Isolation of cardiac myocytes from the adult newt, Notophthalmus viridescens. An electron microscopic and quantitative light microscopic analysis

Tate, John M.; McDonnell, Timothy J.; Oberpriller, John O.

doi:10.1016/0040-8166(87)90049-8

Cited by 7 publications

(4 citation statements)

References 29 publications

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“…Most isolated adult newt myocytes examined by transmission electron microscopy in this study and in a previous one (Tate et al, 1987) demonstrated a n ultrastructure very similar to that of adult amphibian ventricular myocytes in vivo (Lemanski, 1973;Oberpriller et al, 1981;McDonnell and Oberpriller, 1983a). However, some isolated myocytes demonstrated a loss of Z-band material and variable degrees of myofibrillar disorganization.…”

Section: Discussionsupporting

confidence: 70%

“…The method of enzymatic dissociation outlined in this study has permitted a high yield of adult newt ventricular myocytes. As in previously reported methods of dissociation of adult amphibian heart tissue (Tarr and Trank, 1976;Hume and Giles, 1981;Arrio-Dupont and DeNay, 1985;and Tate et al, 1987) the combination of trypsin and collagenase was found to separate viable myocytes successfully. The time required for complete liberation of myocytes from the ventricular tissue routinely was between 5 and 10 hours.…”

Section: Discussionsupporting

confidence: 69%

“…Ventricular myocytes from adult red-spotted newts, Notophthalmus viridescens, were enzymatically iso-lated according to previously published methods (Tate et al, 1987). Briefly, ventricles were removed from anesthetized animals under aseptic conditions and enzymatically separated for 8 to 10 hours at 25°C in a Ca + +and Mg + + -free amphibian wash medium (Freed and Mezger-Freed, 1970) containing 0.5% trypsin (Gibco, Grand Island, NY) and 625 Uiml of CLS I1 collagenase (Worthington, Freehold, NJ).…”

Section: Culture Methodsmentioning

confidence: 99%

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Primary cell culture and morphological characterization of ventricular myocytes from the adult newt, notophthalmus viridescens

Tate

Oberpriller

1989

Anat. Rec.

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Previous work has demonstrated that adult newt cardiac myocytes possess a proliferative ability in response to an experimentally induced injury, in vivo. This study describes an in vitro model in which the proliferative events of the adult cardiac myocyte may be studied. Ventricles were minced and then enzymatically dissociated in a Ca++- and MG++-free salt solution containing 0.5% trypsin and 625 U/ml of CLS II collagenase for 8 to 10 hours at 25 degrees C. Enzyme digests were preplated and then cultured on bovine corneal endothelial-derived basement membrane "carpets" in either serum-free or serum-supplemented modified Leibovitz's medium for up to 30 days. Light and transmission electron microscopic characterization demonstrated that a majority of the myocytes underwent an initial period of disorganization characterized by a "rounding up" of the cell and a loss of myofibrillar organization. Once the myocytes had attached to the culture substratum they began to spread out, underwent a reassembly of their contractile elements, resumed spontaneous contractions, and demonstrated ultrastructural evidence of protein synthesis. Mitosis was observed in several myocytes 8 to 15 days following isolation. In 15-day serum-supplemented and serum-free cultures, 6.5% +/- 0.9% and 8.1% +/- 1.4% of the myocytes were binucleated, respectively. These results demonstrate that adult newt ventricular myocytes can be successfully placed into primary culture and are capable of undergoing mitosis. This work may be considered as a foundation for future investigations which will focus on the mechanisms which control cardiac myocyte proliferation.

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Section: Discussionsupporting

confidence: 70%

Section: Discussionsupporting

confidence: 69%

Section: Culture Methodsmentioning

confidence: 99%

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Primary cell culture and morphological characterization of ventricular myocytes from the adult newt, notophthalmus viridescens

Tate

Oberpriller

1989

Anat. Rec.

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“…Difficulty in isolating high numbers of viable chondrocytes has been reported by other authors, with viable chondrocyte yields of just 19% of the total available in native cartilage, when using an optimised protocol 1 . We felt that one of the reasons could be the incubation with proteolytic enzymes at 37(C. It has been shown in cell isolation techniques for more friable tissues such as pancreas and heart that lowering the isolation temperature increases the yield and functional viability of the cells 12,13 . In this study we compare the chondrocyte yield and viability after enzymatic digestion both at 37(C and at 10(C lower temperature (27(C).…”

Section: Introductionmentioning

confidence: 99%

A low temperature method of isolating normal human articular chondrocytes

Hidvégi

Sales

Izadi

et al. 2006

Osteoarthritis and Cartilage

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From these set of experiments, the method that maximised cell yield without jeopardising cell viability proved to be a two stage process: pre-digestion step using trypsin for 15 min; followed by overnight digestion with a combination of two types of collagenase (types I and II) and at a lower temperature of 27 degrees C. This has resulted in an efficient and robust method of releasing chondrocytes from cartilage, without jeopardising the viability of these cells.

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Long-Term Organ Cultures of Newt Hearts

Piatkowski

Braun

2015

Methods in Molecular Biology

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Adult newts regenerate their hearts after injury by initiating proliferation of cardiac muscle and non-muscle cells. Mechanistic studies in vivo to analyze heart regeneration are challenging due to the long reproduction cycle of newts and the complexity of the genome. Culture of primary newt cells might offer alternative experimental approaches, but monolayers of newt cardiomyocytes and slice cultures of newt hearts show extensive morphological changes during cultivation. Hence, we developed a protocol to culture intact newt hearts in vitro, avoiding major morphological changes of explanted organs during a 5-week cultivation. The model provides improved accessibility and allows manipulation of cultured organs by small molecules and viral vectors. We found that dedifferentiation and S-phase entry of cardiomyocytes, which are hallmarks of cardiac regeneration in vivo, can be recapitulated in cultured hearts in vitro. We reason that long-term organ cultures of newts are a versatile tool for mechanistic studies on organ regeneration.

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Isolation of cardiac myocytes from the adult newt, Notophthalmus viridescens. An electron microscopic and quantitative light microscopic analysis

Cited by 7 publications

References 29 publications

Primary cell culture and morphological characterization of ventricular myocytes from the adult newt, notophthalmus viridescens

Primary cell culture and morphological characterization of ventricular myocytes from the adult newt, notophthalmus viridescens

A low temperature method of isolating normal human articular chondrocytes

Long-Term Organ Cultures of Newt Hearts

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