Hybrid biosensing cellulose‐based scaffolds for imaging‐assisted tissue engineering

Abstract

3D tissue models closely mimic in vivo cell and tissue organization otherwise not seen in conventional cell culture. However, live cell imaging and quantification of cells growing within 3D scaffold are yet to be achieved for success of tissue engineering, cancer biology and regenerative medicine. Here, we aimed to design the environment‐sensitive scaffold material acting both as supporting material and a biosensor for cells growing in it. We chose cellulose‐binding domain of C. fimi CenA protein, termed as CBD (N‐terminal fragment, Ala32‐Thr137) and genetically fused it to either pH (enhanced cyan fluorescent protein, ECFP) or Ca2+ (gCaMP2)‐sensitive proteins. ECFP and gCaMP2 are prospective biosensors for quantitative Fluorescence Lifetime Imaging Microscopy (FLIM). Next, we optimised E. coli expression, purification and folding conditions for these chimeric proteins, achieving yields of ~6 mg/L culture, bright fluorescence (~55% folding rate) and sensitivity to extracellular pH and Ca2+.To assemble the functional biosensing scaffolds using CBD‐fusions, we performed labeling of the three different types of cellulose‐based materials: commercially available nanofibrillar cellulose Growdex™, bacterial cellulose spheres and decellularized plant tissues. Both CBD‐ECFP (pH‐sensitive) and CBD‐gCaMP2 (Ca2+‐sensitive) proteins efficiently labeled scaffolds in concentration‐dependent manner and remained stable under physiological conditions for > 7 days. In a proof‐of‐concept experiments, we tested usability of CBD‐ECFP‐stained Growdex scaffold as an extracellular pH sensor in FLIM: we observed robust and reversible changes in fluorescence lifetime (2.3–2.5 ns) over the range of physiological pH. Further, we tested the biosensing properties of CBD‐ECFP scaffolds, populated with cultured human colon cancer HCT116, mouse embryonic fibroblast (MEF) and primary stem cell‐derived mouse intestinal organoids. All the types of scaffolds were compatible with growth of cells with no evident toxicity, confirmed by experiments with unlabelled scaffolds.In conclusion, we have developed versatile labeling approach for producing biosensing cellulose‐based 3D scaffolds, suitable for different biosensors and types of cell and tissues, including single cells, spheroids and organoids. Such ‘hybrid’ biosensing scaffolds allow for real‐time measurement of extracellular biomolecules using quantitative FLIM microscopy, providing improvements in multi‐parametric imaging of engineered tissue models.Support or Funding InformationScience Foundation Ireland grant SFI 13/SIRG/2144.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.