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.

Three-dimensional culture system enhance the differentiation of mesenchymal stem cells into neural cells in vitro

Nerve injury repair has been an intractable issue wor.ldwide due to the non-renew able property of neurons in vivo.Cell therapy adopting me senchymal stem cells(MSC)induced neurons stands for a promising substitute treatment,which is hindered by the low diffe rentiation efficiency of current approaches.To improve the efficiency of MSC differentiation in vitro,we constructed a three-dimensional(3D)culture system by seeding human bone marrow-de rived MSC(hBMSCs)into collagen-nano fiber scaffolds which are a mixed hydrogel of collagen 1 and silk nanofiber solution and testified the diffe rentiation efficiency from MSC into neurons by chemical or cocktail induction methods.Results indicated MSCs cultured in 3D system showed good viability with neuron-like morphology in both groups.

Hydrogel-supported poly(L-lactic acid) and polystyrene microsphere-based three-dimensional culture systems for in vitro cell expansion

The in vitro expansion of stem cells is important for their application in different life science fields such as cellular tissue and organ repair.An objective of this paper was to achieve static cell culture in vitro through peptide hydrogel-supported microspheres(MSs).The peptides,with their gel-forming properties,microstructures,and mechanical strengths characterized,were found to have good support for the MSs and to be injectable.The internal structures of poly(L-lactic acid)microspheres(PLLA-MSs)and polystyrene microspheres(PS-MSs)made in thelaboratory were observed and statistically analyzed in terms of particle size and pore size,following which the co-cultured MSs with cells were found to have good cell adhesion.In addition,three-dimensional(3D)culturing of cells was performed on the peptide and microcarrier composite scaffolds to measure cell viability and cell proliferation.The results showed that the peptides could be stimulated by the culture medium to self-assembly form a 3D fiber network structure.Under the peptide-Ms composite scaffold-based cell culture system,further enhancement of the cell culture effect was measured.The peptide-Ms composite scaffolds have great potential for the application in 3D cell culture and in vitro cellexpansion.

Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey （Macaca mulatta）

In vitro culture of spermatogonial stem cells （SSCs） has generally been performed using two-dimensional （2D） culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules （including those generated by the SSC niche） and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system （SACS） and a 3D methylcellulose culture system （MCS）. It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we （1） provide a brief overview of the differences in spermatogenesis in rodents and primates, （2） summarize data related to attempts to generate sperm in vitro, （3） report for the first time formation of colonies/clusters of cells and differentiation of meiotic （expression of CREM-1） and postmeiotic （expression of acrosin） germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and （4） indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.

Oxygen tension modulates cell function in an in vitro three-dimensional glioblastoma tumor model

Hypoxia is a typical feature of the tumor microenvironment,one of the most critical factors affecting cell behavior and tumor progression.However,the lack of tumor models able to precisely emulate natural brain tumor tissue has impeded the study of the effects of hypoxia on the progression and growth of tumor cells.This study reports a three-dimensional(3D)brain tumor model obtained by encapsulating U87MG(U87)cells in a hydrogel containing type I collagen.It also documents the effect of various oxygen concentrations(1%,7%,and 21%)in the culture environment on U87 cell morphology,proliferation,viability,cell cycle,apoptosis rate,and migration.Finally,it compares two-dimensional(2D)and 3D cultures.For comparison purposes,cells cultured in flat culture dishes were used as the control(2D model).Cells cultured in the 3D model proliferated more slowly but had a higher apoptosis rate and proportion of cells in the resting phase(G0 phase)/gap I phase(G1 phase)than those cultured in the 2D model.Besides,the two models yielded significantly different cell morphologies.Finally,hypoxia(e.g.,1%O2)affected cell morphology,slowed cell growth,reduced cell viability,and increased the apoptosis rate in the 3D model.These results indicate that the constructed 3D model is effective for investigating the effects of biological and chemical factors on cell morphology and function,and can be more representative of the tumor microenvironment than 2D culture systems.The developed 3D glioblastoma tumor model is equally applicable to other studies in pharmacology and pathology.

Three-dimensional cell-based strategies for liver regeneration

Liver regeneration and the development of effective therapies for liver failure remain formidable challenges in modern medicine.In recent years,the utilization of 3D cell-based strategies has emerged as a promising approach for addressing these urgent clinical requirements.This review provides a thorough analysis of the application of 3D cell-based approaches to liver regeneration and their potential impact on patients with end-stage liver failure.Here,we discuss various 3D culture models that incorporate hepatocytes and stem cells to restore liver function and ameliorate the consequences of liver failure.Furthermore,we explored the challenges in transitioning these innovative strategies from preclinical studies to clinical applications.The collective insights presented herein highlight the significance of 3D cell-based strategies as a transformative paradigm for liver regeneration and improved patient care.

The combined application of stem cells and three-dimensional bioprinting scaffolds for the repair of spinal cord injury

Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and the generation of new scars can make it very difficult for the impaired nervous system to restore its neural functionality.Traditional treatments can only alleviate secondary injuries but cannot fundamentally repair the spinal cord.Consequently,there is a critical need to develop new treatments to promote functional repair after spinal cord injury.Over recent years,there have been seve ral developments in the use of stem cell therapy for the treatment of spinal cord injury.Alongside significant developments in the field of tissue engineering,three-dimensional bioprinting technology has become a hot research topic due to its ability to accurately print complex structures.This led to the loading of three-dimensional bioprinting scaffolds which provided precise cell localization.These three-dimensional bioprinting scaffolds co uld repair damaged neural circuits and had the potential to repair the damaged spinal cord.In this review,we discuss the mechanisms underlying simple stem cell therapy,the application of different types of stem cells for the treatment of spinal cord injury,and the different manufa cturing methods for three-dimensional bioprinting scaffolds.In particular,we focus on the development of three-dimensional bioprinting scaffolds for the treatment of spinal cord injury.

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.

A theory for three-dimensional response of micropolar plates

Through combined applications of the transfer-matrix method and asymptotic expansion technique,we formulate a theory to predict the three-dimensional response of micropolar plates.No ad hoc assumptions regarding through-thickness assumptions of the field variables are made,and the governing equations are two-dimensional,with the displacements and microrotations of the mid-plane as the unknowns.Once the deformation of the mid-plane is solved,a three-dimensional micropolar elastic field within the plate is generated,which is exact up to the second order except in the boundary region close to the plate edge.As an illustrative example,the bending of a clamped infinitely long plate caused by a uniformly distributed transverse force is analyzed and discussed in detail.

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.

Development of a toroidal soft x-ray imaging system and application for investigating three-dimensional plasma on J-TEXT

A toroidal soft x-ray imaging(T-SXRI)system has been developed to investigate threedimensional(3D)plasma physics on J-TEXT.This T-SXRI system consists of three sets of SXR arrays.Two sets are newly developed and located on the vacuum chamber wall at toroidal positionsφof 126.4°and 272.6°,respectively,while one set was established previously atφ=65.50.Each set of SXR arrays consists of three arrays viewing the plasma poloidally,and hence can be used separately to obtain SXR images via the tomographic method.The sawtooth precursor oscillations are measured by T-SXRI,and the corresponding images of perturbative SXR signals are successfully reconstructed at these three toroidal positions,hence providing measurement of the 3D structure of precursor oscillations.The observed 3D structure is consistent with the helical structure of the m/n=1/1 mode.The experimental observation confirms that the T-SXRI system is able to observe 3D structures in the J-TEXT plasma.

Intraoperative application of three-dimensional printed guides in total hip arthroplasty: A systematic review

BACKGROUND Acetabular component positioning in total hip arthroplasty(THA)is of key importance to ensure satisfactory post-operative outcomes and to minimize the risk of complications.The majority of acetabular components are aligned freehand,without the use of navigation methods.Patient specific instruments(PSI)and three-dimensional(3D)printing of THA placement guides are increasingly used in primary THA to ensure optimal positioning.AIM To summarize the literature on 3D printing in THA and how they improve acetabular component alignment.METHODS PubMed was used to identify and access scientific studies reporting on different 3D printing methods used in THA.Eight studies with 236 hips in 228 patients were included.The studies could be divided into two main categories;3D printed models and 3D printed guides.RESULTS 3D printing in THA helped improve preoperative cup size planning and post-operative Harris hip scores between intervention and control groups(P=0.019,P=0.009).Otherwise,outcome measures were heterogeneous and thus difficult to compare.The overarching consensus between the studies is that the use of 3D guidance tools can assist in improving THA cup positioning and reduce the need for revision THA and the associated costs.CONCLUSION The implementation of 3D printing and PSI for primary THA can significantly improve the positioning accuracy of the acetabular cup component and reduce the number of complications caused by malpositioning.

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.

Computer-assisted three-dimensional individualized extreme liver resection for hepatoblastoma in proximity to the major liver vasculature

BACKGROUND The management of hepatoblastoma(HB)becomes challenging when the tumor remains in close proximity to the major liver vasculature(PMV)even after a full course of neoadjuvant chemotherapy(NAC).In such cases,extreme liver resection can be considered a potential option.AIM To explore whether computer-assisted three-dimensional individualized extreme liver resection is safe and feasible for children with HB who still have PMV after a full course of NAC.METHODS We retrospectively collected data from children with HB who underwent surgical resection at our center from June 2013 to June 2023.We then analyzed the detailed clinical and three-dimensional characteristics of children with HB who still had PMV after a full course of NAC.RESULTS Sixty-seven children diagnosed with HB underwent surgical resection.The age at diagnosis was 21.4±18.8 months,and 40 boys and 27 girls were included.Fifty-nine(88.1%)patients had a single tumor,39(58.2%)of which was located in the right lobe of the liver.A total of 47 patients(70.1%)had PRE-TEXT III or IV.Thirty-nine patients(58.2%)underwent delayed resection.After a full course of NAC,16 patients still had close PMV(within 1 cm in two patients,touching in 11 patients,compressing in four patients,and showing tumor thrombus in three patients).There were 6 patients of tumors in the middle lobe of the liver,and four of those patients exhibited liver anatomy variations.These 16 children underwent extreme liver resection after comprehensive preoperative evaluation.Intraoperative procedures were performed according to the preoperative plan,and the operations were successfully performed.Currently,the 3-year event-free survival of 67 children with HB is 88%.Among the 16 children who underwent extreme liver resection,three experienced recurrence,and one died due to multiple metastases.CONCLUSION Extreme liver resection for HB that is still in close PMV after a full course of NAC is both safe and feasible.This approach not only reduces the necessity for liver transplantation but also results in a favorable prognosis.Individualized three-dimensional surgical planning is beneficial for accurate and complete resection of HB,particularly for assessing vascular involvement,remnant liver volume and anatomical variations.

Three-dimensional numerical analysis of plant-soil hydraulic interactions on pore water pressure of vegetated slope under different rainfall patterns

Understanding the pore water pressure distribution in unsaturated soil is crucial in predicting shallow landslides triggered by rainfall,mainly when dealing with different temporal patterns of rainfall intensity.However,the hydrological response of vegetated slopes,especially three-dimensional(3D)slopes covered with shrubs,under different rainfall patterns remains unclear and requires further investigation.To address this issue,this study adopts a novel 3D numerical model for simulating hydraulic interactions between the root system of the shrub and the surrounding soil.Three series of numerical parametric studies are conducted to investigate the influences of slope inclination,rainfall pattern and rainfall duration.Four rainfall patterns(advanced,bimodal,delayed,and uniform)and two rainfall durations(4-h intense and 168-h mild rainfall)are considered to study the hydrological response of the slope.The computed results show that 17%higher transpiration-induced suction is found for a steeper slope,which remains even after a short,intense rainfall with a 100-year return period.The extreme rainfalls with advanced(PA),bimodal(PB)and uniform(PU)rainfall patterns need to be considered for the short rainfall duration(4 h),while the delayed(PD)and uniform(PU)rainfall patterns are highly recommended for long rainfall durations(168 h).The presence of plants can improve slope stability markedly under extreme rainfall with a short duration(4 h).For the long duration(168 h),the benefit of the plant in preserving pore-water pressure(PWP)and slope stability may not be sufficient.

Reviews on Post-earthquake Reconstruction of Qiang Culture: Three-dimensional Pattern

Through the introduction of disaster situation of Qiang Culture after Wenchuan Earthquake, the paper emphasized that carriers of Qiang Culture had been seriously damaged, the inheritance of Qiang Culture had been affected, and the environment for Qiang Culture was difficult to recover. It highlighted that three-dimensional reconstruction of Qiang Culture should stress the core task and timely and effectively rescue endangered cultural heritages of Qiang Nationality from the perspectives of material and spiritual life. It had explained focuses of three-dimensional pattern construction in detail. In terms of spatial reconstruction, it should reconstruct native culture and history while material culture was constructed, and reconstruct Qiang culture highland by depending on aborigines; in terms of cluster reconstruction, it should give support to large tourism enterprises and perfect tourism chain; in terms of ecological reconstruction, it should enhance construction and demonstration of "ecological protection pilot area of Qiang culture"; in terms of development reconstruction, it should realize coordinated unity between protection and development according to classification protection, characteristic protection and key protection, so as to form the virtuous circle of post-disaster recovery protection and sustainable development.

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.

The use of carbon-based particle electrodes in three-dimensional electrode reactors for wastewater treatment

The use of three-dimensional(3D)electrodes in water treatment is competitive because of their high catalytic efficiency,low energy consumption and promising development.The use of particle electrodes is a key research focus in this technology.They are usually in the form of particles that fill the space between the cathode and anode,and the selection of materials used is important.Carbon-based materials are widely used because of their large specific surface area,good adsorption performance,high chemical stability and low cost.The principles of 3D electrode technology are introduced and recent research on its use for degrading organic pollutants using carbon-based particle electrodes is summarized.The classification of particle electrodes is introduced and the challenges for the future development of carbon-based particle electrodes in wastewater treatment are discussed.

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.

Three-dimensional visualization technology for guiding one-step percutaneous transhepatic cholangioscopic lithotripsy for the treatment of complex hepatolithiasis

BACKGROUND Biliary stone disease is a highly prevalent condition and a leading cause of hospitalization worldwide.Hepatolithiasis with associated strictures has high residual and recurrence rates after traditional multisession percutaneous transhepatic cholangioscopic lithotripsy(PTCSL).AIM To study one-step PTCSL using the percutaneous transhepatic one-step biliary fistulation(PTOBF)technique guided by three-dimensional(3D)visualization.METHODS This was a retrospective,single-center study analyzing,140 patients who,between October 2016 and October 2023,underwent one-step PTCSL for hepatolithiasis.The patients were divided into two groups:The 3D-PTOBF group and the PTOBF group.Stone clearance on choledochoscopy,complications,and long-term clearance and recurrence rates were assessed.RESULTS Age,total bilirubin,direct bilirubin,Child-Pugh class,and stone location were similar between the 2 groups,but there was a significant difference in bile duct strictures,with biliary strictures more common in the 3D-PTOBF group(P=0.001).The median follow-up time was 55.0(55.0,512.0)days.The immediate stone clearance ratio(88.6%vs 27.1%,P=0.000)and stricture resolution ratio(97.1%vs 78.6%,P=0.001)in the 3D-PTOBF group were significantly greater than those in the PTOBF group.Postoperative complication(8.6%vs 41.4%,P=0.000)and stone recurrence rates(7.1%vs 38.6%,P=0.000)were significantly lower in the 3D-PTOBF group.CONCLUSION Three-dimensional visualization helps make one-step PTCSL a safe,effective,and promising treatment for patients with complicated primary hepatolithiasis.The perioperative and long-term outcomes are satisfactory for patients with complicated primary hepatolithiasis.This minimally invasive method has the potential to be used as a substitute for hepatobiliary surgery.