Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen
ABSTRACT Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n=14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 microg/ml bovine pituitary extract, 5 microg/ml insulin, 10 ng/ml cholera toxin, and 50 microg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 microg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p<0.05, n=24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p<0.05, n=24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p<0.05, n=16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p<0.05, n=16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.
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- "Then, the semen sample was placed in a Styrofoam cooler with an ice pack in the bottom, and separated by a paper towel, for transport (~15 min) and subsequent preparation for processing in the laboratory. Somatic cells were isolated and cultured as described in Liu et al. (2009) and Nel-Themaat et al. (2008a; b) with minor modifications. Briefly, each semen sample was gently layered over a 3-layer gradient column (2.5 mL each of 20%, 50%, and 90% Percoll®) in a 15 mL conical tube and centrifuged at 400g for 20 min. "
ABSTRACT: Somatic cells in mammalian semen can be a potential source of nuclei for nuclear transfer to produce cloned animals. Somatic cells recovered from avian semen could also provide a source of cells for use in chimera formation or cloning of threatened and endangered birds. This type of assisted reproductive technology is especially important when a genetically unique animal has died and the only viable genetic material available is semen cryopreserved for artificial insemination and in vitro fertilization purposes. The usefulness of somatic cells obtained from fresh and frozen mammalian semen for nuclear transfer has already been evaluated, but still remains to be accomplished for avian semen. A non-invasive, or minimally invasive, technique to conserve avian genetic diversity via somatic cell collection from fresh avian semen, to our knowledge, has not been described. The present study investigated the use of fresh semen samples from domestic chickens (Gallus domesticus) (n = 7), i.e., white leghorn (n = 4) and silkie chickens (n = 3), as a source of somatic cells, specifically fibroblast-like cells and epithelial cells, for cytological analysis and somatic cell gene banking. Ultimately, somatic cell culture was successful for two of six selected samples (33.3%), with two of four white leghorn semen samples yielding cell cultures compared to 0 of 2 silkie chicken semen samples. Further research may include applying this technique to other avian species or address the effects of cryopreservation on the viability and DNA integrity of somatic cells derived in this manner.Avian biology research 12/2013; 6(4):255-260. DOI:10.3184/175815513X13801240386288 · 0.90 Impact Factor
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ABSTRACT: This study investigates the influence of protein concentration in bovine pituitary extract (BPE) on the production of cartilaginous components in chitosan/gelatin (CG) scaffolds. Bovine knee chondrocytes (BKCs) in genipin-crosslinked CG scaffolds were cultured over 28 days for assessing the constructs. Scanning electron micrographs revealed that the pore structure of CG scaffolds was appropriate for cartilage tissue engineering. Also, an addition of BPE into culture medium stimulated the growth of BKCs on the pore surfaces of CG scaffolds. An increase in the protein concentration in BPE enhanced the amounts of BKCs. glycosaminoglycans and collagen. At a constant protein concentration in BPE, a scaffold prepared with a high concentration of biomaterial gels also favored the proliferation of BKCs and secretion of extracellular matrices. The histological and immunochemical staining showed chondrogenesis in the culture of BKCs using CG scaffolds and BPE-containing medium. In addition, type II collagen was synthesized in the constructs, demonstrating the chondrocytic phenotype of proliferated BKCs. Hence, combining CG scaffolds with BPE can be effectual in the regeneration of neocartilage.Journal of the Taiwan Institute of Chemical Engineers 03/2010; DOI:10.1016/j.jtice.2009.08.011 · 3.00 Impact Factor