Foetal Bovine Sera in Cell Culture

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1 Foetal Bovine Sera in Cell Culture Ian Ridgers FAS Cell Culture & Bioreagents 7 th June 2012 ian.ridgers@sial.com

2 What is Serum? Why use FBS? Grades of serum Worldwide demand. Collection Processing QC Testing Selection of Serum Frequently Asked Questions

3 What is Serum? The clear, thin & sticky fluid portion Of the blood that remains after Coagulation. Typical recovery is 50-60% of blood volume Serum contains no blood cells, Platelets or fibrinogen plasma: with anti-coagulating agent serum: without anti-coagulating agent

4 Why use FBS? Historically FBS has been the most widely used growth supplement for cell culture media due to its high content of embryonic growth promoting factors Provides all additional nutritional requirements, NOT provided by Classical Media,. Proteins, electrolytes, lipids, carbohydrates, hormones and enzymes Also growth factors, attachment factors, nutrients for proliferation and differentiation and factors for binding to and inactivating toxic compounds

embryonic growth promoting factors Provides all additional nutritional requirements, NOT provided by Classical

5 Why use FBS? Regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients and trace elements into the cell Buffers the cell culture system against a variety of disruptions to cell growth and toxic effects such as ph change, presence of heavy metals, proteolytic activity and endotoxin. Commonly added to cell culture medium at 2-10%.

micronutrients and trace elements into the cell Buffers the cell culture system against a

6 Grades of Serum USDA Grade FBS Originates from countries certified as free of both BSE and FMD US NZ & Australia Central America e.g. Mexico, Panama, Honduras, Costa Rica, Guatemala (after full USDA safety testing)

7 USDA Grade Can be freely imported into ANY country Product of choice in ALL countries for manufacturing Only the use of this grade allows researchers to send cells, or cell products, to countries with strict import regulations The USDA will not permit the importation of cell cultures, monoclonal antibodies, ascites fluid or bovine serum from countries where rinderpest and foot-and-mouth disease are present unless the imported materials are determined to be virus-free

USDA will not permit the importation of cell cultures, monoclonal antibodies, ascites fluid or bovine serum from

8 Grades of Serum European Grade FBS Dependant on disease status (BSE, FMD) Rest of the World including Europe and S. America

9 European Grade Originates from countries certified by the EU as specific disease free Criteria met by all USDA approved countries Europe South America (Brazil, Uruguay, Argentina) Can be freely moved between all EU member countries and other non- European countries where USDA or FDA regulations are not required

(Brazil, Uruguay, Argentina) Can be freely moved between all EU member

10 Worldwide Demand? L per year

11 Worldwide Demand?

12 Collection Foetuses are harvested and the blood is collected by cardiac puncture under aseptic conditions in disposable sterile bags. Approximately 200ml 2L of blood is collected per foetus All blood collected in one day is pooled

13 Collection Controlling the initial collection is a crucial factor in the quality of the final serum product Collected blood is immediately chilled and kept at 2 8 C through out the process Within 24 hours the blood is allowed to clot and the serum is collected using refrigerated centrifugation At this point the serum is tested for haemoglobin content, bioburden and endotoxin to ensure it meets the required specification Approved serum is then frozen for delivery to the manufacturing facility

is collected using refrigerated centrifugation At this point the serum is tested for haemoglobin content, bioburden and

14 Processing Selected batches of approved serum are thawed under temperature controlled conditions and pooled. Pooled material is transferred via pre-filters to a Raw Serum tank which is constantly stirred. Material is then transferred via 0.2µm filters to a Sterile Serum tank. The serum is constantly stirred to ensure complete homogenity Final filtration is by triple 0.1 µm filters.

Pooled material is transferred via pre-filters to a Raw Serum tank which is constantly stirred.

15 Processing The serum is dispensed into pre-sterilised bottles by an aseptic filling process in a Class 100 environment within a Class 10,000 cleanroom After filling the final product is blast frozen to -20ºC

16 Processing Further processing Heat Inactivation temperature raised to 56ºC for 30 minutes. Destroys Complement to ensure cells are not lysed by antibody binding. Originally developed when adult bovine serum was used. FBS doesn t benefit from heat inactivation. Gamma Irradiation exposure to 60 Co source intended to provide complete assurance of viral inactivation. Charcoal Treatment used to remove the hormones for use in in vitro systems. Used in steroid receptor binding studies, steroid regulation studies. Dialysed studies involving nutritional parameters or incorporation of labelled material require removal of low molecular constituents such as hypoxanthine and thymidine prior to use. This is done by dialysis against 0.85% saline through a 10kDa cutoff membrane. Reduced IgG shown to reduce levels to below 1ug/mL. No negative impact on performance.

Gamma Irradiation exposure to 60 Co source intended to provide complete assurance of viral inactivation. Charcoal Treatment used to remove the hormones for use in in vitro systems.

17 QC Testing FBS is tested at multiple stages throughout the process. FBS undergoes the following testing Physical and Chemical Biochemical Microbiological Biological Performance

18 QC Testing Physical and Chemical testing ph Osmolality Electrophoretic Pattern Total Protein Albumin IgG Globulins Haemoglobin

19 QC Testing Biochemical Testing SMA-24 Microbiological Testing Bacterial and Fungal sterility testing to current USP Mycoplasma Contamination (culture method) Viral Contaminants BVD, IBR, PI3, Rabies, Reovirus, Bovine Adenovirus Type I & V Viral Antibodies determine titre of neutralising antibodies Bacteriophage Endotoxin

method) Viral Contaminants BVD, IBR, PI3, Rabies, Reovirus, Bovine Adenovirus

20 QC Testing Biological Performance Hybridoma Testing to test ability to support growth of mouse myeloma cells and hybridoma cells Cloning Efficiency Testing to test ability to support clonal growth of myeloma cells and/or fusion products Growth promotion assay of MRC-5 cells (fibroblasts) and Hep-2 cells (epithelials)

test ability to support clonal growth of myeloma cells and/or fusion products

21 Selection of Serum Decision is normally based on Performance Cost Specific Customer Requirements A quantity of serum sufficient for a particular time period can then be purchased or reserved. Frequently an annual exercise Can be time consuming

22 Selection of Serum Serum represents an undefined mixture where composition varies from one lot to the next Some cell types and assays are sensitive to the variations in serum performance Studies have shown that some metabolites in serum can show a two to six fold variation in different serum samples Cholesterol levels in serum have been shown to vary from mg/dl. This variability may have the following effects: Stimulation or inhibition of growth Induction of unwanted differentiation

23 Selection of Serum Pre-qualifying and reserving serum is the best way to overcome the problem of lot to lot variability A customer will typically request samples from the major serum suppliers and possibly some of the smaller companies Customer selects an appropriate batch of serum for their individual application Selection is based on the performance of the serum in cell assays performed in the customer s own lab May be a simple as plating efficiency or more advanced testing such as proliferation or differentiation assays

24 Frequently asked questions My FBS contains precipitates Precipitates contain Fibrin (clot forming protein) and lipoproteins. This is a normal characteristic and doesn t affect the performance of the serum To remove particulate, transfer the serum to sterile tubes and briefly centrifuge at 400 g. Then filter the resulting supernatant along with your media. (Attempting to filter heavy flocculence may clog filters). My FBS is turbid Firstly rule out the possibility of contamination. If the serum is sterile then the turbidity is caused by the cryoprecipitation of lipid and or calcium (calcium oxolate crystals). This is normally due to repeated freeze / thaw cycles.

25 Frequently asked questions Why does the colour of my serum vary from lot to lot? The colour is dependant on the haemoglobin concentration in the specific lot and varies from brown to brown-red. How do I heat inactivate FBS? Place in a 56 ºC waterbath for 30 minutes. Water level should be higher than the level of the serum. Monitor the temperature in a reference bottle containing water at the same volume during heat inactivation. Mix the contents of the bottle every 5 minutes to ensure uniform heating. I have a jelly like fraction at the bottom of the bottle Results from improper heat inactivation or, more usually, inactivation without mixing. Protein denaturation is the cause of the jelly. Do not use!

26 Frequently asked questions Can FBS be used past its expiration date? Possibly. While we guarantee FBS from five years from the date of processing, it is most likely viable for several years past that, assuming that it has been stored properly (at -20 C or below), and not subject to multiple thawing/refreezing cycles. How should I thaw FBS? Recommended to thaw at 2-8ºC but may also be thawed at room temperature. Bottles should be swirled as they thaw to mix the contents.

27 Frequently asked questions Why is your FBS is not working with my cells? First, ensure that FBS is recommended for your cell line. For example, calf serum (CS), and not FBS, is advised for NIH/3T3 cells. Check ECACC (and other) sites for media requirements and culturing conditions for specific cell lines. Tissue culture difficulties may be due to a wide variety of problems, such as poor aseptic technique, and suboptimal culturing conditions. Otherwise, due to batch-to-batch variation, certain cell lines may require specific growth and/or attachment factors that may not be present in any particular lot of FBS.

29 Questions?

6 Characterization of Casein and Bovine Serum Albumin

6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function