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Objective:To observe the transparency and tissue structure changes of human corneal stromal lenticules after long-term cryopreservation and explore a simple and feasible method for long-term effective preservation of corneal stromal lenticules.Methods:Two hundred samples of intact human corneal stromal lenticules from 200 eyes were obtained during femtosecond laser small-incision lenticule extraction (SMILE) in Hainan Eye Hospital, Zhongshan Ophthalmic Center from 2013 to 2020.The samples were divided into 1-month, 24-month, 60-month and 80-month group and were stored in an ultra-low temperature freezer for 1, 24, 60 and 84 months respectively at -80 ℃ according to grouping, with 50 samples in each group.Transmittance of the corneal lenticules at wavelength of 300-800 nm was measured with an ultra-micro spectrophotometer and every lenticule was measured for 10 times with a 50 nm interval.The histomorphology and collagen fiber structure of the corneal lenticules were examined by hematoxylin-eosin staining and Masson staining, respectively.The arrangement of collagen fibers and ultrastructure changes of keratocytes in the samples were inspected with a transmission electron microscope.The apoptosis rate of keratocytes was determined by TUNEL staining.The study protocol was approved by an Ethics Committee of Hainan Eye Hospital at Zhongshan Ophthalmic Center (No.2013-003). This study complied with the Declaration of Helsinki.Written informed consent was obtained from each subject before surgery.Results:The corneal lenticules were clear and intact in all groups and no significant difference in the transmittance within 450-800 nm wavelength was seen among the 4 groups (all at P>0.05). Masson staining revealed that the collagen fibers in the lenticules were neatly arranged and tightly packed in the 1-month group.In the 24-month group, interfibrous vacuoles were found in some collagen fibers.The arrangement of the collagen fibers was loose and more vacuoles were displayed in the 60-month group, and the loss of some collagen fibers appeared and the lenticules were thinned in the 84-month group.It was found through hematoxylin-eosin staining that the morphological changes of corneal stromal lenticules corresponded to the alterations of collagen fibers.Transmission electron microscopy showed that in the 1-month group, the collagen fibers of the corneal stroma lenticules were neatly arranged and regular, and the corneal stromal cells were elongated and spindle-shaped, and the nuclear membrane was intact and the cytoplasm was abundant.In the 24-month group, the collagen fibers showed slightly loose arrangement, and the corneal stromal cells were deformed with incomplete nuclear membrane.In the 60-month group, the collagen fibers were in loose and irregular arrangement, and the nuclei were atrophied and deformed.The 84-month group showed disorganized arrangement of collagen fibers, wrinkled and atrophied corneal stromal cells, discontinuous nucleus membrane and nucleoplasmic lysis.TUNEL staining showed that the percentage of apoptotic corneal cells in lenticules was (87.80±1.17)%, (89.50±1.05)%, (89.30±1.51)% and (90.20±1.47)% in the 1-month, 24-month, 60-month and 84-month groups, respectively, with no statistically significant difference found in overall comparison ( F=4.525, P=0.053). Conclusions:The disorder of collagen fibers and apoptosis of keratocytes occur in the human corneal stromal lenticules till 84 months after cryopreservation, however, the transparency and integrity remain excellent.The ultra-low temperature preservation technique provides an effective and simple solution for long-term storage of human corneal stromal lenticule.
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Objective To determine the proper storage environment by comparing the activity of adipose-derived stem cell (ADSC) at 10％ PRP,10％ HS and 0.9％ NaCl.Methods ADSC was isolated and cultured.Then the ADSC was stored in 10％ PRP,10％ HS,and 0.9％ Nacl at room temperature (RT,about 26 ℃C) for 2 hours.After that the proliferation ability,survival rate and differentiation ability of the cells were detected.Finally storage media of ADSC was determined.Results Compared with 0.9％ NaCl,ADSC showed a better proliferation and higher differentiation ability at 10％ PRP and 10％ HS;besides,the survival rates of two groups were higher than the group of 0.9％Nacl.Among these groups,the survive rate of 10％ HS and 10％ PRP were 78.08％ and 75.68％ respectively,while it was 62.34％ in 0.9％ NaCl group.Conclusions 10％ HS is more suitable for short-term preservation of ADSC.
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Objective To evaluate the application value of glycerol blood chocolate soup in the bacteria preservation of Haemophilus influenzae.Methods Haemophilus influenzae strains ATCC10211,ATCC49247 and ATCC49766 were saved into glycerol blood chocolate soup,then it was stored in refrigerator at below -35℃.The preserved strains from the refrigerator at 1 month,3 months,6 months,1 year,2 years were taken out,and dissolved it at 35℃ for 5min,and then immediately transferred it to chocolate agar plate,placed it in the 35℃,CO2 environment overnight.Observed the survival of strains,colony morphology and whether the demand factor test had change.Saved the standard strains ATCC49247 and ATCC49766 2 years for the paper dispersion method drug sensitive test.Results After 2 years of observation,3 strains of Haemophilus influenzae strains grew well in the survival rate of 100%,the colony was colorless,transparent and flat moist,it could be observed gram small negative bacilli by Gram staining, blood agar satellite test was positive,no hemolysis,Columbia satellite test was negative.All traits were the characteristics of Haemophilus influenzae.The drug sensitivity characteristics of ATCC49247,ATCC49766 standard strains were still in line with the requirements of CLSI.Conclusion Glycerol blood chocolate soup is suit for Haemophilus influenzae bacteria preservation.
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BACKGROUND:The relationship between placing time after stem cel preparation and cel survival is the basis of safety and effectiveness for the clinical application. OBJECTIVE: To observe effects of different preservation solutions and different storage time on survival rate of umbilical cord-derived mesenchymal stem cels, and to provide important evidence for identifying effectiveness of stem cels. METHODS:Umbilical cord-derived mesenchymal stem cels were selected to prepare stem cel preparation, which was preserved in physiological saline, medium, medium+physiological saline, physiological saline containing epidermal growth factor, and medium containing low molecular heparin calcium suspension. Cold closet was selected for imitating celular transport conditions. Samples were obtained at 0, 6, 12, 18, 24, 30, 36, 42 and 48 hours. Total cel number and cel survival rate were detected. RESULTS AND CONCLUSION:The difference in cel number and survival rate was not great within 24 hours in each group. Twenty-four hours later, total cel number and survival rate were better in the medium and medium containing low molecular heparin calcium groups than in the physiological saline containing epidermal growth factor, physiological saline, and medium+physiological saline groups. These findings suggest that after stem cel preparation, the cel survival rate can reach more than 90% within 24 hours under refrigerated transport conditions. Nutritional ingredients and proper pH value of preservation solution can make the cel survival rate increased greatly.
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Objective The use of in vivo cryotechnique (IVCT) in combination with electrocardiograph (ECG) to study cardiac microcirculation under different hemodynamic conditions in living mouse.Methods Living mouse heart monitored by electrocardiograph was suffered from IVCT and freezing substitution under normal blood flow,myocardial ischemia or cardiac arrest conditions.Hematoxylin eosin (HE)staining,Schiff's staining and immunofluorescence staining for serum albumin,immunoglobulin were utilized on continuous paraffin sections,respectively.Confocal microscopy and statistical analyses were used.Results Comparing with normal hemodynamics,microvascular red cell volume reduction,morphology changed,myocardial cell glycogen loss,serum albumin ectopic distribution to myocardial cytoplasm,T tubular network failure and spacing width were happened in myocardial iscbemia condition; different shapes of red blood cells,myocardial cells glycogen deficiency,T tubular network failure and interval narrowing were found under cardiac arrest conditions.Conclusions Cardiac microcirculation,pathological changes of myocyte and its surrounding microenviroument in living mouse heart can be immediately captured in situ by the application of IVCT and ECG.
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Apesar de a esplenectomia ser eficaz na abordagem terapêutica de pacientes com hemangioma esplênico, esse procedimento é acompanhado de elevada morbidade e até mortalidade, principalmente devido à sepse, quando realizado em crianças e adolescentes com sistema imunitário ainda imaturo. Para prevenir os efeitos adversos da asplenia, propõe-se neste artigo a esplenectomia parcial, com a retirada apenas da região do hemangioma, mantendo o restante do baço e preservando suas importantes funções.
BACKGROUND: Although splenectomy is helpful in the management of splenic hemangioma, this procedure may be accompanied by a greater morbidity and even mortality, mainly caused by sepsis, when this operation is performed in children and teenagers, due to their immune deficiency. In order to avoid the adverse effects of the asplenism, this paper proposes partial splenectomy to treat splenic hemangioma.
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0.05). The mechanical strength of dacron and thread were not significantly changed. Rb/Rc was approximately 1.2, H/Rc was approximately 1.4 and ?was approximately 10. These parameters accord with standard of prosthetic valve design. Conclusion The stented homograft valve can be cyropreserved by liquid nitrogen and accord with standard of prosthetic valve design.