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A Successful, Reproducible, Ready to use Kit for Human B cell immortalizationA Successful, Reproducible, Ready to use Kit for Human B cell immortalizationDendriticsDendritics--60 avenue Rockefeller60 avenue Rockefeller
www.dendritics.netwww.dendritics.netBackground
Study of peripheral blood B cells physiology are generally limited by:
the low number of B cells (8-10% of PBMC)
ObjectivesObtaining immortalized B cells :
using PBMC as starting
ComingComing
the low number of B cells (8-10% of PBMC)
their low capacity to enter cell cycle
The most successful approaches of these last decades were :
the CD40 system, targeting 1% of B cell population(1),
the EBV infection, targeting 1/106 of B cell population(2;3;4).
A method targeting the IgG+CD22+ B cell subset (5).
using PBMC as starting
reflecting the overall
in sufficient amount
within a short time
using an easily transferable
using an highly reproducible
Ease of useA packaging adapted to the culture procedure
Reagents conditioned in colored vials
One colour for each day of useOne colour for each day of use
Dilution of the total content of each vial in culture medium
PBMC
Experimental
D3-8D-1 D0
An average of 5.104 to 2.105 independent immortalized B cells clones
From D20, 106 cells per well (24w plates) can be obtained
Dendritics process targets up to 20% of the starting B cell populationDendritics process targets up to 20% of the starting B cell population
APPLICATIONS & FIELDSAPPLICATIONS & FIELDS
The crucial condition for success: The crucial condition for success: an an appropriate selection appropriate selection of the donorof the donor
B cell population representative of the overall B cell peripheral repertoirecan be used as biological source for:
Healthy donors
In progressDendritics team is currently focusing on the
Identification and resolution of troubleshootings
Packaging/Guidelines to ease the transfer to any laboratory
an an appropriate selection appropriate selection of the donorof the donorGenomic, transcriptomic and proteomic studies
Patients
For more informations:www.dendritics.net
A Successful, Reproducible, Ready to use Kit for Human B cell immortalizationA Successful, Reproducible, Ready to use Kit for Human B cell immortalization60 avenue Rockefeller60 avenue Rockefeller--FF--69008 Lyon69008 Lyon
www.dendritics.netwww.dendritics.netObjectives
Obtaining immortalized B cells :
starting population
Methodological approachOptimization of the CD40 and the EBV systems:
ComingComing soonsoon!!
starting population
overall B cell repertoire
amount (high number of cells)
time period (< 2 months)
transferable process
reproducible method
replacement of the irradiated murine fibroblasts by inert particles
improvement of the preparation of EBV viral suspension
Generation of new products:a novel monoclonal antibody
a specific B cell growth booster reagent
color vial n° designation volume storage 1 support 1 freeze-dried -20°C2 mAb1 200µl -20°C3 mAb2 200µl -20°C
orange 4 EBV1 2ml -80°C5 support2 freeze-dried -20°C
green
Kit contents
5 support2 freeze-dried -20°C6 mAb3 200µl -20°C7 EBV2 2ml -80°C
60ml -20°C
yellow
booster reagent
½ culture supernatant
procedure
D20D9 D10-20
immortalized B cells clones could be obtained, starting from 107 PBMC
obtained within 2 weeks with a medium change twice a week
Dendritics process targets up to 20% of the starting B cell populationDendritics process targets up to 20% of the starting B cell population
APPLICATIONS & FIELDSAPPLICATIONS & FIELDS
Cancerology, Infectiology
B cell population representative of the overall B cell peripheral repertoirecan be used as biological source for:
B cell physiology
B-cell repertoire
References1- Banchereau J, Rousset F. Nature, 1991, Oct 17;353 (6345):678-92- Rosen A et Al, Nature 1977, May 5, 267 (5606):52-43- Steinitz et al, Nature 1977, Sep 20, 269 (5627):420-24-WO/2004/076677. 2004 Sept. 10. Lanzavecchia A, inventors5-Fraussen et al, J Autoimmun, 2010, Sep;35(2):130-4
Cancerology, Infectiology
Autoimmunity, Allergy
Hematology, Vaccinology
Genomic, transcriptomic and proteomic studies
For more informations:www.dendritics.net
