Anchorage-dependent cells microcarrier cultures

Anchorage-dependent cells are grown in suspension culture by growing the cells attached to small microcarrier beads (100-300 pm) suspended in the culture medium by agitation. There are two types of microcarriers available (Chapter 5, sections 5.8 and 5.9 Griffiths, 1990) the non-porous microcarriers whereby cells are growing only on the outer surface of the beads, and porous microcarriers whereby cells are growing predominantly in the internal porous structure of the microcarrier beads. 

There is no substantial literature on direct sparging of non-porous microcarrier cultures. As is discussed in section 4.6, the difficulty is that the presence of bubbles induces bead flotation, i.e. attachment of beads to bubbles, and the formation of large bead-bubble aggregates that tend to rise and accumulate at the surface of the culture vessel, which is a highly undesirable characteristic. Nevertheless, it is possible slowly to sparge microcarrier cultures without undue cellular injury if suitable surfactants/antifoams (e.g. Pluronic F-68 or Medical Emulsion AF see section 4.6) are used. 

For non-sparged cultures of non-porous microcarriers, cellular injury is likely to be the result of three distinct mechanisms (Papoutsakis, 1991a) interactions 

Aunins JG, Woodson BA, Hale TK Wang Die (1989) Effects of paddle impeller geometry and mass transfer in small-scale animal cell culture vessels. Biotechnology and Bioengineering 34 1127-1132. 

Borys MC Papoutsakis ET (1992) Formation of bridges and large cellular clumps 

---

Kalogerakis, N. and L.A. Behie, "Oxygenation Capabilities of New Basket-Type Bioreactors for Microcarrier Cultures of Anchorage Dependent Cells", Bioprocess Eng., 17, 151-156(1997). 

As previously stated, ammonium in cell culture medium is the product of glutamine metabolism and its spontaneous decomposition at 37°C. Negrotti et al. (1989) showed that the half-life for glutamine at 37°C and pH 7.2 is only 7 days. The effects of ammonium on cell metabolism are observed from concentrations as low as 2 mM, which is easily reached in culture systems (Table 4.2). For example, the spontaneous decomposition of glutamine can result in 0.1 mM ammonia per day (Butler and Spier, 1984). Anchorage-dependent cells grown on microcarriers produce between 2 and 3 mM ammonia after growth in a batch culture (Butler et al.,... 

Anchorage-dependent cells can be grown on solid or macroporous microcarrier beads and these can then be superfused with nutrient medium in the spectrometer. The advantages of the system include direct contact between the perfusate and the cells (see below) and growth of the cells at rates comparable to those found in tissue culture dishes. A disadvantage, with solid beads, is the relatively low cell density obtainable due to the large sample volume occupied by the beads. 

Microcarriers are small solid particles (kept in suspension by stirring) upon which cells may grow as a monolayer. They confer the advantage of large scale suspension cultures on anchorage dependent cells. They thus offer the following advantages. 

As microcarriers provide a good surface area for attachment per unit volume, various types have been routinely used to grow anchorage-dependent cell lines in bioreactors primarily used for suspension cultures. Airlift reactors can also be operated using microcarriers. Wang et al. reported the use of a fluidized-bed... 

This method opened up both industrial production opportunities and allowed research laboratories to easily produce substantial quantities of developmental products. Microcarrier culture is the most versatile, reliable, and characterized procedure for unit volume scale-up of anchorage-dependent cells. It has had widespread use for industrial processes [vaccines, interferon, tPA, and human growth hormone (hGH)] as well as many developmental uses, has been scaled-up to 4000 L, and has the potential for process intensification by perfusion with spin filters or by the use of microporous microcarriers. 

Frequently, hPSCs have been cultmed on microcarriers in suspension because a high surface-to-volume ratio can be achieved for a microcarrier cell culture system. Microcarriers have been used for the scalable cultme of anchorage-dependent cells. 

---