Three-dimensional (3D) bioprinting technologies have great attention in different researching areas such as tissue engineering, medicine, etc. due to its maximum mimetic property of natural biomaterials by providing cell combination, growth factors, and other biomaterials. Bioprinting of tissues, organs, or drug delivery systems emerged layer-by-layer deposition of bioinks. 3D bioprinting technique has some complexity such as choice of bioink combination, cell type, growth, and differentiation. In this study, a composite material in 3D bioprinting studies has been developed for biofabrication of the cell carrying scaffolds namely cryogenic scaffolds. Cryogenic scaffolds are highly elastic and have a continuous interconnected macroporous structure in 3D biomaterials that enable the cell attachment, viability, and proliferation. Freeze-drying cryogelation process for the formation of cryogel scaffolds has been achieved firstly among 3D bioprinting studies. Cryogenic gelatin–hyaluronic acid (Gel–HA)-based 3D-bioprinted scaffolds have been fabricated and characterized by scanning electron microscope (SEM), optical microscope images, tensile tests, determination of swelling degree, and porosity. Then, L929 cells from mouse C3H/An have been attached to cryogenic Gel–HA scaffolds. Cell attachment, viability, and proliferation on cryogenic scaffolds have been investigated for 7 days. The results showed that a combination of 3D bioprinting technologies and cryogenic process provided a new direction on biomedical scaffolds. Graphical Abstract

Background: Enzymes are efficient biocatalysis that catalysis a large number of reactions due to their chemical, regional, or stereo specifities and selectivity. Their usage in bioreactor or biosensor systems has great importance. Carbonic anhydrase enzyme catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. In organisms, the carbonic anhydrase enzyme has crucial roles connected with pH and CO₂ homeostasis, respiration, and transport of CO₂/bicarbonate, etc. So, immobilization of the enzyme is important in stabilizing the catalyst against thermal and chemical denaturation in bioreactor systems when compared to the free enzyme that is unstable at high temperatures and extreme pH values, as well as in the presence of organic solvents or toxic reagents. Nano-scale composite materials have attracted considerable attention in recent years, and electrospinning based all-nanocomposite materials have a wide range of applications. In this study, electrospun nanofibers were fabricated and used for the supporting media for carbonic anhydrase enzyme immobilization to enhance the enzyme storage and usage facilities. Objective: In this article, our motivation is to obtain attractive electrospun support for carbonic anhydrase enzyme immobilization to enhance the enzyme reusability and storage ability in biocatalysis applications. Methods: In this article, we propose electrospun nanofibers for carbonic anhydrase carrying support for achieving our aforementioned object. In the first part of the study, agar with polyacrylonitrile (PAN) nanofibers was directly fabricated from an agar-PAN mixture solution using the electrospinning method, and fabricated nanofibers were cross-linked via glutaraldehyde (GA). The morphology, chemical structure, and stability of the electrospun nanofibers were characterized. In the second part of the study, the carbonic anhydrase enzyme was immobilized onto fabricated electrospun nanofibers. Then, enzyme activity, the parameters that affect enzyme immobilization such as pH, enzyme amount, immobilization time, etc. and reusability were investigated. Results: When the scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis results are combined in the characterization process of the synthesized electrospun nanofibers, the optimum cross-linking time is found to be 8 hours using 5% glutaraldehyde cross-linking agent. Then, thermal stability measurements showed that the thermal stability of electrospun nanofibers has an excellent characteristic for biomedical applications. The optimum temperature value was found 37°C, pH 8 was determined as an optimum pH, and 100 ppm carbonic anhydrase enzyme concentration was found to be optimum enzyme concentration for the carbonic anhydrase enzyme immobilization. According to the kinetic data, carbonic anhydrase immobilized electrospun nanofibers acted as a biocatalyst in the conversion of the substrate to the product in 83.98%, and immobilized carbonic anhydrase enzyme is reusable up to 9 cycles in biocatalysis applications. Conclusion: After applying the framework, we get a new biocatalysis application platform for carbonic anhydrase enzyme. Electrospun nanofibers were chosen as the support material for enzyme immobilization. By using this approach, the carbonic anhydrase enzyme could easily be used in the industrial area by cost-effective advantageous aspects.