MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

3D Collagen Gels:A Promising Platform for Dendritic Cell Culture in Biomaterials Research

The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.

Optimization of Culture Medium and Transfection Method for Head and Neck Squamous Cell Carcinoma Organoids

[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.

Differentiation potential of human adipose tissue derived stem cells into photoreceptors through explants culture and enzyme methods

AIM： To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells （ADSCs） generated by enzyme （EN） and explant （EX） culture methods.METHODS： We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescent （IMF） staining.RESULTS： Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17th day and then the rhodopsin mRNA was detected on the 24th day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31st day. EX orbital ADSCs expressed rhodopsin protein on the 24th day, while EN orbital ADSCs expressed rhodopsin protein on the 31st day. CONCLUSION： Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method.

Comparison of two methods used to culture and purify rat retinal Mller cells

AIM:To study two methods for culturing and purifying Sprague-Dawley(SD)rat retinal Muller cells and determine which one is better.METHODS:The passage culture method of Muller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method.After culturing retinal cells for one month through these two methods,fluorescence-activated cell sorter(FACS),RT-PCR,and immunohistochemistry technology were performed to examine the enrichment and purity of Muller glial cells,and carried out two-sample approximate t test using SSPS 13.0 to further compare the Muller cell positive rate in both methods.RESULTS:The statistical results showed that the purity of Muller cells was 83.2%±5.16%in group A,and the purity was 98.5%±1.08%in group B.The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant(t=-9.178,P【0.005).The results clearly exhibited a difference between the purity of Muller cells cultured by the complete pancreatic enzyme digestion method(group A)and the repeated incomplete pancreatic enzyme digestion method(group B).CONCLUSION:Compared with the complete pancreatic enzyme digestion method,this novel method was more efficient and a higher purity of Muller cells could be obtained using this approach.

Simplified methods to isolate,culture and purify olfactory ensheathing cells

Conventional methods for harvesting, culturing and purifying olfactory ensheathing cells are complicated, time-consuming, and poorly reproducible. Olfactory bulbs were detached from adult Sprague Dawley rats and olfactory ensheathing cells were isolated using shearing, dispersion processes. After the primary cultures reached confluence, the cells were purified using a three-step process. The olfactory ensheathing cells attached and grew rapidly. The purity of the olfactory ensheathing cells increased following the three purification steps, eventually exceeding 95%. These cells could be maintained for an extended period time in culture. This simple, inexpensive, reproducible method of harvesting, culturing and purifying olfactory ensheathing cells shortens the culture cycle and provides sufficient olfactory ensheathing cells of controllable purity.

Comparison on In-Vitro Culture Methods of Yak Endometrial Gland Epithelial Cells

[ Objective] To develop in-vitro culture methods of yak endometrial gland epithelial cells. [Method] The gland epithelial cells were isolated from yak endometrium by explant culture method and digestion culture method, respectively. [ Result] In the first method, the cells isolated from the endometrium explant could merge into monolayer after 8-d culture, and they could be purified by gradation digestion with trypsin. In the second method, the endometrium explant were first digested by collagenase II by incubation at 37℃ for 2.5 h and then further digested in fresh 2 g/L colla- genase II for another 2.5 h. The cell suspension was leached through 74 pm filter and centrifuged at 400 r/min for 5 min. Then the cell pellet was re-suspended, followed by natural sedimentation to collect purified gland epithelial cells. The isolated cells were cytokeratin-positive as detected by immunocytochemical staining, and the positive rate could reach 95%. [Conclusion] The yak endometrial gland epithelial cells can be isolated and purified by both the explant culture method and digestion culture method.

Culture and identification of neonatal rat brain-derived neural stem cells

BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.AIM To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.METHODS First,curved tip operating scissors were used to dissect brain tissues from new born rats(2 to 3 d)and the brain tissues were cut into approximately 1 mm^(3)sections.Filter the single cell suspension through a nylon mesh(200-mesh)and culture the sections in suspensions.Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques.Second,identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation.BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells.Different NSCs specific antibodies(anti-nestin,NF200,NSE and GFAP antibodies)were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.RESULTS Brain derived cells from newborn rats(2 to 3 d)proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging.When BrdU was incorporated into the 5th generation of passaged cells,positive BrdU cells and nestin cells were observed by immunofluorescence staining.After induction of dissociation using 5%fetal bovine serum,positive NF200,NSE and GFAP cells were observed by immunofluorescence staining.CONCLUSION This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

An Innovative Design of Incubator Structure for Cell Culture

In view of the problems of the traditional cell incubator,such as the small range of cell culture types,the inability to adjust the internal space of the incubator according to needs,and the inconvenient sampling,this study innovatively designed a cell incubator structure.It proposed a new design concept that can solve the above-mentioned shortcomings.The cell incubator after the new structural modification can adjust the internal space structure of cell culture by setting the bolt-fixed connection between the fixed plate and the vessel divider.It realizes the cultivation of various cells through refrigeration modules and heating modules.Through setting a sampling hole in the glass inner door,it is favorable for operators to take samples,making cell culture more convenient and efficient.

Repetitive administration of cultured human CD34+cells improve adenine-induced kidney injury in mice

BACKGROUND There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease(CKD).AIM To examine the efficacy of cultured human CD34+cells with enhanced proliferating potential in kidney injury in mice.METHODS Human umbilical cord blood(UCB)-derived CD34+cells were incubated for one week in vasculogenic conditioning medium.Vasculogenic culture significantly increased the number of CD34+cells and their ability to form endothelial progenitor cell colony-forming units.Adenineinduced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice,and cultured human UCB-CD34+cells were administered at a dose of 1×106/mouse on days 7,14,and 21 after the start of adenine diet.RESULTS Repetitive administration of cultured UCB-CD34+cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group.Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group(P<0.01).Microvasculature integrity was significantly preserved(P<0.01)and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group(P<0.001).CONCLUSION Early intervention using human cultured CD34+cells significantly improved the progression of tubulointerstitial kidney injury.Repetitive administration of cultured human UCB-CD34+cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Sequential extraction of RNA,DNA and protein from cultured cells of the same group

BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of life.However,cell samples are often from different culture dishes,resulting in inevitable experimental errors and sometimes poor repeatability.AIM To explore a method to improve the efficiency of ENAP,minimizing errors in ENAP processes,enhancing the reliability and repeatability of subsequent experimental results.METHODS A protocol for the sequential isolation of RNA,DNA,and proteins from the same cultured HepG2 cells using RNAzol reagent is presented here.The first step involves culturing HepG2 cells to the exponential phase,followed by the sequential isolation of RNA,DNA,and proteins from the same cultured cells in the second step.The yield of nucleic acids and proteins is detected in the third step,and their purity and integrity are verified in the last step.RESULTS The procedure takes as few as 3-4 d from the start to quality verification and is highly efficient.In contrast to the existing kits and reagents,which are primarily based on independent isolation,this RNAzol reagent-based method is characterized by the sequential isolation of RNA,DNA,and proteins from the same cells,and therefore saves time,and has low cost and high efficiency.CONCLUSION The RNA,DNA,and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction,polymerase chain reaction,and western blotting,respectively.

A novel primary culture method for high-purity satellite glial cells derived from rat dorsal root ganglion

Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO_2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100β. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100β, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.

Effect of culture methods on individual variation in the growth of sea cucumber Apostichopus japonicus within a cohort and family

There is substantial individual variation in the growth rates of sea cucumber Apostiehopus japonicus individuals. This necessitates additional work to grade the seed stock and lengthens the production period. We evaluated the influence of three culture methods （free-mixed, isolated-mixed, isolated-alone） on individual variation in growth and assessed the relationship between feeding, energy conversion efficiency, and individual growth variation in individually cultured sea cucumbers. Of the different culture methods, animals grew best when reared in the isolated-mixed treatment （i.e., size classes were held separately）, though there was no difference in individual variation in growth between rearing treatment groups. The individual variation in growth was primarily attributed to genetic factors. The difference in food conversion efficiency caused by genetic differences among individuals was thought to be the origin of the variance. The level of individual growth variation may be altered by interactions among individuals and environmental heterogeneity. Our results suggest that, in addition to traditional seed grading, design of a new kind of substrate that changes the spatial distribution of sea cucumbers would effectively enhance growth and reduce individual variation in growth of sea cucumbers in culture.

Modeling One Dimensional Two-Cell Model with Tumor Interaction Using Krylov Subspace Methods

A brain tumor occurs when abnormal cells grow, sometimes very rapidly, into an abnormal mass of tissue. The tumor can infect normal tissue, so there is an interaction between healthy and infected cell. The aim of this paper is to propose some efficient and accurate numerical methods for the computational solution of one-dimensional continuous basic models for the growth and control of brain tumors. After computing the analytical solution, we construct approximations of the solution to the problem using a standard second order finite difference method for space discretization and the Crank-Nicolson method for time discretization. Then, we investigate the convergence behavior of Conjugate gradient and generalized minimum residual as Krylov subspace methods to solve the tridiagonal toeplitz matrix system derived.

EVALUATION OF BIOCOMPATIBILITY OF MATERIALS BY A CELL CULTURE METHOD

Forty-seven kinds of materials were evaluated for their biocompatibility by a cell culture method of direct contact, including polyurethanes, silicone rubbers, polyvinyl chlorides, ethylene-vinyl acetate copolymers, polymethyl methacrylates, polyester, polypropylene, styrenebutadiene copolymer, thermoplastic elastomer, porcelains, and nickel chromium alloy, most of which were made in China as potential biomaterials. Most of the materials tested showed low cytotoxicity except polyvinyl chlorides and polyurethanes.

The Effect of Three Culture Methods on Intensive Culture System of Pacific White Shrimp (Litopenaeus vannamei)

Different culture methods may affect the intensive culture system of Pacific white shrimp(Litopenaeus vannamei) regarding water quality and growth and economic performance.This study evaluated the potential effects of three culture methods through cultivation of juvenile shrimps under consistent tank management conditions for 84 d.The three methods involved shrimp cultivation in different tanks,i.e.,outdoor tanks with cement bottom(mode-C),greenhouse tanks with cement bottom(mode-G) and outdoor tanks with mud-substrate(mode-M).Results showed that water temperature was significantly higher in mode-G than that in mode-C(P < 0.05).In contrast to the other two treatments,mode-M had stable pH after 50 d cultivation of shrimps.In the mid-late period,the average concentrations of TAN,NO2-N,DIP and COD were significantly lower in mode-M and mode-G compared with those in mode-C(P < 0.05).Despite lack of differences in the final shrimp weight among different treatments(P > 0.05),mode-M had significantly higher shrimp yield,survival rate and feed conversion rate(P < 0.05) than other modes.There were significant differences in revenue and net return among different treatments(P < 0.05).These demonstrated that the treatments of mode-G and mode-M were conductive to the intensive culture system of L.vannamei.

Study and promotion of safety culture using mixed methods research

Objective:With a positive safety culture,institutions offer the best quality and safe care to their patients.The objective of this study was to analyze patient safety culture from the perspective of the multidisciplinary team,to identify factors that influence patient safety culture,and to create/promote-jointly with the study participants-strategies for improving processes of change.Methods:The study design represented a mixed methods research approach,with a sequential explanatory design.A multidisciplinary team of workers at a general hospital was eligible for the study.To collect quantitative data,we administered the Safety Attitudes Questionnaire(SAQ).The qualitative phase was accomplished via focus groups(FGs),with participants from the first phase of the study using the principles of deliberative dialogue(DD)as a knowledge-translation strategy.The STROBE guideline was used to develop the study.Results:The overall SAQ score was positive(75.1±10.4).Negative scores were found in the fields of Safety Climate,Working Conditions,and Stress Recognition.Focus group discussions identified the aspects that create a negative impact on safety culture,such as ineffective communication,punitive approach in the event of errors,the lack of commitment and adherence to the protocols,and the non-recognition of the stress and the mistakes.Actions for the promotion of safety culture were developed and implemented during the study.Conclusions:The use of the principles of DD as a strategy for knowledge translation(KT)made it possible to identify and plan for joint actions to generate improvements in safety culture.

On C-E Translation Strategies and Methods of Zhiba Culture for Global Communication

Zhiba culture is a profound culture with a long history,but there are still many problems in the C-E translation of it,such as low popularity,few recipients,and so on,which hinder its communication and development.Therefore,it’s necessary to improve the quality of C-E translation of Zhiba culture.Based on the perspective of communication studies,combining with the principle of“Information First”,the principle of“The Audience First”,the paper explores the existing problems of Zhiba culture C-E translation and hopes to help to improve the present situation,to truly show the charm of Zhiba culture,to make it better accepted by the international community and optimize the global communication effect.

Biotransformation of Gastrodin by Cell Suspension Cultures of Catharanthus roseus

应用长春花 (Catharanthusroseus (L .)G .Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究。经过8d培养形成一个转化产物 ,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇 ,为天麻素水解后形成的甙元。

Productions of Taxol and Related Taxanes by Cell Suspension Cultures of Taxus yunnanensis

A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.