Partial differentiation of rat egg cylinders in serum-free and protein-free medium

Škreb, N.; Bulić, Floriana

doi:10.1016/0012-1606(87)90261-2

Cited by 8 publications

(24 citation statements)

References 11 publications

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“…Based on the previous research with the metabolically poor protein‐free medium and an embryotoxic substance,26, 27, 29, 32 our results with the 3 most extreme regimes discussed below corroborate the idea that in our model a simple simultaneous assessment of survival and overall growth endpoints can efficiently screen for the most deleterious conditions of the microenvironment.…”

Section: Discussionsupporting

confidence: 85%

Influence of hyperthermal regimes on experimental teratoma development in vitro

Bojanac

Rogosic

Sinčić

et al. 2018

Int J Experimental Path

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SummaryWe screened for the impact of hyperthermal regimes varying in the cumulative equivalent minutes at 43°C (CEM43°C) and media composition on tumour development using an original teratoma in vitro model. Rat embryos (three germ layers) were microsurgically isolated and cultivated at the air‐liquid interface. During a two week period, ectodermal, mesodermal and endodermal derivatives developed within trilaminar teratomas. Controls were grown at 37°C. Overall growth was measured, and teratoma survival and differentiation were histologically assessed. Cell proliferation was stereologically quantified by the volume density of Proliferating Cell Nuclear Antigen. Hyperthermia of 42°C, applied for 15 minutes after plating (CEM43°C 3.75 minutes), diminished cell proliferation (P ˂ .0001) and enhanced differentiation of both myotubes (P ˂ .01) and cylindrical epithelium (P ˂ .05). Hyperthermia of 43°C applied each day for 30 minutes during the first week (CEM43°C 210 minutes) impaired overall growth (P ˂ .01) and diminished cell proliferation (P ˂ .0001). Long‐term hyperthermia of 40.5°C applied for two weeks (CEM43°C 630 minutes) significantly impaired survival (P ˂ .005). Long‐term hyperthermia of 40.5°C applied from the second day when differentiation of tissues begins (CEM43°C 585 minutes) impaired survival (P ˂ .0001), overall growth (P ˂ .01) and cartilage differentiation (P ˂ .05). No teratomas survived extreme regimes: 43°C for 24 hours (CEM43°C 1440 minutes), hyperthermia in the scant serum‐free medium (CEM43°C 630 minutes) or treatment with an anti‐HSP70 antibody before long‐term hyperthermia 40.5°C from the second day (CEM43°C 585 minutes). This in vitro research provided novel insights into the impact of hyperthermia on the development of experimental teratomas from their undifferentiated sources and are thus of potential interest for future therapeutic strategies in corresponding in vivo models.

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Section: Discussionsupporting

confidence: 85%

Influence of hyperthermal regimes on experimental teratoma development in vitro

Bojanac

Rogosic

Sinčić

et al. 2018

Int J Experimental Path

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“…To monitor growth, ellipsoid explants major and minor diameters were measured by an ocular micrometer at the beginning of the culture period and four times at the time of the culture medium change (16,17). Measures were included in the formula for ellipse area (A = π × major diameter × minor diameter / 4).…”

Section: Methodsmentioning

confidence: 99%

“…When applied in serum-free conditions to the gastrulating rat embryo-proper (consisting of ectoderm, mesoderm, and endoderm), 5azaC impaired survival, growth, and differentiation (11) but in serum-supplemented conditions it promoted differentiation of muscle (12). In a culture of a younger, pre-gastrulating embryo (consisting of epiblast and hypoblast), it promoted differentiation of muscle, cartilage, blood islands, and neural tissue (13).…”

mentioning

confidence: 99%

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro

et al. 2013

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AimTo establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta.MethodsFischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagle's minimum essential medium (MEM) with 50% rat serum. 30 µmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed.ResultsAt four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P < 0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared.ConclusionOur findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents.

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“…In a variant of the above model, in a serum-free chemically defined culture medium, differentiation of the epidermis proceeded in the same way as in the serum-supplemented medium [Škreb and Bulić , 1987;Bulić -Jakuš et al, 1999]. In our serum-free model, even when a proteinfree medium was used, the epidermis differentiated in almost all explants, but only after a 2-week culture.…”

Section: Introductionmentioning

confidence: 94%

“…The degree of epidermal differentiation in serum-free and serum-supplemented media was investigated in a more precise manner than before by a classical histological analysis and for the first time at the ultrastructural level. Serum-free medium was used supplemented with various substances (holotransferrin, apotransferrin, insulin or Na 2 SeO 3 ) known to promote survival, growth and/or differentiation in this or other culture models [Barnes and Sato, 1980;Bulić -Jakuš et al, 1990;Škreb et al, 1993;Strahinić et al, 1996].…”

Section: Introductionmentioning

confidence: 99%

Chemically Defined Protein-Free in vitro Culture of Mammalian Embryo Does Not Restrict Its Developmental Potential for Differentiation of Skin Appendages

Bulić-Jakuš¹,

Strahinić-Belovari²,

Marić

et al. 2001

Cells Tissues Organs

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In a unique serum- and protein-free chemically defined in vitro culture model of postimplantation mammalian development the epidermis differentiates regularly, although the differentiation of other tissues is impaired due to the lack of the serum. The present study in that model was done to estimate more carefully the degree of epidermal differentiation in defined media supplemented with some growth- or differentiation-stimulating substances. The main objective was to discover by grafting in vivo to the richer environment whether simple protein-free culture conditions restrict an inherent embryonic potential for differentiation of skin appendages. Embryonic parts of E9.5 gastrulating Fischer rat embryos were cultivated for 2 weeks in the protein-free Eagle’s minimum essential medium supplemented with holotransferrin, apotransferrin, insulin and/or Na₂SeO₃ and in controls cultivated in protein-free medium or in serum-supplemented medium. In all experiments there was a high incidence of differentiation of the epidermis. A high level of epidermal differentiation was confirmed for the first time at the ultrastructural level. A well-differentiated cornified layer and cells connected with desmosomes containing keratohyaline masses and cytokeratin filaments were found. A strong immunohistochemical signal for the proliferating cell nuclear antigen was always detected in the basal layer of the epidermis showing that those cells were still able to proliferate. Finally, embryos precultivated for 1 or 2 weeks in the protein-free medium and media supplemented with apotransferrin or serum were grafted under the kidney capsule for an additional 2 weeks. It was discovered that even after spending 2 weeks in the simple protein-free medium in vitro, embryos retained their developmental potential for differentiation of skin appendages (hair and sebaceous glands).

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Partial differentiation of rat egg cylinders in serum-free and protein-free medium

Cited by 8 publications

References 11 publications

Influence of hyperthermal regimes on experimental teratoma development in vitro

Influence of hyperthermal regimes on experimental teratoma development in vitro

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro

Chemically Defined Protein-Free in vitro Culture of Mammalian Embryo Does Not Restrict Its Developmental Potential for Differentiation of Skin Appendages

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