Preparing single cell suspension of rat diaphragm tissues: Mechanical grinding versus mechanical-enzymatic digestion / 中国组织工程研究

BACKGROUND: Flow cytometry is currently the advanced cell analysis technique with single cell suspension as a research basis, but there is no report on the preparation method of single cell suspension of diaphragm tissue. OBJECTIVE: To explore the feasibility of preparing single cell suspension of rat diaphragm tissue by mechanical grinding method and mechanical-enzymatic digestion method and to compare cell number and viability of the cells obtained using different methods. METHODS: The fresh diaphragm tissues of Sprague-Dawley rats were harvested. Based on the mechanical method, trypsin, collagenase I, collagenase II, collagenase IV and their different combinations were used to digest and prepare the single cell suspension of diaphragm tissues. Cell morphology was observed; cell number and viability were determined by trypan blue staining. The living cells, inactivated cells, and cell aggregates were counted, and cell survival rate and concentration of the single cell suspension were calculated. Statistical analyses were performed thereafter. RESULTS AND CONCLUSION: (1) The single cell suspension with better cell dispersion, more complete morphology, clearer boundary, fewer impurities and cell debris, and cleaner background were obtained by mechanical-enzymatic digestion compared with mechanical grinding method. (2) The single cell suspension prepared by simple mechanical grinding method has low number of living cells, high number of inactivated cells, low survival rate and many cell aggregates. (3) The number of living cells and concentration of single cell suspension obtained by same-volume addition of trypsin, collagenase I and collagenase IV mixed enzymes based on the mechanical grinding method were the highest, with (1.0-2.0)×106 cells per 0.1 g diaphragm tissue. There were significant differences between mechanical-enzymatic digestion and mechanical grinding method in terms of living cells, inactivated cells, cell aggregates, cell survival rate and suspension concentration (P < 0.05). Moreover, the single cell suspension prepared by the same-volume addition of trypsin and collagenase IV had higher suspension concentration, higher cell survival rate, and less inactivated cells and cell aggregates. To conclude, the single cell suspension of diaphragm tissues could be prepared successful by both mechanical and mechanical-enzyme digestion methods. Mechanical-enzyme digestion is superior to simple mechanical grinding method, with the best single cell suspension after same-volume addition of trypsin, collagenase I and collagenase IV. This is the preferred method for preparation of single cell suspension of the diaphragm tissue.

Isolation and characterization of mesenchymal stem cells from whole human umbilical cord with a single enzyme approach / 中国医师进修杂志

Objective To explore a new approach to isolate the umbilical cord-mesenchymal stem cells (UC-MSCs) from a whole umbilical cord. Methods Single enzyme was used to digest the whole umbilical cord,and passaged and cultured,draw the growth curve,cell cycle and cell wall antigen on the surface by flow cytometry. In particular inducing system, the adherent cells were induced into adipocytes and osteoblast. Results The mesenchymal stem cells (MSCs) were derived from the whole umbilical cord. The growth curve showed that the cell doubling time was (24.15 ± 0.49) h and the percentage of G1phase cells was 82.66%.The adherent cells expressed the CD93,CD105,CD44,CD29and CD73, and did not express the CD34, CD45, CD31and human leukocyte antigen DR (HLA-DR). UC-MSCs could differentiate into adipocytes and osteoblast. Conclusions Single enzyme approach is a good method to obtain UC-MSCs from whole human umbilical cord,and it provides a theoretical basis for the establishment of MSCs bank and clinical application.

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection.

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10 6 -10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

Culturing Schwann cells by explants and modified enzyme digestion technique / 中华神经医学杂志

Objective To introduce an efficient method for culturing and purifying Schwann cells (SCs) in vitro. Methods Sciatic nerves were harvested from neonatal SD rats and the epineuria were removed. Single enzyme digestion combined with explans and double enzyme digestion were employed, respectively, to digest the nerve tissues following trituration. The proliferation of SCs and degree of purification were evaluated by viable count method and the combination of S-100 labeled monoclonal antibody and SCs. Results Proximally 3.5 × 106 cells were harvested with 96% survival rate and a purity of Schwann cells over 94% by single enzyme digestion combined with explans, however only 3.0×106 cells were gained with the purity being 90% and survival rate being 92% by double enzyme digestion. Conclusion This method yields large amount of viable Schwann cells with high purity and survival rate.

A FAST,ECONOMICAL AND SIMPLIFIED METHOD TO CULTURE AND PURIFY SCHWANN CELLS IN VITRO / 解剖学报

Objective To explore a fast,simplified and economical method for Schwann cells(SCs) cultured and purified in vitro.In this way,a stable and reliable cell sources can be provided in order to study SCs transplanted in vivo. Methods SCs cultures were prepared from the sciatic and brachial nerves of 3 to 5-day-old SD neonatal rats with double enzyme digestion method to acquire dissociated cells and one enzyme digestion method to acquire incomplete digested tissues. Results With the same number of neonatal rats,one enzyme digestion(hemi-explant)method acquired at least two times more SCs than double enzyme digestion(single cell) method did and saved at least 40 minutes.96% of S-100 positive SCs cultured were shown by immunohistochemical staining.Conclusion When the one enzyme digestion method(hemi-explant) is used to culture SCs,sufficient SCs with qualified purity can be acquired in a short time.

Association of Polymorphic Regulatory Region of TIGR Gene with Glaucoma

PURPOSE: To identify the polymorphism in the regulatory region of trabecular meshwork inducible glucocorticoid response(TIGR) gene and evaluate the association of it with glaucoma. METHODS: 5'regulatory region of TIGR gene of 101 normal persons and 91 unrelated glaucoma patients were analyzed by DNA sequencing and restriction enzyme digestion. To know the possible effects of the polymorphism on the transcription rate of TIGR gene, electrophoretic mobility shift assay and luciferase reporter gene assay were performed with cultured cells, and their extracts of trabecular meshwork and ciliary body in which the gene was expressed. RESULTS: Of the 480 bp examined, G to A transition(G-241A) located at 241 bp upstream from transcription start site was identified and its frequency of occurrence was proved to be higher in steroid induced glaucoma patients(18.9%) compared with that in normal population(8.9%), POAG(8.3%) and normal tension glaucoma patients(6.7%, P<0.05). In mobility shift assay, the G-241A probe was proved to have affinity to some DNA-binding proteins and its affinity was revealed to be two times stronger than that of normal sequence. The luciferase activities, however, were observed to be similar in cells transfected with vectors having normal promoter sequence or G-241A containing one. CONCLUSION: The result suggest that G-241A itself is not a cause of steroid-induced glaucoma but is in linkage disequilibrium with the actual causes of the disease.

Restriction enzyme digestion of heterochromatin in Drosophila nasuta.

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types of Drosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while with TaqI, no such undigested band was seen. The AluI resistant 23 kb DNA hybridized in situ specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome of Drosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.

False positives resulted from colony PCR in rapid identification of recombinant clones from phage antibody library / 解放军医学杂志

Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175?10 6 CFU and the content of Fab antibody library was 1.02?10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.