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Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs.

Schenk HC, Krampfl K, Baumgärtner W, Tipold A - BMC Vet. Res. (2012)

Bottom Line: Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation.Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany. henning.schenk@freenet.de

ABSTRACT

Background: The recognition of functional muscular disorders, (e.g. channelopathies like Myotonia) is rising in veterinary neurology. Morphologic (e.g. histology) and even genetic based studies in these diseases are not able to elucidate the functional pathomechanism. As there is a deficit of knowledge and skills considering this special task, the aim of the current pilot study was to develop a canine muscle cell culture system derived from muscle biopsies of healthy client-owned dogs, which allows sampling of the biopsies under working conditions in the daily veterinary practise.

Results: Muscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.

Conclusion: We have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

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Immunohistochemistry of cell pellets of myotube cultures. 3,3′ diaminobenzidintetrahydrochlorid (= DAB) stainings of canine myotubes, Primary antibody: mouse anti-Desmin, secondary antibody: goat-anti-mouse-biotinylated, A, B: (A 100-fold and B 400-fold mag.) primary culture prior to freezing, approximately 70% to 80% of the cells are positive for the muscle-specific intermediate filament desmin. C, D (C 100-fold and D 400-fold mag.) showing a cell culture pellet after freezing and recultivation. Estimated value of desmin positive cells is 40%. Structural integrity of myotubes is intact, but morphology has been lost due to pellet preparation. E: ascites of non-immunised BALB/cJ mice in a dilution of 1:1000 as negative control (100-fold mag.). F: normal canine skeletal muscle tissue as positive control, few vimentin-expressing cells (100-fold mag.). Bar= 100 μm. mag. = magnification.
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Figure 3: Immunohistochemistry of cell pellets of myotube cultures. 3,3′ diaminobenzidintetrahydrochlorid (= DAB) stainings of canine myotubes, Primary antibody: mouse anti-Desmin, secondary antibody: goat-anti-mouse-biotinylated, A, B: (A 100-fold and B 400-fold mag.) primary culture prior to freezing, approximately 70% to 80% of the cells are positive for the muscle-specific intermediate filament desmin. C, D (C 100-fold and D 400-fold mag.) showing a cell culture pellet after freezing and recultivation. Estimated value of desmin positive cells is 40%. Structural integrity of myotubes is intact, but morphology has been lost due to pellet preparation. E: ascites of non-immunised BALB/cJ mice in a dilution of 1:1000 as negative control (100-fold mag.). F: normal canine skeletal muscle tissue as positive control, few vimentin-expressing cells (100-fold mag.). Bar= 100 μm. mag. = magnification.

Mentions: Biopsies were taken during standard surgical procedures from different striated muscle of 16 dogs, stored for one to three days at 4°C in a special transport medium in order to simulate shipping conditions and were further processed (see Table 1). The differential centrifugation steps used to enrich the satellite cells and separate fibroblasts of the currently used method seemed to be the crucial processing steps. Data regarding “id” of the donor, origin of biopsy, age and breed of the animal and further processing of the cultures after the proliferation period of myoblasts are summarised in Table 1. The typical morphology of the muscle cell culture during proliferation and differentiation is demonstrated in Figure 1. Spontaneously occurring contractions of the matured myotubes were documented in video files (Additional file 1: “myotube1.mpg”). These contractions were observed incidentally and no influences on the contraction rate, of age, or breed of donor or duration of storage before processing could be recognized. The initial time necessary for sufficient proliferation varied depending on the age of the biopsy donor. Samples from older dogs (id: 1, 8, 9, 10, 13) needed a longer time period for initiating proliferation (8 to 14 days), whereas samples from younger dogs started to proliferate 2 or 4 days after harvesting. In 6 cases immunofluorescence or immunohistology using a monoclonal antibody directed against the muscle-specific intermediate filament desmin was performed to identify the myotubes (Figures 2 and 3). However, cell morphology was lost due to artifacts obtained during the preparation procedures (e.g. scraping of cells and centrifugation). Therefore, the morphology of the immunohistochemically marked cells (Figure 3) differs in morphology compared to cells labelled by immunofluorescence (Figure 2).


Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs.

Schenk HC, Krampfl K, Baumgärtner W, Tipold A - BMC Vet. Res. (2012)

Immunohistochemistry of cell pellets of myotube cultures. 3,3′ diaminobenzidintetrahydrochlorid (= DAB) stainings of canine myotubes, Primary antibody: mouse anti-Desmin, secondary antibody: goat-anti-mouse-biotinylated, A, B: (A 100-fold and B 400-fold mag.) primary culture prior to freezing, approximately 70% to 80% of the cells are positive for the muscle-specific intermediate filament desmin. C, D (C 100-fold and D 400-fold mag.) showing a cell culture pellet after freezing and recultivation. Estimated value of desmin positive cells is 40%. Structural integrity of myotubes is intact, but morphology has been lost due to pellet preparation. E: ascites of non-immunised BALB/cJ mice in a dilution of 1:1000 as negative control (100-fold mag.). F: normal canine skeletal muscle tissue as positive control, few vimentin-expressing cells (100-fold mag.). Bar= 100 μm. mag. = magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539935&req=5

Figure 3: Immunohistochemistry of cell pellets of myotube cultures. 3,3′ diaminobenzidintetrahydrochlorid (= DAB) stainings of canine myotubes, Primary antibody: mouse anti-Desmin, secondary antibody: goat-anti-mouse-biotinylated, A, B: (A 100-fold and B 400-fold mag.) primary culture prior to freezing, approximately 70% to 80% of the cells are positive for the muscle-specific intermediate filament desmin. C, D (C 100-fold and D 400-fold mag.) showing a cell culture pellet after freezing and recultivation. Estimated value of desmin positive cells is 40%. Structural integrity of myotubes is intact, but morphology has been lost due to pellet preparation. E: ascites of non-immunised BALB/cJ mice in a dilution of 1:1000 as negative control (100-fold mag.). F: normal canine skeletal muscle tissue as positive control, few vimentin-expressing cells (100-fold mag.). Bar= 100 μm. mag. = magnification.
Mentions: Biopsies were taken during standard surgical procedures from different striated muscle of 16 dogs, stored for one to three days at 4°C in a special transport medium in order to simulate shipping conditions and were further processed (see Table 1). The differential centrifugation steps used to enrich the satellite cells and separate fibroblasts of the currently used method seemed to be the crucial processing steps. Data regarding “id” of the donor, origin of biopsy, age and breed of the animal and further processing of the cultures after the proliferation period of myoblasts are summarised in Table 1. The typical morphology of the muscle cell culture during proliferation and differentiation is demonstrated in Figure 1. Spontaneously occurring contractions of the matured myotubes were documented in video files (Additional file 1: “myotube1.mpg”). These contractions were observed incidentally and no influences on the contraction rate, of age, or breed of donor or duration of storage before processing could be recognized. The initial time necessary for sufficient proliferation varied depending on the age of the biopsy donor. Samples from older dogs (id: 1, 8, 9, 10, 13) needed a longer time period for initiating proliferation (8 to 14 days), whereas samples from younger dogs started to proliferate 2 or 4 days after harvesting. In 6 cases immunofluorescence or immunohistology using a monoclonal antibody directed against the muscle-specific intermediate filament desmin was performed to identify the myotubes (Figures 2 and 3). However, cell morphology was lost due to artifacts obtained during the preparation procedures (e.g. scraping of cells and centrifugation). Therefore, the morphology of the immunohistochemically marked cells (Figure 3) differs in morphology compared to cells labelled by immunofluorescence (Figure 2).

Bottom Line: Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation.Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany. henning.schenk@freenet.de

ABSTRACT

Background: The recognition of functional muscular disorders, (e.g. channelopathies like Myotonia) is rising in veterinary neurology. Morphologic (e.g. histology) and even genetic based studies in these diseases are not able to elucidate the functional pathomechanism. As there is a deficit of knowledge and skills considering this special task, the aim of the current pilot study was to develop a canine muscle cell culture system derived from muscle biopsies of healthy client-owned dogs, which allows sampling of the biopsies under working conditions in the daily veterinary practise.

Results: Muscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.

Conclusion: We have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

Show MeSH
Related in: MedlinePlus