15P-1

The 15P-1 cell line was established from testicular cells of transgenic mice that express the large T protein of polyoma virus (PyLT) in the seminiferous epithelium. RefPaquis-Flucklinger V, et al. Expression in transgenic mice of the large T antigen of polyomavirus induces Sertoli cell tumours and allows the establishment of differentiated cell lines. Oncogene 8: 2087-2094, 1993. PubMed: 8393161

This line exhibits characteristics of Sertoli cells, including transcription of the Wilms' tumor (WT1) and Steel genes. RefRassoulzadegan M, et al. Transmeiotic differentiation of male germ cells in culture. Cell 75: 997-1006, 1993. PubMed: 7504588

15P-1 cultures express phagocytic activity evidenced by the internalization of latex beads and the disposal of dead cells. Interaction with germ cells enhances the phagocytic activity of 15P-1 cells. RefGrandjean V, et al. Stage-specific signals in germ line differentiation: control of Sertoli cell phagocytic activity by spermatogenic cells. Dev. Biol. 184: 165-174, 1997. PubMed: 9142992

The cells support the meiotic and postmeiotic differentiation in cocultures of diploid premeiotic germ cells into haploid spermatids expressing the protamine (Prm-1) gene. RefRassoulzadegan M, et al. Transmeiotic differentiation of male germ cells in culture. Cell 75: 997-1006, 1993. PubMed: 7504588

When cocultured with 15P-1 cells, testicular cells explanted from immature 9-day-old animals, before the onset of the first meiosis, generate tetrads of haploid cells with the morphology of round spermatids and initiate protamine transcription. RefRassoulzadegan M, et al. Transmeiotic differentiation of male germ cells in culture. Cell 75: 997-1006, 1993. PubMed: 7504588

The 15P-1 cell line releases protease-sensitive material with broad-spectrum antibacterial activity. The activity of 15P-1 culture medium is increased 10-fold to 50-fold in the presence of fractions enriched in round spermatids and of nerve growth factor. RefGrandjean V, et al. Antimicrobial protection of the mouse testis: synthesis of defensins of the cryptdin family. Biol. Reprod. 57: 1115-1122, 1997. PubMed: 9369178

The KL protein, a ligand of the Kit receptor, is expressed in Sertoli cells and in 15P-1 cells. RefVincent S, et al. Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis. Development 125: 4585-4593, 1998. PubMed: 9778516

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability. 

1. Thaw the vial by gentle agitation in a 32°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2.  
3. 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.
4. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete growth medium and spin at approximately 125 x g for 5 to7 minutes.
5. Resuspend cell pellet with the recommended complete growth medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm² or a 75 cm² culture flask.  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 complete 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).
6. Incubate the culture at 32°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-053mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. 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 32°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 32°C.

Subcultivation Ratio: 1:3 to 1:4
Medium Renewal: Every 2 to 3 days

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.

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