| The low temperature is an important technical means for high-quality preservation of biological samples,and has been widely used in the long-term cryopreservation and clinical applications of active biological samples including cells and tissues.During the cryopreservation,the typical solution penetration phenomenon will exist in important links such as loading.of cryoprotectant,cooling,thawing and elution of cryoprotectant,and the systematic and in-depth research work on it will improve the scientificity of cryopreservation method.However,the research on the interaction characteristics of cryoprotectants and cells during cryopreservation still remain at the level of experimental and theoretical model analysis of single-type cryoprotectants and cell volume change characteristics,and there is still a lack of in-depth understanding of the intracellular penetration behavior of multi-component mixed cryoprotectants and the interaction between crystallization and osmotic behavior inside and outside the cell during cryopreservation.Therefore,in this work,the confocal Raman spectroscopy technique is utilized to establish the basic method for evaluation of the whole process of cryopreservation.Several key crystallization and permeation behavior characteristics,such as the basic characteristics of the cooling process of cryoprotectants,the process of cell loading and eluting the cryoprotectants,the change of intracellular and extracellular concentration during the cryopreservation process and the quenching process of micro droplets,were explored respectively.The quantification of the key crystallization and permeation behavior of cells during cryopreservation was achieved,which provides necessary experimental data and theoretical support for guiding the optimization of the overall scheme of cryopreservation.The detailed research contents of each part are as follows:The Raman spectral characteristics of VS55 solution during the freezing process were firstly studied.A low-temperature confocal Raman spectroscopic test system was built to obtain the Raman spectra of VS55 solution with different different concentrations（100%,75%,50%,25%,0%）during cooling process.It was found that the intensity of different Raman characteristic peaks of VS55 solution showed a good linear relationship with the concentration of solution,and the original source components corresponding to different Raman characteristic peaks in the VS55 mixed solution were identified,including dimethyl sulfoxide,formamide,1,2-propylene glycol and water,etc.By analyzing the Raman spectral characteristics of the VS55 solution system during the crystallization and vitrification process,it is found that the Raman spectral shape of the hydrogen bond range(3000～3800 cm-1)can significantly distinguish the ice crystal phase（crystalline state）and high-density amorphous structure（glassy state）of the water molecule in different VS55 solution after cooling process.Through the peak position shift of the S=O double bond in the dimethyl sulfoxide molecule and the C-N bond of the formamide molecule combined with the change characteristics of the hydrogen bond of the water molecule,the synergistic interaction between the two solute molecules and the hydrogen bond network of water molecules during the crystallization and vitrification process could be discussed.Accordingly,the hypothesis of molecular assembly behavior of water molecules and solute molecules in the VS55 mixed solution during the crystallization and vitrification process were established.Secondly,the permeation behavior of cryoprotectants inside and outside cells at room temperature was studied by Raman spectroscopy.After optimizing the conditions for the Raman spectrum of the active cells loading of cryoprotectants,the acquisition and analysis of intracellular and extracellular Raman spectral curves and Mapping images in different cryoprotectant solution were completed.The Raman Mapping images verified the penetration behavior of the non-permeable protective agent only distributed outside the cell and the osmotic protective agent evenly distributed inside and outside the cell.Through the multivariate spectral image analysis algorithm,the Raman spectral image characteristics of trehalose tending to be distributed on the surface of the cell membrane were found,which revealed the way that trehalose exerted the cryoprotective effect.In addition,a Raman spectrum real-time monitoring system of microfluidic chip for continuous loading and elution of cryoprotectant was built to acquire the Raman spectrum inside and outside of cells during the process of loading and elution of cryoprotectants in real time.It was found that Raman spectroscopy can reflect the changes of intracellular and extracellular cryoprotectant concentration in real time,and it can be used to guide the program optimization and improvement of the loading and elution process of cryoprotectant.In order to achieve accurate quantitative evaluation and analysis of intracellular and extracellular permeation behavior during cryogenic freezing,the Raman spectra of DMSO solutions with different standard volume concentrations（5%,10%,20%,40%,60%,80%）were obtained to calculate the ratio of the peak intensities of the hydrogen bond of water molecules as the internal standard and the intensities of different labeled peaks of DMSO for establishing the Raman spectrum quantitative evaluation method of different volume concentrations of DMSO.Thus,the changes of the concentration of cryoprotectants inside and outside the cell during the permeation process in three different freezing processes（direct slow freezing,slow freezing by nucleation,and rapid freezing by vitrification）were quantitatively analyzed,and the analysis of the permeation behavior during three different cell freezing process was realized.It was found that the essential reasons for the improvement of cell viability in the slow freezing of nucleation methods were the smaller osmotic pressure difference inside and outside the cell and the concentration variation consistent with the primary saturation curve of the DMSO solution system during the freezing process.Finally,the synergistic relationship between extracellular crystallization behavior and intracellular osmotic concentration change process in three different freezing processes is summarized and analyzed,which complements and verifies the concentration change process described in the Two-factor hypothesis.Finally,the confocal Raman spectroscopy is applied to the quenching process of microdroplet.An experimental device for quenching the microdroplets was built to collect Raman spectra of microdroplets after quenching.The internal crystallization and vitrification state of quenched droplets with different concentrations（10%～50%）and different volume sizes（1～10μL）can be distinguished by the Raman spectral characteristics of intramolecular hydrogen bonds.The crystallization degree inside the quenched droplet was quantitatively analyzed by the ratio of the crystallization peak to the hydrogen bond shoulder peak.By obtaining the temperature curve of the microdroplet quenching process,it is found that the greater the concentration and volume of the microdroplet,the longer the time for the quenching process to reach temperature equilibrium,and the lower the cooling rate.In addition,the results of the cell microdroplet quenching and thawing survival rate experiment showed that the cell survival rate decreased significantly with the increase of the volume of the quenched droplet.This is directly related to the increased proportion of crystallization peaks in the Raman measurements and the slower cooling rate of large-volume droplets.For the droplet cryopreservation method with higher concentration（40%～50%）quenched vitrification,the key points are the toxic effect of high concentration and the possible devitrification phenomenon in the thawing process.In view of the current results,the droplet quenching cell cryopreservation method still needs to be further improved.Overall,the confocal Raman spectroscopy technology is comprehensively applied to each key link of cryopreservation process in this work.The basic test methods of Raman spectroscopy under different conditions are established to obtain a large number of experimental datas for quantitative evaluation of crystallization and permeation behavior in the process of cryopreservation,which provides a new evaluation method for in-depth understanding of the essence of different cryopreservation process.However,the application of Raman spectroscopy in the field of cryopreservation is still in its infancy,and comprehensive and thorough work still requires the unremitting efforts of researchers. |
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