||Retinal Pigmented Epithelium; Retina
||19 Year /
||ARPE-19 is a spontaneously arising retinal pigment epithelia (RPE) cell line derived from the normal eyes of a 19-year-old male who died from head trauma in a motor vehicle accident.
||This cell line is a suitable transfection host.
||These cells form stable monolayers, which exhibit morphological and functional polarity. ARPE-19 expresses the RPE-specific markers CRALBP and RPE-65.
The cells exhibit morphological polarization when plated on laminin-coated Transwell-COL filters in medium with a low serum concentration.
They form tight-junctions with transepithelial resistance of monolayers reaching a maximum of 50 to 100 ohms/cm2 after 4 weeks of culture.
||1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 2.5 mM L-glutamine, 0.5 mM sodium pyruvate and fetal bovine serum to a final concentration of 10%.
||Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. T-75 flasks are recommended for subculturing this product.
Remove and discard culture medium.
Briefly rinse the cell layer with PBS without calcium and magnesium 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°C.
NOTE: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 6th edition, published by Alan R. Liss, N.Y., 2010.
|Subculturing Medium Renewal
||Every 2 to 3 days
|Subculturing Subcultivation Ratio
||1:3 to 1:5
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells
||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that
some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes.
4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio).
5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line).
NOTE: It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
||Dunn KC, et al. ARPE-19, A human retinal pigment epithelial cell line with differentiated properties. Exp. Eye Res. 62: 155-169, 1996. PubMed: 8698076
Maidji E, et al. Accessory human cytomegalovirus glycoprotein US9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells. J. Virol. 70: 8402-8410, 1996. PubMed: 8970961
Holtkamp GM, et al. Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells. Clin. Exp. Immunol. 112: 34-43, 1998. PubMed: 9566787
Finnemann SC, et al. Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization. Proc. Natl. Acad. Sci. USA 94: 12932-12937, 1997. PubMed: 9371778
Handa JT, et al. The advanced glycation endproduct pentosidine induces the expression of PDGF-B in human retinal pigment epithelial cells. Exp. Eye Res. 66: 411-419, 1998. PubMed: 9593635
Dunn KC, et al. Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5.. Invest. Ophthalmol. Vis. Sci. 39: 2744-2749, 1998. PubMed: 9856785
Tugizov S, et al. An acidic cluster in the cytosolic domain of human cytomegalovirus glycoprotein B is a signal for endocytosis from the plasma membrane. J. Virol. 73: 8677-8688, 1999. PubMed: 10482621
Orten DJ, et al. Analysis of DNA elements that modulate myosin VIIA expression in humans. Hum. Mutat. 14: 354, 1999. PubMed: 10502787
Rajan PD, et al. Expression of the extraneuronal monoamine transporter in RPE and neural retina. Curr. Eye Res. 20: 195-204, 2000. PubMed: 10694895
Janssen JJ, et al. Retinoic acid delays transcription of human retinal pigment neuroepithelium marker genes in ARPE-19 cells. Neuroreport 11: 1571-1579, 2000. PubMed: 10841379
Udono T, et al. Adrenomedullin in cultured human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 41: 1962-1970, 2000. PubMed: 10845623
||Kátia da Silva Calabrese Instituto Oswaldo Cruz - Fundação Oswaldo Cruz.