Professional Documents
Culture Documents
Paras Yadav1, Annu Yadav1, P. Kumar1, J.S. Arora1, T.K.Datta1, S. De1, S.L. Goswami1, Mukesh Yadav2, Shalini Jain3, Ravinder Nagpal4 and Hariom Yadav3 1Department of Animal Biotechnology, 3Animal Biochemistry Division and 4Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana), India; 2SOS in Chemistry, Jiwaji University, Gwalior-474011, M.P., India
Baculovirus
Baculovirus are present in invertebrates primarily insect species They are not infectious for vertebrates & plants Genome is covalently closed circular double stranded of 134 kbp, due to its small it can accommodate large fragments of foreign DNA They are divided into two groups on the basis of their structure as-: Nucleopolyhedroviruses (NPV) Granuloviruses These NPV are mainly used as expression vectors i.e. Autographa californica NPV (AcMNPV) isolated from the larva of the alfalfa looper
Contd..
Baculovirus expression system based upon the ability to propagate AcMNPV in insect cells Uses many of the protein modification, processing and transport systems present in higher eukaryotic cells. Virus that can be propagated to high titers adapted for growth in suspension cultures obtain large amounts of recombinant protein with relative ease Baculovirus are noninfectious to vertebrates and their promoters are inactive in mammalian cells.
Figure
Tn7R p10 Gent+ Tn7L
Gene of Interest
Gene construct
Contd..
Tn7R GOI Tn7L
128bp M 13 forward
145bp
Bacmid DNA
M 13 reverse
Contd..
PCR amplification using M-13 Forward and Reverse primers If no transposition, then a region a bacmid alone will amplify to gave product of 300bp In condition of transposition then the amplified size will be 2300bp+size of insert Recombinant bacmid is now ready to transfect to insect cell lines
Insect Medium
Graces Insect medium- unsupplemented but contains L-glutamine Graces Insect medium supplementedcontains additional TC yeastolate & Lactalbumin hydrolysate Trichoplusia ni Medium formulation hink (TNM-FH)- contains 10% FBS
1x 105
2 x 105 1x 106 .5-1 x 107 1-2 x 105/ml
1.5-2
3-4 4-5 10 40-500
Cell number may vary depending upon the culture conditions and the health of the culture
Flask size(cm2) sf9 25 4 x 106 sf21 3.8 x 106 High five 3.0 x 106
75
150
1.2 x 107
2.4 x 10
1.1 x 107
2.3 x 107
9.0 x 106
1.8 x 107
Suspension culture
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Perform viable cells count on harvested cells. Inoculate cells at 2 x 105 viable cells/ml into respective culture vessels. Inoculate cultures kept at 25-28 C with loose caps to allow gaseous exchange On day 4 post-planting, aspirate the spent medium from one side of the monolayer & subculture the flask With slower growing cell lines, it may be necessary to feed the flasks on day 3-4 post planting Subculture the flasks when the monolayer reaches 80-100% confluency, approx 2-3 days post planting
Cell density reaches to 2-2.5 x 106 cells/ml they should be diluted to no less than 7 x 105 cells/ml Use a spinner flask with a vertical impeller Culture volume should not exceed half of the volume of the flask Use of surfactant to decrease shearing e.g. Pluronic F-68
Contd..
Not necessary to change medium regularly. Sub culturing requires the removal of cell suspension & the addition of medium Impeller should be rotating regularly Impeller should be submerged 1 cm or more to ensure adequate aeration Cell viability of 95% is required Minimum density of 1 x 106 cells/ml is required
Contd
Keep record of the passage number. After 30 passage or more (2-3 months), cells doubling time increased and also loose their viability and infectivity. Keep a cell log, to do so one should have a knowledge of following; date of initiation of culture, lot number date of passage & passage number density & viability at passage comment on cell appearance medium & its lot number
Sf 9
72 hrs
TNM-FH
Sf 21
24 hrs
Spherical, granular, different in size, firm attachment to surface Spherical, granular, regular in size, loose attachment to surface
TNM-FH
Grow well as monolayer and suspension Grow well as monolayer, also as suspension
High-five
18 hrs
Cryopreseravtion of cells
Freezing cells should be 90% viable and 8090%confluent Freezing medium should have 60% Graces insect medium supplemented with 30%FBS & 10% DMSO
Procedure
Count cells using haemocytometer Placed cryovials on ice & label them Centrifuge cells at 400-600 g for 10 mts at RT. Remove the supernatant Resuspend the cells to the given density in the freezing medium Transfer 1 ml of the cell suspension to sterile cryovials Place at -20 C for 1 hr then transfer to -80 C for 24-48 hrs & then finally store at Liquid nitrogen
Do and Don'ts
Check cells daily until a confluent monolayer is formed. Passage cells at confluency only, as cells will be easy to dislodge & shows better viability Do not overgrow cells, it results in decreased viability Do not splits cells too for. Densities lower than 20% confluency inhibit growth Passage the cells only in log phase, log phase growth can be maintained by splitting cells in 1:5 dilution
Contd..
Possibly keep cultures free of antibiotics in order to be able to recognize the contamination Never use the same media bottle for different Insect cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C Switch on the laminar flow cabinet 20 mts prior to start working Cell cultures which are frequently used should be subcultered & stored as duplicate strains