Formulation of a complex serum-free medium (CSM) for use in the co-culture of mouse embryos with cells of the female reproductive tract

Co-culture of pre-implantation embryos with cells of the reproductive tract requires a medium that is beneficial to both embryos and cells. However, many studies in this area utilize media originally formulated for specific cell lines. In the present study, a complex serum-free medium (CSM) was formulated on the basis of the ionic compositions of existing embryo culture media and mouse oviductal fluid as well as the concentrations of growth factors that appear to benefit mouse embryo development. The study began by investigating the effect of altering the concentrations of K+ ions (0-40 mM) and sulfate ions (0-10 mM) in embryo culture media on the development of 2-cell mouse embryos. Mouse embryos showed improved cell numbers at the blastocyst stage when cultured in 10 mM K+ compared with Whittingham's T6 medium. Embryos were also cultured in T6 supplemented with bovine serum albumin (BSA) containing various concentrations of insulin, insulin-like growth factors I and II, fibroblast growth factor, and epidermal growth factor. Insulin concentrations of 100 ng mL-1 significantly (P less than 0.05) improved the cell numbers of 2-cell embryos cultured to the morulae and blastocyst stages compared with those cultured in T6 + BSA alone. CSM was formulated on the basis of the results of these experiments and was found to support both improved development of 2-cell mouse embryos and the culture of mouse fibroblast and mouse oviduct cells.

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Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

In Vitro Cellular & Developmental Biology ◽

10.1007/bf02620811 ◽

1988 ◽

Vol 24 (11) ◽

pp. 1099-1106 ◽

Cited By ~ 7

Author(s):

Terry L. Riss ◽

Kenneth P. Karey ◽

B. Daniel Burleigh ◽

David Farker ◽

David A. Sirbasku

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Mouse Embryo ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Factor I ◽

Mouse Embryo Fibroblasts ◽

Recombinant Insulin ◽

Human Recombinant Insulin

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Cell-cycle-specific induction of quiescence achieved by limited inhibition of protein synthesis: counteractive effect of addition of purified growth factors

Journal of Cell Science ◽

10.1242/jcs.73.1.375 ◽

1985 ◽

Vol 73 (1) ◽

pp. 375-387

Author(s):

O. Larsson ◽

A. Zetterberg ◽

W. Engstrom

Keyword(s):

Cell Cycle ◽

Protein Synthesis ◽

Growth Factors ◽

Growth Factor ◽

Serum Starvation ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

G1 Cells ◽

Synthesis And Degradation

We have previously shown that Swiss 3T3 cells located in the first part of G1 (post-mitotic G1 cells younger than 4.0 h or G1pm cells) were arrested after 9–10 h in the cell cycle by a short (1-8 h) exposure to serum-free medium or by a short (2-4 h) exposure to low doses of the protein synthesis inhibitor cycloheximide (CH). Kinetic data indicate that such G1pm cells rapidly return to G0 during this brief treatment and thereafter require a preparatory period of 8 h before continuing to G1. Cells older than 4 h, i.e. cells in mid or late G1 are already committed to DNA synthesis (presynthesis or G1ps cells). These cells as well as S and G2 cells were consequently unaffected by the brief serum starvation or the brief treatment with cycloheximide. In the present paper we show that the 10-h intermitotic delay that follows a 1–2 h exposure to serum-free medium can be completely counteracted by the presence of any one of the purified growth factors, epidermal growth factor (EGF), insulin or platelet-derived growth factor (PDGF). In contrast, the intermitotic delay following a longer exposure (8 h) to serum-free medium could no longer be counteracted by EGF or insulin. However, PDGF was still active in this respect. Most interestingly, the 12 h gross intermitotic delay induced by a 4h exposure to CH could be efficiently counteracted by EGF, PDGF or insulin. However, this effect on CH-treated cells could be counteracted by the growth factor only in the presence of 10% serum. This indicates the existence of a cooperative effect between PDGF, EGF or insulin and an unidentified serum factor. The effects on the cell cycle time of brief serum starvation and exposure to CH were compared with the effects on rate of protein synthesis and degradation. Although the effects of serum starvation on protein synthesis and degradation were found to be partially normalized by growth factors, we suggest that growth factors prevent cells from leaving the cell cycle by another mechanism and not merely by affecting the level of overall protein accumulation.

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Regulation of rat ovarian cell growth and steroid secretion

The Journal of Cell Biology ◽

10.1083/jcb.86.2.483 ◽

1980 ◽

Vol 86 (2) ◽

pp. 483-489 ◽

Cited By ~ 11

Author(s):

CC Johnson ◽

WE Dawson ◽

JT Turner ◽

JH Wyche

Keyword(s):

Growth Factors ◽

Cell Division ◽

Growth Factor ◽

Cell Population ◽

Serum Free Medium ◽

Free Medium ◽

Ovarian Cell ◽

Serum Free ◽

Cholesterol Levels ◽

Progesterone Secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.

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Megakaryocytes Cultured from Patients with IMF Produced Significantly Higher mRNA but Not Protein Levels of Fibrosing Grown Factors.

Blood ◽

10.1182/blood.v108.11.3607.3607 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3607-3607

Author(s):

Jen-Chin Wang ◽

Tsong H Chang ◽

Amit Goldberg ◽

Allan D. Novetsky ◽

Steven Lichter ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Growth Factor ◽

Mrna Levels ◽

Serum Free Medium ◽

Free Medium ◽

Myeloid Metaplasia ◽

Serum Free ◽

Protein Levels

Abstract Currently, the prevailing concept concerning the etiology of bone marrow fibrosis in patients with idiopathic myelofibrosis (IMF) is that it results from excessive production of fibrosing growth factors including transforming growth factor beta (TGF-B1), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) from megakaryocytes and monocytes. Since megakaryocytes are difficult to isolate from bone marrow in IMF patients, this concept remains speculative. We obtained megakaryocytes (CD41+ cells) from 10-day in vitro culture of blood CD34+ cells in serum-free medium with thrombopoietin and stem cell factors as described and cultured monocytes from isolating blood CD14+ cells. Then quantitative analyses of fibrosing growth factors at the mRNA and protein levels were obtained. mRNA levels were obtained from real-time RT-PCR technique, and protein levels were obtained from ELISA analysis of the supernatant of CD41+ cells cultured 4 h in serum-free medium. The results showed 1) mRNA levels of TGF-B1, PDGF, and FGF produced by the megakaryocytes were significantly elevated in agnogenic myeloid metaplasia (AMM) compared with those in normal controls (p<0.05). While these growth factors were elevated several-fold in AMM compared with other myeloproliferative disorders (MPD) including essential thrombocythemia and polycythemia vera, they were not statistically significant. 2) mRNA levels of TGF-B1 were higher than levels of PDGF or FGF. 3) The mRNA levels of these growth factors produced from CD14+ cells were not significantly elevated in AMM compared with other MPDs or controls; the AMM mRNA levels were significantly elevated only in some patients. 4) The correlation of mRNA levels of these growth factors with the degree of myelofibrosis in AMM was significant with megakaryocytes (r=0.73) but not with monocytes (r=0.23). 5) ELISA analysis of the growth factors from the cultured megakaryocytes showed that, in most of the patients with AMM and other MPDs and in volunteer controls, the growth factors were undetectable, and only a few patients with AMM had significantly elevated protein levels of these growth factors. We conclude thatin IMF, megakaryocytes but not monocytes are the predominant cells producing fibrosing growth factors, andthe failure of finding increased protein levels of these growth factors in the in vitro system suggest that other factors are necessary to initiate translation of these growth factors in the megakaryoctes, and neutrophil emperipolesis with releasing factors may be important in this process.

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Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3604 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3604-3612 ◽

Cited By ~ 341

Author(s):

C Sell ◽

G Dumenil ◽

C Deveaud ◽

M Miura ◽

D Coppola ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Soft Agar ◽

T Antigen ◽

Insulin Like Growth Factor ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Factor I ◽

Growth Factor I

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.

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Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle

Development ◽

10.1242/dev.115.3.821 ◽

1992 ◽

Vol 115 (3) ◽

pp. 821-826 ◽

Cited By ~ 1

Author(s):

R.C. Larson ◽

G.G. Ignotz ◽

W.B. Currie

Keyword(s):

Cell Cycle ◽

Growth Factors ◽

Growth Factor ◽

Transforming Growth Factor ◽

Platelet Derived Growth Factor ◽

Bovine Embryos ◽

Serum Free Medium ◽

Free Medium ◽

Cell Stage ◽

Serum Free

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.

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