||Loosely Adherent With Floating Clusters
||Derived from a 7 month female Wistar-Furth rat. GH3 cells produce growth hormone faster than the GH1 cell line and also produce prolactin.
||This cell line is a suitable transfection host.
||prolactin; growth hormone (somatotrophin)
||The epithelial-like GH3 clone generates growth hormone at a greater rate than the GH1 cells and also produces prolactin.
Studies on the control of the production of these protein hormones by the GH3 cells have shown that hydrocortisone stimulates the production of growth hormone and inhibits prolactin production.
The cells have been adapted to growth in suspension culture using Eagle's minimum essential medium (spinner) supplemented with 15% horse serum and 2.5% FBS. Under these conditions the cells continue to produce both growth hormone and prolactin
||F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) contains 2 mM L-glutamine and 1500 mg/L sodium bicarbonate 15% horse serum and 2.5% fetal bovine serum.
||Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
Remove and discard culture medium. Sometimes many cells are floating, they can be harvested by centrifugation of medium instead of discarding it.
Add 2.0 to 3.0 mL of 0.25% (w/v) Trypsin- 0.53 mM-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
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.
||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).
||Bancroft FC, et al. Control of growth hormone production by a clonal strain of rat pituitary cells. Stimulation by hydrocortisone. J. Cell Biol. 43: 432-441, 1969. PubMed: 5389137
Tashjian AH Jr., et al. Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82: 342-352, 1968. PubMed: 4951281
Tashjian AH Jr., et al. Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. J. Cell Biol. 47: 61-70, 1970. PubMed: 5513559
Bancroft FC, Tashjian AH Jr.. Growth in suspension culture of rat pituitary cells which produce growth hormone and prolactin. Exp. Cell Res. 64: 125-128, 1971. PubMed: 5541958
Bancroft FC, Tashjian AH Jr.. Control of the production of two protein hormones by rat pituitary cells in culture. In Vitro 6: 180-189, 1970. PubMed: 5535574
Wood WM, et al. Thyroid hormone receptor beta2 promoter activity in pituitary cells is regulated by Pit-1. J. Biol. Chem. 271: 24213-24220, 1996. PubMed: 8798664
||Guilherme Kurtz, Universidade Federal do Rio de Janeiro