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.

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.

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.

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.

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.

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.

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

Chemical Constituents of the Suspension Cell Cultures of Maytenus hookeri

Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.

Study on the Browning in Cell Suspension Culture of Taxus cuspidata

[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.

Detection of Mycoplasma Contamination in Animal Cell Cultures

[ Objective ] To develop a rapid efficient method for detecting mycoplasma contamination in cell cultures. [ Method] A pair of primers was designed according to two highly conserved nucleotide sequences of the 16S RNA from six kinds of mycoplasma that commonly contaminated cells. Then the mycoplasma contamination of 25 cell samples was defected by PCR and DNA fluorescence staining. EResultl When these cell samples were detected by DNA fluorescence staining, the positive rate and probable positive rate were respectively 24% and 16%. And when they were detected by PCR, the positive rate was 36%. [ Condusion] The PCR method is more sensitive and specific than the DNA fluorescence staining, and combining these two methods is the optimal way to detect mycoplasma contamination in cell cultures.

MORPHOLOGICAL MODEL FOR RHEOLOGICAL PROPERTIES OF PLANT CELL SUSPENSION CULTURE SYSTEM

This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheological properties of the system as the effect of the flow behavior index on plant cell concentration are interpreted correctly and the mechanism of the rheological properties of the system is further understood.Therefore the model can be applied in the technological design and optimum conditions of the system and the reformation,evaluation and scale up of reactors.

Selective 3-OH Isomerization of Resibufogenin by Cell Suspension Cultures of Ginkgo biloba

Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus was then transferred aseptically to liquid MSmedium exoge-nously supplemented with appropriate concentration of 6-BA, NAA and 2,4-D to establishsuspension cell culture system. Resibufogenin was administered into the well-grown cell cultures andincubated for 4 d. The products dissolved in the liquid phase of the cultures were extracted andpurified by silica gel column chromatography gradiently eluted with petroleum ether and acetonesystem. Results One transformed product was obtained in 40% yield after 4 d incubation, which wasidentified as 3-epi-resibufogenin on the basis of FAB MS, ~1H NMR and ^(13)C NMR spectroscopicanalysis and corresponding data reported in literature. Conclusion G. biloba suspension cultures canbe used as an enzyme system to biotransform resibufogenin, an animal-originated bufadienolide, into3-epi-resibufogenin.

Cultured meat from muscle stem cells: A review of challenges and prospects

Growing muscle tissue in culture from animal stem cells to produce meat theoretically eliminates the need to sacrifice animals. So-called ＂cultured＂ or ＂synthetic＂ or ＂in vitro＂ meat could in theory be constructed with different characteristics and be produced faster and more efficiently than traditional meat. The technique to generate cultured muscle tissues from stem cells was described long ago, but has not yet been developed for the commercial production of cultured meat products. The technology is at an early stage and prerequisites of implementation include a reasonably high level of consumer acceptance, and the development of commercially-viable means of large scale production. Recent advancements in tissue culture techniques suggest that production may be economically feasible, provided it has physical properties in terms of colour, flavour, aroma, texture and palatability that are comparable to conventional meat. Although considerable progress has been made during recent years, important issues remain to be resolved, including the characterization of social and ethical constraints, the fine-tuning of culture conditions, and the development of culture media that are cost-effective and free of animal products. Consumer acceptance and confidence in in vitro produced cultured meat might be a significant impediment that hinders the marketing process.

Experimental study of bioartificial liver with cultured human liver cells

AIM To establish an extracorporeal bioartificial liver support system (EBLSS) using cultured human liver cells and to study its support effect for fulminant hepatic failure (FHF).METHODS The liver support experiment of EBLSS consisting of aggregates cultured human liver cells, hollow fiber bioreactor, and circulation unit was carried out in dizhepatic dogs.RESULTS The viability of isolated hepatocytes and nonparenchymal liver cells reached 96%. These cells were successfully cultured as multicellular spheroids with synthetic technique. The typical morphological appearance was retained up to the end of the artificial liver experiment. Compared with the control dogs treated with EBLSS without liver cells, the survival time of artificial liver support dogs was significantly prolonged. The changes of blood pressure, heart rate and ECG were slow. Both serum ammonia and lactate levels were significantly lowered at the 3rd h and 5th h. In addition, a good viability of human liver cells was noted after 5 h experiment.CONCLUSION EBLSS playing a metabolic role of cultured human hepatocytes, is capable of compensating the function of the liver, and could provide effective artificial liver support and therapy for patients with FHF.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

Protective Effects of Quercetin on Cadmium-induced Cytotoxicity in Primary Cultures of Rat Proximal Tubular Cells

Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular （rPT） cells. Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 ug/mL quercetin and/or cadmium （2.5, 5.0 umol/L）, in a serum-free medium at 37℃ at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress. Results Exposure of rPT cells to cadmium acetate （2.5, 5.0 umol/L） induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na＋, K＋ -ATPase, Ca2＋ -ATPase, glutathione peroxidase （GSH-Px）, catalase （CAT）, and superoxide dismutase （SOD） activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 ug/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants （non-enzymatic and enzymic） levels. Conclusion The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.