| In vitro cell culture has long been a fundamental technique in cell therapy,transplantation,tissue engineering and regenerative medicine.Conventional adherent cell culture procedure is time-consuming,as it includes cell recovery,digestion,centrifugation,blowing,counting,vaccination,etc.EDTA in digestion buffers can accelerate the detachment of cells from the culture flask and improve the efficiency of digestion.However,EDTA may affect cell viability in a negative manner.Thus,it has to be removed after the digestion is finished.The traditional method is to separate the cells from the solution by centrifugal force to remove the EDTA from the supernatant and add a new medium to disperse the cells by pipette blowing.However,current centrifugal process still faces some disadvantages.For example,centrifugal force damages cells and reduces cell viability;centrifugal operation in the sterile working bench increases the risk of cell contamination;the operation that the cells must be separated through pipette blowing after centrifugation will destroy the cells and is a time-consuming and laborious process.In order to effectively solve the problems caused by centrifugal operation,this paper provides a new strategy that replace centrifugation with filtration.Making full use of the advantages of unidirectional water-transfer ns,cell suspensions can pass through the filter by relying on their own gravity,which effectively avoids the centrifugal force to damage cells.At the same time,cells trapped on the hydrophobic surface of the nonwovens are less likely to adhere on the nonwoven surface and are completely released into the new culture medium.This process successfully eliminates the need for manual pipetting,which can save time and avoid damaging cells.The whole process of filtration and release in the sterile working bench reduces the chance of cell contamination.In view of this,this study prepared PBT/chitosan unidirectional water-transfer nonwovens through the selection of nonwoven filtration substrate,a combination of various technology,performance characterization,and then studied its application potential in cell filtration and release.The preparation steps for producing PBT/chitosan unidirectional water-transfer nonwovens include:(1)preparing PBT nonwoven filtration substrate through meltblown technology;(2)low temperature plasma technology to obtain single-sided hydrophilic properties;(3)coating with chitosan acetic acid solution to prepare PBT/chitosan unidirectional water-transfer nonwovens;(4)stablizing the structure and properties via crosslinking.Finally,PBT/chitosan unidirectional water-transfer nonwovens were used on cell filtration and release study and the results suggest that it works better than centrifugation.Firstly,we studied the meltblown process with the throughput of the polymer flow as a parameter.The results showed that when increasing the throughput from 0.01 to 0.07 g/min,the meltblown fiber diameter increased and the diameter distribution became wider,the average pore size and the maximum pore size gradually increased.Therefore,we prepared the PBT meltblown nonwovens(1 #,2 #,3 #)with the same areal density and thickness by using different meltblown process parameters.Based on the analysis of the fiber diameter,pore size and its distribution,mechanical properties and air permeability of these samples,1 # sample which has smaller fiber diameter,pore size and higher breaking stress was selected for further study.Then,in order to filtrate and release cells effectively,we prepared the PBT/chitosan unidirectional water-transfer nonwovens using 1# sample and chitosan,and studied the optimum process parameters of low temperature plasma treatment and optimum chitosan concentration.The results show that when the treatment time of low temperature plasma is 10 s,the nonwovens have the maximum difference of doublesided wetting properties.The optimal processing parameters of low temperature plasma treatment are as follows: the treatment gas is oxygen,the degree of vacuum is 10% and the treatment time is 10 s.When increasing the concentration of chitosan from 0.01 to 0.04 g/m L,the thickness of the nonwoven does not change much,the areal density and the breaking stresses at both MD and CD increased,while the breaking strains,average pore size,maximum pore size and permeability declined accordingly.In addition,physical and chemical properties of the nonwovens were characterized through scanning electron microscope(SEM),energy dispersive X-ray spectroscopy mapping(EDS-Mapping),Fourier transform infrared spectroscopy(FTIR-ATR),X-ray photoelectron spectroscopy(XPS),differential scanning calorimetry(DSC),thermal gravimetric(TG).The results show that when the concentration of chitosan is 0.04 g/m L,PBT/chitosan unidirectional water-transfer nonwovens exhibit obvious gradient wettability in the thickness direction.Coated surface with chitosan is hydrophilic while uncoated surface without chitosan is hydrophobic.Therefore,the nonwoven has a significant unidirectional water-transfer effect and the water droplets can completely penetrate to the hydrophilic surface within 3 s.Also,the nonwoven has good thermal stability because it won't be decomposed below 200 °C.Finally,PBT/chitosan unidirectional water-transfer nonwovens were assembled into novel cell filter to filtrate and to release the cells.Compared with the centrifugation,we studied the biocompatibility,cell harvest rate,cell viability,and reusable properties of the nonwovens.The results show that PBT/chitosan unidirectional water-transfer nonwovens have good biocompatibility.Due to the uncoated surface of the nonwoven is hydrophobic,the cells are not easily attached on the surface.Therefore,all the cells on the uncoated surface can be easily released by simply swaying the nonwoven.When the concentration of chitosan is 0.04 g/m L,cell harvest rate of the nonwoven is over 99%,which is higher than that obtained by the centrifugation method,and the EDTA in the solution can be effectively removed.The filtration method does not affect cell viability and the nonwoven can be reused.After used repeatedly for 50 times,the nonwoven still maintained the same cell harvest rate and membrane flux,and after irradiation under the UV lamp for a long time,the loss of breaking stresses and strains was low,and the reusable property was good.In summary,PBT/chitosan water-transfer nonwovens were prepared by combining various techniques.We optimized the processing parameters and tested the physical and chemical properties of the nonwoven,after which the nonwoven was assembled into a novel cell filter.In comparison with centrifugation,the PBT/chitosan unidirectional water-transfer nonwovens have obvious advantages in the cell filtration and release. |
PDF Full Text Request