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<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN" "http://www.w3.org/TR/html4/loose.dtd"><html><head><meta http-equiv="Content-Type" content="text/html; charset=UTF-8"><meta name="robots" content="INDEX,NOFOLLOW,NOARCHIVE"><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/"><meta name="citation_journal_title" content="Cytotechnology"><meta name="citation_title" content="Separation of CHO cells using hydrocyclones"><meta name="citation_authors" content="Rodrigo C.V. Pinto, Ricardo A. Medronho, and Leda R. Castilho"><meta name="citation_date" content="2008 January"><meta name="citation_issue" content="1"><meta name="citation_volume" content="56"><meta name="citation_firstpage" content="57"><meta name="citation_doi" content="10.1007/s10616-007-9108-x"><meta name="citation_pdf_url" content="http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2151964&blobtype=pdf"><meta name="citation_abstract_html_url" content="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2151964&rendertype=abstract"><meta name="citation_fulltext_html_url" content="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2151964"><meta name="citation_pmid" content="19002842"><meta name="DC.Title" content="Separation of CHO cells using hydrocyclones"><meta name="DC.Type" content="Text"><meta name="DC.Publisher" content="Springer"><meta name="DC.Contributor" content="Rodrigo C.V. Pinto"><meta name="DC.Contributor" content="Ricardo A. Medronho"><meta name="DC.Contributor" content="Leda R. Castilho"><meta name="DC.Date" content="2008 January"><meta name="DC.Identifier" content="10.1007/s10616-007-9108-x"><meta name="DC.Language" content="en"><link rel="stylesheet" href="corehtml/pmc/css/pmcstatic.css" type="text/css"><link rel="stylesheet" href="corehtml/pmc/css/pmcbase1.css" type="text/css"><link rel="stylesheet" href="corehtml/pmc/css/pmcbars-slateblue.css" type="text/css"><link rel="stylesheet" href="corehtml/pmc/css/pmcbody4.css" type="text/css"><link rel="stylesheet" href="corehtml/pmc/css/pmcrefs1.css" type="text/css"><script type="text/javascript" src="corehtml/pmc/js/jquery-1.3.2.min.js"></script><script type="text/javascript" src="corehtml/pmc/js/jquery.hoverIntent.min.js"></script><script type="text/javascript" src="corehtml/pmc/js/common.js"></script><script type="text/javascript">window.name="mainwindow";</script><link href="http://www.ncbi.nlm.nih.gov/corehtml/jsutils/css/tileshop_pmc.1/tileshop_pmc.1.css" type="text/css" rel="stylesheet"><script type="text/javascript" src="http://www.ncbi.nlm.nih.gov/corehtml/jsutils/utils.1.js"></script><script>utils.jsLoader.sBase = "http://www.ncbi.nlm.nih.gov/corehtml/jsutils/";</script><script type="text/javascript" src="http://www.ncbi.nlm.nih.gov/corehtml/jsutils/tileshop_pmc.1/tileshop_pmc.1.js"></script><script type="text/javascript" src="http://www.ncbi.nlm.nih.gov/corecgi/tileshop/tileshop_data_db.1.js"></script><title>Separation of CHO cells using hydrocyclones</title></head><body class="pmc-body"><div style="height: 0px;"><a id="top" name="top"></a></div><table cellpadding="0" cellspacing="0" width="100%" border="0"><tr valign="top"><td colspan="3"><table border="0" cellspacing="0" cellpadding="0" style="border-collapse: collapse;"><tr><td width="145" valign="top"><img style="border-style: none" usemap="#pmclogo" src="corehtml/pmc/pmcgifs/pmc3_logo_v5.gif" alt="pmc logo image"><map name="pmclogo"><area shape="rect" coords="65,52,137,67" href="fprender.fcgi" alt="Journal List"><area shape="rect" coords="7,52,52,67" href="redirect3.cgi?&&auth=06kxxTF4C0LKlIANIi6aFQHC4HBxBK4kOuTIu_AIL&reftype=other&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CLogo&TO=Entrez%7CSearch%7CAll%20PMC&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/sites/entrez?db=pmc" alt="Search"><area shape="rect" coords="0,0,145,75" href="./" alt="pmc logo image"></map></td><td height="75" valign="top" style="padding-left: 4px;"><div><img align="top" style="border-style: none" src="corehtml/pmc/pmcgifs/logo-cytotech.gif" alt="Logo of cytotech" usemap="#logo-imagemap"><map id="logo-imagemap" name="logo-imagemap"><area shape="rect" alt="springer.com" coords="2,57,108,73" href="redirect3.cgi?&&auth=0pyJtbhlFihCLV-WAhRXMYgvbjdCD4qUpvUDqOAMW&reftype=publisher&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CBanner&TO=Publisher%7COther%7CN%2FA&rendering-type=normal&&http://www.springer.com" onclick="focuswin(pmc_ext)" target="pmc_ext"><area shape="rect" alt="This journal" coords="111,56,203,72" href="redirect3.cgi?&&auth=0jao4rD0k03qYUY__B-fOtUIdbWjlyZf0mv4H1Q-K&reftype=publisher&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CBanner&TO=Publisher%7COther%7CN%2FA&rendering-type=normal&&http://www.springer.com/10616" onclick="focuswin(pmc_ext)" target="pmc_ext"><area shape="rect" alt="Toc Alerts" coords="208,57,289,73" href="redirect3.cgi?&&auth=06OdD92zURYpKpFF-fKS7Ua_Qxhk357tNXzZxn5GH&reftype=publisher&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CBanner&TO=Publisher%7COther%7CN%2FA&rendering-type=normal&&http://www.springer.com/springeralerts/toc-alphabetical" onclick="focuswin(pmc_ext)" target="pmc_ext"><area shape="rect" alt="Submit Online" coords="293,56,393,72" href="redirect3.cgi?&&auth=0xoQJ1nHp2bz1FQjHHxCC8IODsNIA4q_KYncdTABp&reftype=other&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CN%2FA&TO=N%2FA%7CN%2FA%7CN%2FA&rendering-type=normal&&http://www.editorialmanager.com/cyto/" onclick="focuswin(pmc_ext)" target="pmc_ext"><area shape="rect" alt="Open Choice" coords="401,56,497,73" href="redirect3.cgi?&&auth=0Xm82Ay3OlqUhozMfHA2izaB9UfN0KSYZniizzA-I&reftype=publisher&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CBanner&TO=Publisher%7COther%7CN%2FA&rendering-type=normal&&http://www.springer.com/openchoice" onclick="focuswin(pmc_ext)" target="pmc_ext"></map></div></td></tr></table></td></tr></table><table cellpadding="0" cellspacing="3" width="100%" border="0"><tr><td></td><td colspan="2"><div class="navlink-box navlink-box-gray"><a href="fprender.fcgi" class="navlink">Journal List</a><span> > </span><a class="navlink" href="tocrender.fcgi?journal=516&action=archive">Cytotechnology</a><span> > </span><a class="navlink" href="tocrender.fcgi?iid=157542">v.56(1); Jan 2008</a></div></td></tr><tr><td width="145" valign="top" class="sidebar-cell"><div class="sidebar-article-navigation-box"><div class="sidefm-pmclink-item"><a href="articlerender.fcgi?artid=2151964&rendertype=abstract" class="sidefm-pmclink">Abstract</a></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>Full Text</a></div><div class="sidefm-pmclink-item"><a href="picrender.fcgi?artid=2151964&blobtype=pdf" class="sidefm-pmclink">PDF (458K)</a></div><div class="sidefm-pmclink-item"><a href="tocrender.fcgi?iid=157542" class="sidefm-pmclink">Contents</a></div><div class="sidefm-pmclink-item"><a href="tocrender.fcgi?journal=516&action=archive" class="sidefm-pmclink">Archive</a></div></div><div class="sidebar-pubmed-box"><div class="sidebar-pubmed-links-to-box"><form name="pubmedLinks" onsubmit="return pubMedDbLinkSubmit(this)"><div class="sidefm-pmsubhead"><label for="pubmedOption">Related material:</label></div><select name="pubmedOption" id="pubmedOption" class="sidefm-pmart" onchange="return pubMedDbLinkSubmit(this)"><option value="redirect3.cgi?&&auth=0LcSYWHv9CW_xYJFHEZMgCfwgAj56qgGb0cwti5ta&reftype=abs&refto=entrez&reffrom=sidebar&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article|Navigation&TO=Entrez|PubMed|Record&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/pubmed/19002842">PubMed record</option><option selected value="redirect3.cgi?&&auth=0dsBHmWepT2O06HMTrlH9bRPKHL08cohCInjAMQwH&reftype=relart&refto=entrez&reffrom=sidebar&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article|Navigation&TO=Entrez|PubMed|Related%20Records&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed&cmd=Link&dbFrom=PubMed&from_uid=19002842">PubMed related arts</option><option value="redirect3.cgi?&&auth=0f5siJyMrUuVttvMZ_WwFbP_cAccfpAuTmUEhW5F1&reftype=linkout&refto=entrez&reffrom=sidebar&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article|Navigation&TO=Entrez|PubMed|Linkout&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowLinkOut&TermToSearch=19002842">PubMed LinkOut</option></select><br><input type="image" class="sidefm-pmart" alt="GO" src="corehtml/pmc/pmcgifs/go.gif"></form></div><div class="sidebar-pubmed-author-box"><div class="sidefm-pmsubhead">PubMed articles by:</div><div class="sidefm-pmclink-item"><a href="redirect3.cgi?&&auth=04iRm5Z-V8zKhxFp0mFX5Krcpsbe3kKwKIKIVpQQs&reftype=authsrch&refto=entrez&reffrom=sidebar&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CNavigation&TO=Entrez%7CPubMed%7CAuthor%20Search&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term=%20Pinto%2BRC%5Bauth%5D" class="sidefm-pmclink"> Pinto, R.</a></div><div class="sidefm-pmclink-item"><a href="redirect3.cgi?&&auth=02ujHE1cG3kcRZHV63brTR3897moILiOpzlc1R5Fz&reftype=authsrch&refto=entrez&reffrom=sidebar&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CNavigation&TO=Entrez%7CPubMed%7CAuthor%20Search&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term=%20Medronho%2BRA%5Bauth%5D" class="sidefm-pmclink"> Medronho, R.</a></div><div class="sidefm-pmclink-item"><a href="redirect3.cgi?&&auth=06mW6f5DoAXmnBjnQHLt_tsTI1o3SFn5GYDe9qQbk&reftype=authsrch&refto=entrez&reffrom=sidebar&article-id=2151964&issue-id=157542&journal-id=516&FROM=Article%7CNavigation&TO=Entrez%7CPubMed%7CAuthor%20Search&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term=%20Castilho%2BLR%5Bauth%5D" class="sidefm-pmclink"> Castilho, L.</a></div></div></div></td><td rowspan="104" width="20px" height="150" background="corehtml/pmc/pmcgifs/wm-cytotech.gif"><div style="width:20px"></div></td><td valign="top" class="content-cell"><div class="front-matter-section"><table cellspacing="0" cellpadding="0" width="100%"><tr style="vertical-align: top"><td><div class="fm-citation"><div><span class="citation-abbreviation">Cytotechnology. </span><span class="citation-publication-date">2008 January; </span><span class="citation-volume">56</span><span class="citation-issue">(1)</span><span class="citation-flpages">: 57–67. </span></div><div><span class="fm-vol-iss-date">Published online 2007 November 14. </span><span class="fm-vol-iss-date"> </span><span class="fm-vol-iss-date">doi: 10.1007/s10616-007-9108-x.</span></div></div></td><td class="fm-citation-ids"><div class="fm-citation-pmcid"><span class="fm-citation-ids-label">PMCID: </span>PMC2151964</div></td></tr></table><div class="fm-copyright"><a class="int-reflink" href="about/copyright.html">Copyright</a> © Springer Science+Business Media B.V. 2007</div><div class="fm-title">Separation of CHO cells using hydrocyclones</div><div class="contrib-group fm-author">Rodrigo C.V. Pinto,<sup>1</sup> Ricardo A. Medronho,<sup><img src="corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author"></sup><sup>2</sup> and Leda R. Castilho<sup>1</sup></div><div class="fm-affl"><sup>1</sup>Federal University of Rio de Janeiro (UFRJ), COPPE - Chemical Engineering Program, Caixa Postal 68502, CEP 21941-972 Rio de Janeiro, RJ Brazil</div><div class="fm-affl"><sup>2</sup>School of Chemistry, Chemical Engineering Department, Federal University of Rio de Janeiro (UFRJ), CT, Bloco E, CEP 21949-900 Rio de Janeiro, RJ Brazil</div><div class="fm-footnote"><span class="fm-affl">Ricardo A. Medronho, </span><span class="fm-affl">Phone: +55-21-25627635, Fax: +55-21-25627567, Email: <span class="e_id728615">medronho/at/eq.ufr.br</span><script type="text/javascript" language="JavaScript"><!--
try{initUnObscureEmail ("e_id728615", '<a class="ext-reflink" href="' + reverseAndReplaceString('rb.rfu.qe/ta/ohnordem:otliam', '/at/', '@') + '">' + reverseAndReplaceString('rb.rfu.qe/ta/ohnordem', '/at/','@') + '</a>')}catch(e){}
//--></script></span>.</div><div class="fm-footnote"><sup><img src="corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author"></sup>Corresponding author.</div><div class="fm-footnote"></div><div class="fm-pubdate">Received April 3, 2007; Accepted October 20, 2007.</div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"><div class="side-section-group"><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#top" class="sidefm-pmclink" style="text-transform: none;">Top</a></div></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);" style="text-transform: none;"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>Abstract</a></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870765" class="sidefm-pmclink" style="text-transform: none;">Introduction</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id708932" class="sidefm-pmclink" style="text-transform: none;">Materials and methods</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id709529" class="sidefm-pmclink" style="text-transform: none;">Results and discussion</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691345" class="sidefm-pmclink" style="text-transform: none;">Conclusions</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691373" class="sidefm-pmclink" style="text-transform: none;">References</a></div></div></div></td><td class="content-cell"><div class="head1 section-title" style="text-transform: none;" id="id870720">Abstract</div><div class="section-content"><!--article-meta--><div id="id870722" class="p p-first-last">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.</div><div class="p"><span class="kwd-label">Keywords: </span><span class="kwd-text">Animal cells, Cell separation, CHO cells, Hydrocyclones, Perfusion, Retention device</span></div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"><div class="side-section-group"><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#top" class="sidefm-pmclink" style="text-transform: none;">Top</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870720" class="sidefm-pmclink" style="text-transform: none;">Abstract</a></div></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);" style="text-transform: none;"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>Introduction</a></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id708932" class="sidefm-pmclink" style="text-transform: none;">Materials and methods</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id709529" class="sidefm-pmclink" style="text-transform: none;">Results and discussion</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691345" class="sidefm-pmclink" style="text-transform: none;">Conclusions</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691373" class="sidefm-pmclink" style="text-transform: none;">References</a></div></div></div></td><td class="content-cell"><span id="Sec1"></span><div class="head1 section-title" style="text-transform: none;" id="id870765"><a id="Sec1"></a>Introduction</div><div class="section-content"><span id="Sec1"></span><div id="id870775" class="p p-first">Animal cells are widely used for the production of biopharmaceuticals, monoclonal antibodies and vaccines. Continuous animal cell culture processes with cell retention, commonly known as perfusion processes, are characterized by constant fresh medium supply and removal of exhausted medium. Cell retention devices are employed for the separation of cells from spent medium. Thus, the combination of continuous nutrient feeding, metabolites removal and cell retention allows high-cell-density cultures (greater than 10<sup>7</sup> cells/mL) and high productivities (usually at least 10-fold higher than in batch cultures) to be obtained (Woodside et al. <a href="#CR38" rid="CR38" class="cite-reflink bibr popnode">1998</a>; Castilho and Medronho <a href="#CR9" rid="CR9" class="cite-reflink bibr popnode">2002</a>). However, the main concern in perfusion cultures is the technique employed to separate cells from the spent medium. Animal cell separation from the liquid medium is a difficult task because cells are sensitive to mechanical stress, have diameters in the range of 8–40 μm and densities of 1.05–1.14 g/cm<sup>3</sup>, resulting in very low terminal settling velocities (Medronho <a href="#CR24" rid="CR24" class="cite-reflink bibr popnode">2003</a>).</div><div id="id870826" class="p">Adequate cell retention devices must present features such as high separation efficiency, long-term stable operation, no induction of death mechanisms and, preferentially, good resolution in separating viable from non-viable cells. However, the high protein titer in most cell culture media, associated to the small size and density of the cells, causes drawbacks in most of the conventional retention devices (Kretzmer <a href="#CR18" rid="CR18" class="cite-reflink bibr popnode">2002</a>).</div><div id="id870843" class="p">Different devices have been used to perform cell retention in bioreactors, but several limitations have been observed. Filtration-based techniques present problems of filter fouling after a relatively short period of operation (Kawahara et al. <a href="#CR17" rid="CR17" class="cite-reflink bibr popnode">1994</a>; Van Reis and Zydney <a href="#CR35" rid="CR35" class="cite-reflink bibr popnode">2001</a>). Centrifuges may suffer from cell adhesion and clogging, besides their high mechanical complexity and high cost (Jäger <a href="#CR15" rid="CR15" class="cite-reflink bibr popnode">1992</a>; Tokashiki et al. <a href="#CR33" rid="CR33" class="cite-reflink bibr popnode">1990</a>). Sedimentation-based apparatuses are susceptible to cell adhesion, and the high residence time needed to perform the separation exposes the cells to non-controlled environments (Searles et al. <a href="#CR31" rid="CR31" class="cite-reflink bibr popnode">1994</a>; Voisard et al. <a href="#CR36" rid="CR36" class="cite-reflink bibr popnode">2003</a>). Thus, special operation strategies like back-pulsing, to prevent cell build-up at the drain ports, and refrigeration systems, to induce cell aggregation and so enhance particle sedimentation, have to be employed (Lipscomb et al. <a href="#CR20" rid="CR20" class="cite-reflink bibr popnode">2004</a>).</div><div id="id708809" class="p">Hydrocyclones have been proposed in the recent years to promote animal cell separation in batch and perfusion processes (Lübberstedt et al. <a href="#CR21" rid="CR21" class="cite-reflink bibr popnode">2000a</a>, <a href="#CR22" rid="CR22" class="cite-reflink bibr popnode">b</a>; Jockwer et al. <a href="#CR16" rid="CR16" class="cite-reflink bibr popnode">2001</a>). These separation devices present several advantages for use in the biotechnology industry, such as their simplicity and reliability, as well as the absence of moving parts. Furthermore, for cell separation applications, hydrocyclones would require no maintenance, allowing long-term continuous operation of perfusion bioreactors (Castilho and Medronho <a href="#CR9" rid="CR9" class="cite-reflink bibr popnode">2002</a>).</div><div id="id708859" class="p">Previous works using three different commercially available hydrocyclones showed motivating results (Lübberstedt et al<em>.</em><a href="#CR21" rid="CR21" class="cite-reflink bibr popnode">2000a</a>, <a href="#CR22" rid="CR22" class="cite-reflink bibr popnode">b</a>). The best performance was obtained with a 10-mm Dorr-Oliver hydrocyclone, resulting in a separation efficiency of 81%. These experimental values agree with computational fluid dynamics (CFD) predictions that high levels of separation efficiencies for mammalian cells could be achieved with small-diameter hydrocyclones (Medronho et al. <a href="#CR25" rid="CR25" class="cite-reflink bibr popnode">2005</a>). Since the preservation of high cellular viabilities is one main concern regarding animal cell retention devices, Lübberstedt et al. (<a href="#CR21" rid="CR21" class="cite-reflink bibr popnode">2000a</a>) also analyzed the influence of pressure drop on HeLa cell viability. It was shown that cell viability in the concentrated stream (underflow) remained constant for pressure drops varying in the range of 1–4 bar, while the diluted stream (overflow) presented viability losses for pressure drops above 3 bar. Although the shear stress levels inside hydrocyclones are relatively high, the residence times of the cells inside the equipment are very short (in the range of fractions of seconds), specially for those cells being separated in the underflow concentrated cell stream, which is the stream that is recycled to the bioreactor in a perfusion culture. Jockwer et al. (<a href="#CR16" rid="CR16" class="cite-reflink bibr popnode">2001</a>) studied hydrocyclones specially designed for animal cell separation and were able to successfully carry out a 23-day perfusion culture in a 5-L bioreactor with CHO (Chinese hamster ovary) cells.</div><div id="id708927" class="p p-last">The present work investigates the use of different geometries of hydrocyclones that were specially designed for animal cell separation. The separation efficiency of CHO cells, as well as flow rates and cell viability were evaluated. Furthermore, mid-term tests for evaluating if any induction of apoptosis occurred were carried out with the parental CHO.K1 cell line and a recombinant one.</div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"><div class="side-section-group"><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#top" class="sidefm-pmclink" style="text-transform: none;">Top</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870720" class="sidefm-pmclink" style="text-transform: none;">Abstract</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870765" class="sidefm-pmclink" style="text-transform: none;">Introduction</a></div></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);" style="text-transform: none;"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>Materials and methods</a></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id709529" class="sidefm-pmclink" style="text-transform: none;">Results and discussion</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691345" class="sidefm-pmclink" style="text-transform: none;">Conclusions</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691373" class="sidefm-pmclink" style="text-transform: none;">References</a></div></div></div></td><td class="content-cell"><span id="Sec2"></span><div class="head1 section-title" style="text-transform: none;" id="id708932"><a id="Sec2"></a>Materials and methods</div><div class="section-content"><span id="Sec2"></span><div class="sec sec-first"><span id="Sec3"></span><div class="head2 head-separate">Cell lines and cultivation conditions</div><div id="id708950" class="p p-first-last">The CHO.K1 (Chinese hamster ovary) cell line, obtained from DSMZ (German Collection for Microrganisms and Cell Cultures, Braunschweig, Germany), and a recombinant CHO cell line expressing human GM-CSF (granulocyte-macrophage colony stimulating factor), gently provided by Laboratorio de Cultivos Celulares from Universidad Nacional del Litoral (Santa Fe, Argentina), were used. Cells were cultivated in spinner flasks up to a volume of 1 L, in a mixture (1:1) of DMEM and Ham’s F12 media, supplemented with 1% (CHO.K1) and 0.2% (CHO GM-CSF) fetal calf serum (FCS) (Cultilab, Campinas/SP, Brazil). The flasks were incubated at 37 °C and 5% CO<sub>2</sub> and stirred at 50 rpm.</div></div><div class="sec"><span id="Sec4"></span><div class="head2 head-separate">Hydrocyclones</div><div id="id708970" class="p p-first-last">The hydrocyclones (HCs) tested in this work were specially designed to separate animal cells (Fig. <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig1" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">(Fig. 1).</span></span><span style="text-decoration: underline;">1</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig1_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig1_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 1" title="Fig. 1"></div></div></a>). More details on their geometry can be obtained from Deckwer et al. (<a href="#CR11" rid="CR11" class="cite-reflink bibr popnode">2005</a>). Briefly, they have a double tangential inlet, a diameter (D<sub>c</sub>) of 10.0 mm and a choice of two different underflow diameters (D<sub>u</sub>) (2.0 and 3.0 mm) and three different overflow diameters (D<sub>o</sub>) (1.0, 1.5 and 2.0 mm). Hence, six different geometries with varying underflow and overflow diameters were tested.</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Fig1" name="Fig1"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig1" onclick="startTarget(this, 'figure', 1024, 800)" class="icon-reflink figpopup"><div class="thumb-ph"><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig1_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig1_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 1" title="Fig. 1"></div></div><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig1_HTML.gif" class="icon-reflink small-thumb" alt="Fig. 1" title="Fig. 1"></div></div></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig1" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Fig. 1</strong></a><div class="figure-table-caption-in-article"><span>(<strong>a</strong>) Photograph of the hydrocyclone specially designed for animal cell separation; (<strong>b</strong>) Schematic view of the fluid flow inside a hydrocyclone</span></div></div></td></tr></table></div></div><div style="clear:both;"></div></div><div class="sec"><span id="Sec5"></span><div class="head2 head-separate">Experimental set-up</div><div id="id709050" class="p p-first">The cell separation tests were carried out using a 20-L stainless-steel tank containing cell suspension from a 1-L spinner culture diluted to 20 L with PBS (pH 7.2) containing 0.9% NaCl (Fig. <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig2" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">(Fig. 2).</span></span><span style="text-decoration: underline;">2</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig2_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig2_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 2" title="Fig. 2"></div></div></a>). The concentration of the cell suspensions fed to the hydrocyclones was in the range of 6–7 × 10<sup>4</sup> cells/mL. In each experiment, the stainless-steel tank was pressurized with compressed air up to the required pressure, forcing the cell suspension to the hydrocyclone. Underflow and overflow outlets were open at atmospheric pressure, and the flow rate through these orifices was measured to determine the flow ratio (R<sub>f</sub>). Samples (50 mL) were collected from each outlet stream to determine cell concentration and viability. Before cell counting, the samples were concentrated 10-fold to reduce the experimental error.</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Fig2" name="Fig2"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig2" onclick="startTarget(this, 'figure', 1024, 800)" class="icon-reflink figpopup"><div class="thumb-ph"><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig2_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig2_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 2" title="Fig. 2"></div></div><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig2_HTML.gif" class="icon-reflink small-thumb" alt="Fig. 2" title="Fig. 2"></div></div></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig2" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Fig. 2</strong></a><div class="figure-table-caption-in-article"><span>Experimental set-up of the 20-L stainless-steel tank with two manometers (1), cell suspension (2), a valve (3) and the hydrocyclone (4)</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div id="id709102" class="p">Further separation tests were carried out using hydrocyclone configurations 3020 (underflow diameter: 3.0 mm; overflow diameter: 2.0 mm) and 2010 (underflow diameter: 2.0 mm; overflow diameter: 1.0 mm) with the purpose of assessing the possible mid-term effects of hydrocycloning on cell viability. High-viability cell suspensions (>98%), cultured in 500-mL spinner flasks, were fed under sterile conditions to the hydrocyclones using a low-pulse peristaltic pump (Watson Marlow, model 520 U with pumphead 505 L), at a pressure drop of 1 bar. After one passage through the hydrocyclone, the underflow stream (cell-concentrated stream) was collected in a sterile spinner flask, which was incubated for 48 h. Samples were collected for evaluation of LDH, apoptosis and cell growth in intervals of 0, 3, 6, 24 and 48 h after passage through the hydrocyclone.</div><div id="id709112" class="p">The cell separation efficiency E was calculated as the fraction of cells (in number) recovered in the underflow (Eq. 1) (Castilho and Medronho <a href="#CR10" rid="CR10" class="cite-reflink bibr popnode">2008</a>): <a id="Equ1" name="Equ1"></a><div class="disp-formula"><table width="100%" border="0" cellspacing="5" cellpadding="5"><tr><td style="width:95%"><div class="eqn-image"><img src="picrender.fcgi?artid=2151964&blobtype=equ&blobname=M1" style="vertical-align: middle; background: #F8F8F8;padding: 2pt;border-style: none;" alt="equation M1" title="equation M1"></div></td><td style="width:5%"><div class="eqn-id"><span style="white-space: nowrap;">1</span></div></td></tr></table></div>where Q and Q<sub>u</sub> are the flow rates and X and X<sub>u</sub> are the cell concentrations, as cell number per unit volume, of the feed and concentrated streams, respectively.</div><div id="id709153" class="p p-last">The flow ratio R<sub>f</sub> was evaluated using Eq. 2. <a id="Equ2" name="Equ2"></a><div class="disp-formula"><table width="100%" border="0" cellspacing="5" cellpadding="5"><tr><td style="width:95%"><div class="eqn-image"><img src="picrender.fcgi?artid=2151964&blobtype=equ&blobname=M2" style="vertical-align: middle; background: #F8F8F8;padding: 2pt;border-style: none;" alt="equation M2" title="equation M2"></div></td><td style="width:5%"><div class="eqn-id"><span style="white-space: nowrap;">2</span></div></td></tr></table></div>The cell number balances (comparison of the amount of cells leaving the hydrocyclone through the underflow and overflow streams with the amount of cells fed to the equipment) closed satisfactorily, with an average deviation of ±9.9%, which is approximately the same deviation associated to the cell counting technique in Neubauer chambers used for quantification of cell concentration in the different streams.</div></div><div class="sec"><span id="Sec6"></span><div class="head2 head-separate">Experimental design</div><div id="id709187" class="p p-first-last">A statistical experimental design was employed to evaluate the effects of three variables (overflow diameter, D<sub>o</sub>, underflow diameter, D<sub>u</sub>, and pressure drop, ΔP). A fractional factorial experimental design (2<sup>3–1</sup>), shown in Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab1" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">Table 1,</span></span><span style="text-decoration: underline;">1</span><div class="large-thumb-canvas"></div></a>, was used, and allowed obtaining mathematical models describing the effects of each variable and of the interactions among them on the responses (separation efficiency, flow rate and flow ratio). Four additional experiments were added to assess the experimental error using the central points of D<sub>o</sub> and pressure drop. Statistical analysis of the results and mathematical modeling were accomplished with STATISTICA 6.0 (StatSoft, Tulsa, USA), using the non-linear parameter estimation module with least square objective function.</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab1" name="Tab1"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab1" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 1" title="Table 1"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab1" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 1</strong></a><div class="figure-table-caption-in-article"><span>Fractional factorial experimental design</span></div></div></td></tr></table></div></div><div style="clear:both;"></div></div><div class="sec"><span id="Sec7"></span><div class="head2 head-separate">Cell quantification and viability assays</div><div id="id709419" class="p p-first-last">Cell quantification was carried out using a haemocytometer and an optical microscope. Dead cells were determined by the trypan blue exclusion method (Kuchler <a href="#CR19" rid="CR19" class="cite-reflink bibr popnode">2000</a>). Total cells were quantified by counting total cell nuclei (Sanford et al<em>.</em><a href="#CR30" rid="CR30" class="cite-reflink bibr popnode">1950</a>).</div></div><div class="sec"><span id="Sec8"></span><div class="head2 head-separate">Determination of cell size distribution</div><div id="id709455" class="p p-first-last">A particle size analyzer (Malvern Mastersizer) was used to determine cell size distribution of a suspension of CHO.K1 cells grown in 1% FCS.</div></div><div class="sec"><span id="Sec9"></span><div class="head2 head-separate">Lactate dehydrogenase (LDH) activity determination</div><div id="id709467" class="p p-first-last">After centrifugation of samples at 250<em>g</em> for 4 min, lactate dehydrogenase activity in supernatants was determined at room temperature by following the conversion of NADH to NAD<sup>+</sup> by reduction of pyruvate to lactate, which is catalyzed by LDH. NADH concentration was measured at 340 nm (Racher et al. <a href="#CR29" rid="CR29" class="cite-reflink bibr popnode">1990</a>). The reaction was initiated by the addition of 1 mL of supernatant to a mixture of 0.4 mL of 2.4 mM pyruvate and 0.025 mL of 6 mM NADH in 0.1 M phosphate buffer (pH 7.2). One activity unit (U) is defined as the amount of enzyme that catalyzes the consumption of 1 μmol of NADH per minute, under the assay conditions.</div></div><div class="sec sec-last"><span id="Sec10"></span><div class="head2 head-separate">Determination of apoptosis and necrosis</div><div id="id709501" class="p p-first-last">Normal, apoptotic and necrotic cells were determined according to visible aberrations of the chromatin using an epifluorescence microscope (Nikon, TS100F). Two fluorescent dyes that bind to DNA (acridine orange and ethidium bromide, both from Fluka) were used. 4 μL of a solution containing 100 μg/mL of each of both dyes was added to 100 μL culture suspension (5 × 10<sup>5</sup>–5 × 10<sup>6</sup> cells/mL). The samples were examined using a 40× objective with epi-illumination and a combined filter-set for fluorescein (Mercille and Massie <a href="#CR26" rid="CR26" class="cite-reflink bibr popnode">1994</a>). Five different cell physiological states were identified: viable non-apoptotic (VNA), viable apoptotic (VA), non-viable apoptotic (NVA), necrotic (NEC) and chromatin-free (CF).</div></div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"><div class="side-section-group"><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#top" class="sidefm-pmclink" style="text-transform: none;">Top</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870720" class="sidefm-pmclink" style="text-transform: none;">Abstract</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870765" class="sidefm-pmclink" style="text-transform: none;">Introduction</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id708932" class="sidefm-pmclink" style="text-transform: none;">Materials and methods</a></div></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);" style="text-transform: none;"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>Results and discussion</a></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691345" class="sidefm-pmclink" style="text-transform: none;">Conclusions</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691373" class="sidefm-pmclink" style="text-transform: none;">References</a></div></div></div></td><td class="content-cell"><span id="Sec11"></span><div class="head1 section-title" style="text-transform: none;" id="id709529"><a id="Sec11"></a>Results and discussion</div><div class="section-content"><span id="Sec11"></span><div class="sec sec-first"><span id="Sec12"></span><div class="head2 head-separate">Influence of pressure drop and outlet diameters on hydrocyclone performance</div><div id="id709547" class="p p-first">CHO.K1 cell suspensions, previously grown in DMEM/F12 medium containing 1% FCS and presenting cell viability above 95%, were fed from a pressurized 20-L steel tank to the six different configurations of the specially designed hydrocyclones, at pressures of 1, 2 and 3 bar. The experiments followed a fractional factorial design. The hydrocyclone geometries 2015 (D<sub>u</sub> = 2.0 mm and D<sub>o</sub> = 1.5 mm) and 3015 (D<sub>u</sub> = 3.0 and D<sub>o</sub> = 1.5) were analyzed in duplicate to evaluate the experimental error. The results are shown in Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab2" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">Table 2</span></span><span style="text-decoration: underline;">2</span><div class="large-thumb-canvas"></div></a> in terms of feed flow rate (Q), flow ratio (R<sub>f</sub>), total separation efficiency (E) and cell viability loss in the underflow stream (ΔV<sub>u</sub> = viability in the feed minus viability in the underflow).</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab2" name="Tab2"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab2" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 2" title="Table 2"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab2" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 2</strong></a><div class="figure-table-caption-in-article"><span>Separation of CHO.K1 cells grown in DMEM/F12 medium (1% FCS)</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div id="id689331" class="p">High cell separation efficiencies, varying from 97.9% to 99.9%, were obtained for 5 out of 6 hydrocyclone configurations. The geometry 3010 (D<sub>u</sub> = 3.0 mm and D<sub>o</sub> = 1.0 mm) presented a flow ratio of 100%, diverting the total feed flow to the underflow, indicating that this configuration is inappropriate to perform any kind of cell separation. Loss of viability was within the range of 2.9–9.1% for all configurations with exception of HC 2020, which presented a viability loss of 14.4%.</div><div id="id689346" class="p">Hydrocyclone separation efficiencies for CHO.K1 cells shown in Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab2" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">Table 2</span></span><span style="text-decoration: underline;">2</span><div class="large-thumb-canvas"></div></a> are higher than the efficiencies obtained with other devices described in the literature. Wen et al. (<a href="#CR37" rid="CR37" class="cite-reflink bibr popnode">2000</a>) employed a two-step sequential sedimentation device for the retention of an IgG-producing hybridoma, achieving a separation efficiency of 88%. In CHO-cell perfusion cultures with spin-filters, the efficiencies attained were in the range of 75–95% (Iding et al. <a href="#CR14" rid="CR14" class="cite-reflink bibr popnode">2000</a>), as compared to a minimum efficiency of 97.9% found for hydrocyclones in the present work. Furthermore, unlike hydrocyclones, fouling and clogging of spin-filter meshes is considered a major problem, which may cause premature interruption of the culture process (Esclade et al. <a href="#CR13" rid="CR13" class="cite-reflink bibr popnode">1991</a>; Deo et al<em>.</em><a href="#CR12" rid="CR12" class="cite-reflink bibr popnode">1996</a>; Voisard et al. <a href="#CR36" rid="CR36" class="cite-reflink bibr popnode">2003</a>). Furthermore, when compared to other hydrocyclone geometries, the efficiencies found in the present work are higher than the best efficiency (81%) obtained for HeLa cells by Lübberstedt et al. (<a href="#CR22" rid="CR22" class="cite-reflink bibr popnode">2000b</a>). These authors used commercial hydrocyclones designed for conventional (non-biological) solid-liquid separations (a 7-mm Bradley, a 10-mm Mozley and a 10-mm Dorr-Oliver hydrocyclone), which have geometric proportions that are different from those of the hydrocyclones used in this work. These HCs were specially designed for the separation of animal cells (Deckwer et al. <a href="#CR11" rid="CR11" class="cite-reflink bibr popnode">2005</a>) based on the knowledge of how geometric proportions of hydrocyclones affect separation efficiency (Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab3" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7.5em; border: dashed 1px green;">(Table 3).</span></span><span style="text-decoration: underline;">3</span><div class="large-thumb-canvas"></div></a>). A comparison of some geometric proportions of the hydrocyclone used in this work (Deckwer et al. <a href="#CR11" rid="CR11" class="cite-reflink bibr popnode">2005</a>) with those of the HCs used by Lübberstedt et al. (<a href="#CR21" rid="CR21" class="cite-reflink bibr popnode">2000a</a>, <a href="#CR22" rid="CR22" class="cite-reflink bibr popnode">b</a>) is given in Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab4" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">Table 4.</span></span><span style="text-decoration: underline;">4</span><div class="large-thumb-canvas"></div></a>. According to Tables <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab3" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7.5em; border: dashed 1px green;">Tables 3</span></span><span style="text-decoration: underline;">3</span><div class="large-thumb-canvas"></div></a> and <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab4" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -1.5em; border: dashed 1px green;">and4,</span></span><span style="text-decoration: underline;">4</span><div class="large-thumb-canvas"></div></a>, a low D<sub>o</sub>/D<sub>c</sub> range and a high D<sub>u</sub>/D<sub>c</sub> range were chosen for the specially designed hydrocyclones with the aim of maximizing their separation efficiency.</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab3" name="Tab3"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab3" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 3" title="Table 3"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab3" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 3</strong></a><div class="figure-table-caption-in-article"><span>Effects of increases in the values of geometric and operational variables on the capacity (feed flow rate) and separation efficiency (Eq. 1) of hydrocyclones (+: increase, −: decrease). Adapted from Matta and Medronho (<a href="#CR23" rid="CR23" class="cite-reflink bibr popnode">2000</a>)</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab4" name="Tab4"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab4" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 4" title="Table 4"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab4" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 4</strong></a><div class="figure-table-caption-in-article"><span>Comparison of some geometrical proportions of the Bradley hydrocyclone family (Bradley and Pulling <a href="#CR6" rid="CR6" class="cite-reflink bibr popnode">1959</a>), two commercial hydrocyclone designs (Richard Mozley Ltd., and FLSmidth Dorr-Oliver Eimco) and the hydrocyclone used in this work (Deckwer et al.</span><a class="side-caption" style="font-size: 100%;" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab4" onclick="startTarget(this, 'figure', 1024, 800)"> (more ...)</a></div></div></td></tr></table></div></div><div style="clear:both;"></div><div id="id689894" class="p">The viability losses found in the present work were slightly higher than those obtained with commercial Dorr-Oliver hydrocyclones for HeLa cells (Lübberstedt et al. <a href="#CR21" rid="CR21" class="cite-reflink bibr popnode">2000a</a>), but lower than those obtained by Jockwer et al. (<a href="#CR16" rid="CR16" class="cite-reflink bibr popnode">2001</a>) for the separation of CHO cells cultured in serum-free medium, using the same type of hydrocyclone as in this work. Just in the case of HC 2020, viability loss was identical (14.4%) in this study and in that by Jockwer et al. (<a href="#CR16" rid="CR16" class="cite-reflink bibr popnode">2001</a>). These results suggest that there may be a difference between HeLa and CHO cells regarding sensitivity to shear generated inside hydrocyclones. Furthermore, comparing the present results with those obtained for CHO cells grown in serum-free medium (Jockwer et al. <a href="#CR16" rid="CR16" class="cite-reflink bibr popnode">2001</a>), the data also suggest that the presence of seric proteins could be attenuating the effects of the hydrodynamic stress on cells maintained in serum-supplemented medium, as previously observed by Van Der Pol et al. (<a href="#CR34" rid="CR34" class="cite-reflink bibr popnode">1990</a>).</div><div id="id689955" class="p">Suspension cultures of CHO.K1 cells naturally present aggregates that can be easily visualized. Using a Malvern particle size analyzer, the size distribution of CHO.K1 cells in the feed was determined (Fig. <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig3" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">(Fig. 3),</span></span><span style="text-decoration: underline;">3</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig3_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig3_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 3" title="Fig. 3"></div></div></a>), and it was possible to identify two distinct populations with mean diameters of 12 μm, representing isolated cells, and 110 μm, indicating aggregates. Oxygen and nutrient diffusion inside these clumps is limited, creating inadequate conditions for the innermost cells, resulting in non-viable cells that cannot be detected by the trypan blue technique, which in turn leads to over-estimated viability values. However, due to the high shear rates inside the hydrocyclone, cell clump disaggregation could be an important factor leading to a decrease in cell culture viability that is rather apparent, since only individual cells or small clumps (3–6 cells) had been previously visualized and counted in the haemocytometer by the trypan blue technique. Some authors (Mercille et al. <a href="#CR27" rid="CR27" class="cite-reflink bibr popnode">1994</a>; Castilho et al. <a href="#CR8" rid="CR8" class="cite-reflink bibr popnode">2002</a>) have shown that DNAse treatment of CHO cell cultures can disaggregate cell clumps, significantly increasing the countable concentration of non-viable cells. Therefore, one important factor that may have influenced the increase in non-viable cell concentration in the underflow stream is the disaggregation of clumps. Visually, it was possible to observe that the number and size of cell clumps collected in the hydrocyclone underflow stream were lower than in the feed before hydrocycloning.
</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Fig3" name="Fig3"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig3" onclick="startTarget(this, 'figure', 1024, 800)" class="icon-reflink figpopup"><div class="thumb-ph"><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig3_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig3_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 3" title="Fig. 3"></div></div><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig3_HTML.gif" class="icon-reflink small-thumb" alt="Fig. 3" title="Fig. 3"></div></div></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig3" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Fig. 3</strong></a><div class="figure-table-caption-in-article"><span>(<strong>a</strong>) Size distribution of CHO.K1 cells cultured in spinner flasks in DMEM/F12 medium supplemented with 1% FCS. (<strong>b</strong>) Cell clumps stained with acridine orange and ethidium bromide observed under a fluorescence microscope</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div id="id690035" class="p">The results shown in Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab2" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">Table 2</span></span><span style="text-decoration: underline;">2</span><div class="large-thumb-canvas"></div></a> were also used to determine empiric mathematical models to describe separation efficiency (E), flow ratio (R<sub>f</sub>), and flow rate (Q) as a function of the geometric variables (D<sub>u</sub> and D<sub>o</sub>) and the pressure drop (ΔP).</div><div id="id690061" class="p">Equation 3 shows the model for separation efficiency (E), which presented a correlation coefficient (R) of 0.988. This correlation has the form of a typical polynomial equation, as commonly used in experimental design analyses, and only includes the terms that presented statistical significance. <a id="Equ3" name="Equ3"></a><div class="disp-formula"><table width="100%" border="0" cellspacing="5" cellpadding="5"><tr><td style="width:95%"><div class="eqn-image"><img src="picrender.fcgi?artid=2151964&blobtype=equ&blobname=M4" style="vertical-align: middle; background: #F8F8F8;padding: 2pt;border-style: none;" alt="equation M4" title="equation M4"></div></td><td style="width:5%"><div class="eqn-id"><span style="white-space: nowrap;">3</span></div></td></tr></table></div>where D<sub>o</sub> and D<sub>u</sub> are in centimetres, ΔP in bar and Q in L min<sup>−1</sup>.</div><div id="id690096" class="p">The models for flow ratio (Eq. 4) and flow rate (Eq. 5) presented correlation coefficients of 0.977 and 0.961, respectively. The structure of these equations was based on classical hydrocyclone models (Plitt <a href="#CR28" rid="CR28" class="cite-reflink bibr popnode">1976</a>; Coelho and Medronho 2000). <a id="Equ4" name="Equ4"></a><div class="disp-formula"><table width="100%" border="0" cellspacing="5" cellpadding="5"><tr><td style="width:95%"><div class="eqn-image"><img src="picrender.fcgi?artid=2151964&blobtype=equ&blobname=M5" style="vertical-align: middle; background: #F8F8F8;padding: 2pt;border-style: none;" alt="equation M5" title="equation M5"></div></td><td style="width:5%"><div class="eqn-id"><span style="white-space: nowrap;">4</span></div></td></tr></table></div><a id="Equ5" name="Equ5"></a><div class="disp-formula"><table width="100%" border="0" cellspacing="5" cellpadding="5"><tr><td style="width:95%"><div class="eqn-image"><img src="picrender.fcgi?artid=2151964&blobtype=equ&blobname=M6" style="vertical-align: middle; background: #F8F8F8;padding: 2pt;border-style: none;" alt="equation M6" title="equation M6"></div></td><td style="width:5%"><div class="eqn-id"><span style="white-space: nowrap;">5</span></div></td></tr></table></div>where D<sub>o</sub> and D<sub>u</sub> are in centimetres, ΔP in bar and Q in L min<sup>−1</sup>.</div><div id="id690156" class="p p-last">Figure <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig4" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7.5em; border: dashed 1px green;">Figure 4</span></span><span style="text-decoration: underline;">4</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig4_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig4_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 4" title="Fig. 4"></div></div></a> compares the predicted values with the experimental data, considering a confidence level of 98%, for the efficiency, flow ratio and flow rate. These three models show that although the efficiencies were high in all cases, the flow rate and flow ratio varied considerably, resulting in important differences in overflow flow rate, for example. Thus, these models allow predicting the effects of changes in D<sub>o</sub>, D<sub>u</sub> and ΔP within the tested ranges on variables that are of great relevance in establishing e.g. a perfusion process, such as harvest flow rate.
</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Fig4" name="Fig4"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig4" onclick="startTarget(this, 'figure', 1024, 800)" class="icon-reflink figpopup"><div class="thumb-ph"><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig4_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig4_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 4" title="Fig. 4"></div></div><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig4_HTML.gif" class="icon-reflink small-thumb" alt="Fig. 4" title="Fig. 4"></div></div></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig4" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Fig. 4</strong></a><div class="figure-table-caption-in-article"><span>Separation of CHO.K1 cells. Experimental data versus data predicted by the models for: (<strong>a</strong>) separation efficiency; (<strong>b</strong>) flow ratio; (<strong>c</strong>) flow rate. Dotted lines show a confidence level of 98%</span></div></div></td></tr></table></div></div><div style="clear:both;"></div></div><div class="sec sec-last"><span id="Sec13"></span><div class="head2 head-separate">Mid-term effects of hydrocycloning on cell viability and apoptosis induction</div><div id="id690226" class="p p-first">In order to evaluate any mid-term effects of hydrocycloning on the cells, fluorescence microscopy with acridine orange and ethidium bromide was used as a sensitive technique to determine cell viability and to assess any possible induction of programmed cell death (apoptosis). Two hydrocyclone configurations (HC 3020 and HC 2010) were tested at 1 bar using a peristaltic pump, in order to mimic the set-up in a perfusion bioreactor. The CHO.K1 cell line was used in tests carried out with HC 3020 (Table <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab5" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7.5em; border: dashed 1px green;">(Table 5)</span></span><span style="text-decoration: underline;">5</span><div class="large-thumb-canvas"></div></a>) and in a control test, in which cell suspension was pumped through the test system without the hydrocyclone. This control test gave evidence that the pumping system had no significant influence on the cells (data not shown). Due to the good results observed in these tests, recombinant CHO cells expressing GM-CSF were also tested for separation efficiency, viability and apoptosis with HC 3020 and HC 2010 (Tables <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab6" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -8em; border: dashed 1px green;">(Tables 6</span></span><span style="text-decoration: underline;">6</span><div class="large-thumb-canvas"></div></a> and <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab7" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -1.5em; border: dashed 1px green;">and7,</span></span><span style="text-decoration: underline;">7</span><div class="large-thumb-canvas"></div></a>, respectively).
</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab5" name="Tab5"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab5" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 5" title="Table 5"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab5" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 5</strong></a><div class="figure-table-caption-in-article"><span>CHO.K1 culture conditions before (“Culture”) and after (0, 3, 6, 24, 48 h) hydrocycloning (HC 3020) at a pressure drop of 1 bar</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab6" name="Tab6"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab6" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 6" title="Table 6"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab6" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 6</strong></a><div class="figure-table-caption-in-article"><span>CHO-GMCSF culture conditions before (“Culture”) and after (0, 3, 6, 24, 48 h) hydrocycloning (HC 3020) at a pressure drop of 1 bar</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Tab7" name="Tab7"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab7" onclick="startTarget(this, 'table', 1024, 800)"><div class="thumb-ph"><img src="corehtml/pmc/pmcgifs/table-icon.gif" class="icon-reflink" style="border: 1px solid;" alt="Table 7" title="Table 7"></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab7" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Table 7</strong></a><div class="figure-table-caption-in-article"><span>CHO-GMCSF culture conditions before (“Culture”) and after (0, 3, 6, 24, 48 h) hydrocycloning (HC 2010) at a pressure drop of 1 bar</span></div></div></td></tr></table></div></div><div style="clear:both;"></div><div id="id691049" class="p">The separation of both cell lines with hydrocyclones was accomplished with high separation efficiencies (>97%) and low viability losses (<7%), maintaining cell culture viability above 92%. Previous studies have shown that some cell lines are susceptible to hydrodynamic stress, presenting a significant decrease in cell viability when subject to stress levels above a threshold limit (Born et al. <a href="#CR5" rid="CR5" class="cite-reflink bibr popnode">1992</a>). However, due to the low viability drops observed in this work, the viability of CHO cells separated by hydrocyclones was kept above 92%, which is higher than the cell viability obtained for most cell retention devices reported by Voisard et al. (<a href="#CR36" rid="CR36" class="cite-reflink bibr popnode">2003</a>). In this review article, a compilation of 21 works using different cell retention devices showed that in only two cases cell viability was kept at values higher than the minimum viability (92%) obtained in the present work. In those two works cited by Voisard et al. (<a href="#CR36" rid="CR36" class="cite-reflink bibr popnode">2003</a>), the cell viability was 95%.</div><div id="id691090" class="p">LDH activity released to the supernatant of the CHO.K1 culture (Fig. <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig5" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">(Fig. 5a)</span></span><span style="text-decoration: underline;">5</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 5" title="Fig. 5"></div></div></a>a) presented an increase up to 24 h after hydrocycloning, becoming stable within 48 h. For the recombinant cell line, rather stable values were observed between 0 and 24 h, with a tendency to decrease within 48 h (Fig. <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig5" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">(Fig. 5b</span></span><span style="text-decoration: underline;">5</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 5" title="Fig. 5"></div></div></a>b and c). In all cases, the LDH values were in agreement with the trypan blue viability data, with increases in LDH activity released to the supernantant correlating to decreases in cell viability. These increases in LDH activity could also be related to the disaggregation of cell clumps, as discussed previously. Due to the break up of clumps upon hydrocycloning, dead cells become freely suspended in the medium, increasing the release of LDH to the supernatant.
</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Fig5" name="Fig5"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig5" onclick="startTarget(this, 'figure', 1024, 800)" class="icon-reflink figpopup"><div class="thumb-ph"><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 5" title="Fig. 5"></div></div><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig5_HTML.gif" class="icon-reflink small-thumb" alt="Fig. 5" title="Fig. 5"></div></div></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig5" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Fig. 5</strong></a><div class="figure-table-caption-in-article"><span>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: (<strong>a</strong>) CHO.K1 cells, HC 3020; (<strong>b</strong>) CHO-GMCSF cells, HC 3020; (<strong>c</strong>) CHO-GMCSF cells, HC</span><a class="side-caption" style="font-size: 100%;" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig5" onclick="startTarget(this, 'figure', 1024, 800)"> (more ...)</a></div></div></td></tr></table></div></div><div style="clear:both;"></div><div id="id691157" class="p">Samples collected from the overflow stream showed a lower viability and, thus, a higher relative amount of dead cells when compared to the underflow stream (samples designated “0 h” in Tables <a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab5" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7.5em; border: dashed 1px green;">Tables 5</span></span><span style="text-decoration: underline;">5</span><div class="large-thumb-canvas"></div></a>–<a href="articlerender.fcgi?artid=2151964&rendertype=table&id=Tab7" style="text-decoration:none;" onclick="startTarget(this, 'true', 1024, 800)" class="fig-table-link table"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -4.5em; border: dashed 1px green;">–7).</span></span><span style="text-decoration: underline;">7</span><div class="large-thumb-canvas"></div></a>). These data give evidence that the hydrocyclones showed a preferential retention of viable cells, separating non-viable cells preferentially in the overflow stream, which in a perfusion run would be the harvest stream. Acoustic filters also presented preferential separation of viable hybridoma cells, and this was credited to the decrease of the mean diameter of dead cells (Batt et al. <a href="#CR4" rid="CR4" class="cite-reflink bibr popnode">1990</a>; Lipscomb et al. <a href="#CR20" rid="CR20" class="cite-reflink bibr popnode">2004</a>). However, it is probable in the present case that not only the alterations in size, but also the changes in density have influenced cell separation. Whereas apoptotic cells usually have a decreased size, it is known that necrotic cells are prone to experience an increase in size due to cell swelling after membrane breakdown (Al-Rubeai <a href="#CR3" rid="CR3" class="cite-reflink bibr popnode">1998</a>; Buja et al. <a href="#CR7" rid="CR7" class="cite-reflink bibr popnode">1993</a>). However, due to loss of intracellular components, the density of necrotic cells becomes lower than that of living cells. In both cases (decrease in diameter or in cell density), the terminal settling velocity of the cells decreases, favoring the separation of dead cells in the overflow stream. A similar behaviour has been observed in inclined settlers and Centritech centrifuges (Batt et al. <a href="#CR4" rid="CR4" class="cite-reflink bibr popnode">1990</a>; Takagi et al. <a href="#CR32" rid="CR32" class="cite-reflink bibr popnode">2000</a>; Lipscomb et al. <a href="#CR20" rid="CR20" class="cite-reflink bibr popnode">2004</a>), which are equipment that also separate particles based on their terminal velocity.</div><div id="id691264" class="p p-last">The cells were also monitored within the first 48 h after passage through the hydrocyclone by fluorescence microscopy to evaluate any possible induction of apoptosis. According to the literature, exposure to high levels of hydrodynamic stress can induce cell death mechanisms such as apoptosis and necrosis, depending on its intensity and frequency (Al-Rubeai et al. <a href="#CR1" rid="CR1" class="cite-reflink bibr popnode">1995a</a>, <a href="#CR2" rid="CR2" class="cite-reflink bibr popnode">b</a>). Analysis of cell samples taken along 48 h, upon labelling with ethidium bromide (BE) and acridine orange (AO), showed insignificant levels of apoptotic cells (Fig. <a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig6" style="text-decoration:none;" onclick="startTarget(this, 'figure', 1024, 800)" class="fig-table-link fig figpopup"><span style="position: relative;text-decoration:none;"> <span style="cursor: hand; text-decoration:none; position: absolute; background-color: transparent; color: transparent; opacity: 0; filter: alpha(opacity=0); left: -7em; border: dashed 1px green;">(Fig. 6).</span></span><span style="text-decoration: underline;">6</span><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig6_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig6_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 6" title="Fig. 6"></div></div></a>). Thus, the present results indicate that the shear stress inside the hydrocyclones, combined to the short residence times of cells inside the equipment, does not induce apoptotic mechanisms. Considering that the apoptosis assay using fluorescence microscopy with BE/AO is significantly more sensitive than the trypan blue method, these results indicate that hydrocyclones can most probably be used as separation devices in long-term runs, e.g. in perfusion processes.
</div><div style="margin: 1em; margin-right: 2em; border: 1px solid #999999;border-left: 1px solid #AAAAAA;border-bottom: 1px solid #AAAAAA;"><div style="border: 3px solid #F0F0F0; border-left: 1px solid #F8F8F8; border-bottom: 1px solid #F8F8F8;"><a id="Fig6" name="Fig6"></a><table border="0" cellpadding="0" cellspacing="0" style="clear:both; width: 100%;"><tr valign="top" align="left"><td class="thumb-cell"><a href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig6" onclick="startTarget(this, 'figure', 1024, 800)" class="icon-reflink figpopup"><div class="thumb-ph"><div class="large-thumb-canvas"><div class="large-thumb-canvas-1"><img hires="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig6_HTML.jpg" src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig6_HTML.gif" style="border: 1px solid" class="icon-reflink large-thumb" alt="Fig. 6" title="Fig. 6"></div></div><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img src="picrender.fcgi?artid=2151964&blobname=10616_2007_9108_Fig6_HTML.gif" class="icon-reflink small-thumb" alt="Fig. 6" title="Fig. 6"></div></div></div></a></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig6" onclick="startTarget(this, 'figure', 1024, 800)"><strong>Fig. 6</strong></a><div class="figure-table-caption-in-article"><span>Monitoring of cells before (“Culture”) and after hydrocycloning (0, 3, 6, 24 and 48 h): (<strong>a</strong>) CHO.K1 cells, HC 3020; (<strong>b</strong>) CHO-GMCSF, HC 3020; (<strong>c</strong>) CHO-GMCSF, HC 2010. VNA: viable non-apoptotic cells; VA: viable apoptotic cells; NVA:</span><a class="side-caption" style="font-size: 100%;" href="articlerender.fcgi?artid=2151964&rendertype=figure&id=Fig6" onclick="startTarget(this, 'figure', 1024, 800)"> (more ...)</a></div></div></td></tr></table></div></div><div style="clear:both;"></div></div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"><div class="side-section-group"><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#top" class="sidefm-pmclink" style="text-transform: none;">Top</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870720" class="sidefm-pmclink" style="text-transform: none;">Abstract</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870765" class="sidefm-pmclink" style="text-transform: none;">Introduction</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id708932" class="sidefm-pmclink" style="text-transform: none;">Materials and methods</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id709529" class="sidefm-pmclink" style="text-transform: none;">Results and discussion</a></div></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);" style="text-transform: none;"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>Conclusions</a></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691373" class="sidefm-pmclink" style="text-transform: none;">References</a></div></div></div></td><td class="content-cell"><span id="Sec14"></span><div class="head1 section-title" style="text-transform: none;" id="id691345"><a id="Sec14"></a>Conclusions</div><div class="section-content"><span id="Sec14"></span><div id="id691355" class="p p-first-last">In the present work, the separation of CHO cells with hydrocyclones was evaluated. The separation efficiencies attained in all cases were high (>97%), and low drops in cell viability were observed. Tests performed with two CHO cell lines (wild-type CHO.K1 and recombinant CHO-GMCSF) have suggested that apoptotic cell death mechanisms are not activated upon passage through the hydrocyclones tested. Furthermore, the results suggest the same behaviour for both cell lines, indicating that the expression of a heterologous protein has not affected cell sensitivity to the hydrodynamic stress generated inside the hydrocyclones. Due to the high separation efficiency, the absence of apoptosis induction and the lack of moving parts, it can be postulated that hydrocyclones are suitable devices for cell retention in perfusion bioreactors.</div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"> </td><td class="content-cell"><div class="head1 section-title" style="text-transform: none;" id="id691366">Acknowledgements</div><div class="section-content"><div class="sec"><div id="id691370" class="p">The authors wish to thank the Brazilian funding agencies CNPq, FAPERJ and FINEP for financial support.</div></div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"> </td><td class="content-cell"><div class="head1 section-title" style="text-transform: none;" id="id799205">Abbreviations</div><div class="section-content"><div class="sec"><div class="def-list"><table border="0" cellspacing="4"><tr><td></td></tr><tr><td>D<sub>c</sub></td><td>Hydrocyclone diameter (diameter of the cylindrical part)</td></tr><tr><td></td></tr><tr><td>D<sub>i</sub></td><td>Inlet diameter</td></tr><tr><td></td></tr><tr><td>D<sub>o</sub></td><td>Overflow diameter</td></tr><tr><td></td></tr><tr><td>D<sub>u</sub></td><td>Underflow diameter</td></tr><tr><td></td></tr><tr><td>E</td><td>Separation efficiency</td></tr><tr><td></td></tr><tr><td>HC</td><td>Hydrocyclone</td></tr><tr><td></td></tr><tr><td>L</td><td>Hydrocyclone total length</td></tr><tr><td></td></tr><tr><td><a id="IEq1" name="IEq1"></a><img src="picrender.fcgi?artid=2151964&blobtype=equ&blobname=M7" style="vertical-align: middle; background: #F8F8F8;padding: 2pt;border-style: none;" alt="equation M7" title="equation M7"></td><td>Vortex finder length</td></tr><tr><td></td></tr><tr><td>Q</td><td>Feed flow rate</td></tr><tr><td></td></tr><tr><td>Q<sub>u</sub></td><td>Underflow flow rate</td></tr><tr><td></td></tr><tr><td>R<sub>f</sub></td><td>Flow ratio</td></tr><tr><td></td></tr><tr><td>X</td><td>Cell concentration</td></tr><tr><td></td></tr><tr><td>X<sub>u</sub></td><td>Underflow cell concentration</td></tr><tr><td></td></tr><tr><td>ΔP</td><td>Pressure drop</td></tr><tr><td></td></tr><tr><td>ΔV<sub>u</sub></td><td>Viability loss (cell viability in the feed minus viability in the underflow stream)</td></tr><tr><td></td></tr><tr><td>θ</td><td>Angle of the conical part</td></tr></table></div><br style="clear:both;"></div></div></td></tr><tr valign="top"><td class="sidebar-cell" width="145"><div class="side-section-group"><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#top" class="sidefm-pmclink" style="text-transform: none;">Top</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870720" class="sidefm-pmclink" style="text-transform: none;">Abstract</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id870765" class="sidefm-pmclink" style="text-transform: none;">Introduction</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id708932" class="sidefm-pmclink" style="text-transform: none;">Materials and methods</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id709529" class="sidefm-pmclink" style="text-transform: none;">Results and discussion</a></div></div><div style="text-transform: none;;"><div class="sidefm-pmclink-item"><a href="#id691345" class="sidefm-pmclink" style="text-transform: none;">Conclusions</a></div></div><div class="sidefm-pmccurrent-item"><a class="sidefm-pmclink" href="javascript:return(false);" style="text-transform: none;"><span class="sidebar-menu-square-image-holder"><img src="corehtml/pmc/pmcgifs/square.gif" alt=">" style="border-style: none;"></span>References</a></div></div></td><td class="content-cell"><span id="Bib1"></span><div class="head1 section-title" style="text-transform: none;" id="id691373"><a id="Bib1"></a>References</div><div class="section-content"><div class="back-matter-section" id="reference-list"><ul class="back-ref-list-1st-line-shifted"><li id="CR1"><div class="citation"></div><div class="citation">Al-Rubeai M, Singh RP, Goldman MH, Emery AN (1995a) Death mechanisms of animal cells in conditions of intensive agitation. 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