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Research Papers

# A Dilution-Filtration System for Removing Cryoprotective Agents

[+] Author and Article Information
Xiaoming Zhou

School of Mechatronics Engineering, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, Chinaxiao-ming_zhou@hotmail.com

Zhong Liu

Anhui Blood Center, Hefei, Anhui 230022, Chinaliuzhong126@yahoo.com.cn

Zhiquan Shu

Department of Mechanical Engineering, University of Washington, Seattle, WA 98195zqshu@u.washington.edu

Weiping Ding

Department of Mechanical Engineering, University of Washington, Seattle, WA 98195wpding@u.washington.edu

Pingan Du

School of Mechatronics Engineering, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, Chinadupingan@uestc.edu.cn

JaeHyun Chung

Department of Mechanical Engineering, University of Washington, Seattle, WA 98195jae71@u.washington.edu

Carolyn Liu

Swedish Physicians Pine Lake Clinic, Sammamish, WA 98075carolynLiu@comcast.net

Shelly Heimfeld

Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA98109sheimfel@fhcrc.org

Dayong Gao1

Department of Mechanical Engineering, University of Washington, Seattle, WA 98195dayong@u.washington.edu

1

Corresponding author.

J Biomech Eng 133(2), 021007 (Jan 31, 2011) (7 pages) doi:10.1115/1.4003317 History: Received May 10, 2010; Revised November 25, 2010; Posted December 22, 2010; Published January 31, 2011; Online January 31, 2011

## Abstract

In most cryopreservation applications, the final concentrations of cryoprotective agents (CPAs) must be reduced to biocompatible levels. However, traditional methods for removing CPAs usually have disadvantages of operation complexity, time consumption, and ease of contamination, especially for the applications involving large volumes of cell suspensions. A dilution-filtration system, which involves pure ultrafiltration for separation, was developed for continuous, automatic, and closed process of removing CPAs. To predict the optimal protocols under given experimental conditions, a theoretical model was established first. Cell-free experiments were then conducted to investigate the variation in CPA concentration during the process, and the experimental data were compared with the theoretical values for the validation of the model. Finally, ten units $(212.9 ml/unit±9.5 ml/unit)$ of thawed human red blood cells (cryopreserved with 40% (w/v) glycerol) were deglycerolized using the theoretically optimal operation protocols to further validate the effectiveness and advantage of the system. In the cell-free experiments, glycerol was continuously removed and the concentration variations fitted the simulated results quite well. In the in-vitro experiments, glycerol concentration in RBC suspension was reduced to $5.57 g/l±2.81 g/l$ within an hour, and the cell count recovery rate was $91.19%±3.57%$, $(n=10)$, which proves that the system is not only safe for removing CPAs, but also particularly efficient for processing large-scale samples. However, the operation parameters must be carefully controlled and the optimal protocols should be specialized and various from case to case. The presented theoretical model provides an effective approach to find out the optimal operation protocols under given experimental conditions and constrains.

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## Figures

Figure 1

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.

Figure 2

Theoretical modeling of the system. (a) The overall system and (b) a control volume.

Figure 3

Simulated glycerol concentration variation and cell volume excursion in CV1 (initially at the diluting point) during a dilution-filtration process

Figure 4

Variations in time cost (real line and left Y-axis) and maximum cell volume (dash line and right Y-axis) with blood or diluent flow rates as parameters

Figure 5

Variations in glycerol clearance (real line and left Y-axis) and maximum cell volume (dash line and right Y-axis) with glycerol concentration as a parameter

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