Technical Note XerumFree™ XF205 Subject: TNC BIO’s ACGS Approach Background In the in vivo situation, cells are bathed by the extracellular fluid which can be divided into two major sub compartments, the interstitial fluid and the blood plasma. Plasma and interstitial fluid are very similar, as they communicate freely through pores and intercellular clefts in capillary endothelium. The interstitial fluid collects also all the factors and proteins that are synthesized and secreted by the cells. TNC BIO BV High Tech Campus 1E 5656 AE Eindhoven The Netherlands T +31 4030 400 80 [email protected] www.tncbio.com Thus the interstitial fluid accumulates the chemical compounds of the plasma on one hand and those released by the cells on the other. Consequently, an ideal composition of a culture medium would consist of plasma components AND cell culturederived components. ACGS Approach Apart from waste metabolic products, cells in culture produce all sorts of autocrine growth factors, cytokines and attachment and spreading factors, many of them involved in autocrine control of attachment, spreading, growth and proliferation. However these factors are absent at the seeding stage, just after the cell dissociation step. This is the principal reason why many cell types have some difficulties to adapt to growth in the absence of serum (which supplies also attachment and growth promoting factors). Obviously, the first thought is how to supplement the cells with these autocrine agents when passing the cells. 1/3 Technical Note XerumFree™ XF205 The absence of autocrine adhesion and growth promoting factors at the seeding stage may be compensated by an easy step which consist in supplying the cell culture with ‘spent medium’ or better medium that has been incubated with the cells at former passages. In this procedure, all autocrine attachment and growth factors are collected in the cell-free culture supernatant (CFCS), and supplied back to the cells at the seeding stage. CFCS may be used as such or further processed by desalting and/or concentration which eliminates also all the low molecular weight cellular waste metabolites that are present. Suggested ACGS Protocol during the adaptation of cell cultures to serum-free growth This protocol can be applied to the direct adaptation method as well as to the sequential “weaning” method. A. Cell cultures: - growing in 10% FBS cell culture medium, or - growing at the 1.5 % XF205 + 2.5% FBS stage (see instructions for use XerumFree) All cell cultures should be near confluence when used in the procedure B. Preparation of the culture surfaces for the first serum-free passage: As no ACGS is available at this time point, the culture vessels must be coated with adequate attachment factors. This can be done by using commercially available coating kits (see TNCbio’s Tech Note Cell Attachment, or contact us for a copy). This type of coating is interesting when all animal-derived material is to be avoided. This is not the case at this stage (the cells have been in contact with FBS until now). We recommend a very efficient, albeit not animal component-free coating which consists in - dispensing a minimal amount of FBS in the culture vessel, enough to cover the entire cell culture surface, e.g. 500 μl for a T75 flask - incubation overnight at 37° C - rinsing twice with PBS or fresh culture medium - [optional, recommended] put aside one flask for total protein determination The protein assay allows to determine how much FBS protein has remained anchored to the cell culture surfaces. Here we leave it up to the researcher to carry out this total protein assay or not and by which method. C. Pass the cells from their serum containing medium (10% or 2.5% FBS) directly into cell culture medium made up with Basal Medium + 2% Xerum-free XF205 D. Feed cells fresh medium according to your standard medium change scheme. When cells are growing in the mid-log phase START COLLECTING ALL SPENT MEDIA E. Prepare Cell-Free Cell culture Supernatants (CFCS) by - sterile filtration of the collected spent media, - store at 4°C or frozen Optionally, the CFCS may be further processed, depending on which degree of standardization is sought: - desalting step to eliminate the inorganic compounds - concentration The Centriprep centrifuge columns available from Merck Millipore allow for a concomitant desalting and concentration step. The recommended molecular weight cut-off for this application is 3.000 D. For any questions related to this Technical Note please contact TNCbio, [email protected] 2/3 Technical Note XerumFree™ XF205 F. Prepare ACGS Media consisting of - 58% fresh basal medium - 40% CFCS (unconcentrated) - 2% XerumFree XF205 or - 94% fresh basal medium - 4% CFCS (10 times concentrated) - 2% XerumFree XF205 G. When cells are confluent and when enough CFCS has been collected replate the cells with ACGS medium H. Continue to collect all SPENT MEDIA as under D. and prepare CFCS for the next passage Once the cells are successfully grown in the ACGS Medium they may be weaned off by slowly decreasing the proportion of CFCS added to the culture medium. The described method is somewhat more laborintensive than the more common adaptation methods. The advantage is that it allows for a more smooth transition from the over-rich FBS cell culture environment to a more basic serumfree milieu, also in the case of more ‘recalcitrant’ or tedious cell types. For any questions related to this Technical Note please contact TNCbio, [email protected] 3/3
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