A Study On Microfluidic Technology Fabrication And Cryopreservation Of Bone Marrow Stromal Cells/alginate Microcapsules

Objective:To fabricate bone marrow stromal cells alginate microgel(BMSC/ALG microgel)by one-step microfluidic approach,explore how the preparation process and the three-dimensional micro-environment influence the cell proliferation、osteogenic potentiality and cryopreservation.To further explore the application of BMSC/ALG microgel in tissue engineering.Methods:1.A study on the cell proliferation and osteogenesis potentiality of BMSC/ALG microgels.To fabricate BMSC/ALG microgel by one-step microfluidic approach,seeding cells and BMSC/ALG microgels with 5*10^5/cm2 cell density in cell culture plate,respectively for common cultivation and osteogenesis induction cultivation.Using optical biological microscope and laser confocal microscopy in observation of the survival and growth of cells.Using CCK-8 to detect the proliferation of cells.Detect the activity of ALP(alkaline phosphatase).Using optical biological microscope,Fourier transform infrared spectroscopy(IR),scanning electron microscope(SEM),energy dispersive X-ray spectrum(EDS)to detecte the mineralization of BMSC/ALG microgel.2.A study on the cryopreservation of BMSC/ALG microgels.To fabricate BMSC/ALG microgel by one-step microfluidic approach.Using four different volume fraction of DMSO concentrations(2.5%,5%,7.5%,10%),and put in 4 ℃ for 45 min min,15 min,30 min and 0 min before cooling,to receive 16 groups.Using 106/ml cells concentration to cryopreservation.Recovery cells after 7 days,using Calcein-AM/PI staining to detect cell survival,using trypan blue staining to detect cell membrane integrity,using CCK-8 to test the cell activity after culture 24 hours.Result:1.A study on the cell proliferation and osteogenesis potentiality of BMSC/ALG microgels.The results show that the process have some influence on BMSC survival,before production,cells with a high survival rate(98.367% + /-0.715)but appeared in the cell survival rate after making some fall,but remained at a high level(86.167% + /-2.135).Using optical microscope and laser confocal microscope to observe BMSC/ALG microgels,the cell cluster size from the initial((?) = 15.106±1.321μm)increase with the cultivate time,reach the peak value at Day8((?) = 37.419±2.963μm).CCK-8 test shows that in the initial,although the cell activity of GEL group was slightly lower than that in CON group,there was no significant difference between the two groups in the following observation period.After the culture of BMSC/ALG microgel with osteogenesis induction medium,the mineralized deposition in the was observed from 7 days after induction.The infrared spectra(IR)were used to detect osteogenesis induction BMSC/ALG microgel after 21 days,shows the appearance of phosphate crystals and Ⅰ type collagen,Scanning electron microscopy(SEM)observed a large number of hydroxyapatite crystals on the surface of gels,Energy scattering spectrum analysis(EDS)shows that compared to the initial state of the BMSC/ALG microgel,Ca element and P element content increased significantly,shows that within the BMSC in microgel maintained osteogenesis activity.After osteogenesis cultivation,the ALP activity in the GEL group was increased faster than the CON group,and reached the peak value earlier(GEL group Day6:1.782 ± 0.120U/L;CON group Day12:1.890 ± 0.11 U /L),and the ALP activity standardized by whole DNA concentration shows that the ALP activity in GEL group also reached the peak value earlier(Day6: 57.00 ±5.90U/ng DNA;CON group Day12: 53.13±2.70 U/ng DNA).2.A study on the cryopreservation of BMSC/ALG microgels.Calcein-AM/PI staining showed that the freezing process significant influence the cell survival rate.The pretreatment time of the DMSO was significantly affected by the survival rate of the treatment group using 2.5% and 5% volume fraction DMSO.There was no significant difference in cell survival in the treatment group using 7.5% and 10% volume fraction DMSO.Trypan Blue test shows that the cell membrane integrity rate in the group use of 2.5% and 7.5% volume fraction of DMSO associated increased with DMSO concentration.There was no significant difference in cell membrane integrity between the treatment group using the 7.5% volume fraction DMSO and the treatment group using the 10% volume fraction DMSO.CCK-8 test shows that in the treatment group using 2.5% and 5% volume fraction of DMSO,experimental groups are significant lower compared to control group.And there was no significant difference in cell activity between the experimental group and the control group in 7.5% and 10% volume fraction of DMSO treatment group.Conclusion:(1)The growth and proliferation activity of BMSC/ALG microgels was maintained,and the alginate materials of gels supported the cells well.The BMSCS cultured in the microgel showed a high osteogenic differentiation activity,and the local microenvironment had the effect of promoting the osteogenic differentiation of BMSC.The alginate gel network provides a basis for mineral deposition and promotes the process.(2)The programmed freezing protocol is suitable with BMSC/ALG microgels.The optimized parameter of BMSC/ALG microgels is 7.5% volume fraction of DMSO,20% volume fraction of FBS and 30 mins preloading time in 4℃.