Enzymatic Cell Isolation and Explant Cultures of Rat Calvarial Osteoblast Cells

Osteoblast cells were isolated from the calvarial bones of newborn Wistar rats and cultured in vitro via both collagenase digestion method and explant technique, and a comparative study was carried out on the two culture methods. The biologic charwteristics of the osteoblast cells were studied via cell number counting, morphology observation, alkaline phosphatase staining of the cells and alizarine- red staining of the calcified nodules. The results show that osteoblast cells can be cultured in vitro via collagenase digestion method and explant technique, and the obtained cells ure of good biologic characteristics. In comparison with the explant technique, the operative procedure of the enzymatic digestion method is more complicated. The digestion time must be carefully controlled. However, with this method, one can obtain a lager number of cells in a short time. The operative procedure of the explant technique is simpler, but it usually takes longer time to obtain cells of desirable number.

Plenty more room on the glass bottom： Surface functionalization and nanobiotechnology for cell isolation

Surface functionalization is a widely adopted technique for surface modification which allows researchers to customize surfaces to integrate with their research. Surface functionalization has been used recently to adapt surfaces to integrate with biological materials specifically to isolate cells or mimic biological tissues through cell patterning. Cell isolation and cell patterning both can be integrated with extant techniques or surfaces to customize the research to whatever needs to be tested. Substrates such as metals, biologically mimicking surfaces, environmental responsive surfaces, and even three-dimensional surfaces such as hydrogels have all been adapted to allow for functionalization for both patterning and isolation. In this review we have described both the advantages and disadvantages of these techniques and the related chemistries to better understand these tools and how best to apply them in the hope that we can further expand upon the research in the field.

Cell culture isolation can miss the laboratory diagnosis of HSV ocular infection

AIMWe compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease.

Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas

Primary cultures of pancreatic stellate cells（PSCs） remain an important basis for in vitro study.However,effective methods for isolating abundant PSCs are currently lacking.We report on a novel approach to isolating PSCs from normal rat pancreases and human pancreatic ductal adenocarcinoma（PDAC） tissue.After anaesthesia and laparotomy of the rat,a blunt cannula was inserted into the pancreatic duct through the anti-mesentery side of the duodenum,and the pancreas was slowly infused with an enzyme solution until all lobules were fully dispersed.The pancreas was then pre-incubated,finely minced and incubated to procure a cell suspension.PSCs were obtained after the cell suspension was filtered,washed and subject to gradient centrifugation with Nycodenz solution.Fresh human PDAC tissue was finely minced into 1×1×l mm^3 cubes with sharp blades.Tissue blocks were placed at the bottom of a culture plate with fresh plasma（EDTA-anti-coagulated plasma from the same patient,mixed with CaCL） sprinkled around the sample.After culture for 5-10 days under appropriate conditions,activated PSCs were harvested.An intraductal perfusion of an enzyme solution simplified the procedure of isolation of rat PSCs,as compared with the multiple injections technique,and a modified outgrowth method significantly shortened the outgrowth time of the activated cells.Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period,thus facilitating future PSC-related research.

Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system

BACKGROUND: The demand for the clinical use of hepa- tocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes. METHODS: The domestic extracorporeal circulating perfu- sion apparatus was used to isolate and harvest swine hepato- cytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and ob- served by the trypan blue exclusion test, biochemical mea- surements, phase contrast microscopy and transmission electron micrography (TEM). RESULTS: The total yield of isolated hepatocytes reached to 1.5(±0.4)×l010 per liver with a viability of 92(±5)%, and the purity of hepatocytes reached to 98% Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotrans- ferase ( AST ), lactate dehydrogenase ( LDH ), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly. CONCLUSION: This method of procuring swine hepato- cytes could get high quality cells with active metabolic function.

Simplified isolation and spheroidal aggregate culture of rat hepatocytes

ＳｉｍｐｌｉｆｉｅｄｉｓｏｌａｔｉｏｎａｎｄｓｐｈｅｒｏｉｄａｌａｇｇｒｅｇａｔｅｃｕｌｔｕｒｅｏｆｒａｔｈｅｐａｔｏｃｙｔｅｓＷＡＮＧＹｉｎｇＪｉｅ，ＬＩＭｅｎｇＤｏｎｇ，ＷＡＮＧＹｕＭｉｎ，ＤＩＮＧＪｉａｎ，ａｎｄＮＩＥＱｉｎｇＨｅＳｕ...

Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox （MEQ）, 3-methyl-2-quinoxalinacetyl-l,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ＇s metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap （LC-LTQ-Orbitrap） mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we fotmd that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

Inhibition of pancreatic stellate cell activity by adipose-derived stem cells

BACKGROUND：Pancreatic stellate cells（PSCs）play a critical role in the development of pancreatic fibrosis.In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells（ADSCs）on activation and proliferation of PSCs.METHODS：Pancreatic tissue was obtained from SpragueDawley rats for PSCs isolation.Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs prolifera- tion and apoptosis were determined using CCK-8 and flow cytometry, respectively, a-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor （NGF）, interleukin-10 （IL-10） and transforming growth factor-ill （TGF-[31）] in conditioned medium were detected by ELISA. Gene expression （MMP-2, MMP-9 and TIMP-1） was analyzed using qRT-PCR. RESULTS： This method produced 17.6_＋6.5 ~ 103 ceils per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhib- ited PSCs proliferation and induced PSCs apoptosis. Moreover, a-SMA expression was significantly reduced in PSCs＋ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins （e.g., MMP-2 and MMP-9） was upregulated and anti-fibrinolytic protein （TIMP-1） was downregulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the coculture conditioned medium compared with those in the PSC- only culture medium. CONCLUSIONS： This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.

IN VITRO ANTI-HEPATOMA EFFECTS OF MONOCYTES AND KUPFFER CELLS ISOLATED FROM HEPATOMA PATIENTS AFTER TREATMENT WITH BIOLOGICAL IMMUNE STIMULANTS

Monocytes (MC), lymphocytes (LC) and Kupffer cells (KC) were isolated respectively from blood and surgical liver samples of patients suffering from he-patocellular carcinoma (HCC). 13 patients were given BCG, mixed bacterium vaccine (MBV) and human white blood cell interferon (IFN), the other 3 patients were not treated with any biological immune stimulants (BIS) and served as controls. The cytosta-tic and cytotoxic effects of MC and KC on human hepatoma SMMC-7721 (TC) were assayed in vitro and the numbers of T total (Tt), T helper (Th) and T suppressor (Ts) cells were counted using CD monoclonal antibody immunofluorescence. The results were as follows: (1) On the 7th day after the first administration of BIS, the cytostatic and cytotoxic effects of MC on TC showed obvious increase over pre-administration. The activity of BIS was 1 ?5 times as high as that in the controls. (2) After 3 administrations, the cytostatic effect of MC on TC increased to the normal level (84%), while the controls remained as before (45%). (3) On the 7th day after first administration, cytostatic and cytotoxic effects of KC on TC were 0.5 and 1 times higher respectively than those of the controls. (4) The numbers of Tt and Th of patients given BIS increased continuously; on the contrary Ts decreased in number. These results indicate that combined use of BCG, MBV and IFN can actively enhance the immune anti-hepatoma function of patients suffering from HCC.

An effective technique for isolating adult activated Schwann cells

BACKGROUND： Schwann cells （SCs） are neuroglial cells of peripheral nerve and play a key role in repairing peripheral nerve injury; therefore, it provides an important evidence for transplantation of SCs which are characterized by active proliferation and adult high-purity in vitro after nerve injury in clinic, and also develops a new therapeutic way for nerve injury.OBJECTEVE： To investigate an effective technique for isolating adult activated Schwann cells,DESIGN： Controlled observational study.SETTING： Mudanjiang Medical College.MATERIALS： The experiment was completed at the Department of Medical Genetics of Harbin Medical University from March 2003 to April 2005. Health female Wistar rats, aged 2 months, weighting 150-160 g, were randomly divided into 3 groups with 5 in each group.METHODS： The right sciatic nerves from 15 Wistar rats were exposed and transected at the mid thigh under pentobarbital anesthesia （4 mg/kg, Lp）. Seven days later, the distal segments of the predegenerated nerves were removed and used to produce adult Schwann cell cultures. The distal segment of the predegenerated nerve, 20 mm in length, was resected. The nerve was cut into pieces 1 mm in length and incubated for 3 hours under CO2 at 37 ℃ with an enzyme mixture of 0.05% collagenase/dispase. Rats were divided into 3 groups： ① Group 1： The nerve fragments were explanted in poly-L-lysine and laminin-coated dishes with BS medium from the 1st to the 6th day, On the 6^th day, the fragments were removed into a new poly-L-lysine-laminin-coated dish and the BS medium was changed to BS with 10% FBS, The nerve fragments were replaced repeatedly in the same way in new dishes on the 12^th and the 18th days. ② Group 2： For the first 3 days, the nerve fragments were fed with BS with 10% FBS. This medium was changed to BS medium on the third day. The nerve fragments were removed to another dish on day 6 and BS medium was changed to BS with 25 mI.JL FBS. Hereafter the culture method was the same as for group 1. ③ Group 3： For the first 6 days, nerve fragments were incubated in a dish not coated with poly-L-lysine and laminin, in BS medium supplemented with 8×10^7 U/L of penicillin-streptomycin. On the 6th day, the nerve fragments were removed to a poly-L-lysine-laminin-coated dish and cultured in BS with 25 mI.JL FBS, On the 12th day, the nerve fragments were explanted a second dish and fed with BS containing 100 mL/L FBS. On the 18^th day, they were explanted to a third poly-L-lysine-laminin-coated dish, SCs were obtained from all 3 dishes on the 21st day, Finally, purity and density of SCs were identified and proliferation index was calculated at the same time.MAIN OUTCOME MEASURES ： Purity and density of SCs cultured with various methods in the three groups for 21 days.RESULTS ： ① Isolation and proliferation of SCs： In the group 1, they increased in number after 4 days and both purity and density of cultured SCs were significantly higher than those from group 2. In the group 2, there were few fibroblasts. In the group 3, both purity and density of cultured SCs were remarkably higher than in those from groups 1 or 2. Then optimal proliferation was soon seen and the rapid expansion of SC populations suppressed the development of contaminating fibroblasts. On the 21st day, SCs proliferated to achieve maximal density and were too crowded to be counted. With Chi-square test, the data of the purity and the density were analyzed from groups 1 to 3, the result indicated X^2=430.47, P 〈 0.05. ② Characterization and proliferation rate of SCs： Immunostaining for S100 protein was evident in the cell soma and the processes of all three groups in cultures of SCs. SCs in vitro demonstrated typical bior tri-polar morphology, had oval nuclei, and stained brightly for $100. The proliferation rate of SCs was assessed with double fluorescence staining for BrdU and S100 on the 21^st day of all three groups in cultures. About 40%-50% of the total SCs in the each group showed BrdU incorporation.CONCLUSION： The method is to use predegeneration in vivo, differential speed culture supplemented with the penicillin-streptomycin in low concentration, and changing of the concentration of FBS in the BS medium from 0 to 100 mL/L. This method allows remarkable suppression of fibroblast growth and attainment of SC proliferation and purity, in a short time, from adult nerves.

A NEW METHOD OF ISOLATING KUPFFER CELLS FROM BIOPSY TISSUE OF RAT LIVER

The isolation of a high yield and purity of Kupffer cells has been reported in detail.1 This paper reports into the research about isolation Kupffer cells from biopsy tissue of liver. This method includes 5 important steps: (1) take fresh liver tissue, and mince with scissors. (2) spin at low speed to wash off red blood cells. (3) digest in collagenase for suitable time. (4) isolate Kupffer cells on a percoll density gradient. (5) cell charaterization was observed by N.S.E stain and peroxidatic activity with lumino-meter measurement and phagocytosis with latex beads.2.3

Plant regeneration of protoplasts isolated from suspension cells derived from leaf blale of Oryza sative

We used the leaf blade of rice (cultivars were Nonghu 6, Sugeng 2, Huyou 2 and Hanfeng) as initial material for protoplast culture, and a great number of regenerated plants were obtained. Rice seeds were sterilized and germinated. The immature leaves were cut into 3-5 mm pieces when the third or forth leaf appeared. Leaf pieces were inoculated on MS medium with 2,4-D 4 mg/1, NAA 2mg/1 and IAA Img/1. After 2 wk culture, calli were induced and subcultured once or twice for multiplication. 3-5 g calli were transferred to the modified MS liquid medium with 2,4-D 2 mg/1 and KT 0.5mg/1 for suspension culture. Embryogenic cell suspension was established after 2 mo culture. The effect of the growth period of suspension cells on the

Construction of Polymeric DNA Network and Application for Cell Manipulation

Comprehensive Summary Deoxyribonucleic acid(DNA)is a biomacromolecule,as well as a polymeric material,whose sequences with different manipulative structures enable them to implement a series of functions,such as reorganization,target,and catalysis.Compared to existing traditional materials incapable of multifunctional integration,the polymeric DNA network is a form of material that can achieve functional integration while maintaining specific DNA properties.Furthermore,precise target enabled by DNA network is one of the most essential components of cellular manipulation.Hence,the DNA network is indispensable and irreplaceable to cell manipulation that it is a versatile tool for the understanding of basic laws of living life and treatments of diseases,such as cell isolation,cell delivery,and cell interference.Herein,the construction of polymeric DNA network is briefly introduced from the aspects of assembly modules,construction methods,and properties.

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability

Single-cell analysis in endometrial research

Human endometrium undergoes dynamic shedding,regeneration,and differentiation,with remarkable changes in gene expression across the menstrual cycle.The development of a receptive endometrium within a particular time frame(window of implantation)is critical for successful embryo implantation.To understand the role of the endometrium in human fertility and regenerative biology,transcriptomic characterization of the endometrium has traditionally been pursued at the tissue bulk level using microarray and next-generation sequencing.Owing to the rapid development of single-cell RNA sequencing technology,researchers have uncovered heterogeneous molecular activities in individual cells masked by bulk analysis.In this review,we opted to mainly focus on single-cell analysis in endometrial research and introduce basic knowledge of single-cell RNA sequencing and the isolation of single cells from endometrial cells.We also discussed how single-cell approaches are used to understand the transformation and regeneration of the endometrium physiologically and uncover endometrial factors that contribute to uterine pathology.

A dual V_t disturb-free subthreshold SRAM with write-assist and read isolation

This paper presents a new dual V_t 8 T SRAM cell having single bit-line read and write, in addition to Write Assist and Read Isolation（WARI）. Also a faster write back scheme is proposed for the half selected cells. A high V_t device is used for interrupting the supply to one of the inverters for weakening the feedback loop for assisted write. The proposed cell provides an improved read static noise margin（RSNM） due to the bit-line isolation during the read. Static noise margins for data read（RSNM）, write（WSNM）, read delay, write delay, data retention voltage（DRV）, leakage and average powers have been calculated. The proposed cell was found to operate properly at a supply voltage as small as 0.41 V. A new write back scheme has been suggested for half-selected cells,which uses a single NMOS access device and provides reduced delay, pulse timing hardware requirements and power consumption. The proposed new WARI 8 T cell shows better performance in terms of easier write, improved read noise margin, reduced leakage power, and less delay as compared to the existing schemes that have been available so far. It was also observed that with proper adjustment of the cell ratio the supply voltage can further be reduced to 0.2 V.