Three-dimensional Culture System Combined With RCCS Promotes Platelet Generation From Cord Blood Stem Cells

Objective:To culture CD34+cells in3-dimensional cell-culture system(3D), which is constructedwith3-dimensional chitosan-gelatin scaffold and rotary cell culture system(RCCS), andobserve the differentiation of umbilical cord blood stem cell(UCBSC) to platelet.Methods:1. Chitosan-gelatin scaffold of varied concentration were lyophilized for preparation.The surface feature of the chitosan-gelatin scaffold was observed by scanning electronmicroscope, and the bore diameter, available pore space, water-absorbing quality, andcompatibility for the UCBSC growing well in the chitosan-gelatin scaffold weredetermined.2. Umbilicus CD34+cells were screened by magnetic beads and transferred to3-dimensional chitosan-gelatin scaffold which was placed in RCCS. The morphology of thecells were observed by inverted and scanning electron microscope. The activation and theexpression of the cells were detected by MTT or flow cytometry, respectively. Comparedwith the2-dimensional cell-culture system, morphological observation and functionalmeasurement of the platelets obtained from the UCBSC were implemented in the3-dimensional cell-culture system.Results:1. The3-dimensional scaffold culture system, which is made of0.5g chitosan and0.5ggelatin, and prepared at-30°C, was successfully constructed. For the scaffold, the borediameter is (86±21)μm, the water-absorbing rate is (87.62±10.19)%, the available porespace rate is (92.56±1.12)%, and the cells are being growing well without toxic and sideeffect.2. The umbilicus CD34+cells screened by magnetic beads were inoculated in2D and3Dcell-culture system simultaneously at the density of5×104/ml. Under the scanning electronmicroscope, the cells grew well in the both systems; the proliferation times of the CD34+cells in3D system achieved ((87.02±4.35),8thd), compared with ((20.78±5.03,5thd))in2D system. There was significant difference between the two groups(P<0.05). Theexpression of CD41and CD61on the surface of the cells cultured at7days and14dayswere detected by flow cytometry. The double positive expression in3D system was at arelatively higher level (28.70±1.75)%at7d and (82.40±2.91)%at14d, as compared withthe2D system (27.18±2.63)%at7d and (80.33±3.56)%at14d. Adhering rate of theplatelets cultured in the3D system was achieved a relatively higher level(43.05±1.93)%compared with (41.37±1.06)%in the2D system, but without significant differencesbetween them. The platelets obtained from the both systems were possessed of the ability ofaggregation, but the aggregation rate of the platelets in3D system was relatively higher thanthat in2D system. Meanwhile, the CD62p+cells account for (73.82±1.01)%of the platletscultured in3D system and for (75.26±2.57)%in the2D system, which was notsignificantly different.Conclusion：1. The3-dimensional scaffold was successfully constructed by chitosan-gelatin forUCBSC growing.2. The3-dimensional cell-culture system made of chitosan-gelatin scaffold and RCCSis good for UCBSC proliferation, and it should be a good way for amplifying platelets invitro.