Separation of CHO cells using hydrocyclones

Abstract

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

Fig. 1

(a) Photograph of the hydrocyclone specially designed for animal cell separation; (b) Schematic view of the fluid flow inside a hydrocyclone

Fig. 2

Experimental set-up of the 20-L stainless-steel tank with two manometers (1), cell suspension (2), a valve (3) and the hydrocyclone (4)

Fig. 3

(a) Size distribution of CHO.K1 cells cultured in spinner flasks in DMEM/F12 medium supplemented with 1% FCS. (b) Cell clumps stained with acridine orange and ethidium bromide observed under a fluorescence microscope

Fig. 4

Separation of CHO.K1 cells. Experimental data versus data predicted by the models for: (a) separation efficiency; (b) flow ratio; (c) flow rate. Dotted lines show a confidence level of 98%

Fig. 5

Cell viability and LDH activity in the supernatant before (“Culture”) and after (0, 3, 6, 24 and 48 h) hydrocycloning at a pressure drop of 1 bar: (a) CHO.K1 cells, HC 3020; (b) CHO-GMCSF cells, HC 3020; (c) CHO-GMCSF cells, HC 2010

Fig. 6

Monitoring of cells before (“Culture”) and after hydrocycloning (0, 3, 6, 24 and 48 h): (a) CHO.K1 cells, HC 3020; (b) CHO-GMCSF, HC 3020; (c) CHO-GMCSF, HC 2010. VNA: viable non-apoptotic cells; VA: viable apoptotic cells; NVA: non-viable apoptotic cells; NEC: necrotic cells; and CF: chromatin-free cells