Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.RESULTSWe established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSIONThe maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

CLONAL PROLIFERATION AND LONG-TERM CULTURE OF MALIGNANT LYMPHOMA CELLS UNDER SERUM-FREE CULTURE CONDITIONS

We developed a serum-free culture system that promoted the growth of B cell colonies in peripheral blood, bone marrow, lymph nodes and cerebrospinal fluid (CSF) from 7 out of 8 patients with non-Hodgkin's lymphomas of B cell type. The culture cells were pretreated with or without galactose oxi-dase (GO) prior to plating. Colony growth was best supported with BCGF. A moderate increment was observed with rIL-3, as well as rIL-1β and even to a lesser degree, by rlL-2, while B cell stimulating factor-2 (rBCSF-2) and rlL-1β did not show significant activity. rGM-CSF and rG-CSF had little effect, while rM-CSF enhanced the formation of lymphoma colonies. The cells from different patients had different requirements for Staphylococcus aureus protein A and GO pretreatment. It reflected the differences in activation and differentiation status and surface properties of lymphoma cells from different patients. The cells from CSF of one patient were successfully maintained in serum-free culture medium supplemented with 10% BCGF or 5% PHA-LCM for more than 4 months. The long-term culture cells were EBV negative, phenotypically consistent with B cells and gene rearrangements for JH, Kappa and myc. This serum-free culture system allowed extensive analysis of the growth requirements for clonogenic precursors.

In vitro long-term culture and differentiation of mouse spermatogonia stem cells

The studies from 7 or 8 days old mice were used to prepare the spermatogonial stem cells. The isolation and purification of spermatogonial stem cells were done by means of the Percoll discontinual density gradient concentration. The cells of the 3rd band were collected and cultured in vitro in DMEM supplemented with 2.5% or 10% fetal bovine serum （FBS）. The results showed that with the feeder layer and 2.5% serum, the spermatogonial stem cells could proliferate, differentiate last more than 4 months.

6 Kinds of Isozymes after Long-term Subculture of Emmenopterys henryi Oliv.

[Objective] The aim was to study the changes of zymography in 6 kinds of isozymes after long-term subculture of Emmenopterys henryi Oliv.. [Method] Non-denaturing polyacrylamide gel electrophoresis was used to analyze isozyme patterns such as esterase (EST),acid phosphatase (ACP),ATP enzyme (ATPase),amylase (AMY),superoxide dismutase (SOD) and peroxidase (POD) in long-term subculture callus of Emmenopterys henryi Oliv. [Result] The research showed that there were differences among the 6 kinds of isozymes in embryogenic callus and non-embryogenic callus of Emmenopterys henryi Oliv.,and both levels could be taken as the basis for identification,the EST,ACP,and POD of non-embryogenic callus were significantly higher than embryogenic callus. The browning of non-embryogenic callus was non-level in the AMY,SOD and POD isozymes when was compared with normal non-embryogenic callus,while the EST,ACP and ATPase isozymes decreased; When the browning of embryogenic callus was contrasted with normal embryogenic callus,EST isozyme increased and the other 5 kinds of enzymes decreased. [Conclusion] The study provided theoretical basis for research morphological difference and browning of long-term tissue culture of Emmenopterys henryi Oliv..

Preparation of lactic acid bacteria compound starter cultures based on pasting properties and its improvement of glutinous rice flour and dough

The effects of 5 lactic acid bacteria(LAB)fermentation on the pasting properties of glutinous rice flour were compared,and suitable fermentation strains were selected based on the changes of viscosity,setback value,and breakdown value to prepare LAB compound starter cultures.The results revealed that Latilactobacillus sakei HSD004 and Lacticaseibacillus rhamnosus HSD005 had apparent advantages in increasing the viscosity and reducing the setback and breakdown values of glutinous rice flour.In particular,the compound starter created using the two abovementioned LAB in the ratio of 3:1 had better performance than that using a single LAB in improving the pasting properties and increasing the water and oil absorption capacity of glutinous rice flour.Moreover,the gelatinization enthalpy of the fermented samples increased significantly.For frozen glutinous rice dough stored for 28 days,the viscoelasticity of frozen dough prepared by compound starter was better than that of control dough,and the freezable water content was lower than that of control dough.These results indicate that compound LAB fermentation is a promising technology in the glutinous rice-based food processing industry,which has significance for its application.

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells(ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional(3-D) self-assembling scaffolds and compared with traditional two-dimentional(2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate(PEG-4-Acr) and thiolfunctionalized dextran(Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoB lue(PB) assays. Genetic expression of pluripotency markers(Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D cultureconditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining(Oct4 and Nanog) and western blot analysis(Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH(1:1 v/v) to a final concentration of 5%(w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels(P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury(mesoderm), NCAM(ectoderm), and GATA4(endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.

Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures(RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

Investigation of the Acceptability of Cultured Meat in University Students

Background: Over the past 20 years, cultured meat has drawn a lot of public attention as a potential solution to issues with animal husbandry, including inadequate use of natural sources, improper animal welfare practices, and possible risks to public health and safety. The novel method of producing meat through culture reduces the need for animals to produce muscle fiber, thereby obviating the necessity for animal slaughter. Apart from its ethical advantages, cultured meat presents a possible way to fulfill the expanding need for food among growing populations. The purpose of this research was to find out whether Turkish students would be willing to pay for and accept cultured meat. Technique: Method: 371 university students who willingly consented to fill out a questionnaire and provide demographic data make up the research sample. Questions from previous studies on the acceptability of cultured meat were compiled to create the survey. The research’s data collection took place in March and April of 2022. The research was completed in June 2022 after the data had been processed and analyzed. Results: The results showed that the majority of participants were female and had omnivorous eating habits. Based on the results of the Bonferroni correction test, students with a higher intention to purchase and consume cultured meat were those who received economics and business education. Students with two years of university education had a higher overall survey score than those with four years of education (p < 0.05). Furthermore, it is discovered that there is a negative correlation between the participants’ ages and their Factor 2 (using cultured meat as an alternative to industrial meat) and Factor 3 (consuming and purchasing it) section points (r = -109, p = 0.036) (r = -0.121, p = 0.019). In conclusion, university students generally have a negative outlook on health-related issues, such as eating cultured meat as an alternative.

The Philosophy of He(和):Examined from the Perspectives of Technology and Culture

Pre-modern Chinese crafts,such as iron smelting,cookery,medicine,and the production of vehicles,bows,and arrows indicate the traditional Chinese view of technology as being organic,holistic,and comprehensive.This view of technology is guided by the concept of he(和)and employs the means and methods of he,thus achieving the purport of he.In Chinese,the character he(和)possesses positive connotations.It originated from the meaning of"to season;to add flavoring to"(调和)and that of flavors being"perfectly harmonious"(和美).From this sensory experience,he gradually extended to the abstract levels of materiality,humanity,sociality,"order"(wei位),and "power,situation,force"(shi势).Finally,he was elevated to the supreme level of"qi of great harmony"(taihe zhi qi太和之气),which is comparable to the concept of dao(道).The philosophy of he has exerted wide impact on such areas as technology,art,national character,cultural psychology,and behavior patterns,and has become an integral part of China's inherent culture.The paradoxes and deviations of he hold their own profound philosophical implications that merit further exploration.As humanity confronts significant challenges,such as how we can coexist with others,with technology,and with nature,the ancient Eastern wisdom embodied in he continues to possess practical characteristics and value.

Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

Effects of Different Culture Conditions and Growth States on the Structure and Hemostatic Properties of Coscinodiscus sp.

As a renewable marine inorganic material,Coscinodiscus sp.has significant potential in the field of rapid hemostasis.However,the low yield of Coscinodiscus sp.seriously limits the application.In this study,two new culture modes were adopted to increase the production of Coscinodiscus sp.,the effect of changes in culture conditions and growth status on the hemostatic activity of diatoms was detected.To prevent Coscinodiscus sp.from sinking in culture,the suspension culture mode was realized by adding0.5%agar.The semi-continuous high nutrient concentration culture mode increased the cell density of Coscinodiscus sp.to 11000cells mL^(-1)and shorten the culture cycle to 5 d.In terms of coagulation activity,the addition of frustules reduced the in vitro coagulation time by half and the activation time of coagulation by 70%.The hemolysis rate and cytotoxicity of frustules harvested in the two culture modes did not change significantly.The results showed that suspension culture mode and high nutrient concentration culture mode only changed the growth state of Coscinodiscus sp.,while the hemostatic performance remained stable.

Current status and challenges for cell-cultured milk technology: a systematic review

Cellular agriculture is an innovative technology for manufacturing sustainable agricultural products as an alternative to traditional agriculture.While most cellular agriculture is predominantly centered on the production of cultured meat,there is a growing demand for an understanding of the production techniques involved in dairy products within cellular agriculture.This review focuses on the current status of cellular agriculture in the dairy sector and technical challenges for cell-cultured milk production.Cellular agriculture technology in the dairy sector has been classified into fermentation-based and animal cell culture-based cellular agriculture.Currently,various companies synthesize milk components through precision fermentation technology.Nevertheless,several startup companies are pursuing animal cell-based technology,driven by public concerns regarding genetically modified organisms in precision fermentation technology.Hence,this review offers an up-to-date exploration of animal cell-based cellular agriculture to produce milk components,specifically emphasizing the structural,functional,and productive aspects of mammary epithelial cells,providing new information for industry and academia.

An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells' autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.

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.

Happiness and harmony through health culture diplomacy as integrative maternal-child health nursing

For this editorial,our colleague Dr.Naeema Hasan Al Qasseer,former World Health Organization(WHO)Senior Scientist of Nursing and Midwifery joins me to add her wisdom and experience on the topic of integrative maternal-child health nursing.

Comparative Study of the Widal Test against Stool Culture in the Diagnosis of Suspected Cases of Typhoid Fever in Some Low Income Communities in the Adamawa Region of Cameroon

Introduction: Infectious diseases constitute a major concern of public health in developing countries. Facilities and well trained staff have been shown to be one of the major obstacles in the rapid and quality diagnosis of these diseases. As such, we carried out an analysis to compare the Widal test and stool culture to identify febrile patients with Salmonella infection. Method: A cross sectional study was conducted to diagnose salmonella infection with out-patients who demonstrated signs of salmonella infection. Serum was harvested from blood collected from 368 (Vina = 234, Mayo Banyo 65, and Djerem = 69) patients accompanied by stool, Widal test was conducted on the spot and stool was taken to a reference laboratory for culture using standard microbiological methods, sociological set up was calculated in percentages, prevalence was calculated using excel while statistical difference was calculated using SPSS. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to compare the Widal test against stool culture. Results: A total of 368 (50.8% females and 49.2% males) participants took part in the survey. Salmonella prevalence (66.24%) in stool culture in the Vina division was significantly different (p 0.05). The sensitivity,specificity, PPV, and NPV of slide agglutination test against stool culture varied from different areas (Vina: 51.6%, 73.62%, 79.21% and 43.61%;Mayo Banyo: 60.53%, 77.78%, 79.31% and 58.33%;Djerem: 53.18%, 83.73% 73.91% and 67.39%) respectively. Slide agglutination test has a fair agreement with the stool culture (kappa, Vina = 0.202;Mayo Banyo = 0.37 and Djerem = 0.38). Conclusion: Generally, in the three areas of study, the Widal test had a fair correlation with the stool culture;This means the Widal test should not be used alone but in combination with stool culture in the detection of salmonella infections.

Historiography and Scientific Research of Religious Buildings of the Cucuteni-Trypillian Culture

The purpose of this article is to reveal the historiography and current state of research related to the cult buildings of the Eneolithic period of the Cucuteni-Trypillian cultural community.The article describes the historical path of archaeological discoveries,from the first Trypillian sanctuaries to the discovery of the largest Nebelivka temple complex in Ukraine in 2012.At the same time,the work partially raises the issue of religious beliefs of the ancient farmers of Central Europe in connection with the discovery of their sacred buildings.Since special scientific works have not comprehensively addressed this issue and are still fragmentary in nature,it is important at the present stage of research to make some coverage and systematization of existing materials on this issue.

Tourism Mode of Tea Culture Museum in Guizhou:A Case Study of Meitan Modern Tea Culture Museum Settlement

Since the 14 th Five-Year Plan,tea culture has become very popular.However,due to the lack of brand awareness and overall planning,and the mechanical application of the model outside the province and other tourism products,Guizhou Tea Culture Tourism presents a"strong tea,light culture,weak tourism".In view of this,in order to realize the positioning goal of"building a national demonstration zone for the integration of tea culture and tourism"in the 14 th Five-Year Plan of Guizhou tea industry,and explore the development mode of integration of tea culture and tourism with Guizhou characteristics,this study takes museum tourism as the breakthrough point,Meitan Tea Museum settlement as the research object,and collects Meitan Tea Museum tourism resources.This paper uses SWOT analysis to evaluate the Meitan Tea Culture Museum Tourism model,and provides some recommendations for the development of tea culture museum tourism in Guizhou Province.

AIGC Related Context: A New Communication Culture For Human

The emergence and rapid development of Artificial Intelligence Generated Content(AIGC)technology is revolutionizing human communication and cultural landscapes.This paper explores the impact of AIGC on society,identifying three distinct cultural paradigms:AIGC Blended Culture,AIGC Based Culture,and AIGC Polluted Culture.AIGC Blended Culture represents a synergistic collaboration between AI and human creativity,enhancing content creation efficiency,promoting cross-cultural communication,and enriching human experiences.AIGC Based Culture signifies a shift where AI becomes the core driver of cultural production and dissemination.This leads to innovative cultural industry models,expanded development fields,and improved cultural product quality and diversity.However,AIGC Polluted Culture arises from the misuse of AIGC to produce or disseminate harmful content.The paper proposes countermeasures and recommendations to maximize the benefits of AIGC while mitigating potential risks.This involves fostering collaboration between AI and humans,focusing on talent cultivation,establishing and improving laws and regulations,and developing solutions to detect and mitigate harmful content.Additionally,promoting responsible innovation and ethical AI development is crucial.In conclusion,AIGC represents a transformative force reshaping human communication and culture.While offering immense opportunities for innovation and efficiency,it also presents challenges that require careful consideration and proactive measures to ensure its positive and sustainable impact on society.

Tourism-Oriented Cultural Product Design: A Case Study of Yami Culture in Lanyu

Due to the prevalence of modern commerce and mainstream culture, many indigenous cultures around the world are rapidly diminishing. These cultures, with their unique primitiveness distinct from modern culture, often attract tourists. This study integrates the indigenous Yami culture of Lanyu Island, Taiwan Region, into innovative designs from the perspective of tourists’ understanding of the local culture. In this way, tourists can experience the harmonious coexistence of the Yami tribe and nature, while these culturally rich products foster deeper experiential and emotional resonance. This study employs the Mandala thinking method for concept expansion and the Osborn checklist method for the analysis and transformation of design elements. Subsequently, designers were invited to conduct on-site travel experiences, leading to the creation of a series of Yami cultural products. Through the evaluation and validation of cultural product assessment indicators, functional commercialized products were ultimately produced. This study aims to establish a design process for cultural products through this design case. It seeks to help users recall their Lanyu Island travel experiences and appreciate the often-overlooked cultural beauty, thereby raising awareness of cultural preservation.