mIMCD-3 cell line細胞株
Organism  Mus musculus, transgenic, mouse, transgenicTissue  kidney, medulla/collecting ductCell Type  SV40 transformedProduct Format  frozenMorphology  epithelialCulture Properties  adherentBiosafety Level  2[Cells contain Papovavirus]Derivation  mIMCD-3 is an inner medullary collecting duct (IMCD) cell line derived in 1991 by Michael Rauchman from a mouse transgenic for the early region of SV40 [Tg(SV40E)bri/7].A tubule from the terminal one-third of the IMCD was microdissected and placed in culture.Confluent cells were subcultured and cloned using cloning cylinders.Comments  This is a polarized epithelial cell line which retains many differentiated characteristics of the terminal IMCD including inhibition of apical to basal sodium flux by amiloride and by atrial natriuretic peptide (ANP).The cells possess an amiloride sensitive sodium channel as determined by Western blot analysis, and accumulate the major organic osmolytes (inositol, sorbitol, betaine and glycerophosphorylcholine) in response to hypertonic stress.The cells secrete endothelin and form tubules and tight junctions.mIMCD-3 cells are responsive to Hepatocyte Growth Factor (HGF), and are readily adaptable to growth in hypertonic medium supplemented with NaCl and urea up to 910 mosmol/kg H20. These extreme osmotic conditions exist in the renal medulla in vivo, but are known to be lethal to most other cells.Complete Growth Medium  The base medium for this cell line is ATCC-formulated DMEM:F12 Medium Catalog No. 30-2006. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.Subculturing  Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.    Remove and discard culture medium.    Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.    Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.    Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.    Add appropriate aliquots of the cell suspension to new culture vessels.    Incubate cultures at 37°CSubculture Ratio: 1:4 to 1:8Medium Renewal: 2 to 3 times a week.Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.Cryopreservation  Freeze medium: Complete growth medium, 95%; DMSO, 5%Storage temperature: liquid nitrogen vapor temperatureCulture Conditions  Temperature: 37°CAtmosphere: air, 95%; carbon dioxide (CO2), 5%Year of Origin  1991References  Rauchman MI, et al. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am. J. Physiol. 265: F416-F424, 1993. PubMed: 8214101
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