Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 µm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.

Isolation, culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85 +/- 4.6%), and a correspondingly low proportion K3 positive cells (15 +/- 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean +/- SE, n=10). Cultures reached full confluency after 17.3 +/- 1.2 days when the medium was supplemented with human EGF, while 21.7 +/- 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.