cCDCs were cultured in IMDM with 20% FBS media and passaged every 3–5 days. Canine CSps were then collected from the low-attachment flasks and replated onto fibronectin-coated surface to produce adherent cCDCs. Phase-bright canine cardiospheres (CSps) started to form in 3–7 days. The cells were then seeded into an ultra-low-attachment flask (Corning) at a density of 100,000 cells/cm 2 and cultured in IMDM with 10% FBS. of incubation with TrypLE Select™ (Life Technologies). Once these outgrowth cells are about 70–80% confluent, they were harvested by 5–10 min. In about one week, cells started to outgrow from the tissue explants. The cultures were maintained in 25–30 ml IMDM with 20% FBS, and media change was performed every other day. Approximately 50 pieces of tissue explants were then placed onto a fibronectin-coated plate with approximately 1.5 cm between each explant and covered with 2 ml of IMDM with 20% FBS overnight to aid the attachment. After that, the tissue samples were further minced into smaller tissue explants (~ 0.5 × 0.5 mm) before plating.
Iscove's modified Dulbecco's medium (IMDM Life Technologies) containing 20% foetal bovine serum (FBS Corning, Corning, NY, USA) is then added to the sample to inactivate the collagenase. The tissue sample was then cut into smaller biopsy-sized pieces and washed three times with PBS, followed by enzymatic digestion at 37☌ in 5 mg/ml collagenase IV solution (Sigma-Aldrich, St.
An approximately 6 × 6 mm piece of myocardial tissue was separated and washed with phosphate-buffered saline (PBS) (Life Technologies, Carlsbad, CA., USA). Briefly, this tissue was surgically collected from the right ventricle of a 2-year-old canine beagle. Materials and methods Derivation and culture of cCDCsĬanine CDCs were generated and expanded as described 3, 12 from myocardial specimens of a healthy male beagle dog 8, 13. The regenerative potential of CDCs has also been demonstrated in non-ischaemic cardiomyopathy 10, 11, and thus, we hypothesize intracoronary CDC therapy can provide a safe and effective intervention for DCM in addition, successful implementation of this intervention in a canine model of DCM will provide valuable insight into our ability to apply this technique to treat DCM in human beings.
#FLOWJO CARRBORO NC TRIAL#
A recent clinical trial indicates that CDC therapy benefits patients with mild-to-moderate myocardial infarction (MI) 9. Over the last 7 years, our laboratory has been studying CDCs as a source to generate therapeutic cardiac progenitor cells for ischaemic heart diseases. Dog and human DCMs share significant clinical similarities including but not limited to ascites, rhythm disruption, dyspnoea, syncope and sudden death 5- 7, which inspired us to ask the following: can we use Doberman pinschers with DCM as a spontaneously occurring, clinically relevant large animal model of cardiomyopathy from which to translate cell-based therapies for humans? The safety of adult stem cell therapy for heart diseases has been well established in humans 8. For instance, in Doberman pinschers, the DCM appears to represent a major cause of death 4. Interestingly, naturally occurring cardiomyopathy also affects the well-being of domestic dogs. One dilemma is that many naturally occurring cardiomyopathies in humans cannot be modelled effectively in the laboratory. However, translation to human trials necessitates large animal studies ( e.g. Small animal (rodent) studies are widely adopted for initial proof of concept 3. Stem cell transplantation is a promising therapeutic strategy for acute or chronic cardiomyopathy, considering current treatments for human DCM usually involve intense drug regimen and/or invasive implantable devices 2. Heart diseases remain the number one killer in western countries 1.
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