Magnetomitotransfer: An efficient way for direct mitochondria transfer into cultured human cells.

In the course of mitochondrial diseases standard care mostly focuses on treatment of symptoms, while therapeutic approaches aimed at restoring mitochondrial function are currently still in development. The transfer of healthy or modified mitochondria into host cells would open up the possibilities of new cell therapies. Therefore, in this study, a novel method of mitochondrial transfer is proposed by anti-TOM22 magnetic bead-labeled mitochondria with the assistance of a magnetic plate. In comparison to the passive transfer method, the magnetomitotransfer method was more efficient at transferring mitochondria into cells (78-92% vs 0-17% over 3 days). This transfer was also more rapid, with a high ratio of magnetomitotransferred cells and high density of transferred mitochondria within the first day of culture. Importantly, transferred mitochondria appeared to be functional as they strongly enhanced respiration in magnetomitotransferred cells. The novel method of magnetomitotransfer may offer potential for therapeutic approaches for treatment of a variety of mitochondria-associated pathologies, e.g. various neurodegenerative diseases.

Decellularized ovine esophageal mucosa for esophageal tissue engineering.

BACKGROUND: The success of a tissue engineered construct is greatly influenced by the choice of scaffold material. Decellularized esophageal matrix is a promising material for esophageal tissue engineering. The aim of this study was to develop a decellularized ovine esophageal mucosa and to investigate the effect of decellularization on the appearance of the resulting tissue. METHODS: Ovine esophagi were decellularized by combination of agitation, enzymatic digestion and treatment with 1 and 5% sodium dodecyl sulphate (SDS). Efficiency of decellularization was assessed by histological examination and DNA quantification. Finally, the effect of decellularization on tissue morphology was investigated by scanning electron microscopy. RESULTS: Decellularization resulted in a highly efficient removal of cells and DNA content. Electron microscopic investigation revealed a denuded, rough, undulating surface with preserved papillary structures. Individual papillae had a length of approximately 55 µm and were present at a density of 1332 µm/mm(2). Closer examination revealed a tightly packed sponge-like appearance with pores in the region of 300 nm. Numerous, intact collagen fibres were also visible. CONCLUSION: Ovine esophagus can be successfully decellularized through treatment with SDS. The resulting decellularized mucosal surface possesses a rough, 2D surface with a well preserved extracellular matrix. Such a material may be of advantage in tissue engineering of the esophagus. Characterisation of the papillary layer gives important insight into the suitability of decellularized esophageal mucosa for use in esophageal reconstruction and also provides morphological information which may help in the design of synthetic, biomimetic materials.

Effects of sodium hydroxide exposure on esophageal epithelial cells in an in vitro ovine model: implications for esophagus tissue engineering.

BACKGROUND: Esophagus tissue engineering holds promises for esophageal replacement after severe caustic injuries. The aim of this study was to determine whether viable esophageal epithelial cells could be isolated from an esophagus exposed to varying concentrations of alkali with regard to number, viability, and morphology during in vitro culture. METHODS: Ovine esophagi were exposed to phosphate-buffered saline 2.5%, 15%, or 25% sodium hydroxide (NaOH). The effect of NaOH concentrations on epithelial damage was assessed histologically. Esophageal epithelial cells were then isolated, and cell count and viability were investigated. Finally, cell number, viability, and morphology of esophageal epithelial cells were determined for 24 days of in vitro culture. RESULTS: Histologic analysis showed a progressive destruction of the epithelium proportional to increasing NaOH concentrations. Esophagi treated with phosphate-buffered saline and 2.5% NaOH showed significantly higher viable cell counts after isolation and culture in comparison with those treated with 15% to 5% NaOH. CONCLUSION: The evidence presented in this study indicates that epithelial biopsies from an esophagus exposed to low concentrations (2.5%) of NaOH will still yield large numbers of viable cells suitable for tissue engineering applications. In cases of exposure to higher concentrations (15%-25%), alternative cell sources for epithelial regeneration, such as stem cells, will be necessary for tissue engineering applications.

In vitro effect of bethanechol and suberyldicholine on regions of guinea pig esophagus.

BACKGROUND: Tissue engineering and regenerative medicine is envisaged as the future option for esophageal replacement; however, engineering of a functional esophagus is impeded by the limited understanding of the anatomical complexity of this dynamic muscular organ. The aim of this study was to characterize the function of native esophageal tissue and determine differences in functional response to stimulation between anatomical sites. MATERIALS AND METHODS: The in-vitro response of guinea pig esophageal preparations, from various anatomical sites, to muscle agonists was investigated. Esophageal strips were exposed to bethanechol, an agonist of muscarinic receptors located on smooth muscle, and suberyldicholine, an agonist of nicotinic receptors located on striated muscle, within a Schuler organ bath, to determine the contractile response on the various segments of the esophagus. RESULTS: The esophagus responded in a reliable and consistent manner to agonist stimulation. Bethanechol dose response curves were constructed with doses of 10 to 300 µM. The average maximal contractions to bethanechol exposure were 4.51, 4.80, 5.55, and 9.15 mN for upper, upper middle, lower middle, and lower esophageal segments, respectively. Responses to singular stimulation with 30 µM suberyldicholine in the presence of tetrodotoxin (100 µM) gave average contractions of 1.07, 0.84, 2.60, and 3.02 mN for upper, upper middle, lower middle, and lower esophageal segments, respectively. Bethanechol and suberyldicholine-induced responses were greater in the lower esophagus in comparison to the upper esophageal segments. CONCLUSION: These findings pave the way for the use of an in-vitro bethanechol and suberyldicholine-induced contraction model for future assessment of engineered esophageal tissue.