Cellulose-based scaffolds for fluorescence lifetime imaging-assisted tissue engineering.

Quantitative measurement of pH and metabolite gradients by microscopy is one of the challenges in the production of scaffold-grown organoids and multicellular aggregates. Herein, we used the cellulose-binding domain (CBD) of the Cellulomonas fimi CenA protein for designing biosensor scaffolds that allow measurement of pH and Ca2+ gradients by fluorescence intensity and lifetime imaging (FLIM) detection modes. By fusing CBD with pH-sensitive enhanced cyan fluorescent protein (CBD-ECFP), we achieved efficient labeling of cellulose-based scaffolds based on nanofibrillar, bacterial cellulose, and decellularized plant materials. CBD-ECFP bound to the cellulose matrices demonstrated pH sensitivity comparable to untagged ECFP (1.9-2.3 ns for pH 6-8), thus making it compatible with FLIM-based analysis of extracellular pH. By using 3D culture of human colon cancer cells (HCT116) and adult stem cell-derived mouse intestinal organoids, we evaluated the utility of the produced biosensor scaffold. CBD-ECFP was sensitive to increases in extracellular acidification: the results showed a decline in 0.2-0.4 pH units in response to membrane depolarization by the protonophore FCCP. With the intestinal organoid model, we demonstrated multiparametric imaging by combining extracellular acidification (FLIM) with phosphorescent probe-based monitoring of cell oxygenation. The described labeling strategy allows for the design of extracellular pH-sensitive scaffolds for multiparametric FLIM assays and their use in engineered live cancer and stem cell-derived tissues. Collectively, this research can help in achieving the controlled biofabrication of 3D tissue models with known metabolic characteristics. STATEMENT OF SIGNIFICANCE: We designed biosensors consisting of a cellulose-binding domain (CBD) and pH- and Ca2+-sensitive fluorescent proteins. CBD-tagged biosensors efficiently label various types of cellulose matrices including nanofibrillar cellulose and decellularized plant materials. Hybrid biosensing cellulose scaffolds designed in this study were successfully tested by multiparameter FLIM microscopy in 3D cultures of cancer cells and mouse intestinal organoids.

Three-Dimensional Tissue Models and Available Probes for Multi-Parametric Live Cell Microscopy: A Brief Overview.

In recent years, the advances in tissue engineering and regenerative medicine have resulted in introduction of novel 3D tissue models, materials and methods to the regular practice of cell biologists, material scientists and specialists from related areas. 3D tissue models allow mimicking in vivo cell and tissue organization. However, the efficient work in three dimensions has significant challenges, such as compatibility with conventional cell biology methods, live cell imaging and quantification readouts. Here, we briefly discuss the applicability of 3D tissue models to different live cell microscopy modalities and the available range of fluo- and phosphorescent probes and sensors allowing for multi-parametric imaging.

Metallochelate Coupling of Phosphorescent Pt-Porphyrins to Peptides, Proteins, and Self-Assembling Protein Nanoparticles.

Specific and reversible metallochelate coupling via nitrilotriacetate (NTA) moiety is widely used for immobilization, purification, and labeling of oligo(histidine)-tagged proteins. Here, we evaluated this strategy to label various peptides and proteins with phosphorescent Pt-porphyrin derivatives bearing NTA group(s). Zn(2+) complexes were shown to have minimal effect on the photophysics of the porphyrin moiety, allowing quenched-phosphorescence sensing of O2. We complexed the PtTFPP-NTA conjugate with His-containing peptide that can facilitate intracellular loading, and observed efficient accumulation and phosphorescent staining of MEF cells. The more hydrophilic PtCP-NTA conjugate was also seen to form stable complexes with larger polypeptide constructs based on fluorescent proteins, and with subunits of protein nanoparticles, which retained their ability to self-assemble. Testing in phosphorescence lifetime based O2 sensing assays on a fluorescence reader and PLIM microscope revealed that phosphorescent metallochelate complexes perform similarly to the existing O2 probes. Thus, metallochelate coupling allows simple preparation of different types of biomaterials labeled with phosphorescent Pt-porphyrins.

Cost-effective therapeutic hypothermia treatment device for hypoxic ischemic encephalopathy.

Despite recent advances in neonatal care and monitoring, asphyxia globally accounts for 23% of the 4 million annual deaths of newborns, and leads to hypoxic-ischemic encephalopathy (HIE). Occurring in five of 1000 live-born infants globally and even more in developing countries, HIE is a serious problem that causes death in 25%-50% of affected neonates and neurological disability to at least 25% of survivors. In order to prevent the damage caused by HIE, our invention provides an effective whole-body cooling of the neonates by utilizing evaporation and an endothermic reaction. Our device is composed of basic electronics, clay pots, sand, and urea-based instant cold pack powder. A larger clay pot, lined with nearly 5 cm of sand, contains a smaller pot, where the neonate will be placed for therapeutic treatment. When the sand is mixed with instant cold pack urea powder and wetted with water, the device can extract heat from inside to outside and maintain the inner pot at 17°C for more than 24 hours with monitoring by LED lights and thermistors. Using a piglet model, we confirmed that our device fits the specific parameters of therapeutic hypothermia, lowering the body temperature to 33.5°C with a 1°C margin of error. After the therapeutic hypothermia treatment, warming is regulated by adjusting the amount of water added and the location of baby inside the device. Our invention uniquely limits the amount of electricity required to power and operate the device compared with current expensive and high-tech devices available in the United States. Our device costs a maximum of 40 dollars and is simple enough to be used in neonatal intensive care units in developing countries.