two-phase (i.e., gas–liquid) culture system composed of CO2-

enriched air and aqueous culture medium, closed inside the dispos-

able bag-like container. Continuously generated waves cause agita-

tion of large volumes of medium and facilitate to disperse gases,

nutrients, extracellularly secreted bioproducts in the liquid phase

[6–8], what finally enhances the homogeneity of whole waving

culture system.

To enhance the proliferation yield of adherent animal cells

exhibiting anchorage-dependency toward solid surface, that is,

majority of mammalian, avian, fish, and insect cells, an easy access

of biomass to solid-state surface must be provided [9]. Microcar-

riers, in the form of μm-/mm-scale beads made of biocompatible

polymer, characterized by large surface-to-volume ratio, are recog-

nized as the most efficient proliferation supporters in cultures of

adherent cells [10, 11]. Moreover, depending on the type of

applied microcarriers, adherent cells may grow in monolayered

form on the outer surface of beads or migrate inside micropores

of porous beads suspended in the culture medium [12, 13].

The chondrocytes and cartilage tissue are responsible for the

skeleton stabilization and connecting elements of the skeletal sys-

tem. Due to slow in vivo regeneration of joint cartilage, the most

effective way to rebuild degraded cartilage is chondrocyte reim-

plantation into damaged joints [14, 15]. Currently, new methods

for efficient and low-cost in vitro cultures of chondrocytes are

seeking with a wide scope of possible applications of bioprocessed

chondrocytes in medicine [15, 16].

In this chapter, a detailed methodology for efficient prolifera-

tion of CP5 chondrocytes on Cytodex 3 microcarrier beads, per-

formed in ReadyToProcess WAVE 25 disposable bioreactor has been

presented. Some procedures useful for daily monitoring the prolif-

eration of CP5 chondrocytes, by measurement of density, viability

of cells and their metabolic activity, the specific glucose consump-

tion rate, the level of lactate dehydrogenase activity in culture

medium, as well as levels of DO and pH, have been comprehen-

sively described in detail.

2

Materials

All culture media components must be certified as cell culture

approved. All applied liquid media must be prepared under sterile

conditions. Inoculation of the disposable bag-like container with

cells must be performed in a biological safety cabinet under sterile

conditions. Cell-containing samples harvested from the disposable

culture bag must be sterilely handled. Analysis of harvested cell-free

samples of culture medium may be proceeded outside biological

safety cabinet, but in a reasonably clean-class laboratory, as it is

usually provided for cell or tissue culture.

146

Kamil Wierzchowski and Maciej Pilarek