PROCEDURE PROPAGATION AND SUBCULTIVATION OF HUMAN DIPLOID CELLS IN 150cm2
This method describes the establishment and subcultivation of 150-cm2 cell cultures originating from a human diploid working cell bank. The harvests of these cultures will be used to seed further similar vessels or microcarrier culture systems. Ampoule vial of MRC-5 WI-38 Trypsin in phosphate-buffered saline PBS Dulbecco's modified Eagle's medium DMEM with 10 foetal bovine serum FBS 150-cm2 plastic culture vessels with a pipette and pro-pipetter, into a 150-cm2 100 ml of DMEM with 10 FBS. at 37...
Supplementary Procedure Microbiological Culture
1. Using a sterile swab, harvest adherent cells. Resuspend them in the culture medium to a concentration of about 5 x 10s cells ml-1. 2. Test the suspension cells direct at about 5 x 105 cells ml4. 3. Inoculate agar plate with 0.1 ml of the test sample. 4. Inoculate broth with 0.2 ml of the test sample. 5. Incubate test plate under anaerobic conditions Gaspak, BBL Microbiology Systems, Cockeysville. MD, USA at 36 1 C for 21 days. 6. At approximately 7 and 14 days post-inoculation, subculture...
Mtt Assay
Ulk' ita jl a JL JLKjKJrA M. jl 5. Read plate on a micro-ELISA reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. Plates should be read within 5 min of adding acidified propan-2-ol. The MTT assay can also be used to quantify cell activation by determining the maximal velocity V of the reaction Gerlier amp Thomasset, 1986 . Alternatively, in order to compare two states of activation of a given cell, the MTT formazan produced by the same number of viable cells can be...
Lactate assay
Batch, repeated batch feed and harvest modes Glucose consumption rate GCR GCR MFR Gc - G3 - F V 5 Af
Initiating a chemostat culture
Cells can be inoculated at a normal inoculum level, e.g. 1-2 x 10s cells ml4, and grown as a batch culture until the mid-exponential phase of growth. The supply of medium from the feed reservoir can then be started at a flow rate that gives the required dilution rate. In order that the cells are not washed out of the fermenter, the initial dilution rate should be below the maximum specific growth rate of the cells, although too low a dilution rate may result in low cell viability Figure 5.6.1...
Pilotscale Suspension Culture Of Hybridomas An Overview
The most common cell culture systems developed for pilot- and commercial-scale production of monoclonal antibodies MAbs are hollow-fibre and ceramic matrix modules, stirred bioreactors and airlift fermenters. These systems allow cultivation of cells in batch, fed-batch, continuous or perfusion mode. The selection of a culture system and culture mode for the large-scale production of a particular MAb should take into account the growth and antibody-production characteristics of the particular...
CELL LINE Vera
The Vero cell line was derived from the kidney of a normal jA.fnc3.11 green monkey and is susceptible to a wide range of viruses, including polio, rubella, arboviruses and reoviruses Simizu amp Terasima, 1988 . The Vero monkey kidney cell line has been used for the industrial production of viral vaccines in prcfcrcncc to pnni ciry monkey kidney cells because of availability and the reduced risk of contamination by endogenous viruses. Vero cells have been used for the production of the polio...
Discussion Tdl
Apoptosis was first described in 1972 Kerr et al, 1972 . Since then it has become apparent that this mode of cell death plays a role in a number of important physiological processes. Thus it is advantageous to be able to recognize when this mode Figure 4,4.4 Percoll gradient for isolation of apoptotic cells from an LH-60 cell culture. of death is occurring. Investigations into the mechanistic aspects of apoptosis have revealed a regulatory role of different ions m this process. For cxEmplc...
Procedure Culture Of Suspension Cells In A Spinner Flask
to as low a level as possible 0.5-5 PThe use of HEPES buffer to stabilize the pH during the setting-up procedure is beneficial. Figure 5.3.1 a A typical spinner flask with magnetic spinner bar. b A Techne flask with asymmetric stirring rod. Figure 5.3.1 a A typical spinner flask with magnetic spinner bar. b A Techne flask with asymmetric stirring rod. Pluronic F-68 polyglycol BASF, Wyandot can be used at 0.1 to protect cells against mechanical damage, especially at reduced serum concentrations....
Additives For Microcarrier Cultures
As already discussed, theory and experimental evidence suggest that an increased medium viscosity will reduce damage of cells on microcarriers because of the reduced intensity and frequency of interactions that cause cell damage in microcarrier cultures. Among the possible additives that can increase medium viscosity with no apparent detrimental effects on the cells, dextran up to 0.3 , w v has been found beneficial to these cultures. A limited number of experiments have shown that MCs and CMCs...
Spinner Flask Culture
This technique originated by placing a silicone or Te central ring into a glass vessel that is placed on a approach is still used, it is preferable to use a the cells and microcarriers will be destroyed by the grinding action of the let just above the bottom of the flask. A typical spinner flask is shown in 5.3.1, and has a stirrer shaft containing the magnet fitted to the bottle top. In addition, side arms are fitted in order to add cells, change medium or gas with oxygen or C02-enriched air...
Miniaturized Colorimetric Methods For Determining Cell Number
Rapid and accurate assessment of viable cell number is an important requirement in many experimental situations involving animal cell culture, e.g. measurement of growth factor activity, serum batch testing and toxicity assays. Colony-forming efiiciency clonogenic assays are the most reliable methods for assessing viable cell number. These methods are time-consuming, however, and become impractical when many samples have to be analysed e.g. in toxicity assessment where several chemicals at a...
Volumetric Scale Up
MICROCARRIER STIRRED TANKS AIRLIFT EFFICIENCY MODIFICATIONS e.g. Spin-filter Figure 5.1.1 Scale-up options - the decision process. ADC anchorage-dependent cells. 2. Low-volume under 11 but very high density 1-2 x 108 ml4 heterogeneous systems e.g. hollow-fibre bioreactors . 3. Intermediate systems using a spin-filter to increase cell density to 1-2 x 1Q7 ml1 but to a volumetric scale of 500 1 only. 4. High-density 1 x 108 ml4 scaleable systems to 2001 based on porous microcarriers. Medium cell...
Procedure Roller Bottle Culture Of Animal Cells
Standard tissue culture media and reagents Roller bottles e.g. 23 x 12-cm plastic bottle with surface area of 1400 cm2 from, for example, Becton Dickinson, Sterilin Roller bottle apparatus with speed control between 5 and 60 rph e.g. Wheaton, Bellco, New Brunswick 1. Add approximately 1 ml of complete culture medium at 37 C per 5-cm2 culture area. 2. Add 5 C02 in air to headspace. 4. Seal bottle and place in roller culture apparatus. 5. Rotate bottle at 12-24 rph for the initial attachment...
Neutral Red Nr Assay
Neutral red 3-amino-7-dimethyl-2-methylphenazine hydrochloride is a water-soluble, weakly basic, supravital dye that accumulates in lysosomes of viable cells. The neutral red NR assay is an in vitro cell viability test that was developed and extensively studied for in vitro cytotoxicity determinations Babich amp Borenfreund, 1990 . Earlier in vitro tissue culture studies using the NR dye were developed for assessments of viral cytopathogenicity Finter, 1969 and for immunotoxicity assays...
Cell Lime Gh3
The GH3 cell line was derived from the pituitary tumour of a 7-month-old female Wistar-Furth rat Tashjian et al, 1968 and is used for the study of hormones. The GH3 clone produces growth hormone at a greater rate than GH1 cells, a clone obtained from the same primary culture, and also produces prolactin. Hydrocortisone can be used to stimulate hormone production and also inhibits the production of prolactin Bancroft et al, 1969 . The cells are epithelial-like in morphology but can be adapted to...
Background Reading
Batt BC amp Kompala DS 1989 A structured kinetic modeling framework for the dynamics of hybridoma growth and monoclonal antibody production in continuous suspension culture. Biotechnology and Bioengineering 34 515-531, Bree MA, Dhurjati P, Geoghedan Jr RF amp Robnett B 1988 Kinetic modelling of hybridoma cell growth and immunoglobulin production in a large-scale suspension culture. Biotechnology and Bioengineering 32 1067-1072. Dalili M, Sayles GD amp Ollis DF 1990 Glutamine-limited batch...
Index
National Institute for Medical Research, Mill Hill, London NW7 IAA, UK Section 1.2 National Cell and Tissue Culture Centre, Bioscience Ireland, School of Biological Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland Section 2.6 Department of Biochemistry, University College Cork, Lee Makings, Prospect Row, Cork, Ireland Section 4.4 American Type Culture Collection, Cell Culture Department, PO Box 1549, Manassas, Virginia 20108 1549, USA Section 1.8 National Cell and Tissue Culture...
Procedure Amino Acid Analysis
Several techniques have been developed to analyse amino acids. The following procedure describes one of the most frequently used methods for the determination of amino acid concentration, the PITC method. Free amino acids in medium samples are derivatized with phenylisothiocyanate PITC to produce phenyl-thiocarbamyl PTC amino acids. After separation by reverse-phase HPLC, the PTC amino acids are detected using a UV spectrophotometer at 254 nm. The amino acids alanine, arginine, asparagine,...
Anchoragedependent Cells Microcarrier
Anchorage-dependent cells are grown in suspension culture by growing the cells attached to small microcarrier beads 100-300 xm suspended in the culture medium by agitation. There are two types of microcarriers available Chapter 5, sections 5.8 and 5.9 Griffiths, 1990 the non-porous microcarriers whereby cells are growing only on the outer surface of the beads, and porous microcarriers whereby cells are growing predominantly in the internal porous structure of the microcarrier beads. For porous...
Procedure Dma Stain
This stain is heat and light sensitive. The toxic properties of Hoechst 33258 Take 22.2 ml of citric acid 0.1 M and 27.8 ml of disodium phosphate 0.2 M 15 psi for 15 min. Cool to 50 C and mix 8 ml per 5 cm diameter Petri dish. Store at 4 C in sea Hoechst stain stock solution Hoechst stain working solution prepare fresh as required Fluorescence microscope equipped with epifluorescence e.g. Zeiss and 340-380 nm excitation filter and 430 nm suppression filter Note Both methods used for the...
Extended Cell Bank
From the regulatory point of view, the characterization requirements for the cells will depend on the bulk culture process to be used. This will define the number of PDs set within the limits discussed above that the cells will undergo. An additional regulatory requirement is that the cells should be passaged beyond the anticipated PD level achieved at the end of each production run. This procedure should therefore establish the stability of cells well beyond the normal working limits....
a c nryecT yp PT TT TT TR F SIT TTFAPFS
J a J A A VJ J v_y 1 j V__' -1 A A .A v WJP A A .Z I-VIII Represent different surface chemistries flaunt 5 5 1 Growth of MRC-5 cells on experimental surfaces with varying chemistries. The tissue culture surface is therefore not a coating, but a surface-modified polystyrene having random functional groups covalently bound to the surface. Tissue co-culture i.e. cytoplasmic contact and 8-10 xm can be used for cell invasion studies. It is recommended that the manufacturer be contacted about the...
References
Babich H amp Babich JP 1997 Sodium lauryl sulfate and triclosan in vitro cytotoxicity studies with gingival cells. Toxicology Letters 91 189-196. Babich H amp Borenfreund E 1987 Structure-activity relationship SAR models established in vitro with the neutral red cytotoxicity assay. Toxicology In Vitro 1 3-9. Babich H amp Borenfreund E 1990 Application of the neutral red cytotoxicity assay to in vitro toxicology. Alternatives to Laboratory Animals 18 129-144. Babich H amp Borenfreund E 1991...
Bacteria And Fungi
For any reasonable quality control programme involving cell lines it is absolutely necessary to include routine culture tests for bacteria and fungi. Most often the presence of such contaminants will be obvious even to the novice. This is especially true when the cultures are set up in the absence of all antibiotics - a practice that is strongly recommended. However, while overt microbial contamination may be more common, many organisms will grow slowly in the usual cell cultures, sometimes...