geometries and surface chemical bonding treatments give researchers enhanced flexibility. The Z®-RP BIOREACTOR System allows for in vitro extracellular matrix (ECM) studies that mimic the natural environment of cells. Also, long-term perfusion operation allows for both the highly economical recovery of excreted biochemicals and the semi-continuous extraction of cells such as stable stem cell lines, while eliminating the need for thousands of rolling bottles. Additionally, with the 3-D structure it is reported to induce specific detoxification abilities. In this apparatus 3D-grown Hepatoma cell lines are candidates for drug testing.

THE CHALLENGE
The need to grow cells in high densities for long periods of time presents a great challenge. Allowing adherent cells to grow into a truly natural 3-D structure with minimal use of growth media and high cell densities is part of the target criteria. The Background section of this report reviews the 2-D techniques for cell culture. The equipment reviewed here starts with a discussion of the novel SPONCERAM® based scaffold (doped ceramic oxides) and then the Z®RP BIOREACTOR System that has demonstrated the ability to culture cells continuously for twelve months. An example with hepatoma cells validates the equipment while demonstrating how this equipment can help in drug discovery, stem cell culturing, and regenerative medicine applications.

BACKGROUND
Equipment selection depends upon the cells being cultured, whether they are (1) free cells or (2) anchored/adherent cells and (3) the quantity to be produced.

For free cells the traditional bioreactor, fermenter, or chemostat can grow cells such as bacteria, algae, yeast, et al. Also disposable equipment such as wave bags may be used. Volumes from very minute up to thousands of liters, such as in the brewing industry, can be produced. This class of equipment requires cells with structural integrity that can resist vigorous stirring and sparing of gases without damage.

One concern here is that the cell densities may be very low, often below one percent by weight. For recovery, such as for intra-cellular enzymes, this would necessitate kill, dewatering, re-suspension, cell breakage (ref: Goldberg) removal of cell debris, and then purification. 3-D cell structures are not formed by such individual cells.

The early culturing of adherent cells has been within petri dishes on agar. Limited space and batch operation have curtailed the length of culture time and the cell densities attainable. The 2-D layering forces an abnormal geometry upon the cells, causing their interactions and aging to be unnatural to accommodate this non-3-D structure. Harvesting of the cells risks contaminating the balance, and quantities recovered are small. Use of clusters of T-flasks allows for greater quantities, but still mandates handling hundreds of individual vessels. Gels and other substrates can induce somewhat thicker masses "2.5-D" but not fully 3-D.

Improvement to rolling bottles offers surfaces that are specific for a given adherent cells line. The gentle rolling allows them to affix to the walls while being dipped in and out of the liquid nutrients then back to the atmosphere. The ganging of hundred or thousand of bottles can yield industrial quantities of needed products. Thermal controls, gas monitoring, and rotation may be accomplished by placement into control chambers.

All bottles are discrete, so any contamination can be isolated to limit losses. However, the size of and costs to build and maintain GMP rooms are high. Disposal of spent bottles has become a matter of concern, as has the need to manually handle great numbers of items. Cells densities of 107 are achievable in bottles, but not very useful for the study of Extracellular Matrixes (ECM), nor for testing various scaffold materials and geometries.

A rotating bottle with continuous feed/discharge was developed for NASA over a decade ago. The free falling cells were timed to match rotation, thus giving a "zero gravity" effect (ref: NASA). Here the cells were either freely suspended in the liquid or could be anchored to beads, but no large macrostructures were developed.

DEVELOPMENT OF THE DOPED CERAMIC OXIDE SUPPORT - SPONCERAM®
The initial work to produce a suitable scaffold was within the ceramic manufacturing company, HiPer-Group of Oberkrämer, Germany (near Berlin). By working with doped ceramic oxides, Dr. Hans Hoffmeister developed porous, high surface area structures (up to several hundred square meters in a 500 ml volume) having both macro-pores and micro-pores for cell anchoring (tradename SPONCERAM®). The surface lends itself to various custom moiety treatments that would allow for cells of differing strains to be able to grow upon its matrix. Demonstration segments were used to validate the ability of cells to grow (ref: Hoffmeister).

Small samples or plugs of one inch (2.54 cm) are available for researchers to determine their suitability for any particular cell line. The cells after seeding tend to migrate and anchor at the edges of the pores. Then as growth continues the remainder of the surfaces is coated. The interesting point is, though cell densities approach that of the liver (1010), the matrix does not starve the interior cells. Extended apoptosis is not evident.

Having mastered the technique of forming the SPONCERAM® scaffold, (Figure 1 - Photograph of Scaffold) the next task was to construct a suitable bioreactor that would take advantage of these new materials.
DEVELOPMENT OF THE NOVEL 3-D BIOREACTOR
Three components of the Z®RP BIOREACTOR System (REACTOR, GMP BREEDER, and CONTROLLER) are integrated into one package. (Figure 2 - Photograph of System)

Z®RP BIOREACTOR
Being the heart of the system the Z®RP BIOREACTOR is usually operated in perfusion mode and hosts large amounts of cells in very small volumes. The key component is a magnetically coupled rotating axis mounted with the cell- or tissue-carrier of choice exposing cells to the media and the over layer of gas alternately by a gentle dipping action. Fresh gases and nutrients are continuously fed while compounds of economic value and metabolic wastes are expelled. Cells can be extracted in semi-batch operations.

Various supports have be installed in the Z® RP BIOREACTOR including materials from highly porous Sponceram® discs all the way to implantable scaffolds. This presents researchers with a vast variety of culturing options. In all configurations, the rotating bed has shown that excellent aeration and feeding is occurring. The gentle rotational motion stimulates cells and tissues to adhere and proliferate rapidly without being stressed by shear forces. Cell populations stay viable and develop large areas of extracellular matrixes. Three-dimensional, high-density cultivation can last many months without loosing viability or expression productivity. (Figure 3 - Photograph of Chinese Hamster Ovaries CHO 200 days; 100 grams) The longest continuous test of about twelve (12) months was purposely terminated due to costs of media. Harvesting of adherent cells is achieved by following specific rotation programs in combination with detaching solutions. Any blockage of the air/nutrients streams or mechanical interference with moving parts by the cells is not evident. (Figure 4 - Photograph of CHO close up)

Z®RP GMP BREEDER (Incubator)
The Z® RP GMP BREEDER is a Class 100 enclosure that maintains sterility and thermal stability by laminar air flow. The compact (3' x 3' x 3') sterile and temperature controlled chamber protects the Z®RP BIOREACTOR from contamination with UV lights, HEPA filters, and slightly excess air pressure. This allows the user to conduct all necessary manipulations - e. g. preparation of cell and tissue samples, inoculation, change of media container, tube change, sensor exchange, cell harvesting - in a sterile and closely controlled environment. The use of thermally controlled air is more stable than liquids such as those around glass reactors.

Z®RP CONTROLLER
The Z®RP CONTROLLER monitors the operation of all cell- and tissue-culturing processes conducted in Z®RP BIOREACTOR and GMP BREEDERS. System configurations and process parameters are accessible by the integrated Siemens touch screen. The unit contains mass flow meters, peristaltic pumps, monitors, and instrumentation. All relevant cultivation data are logged and evaluated on a personal computer with standard software that meets GMP standards (ref: Suck).

CRITICAL DEMONSTRATION - HEPATOMA CELL
Human hepatoma cells may provide an excellent model to predict toxic effects of pharmaceuticals on the human body, avoiding animal testing. Recent work performed in Germany compared 2-D and 3-D cell cultures of hepatoma cells (HepG2) for their xenobiotic metabolizing function. This was done by measuring EROD, a biomarker that indicates a liver cell is digesting a toxin through the cytochrome pathway.Dr. Hoffmeister stated "Measuring the amount of this biomarker should directly correlate with the toxic effect of a substance incubated with the liver cell." Normally, liver cells do not activate that pathway, but the 3-D culture induces this detoxification ability and, therefore, makes hepatoma cell lines a candidate for drug testing (currently only primary hepatic cells have been used).

He continues, "Hepatoma cells, as well as many other types, arrange in a 3-D fashion including embedding themselves in extracellular matrix and develop characteristics of primary cells, i.e., change their metabolism from an undifferentiated cell line to a metabolism close to a primary cells."

The study data show that maximum EROD activity on the Sponceram was 2.23-fold on day 12 versus the activity of monolayer cells on day 7 of cultivation. This demonstrated that 3-D cultivation resulted in improved functional characteristics versus monolayer culture.Dr. Goepfert and Dr. Hoffmeister presented this study at the "ESACT" conference in Dublin (June, 2009). The cells were grown on SPONCERAM® discs in a 500 mL Z®RP BIOREACTOR System (distributed in North America by GLEN MILLS INC., Clifton, NJ).

CONCLUSIONS - OBJECTIVES ATTAINED
Many cells that are anchorage dependent may be cultured on SPONCERAM®. This allows for the production of cell lines for biopharmaceutical production, stem cells for cell therapy, and primary human cells for tissue engineering.

The Z®RP BIOREACTOR Systems has, in addition to the 500ml and 5,000ml BIOREACTORS, a 50ml disposable units for individual patients or small-scale experiments.

Other future applications for this carrier include the manufacture of large tissue pieces for patient-specific bone/cartilage for arthritis implants.

Reduction of culture bottles usages started with inoculation of 107 cells into the Z®P BIOREACTOR where the final density of 1010 can be achieved. When compared with bottles where 107 is a good final density, one Z®RP can replace 1,000 bottles (103). Thus presenting a great savings in space, bottles, and disposal costs.

In summary, the accomplishments include: (1) GMP compliant technology for the expansion of stem cells and other primary cell lines, (2) Production of bulk amounts of customers cells suitable for therapeutic use, and (3) Contract manufacturing of complex recombinant glycoproteins.

REFERENCES
Goldberg, S. I., "Mechanical Cell Disruption Techniques", Posch, A., Ed. 2-D PAGE, Humana Press, 2008, pp 2-24.NASA: http://science.nasa.gov/NEWHOME/br/bioreactor.htm

Hoffmeister, H., Sponceram® Carrier Discs for Adherent Cell Culture, Rev 103, ZELLWERK GmbH, Germany, 2008, pp 1-2.

Suck, Hoffmeister, et al., "Cultivation of MC3T3-E1 Cells", Journal of Biomedical Materials Research Part A, DO1, 10.1002, pp 269-275.