**Theory of optimal operation protocol**

122 Current Frontiers in Cryobiology

contamination (Valeri et al, 2001), but the expensive cost limits their application in many areas. Recently, Dialysis was considered as an alternative method by some researchers (Castino et al,1996; Arnaud et al 2003; Ding et al 2007,2010). It can remove CPAs efficiently; however, due to the non-uniformity of distribution of hollow fibers, the mass transport in dialyzer is too complicated to be controlled, especially in the unsteady state. In addition, dialysis method is not efficient to remove large molecular substances (Daugirdas, et al, 2006), such as cell fragment and the released protein from broken cells. These factors limit

In clinic, hemofiltration, which involves dilution and filtration to remove toxins from blood, has been proved to have better controllability as well as ability of removing large molecular substances than hemodialysis (Daugirdas, et al, 2006). By referencing to hemofiltration, a dilution-filtration system is developed recently for removing CPAs (Zhou et al, 2011). The closed system helps to avoid contamination to cells, and the continuous and automatic process could provide particular advantage in efficiency especially for large-scale samples.

A dilution-filtration system is developed as shown in Fig.16 (Hemofilter: Plasmflo TM AP-05H/L, ASAHI; Pumps: 400F/M1, Watson-Marlow; silicone tubing: 985-75, Pall). For removing CPAs, thawed cell suspension is first transferred into the special blood bag (made by an infusion bag). Then, the suspension is driven by the blood pump to flow circularly among the bag, the mixer and the hemofilter. While going through the mixer, the suspension is quickly diluted by diluent, and the dilution ratio can be controlled to prevent lysis. In the hemofilter, extracellular solution containing CPA is partly ultrafiltrated while cells keep inside. Along with the circulation goes on, CPAs in cell suspension can be removed continuously. The whole process is conducted automatically in a closed system, and thus it is hopeful for this

Fig. 16. Principle of the dilution-filtration system. Cell suspension is diluted and ultrafiltrated during circulating in the system, and then the CPAs inside can be continuously removed.

method to reduce human labor as well as the risk of contamination significantly.

the use of dialysis method in some applications.

**2.3 Materials and methods** 

**Technical Design** 

The related research work is introduced in the following.

Optimal operation protocol is defined here as the processes that minimize the operation time (to a final CPA concentration below 10g/L) as well as the osmotic cell volume excursion. A theoretical model was developed to predict the optimal operation protocols under the given experimental conditions (initial CPA concentration, cell density and total volume of cell suspension) and practical constraints. The detailed considerations for this procedure are described below.
