Emerging developments in tissue engineering and cell technology.

Tissue engineering is moving rapidly from potential to accepted medical practice. Use of human diploid fibroblast cells to form three-dimensional dermal replacement tissue is described. The cell source of these products is fully tested and the products may be frozen and stored prior to use. A summary of pilot clinical trials for Dermagraft()-Transitional Covering and Dermagraft()-Ulcer show the feasibility of this approach. A development program for cartilage tissue has resulted in both reconstructive and orthopedic applications that have been demonstrated in preclinical animal studies. Cardiovascular applications to prosthetic biological heart valve constructs are also discussed.

Manufacture of biopharmaceutical proteins by mammalian cell culture systems.

In the last several years, dramatic advances have been in the development of new biopharmaceuticals including monoclonal antibodies for diagnosis and treatment and such genetically engineered proteins as tPA, Factor VIIIc, erythropoietin and soluble CD4, an anti-AIDS protein. Currently, there are several hundred such candidate drugs in human clinical trials. In most cases, these protein-based drugs will require manufacture by mammalian cell culture due to the inability of lower organisms to properly glycosylate, fold, make correct disulfide bonds and secrete active biomolecular forms. The need for large scale production from cell culture will greatly increase as more of the products in clinical trials are approved for commercial production. This will require significant reduction in manufacturing costs per gram, concomitant with increased capacity to hundreds or perhaps even thousands of kilograms annually. As an example, Invitron's multi-reactor manufacturing facility has operated at greater than one-half million liters per year and has experience with more than 250 mammalian cell lines for producing protein drug products.

Manufacture of pharmaceutical proteins from hybridomas and other cell substrates.

Murine monoclonal antibodies are now routinely produced from highly expressing hybridoma cell lines for human clinical applications. Epstein Barr Virus transformed human lymphocytes are used to produce human monoclonal antibodies but these cells have generally exhibited low expression and poor stability in culture when compared with typical murine hybridomas. Alternative cells are therefore finding wider usage. Backfusions and tribrids are being used to increase and/or prolong the expression of monoclonal antibodies. In addition, genetically engineered cells producing antibodies with human constant and murine variable regions may reduce antigenicity of the chimeric protein used for injection. Pharmaceutical acceptability of the final product is also strongly dependent on culture and purification methods that are used in the manufacturing process. Large scale perfusion technology has allowed high density, efficient monoclonal production in the absence of antibiotics in low serum or serum free media. These methods reduce the cell protein/DNA challenge to the purification process by dead and dying cells and also prevent product degradation by reducing product residence time within the bioreactor. Gentle purification procedures that avoid extremes of pH and salt concentration can preserve structural integrity and biological activity of the protein and can provide high yields and purity in excess of 99%. Such purification procedures alone or in combination with specific viral inactivation steps can be validated to have the capability overall to reduce virus concentration by a factor of 10(8) to 10(12). The combination of carefully developed cell substrates, with advanced perfusion culture and purification technology can consistently produce acceptable protein-based pharmaceutical products in compliance with cGMP's.

The recovery of highly purified biopharmaceuticals from perfusion cell culture bioreactors.

The impact of continuous perfusion cell culture technology on production of one and two chain human rtPA, as well as monoclonal IgG and IgM, will be reviewed. Perfusion as compared to batch systems improve the quality of the product in the conditioned media in terms of biological activity and structural integrity. This significantly increases downstream recovery and daily production output of final purified material. These findings are a consequence of the ability of perfusion technology to 1) maintain cells intact at high cell density; 2) effectively reduce serum content to approximate serum free conditions; and 3) minimize residency time of labile product within the 37 degrees C environment of the bioreactor.

Large-scale mammalian cell culture: Design and use of an economical batch suspension system.

Large-scale mammalian cell culture in the absence of antibiotics requires stringent conditions of sterility for all vessels, procedure, and systems used. Application of existing fermentation technology suffers from the differences between mammalian and bacterial cultures. Relatively simple and inexpensive 100-L vessels have been designed specifically for medium storage and antibiotic-free mammalian cell culture. These vessels are portable and sterilized in a 2 x 3 x 5 ft conventional or VACUMATIC autoclave. They consist of 30-gal 316 stainless-steel sanitary process drums whose heads have been modified to meet the rapid pressure changes that occur during autoclaving. The vessels incorporate systems for aseptic introduction and removal of both liquids and gases required for inoculation, growth, and harvesting of cell suspensions. A two-disk vibromixer is used for agitation with inoculation at a laminar flow hood and incubation in a warm room. These vessels have been used for culture of one rat and eight human tumor lines for over 2 x 10(5) L of suspension.

Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures.

A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10(9) cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O2 and CO2 were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10- to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4- ad 40-liter reactor also has been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10 x 10(6) cells/ml with a total yield of 8.7 x 10(11) cells from 360 liters of medium.

Cell aggregate suspension culture for large-scale production of biomolecules.

A method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm2 flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1 x 10(10) cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.

Establishment of heteroploid cell lines from mouse peritoneal exudate cells.

Proliferation was observed during in vitro cultivation of peritoneal exudate cells that had been educed from a C3H mouse with Freund's incomplete adjuvant. These cells were successfully subcultured by release with trypsin-EDTA solution and are now at passage 108 after 22 months in culture. Using this technique, 12 other rapidly growing peritoneal exudate cultures were obtained, whereas 10 cultures not educed with adjuvant did not proliferate. Characteristics of four adjuvant-induced cell lines established in culture include: rapid attachment to glass, doubling time in culture of 18 to 19 hr, phagocytosis of colloidal carbon, enhanced phagocytosis of specifically sensitized bacteria, epithelium-like morphology, and retention of C3H histocompatible specificities. These cell lines had widely varying chromosome distributions with modes from 37.3 +/- 2.4 to 82.6 +/- 2.30, but inoculation of 10(7) cultured cells into syngeneic animals did not produce tumors. Procedures described for the reproducible establishment of peritoneal exudate cell lines did not require use of conditioned media or exogenous viral infection.