(367g) Bioprinting with Chitosan-Gelatin Thermo-Sensitive Hydrogels

Authors: 
Singarapu, K., Oklahoma State University
Madihally, S. V., Oklahoma State University

Conventional cell-seeding methods are inadequate in the development of in vitro tissues, because they involve random placement of cells and therefore lack the precision necessary for spatial control.  Bioprinting is a novel technology, which can be used to pattern scaffolding biomaterials and cells with high precision to build 3D structures in vitro.  However, the limitation of this approach is providing a conducive environment during and after the printing process.  Some have used various biomaterials that change from solution phase to hydrogel phase with the initiation of chemical reactions by photonic energy.  We evaluated the possibility of using thermo-sensitive chitosan-gelatin hydrogels formed at physiological conditions.  Hydrogels were prepared with chitosan-gelatin along with Transglutaminase and DMEM powder in presence of 2-glycerol phosphate in sterile condition.  We conducted a parametric study of printing speed and needle length to analyze and quantify cell viability.  Cell viability can be controlled by varying the parameters such as printing speed and needle length.  Cell viability of three different cell types was evaluated at day 2 and day 5.  This study gave percentage of live, apoptotic, and necrotic cells to the process parameters.  The increase in percentage of injured cells at day 5 indicated necessity to study the effects of cell density and also study about hydrogel materials.  Material composition and architecture of the hydrogels can influence the fate and function of the cells.  Material architecture studied using scanning electron microscope (SEM) shown that the hydrogels are porous. 

Human umbilical vein endothelial cells (HUVEC-2) were cultured in Medium 200PRF supplemented with LSGS following vendor’s protocols (BD Biosciences).  The final concentrations of the components in the supplemented medium are: fetal bovine serum (FBS) 2% v/v, hydrocortisone 1 µg/mL, human epidermal growth factor 10 ng/ml, basic fibroblast growth factor (bFGF) 3 ng/mL, and heparin 10 µg/mL.  Cells were maintained at 37°C, 5% CO2/95% air, humidified cell culture incubator and fed with fresh medium every 36 hours.  When confluent, cells were dissociated with trypsin and neutralized with trypsin neutralizer solution. 

IMR-32 neuroblasts and HepG2 (HB-8065) cells (ATCC) were cultured with media containing EMEM following vendor’s protocol.  In brief, cells were maintained in EMEM containing 10% FBS at 37°C, 5% CO2/95% air, humidified cell culture incubator and fresh media was supplemented every two days.  When confluent, cells were detached from TCP using trypsin and neutralized with growth media.  All viable cells were counted using trypan blue dye exclusion assay. 

Cell viability was evaluated using live/dead cell viability assay kit (L34951, life technologies) according to manufactures protocol.  Briefly, Percentages of late apoptotic and necrotic (low level green-stained with SYTOX green), and metabolically active/live (red-stained with C12-resazurin red), cells were assessed using live/dead cell viability assay kit.  Samples were processed according to the manufacturer's recommendations.  Briefly, cell-laden hydrogels were printed, cultured, and detached from TCP using trypsin.  Cells were centrifuged and dispersed in 100 µL PBS.  One µL of 50 µM C12-resazurin red stain and 1 µL of 1 µM SYTOX green stain were added to 100 µL of cell suspension.  Cells were incubated at 37°C in an atmosphere of 5% CO2 for 15 minutes.  The stained cells were analyzed according to the vendor’s protocol using FACSCalibur flow cytometer.

The results showed that decreased the residence time of the cells during bioprinting (by decreased needle length) increased the cell viability.  To understand the effect of printing speed on cell viability, cell-laden hydrogels were printed varying volume ejected through the needle over time.  The results showed that the cell viability is a strong function of printing speed. Decreased printing speed increased cell viability.  However, at day 5, decreased cell viability was observed, probably due to very high cell density; therefore the cell viability will be evaluated at lower cell density.  Cells seeded in hydrogels showed lower cell viability compared to the hydrogels printed at lower speeds; which indicated that this bioprinting technique did not cause injury to the cells.  These results also suggested that there is a necessity to improve/optimize the hydrogel material combinations.  Separately we also traced the cells with fluorescence and SEM pictures showed that the hydrogels are porous. 

In summary, CG hydrogels provide significant potential for bioprinting.  We demonstrated that this bioprinting technique can be used to control cell viability by varying process parameters such as printing speed and needle length.  This approach had shown that with lower printing and smaller needle length we can have viable cells to form in vitro vascularization.  Future studies will focus on testing stability of hydrogels by optimizing the combinations of hydrogel materials and evaluating cell functioning.  Improving cell viability by varying cell density during bioprinting will be evaluated.