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.

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.

Tailoring Iron-Ion Release of Cellulose-Based Aerogel-Coated Iron Foam for Long-Term High-Power Microbial Fuel Cells

The presence of iron(Fe) has been found to favor power generation in microbial fuel cells(MFCs). To achieve long-term power production in MFCs, it is crucial to effectively tailor the release of Fe ions over extended operating periods. In this study, we developed a composite anode(A/IF) by coating iron foam with cellulose-based aerogel. The concentration of Fe ions in the anode solution of A/IF anode reaches 0.280 μg/mL(Fe^(2+) vs. Fe^(3+) = 61%:39%) after 720 h of aseptic primary cell operation. This value was significantly higher than that(0.198 μg/mL, Fe^(2+) vs. Fe^(3+) = 92%:8%) on uncoated iron foam(IF), indicating a continuous release of Fe ions over long-term operation. Notably, the resulting MFCs hybrid cell exhibited a 23% reduction in Fe ion concentration(compared to a 47% reduction for the IF anode) during the sixth testing cycle(600-720 h). It achieved a high-power density of 301 ± 55 mW/m^(2) at 720 h, which was 2.62 times higher than that of the IF anode during the same period. Furthermore, a sedimentary microbial fuel cell(SMFCs) was constructed in a marine environment, and the A/IF anode demonstrated a power density of 103 ± 3 mW/m^(2) at 3240 h, representing a 75% improvement over the IF anode. These findings elucidate the significant enhancement in long-term power production performance of MFCs achieved through effective tailoring of Fe ions release during operation.

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.

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.

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.

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.