Surface immobilization of anchorage‐dependent mammalian cells

Abstract

Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (< or = 2-3 h) attachment of inoculated cells (> or = 95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.

Rabies virus production in non‐woven polyester fabric (NWPF) packed‐bed reactors

“…In addition, the variation in pH value was controlled by a pH meter (Microprocessor pH/ION Meter, pMX 2000, WTW, Werkstatten, Welheim, Germany) and, depending upon the variation in pH, addition of fresh medium was carried out as indicated in the Results and discussion section. Since the standard trypsin‐treatment techniques were not satisfactory for counting cells growing on the fibre discs, an alternative counting technique based on the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2 H ‐tetrazolium bromide (MTT) assay was employed [11].…”

Utilizing the macroporous packed bed for insect cell/baculovirus expression

“…For example, the development of adequate inoculation protocol [13,14], toxicity problems associated with high bead concentration [15] and the extreme sensitivity of adsorbed cells to mixing and bubble shear [14,16] are concerned. Hollow ®ber and ceramic cartridges have inherent problems of mixing and`inoculation' and require elaborate circulation, oxygen transfer and control system [11]. Problems such as diffusivity coef®cient decrease in polymer gel, cut-off of larger molecular weight nutrients and toxicity of polymer exist in encapsulation techniques [17,18].…”

“…PUF was used in some investigations [21,22]. It is widely accepted that the cells inoculated into the PUF packed bed become readily entrapped within pores [11]. Compared with agarose bead, alginate bead [23] and polylysine capsules [24] which often require more effort on materials treatment, there is no need to activate immobilized cells or support before inoculating cells into PUF packed bed.…”

Utilizing the macroporous media for insect cell/baculovirus expression

“…However, some problems limit this culture system including the development of suitable inoculation protocol [13], toxicity problems associated with high bead concentration [14] and shear stress exposing to cells attached onto microcarrier surface [15]. Other immobilization techniques like hollow-®ber and ceramic cartridges have inherent problems of mixing, inoculation, and require forced circulation for oxygen transfer [11]. Encapsulation by polyelectrolyte complexation like alginate-polylysine [16], cross-linked albumin [17] still have some problems as polymer toxicity [18] and diffusion limitation [19].…”

“…Cells¯ow through porous substratum, get captured physically and then grow within matrices of the porous support. Polyester mat [11], polyvinyl formal (PVF) resin [20] and polyurethane foam (PUF) [21] have been employed for human carcinoma cell line (HeLa), mouse myeloma MPC-11 cell and monkey kidney cells cultivation.…”